X-ray-induced recombination during oogenesis in the silkworm (Bombyx mori L.)

Summary The physical induction of recombinational events has been studied in the female silkworm (Bombyx mori), in which crossing-over does not normally occur. Female silkworms heterozygous in the trans type of two egg-color genes,pe (V-0.0) andre (V-31.7), received a single dose of X-rays (1000 R) at various developmental stages. Then they were crossed to marked males homozygous for both genes. The results indicated that X-rays increase the occurrence of recombinational events in silkworm females from first instar larvae to late stage pupae. The spontaneous frequency of exchange type recombinants in the control series was 2.5 x 10^–5, while after irradiation the frequency of these recombinants was up to 38.8 x^10–5. The sensitive stage to X-ray-induced recombinational events was late stage larvae from fourth to fifth instar. Exchange (cross-over) type recombinants were about three times more frequent than interchange types among the 122 recombinants recovered. The biological significance of the present finding is discussed.     

Enzymatic synthesis of poly-β-hydroxybutyrate inZoogloea ramigera

The enzyme activity synthesizing poly--hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction ofZoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction, when the bacterium grew dispersedly in a glucose-starved medium.The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose.PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated withd(-)--hydroxybutyryl CoA in vitro.Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity ford(-)--hydroxybutyryl CoA, and showed the same pH optimum at 7.0.Non-Standard Abbreviations PHB poly--hydroxybutyrate     

The peripheral inhibition of the tarsal sugar receptor by sodium chloride in the proboscis extension response of the blowfly,Phormia regina M.

Summary The proboscis extension reponse of the blowfly during stimulation of the tarsal sugar receptors was inhibited by the presence of NaCl. Acceptance thresholds for sucrose in various concentrations of NaCl were measured. The median acceptance thresholds for sucrose in mixtures of 0.01, 0.25, 0.5 and 1.0 M NaCl were 1.8 × 10^–3, 6.0 × 10^–3, 1.2 × 10^–2, and 2.0 × 10^–2 M, respectively. Concentration-response curves for sucrose in the tarsal D-type sugar receptor shifted to the right under the existence of high concentration of NaCl. Number of impulses per D-type sugar receptor at the median acceptance thresholds described above were 7.5, 8.4, 6.8 and 10.4 for the first 0.1 s of stimulation, respectively. The average number was 8.2 impulses per 0.1 s. Comparisons were made between the behavioral acceptance thresholds (1) on one leg exposed to sucrose mixed with 0.01 M NaCl and (2) on two contralateral legs, one of which was exposed to sucrose in 0.01 M NaCl and the other to 0.5 M NaCl alone. The acceptance thresholds from two experiments agreed with each other. The median threshold value was 1.7 × 10^–2 M sucrose. Behavioral inhibition by NaCl in mixtures with sucrose can be explained by its peripheral inhibition of sugar receptors.This research was supported in part by ITO foundation and Scientific Research Fund from the Ministry of Education of Japan.     

Studies on a phospholipase B from Penicillium notatum. Purification, properties, and mode of action.

1. Phospholipase B which hydrolyzes both the acyl ester bonds of diacylphospholipids (diacyl-hydrolase) and the acyl ester bond of monoacylphospholipids or lysophospholipids, [monoacyl-hydrolase or lysophospholipase, EC 3.1.1.5] was purified from Penicillium notatum about 2000-fold over the crude extract. The final preparation was homogeneous on disc electrophoresis. The apparent molecular weight, determined by gel filtration on Sephadex G-200, was about 116,000. The isoelectric point was pH 4.0. 2. The purified enzyme was a glycoprotein. The carbohydrate content was approximately 30%, consisting of mannose, glucose, and glucosamine. The amino acid composition was also determined. 3. The ratio of monoacyl-hydrolase to diacyl-hydrolase activities was influenced by the physical state of the substrate in the assay system. It was about 1 : 1 or 100 : 1 in the presence of absence of Triton X-100, respectively, and the latter value remained constant throughout the purification procedures. 4. Both enzyme activities had the same pH optimum, 4.0, and were heat-labile. None of the metals tested had any effect on either activity except for Fe2+ and Fe3+. Diisopropyl fluorophosphate at relatively high concentrations completely inhibited both enzyme activities. 5. The Michaelis-Menten constants (Km) of the enzyme for egg lecithin were about 1.5 and 25 mM in the absence and presence of Triton X-100, respectively. The Km value for dicaproyllecithin was 9.8 mM in the absence of Triton X-100. 6. Using a mixture of 1-[14C]stearoyl-lecithin and 2-[14C]oleoyl-lecithin in the presence of Triton X-100 as a substrate, it was found that the P. notatum phospholipase B attacked the acyl ester bonds sequentially, first the 2-acyl and then 1-acyl groups.     

