Anatomy and physiology of hamstring injury.

The hamstring muscles were analyzed anatomically and physiologically to clarify the specific reasons for the incidence of muscle strain of the hamstrings. For the anatomical study, hamstring muscles of 13 embalmed cadavers were dissected. For the physiological study, the knee flexor torque and surface electromyographic (EMG) signals were measured during isometric contraction of hamstring muscles in 10 healthy adults. The biceps femoris muscle long head (BF-L) and semimembranosus muscle (SM) had hemi-pennate architecture and their fiber length per total muscle length (FL/TML) was smaller than that of semtendinosus muscle (ST) and biceps femoris muscle short head (BF-S) with other architecture. The decrease of total muscle length per fiber length (ΔTML/FL) was larger in BF-L and SM than in ST and BF-S. The EMG activities at 0° of knee angle were at maximal compared with other knee angles and were of similar level in BF-L, in SM and in ST, whereas they were considerably smaller in BF-S. The EMG at 0° of knee angle activity per physiological cross-sectional area (PCSA) was about 1.6 times greater in BF-L than in SM. These results indicate the highest risk of muscle strain was in BF-L followed by SM.

A small nuclear RNA, U5, can transform cells in vitro.

Low-molecular-weight RNA exhibiting transforming potential was identified in chemically induced lymphoma cells by the transformation of mink lung cells after transfection. The RNA was sequenced by the direct chemical method and was shown to be a small nuclear RNA, U5. The transforming potential of the RNA was further studied in quantitative transformation assays using 3Y1, a rat fibroblastic cell line. Transformed foci appeared with a latency of 3 to 4 weeks after transfection. U5-transformed 3Y1 cells frequently carried an amplified c-myc oncogene. In addition, U5 induced chromosome aberrations in transfected cells, indicating that the RNA acts as a clastogen. Transforming and clastogenic potentials were specifically inactivated when U5 was incubated with RNase H in the presence of a complementary oligonucleotide. We discuss a possible mechanism of U5-induced cell transformation.

The C-terminal region, Arg201-Gln209, of glutathione S-transferase P contributes to stability of the active-site conformation.

The C-terminal region of rat glutathione S-transferase P (GST-P) was deleted by either carboxypeptidase (CPase) A and B or site-specific truncation to evaluate the role of the region in the catalytic mechanism. The C-terminal sequence from the 201st to 209th amino-acid residues is Arg-Pro-Ile-Asn-Gly-Asn-Gly-Lys-Gln. When seven of the C-terminal amino-acid residues from the C-terminus were removed by the CPases, the catalytic activity decreased in parallel with the amino-acid removal, amounting to less than 5% of that of the wild-type GST-P. On the other hand, a decrease of the catalytic activity was observed in a different manner when the C-terminal sequence was site-specifically truncated. The VmaxGSH/KmGSH values of the mutants withthree (GSTd207-209), four (GSTd206-209) and seven (GSTd203-209) C-terminal amino-acid residues deleted, were comparable or similar to that of the wild-type GST-P, whereas those of five (GSTd205-209), six (GSTd204-209), and eight (GSTd202-209) amino-acid residue-truncated mutants decreased to 43%, 40%, and 19% of that of the wild-type GST-P, respectively. Similar results were obtained as for VmaxCDNB/KmCDNB. The nine amino-acid residue-truncated mutant showed no catalytic activity. Heat treatment at 50 degrees C for 5 min had little effect on the catalytic activities of the wild-type GST-P and GSTd204-209, whereas those of GSTd207-209, GSTd206-209, GSTd203-209 and GSTd202-209 decreased to 22%, 27%, 18% and 10%, respectively, compared to the catalytic activity of the non-treated enzymes. Considering these results, it is concluded that the C-terminal region, Arg201-Gln209, has an important role in stabilizing the active-site conformation.

Nucleotide sequence of 5 S rRNA from gonads of a Japanese ascidian, Holocynthia roretzi.

The nucleotide sequence of 5 S rRNa from gonads of an ascidian Holocynthia roretzi has been determined. The sequence is almost equally related to those of vertebrates and most of the multicellular animal groups. The secondary structure of this 5 S rRNA fits in with the general structural model for multicellular 5 S rRNA.

Alterations in transcription factor-binding activities to fibronectin promoter during aging of vascular endothelial cells.

