Synthesis of citrulline from ornithine by the small intestinal mucosa of cattle

Three segments of cattle small intestine (duodenum, upper jejuno‐ileum and lower jejuno‐ileum) were examined in an in vitro system for activity of ornithine carbamoyltransferase (OCT; EC 2.1.3.3) which is involved in the synthesis of citrulline (Cit) from ornithine (Orn). The mucosa of the three segments of small intestine was collected from Japanese black cattle, homogenized and then centrifuged. The supernatant fraction was used as the crude OCT enzyme solution. The OCT activity was assayed by the production of Cit from Orn determined directly by HPLC. The optimal pH and temperature for OCT activities in the duodenum, upper jejuno‐ileum and lower jejuno‐ileum of cattle small intestine were 7.47 and 39°C. Little difference was observed between the three segments. The OCT activity in cattle kidney was also examined for comparison, and almost no activity was found. The OCT activities in crude enzyme solutions of the three segments of small intestine were stable for up to one month of storage at ?20°C in Tris HCl buffer solution. Finally, the role of the small intestine in supplying Cit as a precursor for arginine synthesis in cattle kidney was discussed.     

A quantitative study on arginine synthesis from argininosuccinic acid and citrulline by crude enzymes of cattle kidney

In vitro experiments were conducted to examine the synthesis of arginine (Arg) from argininosuccinic acid (ASA) and citrulline (Cit) by crude enzymes of cattle kidney cortex. Kidney samples, collected from Japanese black cattle, were homogenized in KCl solution (ice‐cold), and centrifuged at 27 000 × g for 20 min at 4°C, and the supernatant fluid was used as a crude enzyme solution. The enzyme solution was incubated at 39°C in Tris HCl buffer with 15 mmol/L ASA or with 10 mmol/L Cit in the presence of 10 mmol/L aspartic acid (Asp), 10 mmol/L ATP and 5 mmol/L MgCl₂ to examine the activities of two enzymes, argininosuccinate lyase and argininosuccinate synthetase, which work at the terminal steps of Arg biosynthesis. The production of Arg from ASA, or ASA and Arg from Cit by argininosuccinate lyase and argininosuccinate synthetase activities, respectively, were determined directly by the HPLC method. The optimum pH for argininosuccinate lyase activity was 7.85. Unfortunately, the optimum pH for argininosuccinate synthetase activity could not be determined because no inhibitor of argininosuccinate lyase was used in the Cit incubation, so the ASA produced from Cit spontaneously converted to Arg during incubation with Cit. The maximum production of ASA from Cit was found at pH 6.45 under these conditions. We observed the optimum pH for the synthesis of Arg from Cit at 7.7. The production of Arg from ASA or Cit was quantitatively determined as 14.4 or 8.83 µmol/g kidney tissue/h, respectively, at the optimal pH values. This suggests that the daily production of Arg from ASA or Cit by the kidney might be sufficient to cover the daily requirement of Arg in cattle.     

Actions of glucocorticoid and their regulatory mechanisms in the ovary

Glucocorticoid (G) directly modulates ovarian functions through binding to G receptor. The actions of G are both agonistic and antagonistic depending on the developmental stage of follicles and corpora lutea (CL). During follicular maturation, G suppresses follicular differentiation by downregulating expression of P450 aromatase and luteinizing hormone (LH) receptor in granulosa cells. During ovulation, G protects the ovulatory follicle from inflammatory damage and promotes luteinization, ensuring a smooth transition of the follicle to CL. Throughout life the ovary is exposed to periodic and sporadic waves of G. The Ovary appears to cope with this situation by locally modulating levels of active G. The primary regulatory mechanism consists of two isoforms of 11β‐hydroxysteroid dehydrogenase (11βHSD) that catalyze conversion between active and inactive G. During follicular maturation the levels of active G are suppressed by the dehydrogenase activity of 11βHSD, whereas during the ovulatory process, levels of active G are further increased by the oxo‐reductase activity of 11βHSD. The expression of these enzymes is under the control of gonadotrpins and local regulatory factors such as cytokines, allowing the mechanism to act in coordination with major reproductive events. Thus the G system is an integral part of ovarian physiology, which ensures that the ovary experiences only beneficial effects of G.     

