Fluorescence study of thymus lymphocytes of X-irradiated mice and their progeny.

The mechanisms of changes in the ultra-violet fluorescence (U.V.F.) intensity of mouse thymus lymphocytes 24 hours and 30 days after whole-body X-irradiation have been studied. The thymus lymphocytes of the first generation offspring (F1) from X-irradiated males and unirradiated females were also investigated. At 24 hours after irradiation the U.V.F. intensity decreased for small doses (50 and 65 rad) and increased for doses of more than 100 rad. The changes in U.V.F. intensity were related to a size-independent mechanism. It was found that the U.V.F. increase for doses of 100-700 rad was not connected with the appearance of non-viable (eosin test) cells. The changes in U.V.F. intensity and cellular composition of the thymus were the same 30 days after irradiation and for F1 mice. The increase in U.V.F. intensity was about 14% and did not depend on dose between 50 and 500 rad. About one-half of this increase was connected with an increase in the proportion of medium and large lymphocytes in the thymus. The rest of the effect was related to a size-independent mechanism.     

Photobiological behaviour of bacteria and phages supplemented with aza-analogoues of nucleic acid bases.

The photochemical stability of anomalous nucleic acid bases of the azatype, 5-azacytosine (I), 5-azacytidine (II), 6-azacytosine (III), 6-azacytidine (IV), 6-azathymine (V), 6-azauracil (VI), and 8-aza-adenine (VII) to U. V. light of the wavelength 254 nm differs from the U. V. stability of the normal constituents. Changes of the U.V. inactivation of Escherichia coli K12 C600, E. coli B, Bacillus cereus, as well as E. coli phages gamma cb2 and gamma b2b5 supplemented with azaderivatives prior to irradiation were investigated. It was found that I, II, III, IV, and VII are more, V and VI less sensitive to U. V. light compared with corresponding natural nucleic acid bases. Their changed U. V. sensitivities are reflected in the survival curves after U. V. -irradiation in as far as azabases are incorporated into the nucleic acids in vivo. This explains the increase in U.V. sensitivity of E. coli K12 C600, E. coli B, and B. cereus supplemented with I, II, III, IV, and VII and the decrease in U.V. sensitivity of Streptococcus faecalis supplemented with V (the latter information was taken from Gunther and Prusoff 1967). The lack of any significant influence on inactivation curves of E. coli K12 C600 by V and VI, and on E. coli phages gamma cb2 and gamma c2b5 by II, is discussed in terms of too small incorporation rates. No discrimination was put forward with respect to DNA and RNA incorporation.     

High-light stress and photoprotection in Umbilicaria antarctica monitored by chlorophyll fluorescence imaging and changes in zeaxanthin and glutathione

The effect of high light on spatial distribution of chlorophyll (Chl) fluorescence parameters over a lichen thallus (Umbilicaria antarctica) was investigated by imaging of Chl fluorescence parameters before and after exposure to high light (1500 micro mol m (-2) s (-1), 30 min at 5 degrees C). False colour images of F (V)/F (M) and Phi (II) distribution, taken over thallus with 0.1 mm (2) resolution, showed that maximum F (V)/F (M) and Phi (II) values were located close to the thallus centre. Minimum values were typical for thallus margins. After exposure to high light, a differential response of F (V)/F (M) and Phi (II) was found. The marginal thallus part exhibited a loss of photosynthetic activity, manifested as a lack of Chl fluorescence signal, and close-to-centre parts showed a different extent of F (V)/F (M) and Phi (II) decrease. Subsequent recovery in the dark led to a gradual return of F (V)/F (M) and Phi (II) to their initial values. Fast (30 min) and slow (1 - 22 h) phase of recovery were distinguished, suggesting a sufficient capacity of photoprotective mechanisms in U. antarctica to cope with low-temperature photoinhibition. Glutathione and xanthophyll cycle pigments were analyzed by HPLC. High light led to an increase in oxidized glutathione (GSSG), and a conversion of violaxanthin to zeaxanthin, expressed as their de-epoxidation state (DEPS). The responses of GSSG and DEPS were reversible during subsequent recovery in the dark. GSSG and DEPS were highly correlated to non-photochemical quenching (NPQ), indicating involvement of these antioxidants in the resistance of U. antarctica to high-light stress. Heterogeneity of Chl fluorescence parameters over the thallus and differential response to high light are discussed in relation to thallus anatomy and intrathalline distribution of the symbiotic alga Trebouxia sp.     

