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N Hoffman Dr. Neal Hoffman, Medical Director of the Adolescent AIDS Program at Montefiore Hospital, discusses special care considerations for HIV-positive adolescents. General health maintenance and STD treatment encompass about 75 percent of the care provided through the clinic. The program has been expande... ( view more )d to serve people up to the age of 24, unlike many programs which end at 21. Dr. Hoffman believes that many in the program are just developing their sense of trust in the doctors and it is counterproductive to arbitrarily eliminate them from the program. Teenagers tend to be less compliant about taking their medications, and have great fears of disclosing their HIV status to partners and family members. Mandatory partner notification programs are not a viable alternative, as many of the clients have limited sexual experience and their confidentiality is compromised by telling their partners. The program is part of the AMHARN study, a five-year longitudinal study of disease progression in adolescent cohorts funded by the National Institutes of Health (NIH). ( view less ) D R Hoffman A series of control, IgE myeloma containing and atopic sera were tested by the radioallergosorbent test using identical lots of commercially available reagents following three different RAST protocols: Phadebas, Fadal-Nalebuff and that used in our own laboratories. In addition, a new RAST tracer wa... ( view more )s evaluated in the Phadebas and Fadal-Nalebuff methods. The Phadebas technique, using the standard 0.35 PRU cut-off, was the least sensitive at 68% but its sensitivity was raised to 84% using an 0.14 PRU cut-off. The specificity was 100% at 0.35 and 95% at 0.14 PRU. Our laboratory's technique gave a sensitivity of 84% and a specificity of 100%. The Fadal-Nalebuff method had the highest sensitivity at 92% but the lowest specificity at 92%. The Fadal-Nalebuff technique leads to a significant number of false positive scores, particularly in sera with IgE > 500 U/ml or with RAST discs that have significant non-specific binding. The Fadal-Nalebuff method can be used reliably if modified to include suitable negative controls for each antigen tested. ( view less ) T McDonald,W E Hoffman,R Berkowitz During rapid opioid detoxification, increased sympathetic activity and a greater median frequency (MF) of activity on electroencephalography (EEG) have been reported. The purpose of this study was to evaluate a new index for detoxification that combines sympathetic activity and MF data. After infor... ( view more )med consent was obtained, eight patients were sedated with propofol. The MF of EEG activity derived from frontal electrodes was determined. Heart rate variability was evaluated in 256-second segments by power spectral analysis, and sympathetic activity was determined by the low frequency component. The Hoffman Index for narcotic detoxification was weighted 70% to sympathetic activity and 30% to MF to normalize the difference in scales and to provide adequate weight to the sympathetic component. Opioid detoxification was produced by infusion of 25 mg naloxone for 30 minutes, followed by a 24-hour infusion of 1 mg per hour. The MF showed a rapid increase during high-dose infusion of naloxone, but the peak response occurred 1 to 2 hours later. Sympathetic activation and the Hoffman Index increased more slowly after the start of naloxone infusion, but peak increases in all components occurred at approximately the same time. The peak increases in Hoffman Index (110% of baseline), MF (260%), and sympathetic activity (304%) during administration of naloxone were significant and correlated with respect to time (r = 0.89-0.94). The Hoffman Index showed an early increase related to MF and a well-defined peak response indicative of sympathetic and MF activity. The behavior of the Hoffman Index in relation to the MF and sympathetic activity more clearly indicated the onset of opioid detoxification and the maximum response to opioid reversal than did MF or sympathetic activity alone. ( view less ) Judith M Nocek,Shelby L Hatch,Jennifer L Seifert,Gregory W Hunter,David D Thomas,Brian M Hoffman In this paper, we describe the first observations of photoinitiated interprotein electron transfer (ET) within sol-gels. We have encapsulated three protein-protein complexes, specifically selected because they represent a full range of affinities, are sensitive to different types of dynamic process... ( view more )es, and thus are expected to respond differently to sol-gel encapsulation. The three systems are (i) the [Zn, Fe(3+)L] mixed-metal hemoglobin hybrids, where the alpha(1)-Zn and beta(2)-Fe subunits correspond to a "predocked" protein-protein complex with a crystallographically defined interface (Natan, M. J.; Baxter, W. W.; Kuila, D.; Gingrich, D. J.; Martin, G. S.; Hoffman, B. M. Adv. Chem. Ser. 1991, 228 (Electron-Transfer Inorg., Org., Biol. Syst.), 201-213), (ii) the Zn-cytochrome c peroxidase complex with cytochrome c, [ZnCcP, Fe(3+)Cc], having an intermediate affinity between its partners (Nocek, J. M.; Zhou, J. S.; De Forest, S.; Priyadarshy, S.; Beratan, D. N.; Onuchic, J. N.; Hoffman, B. M. Chem. Rev. 1996, 96, 2459-2489), and (iii) the [Zn-deuteromyoglobin, ferricytochrome b(5)] complex, [ZnDMb, Fe(3+)b(5)], which is loosely bound and highly dynamic (Liang, Z.