Calcium-activated neutral protease and its endogenous inhibitor Activation at the cell membrane and biological function

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca²⁺ and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.     

Effect of water deprivation on the centrally-mediated hypertensive response to clonidine in freely moving, normotensive rats

Effects of water deprivation on pressor responses to centrally and peripherally administered clonidine was investigated in freely moving, normotensive rats with chronic guide cannula and catheters implanted into the abdominal aorta via the femoral artery. In normal hydrated rats, intracerebroventricularly (i.c.v.) injected clonidine (10 and 20 micrograms) produced a dose-dependent and long-lasting rise in mean blood pressure (MBP) concomitant with a decrease in heart rate. However, a significant depressor response was not observed for up to 90 min. In 48 hr dehydrated rats, the pressor response to i.c.v. injected clonidine (10 and 20 micrograms) was significantly depressed and a depressor response appeared. Intravenously (i.v.) administered clonidine (25 micrograms/kg) in normal hydrated rats also produced a long-lasting pressor response following an initial rapid rise in MBP. The long-lasting pressor response to i.v. injected clonidine was abolished after water deprivation for 48 hr, whereas the initial rise in MBP was less affected. These results suggest that clonidine elicits centrally-mediated pressor response, which is influenced by body fluid volumes.     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels

The effects of hyaluronic acid (HA) on the proliferation and chondroitin sulfate (CS) synthesis of chondrocytes embedded in collagen gels were examined. Articular cartilage was isolated from the humerus, femur, and tibia of 21 10-week-old Japanese white rabbits. Chondrocytes isolated by collagenase digestion were embedded in type I collagen gels and cultured in Dulbecco's modified Eagle's medium (DMEM) with various doses of HA for 4 weeks. Histological and biochemical evaluations were performed at postculture weeks 1, 2, 3, and 4. For biochemical evaluations, isomers such as chondroitin 6-sulfate (delta(di)-6S) and chondroitin 4-sulfate (delta(di)-4S) synthesized by cultured chondrocytes were determined by high performance liquid chromatography (HPLC) combined with fluorometry. Morphological and histological studies demonstrated that HA-treated chondrocytes in collagen gel proliferated profusely while maintaining their phenotype. At postculture week 4, 0.1 mg/ml of HA induced an eightfold increase in cell counts compared with HA pretreatment values, or 1.5-fold more than control group. Synthesis of delta(di)-6S (delta(di)-6S content/cell) in groups treated with 0.01 and 0.1 mg/ml of HA significantly increased, while gel accumulation rates in groups treated with 0.1 and 1.0 mg/ml of HA scored significantly higher values than other groups. In collagen gel culture, HA enhanced the proliferation and delta(di)-6S synthesis of chondrocytes while maintaining their phenotype. In clinical application, since the supply of autologous chondrocytes for transplantation is not unlimited, the HA-treated culture method may be useful for increasing the number of chondrocytes and thus improving the quality of implants.     

The biology of IL-12: coordinating innate and adaptive immune responses

Cytokines play critical roles in regulating all aspects of immune responses, including lymphoid development, homeostasis, differentiation, tolerance and memory. Interleukin (IL)-12 is especially important because its expression during infection regulates innate responses and determines the type and duration of adaptive immune response. IL-12 induces interferon-gamma (IFN-gamma) production by NK, T cells, dendritic cells (DC), and macrophages. IL-12 also promotes the differentiation of na?ve CD4+ T cells into T helper 1 (Th1) cells that produce IFN-gamma and aid in cell-mediated immunity. As IL-12 is induced by microbial products and regulates the development of adaptive immune cells, IL-12 plays a central role in coordinating innate and adaptive immunity. IL-12 and the recently identified cytokines, IL-23 and IL-27, define a family of related cytokines that induce IFN-gamma production and promote T cell expansion and proliferation.