Identification of encephalitogenic V beta-4-bearing T cells in SJL mice. Further evidence for the V region disease hypothesis?

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by T cells bearing TCR of restricted heterogeneity. Thus, in the murine PL strain, V beta-8.2 is used by 80% of the encephalitogenic T cells. This observation has led to the successful prevention and reversal of EAE by the in vivo use of mAb directed to these restricted gene products. In SJL mice, the V beta-17a gene product has been shown to be used by approximately 50% of encephalitogenic T cells subsequent to immunization with a myelin basic protein (MBP)-derived peptide. However, the other V beta genes used by encephalitogenic T cells in SJL EAE have remained uncharacterized. We now report, for the first time, the beta-chain-encoding DNA sequence of two encephalitogenic, MBP-reactive, SJL-derived T cell clones. These clones which are specific for H-2s and the carboxyl-terminus (amino acid 92-103) of MBP, use TCR encoded by V beta-4. In addition, we demonstrate that the transfer of EAE by a heterogenous SJL-derived encephalitogenic T cell line can be prevented using an anti-V beta-4 antibody in vivo. V beta-4 usage has been previously described in a H-2u/MBP amino-terminus-reactive encephalitogenic T cell. The present findings may thus further support the "V region-disease" hypothesis.     

Developmental Expression of HNK-1-Reactive Antigens in Rat Cerebral Cortex and Molecular Heterogeneity of Sulfoglucuronylneolactotetraosylceramide in CNS Versus PNS

Monoclonal antibody HNK-1 reacts with a carbohydrate epitope present in proteins, proteoglycans, and sulfoglucuronylglycolipids (SGGLs). On high-performance TLC plates, SGGLs of the CNS from several species migrated consistently slower than those from the PNS, a result indicating possible differences in the structures. The structural characteristics of the major SGGL, sulfoglucuronylneolactotetraosylceramide (SGGL-1), from CNS was compared with those of SGGL-1 from PNS. Although the composition, sequence, and linkages of the carbohydrate moiety of the SGGL-1 species were identical, SGGL-1 from CNS contained mainly short-chain fatty acids, 16:0, 18:0, and 18:1, amounting to 85% of the total fatty acids, whereas SGGL-1 from PNS contained large proportions (59%) of long-chain fatty acids (greater than 18:0). These differences in the fatty acid composition accounted for the different migration pattern observed. The developmental expression of SGGLs and HNK-1-reactive proteins was studied in rat cerebral cortex between embryonic day (ED) 15 to adulthood. SGGLs in the rat cortex were maximally expressed around ED 19 and almost completely disappeared by postnatal day (PD) 20. This expression was contrary to their increasing expression in the cerebellum and sciatic nerve with postnatal development. Six to eight protein bands with a molecular mass of greater than 160 kDa were HNK-1 reactive in the rat cerebral cortex at different ages. The major HNK-1 reactivity to the 160-kDa protein band seen in ED 19 to PD 10 cortex decreased and completely disappeared from the adult cortex, whereas several other proteins remained HNK-1 reactive even in the adult. Western blot analyses of the neural cell adhesion molecules (N-CAMs) during development of the rat cortex with a polyclonal anti-N-CAM antibody showed that the major HNK-1-reactive protein bands were not N-CAMs. Between PD 1 and 10, 190-200-kDa N-CAM was the major N-CAM, and between PD 15 to adulthood, 180-kDa N-CAM was the only N-CAM present in the rat cortex.     

Strong electrostatic loop-helix interactions in bundle motif protein structures.

Based on CHARMM potential (Brooks et al., 1983) an energetic analysis has been carried out for four typical 4-alpha-helix bundle proteins, i.e., methemerythrin, cytochrome b-562, cytochrome c', and bovine somatotropin. The bovine somatotropin possesses long loops, but all the other three proteins have short loops. It was found that in all these four 4-alpha-helix bundle motif structures the interaction between loops and helices was much stronger than the interaction among the four helices themselves. Particularly for the electrostatic interaction energy, the loop-helix interaction is overwhelmingly stronger than the interhelix interaction although the latter involves the favorable helix dipole interaction due to the antiparallel arrangement of neighboring alpha-helices. The present study indicates that such a conclusion holds true regardless of what loops, long or short, are in the 4-alpha-helix bundle protein, and also regardless of which empirical potential, ECEPP or CHARMM, is used for calculations although in CHARMM the electrostatic energy is much more heavily emphasized than in ECEPP. Therefore, no appropriate conclusion can be drawn in arguing whether the dipole interaction among the four alpha-helices play a stabilizing role or destabilizing role for a 4-alpha-helix bundle protein without taking into consideration the effect of interaction between helices and loops. The calculated results reported here provide, from a different point of view, insights that might be useful for revealing the essence of the driving forces during the folding of proteins.     

Monte Carlo simulation studies on the prediction of protein folding types from amino acid composition.