-X.; Nocek, J.; Huang, K.; Hayes, R. T.; Kurnikov, I. V.; Beratan, D. N.; Hoffman, B. M. J. Am. Chem. Soc. 2002, 124, 6849-6859. Intersubunit ET within the hybrid does not involve second-order processes or subunit rearrangements, and thus is influenced only by perturbations of high-frequency motions coupled to ET. For the latter two complexes, sol-gel encapsulation eliminates second-order processes: protein partners encapsulated as a complex must stay together throughout a photoinitiated ET cycle, while proteins encapsulated alone cannot acquire a partner. It further modulates intracomplex motions of the two partners. ( view less ) P Hoffman,M B Yaffe,B L Hoffman,S Yei,W S Wold,C Carlin We have characterized the biosynthesis and processing of a 91 amino acid hydrophobic integral membrane protein encoded by human group C adenoviruses which down-regulates the EGF receptor (Carlin, C. R., Tollefson, A. E., Brady, H. A., Hoffman, B. L., and Wold, W. S. M. (1989) Cell 57, 135-144). Pre... ( view more )vious studies have shown that two immunologically related proteins are produced in vivo, a 13.7-kDa protein encoded by E3 message f and a 11.3-kDa protein derived from 13.7 kDa by proteolysis (Hoffman, B. L., Ullrich, A., Wold, W. S. M., and Carlin, C. R. (1990) Mol. Cell. Biol. 10, 5521-5524; Tollefson, A. E., Krajcsi, P., Yei, S., Carlin, C. R., and Wold, W. S. M. (1990) J. Virol. 64, 794-801). We report here that the 13.7- and 11.3-kDa proteins form intermolecular disulfide bonds cotranslationally at Cys-31 and tend to migrate as high molecular weight aggregates under nonreducing conditions. Both proteins are also present at the cell surface, as evidenced by specific immunoprecipitation from intact monolayers enzymatically labeled with 125I. Moreover, an antiserum specific for a putative extracellular epitope recognizes the same viral proteins as antibodies directed against a C-terminal synthetic 15-mer. The 13.7- and 11.3-kDa proteins are detected at early time points during pulse-chase radiolabeling of infected cells, do not undergo any further changes in molecular weight, and focus at their predicted isoelectric points (7.4 and 7.2, respectively). Identical results are obtained in stable transfectants constitutively expressing only 13.7 and 11.3 kDa, suggesting that biosynthesis and processing is not dependent on other viral proteins. These results have been incorporated into a computer-based model to predict the orientation of 13.7 and 11.3 kDa in the lipid bilayer. This model provides a basis for testing predictions regarding the topology of the viral proteins, as well as putative interactions with heterologous proteins in the microenvironment of the plasma membrane that cause down-regulation of the epidermal growth factor receptor. ( view less ) P Hoffman,P Rajakumar,B Hoffman,R Heuertz,W S Wold,C R Carlin We have reported previously that human group C adenoviruses down-regulate the epidermal growth factor (EGF) receptor (EGF-R) (C. R. Carlin, A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. S. M. Wold, Cell 57:135-144, 1989). Expression of a 13.7-kDa protein encoded by a gene in the E3 transcript... ( view more )ion unit is necessary and sufficient for this effect (Carlin et al., Cell, 1989; B. L. Hoffman, A. Ullrich, W. S. M. Wold, and C. R. Carlin, Mol. Cell. Biol. 10:5521-5524, 1990). We show here that EGF-R down-regulation is accelerated in cells which overexpress the receptor when these cells are infected with virus mutants that overproduce the 13.7-kDa protein compared with wild-type virus. This is in contrast to EGF stimulation, for which others have shown that high concentrations of ligand are associated with low rates of receptor internalization in EGF-R-overexpressing cells (D. Kuppuswamy and L. J. Pike, J. Biol. Chem. 264:3357-3363, 1989; H. S. Wiley, J. Cell Biol. 107:801-810, 1988). We also show that the E3 protein is not present in media conditioned by infected cells and that it does not induce secretion of an EGF-like autocrine factor. Moreover, while mature membrane-bound EGF-R is down-regulated, the precursor of the membrane-bound form is not. Adenovirus infection also does not affect receptor-related molecules expressed in the secretory pathway. Interestingly, adenovirus-induced down-regulation is not regulated by concentrations of EGF associated with a slow rate of internalization in A431 cells. This suggests that 13.7-kDa protein expression triggers receptor entry by a novel ligand-independent pathway or, alternatively, that it compensates for a cellular factor that may be rate limiting during EGF-mediated endocytosis. ( view less ) H J Kim,B R Zeeberg,F H Fahey,A N Bice,E J Hoffman,R C Reba We have developed a three-dimensional computer simulation of SPECT imaging. We have applied the simulation procedure to the realistic mathematical