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Collagen-related peptides, Gly-Pro-Arg and its analogues, were examined for their inhibitory effects on platelet aggregation induced by the addition of ADP. Human platelet aggregation was suppressed by more than 50% with each of Gly-Pro-Arg and such Gly-Pro-Arg-containing peptides as Gly-Pro-Arg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Pro-Pro, and Gly-Pro-Arg-Pro-Pro-Pro at a concentration of 0.3 mM. The inhibitory effects of these peptides were about 10 times higher in human PRP than in rat PRP. Other Gly-Pro-Arg analogues such as Sar-Pro-Arg, Gly-Pro-Lys, Gly-Ala-Arg, and Ala-Gly-Pro-Arg had no inhibitory effect at a concentration from 0.1 to 0.8 mM even in human PRP. Intravenous and oral administrations of Gly-Pro-Arg and enzymatic hydrolysates of collagen suppressed the decrease in platelet count for endotoxin-induced DIC in rats. Collagen itself has been regarded as a potent inducer of platelet aggregation, but these findings suggest that collagen-related peptides and enzymatic hydrolysates of collagen prevent platelet aggregation.

We investigated the effects of a chicken collagen hydrolysate (CCH) on the circulation system in humans. A total of 58 subjects with either mild hypertension (systolic blood pressure (SBP) of 140-159 mmHg or diastolic blood pressure (DBP) 90-99 mmHg) or high-normal blood pressure (SBP 130-139 mmHg or DBP 85-89 mmHg) were assigned to two groups, one involving a placebo and the other, the test food (including CCH of 2.9 g/d). The parameters related to each subject's circulation system were monitored over the study period of 18 weeks. The Δbrachial-ankle pulse wave velocity (baPWV), an indicator of arterial stiffness and marker of vascular damage, was significantly lower in the test food group than in the placebo group during the treatment period. The blood pressure in the test food group was also significantly lower than that in the placebo group, while the serum nitrogen oxide was higher in the test food group after the treatment. These results suggest that CCH exerted modulatory effects on the human circulation system.

The ammonia-oxidizing chemoautotrophic bacterium Nitrosomonas europaea possesses prominant succinate-reducing activity of succinyl-Coenzyme A synthetase (SCS, EC 6.2.1.5). SCS was purified as an electrophoretically homogeneous protein from Nitrosomonas europaea strain ATCC 25978 about 275-fold, with a 3.9% activity yield. The molecular mass of the native enzyme was estimated to be about 130 kDa by gel filtration, whereas SDS-PAGE gave two protein bands with M r values of 29 (α) and 36 kDa (β). The isoelectric point of the enzyme was 5.3. The apparent K m values of the enzyme for ATP, succinate and CoA were 0.4 mM, 5 mM and 0.1 mM, respectively. The pH and temperature optima of the SCS were about 5.0 and 55°C, respectively. The SCS was stable in the pH range of 8.0–10.0 and up to 70°C. The enzyme was thermostable; 50% of the enzyme activity was retained at 90–100°C for 10 min. The SCS was activated by Mg 2+ at 1.0–100 mM, but inhibited by Cu 2+ (0.1 mM) and SDS (1.0 mM). The enzyme utilized ATP as the preferred substrate.

High pressure at 101.3 to 506.6 MPa was applied to bovine liver and fractioned lysosomes to examine its effects on the lysosomes and the enzymes involved. Carboxypeptidase was completely inactivated at 405.3 MPa. The activities of about 60, 70, 30, and 50% of acid phosphatase and cathepsins B, D, and H, respectively, remained after pressurization at 506.6 MPa. Pressurization of the liver at 506.6 MPa or lower caused changes in the subcellular distribution of lysosomal enzymes. Acid phosphatase and cathepsins B and H were released from fractioned lysosome by treatment higher than 202.7 MPa and cathepsin D at 101.3 MPa. These findings indicate that high pressure at 101.3 to 202.7 MPa induces destruction of the lysosome membranes and leakage of the lysosomal enzymes.