Effects of desiccation on the excitation energy distribution from phycoerythrin to the two photosystems in the red alga Porphyra perforata

Low temperature (77°K) fluorescence emission and excitation spectra were recorded for wet and desiccated thalli of Porphyra perforata . The photosystem I (F730) and photosystem II (F695) fluorescence emission kinetics during photosystem II trap closure were also recorded at 77°K. Desiccation induced a lowering of the fluorescence yield over the whole emission spectrum but the decrease was most pronounced for the photosystem II fluorescence bands, F688 and F695. It was shown that the desiccation-induced changes of the phycoerythrin sensitized emission spectrum were due to 1) a decrease in the fluorescence yield of the photosystem I antenna, 2) an even stronger decrease in the fluorescence of photosystem II, which was mediated by an increased spillover (k_T(II→I)) of excitation to photosystem I and an increase in the absorption cross section, α, for photosystem I. We hypothesize that the increase of both k_T(II→I) and α are part of a mechanism by which the desiccation-tolerant, high light exposed, Porphyra can avoid photodynamic damage to photosystem II, when photosynthesis becomes inhibited as a result of desiccation during periods of low tide.     

Abp1p and Fyn SH3 domains fold through similar low-populated intermediate states

Src homology 3 (SH3) domains are small modules that are thought to fold via a two-state mechanism, without the accumulation of significant populations of intermediate states. Relaxation dispersion NMR studies of the folding of G48V and G48M mutants of the Fyn SH3 domain have established that, at least for these modules, folding proceeds through the formation of a transient on-pathway intermediate with an equilibrium population of 1-2% that can be readily detected [Korzhnev, D. M., et al. (2004) Nature 430, 586-590]. To investigate the generality of this result, we present an (15)N relaxation dispersion NMR study of a pair of additional SH3 domains, including a G48V mutant of a stabilized Abp1p SH3 domain that shares 36% sequence identity with the Fyn SH3 module, and a A39V/N53P/V55L mutant Fyn SH3 domain. A transient folding intermediate is detected for both of the proteins studied here, and the dispersion data are well fit to a folding model of the form F <--> I <--> U, where F, I, and U correspond to folded, intermediate, and unfolded states, respectively. The temperature dependencies of the folding/unfolding rate constants were obtained so that the thermodynamic properties of each of F, I, and U could be established. The detection of I states in folding pathways of all SH3 domains examined to date via relaxation dispersion NMR spectroscopy indicates that such intermediates may well be a conserved feature in the folding of such domains in general but that their transient nature along with their low population makes detection difficult using more well-established approaches to the study of folding.     

Abbreviated Half-Lives and Impaired Fuel Utilization in Carnitine Palmitoyltransferase II Variant Fibroblasts

Carnitine palmitoyltransferase II (CPT II) deficiency is one of the most common causes of fatty acid oxidation metabolism disorders. However, the molecular mechanism between CPT2 gene polymorphisms and metabolic stress has not been fully clarified. We previously reported that a number of patients show a thermal instable phenotype of compound hetero/homozygous variants of CPT II. To understand the mechanism of the metabolic disorder resulting from CPT II deficiency, the present study investigated CPT II variants in patient fibroblasts, [c.1102 G>A (p.V368I)] (heterozygous), [c.1102 G>A (p.V368I)] (homozygous), and [c.1055 T>G (p.F352C)] (heterozygous) + [c.1102 G>A (p.V368I)] (homozygous) compared with fibroblasts from healthy controls. CPT II variants exerted an effect of dominant negative on the homotetrameric proteins that showed thermal instability, reduced residual enzyme activities and a short half-life. Moreover, CPT II variant fibroblasts showed a significant decrease in fatty acid β-oxidation and adenosine triphosphate generation, combined with a reduced mitochondrial membrane potential, resulting in cellular apoptosis. Collectively, our data indicate that the CPT II deficiency induces an energy crisis of the fatty acid metabolic pathway. These findings may contribute to the elucidation of the genetic factors involved in metabolic disorder encephalopathy caused by the CPT II deficiency.     