Hoffman three-dimensional brain model to generate the projection data (in the absence of attenuation, scatter, or noise) of both a parallel-hole and a m... ( view more )ultidetector SPECT system with point-focusing collimators. The simulated projection data were then reconstructed using standard software. The projection data resulting from the distribution of grey matter alone, or grey and white matter, were simulated. The results of these simulations indicate the existence of significant qualitative and quantitative artifacts in reconstructed human brain images. For example, the reconstructed values for grey matter along a cortical circumferential profile in a transverse slice through the basal ganglia varied by a factor of 2.40 (parallel-hole) and 2.99 (point-focusing), although the original grey matter values were identical in all cortical regions in the model. We have compared the simulated reconstructed images with those obtained by imaging the physical three-dimensional Hoffman brain phantom, which was constructed based upon the same set of data from which the mathematical three-dimensional Hoffman brain model was derived. Although the simulation did not include all of the degrading factors present in the physical imaging, the two images were in good agreement, indicating the applicability of the simulation to a realistic situation and the importance of the detector resolution effect. ( view less ) John J L Morton,Nicholas S Lees,Brian M Hoffman,Stefan Stoll A recent article by Yang and Hoffman [T.-C. Yang, B.M. Hoffman, J. Magn. Reson., 181 (2006) 280] presents a 'Davies/Hahn ENDOR multi-sequence' in which the alpha and beta peaks of an electron-nuclear double resonance (ENDOR) spectrum can be distinguished. This represents one instance of a family of... ( view more ) ENDOR sequences which have no initial microwave inversion pulse, and which can reveal information about nuclear relaxation rates and the signs of hyperfine coupling constants. Here we discuss the more general set of such sequences, which we refer to as Saturated Pulsed ENDOR, and show how signal sensitivity can be optimised within the context of this new technique. Through simulations, we compare its performance to other techniques based on Davies ENDOR, and experimentally illustrate its properties using the non-heme Fe enzyme anthranilate dioxygenase AntDO. Finally, we suggest a protocol for extracting both the magnitude and sign of the hyperfine tensor using a combination of ENDOR techniques. ( view less ) Robert M HoffmanIn vivo imaging with green fluorescent protein (GFP) and other fluorescent proteins is revolutionizing cancer biology and other fields of in vivo biology (Hoffman, 2005; Hoffman and Yang, 2006a,b,c). Our laboratory pioneered the use of GFP for in vivo imaging in 1997 (Chishima et al., 1997). This c... ( view more )hapter highlights recent developments from our laboratory on both macro and micro in vivo imaging by using fluorescent proteins. ( view less ) Paula L Hoffman,Michael Miles,Howard J Edenberg,Wolfgang Sommer,Boris Tabakoff,Jeanne M Wehner,Joanne Lewohl This article represents the proceedings of a symposium at the 2002 joint RSA/ISBRA Conference in San Francisco, California. The organizer was Paula L. Hoffman and the co-chairs were Paula L. Hoffman and Michael Miles. The presentations were (1) Introduction and overview of the use of DNA microarray... ( view more )s, by Michael Miles; (2) DNA microarray analysis of gene expression in brains of P and NP rats, by Howard J. Edenberg; (3) Gene expression patterns in brain regions of AA and ANA rats, by Wolfgang Sommer; (4) Patterns of gene expression in brains of selected lines of mice that differ in ethanol tolerance, by Boris Tabakoff; (5) Gene expression profiling related to initial sensitivity and tolerance in gamma-protein kinase C mutants, by Jeanne Wehner; and (6) Gene expression patterns in human alcoholic brain: from microarrays to protein profiles, by Joanne Lewohl. ( view less ) F Fritz,E M Howard,M M Hoffman,P D Roepe Recently [Hoffman, M. M., and Roepe, P. D. (1997) Biochemistry 36, 11153-11168] we presented evidence for a novel Na+- and Cl--dependent H+ transport process in LR73/hu MDR 1 CHO transfectants that likely explains pHi, volume, and membrane potential changes in eukaryotic cells overexpressing the hu... ( view more ) MDR 1 protein. To further explore this process, we have overexpressed human MDR 1 protein in yeast strain 9.3 following a combination of approaches used previously [Kuchler, K., and Thorner, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2302-2306; Ruetz, S., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11588-11592]. Thus, a truncated hu MDR 1 cDNA was cloned behind a tandem array of sterile 6 (Ste6) and alchohol dehydrogenase (Adh) promoters to create the yeast expression vector pFF1. Valinomycin resistance of intact cells and Western blot analysis with purified yeast plasma membranes confirmed the overexpression of full length, functional, and properly localized hu MDR 1 protein in independently isolated 9.3/pFF1 colonies. Interestingly, relative valinomycin resistance and growth of the 9.3/hu MDR 1 strains are found to strongly depend on the ionic composition of the growth medium. Atomic absorption reveals significant differences in intracellular K+ for 9.3/hu MDR 1 versus control yeast. Transport assays using [3H]tetraphenylphosphonium ([3H]TPP+) reveal perturbations in membrane potential for 9.3/hu MDR 1 yeast that are stimulated by KCl and alkaline pHex. ATPase activity of purified plasma membrane fractions from yeast strains and LR73/hu MDR 1 CHO transfectants constructed previously [Hoffman, M. M., et al. (1996) J. Gen. Physiol. 