An Endophytic Actinomycete, Streptomyces sp. AOK-30, Isolated from Mountain Laurel and Its Antifungal Activity

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of mountain laurel, young plants of mountain laurel (Kalmia latifolia L.) were used as an isolation source. From a total of 73 actinomycetes isolates obtained from leaves, stems and roots of test plants, Streptomyces sp. AOK-30 was selected, because i) it had a broad and intense antimicrobial spectrum against various yeasts and fungal pathogens of Ericaceae, ii) it grew on the multiplication and rooting media for tissue culture for this plant, and iii) the tissue-cultured seedlings that had been treated with this isolate became resistant to Pestalotia disease without any adverse growth effects such as malformation, dwarfing, discolorization and defoliation. This isolate was identified as Streptomyces sp., based on cultural, physiological and morphological characteristics. The present results indicate that isolate AOK-30 is a potential biocontrol agent against diseases of mountain laurel. Received 10 September 2002/ Accepted in revised form 22 November 2002     

Effect of oral administration of L-pipecolic acid on food intake in chicks and mice

It has been demonstrated that L-pipecolic acid (L-PA), a major metabolic intermediate of L-lysine (L-Lys) in the brain, is involved in the functioning of the γ-aminobutyric acid-ergic system. Previous study has shown that intracerebroventricular injection of L-PA suppressed feeding and induced sleep-like behavior in neonatal chicks. The present study examines whether the action of L-PA was induced by gavage in both chicks and mice. Oral administration of L-PA significantly inhibited food intake at 2 h after treatment in neonatal chicks, although no significant effect of L-Lys was detected. In mice, oral L-PA suppressed food intake compared to the control after 2 h of treatment. It was concluded that L-PA was effective for suppression of food intake after oral administration in both avian and mammalian species.     

Oridonin enhances phagocytosis of apoptotic U937 cells by macrophage-like cells

AIM: To study the effect of oridonin on the phagocytosis of apoptotic U937 cells by macrophage-like cells. METHODS: DNA agarose gel electrophoresis, Giemsa staining, Hoechst 33258 staining and photomicroscopical observation were used. RESULTS: UV irradiation (2.4 J/cm2, 4 min) induced U937 cell apoptosis. Marked DNA fragmentation in agarose gel electrophoresis was observed. Oridonin augmented phagocytosis of apoptotic U937 cells by U937 cell-derived macrophages in a time- and dose-dependent manner. However, less effect on synthetic fluoresbrite micropheres was observed. The oridonin-augmented phagocytosis was attenuated by anti-human TNFα or anti-human IL-1β antiserum. In addition, the similar effect of phagocytosis was observed in oridonin-treated human monocyte-derived macrophages at 4 day maturation. CONCLUSION: Oridonin enhances phagocytosis of apoptotic U937 cells by macrophage-like cells. The releases of TNFα and IL-1β are involved in this mechanism.     

Pipecolic acid in rumen fluid and plasma in ruminant animals

A survey was conducted to investigate the physiological levels of pipecolic acid (Pip) in rumen fluid and plasma of ruminants such as goats and cattle in the presence or absence of rumen protozoa. The concentration of Pip was determined using HPLC. Basal Pip levels in the rumen fluid and plasma of normal faunated animals were 21 ± 8 and 2.3 ± 1.3 µM, respectively, and levels increased 1–2 h after feeding. The Pip levels in the rumen fluid and plasma of faunated goats and cattle were significantly higher than those of defaunated goats and unfaunated cattle. A small amount of Pip was also found in the rumen fluids of the defaunated and unfaunated animals; this appeared to be derived from feeds such as hay cube and corn silage. The results obtained in the present study suggest that a significant amount of rumen‐produced Pip is likely to be absorbed into the plasma of the host animals and that rumen protozoa significantly enhance the concentration of Pip in the rumen fluid and plasma of ruminant animals.     

