Discharge properties of human motor units during sustained contraction at low level force.

The characteristic of discharge behaviors of motor units (MUs) during low level contraction was investigated. The discharge of MUs in the m. vastus medialis was observed during the sustained contraction at 4 different levels below 10% MVC (2, 4, 8 and 10% MVC) for 15 min. The spike interval of all observed MUs gradually elongated during an initial several minutes of the contraction and the characteristic discharge patterns following the elongation were observed. i.e. continuous discharge throughout the contraction (CONT), decruitment (D-N), and re-recruitment following decruitment (D-REC). The relationship between recruitment threshold force (F(th)) and discharge pattern was not significant at 2% MVC but, at 10% MVC, there were significant differences in F(th) between D-N and CONT, and between D-REC and CONT MU populations. In pooled data, the MUs with the shorter mean spike interval at the beginning of the contraction (MSI(0), below 90 ms) tend to discharge continuously, but the MUs with longer MSI(0) showed various discharge patterns. In conclusion, during low level contraction MUs discharge characteristically, and the MU with high excitation levels tend to discharge continuously, but individual MU represents an intrinsic discharge pattern at not a high excitation level.

Restoration of immunocyte functions by thymosin alpha1 in cyclophosphamide-induced immunodeficient mice.

Thymosin alpha1 (Talpha1) is an oligopeptide hormone originally isolated from the thymus gland, and has been reported to have stimulating effects on the differentiation of T cells and NK cells. These immunostimulating properties have been considered to be useful for improving immune disorders associated with various diseases including cancer, AIDS and hepatitis. Here, we characterized immunostimulating properties of Talpha1 in experimental immunodeficiency of mice that was induced by the administration of cyclophosphamide (CY). Repeated injection of 30-300 microg/kg/day of Talpha1 after CY-treatment significantly accelerated the restoration of the reduced number of CD4+CD8+ T cells in the thymus. Talpha1 administration was effective in restoring the suppressed activities of helper T cells and cytotoxic T cells in CY-treated mice. Talpha1 also had stimulating effects on reduced activity of lymphokine-activated killer cells in CY-treated mice. These results indicate that Talpha1 is stimulatory for both humoral and cellular immune responses, thus providing the immunological basis for the clinical benefit of this compound.

Application of chemical selective cleavage methods to analyze post-translational modification in proteins.

Three chemical specific cleavage reactions, one for the carboxyl side of aspartyl peptide bonds, one for the carboxyl side of asparaginyl peptide bonds and another for the amino side of seryl/threonyl peptide bonds have been recently established. Additionally, these reactions simultaneously react on several post-translationally modified groups in peptides or proteins. The modified groups cover the external modifications N-formyl, N-acetyl, N-pyroglutamyi residues and C-terminal-alpha amide, as well as the internal modifications such as O-acetyl serine, phosphorylated serine/tyrosine, sulfonylated tyrosine, glycosylated serine/threonine and glycosylated asparagine. These three cleavage reactions relate to key amino acids for modifications, deamidation for asparagine, phosphorylation and acetylation for serine, and glycosylation for asparagine, serine and threonine. The chemical reactions on these modifications change the peptide mapping pattern, and information from these reactions may contribute characterization and location of post-translational modified groups in the protein.

Purification of ferredoxins and their reaction with purified reaction center complex from the green sulfur bacterium Chlorobium tepidum.