108, 295-313] was compared. MDR 1 ATPase activity exhibits a higher pH optimum and different salt dependencies, relative to yeast H+ ATPase. Inside-out plasma membrane vesicles (ISOV) fabricated from 9.3/hu MDR 1 and control strains were analyzed for formation of H+ gradients +/- verapamil. Similar pharmacologic profiles are found for verapamil stimulation of MDR 1 ATPase activity and H+ pumping in 9.3/hu MDR 1 ISOV. In sum, these experiments strongly support the notion that hu MDR 1 catalyzes H+ transport in some fashion and lowers membrane potential in yeast when K+ contributes strongly to that potential. In the accompanying paper [Santai, C. T., Fritz, F., and Roepe, P. D. (1999) Biochemistry 38, XXXX-XXXX] the effects of ion gradients on H+ transport by hu MDR 1 are examined. ( view less ) T Chishima,M Yang,Y Miyagi,L Li,Y Tan,E Baranov,H Shimada,A R Moossa,S Penman,R M Hoffman Metastasis is the ultimate life-threatening stage of cancer. The lack of accurate model systems thwarted studies of the metastatic cell's basic biology. To follow continuously the succeeding stages of metastatic colony growth, we heritably labeled cells from the human lung adenocarcinoma cell line ... ( view more )ANIP 973 with green fluorescent protein (GFP) by transfection with GFP cDNA. Labeled cells were then injected intravenously into nude mice, where, by 7 days, they formed brilliantly fluorescing metastatic colonies on mouse lung [Chishima, T., Miyagi, Y., Wang, X., Yang, M., Tan, Y., Shimada, H., Moossa, A. R. & Hoffman, R. M. (1997) Clin. Exp. Metastasis 15, 547-552]. The seeded lung tissue was then excised and incubated in the three-dimensional sponge-gel-matrix-supported histoculture that maintained the critical features of progressive in vivo tumor colonization while allowing continuous access for measurement and manipulation. Tumor progression was continuously visualized by GFP fluorescence in the same individual cultures over a 52-day period, during which the tumors spread throughout the lung. Histoculture tumor colonization was selective for lung cancer cells to grow on lung tissue, because no growth occurred on histocultured mouse liver tissue, which was also observed in vivo. The ability to support selective organ colonization in histoculture and visualize tumor progression by GFP fluorescence allows the in vitro study of the governing processes of metastasis [Kuo, T.-H., Kubota, T., Watanbe, M., Furukawa, T., Teramoto, T., Ishibiki, K., Kitajima, M., Moossa, A. R., Penman, S. & Hoffman, R. M. (1995) Proc. Natl. Acad. Sci. USA 92, 12085-12089]. The results presented here provide significant, new opportunities to understand and to develop treatments that prevent and possibly reverse metastasis. ( view less ) R J Gurbiel,P E Doan,G T Gassner,T J Macke,D A Case,T Ohnishi,J A Fee,D P Ballou,B M Hoffman Continuous wave electron nuclear double resonance (CW ENDOR) spectra of [delta-15N,epsilon(-14)N]histidine-labeled phthalate dioxygenase (PDO) from Pseudomonas cepacia were recorded and found to be virtually identical to those previously recorded from [delta,epsilon-15N2]histidine-labeled protein [... ( view more )Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. Thus, the two histidine residues, previously shown to ligate one of the irons in the cluster [cf. Gurbiel et al. 1989)], both coordinate the metal at the N(delta) position of their imidazole rings. Pulsed ENDOR studies showed that the "remote", noncoordinating nitrogen of the histidine imidazole ring could be observed from the Rieske protein in a sample of Rhodobacter capsulatus cytochrome bc1 complex uniformly labeled with 15N but not in a sample of PDO labeled with [delta-15N,epsilon-14N]histidine, but this atom was easily observed with a sample of Rh. capsulatus cytochrome bc1 complex that had been uniformly labeled with 15N; this confirmed the conclusion from the CW ENDOR studies that ligation is exclusively via N(delta) for both ligands in the PDO center. Modifications in the algorithms previously used to simulate 14N ENDOR spectra permitted us to compute spectra without any constraints on the relative orientation of hyperfine and quadrupole tensors. This new algorithm was used to analyze current and previously published spectra, and slightly different values for the N-Fe-N angle and imidazole ring rotation angles are presented [cf. Gurbiel et al. (1989) Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., and Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. This analysis has permitted