In the methodology development for statistical prediction of protein structures, the founders of different methods usually selected different sets of proteins to test their predicted results. Therefore, it is hard to make a fair comparison according to the results they reported. Even if the predictions by different methods are performed for the same set of proteins, there is still such a problem: a method better that the other for one set of proteins would not necessarily remain so when applied to another set of proteins. To tackle this problem, a Monte Carlo simulation method is proposed to establish an objective criterion to measure the accuracy of prediction for the protein folding type. Such an objective accuracy is actually corresponding to the asymptotical limit genereated during the Monte Carlo simulation process. Based on that, it has been found that the average objective accuracy for predicting the all-alpha, all-beta, alpha + beta, and alpha/beta proteins by the least Euclid's distance method (Nakashima, H., K. Nishikawa, and T. Ooi. 1986. J. Biochem. 99:152-162) is 73.0% and that by the least Minkowski's distance method (Chou, P.Y. 1989. Prediction in Protein Structure and the Principles of Protein Conformation. Plenum Press. New York. 549-586) is 70.9%, indicating that the former is better than the latter. However, according to the original reports, the latter claimed a rate of correct prediction with 79.7% but the former with only 70.2%, leading to a completely opposite conclusion. This indicates the necessity of establishing an objective criterion, and a comparison is meaningful only when it is based on the objective criterion. The simulation method and the idea developed here also can be applied to examine any other statistical prediction methods.     

Evidence that the acidic polysaccharide secreted by Agrobacterium radiobacter (ATCC 53271) has a seventeen glycosyl-residue repeating unit.

The extracellular anionic polysaccharide produced by the bacterium Agrobacterium radiobacter (ATCC 53271) contains D-galactose, D-glucose, and pyruvic acid in the molar ratio 2:15:2. Analysis of the methylated polysaccharide indicated the presence of terminal, non-reducing glucosyl, 3-, 4-, 6-, 2,4-, and 4,6-linked glucosyl residues, 3-linked 4,6-O-[(S)-1-carboxyethylidene]glucosyl residues, and 3-linked galactosyl residues. Partial acid hydrolysis of the methylated polysaccharide, followed by reduction with NaB2H4 and then O-ethylation, gave a mixture of alkylated oligoglycosyl alditols that were separated by reversed-phase h.p.l.c. and analyzed by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. Smith degradation of the polysaccharide gave three diglycosyl alditols that were separated by semi-preparative, high-pH anion-exchange chromatography, and were analyzed by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. The polymer obtained by NaBH4 reduction of the periodate-oxidized polysaccharide was methylated, and the noncyclic acetals were hydrolyzed with aq. 90% formic acid to generate a mixture of partially O-methylated mono- and di-glycosyl alditols. The partially O-methylated oligoglycosyl alditols were O-ethylated. The resulting alkylated oligoglycosyl alditols were separated by reverse-phase h.p.l.c. and then characterized by 1H-n.m.r. spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis. The results from the studies described here provide strong evidence that the acidic polysaccharide secreted by A. radiobacter (ATCC 53271) has a heptadecasaccharide repeating unit.     

Energy-optimized structure of antifreeze protein and its binding mechanism.

A combination of Monte Carlo simulated annealing and energy minimization was utilized to determine the conformation of the antifreeze protein from the fish winter flounder. It was found from the energy-optimized structure that the hydroxyl groups of its four threonine residues, i.e. Thr2, Thr13, Thr24, Thr35, are aligned on almost the same line parallel to the helix axis and separated successively by 16.1, 16.0 and 16.2 A, respectively, very close to the 16.6 A repeat spacing along [0112] in ice. Based on such a space match, a zipper-like model is proposed to elucidate the binding mechanism of the antifreeze protein to ice crystals. According to the current model, the antifreeze protein may bind to an ice nucleation structure in a zipper-like fashion through hydrogen bonding of the hydroxyl groups of these four Thr residues to the oxygen atoms along the [0112] direction in ice lattice, subsequently stopping or retarding the growth of ice pyramidal planes so as to depress the freeze point. The calculated results and the binding mechanism thus derived accord with recent experimental observations. The mechanistic implications derived from such a special antifreeze molecule might be generally applied to elucidate the structure-function relationship of other antifreeze proteins with the following two common features: (1) recurrence of a Thr residue (or any other polar amino acid residue whose side-chain can form a hydrogen bond with water) in an 11-amino-acid period along the sequence concerned; and (2) a high percentage of Ala residue component therein. Further experiments are suggested to test the ice binding model.     

High-resolution NMR studies of chimeric DNA-RNA-DNA duplexes, heteronomous base pairing, and continuous base stacking at junctions.