The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes

The ratios of the oxidative phosphorylation complexes NADH:ubiquinone reductase (complex I), succinate:ubiquinone reductase (complex II), ubiquinol:cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and F1F0-ATP synthase (complex V) from bovine heart mitochondria were determined by applying three novel and independent approaches that gave consistent results: 1) a spectrophotometric-enzymatic assay making use of differential solubilization of complexes II and III and parallel assays of spectra and catalytic activities in the samples before and after ultracentrifugation were used for the determination of the ratios of complexes II, III, and IV; 2) an electrophoretic-densitometric approach using two-dimensional electrophoresis (blue native-polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis) and Coomassie blue-staining indices of subunits of complexes was used for determining the ratios of complexes I, III, IV, and V; and 3) two electrophoretic-densitometric approaches that are independent of the use of staining indices were used for determining the ratio of complexes I and III. For complexes I, II, III, IV, and V in bovine heart mitochondria, a ratio 1.1 +/- 0.2:1.3 +/- 0.1:3:6.7 +/- 0.8:3.5 +/- 0.2 was determined.     

Father-male offspring transmission of seasonal variations in testicular diameter and percentage of abnormal sperm in the Ile-de-France ram. 1. Male offspring born in February

An experiment was conducted on the Ile-de-France (IF) breed to determine if the more or less important sensitivity of the ram to photoperiodism came under genetic control. Five base breed unrelated rams (greater than or equal to 4 yr old), were chosen for this study: 2 good (I and II), 2 bad (III and IV), and an intermediate sire (V). Ram I, which died a few years before the experiment began, was selected on both the low amplitude of its sperm production during a 6-month period of artificial lighting and the very high fertilizing capacity of its sperm in spring. Rams II to V were controlled for 12 (percentage of morphologically abnormal spermatozoa, AM) or 14 (maximum antero-posterior scrotal diameter of both testes, DT) consecutive months. Following this period of control, seasonal variation in rams was assessed as follows: low (ram II), high (rams III and IV) and intermediate (ram V) seasonal variation rams. Breeding (artificial insemination) of these 5 rams to IF ewes resulted in 18 male offspring born in February and distributed as follows: rams I to III: 4 male offspring/ram (families 1 to 3, F1 to F3), rams IV and V: 3 male offspring/ram (families 4 and 5, F4 and F5). The 18 animals were controlled once a week (DT and AM) from 8, 5 to 46 months of age (7 periods, P1 to P7). Regarding DT, mean DT was higher in F1 and F2 than those of the whole population (WP) (represented by a discontinuous line of ordinate 5.0 in Graph 3), but F2 was closer to this population than F1. On the other hand, they were lower in the rams of F3 and F4. Differences between F3 or F4 and WP were lower in autumn than in spring. All families showed significant differences during the experimental periods except at P7 for pairs 3-5 and 4-5. Distances between F1, F2, F3 were always different (P less than 0.01 or P less than 0.001), whatever the size of the population (n = 3 or 4). Weekly F5 values varied in an opposite way to those of F3 and F4: increase in spring and decrease in autumn. Regarding AM, families did not differ as much as in DT (graph 4). Weekly variations in AM were also stronger. However, mean AM was almost always lower in F1 males and somewhat higher in F3 males to that of WP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hemostatic effect of a heat-treated factor VIII concentrate (Haemate P) in von Willebrand's disease