us to refine the proposed structure of the [2Fe-2S] Rieske-type cluster and rationalize some of the properties of these novel centers. Although the spectra of cytochrome bc1 complex from Rh. capsulatus are of somewhat lower resolution than those obtained with samples of PDO, our analysis nevertheless permits the conclusion that the geometry of the cluster is essentially the same for all Rieske and Rieske-type proteins. Structural constraints inferred from the spectroscopic results permitted us to apply the principles of distance geometry to arrive at possible three-dimensional models of the active site structure of Rieske protein from Rh. capsulatus. Results from this test case indicate that similar procedures should be generally useful in metalloprotein systems. We also recorded the pulsed and CW ENDOR spectra of 57Fe-labeled PDO, and the resulting data were used to derive the full hyperfine tensors for both Fe(III) and Fe(II) ions, including their orientations relative to the g tensor. The A tensor of the ferric ion is nominally isotropic, while the A tensor of the ferrous ion is axial, having A(parallel) > A(perpendicular); both tensors are coincident with the observed g tensor, with A(parallel) of the ferrous ion lying along the maximum g-value, g1. These results were examined using refinements of existing theories of spin-coupling in [2Fe-2S]+ clusters, and it is concluded that current theories are not adequate to fully describe the experimental results. ( view less ) M L Braunstein,D D Hoffman,A Saidpour Three experiments were conducted to test Hoffman and Richards's (1984) hypothesis that, for purposes of visual recognition, the human visual system divides three-dimensional shapes into parts at negative minima of curvature. In the first two experiments, subjects observed a simulated object (surfac... ( view more )e of revolution) rotating about a vertical axis, followed by a display of four alternative parts. They were asked to select a part that was from the object. Two of the four parts were divided at negative minima of curvature and two at positive maxima. When both a minima part and a maxima part from the object were presented on each trial (experiment 1), most of the correct responses were minima parts (101 versus 55). When only one part from the object--either a minima part or a maxima part--was shown on each trial (experiment 2), accuracy on trials with correct minima parts and correct maxima parts did not differ significantly. However, some subjects indicated that they reversed figure and ground, thereby changing maxima parts into minima parts. In experiment 3, subjects marked apparent part boundaries. 81% of these marks indicated minima parts, 10% of the marks indicated maxima parts, and 9% of the marks were at other positions. These results provide converging evidence, from two different methods, which supports Hoffman and Richard's minima rule. ( view less ) D W Coalson,J O Mecham,P H Stern,R M Hoffman Methionine (Met) dependence--i.e., the inability of cultured cells to grow when Met is replaced by its immediate precursor homocysteine (Met-Hcy+ medium)--is a frequent component of the oncogenically transformed phenotype. Normal cells, on the other hand, grow in this medium. There have been report... ( view more )s [Hoffman, R. M. & Erbe, R. W. (1976) Proc. Natl. Acad. Sci. USA 73, 1523-1527; Hoffman, R. M., Jacobsen, S. J. & Erbe, R. W. (1978) Biochem. Biophys. Res. Commun. 82, 228-234] of normal or higher rats of Met biosynthesis in Met-dependent cells and a postulation that Met-dependent cells are deficient in utilization of endogenously synthesized Met as opposed to exogenously supplied Met. To answer the critical question of what biochemical reaction(s) requires preformed Met in Met-dependent cels, we labeled cells with Met-free [35S]Hcy or [35S]Met and determined the levels of Met, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy). We report here experiments that demonstrate that Met-dependent cells synthesize a normal amount of endogenously synthesized Met and are deficient in utilizing this Met for AdoMet synthesis. In contrast, exogenously supplied Met is utilized normally for AdoMet biosynthesis. The ratio of AdoMet to AdoHcy is low in Met-dependent cells growing in Met-Hcy+ medium but is normal in Met+Hcy- medium. We determined that the low AdoMet/AdoHcy ratio probably limits growth of Met-dependent cells in Met-Hcy+ medium. ( view less ) R M Hoffman,S J Jacobsen,R W Erbe Many transformed and malignant cells, unlike normal cells, do not grow when methionine in the growth medium is replaced by its immediate precursor homocysteine [Chello, P. & Bertino, J. (1973) Cancer Res. 33, 1898-1904 and Hoffman, R. & Erbe, R. (1976) Proc. Natl. Acad. Sci. USA 73, 1523-1527]. Rar... ( view more )e cells from those populations revert to methionine independence [Hoffman, R., Jacobsen, S. & Erbe, R. (1978) Biochem. Biophys. Res. Commun. 