Previously, we showed that the expression of the fibronectin (FN) gene is enhanced during aging of human endothelial cells and fibroblasts. To elucidate the mechanism, we explored binding proteins to the FN promoter. The promoter contains sites for the general transcription factors: CAAT-binding transcription factor (CTF), promoter-specific transcription factor-1 (Sp1), and transcription factor-IID (TFIID). The promoter also contains sites for inducible transcription factors, cAMP-responsive element (CRE)-binding protein (CREB), and Activator protein 2 (AP-2). We synthesized 10 different oligonucleotides for these and other potential transcription factor-binding sites. Using these oligonucleotides, we searched for binding proteins in young and old endothelial cells by electrophoretic mobility shift and supershift assays. Our results showed that AP-1 decreased with aging, but Sp1 and CREB1 were unaffected. However, decreased binding activities to CRE at positions -170 and -415 were shown in old cells. This could be explained by the decrease of AP-1 because these CREs bound not only CREB1 but also AP-1. Moreover, we observed that the binding activities of TFIID, CTF, and binding proteins to -40, -120 and -260 regions increased. These differential changes may cause the enhancement of FN expression in senescent cells.

Senescent cells are resistant to death despite low Bcl-2 level.

Cellular senescence may be an antioncogenic event. Bcl-2 is a product of the oncogene, bcl-2, but it may also participate in cellular senescence. To investigate the role of Bcl-2, we analyzed the level of Bcl-2 during aging of normal human fibroblasts by immunoblot analysis and found that its level was highly suppressed in four normal senescent fibroblast strains. This result suggests that senescent cells are more sensitive to induction of death than young cells. Nevertheless, senescent cells showed more resistance to death induced by hydrogen peroxide than young cells, according to LDH assay. Because the balance among antiapoptotic and proapoptotic proteins is an important factor for the determination of cell death, we examined levels of other Bcl-2 family and apoptosis-regulating proteins, but observed no reasonable change to explain the resistance. During the course of the death induction experiment, we obtained results showing that growth-arrested cells were also resistant to death, and this was further confirmed by a BrdU-labeling experiment. As senescent cells are stopped permanently in the G0/G1-phase of the cell cycle, our data strongly suggest that this is the cause of resistance to death in senescent cells.

Enhanced expression of mitochondrial genes in senescent endothelial cells and fibroblasts.

It has been suggested that some mitochondrial genes are important in cellular senescence. In order to identify the mitochondrial genes that are involved in cellular senescence, we have constructed a cDNA library from senescent human vascular endothelial cells and isolated 86 senescence-specific cDNA clones by differential screening. Among the clones, we identified four distinct mitochondrial genes including NADH dehydrogenase subunit 2 (ND2), ND3, ATPase 6 and 16S ribosomal RNA. We then compared the levels of expression of these genes in young and senescent cells by using two endothelial and two fibroblast cell strains. Northern blot and slot blot hybridization confirmed that the expression levels of ND3, ATPase 6 and 16S rRNA were elevated in senescent cells of all four strains. The expression level of ND2 was also elevated during cellular senescence in three of the four strains. Because mitochondria are actively involved in oxidative phosphorylation and respiratory functions, the altered expression levels of these genes may participate in aging processes.

Expression of endothelin, fibronectin, and mortalin as aging and mortality markers.

Studies on fibronectin, endothelin-1, and mortalin from our laboratory are reviewed here. Fibronectin expression has been analyzed as upregulated during in vitro serial passaging of human fetal lung and neonatal foreskin fibroblasts as well as umbilical vein endothelial cells. In vivo aging of skin fibroblasts, as well as aortic endothelial cells, are also accompanied by upregulation of fibronectin expression. Fibronectin promoter binding proteins from young and old cell nuclear extracts were further explored by gel retardation assay. Preliminary analyses have detected age-related differential binding activities with respect to AP-1, CRES, TFIID, CTF, and AP-2 regions, whereas Sp1 binding proteins remain unaltered. Endothelin-1 expression is also seen as upregulated during in vitro and in vivo aging of endothelial cells. This can contribute to the hypertension commonly observed in elderly patients. Mortalin, a novel member of hsp 70 family of proteins, was initially identified by virtue of its association with a cellular mortal phenotype. Subsequently, normal cells and the ones with an immortal phenotype have been found to have differential subcellular localization of this protein. Antiproliferative activity of this protein in normal cells and the deregulation of expression in transformed cells is observed which suggests the association of mortalin in pathways that determine cellular divisional phenotype.

Unique action determinants of double acting topoisomerase inhibitor, TAS-103.