Stereochemical basis for the insecticidal activity of carbamoylated and acylated pyrazolines

Methyl 3-(4-chlorophenyl)-1-[N-(4-chlorophenyl)carbamoyl]-4-methyl-2-pyrazoline-4-carboxylate was converted to corresponding (1R)- and (1S)-phenethyl esters via its carboxylic acid and acid chloride at the C-4 atom to separate the diastereomers. Their configurations were confirmed by X-ray analysis. Both isomers of the (1R)methylbenzyl ester were subjected to transesterification with sodium methoxide to obtain enantiomers of the starting methyl ester. Their insecticidal activity was measured against American cockroaches (Periplaneta americana (L.)) by injection and against house flies (Musca domestica L.) by topical application under various synergistic conditions with metabolic inhibitors. The activity values of the four α-methylbenzyl esters and the R-isomer of the starting methyl ester were similar. The S-enantiomer of the methyl ester was about 10 and 100 times more active than the R-isomer against the cockroach and the fly, respectively. Some N-arylacetyl and N-aryloxyacetyl derivatives of the starting N-(4-chlorophenyl)carbamoyl compound gave very low activity. Conformation-energy profiles for some compounds suggested that the conformation of substituents on the N-1 atom in the pyrazoline ring has a specific role for the potential insecticidal effects.     

Identifying potential habitat distribution of the neon flying squid (Ommastrephes bartramii) off the eastern coast of Japan in winter

Habitat suitability index (HSI) models were applied to identify the potential habitat distribution of the neon flying squid (Ommastrephes bartramii) off the eastern coast of Japan during winter. We used an ocean reanalysis product, a satellite‐derived dataset, and commercial fisheries data during 2003–2008 to develop the HSI models, and illustrated the characteristics of the ocean environments at the fishing ground of the neon flying squid, focusing on a typical fishing ground formation event in 2006. The estimated HSI fields of the neon flying squid using three‐dimensional (3D) ocean environmental parameters showed a clear relationship between the squid habitat and the edge of a warm core ring south of the Oyashio water; this is considered a key characteristic of fishing ground formation, as noted in Sugimoto and Tameishi (Deep‐Sea Research, 39, 1992 and S183). This result suggests that mixing of the warm and nutrient‐poor Kuroshio water and the cold and nutrient‐rich Oyashio water at the edge of the ring could provide favorable conditions for the foraging of the neon flying squid. The warm water condition in the subsurface layers could be a further advantage to the formation of a stable fishing ground for the neon flying squid. Comparison of the Akaike Information Criteria among a satellite‐data‐based model, a reanalysis‐based model using the same parameters as the satellite‐based model, and a reanalysis‐based model using 3D ocean environmental parameters, showed an apparent improvement in the performance of the reanalysis‐based model using the 3D parameters, reproducing realistic features of the squid fishing ground during the winter of 2006.     

Tetrodotoxin accumulation conflicts with low salinity tolerance in juvenile tiger puffer Takifugu rubripes

We evaluated whether bearing tetrodotoxin (TTX) affects salinity stress in the juvenile tiger puffer Takifugu rubripes. Juveniles of hatchery-reared non-toxic T. rubripes [body weight (BW): 1.7?±?0.2 g, n?=?120] were divided into six tanks and acclimatized to salinity (8.5 ppt) that is equivalent to blood osmolality. Fish in three tanks were fed non-toxic diet, and those in the other three tanks were fed a TTX-containing diet (356 ng/g diet) three times a day until satiation. In each diet treatment, salinity of one tank was kept at 8.5 ppt, and the other two tanks were adjusted to either 1.7 or 34.0 ppt, and fish were reared for another 33 days. Then, we compared survival, growth, TTX accumulation, plasma osmolality, plasma cortisol, and glucose levels among treatments. We detected TTX only in the fish in the TTX-diet groups. Survival was highest at 8.5 ppt (70%) and lowest at 1.7 ppt in the TTX-diet group (20%). The BW was greater at 8.5 ppt, and plasma osmolality was significantly higher at 34.0 ppt than at any other salinities. Plasma cortisol level was significantly higher but glucose level was lower at 1.7 ppt. Possessing TTX at a low salinity may be lethal to tiger puffer juveniles.