82, 228-234]. We report here that methionine-independent revertants of both human fibroblasts transformed by simian virus 40 and malignant rat fibroblasts concomitantly revert for some of the properties associated with the transformed state. Of the 13 methionine-independent revertants described here, 5 showed increased anchorage dependence as reflected by reduced cloning efficiences in methylcellulose; 8 showed an increased serum requirement for optimal growth; 8 showed decreased cell density in medium containing high serum; and 3 altered their cell morphology significantly. Eight of the 13 have increased chromosome numbers. All lines tested contained immunologically identifiable tumor antigen of simian virus 40. Thus by selecting for methionine independence it is possible to select for heterogeneous transformation revertants, indicating further a relationship between altered methionine metabolism and oncogenic transformation. Therefore a positive metabolic method to select for transformation revertants has been developed, and its use has resulted in selection of human transformation revertants. ( view less ) J W Brown,E A Sirlin,A M Benoit,J M Hoffman,R A Darnall Activation of 5-HT(1A) receptors in the medullary raphé decreases sympathetically mediated brown adipose tissue (BAT) thermogenesis and peripheral vasoconstriction when previously activated with leptin, LPS, prostaglandins, or cooling. It is not known whether shivering is also modulated by medullar... ( view more )y raphé 5-HT(1A) receptors. We previously showed in conscious piglets that activation of 5-HT(1A) receptors with (+/-)-8-hydroxy-2-(dipropylamino)-tetralin (8-OH-DPAT) in the paragigantocellularis lateralis (PGCL), a medullary region lateral to the raphé that contains substantial numbers of 5-HT neurons, eliminates rapid eye movement (REM) sleep and decreases shivering in a cold environment, but does not attenuate peripheral vasoconstriction. Hoffman JM, Brown JW, Sirlin EA, Benoit AM, Gill WH, Harris MB, Darnall RA. Am J Physiol Regul Integr Comp Physiol 293: R518-R527, 2007. We hypothesized that, during cooling, activation of 5-HT(1A) receptors in the medullary raphé would also eliminate REM sleep and, in contrast to activation of 5-HT(1A) receptors in the PGCL, would attenuate both shivering and peripheral vasoconstriction. In a continuously cool environment, dialysis of 8-OH-DPAT into the medullary raphé resulted in alternating brief periods of non-REM sleep and wakefulness and eliminated REM sleep, as observed when 8-OH-DPAT is dialyzed into the PGCL. Moreover, both shivering and peripheral vasoconstriction were significantly attenuated after 8-OH-DPAT dialysis into the medullary raphé. The effects of 8-OH-DPAT were prevented after dialysis of the selective 5-HT(1A) receptor antagonist WAY-100635. We conclude that, during cooling, exogenous activation of 5-HT(1A) receptors in the medullary raphé decreases both shivering and peripheral vasoconstriction. Our data are consistent with the hypothesis that neurons expressing 5-HT(1A) receptors in the medullary raphé facilitate spinal motor circuits involved in shivering, as well as sympathetic stimulation of other thermoregulatory effector mechanisms. ( view less ) Martin D Hoffman,Debi Rufi Hoffman OBJECTIVE: To determine whether a single session of exercise of appropriate intensity and duration for aerobic conditioning has a different acute effect on mood for nonexercisers than regular exercisers. DESIGN: Repeated-measures design. SETTING: Research laboratory. PARTICIPANTS: Adult nonexercise... ( view more )rs, moderate exercisers, and ultramarathon runners (8 men, 8 women in each group). INTERVENTIONS: Treadmill exercise at self-selected speeds to induce a rating of perceived exertion (RPE) of 13 (somewhat hard) for 20 minutes, preceded and followed by 5 minutes at an RPE of 9 (very light). MAIN OUTCOME MEASURE: Profile of Mood States before and 5 minutes after exercise. RESULTS: Vigor increased by a mean +/- standard deviation of 8+/-7 points (95% confidence interval [CI], 5-12) among the ultramarathon runners and 5+/-4 points (95% CI, 2-9) among the moderate exercisers, with no improvement among the nonexercisers. Fatigue decreased by 5+/-6 points (95% CI, 2-8) for the ultramarathon runners and 4+/-4 points (95% CI, 1-7) for the moderate exercisers, with no improvement among the nonexercisers. Postexercise total mood disturbance decreased by a mean of 21+/-16 points (95% CI, 12-29) among the ultramarathon runners, 16+/-10 points (95% CI, 7-24) among the moderate exercisers, and 9+/-13 points (95% CI, 1-18) among the nonexercisers. CONCLUSIONS: A single session of moderate aerobic exercise improves vigor and decreases fatigue among regular exercisers but causes no change in these scores for nonexercisers. Although total mood disturbance improves postexercise in exercisers and nonexercisers, regular exercisers have approximately twice the effect as nonexercisers. This limited postexercise mood improvement among nonexercisers may be an important deterrent for persistence with an exercise program. ( view less ) Krishnakumar Kizhatil,Jonathan Q Davis,Lydia Davis,Jan Hoffman,Brigid L M Hogan,Vann Bennett E-cadherin is a ubiquitous component of lateral membranes in epithelial tissues and is required to form