O6-methylguanine-DNA methyltransferase (MGMT), gamma-glutamylcysteine synthetase (gamma-GCS), and glutathione (GSH) are found to participate in resistance to TAS-103, a topoisomerase I/II inhibitor. In 13 human cancer cell lines, MGMT expression correlated with IC50 for TAS-103, whereas gamma-GCS expression inversely correlated with the IC50 value, suggesting MGMT may work to decrease TAS-103 activity but gamma-GCS may increase it. A reduced gamma-GCS and GSH, and an increased MGMT were associated with the development of resistance in A549 and DLD cells, and gamma-GCS inhibition by buthionine sulphoximine increased the TAS-103 resistance, whereas MGMT inhibition by both O6-benzyl-guanine and MGMT-antisense transfection sensitized cells to TAS-103.

Effect of water-soluble iodinated contrast media on pressure-flow relationship of red cell suspension.

The effect of radiological contrast media on blood flow through a vascular network was investigated, taking physical and physiological conditions such as osmolality into account. The perfusion of the bullfrog's hind limbs was performed, with a slight modification of the vertical tube method. The effect of contrast media on red cell deformability was studied by perfusion with erythrocyte suspensions in glutaraldehyde-fixed hind limbs. The echinocytic shape change induced by metrizamide and hypertonic iothalamate solutions caused a marked increase in resistance to flow. When the perfusion with erythrocyte suspension was performed using intact hind limbs, the pressure-flow relationship was influenced by contrast media effects on both red cell deformability and the vascular bed. Ioxaglate had less rheologic effect on the pressure-flow relationship than metrizamide or iothalamate. It could be concluded that contrast media should be isotonic, of low viscosity and chemotoxicity, and that ioxaglate was preferable to metrizamide and iothalamate at equal iodine content.

Characterization of active derivatives produced by acetamidination and selective autolysis of bovine trypsin.

A commercial preparation of bovine trypsin was treated with methyl acetimidate-HCl, and most of the lysine residues were converted to trypsin-resistant residues retaining their cationic charges. The modified preparation was then fractionated by ion-exchange chromatography on SE-Sephadex C-50 into two active components, amidinated alpha- and amidinated beta-trypsins. The latter component (Am-beta-trypsin), which consisted of a single polypeptide chain, was allowed to autolyze at pH 7.8, 25 degrees C for 3.5 h and a new active component named Am-delta-trypsin was isolated from the autolysate. Several lines of experimental evidence indicated that Am-delta-trypsin was derived by primary cleavage of the bond Arg105-Val106. Cleavage at Arg55-Leu56, on the other hand, appeared to lead to inactivation of Am-beta-trypsin. The kinetic properties of the catalytic hydrolyses of synthetic substrates and the affinity to Gly-Gly-Arg immobilized on Sepharose were compared among Am-delta-, Am-beta-, and Am-alpha-trypsins. Am-delta-trypsin resembled Am-beta-trypsin in these properties, but did not resemble Am-alpha-trypsin which had a cleavage at Lys131-Ser132.

A simple photometric method for determination of the activity of pyrocin R1.

A simple photometric method for rapid and accurate determination of the activity of pyocin R1, a bacteriocin produced by Pseudomonas aeruginosa strain P15, has been developed. This method is based on the turbidity-decrease observed when the bacteriocin is added to a suspension of sensitive bacteria P. aeruginosa strain P11. Optimum conditions for the turbidity-decreasing activity of pyocin R1 are in 0.01 M Tris-HCl buffer containing 0.2 M NaCl (pH 7.5) at 37 degrees C. A good correlation was found between the dose of pyocin R1 and the rate of the turbidity-decrease (with a correlation coefficient of more than 0.98). The amount of pyocin R1 required for this assay is nearly the same as that used for the conventional colony-counts method. The assay for one sample takes less than 3 min, whereas an overnight wait is necessary for the conventional method. This method is shown to be very suitable for following the time course of activity change observed when pyocin R1 is treated with various chemicals, including receptor substances obtained from sensitive cells. The turbidity-decrease assay was also found to be applicable to the determination of activities of other R-type pyocins.

Comparative study on fibers isolated from four R-type pyocins, phage-tail-like bacteriocins of Pseudomonas aeruginosa.