A heat-treated factor VIII (F VIII) concentrate (Haemate P) has been administered to patients with various types of von Willebrand's disease (vWD). The 4 activities of F VIII/vWF as well as change in the multimeric structure of vWF were then studied. In 4 patients with type I vWF who were given a Ristocetin cofactor (Rcof) dose of 42-78 U/kg, there was a clear reduction of the bleeding time and an increase of F VIII: C, F VIII: Ag, Rcof and vWF: Ag for several hours. The recovery of Rcof. after 1 h was 50-75%. Although the multimeric composition of vWF in these patients was similar to that of normal plasma, the density of each multimer band was very low. After infusion, however, the density of all multimer bands increased for several hours, to decrease again after 24 h. In 4 patients with type II A vWD who received a dose of Rcof of 55-76 U/kg, the 4 activities of F VIII/vWF increased similarly as was the case in type I. All patients had only 3-4 smaller multimer bands. New larger and intermediate multimers appeared for several hours after infusion of the preparation. Two patients with type III vWD who received doses of Rcof of 52 and 65 U/kg showed also a similar increase in the 4 activities of F VIII/vWF after infusion. All the multimers lacking in these patients appeared for several hours after infusion.     

The involvement of transforming growth factor-beta1 secretion in urotensin II-induced collagen synthesis in neonatal cardiac fibroblasts

As the most potent vasoconstrictor in mammals, urotensin II (U II) has recently been demonstrated to play an important role in adverse cardiac remodeling and fibrosis. However, the mechanisms of U II-induced myocardial fibrosis remain to be clarified. We postulated that U II alters transforming growth factor-beta1 (TGF-beta1) expression, and thereby modulates cardiac fibroblast collagen metabolism. Experiments were conducted using cardiac fibroblast from neonatal Wistar rats to determine the expression of TGF-beta1, and the role of U II receptor UT in this process. The functional role of TGF-beta1 and UT in modulating U II effects on type I, III collagen mRNA expression and 3H-proline incorporation was also analyzed. TGF-beta1 gene and protein expression were consistently identified in quiescent cardiac fibroblasts. U II increased the expression of TGF-beta1 mRNA and protein in a time-dependent manner. This effect was UT mediated, because UT antagonist urantide abolished U II-induced TGF-beta1 expression. U II-induced increase in type I, III collagen mRNA expression and 3H-proline incorporation were both inhibited by a specific TGF-beta1 neutralizing antibody and UT receptor antagonist urantide. Hence, our results indicate that TGF-beta1 is upregulated in cardiac fibroblasts by U II via UT and modulates profibrotic effects of U II. These findings provide novel insights into U II-induced cardiac remodeling.     

Steady-state kinetic properties of FoF1-ATPase: the pH effect.

1. The kinetic properties of FoF1-ATPase from submitochondrial particles isolated from rat heart were studied, with emphasis to the pH effect. The velocity data were treated according to the Hill equation, and the results were discussed on the basis of the knowledge on the soluble F1-ATPase properties. 2. Three kinetic phases were observed in the range of pH 6.0-8.5, with apparent dissociation constant values (K0.5) of 0.001, 0.04 and 1.5 mM (respectively sites I, II and III) at pH 7.0. Their contribution to the total activity of the enzyme were pH-dependent on the range of 6.0-7.0, but not from 7.0 to 8.5, where the maximal velocity (V) for site III was some 4-fold larger than for site II, and the total V of sites II and III was some 40-fold larger than V assumed for site I. Therefore, two catalytic sites seem to participate significantly in the catalysis at steady-state condition. 3. Azide increased the sites II and III K0.5 values as well as decreased the site III V. In the presence of bicarbonate these two sites were not distinguishable, and the kinetic parameters at pH 7.0 were similar to those for sites II and III combined. Both azide and bicarbonate did not have a significant effect on site I, and this behavior was not pH-dependent. 4. The studies on the effect of pH on the kinetic parameters showed the following results: (1) the optimum pH for V was around 8.5; (2) decrease in the K0.5 values at pH below 7.0 for site II, and increase at pH over 7.0 for sites II and III; (3) in the pH range of 6.0-8.5 the Hill coefficient increased for site II, decreased for site III, and an intermediary effect was observed for the sites II and III combined, with a Michaelis-Menten behavior in the highest affinity pH, which was found in the physiological range.     