Ammonia-oxidizing chemoautotrophic Nitrosomonas europaea ATCC 25978 exhibited remarkable oxaloacetate-oxidative activity. Citrate(si)-synthase [EC 4.1.3.7.] was purified as an electrophoretic homogeneous protein. The molecular weight of the enzyme was estimated to be about 295, 000 (αxβy) by gel filtration, suggesting that the enzyme consisted of two different subunits (α; 65, 000, β; 75, 000), as demonstrated by SDS-PAGE. The N-terminal sequences of α and β type enzyme proteins were Ala-Leu-Val-Ser-Leu-Arg-Gln-Leu-Leu. The isoelectric point of the enzyme was pH 5.2. The enzyme was stable up to 35°C and in a pH range between 6.0-8.0. The optimum pH for the reaction was 6.5-8.5 and the optimum temperature was 30-40°C. The apparent Km values for oxaloacetate and acetyl-CoA were about 0.025 and 0.080mM, respectively. The enzyme was not inhibited by ATP (1mM), NADH (1mM) or 2-oxoglutamic acid (10mM). Activation by metal ions (0.1mM) was not observed.

Ammonia-oxidizing chemoautotrophic Nitrosomonas europaea exhibited the NADP-specific isocitrate dehydrogenase (ICDH) activity. Isocitrate-oxidizing activity was found in two fractions (ICDH-FI and ICDH-FII) on a DEAE-Sepharose CL-6B. These enzyme proteins with isocitrate-oxidizing activity were further purified as electrophoretic homogeneity using gel filtration (Cellulofine GCL-2000-m) and then ion exchange chromatography (Mono Q). These enzymes were purified about 69-fold for ICDH-FI and about 140-fold for ICDH-FII. The molecular weights were determined as both 82, 000 for both, using gel filtration and SDS-PAGE of enzymes. The isoelectric points of ICDH-FI and ICDH-FII were pH 5.8 and 6.2, respectively.The pH and temperature optima for ICDH-FI and ICDH-FII were pH 8.5 and 8.0, respectively, and about 55°C for both enzymes. The enzymes were stable up to 45°C and acted specifically on D- or DL-isocitrate and not on any other organic acids tested. The apparent Km values of ICDH-FI for D- and DL-isocitrate were 15.0μM and 17.5μM, respectively and Km values of ICDH-FII for D- and DL-isocitrate were 13.0μM and 11.5μM, respectively. Both enzymes were strongly activated by Mn2+ and Mg2+, and inhibited by Cu2+, Ni2+, Zn2+ and Hg2+. They were strongly inhibited by p-chloromercuribenzoate.

High pressures of 1,000 to 5,000 atm were applied to beef rounds to study the effects of pressure on the proteolytic activities involved. Acid protease was scarcely inactivated, and neutral protease was slightly inactivated at 4,000 atm or higher. Aminopeptidase and carboxypeptidase were completely inactivated at 5,000 and 4,000 atm, respectively. The autolytic activity of the extract from pressure-treated beef at pH 3.0 to 6.0 increased with increasing applied pressure, while that at pH 6.5 to 8.0 showed two maxima at 3,000 and 5,000 atm. The increase in free amino acid content was amplified by pressurization at 1,000 to 3,000 atm, and the tryptic digestibility of the extract from the pressurized beef was increased at 4,000 atm or higher. These results suggest that high-pressure treatment modulates the proteolytic activities of meat to improve its quality.

A simple and rapid method using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of 130 veterinary drugs and their metabolites in bovine, porcine, and chicken muscle was developed. The drugs (1 to 10 ng/g, in muscle) were extracted from bovine, porcine, or chicken muscles with acetonitrile-methanol (95:5, v/v), and the extracts were delipidated with n-hexane saturated with acetonitrile. The extracts were evaporated, dissolved with methanol, analyzed by liquid chromatography with gradient elution on a C18 column, and determined by electrospray ionization tandem mass spectrometry. The detection limits ranged from 0.03 to 3 ng/g. The quantitation limits ranged from 0.1 to 10 ng/g. One hundred eleven, 122, and 123 drugs from bovine, porcine, and chicken muscle respectively showed recoveries between 70 and 110%.

The properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) of an obligate chemoautotrophic bacterium, Nitrosomonas europaea ATCC 25978 were studied. The pH and temperature optima of the PEPC activity were about 8.0–8.5 and 45–50°C, respectively. The PEPC was stable in the pH range 6.0–10.0 and up to 35°C. It required Mg2+ (20 mM) or Mn2+ (10 mM), and was activated by acetyl-CoA (0.16 mM). SDS (1 mM), pHMB (1 mM) and l -malate (30 mM) were found to be inhibitory against the PEPC activity, while l -aspartate, FBP and various nucleotides had no effect.

The brain prolylendopeptidase (PEP) inhibitory activity of synthetic peptides related to the bovine brain-derived PEP inhibitor, GFAP-(38–55) (MPPPLPARVDFSLAGALN), was investigated. Homologous peptides such as MPPPLPTRVDFSLAGALN (human type) and MTPPLPARVDFSLAGALN (mouse type) also inhibited PEP. Among various synthetic fragments of GFAP-(38–55), only MPPPLP had a Ki essentially the same as that of GFAP-(38–55). Similar synthetic proline-rich peptides such as synapsin fragments and SH3 domain-binding peptides had more or less inhibitory activity.

An aminopeptidase P, capable of hydrolyzing oligoproline, was isolated from the homogenate of bovine brain. The molecular mass of the enzyme was 140 kDa by gel filtration. The enzyme was activated by Mn2+ and inhibited by o-phenanthroline. The enzyme hydrolyzed substrates such as Pro-Pro-Pro-Pro, Pro-Pro-Pro, and Pro-Pro to proline, and cleaved N-terminal amino acids from peptides containing penultimate prolines such as bradykinin and neuropeptide Y.

The title compound has been synthesized efficiently from 2-deoxy-D-ribose. The synthesis involves : 1) an aldol like carbon-carbcr. bond formation of (S)-3, 5-dibenzyioxy-2-pentanor.e (8) with a lithium enolate of methyl propanoate, and 2) O-de-benzylation of the aldol adducts (11) for a γ-lactonization followed by β-elimination of the desired butenolide skeleton.

The relationship between proliferation and differentiation in Dictyostelium discoideum Ax-2 was analyzed with reference to the cell-cycle position at the onset of starvation, using cells synchronized by temperature shift (11.5°C-22.0°C). To examine how far Ax-2 cells at any particular phase of the cell cycle are able to progress through the cycle in response to nutritional deprivation, we measured temporal changes in cell number and nuclearity after starvation. Nuclear DNA synthesis in synchronously developing cells was also monitored by pulse-labeling with [methyl-3H]thymidine. Increase in cell number and subsequent DNA synthesis occurred in cells just before mitosis (referred to as TO.5 cells and T1 cells; 0.5 h and 1 h after the shift-up from 11.5° C to 22.0° C respectively), but not in T3, T5, or T7 cells. When T1 cells were incubated for 6 h in the absence of external nutrients, they (T1 + 6 cells) exhibited developmental features similar to T7 cells, which most rapidly acquired chemotactic sensitivity to 3′,5′-cyclic adenosine monophosphate (cAMP) and EDTA-resistant cohesiveness after starvation. Thus, it is quite likely that Ax-2 cells may progress through the cell cycle to a particular point (possibly the cell-cycle position of T7 cells), irrespective of the presence or absence of nutrients, and enter the differentiation phase from this point under conditions of nutritional deprivation. There was no difference in the ratio of prestalk to prespore cells in migratory pseudoplasmodia derived from cells that had been starved at other cell-cycle positions. The possible significance of cell-cycle-dependent variations in the timing of differentiation is discussed in relation to pattern formation and cell-type proportioning.