Four ferredoxin (Fd) fractions, namely, FdA-D were purified from the green sulfur bacterium Chlorobium tepidum. Their absorption spectra are typical of 2[4Fe-4S] cluster type Fds with peaks at about 385 and 280 nm and a shoulder at about 305 nm. The A(385)/A(280) ratios of the purified Fds were 0.76-0.80. Analysis of the N-terminal amino acid sequences of these Fds (15-25 residues) revealed that those of FdA and FdB completely agree with those deduced from the genes, fdx3 and fdx2, respectively, found in this bacterium (Chung and Bryant, personal communication). The N-terminal amino acid sequences of FdC and FdD (15 residues) were identical, and agree with that deduced from the gene fdx1 (Chung and Bryant, personal communication). The A(385) values of these Fds were unchanged when they were stored for a month at -80 degrees C under aerobic conditions and decreased by 10-15% when they were stored for 6 days at 4 degrees C under aerobic conditions, indicating that they are not extremely unstable. In the presence of Fd-NADP(+) reductase from spinach, and a purified reaction center (RC) preparation from C. tepidum composed of five kinds of polypeptides, these Fds supported the photoreduction of NADP(+) at room temperature with the following K(m) and V(max) (in micromol NADP(+) micromol BChl a(-1) h(-1)): FdA, 2.0 microm and 258; FdB, 0.49 microM and 304; FdC, 1.13 microM and 226; FdD, 0.5 microM and 242; spinach Fd, 0.54 microM and 183. The V(max) value of FdB was more than twice that previously reported for purified RC preparations from green sulfur bacteria.

Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging.

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references.

Comparative analysis of brain proteins from p53-deficient mice by two-dimensional electrophoresis.

p53 is a tumor suppressor protein that regulates many cellular processes including the cell cycle, DNA repair, and apoptosis. It also serves as a critical regulator of neuronal apoptosis in the central nervous system (CNS). To elucidate the role of p53 in the CNS, brain proteins of p53 knock-out mice (p53-/-) were analyzed by two-dimensional gel electrophoresis (2-DE) and compared with those from p53 wild type (p53+/+) mice. Six types of brain tissue (temporal cortex, cerebellum, hippocampus, striatum, olfactory bulb, and cervical spinal cord) and other control tissues (lung and blood) from 18-week-old non-stress-induced mice were analyzed. The morphology of brains from p53-/- mice appeared to be normal and identical to that of p53+/+ mice, although lungs showed diffuse tumors that may have been caused by p53 deficiency. Comparative 2-D gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53-/- specific, whereas 12 of 1008 spots from lung tissue were p53-/- specific. N-terminal amino acid sequence was determined for p53-/- specific proteins. In all brain tissues from p53-/- mice, a newly identified mouse mitochondrial NADH-ubiquinone oxidoreductase 24 kDa subunit showed decreased expression, and apolipoprotein A1 acidic forms showed increased expression. In addition, brain-type creatine kinase B chain and tubulin beta-5 N-terminal fragment were increased in the p53-/- cerebellum, and a new protein in mouse, hydroxyacylglutathione hydrolase (glyoxalase II) was decreased in the temporal cortex of p53-/- mice. The alterations in protein expression identified in this study may imply a p53-related brain function. This is the first proteomic analysis on the p53-/- mouse brain, and further information based on this study will provide new insights into the p53 function in the CNS.

Cloning, expression of the psbU gene, and functional studies of the recombinant 12-kDa protein of photosystem II from a red alga Cyanidium caldarium.

The encoding extrinsic 12-kDa protein of oxygen-evolving PS II complex from a red alga, Cyanidium caldarium, was cloned and sequenced by means of PCR and a rapid amplification of cDNA ends (RACE) procedure. The gene encodes a putative polypeptide of 154 amino acids with a calculated molecular mass of 16,714 Da. The full sequence of the protein includes two characteristic transit peptides, one for transfer across the chloroplast envelope and another for targeting into the thylakoid lumen. This indicates that the protein is encoded in the nuclear genome. The mature protein consists of 93 amino acids with a calculated molecular mass of 10,513 Da. The cloned gene was successfully expressed in Escherichia coli and the resulting protein was purified, reconstituted to CaCl2-washed PS II complex together with the other extrinsic proteins of 33 and 20 kDa and cyt c-550. The recombinant 12-kDa protein bound completely with the PSII complex, which resulted in a restoration of oxygen evolution equal to the level achieved by binding of the native 12-kDa protein.

Specific cleavage of amino side chains of serine and threonine in peptides and proteins with S-ethyltrifluorothioacetate vapor.