Exploring sequence/folding space: folding studies on multiple hydrophobic core mutants of ubiquitin

The stability, dynamic, and structural properties of ubiquitin and two multiple hydrophobic core mutants were studied. One of the mutants (U4) has seven substitutions in the hydrophobic core (M1L, I3L, V5I, I13F, L15V, V17M, and V26L). On average, its side chains are larger than the wild-type, and it can thus be thought of as having an overpacked core. The other mutant (U7) has two substitutions (I3V and I13V). On average, it has smaller side chains than the wild-type, and it can therefore be considered to be underpacked. The three proteins are well-folded and show similar backbone dynamics (T(1), T(2), and HNOE values), indicating that the regular secondary structure extends over the same residue ranges. The crystallographic structure of U4 was determined. The final R(factor) and R(free) are 0.198 and 0.248, respectively, at 2.18 A resolution. The structure of U4 is very similar to wild-type ubiquitin. Remarkably, there are almost no changes in the positions of the C(alpha) atoms along the entire backbone, and the hydrogen-bonding network is maintained. The mutations of the hydrophobic core are accommodated by small movements of side chains in the core of mutated and nonmutated residues. Unfolding and refolding kinetic studies revealed that U4 unfolds with the highest rates; however, its refolding rate constants are very similar to those of the wild-type protein. Conversely, U7 seems to be the most destabilized protein; its refolding rate constant is smaller than the other two proteins. This was confirmed by stopped-flow techniques and by H/D exchange methodologies. This work illustrates the possibility of repacking the hydrophobic core of small proteins and has important implications in the de novo design of stable proteins.     

NEUROCHEMISTRY OF THE MUCOPOLYSACCHARIDOSES: BRAIN GLYCOSAMINOGLYCANS IN NORMALS AND FOUR TYPES OF MUCOPOLYSACCHARIDOSES

Glycosaminoglycan content, composition and molecular weight distribution were determined in cerebral gray and white matter, liver and spleen from normals and 7 patients with mucopolysaccharidosis; 4 were of Type I (Hurler), one Type II (Hunter), one Type IIIA (Sanfilippo A) and one Type V (Scheie). There was a 3 to 4-fold increase in glycosaminoglycan content of the brains from patients with mucopolysaccharidosis Type I, II and IIIA, but only a 40% increase in the Type V patient. Partially degraded dermatan sulfate accounted for most of the increase in Types I, II and V. Highly fragmented heparan sulfate was the major glycosaminoglycan in the brain of the Type IIIA patient and was also a sizable component in Types I and II. Remarkably, the changes in the brain glycosaminoglycans of the Type V patient were minimal. He also was of normal intelligence     

Physiological evidence for an interaction between helix XI and helices I, II, and V in the melibiose carrier of Escherichia coli

In a previous study 23 residues in helix XI of the cysteine-less melibiose carrier were changed individually to cysteine. Several of these cysteine mutants (K377C, A383C, F385C, L391C, G395C) had low transport activity and they were white on melibiose MacConkey fermentation plates. After several days of incubation of these white clones on melibiose MacConkey plates a rare red mutant appeared. The plasmid DNA was then isolated and sequenced. The two second site revertants from K377C were I22S and D59A. This change of aspartic acid to a neutral residue suggests that physiologically there is an interaction between K377 and D59 (possibly a salt bridge). The revertants from A383C were in positions 20 (F20L) and 22 (I22S and I22N). Revertants of F385C were intrahelical changes (I387M and A388G) and a change in C-terminal loop (R441C). Revertants of L391C were in helix I (I22N, I22T and D19E) and helix V (A152S). Revertants of G395C were in helix I (D19E and I22N). We suggest that there is an interaction between helix XI and helices I, II, and V and proximity between these helices.     