Five R-type pyocins have been reported which are almost identical with one another in their morphology and subunit composition, though distinct in receptor-binding specificity. We isolated fibers from pyocin R2, R3, and R4 by essentially the same procedure as used in our previous isolation of pyocin R1 fiber with unimpaired receptor-binding ability. All the isolated fibers including R1 fiber were indistinguishable from one another, in terms of electron microscopic observation and subunit composition analysis by SDS gel electrophoresis. They consisted mainly of Subunit No. 2 (Mw 71,000) and No. 9 (31,000) proteins. Although Subunit No. 9 protein in every fiber was susceptible to trypsin and afforded a fragment with the same molecular weight (about 19,000) detectable in the SDS gel, Subunit No. 2 protein was cleaved with trypsin only after the fiber had been treated with an organomercurial, 4-(p-sulfophenylazo)-2-mercuriphenol. The cleavage of Subunit No. 2 protein proceeded to give several fragments with molecular weights ranging from 64,000 to 34,000, and the fragmentation patterns were electrophoretically distinct at least among R1 fiber, R3 fiber, and others (R2 and R4 fibers). The results indicate that Subunit No. 2 proteins of these fibers are different from one another in the structure surrounding trypsin-susceptible peptide bonds. Immunological investigations with anti-R1 fiber antibodies provided some additional information on the difference among R-type pyocins at the fiber level.

Disulfide bridge structure of ascidian trypsin inhibitor I: similarity to Kazal-type inhibitors.

The primary structures of ascidian trypsin inhibitors (iso-inhibitors I and II) were reported in the preceding paper (Kumazaki, T. et al. (1990) J. Biochem. 107, 409-413). Both of them have eight half-cystines in a molecule composed of 55 amino acid residues with a sequence showing no extensive homology to other known protease inhibitors. To locate the four disulfide bridges in the molecule, native inhibitor I was digested with thermolysin to yield cystine-containing peptides. The peptides were separated from each other by reversed-phase HPLC. A core peptide still containing six closely located half-cystines (e.g. -Cys-Arg-Cys and -Cys-Cys-) was further digested with Streptomyces griseus trypsin for cleavage of the Arg-Cys bond. On the other hand, the Cys-Cys bond was split by applying manual Edman degradation to the core peptide. Amino acid composition analyses of the resulting cystine peptides allowed us to define the whole disulfide bridge structure in the parent molecule. The topological relation between the disulfide loops and the reactive site suggested that the ascidian trypsin inhibitor may be classified as a member of the Kazal-type inhibitor family.

Inhibition of Streptomyces griseus metallo-endopeptidase II (SGMPII) by active-site-directed inhibitors.

Inactivation of Streptomyces griseus metallo-endopeptidase II (SGMPII) by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 was studied kinetically. These reagents cause irreversible inhibition of the enzyme in a pseudo-first order reaction, and the inhibition reaction exhibits saturation kinetics. The second-order rate constants for inactivation of SGMPII by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 were measured to be 0.12 and 8.9 M-1.s-1, respectively. The order of affinities of metallo-endopeptidases towards these irreversible inhibitors is thermolysin > SGMPII > Pseudomonas aeruginosa elastase. A competitive inhibitor of SGMPII, L-Val-L-Trp, protects the enzyme against inactivation by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 in a competitive manner. Furthermore, the pH profile of the inactivation closely resembles that for the hydrolysis of synthetic peptide substrates by the enzyme. These findings suggest that these reagents bind reversibly and react irreversibly at the active site of the enzyme.

Identification of the reactive site of ascidian trypsin inhibitor.

A trypsin inhibitor from hemolymph of the solitary ascidian, Halocynthia roretzi, has been reported to be present in two forms, ATI-I and ATI-II. ATI-I consists of a single polypeptide chain with a unique sequence of 55 amino acid residues, while ATI-II has two chains that seem to be derived from ATI-I by cleavage at the Lys16-Met17 bond [Kumazaki, T., Hoshiba, N., Yokosawa, H., and Ishii, S. (1990) J. Biochem. 107, 409-413]. ATI-II (as modified inhibitor) was proved to be produced by incubation of ATI-I (as virgin inhibitor) with a catalytic amount of bovine trypsin. The tryptic hydrolysis at the Lys16-Met17 bond in virgin inhibitor showed a maximum velocity at around pH 3.5. On the other hand, acid treatment of a complex prepared by mixing equimolar quantities of trypsin and the the modified inhibitor yielded free trypsin and the virgin inhibitor. The results of chemical analyses indicated that the Lys16-Met17 bond that had been cleaved in ATI-II was resynthesized by the acid treatment. These findings strongly suggest that the Lys16-Met17 bond is the reactive site of ATI for trypsin.

Amino acid sequence and disulfide-bridge location of canine haptoglobin.