A vapor of S-ethyltrifluorothioacetate was found to specifically cleave the amino side of serine and threonine peptide bonds. The cleavage reactions were carried out at 50 degrees C for 6 h-24 h or at 30 degrees C for 24 h. When vapors were generated in a solution containing several conventional organic solvents, the cleavage reactions were reduced or stopped, or modification took place. When the reagent vapor was made in an aqueous solution, the cleavage reaction at glycine residues was enhanced. This reagent did not oxidize any amino acid residues, such as methionine, cysteine and tryptophan. The cleavage was also effective on proteins on membranes blotted or electroblotted from polyacrylamide gels. This method therefore may be used for the peptide mass fingerprinting [Patterson, S. D. (1995) Electrophoresis 16, 1104-1114] after two-dimensional electrophoresis.

Additional possible tools for identification of proteins on one- or two-dimensional electrophoresis.

Additional, essentially chemical, identification methods of proteins in polyacrylamide gel electrophoresis are described. Two cleavages of peptide bonds were used at the C-side of aspartic acid with a 0.2% pentafluoropropionic acid (PFPA) aqueous vapor at 90 degrees C for 4-16 h, and the N-side of serine/threonine with an S-ethyl trifluorothioacetate vapor at 50 degrees C for 6-24 h. The products were analyzed by mass spectrometry-peptide mass fingerprinting. A new type of C-terminal sequencing at multisites of protein was introduced. An aqueous vapor of 90% PFPA at 90 degrees C for 2-16 h provided cleavages at the C-side of aspartic acid and the N-side of serine/threonine and simultaneous successive truncation at the C-termini of the cleaved fragments. The product resulted in C-terminal sequences at multisites in proteins by mass spectrometric analysis. The following chemical deblocking methods were used. Anhydrous hydrazine vapor at -5 degrees C for 8 h deblocked the N-formyl group, and the vapor at 20 degrees C for 4 h deblocked pyrrolidone carboxylate. N-acetylserine/threonine was deblocked by aqueous vapor of 75% PFPA at 50 degrees C for 1 h, followed by reaction with p-sulfophenylisothiocyanate at pH 6.0. These methods were applied to a variety of protein spots on polyacrylamide gels. A new stepwise C-terminal sequencing of protein from polyacrylamide gels is also described.

Intramolecular cross-linking of the extrinsic 33-kDa protein leads to loss of oxygen evolution but not its ability of binding to photosystem II and stabilization of the manganese cluster.

The extrinsic 33-kDa protein of photosystem II (PSII) was intramolecularly cross-linked by a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The resulting cross-linked 33-kDa protein rebound to urea/NaCl-washed PSII membranes, which stabilized the binding of manganese as effectively as the untreated 33-kDa protein. In contrast, the oxygen evolution was not restored by binding of the cross-linked protein, indicating that the binding and manganese-stabilizing capabilities of the 33-kDa protein are retained but its reactivating ability is lost by intramolecular cross-linking of the protein. From measurements of CD spectra at high temperatures, the secondary structure of the intramolecularly cross-linked 33-kDa protein was found to be stabilized against heat treatment at temperatures 20 degrees C higher than that of the untreated 33-kDa protein, suggesting that structural flexibility of the 33-kDa protein was much decreased by the intramolecular cross-linking. The rigid structure is possibly responsible for the loss of the reactivating ability of the 33-kDa protein, which implies that binding of the 33-kDa protein to PSII is accompanied by a conformational change essential for the reactivation of oxygen evolution. Peptide mapping, N-terminal sequencing, and mass spectroscopic analysis of protease-digested products of the intramolecularly cross-linked 33-kDa protein revealed that cross-linkings occurred between the amino group of Lys48 and the carboxyl group of Glu246, and between the carboxyl group of Glu10 and the amino group of Lys14. These cross-linked amino acid residues are thus closely associated with each other through electrostatic interactions.

A uniformly cleaved epitaxically grown diamond crystal for synchrotron radiation.

A homoepitaxic single-crystal diamond (111) film grown by microwave-assisted chemical vapour deposition (CVD) and fractured along the [110] directions to form small triangles was investigated by X-ray double-crystal topography. The X-ray topographic image showed that all parts of the cleaved CVD diamond film sections uniformly reflected X-rays at the peak position of the rocking curve, which was measured in the Bragg case. Furthermore, no bending effect was observed and the CVD diamond film appeared to be more perfect than and showed higher integrated intensity than the natural diamond substrate.