the first lateral membrane domains in development. Here, we identify ankyrin-G as a molecular partner of E-cadherin and demonstrate that ankyrin-G and beta-2-spectrin are required for accumulatio... ( view more )n of E-cadherin at the lateral membrane in both epithelial cells and early embryos. Ankyrin-G binds to the cytoplasmic domain of E-cadherin at a conserved site distinct from that of beta-catenin. Ankyrin-G also recruits beta-2-spectrin to E-cadherin-beta-catenin complexes, thus providing a direct connection between E-cadherin and the spectrin/actin skeleton. In addition to restricting the membrane mobility of E-cadherin, ankyrin-G and beta-2-spectrin also are required for exit of E-cadherin from the trans-Golgi network in a microtubule-dependent pathway. Ankyrin-G and beta-2-spectrin co-localize with E-cadherin in preimplantation mouse embryos. Moreover, knockdown of either ankyrin-G or beta-2-spectrin in one cell of a two-cell embryo blocks accumulation of E-cadherin at sites of cell-cell contact. E-cadherin thus requires both ankyrin-G and beta-2-spectrin for its cellular localization in early embryos as well as cultured epithelial cells. We have recently reported that ankyrin-G and beta-2-spectrin collaborate in biogenesis of the lateral membrane ( Kizhatil, K., Yoon, W., Mohler, P. J., Davis, L. H., Hoffman, J. A., and Bennett, V. (2007) J. Biol. Chem. 282, 2029-2037 ). Together with the current findings, these data suggest a ankyrin/spectrin-based mechanism for coordinating membrane assembly with extracellular interactions of E-cadherin at sites of cell-cell contact. ( view less ) Lauren A Fine,Laura D Hoffman,Mark D HoffmanThe association of Behçet's disease with myelodysplastic syndromes has been established in the hematologic, gastrointestinal, and medical genetics literature; trisomy 8 is a feature present in 70% of these reports. Because trisomy 8 is ordinarily present in only 14% of patients with myelodysplastic... ( view more ) syndrome, this is a significant overrepresentation. We describe a patient with recurrent aphthous stomatitis who was subsequently diagnosed with trisomy 8-positive myelodysplastic syndrome after an unexplained macrocytosis was further investigated. ( view less ) James J Lai,John M Hoffman,Mitsuhiro Ebara,Allan S Hoffman,Claude Estournès,Alain Wattiaux,Patrick S Stayton A stimuli-responsive magnetic nanoparticle system for diagnostic target capture and concentration has been developed for microfluidic lab card settings. Telechelic poly(N-isopropylacrylamide) (PNIPAAm) polymer chains were synthesized with dodecyl tails at one end and a reactive carboxylate at the o... ( view more )pposite end by the reversible addition fragmentation transfer technique. These PNIPAAm chains self-associate into nanoscale micelles that were used as dimensional confinements to synthesize the magnetic nanoparticles. The resulting superparamagnetic nanoparticles exhibit a gamma-Fe2O3 core ( approximately 5 nm) with a layer of carboxylate-terminated PNIPAAm chains as a corona on the surface. The carboxylate group was used to functionalize the magnetic nanoparticles with biotin and subsequently with streptavidin. The functionalized magnetic nanoparticles can be reversibly aggregated in solution as the temperature is cycled through the PNIPAAm lower critical solution temperature (LCST). While the magnetophoretic mobility of the individual nanoparticles below the LCST is negligible, the aggregates formed above the LCST are large enough to respond to an applied magnetic field. The magnetic nanoparticles can associate with biotinylated targets as individual particles, and then subsequent application of a combined temperature increase and magnetic field can be used to magnetically separate the aggregated particles onto the poly(ethylene glycol)-modified polydimethylsiloxane channel walls of a microfluidic device. When the magnetic field is turned off and the temperature is reversed, the captured aggregates redisperse into the channel flow stream for further downstream processing. The dual magnetic- and temperature-responsive nanoparticles can thus be used as soluble reagents to capture diagnostic targets at a controlled time point and channel position. They can then be isolated and released after the nanoparticles have captured target molecules, overcoming the problem of low magnetophoretic mobility of the individual particle while retaining the advantages of a high surface to volume ratio and faster diffusive properties during target capture. ( view less ) Martin D Hoffman,Debi Rufi HoffmanAerobic exercise can cause an acute improvement in mood as well as a reduction in the perception of pain from a painful stimulus. Regular exercise training also may offer some protection from depression, is clinically useful in treating certain psychiatric and chronic pain conditions, and may allow... ( view more ) for an enhancement of the acute improvements in mood from a single exercise session. The utility of aerobic exercise training for improving mood disturbances and pain perception among patients with chronic pain requires further