The complete amino acid sequence and disulfide-bridge location of canine haptoglobin (Hp) were determined by analyzing various fragments produced chemically and/or enzymatically. Canine Hp consists of two light (L) and two heavy (H) chains with 83 and 245 amino acid residues, respectively. It has three potential oligosaccharide-binding sequences, Asn-X-Ser/Thr, one in the L chain and two in the H chain. All of them are glycosylated. Comparison of the amino acid sequences between canine Hp and human Hp shows 68 and 85% homology for L chains and H chains, respectively. About 20% of the canine L chain still retains a carboxyl-terminal arginine residue, which is completely removed during maturation in human L-chain. The half-cystine residue at position 15 in the L chain, which participates in the inter-L chain disulfide bridging in human Hp, has been replaced by a leucine residue in canine Hp. Therefore, an LH unit in canine Hp may be joined to another LH unit by a noncovalent (mainly hydrophobic) interaction to form the complete molecule. The disulfide bridges in canine Hp link Cys-34L to Cys-68L, Cys-72L to Cys-105H, Cys-148H to Cys-179H, and Cys-190H to Cys-220H, as in the case of human Hp.

Affinity chromatography on immobilized anhydrotrypsin: general utility for selective isolation of C-terminal peptides from protease digests of proteins.

Recently we have succeeded in the efficient isolation of the C-terminal peptides from tryptic digests of the tail sheath protein (with C-terminal Gly) and the tube protein (with C-terminal Glu) of bacteriophage T4, by taking advantage of a unique property of immobilized anhydrotrypsin, that is, a strong specific affinity for peptides containing Arg or Lys residues at their C-termini. In this study, the utility of affinity chromatography on immobilized anhydrotrypsin was further demonstrated in the cases of Streptomyces subtilisin inhibitor (as a reduced and S-carboxymethylated form, with C-terminal Phe) and alpha 1-antitrypsin (with C-terminal Lys). By subjecting a tryptic digest of the former protein and a chymotryptic digest of the latter protein to the affinity chromatography, the C-terminal peptides were specifically recovered in the breakthrough fraction and in the adsorbed fraction, respectively. It was further shown that immobilized anhydrotrypsin can also adsorb peptides with C-terminal S-aminoethyl-Cys residues and exerts adsorptive ability even toward the peptides in solution containing urea at a high concentration if appropriate precautions are taken. These findings suggest the general utility of this simple method for C-terminal peptide isolation, which is extremely helpful for studies to confirm amino acid sequences deduced from nucleotide sequences of the cDNA (or genomic DNA) of proteins.

Isolation and characterization of pyocin R1 fibers.

By a mild alkaline treatment, pyocin R1 was disassembled into its structural parts, a contracted sheath (and its fragments), a core, and fibers. An alkaline sucrose density gradient centrifugation after this treatment was effective in obtaining fiber-density fractions. The pooled fractions were treated with IgGs against isolated sheaths and isolated cores, simultaneously, and then chromatographed on DEAE-Sepharose CL-6B. The final preparation of fibers purified in this way was confirmed to be homogeneous by electron microscopic observation and an immuno-precipitation reaction. The isolated fiber was found to consist of two major subunit proteins, No. 2 and No. 9, with molecular weights of 71,000 and 31,000, respectively. The fiber exhibited the ability to be adsorbed on sensitive bacterial cells (pseudomonas aeruginosa P14), and to protect against the inactivation of pyocin R1 by a lipopolysaccharide preparation from the bacteria.

Affinity chromatography of trypsin and related enzymes. II. An affinity adsorbent containing glycylglycyl-L-arginine.

An affinity adsorbent for trypsin [EC 3.4.21.4] (GGA Sepharose) was prepared. Glycylglycyl-L-arginine (GGA) was synthesized by a simple procedure and was immobilized on agarose gel. This adsorbent proved to have essentially the same characteristics as AP Sepharose, which is an affinity adsorbent containing tryptic peptides of protamine (1). GGS Sepharose was specific for native trypsin and had a stronger affinity at lower pH's (6-5) than at the optimum pH of trypsin action (8.2). It also proved to be suitable for analytical experiments because of its relatively weak affinity. By comparison of the elution profiles of trypsin from GGA Sepharose under various conditions, the nature of the interaction of trypsin with the adsorbent could be studied. It was found that alpha- and beta-trypsin could be distinguished. In the presence of arginine and N-substitute arginines, the elution of trypsin was accelerated. From the extents of the accelerating effects, the affinities of these compouunds could be compared.