Two symmetrical DNA-RNA-DNA duplex chimeras, d(CGCG)r(AAUU)d(CGCG) (designated rAAUU) and d(CGCG)r(UAUA)d(CGCG) (designated rUAUA), and a nonsymmetrical chimeric duplex, d(CGTT)r(AUAA)d(TGCG)/d(CGCA)r(UUAU)d(A ACG) (designated rAUAA), as well as their pure DNA analogues, containing dU instead of T, have been synthesized by solid-phase phosphoramidite methods and studied by high-resolution NMR techniques. The 1D imino proton NOE spectra of these d-r-d chimeras indicate normal Watson-Crick hydrogen bonding and base stacking at the junction region. Preliminary qualitative NOESY, COSY, and chemical shift data suggest that the internal RNA segment contains C3'-endo (A-type) sugar conformations except for the first RNA residues (position 5 and 17) following the 3' end of the DNA block, which, unlike the other six ribonucleotides, exhibit detectable H1'-H2' J coupling. The nucleosides of the two flanking DNA segments appear to adopt a fairly normal C2'-endo B-DNA conformation except at the junction with the RNA blocks (residues 4 and 16), where the last DNA residue appears to adopt an intermediate sugar conformation. The DNA-RNA junction residues exhibit quite different COSY, chemical shift, and NOE behavior, but these effects do not appear to propagate into the DNA or RNA segments. The circular dichroism spectra of these d-r-d chimeras also display a mixture of characteristic A-type and B-type absorption bands. The data indicate that A-type and B-type conformations can coexist in a single short continuous nucleic acid duplex, but our results differ somewhat from previous theoretical model studies.     

Energetic approach to the folding of alpha/beta barrels

The folding of a polypeptide into a parallel (alpha/beta)8 barrel (which is also called a circularly permuted beta 8 alpha 8 barrel) has been investigated in terms of energy minimization. According to the arrangement of hydrogen bonds between two neighboring beta-strands of the central barrel therein, such an alpha/beta barrel structure can be folded into six different types: (1) left-tilted, left-handed crossover; (2) left-tilted, right-handed crossover; (3) nontilted, left-handed crossover; (4) nontilted, right-handed crossover; (5) right-tilted, left-handed crossover; and (6) right-tilted, right-handed crossover. Here "tilt" refers to the orientational relation of the beta-strands to the axis of the central beta-barrel, and "crossover" to the beta alpha beta folding connection feature of the parallel beta-barrel. It has been found that the right-tilted, right-handed crossover alpha/beta barrel possesses much lower energy than the other five types of alpha/beta barrels, elucidating why the observed alpha/beta barrels in proteins always assume the form of right tilt and right-handed crossover connection. As observed, the beta-strands in the energy-minimized right-tilted, right-handed crossover (alpha/beta)8-barrel are of strong right-handed twist. The value of root-mean-square fits also indicates that the central barrel contained in the lowest energy (alpha/beta)8 structure thus found coincides very well with the observed 8-stranded parallel beta-barrel in triose phosphate isomerase (TIM). Furthermore, an energetic analysis has been made demonstrating why the right-tilt, right-handed crossover barrel is the most stable structure. Our calculations and analysis support the principle that it is possible to account for the main features of frequently occurring folding patterns in proteins by means of conformational energy calculations even for very complicated structures such as (alpha/beta)8 barrels.     

Cyclic AMP induces activity increase of kinase FA (a transmembrane signal of insulin) during NG108-15 hybrid cell differentiation

The ATP.Mg-dependent protein phosphatase activating factor (protein kinase FA) has been identified to exist in neuroblastoma x glioma hybrid 108-15 cells (NG108-15 cells). More importantly, when NG cells were induced to differentiate with N6, O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (dibutyryl cAMP), the cellular activity of kinase FA was found to increase dramatically. Time course study further revealed that induction of differentiation in NG cells by dibutyryl cAMP treatment increased the FA activity to over 3 times the levels found in undifferentiated cells and in a linear day-dependent manner, indicating that the FA activity level is correlated with the state of differentiation of NG108-15 cells. This is the first report providing initial evidence that protein kinase FA (a transmembrane signal of insulin) is involved in the induction of neuronal cell differentiation.     

Systems for exposing mice to 2,450-MHz electromagnetic fields

Two systems for exposing mice to 2,450-MHz electromagnetic fields are described. In a waveguide system, four mice were placed in a Styrofoam cage and exposed dorsally to circularly polarized electromagnetic fields. The temperature and humidity in the mouse holder were kept constant by forced-air ventilation. For 1-W input power to the waveguide, the average specific absorption rate (SAR) was determined by twin-well calorimetry to be 3.60 ± 0.11 (SE) W/kg in 27-g mice. The maximum SAR at the skin surface determined thermographically was 8.36 W/kg in the head of the mouse. The second system was a miniature anechoic chamber. Six mice were irradiated dorsally to far field plane waves. Copper shielding and high-temperature absorbing material were lined inside the chamber to accommodate the high input power. The air ventilation at the location of the mice was separately controlled so that any heating in the absorber would not affect the animals. For 1-W input power, the average SAR was 0.17 ± 0.01 W/kg and the maximum SAR at the skin surface was 0.41 W/kg in the animal when irradiated with body axis parallel to the E field; the SARs were 0.11 ± 0.01 W/kg and 0.64 W/kg, respectively, when irradiated perpendicular to the E field.