Identification of domains on the 43 kDa chlorophyll-carrying protein (CP43) that are shielded from tryptic attack by binding of the extrinsic 33 kDa protein with photosystem II complex.

The structural association of the spinach 33 kDa extrinsic protein with the 43 kDa chlorophyll-carrying protein (CP43) in oxygen-evolving photosystem II (PS II) complexes was investigated by comparing the peptide mappings and N-terminal sequences of the trypsin-digested products of NaCl-washed PS II membranes, which bind the 33 kDa protein, with those of CaCl2-washed PS II membranes, which lack the 33 kDa protein. (1) Peptide from N-terminus to Arg26 of CP43, which is exposed to stromal side, was digested in both PS II membranes, independent of binding of the 33 kDa protein. (2) Peptide bond of Arg357-Phe358 located in the large extrinsic loop E of CP43, which is exposed to lumenal side, was cleaved by trypsin in CaCl2-washed PS II membranes but not in NaCl-washed PS II membranes. This indicates that the region around Arg357-Phe358 in loop E of CP43 is shielded from tryptic attack by binding of the 33 kDa protein to PS II. (3) Trypsin treatment of CaCl2-washed PS II membranes also cleaved peptide bond between Lys457 and Gly458 in C-terminal region of CP43, while no cleavage of this region was detected by trypsin treatment of NaCl-washed PS II membranes. This implies that a conformational change of the C-terminal region of CP43 which is exposed to stromal side occurred upon removal of the 33 kDa protein, which makes the C-terminal region accessible to trypsin. (4) Release of peptide from Gln60 to C-terminus of the alpha-subunit of cytochrome b-559 was detected only in trypsin treatment of CaCl2-washed PS II membranes, indicating that the C-terminal region of this subunit is shielded from tryptic attack by binding of the 33 kDa protein. (5) The PS II membranes, in which Arg357-Phe358, Lys457-Gly458 of CP43 and the C-terminal part of the cytochrome b-559 alpha-subunit had been cleaved by trypsin, was no longer able to bind the 33 kDa protein. This strongly suggests that a domain in loop E of CP43 and/or the C-terminal region of the cytochrome b-559 alpha-subunit are necessary for binding of the extrinsic 33 kDa protein to PS II.

Identification of domains on the extrinsic 33-kDa protein possibly involved in electrostatic interaction with photosystem II complex by means of chemical modification.

The extrinsic 33-kDa protein of photosystem II (PSII) was modified with various reagents, and the resulting proteins were checked for the ability to rebind to PSII and to reactivate oxygen evolution. While modification of more than eight carboxyl groups of aspartyl and glutamyl residues with glycine methyl ester did not affect the rebinding and reactivating capabilities, modification of amino groups of lysyl residues with either N-succinimidyl propionate or 2, 4,6-trinitrobenzene sulfonic acid or modification of guanidino groups of arginyl residues with 2,3-butanedione resulted in a loss of rebinding and reactivating capabilities of the 33-kDa protein. Moreover, the number of lysyl and arginyl residues susceptible to modification was significantly decreased when the protein was bound to PSII as compared with when it was free in solution, whereas the number of carboxyl groups modified was little affected. These results suggested that positive charges are important for the electrostatic interaction between the extrinsic 33-kDa protein and PSII intrinsic proteins, whereas negative charges on the protein do not contribute to such interaction. By a combination of protease digestion and mass spectroscopic analysis, the domains of lysyl residues accessible to N-succinimidyl propionate or 2,4, 6-trinitrobenzene sulfonic acid modification only when the 33-kDa protein is free in solution were determined to be Lys4, Lys20, Lys66-Lys76, Lys101, Lys105, Lys130, Lys159, Lys186, and Lys230-Lys236. These domains include those previously reported accessible to N-hydroxysuccinimidobiotin only in solution (Frankel and Bricker (1995) Biochemistry 34, 7492-7497), and may be important for the interaction of the 33-kDa protein with PSII intrinsic proteins.