investigation. ( view less ) Zakaria Messieha,Ranga Chelva Ananda,Ian Hoffman,William Hoffman We assessed the safety of general anesthesia for dental treatment of special needs patients as it related to American Society of Anesthesiology Physical Status (ASAPS) classification, procedure, and other factors. After Institutional Review Board review and approval, special needs patients who were... ( view more ) admitted to the outpatient surgical operating room for comprehensive dental rehabilitation (CDR) under general anesthesia within a period of 5 years had their medical records evaluated retrospectively for intraoperative and postoperative complications both related to anesthesia and surgery. All records were evaluated by an independent evaluator who tabulated the patients' age, gender, ASAPS, and duration of procedure. N = 363, age mean = 46.93 +/- 16.835 years, age median = 48 years, male patients = 180, female patients = 183, ASAPS I =183, ASAPS II = 127, ASAPS III = 53, duration of surgery mean = 140.631 +/- 23.104 minutes, duration of surgery median time = 142.000 minutes, and number of complications = 2. One complication resulted in an ASAPS I 16-year-old boy, which was airway related, and a second was an ASAPS III 22-year-old woman, which was surgically related. Both led to unplanned inpatient admissions and were treated successfully with no residual morbidity. Dental rehabilitation of special needs patients under general anesthesia is safe. While morbidity is very low, larger studies are needed to establish risk versus benefit stratification among this patient population. ( view less ) Eunkyung An,Xiaoning Lu,Jessica Flippin,Joseph M Devaney,Brian Halligan,Eric P Hoffman,Eric Hoffman,Nataly Strunnikova,Karl Csaky,Yetrib Hathout Age-related macular degeneration (AMD) is characterized by progressive loss of central vision, which is attributed to abnormal accumulation of macular deposits called "drusen" at the interface between the basal surface of the retinal pigment epithelium (RPE) and Bruch's membrane. In the most severe... ( view more ) cases, drusen deposits are accompanied by the growth of new blood vessels that breach the RPE layer and invade photoreceptors. In this study, we hypothesized that RPE secreted proteins are responsible for drusen formation and choroidal neovascularization. We used stable isotope labeling by amino acids in cell culture (SILAC) in combination with LC-MS/MS analysis and ZoomQuant quantification to assess differential protein secretion by RPE cell cultures prepared from human autopsy eyes of AMD donors (diagnosed by histological examinations of the macula and genotyped for the Y402H-complement factor H variant) and age-matched healthy control donors. In general, RPE cells were found to secrete a variety of extracellular matrix proteins, complement factors, and protease inhibitors that have been reported to be major constituents of drusen (hallmark deposits in AMD). Interestingly, RPE cells from AMD donors secreted 2 to 3-fold more galectin 3 binding protein, fibronectin, clusterin, matrix metalloproteinase-2 and pigment epithelium derived factor than RPE cells from age-matched healthy donors. Conversely, secreted protein acidic and rich in cysteine (SPARC) was found to be down regulated by 2-fold in AMD RPE cells versus healthy RPE cells. Ingenuity pathway analysis grouped these differentially secreted proteins into two groups; those involved in tissue development and angiogenesis and those involved in complement regulation and protein aggregation such as clusterin. Overall, these data strongly suggest that RPE cells are involved in the biogenesis of drusen and the pathology of AMD. ( view less ) Mitsuhiro Ebara,John M Hoffman,Allan S Hoffman,Patrick S Stayton We report here a reversible microchannel surface capture system for stimuli-responsive grafted bioanalytical beads. Poly(N-isopropylacrylamide) (PNIPAAm) was grafted onto polydimethylsiloxane (PDMS) surfaces by a UV-mediated graft polymerization from a photoinitiator that was preadsorbed in the cha... ( view more )nnel wall. The surface grafting density and resulting switchable hydrophilic/hydrophobic properties were controlled by varying the photo-illumination times and/or the initiator concentration. At limiting PNIPAAm-graft densities, the surfaces demonstrated minimal contact angles of 35 degrees below the lower critical solution temperature (LCST) and maximal contact angles of 82 degrees above it. These contact angles could be varied depending on the graft density. The surface grafts are spatially limited to the photo-illuminated region to define where the trap is constructed. The surface traps capture PNIPAAm-grafted nanobeads uniformly above the LCST and facilitate their rapid release as the temperature is reversed to below the LCST. This dual surface trap and injectable chromatography system could be useful in many applications, such as affinity separations, immunoassays, and enzyme bioprocesses, by providing for the controlled capture and release of chromatography beads. ( view less )
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