Overall Prevalence and Distribution of Knockdown Resistance (kdr) Mutations in Aedes aegypti from Mandalay Region,Myanmar

Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) of mosquitoes confer resistance to insecticides. Although insecticide resistance has been suspected to be widespread in the natural population of Aedes aegypti in Myanmar, only limited information is currently available. The overall prevalence and distribution of kdr mutations was analyzed in Ae. aegypti from Mandalay areas, Myanmar. Sequence analysis of the VGSC in Ae. aegypti from Myanmar revealed amino acid mutations at 13 and 11 positions in domains II and III of VGSC, respectively. High frequencies of S989P (68.6%), V1016G (73.5%), and F1534C (40.1%) were found in domains II and III. T1520I was also found, but the frequency was low (8.1%). The frequency of S989P/V1016G was high (55.0%), and the frequencies of V1016G/F1534C and S989P/V1016G/F1534C were also high at 30.1% and 23.5%, respectively. Novel mutations in domain II (L963Q, M976I, V977A, M994T, L995F, V996M/A, D998N, V999A, N1013D, and F1020S) and domain III (K1514R, Y1523H, V1529A, F1534L, F1537S, V1546A, F1551S, G1581D, and K1584R) were also identified. These results collectively suggest that high frequencies of kdr mutations were identified in Myanmar Ae. aegypti, indicating a high level of insecticide resistance.     

Inhibitory properties of cystatin F and its localization in U937 promonocyte cells

Cystatin F is a recently discovered type II cystatin expressed almost exclusively in immune cells. It is present intracellularly in lysosome-like vesicles, which suggests a potential role in regulating papain-like cathepsins involved in antigen presentation. Therefore, interactions of cystatin F with several of its potential targets, cathepsins F, K, V, S, H, X and C, were studied in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values ranging from 0.17 nM to 0.35 nM, whereas cathepsins S and H were inhibited with 100-fold lower affinities (Ki approximately 30 nM). The exopeptidases, cathepsins C and X were not inhibited by cystatin F. In order to investigate the biological significance of the inhibition data, the intracellular localization of cystatin F and its potential targets, cathepsins B, H, L, S, C and K, were studied by confocal microscopy in U937 promonocyte cells. Although vesicular staining was observed for all the enzymes, only cathepsins H and X were found to be colocalized with the inhibitor. This suggests that cystatin F in U937 cells may function as a regulatory inhibitor of proteolytic activity of cathepsin H or, more likely, as a protection against cathepsins misdirected to specific cystatin F containing endosomal/lysosomal vesicles. The finding that cystatin F was not colocalized with cystatin C suggests distinct functions for these two cysteine protease inhibitors in U937 cells.     

Substrate specificity of nickel/cobalt permeases: insights from mutants altered in transmembrane domains I and II

HoxN, a high-affinity, nickel-specific permease of Ralstonia eutropha H16, and NhlF, a nickel/cobalt permease of Rhodococcus rhodochrous J1, are structurally related members of the nickel/cobalt transporter (NiCoT) family. These transporters have an eight-helix structure and are characterized by highly conserved segments with polar or charged amino acid residues in transmembrane domains (TMDs) II, III, V, and VI. Two histidine residues in a Ni2+ binding motif, the signature sequence of NiCoTs, in TMD II of HoxN have been shown to be crucial for activity. Replacement of the corresponding His residues in NhlF affected both Co2+ and Ni2+ uptake, demonstrating that NhlF employs a HoxN-like mechanism for transport of the two cations. Multiple alignments of bacterial NiCoT sequences identified a striking correlation between a hydrophobic residue (Val or Phe) in TMD II and a position in the center of TMD I occupied by either an Asn (as in HoxN) or a His (as in NhlF). Introducing an isoleucine residue at the latter position strongly reduced HoxN activity and abolished NhlF activity, suggesting that a Lewis base N-donor moiety is important. The Asn-to-His exchange had no effect on HoxN, whereas the converse replacement reduced NhlF-mediated Ni2+ uptake significantly. Replacement of the entire TMD I of HoxN by the respective NhlF segment resulted in a chimera that transported Ni2+ and Co2+ with low capacity. The Val-to-Phe exchange in TMD II of HoxN led to a considerable rise in Ni2+ uptake capacity and conferred to the variant the ability to transport Co2+. NhlF activity dropped in response to the converse mutation. Our data predict that TMDs I and II in NiCoTs spatially interact to form a critical part of the selectivity filter. As seen for the V64F variant of HoxN, modification of this site can increase the velocity of transport and concomitantly reduce the specificity.     

Regulation of MHC protein expression in pancreatic beta-cells by interferon-gamma and tumor necrosis factor-alpha

Isolated human and mouse pancreatic islet cells and the rat insulinoma cell line RIN-m5F were used to examine the ability of recombinant interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) to regulate the expression of the class I and class II major histocompatibility (MHC) surface proteins and mRNA in beta-cells. Each cytokine increased significantly the expression of class I MHC proteins as determined by double indirect immunofluorescence microscopy and flow cytofluorimetric analysis. In the RIN-m5F cells, this increase in surface expressed class I MHC proteins was mirrored by an increase in the level of class I MHC mRNA. The order of potency of the cytokines on class I MHC expression was TNF-alpha plus IFN-gamma greater than or equal to IFN-gamma greater than or equal to TNF-alpha. While IFN-gamma or TNF-alpha alone were without effect, in combination they were found to induce class II MHC proteins on 30-40% of human or murine beta-cells. In contrast, IFN-gamma plus TNF-alpha did not induce detectable class II MHC proteins or mRNA in the RIN-m5F cells. These findings indicate that 1) TNF-alpha, in addition to IFN-gamma, upregulates the expression of beta-cell class I MHC proteins and mRNA, and 2) more than one signal is required for the induction of class II MHC proteins on beta-cells. The ability of IFN-gamma plus TNF-alpha to induce class II MHC proteins on only a fraction of the normal beta-cell population and not on RIN-m5F cells suggests that this response is related to the differentiation state of the beta-cell.     

Identification of a collapsed intermediate with non-native long-range interactions on the folding pathway of a pair of Fyn SH3 domain mutants by NMR relaxation dispersion spectroscopy

Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central beta-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal beta-strands that exist in the folded state (F). Initial studies focused on mutants where Gly48 is replaced; in an effort to establish whether this intermediate is a general feature of Fyn SH3 folding a series of 15N relaxation experiments monitoring the folding of Fyn SH3 mutants N53P/V55L and A39V/N53P/V55L are reported here. For these mutants as well, folding proceeds through an on-pathway intermediate with similar features to those observed for G48M and G48V Fyn SH3 domains. However, the 15N chemical shifts extracted for the intermediate indicate pronounced non-native contacts between the NH2 and COOH-terminal regions not observed previously. The kinetic parameters extracted for the folding of A39V/N53P/V55L Fyn SH3 from the three-state folding model F<-->I<-->U are in good agreement with folding and unfolding rates extrapolated to zero denaturant obtained from stopped-flow experiments analyzed in terms of a simplified two-state folding reaction. The folding of the triple mutant was studied over a wide range of temperatures, establishing that there is no difference in heat capacities between F and I states. This confirms a compact folding intermediate structure, which is supported by the 15N chemical shifts of the I state extracted from the dispersion data. The temperature-dependent relaxation data simplifies data analysis because at low temperatures (< 25 degrees C) the unfolded state (U) is negligibly populated relative to I and F. A comparison between parameters extracted at low temperatures where the F<-->I exchange model is appropriate with those from the more complex, three-state model at higher temperatures has been used to validate the protocol for analysis of three-site exchange relaxation data.