Ocular clocks are tightly coupled and act as pacemakers in the circadian system of Japanese quail

Our previous studies showed that the eyes of Japanese quail contain a biological clock that drives a daily rhythm of melatonin synthesis. Furthermore, we hypothesized that these ocular clocks are pacemakers because eye removal abolishes freerunning rhythms in constant darkness (DD). If the eyes are indeed acting as pacemakers, we predicted that the two ocular pacemakers in an individual bird must remain in phase in DD and, furthermore, the two ocular pacemakers would rapidly regain coupling after being forced out of phase. These predictions were confirmed by demonstrating that 1) the ocular melatonin rhythms of the two eyes maintained phase for at least 57 days in DD and 2) after ocular pacemakers were forced out of phase by alternately patching the eyes in constant light, two components of body temperature were observed that fused into a consolidated rhythm after 5-6 days in DD, showing pacemaker recoupling. The ability to maintain phase in DD and rapidly recouple after out-of-phase entrainment demonstrates that the eyes are strongly coupled pacemakers that work in synchrony to drive circadian rhythmicity in Japanese quail.     

Novel metal-chelate affinity adsorbent for purification of immunoglobulin-G from human plasma

Metal-chelating ligand and/or comonomer 2-methacrylolyamidohistidine (MAH) was synthesized by using methacryloyl chloride and L-histidine methyl ester. MAH was characterized by NMR and FTIR. Spherical beads with an average diameter of 75-125 microm were produced by suspension polymerization of methylmethacrylate (MMA) and MAH carried out in an aqueous dispersion medium. Poly(MMA-MAH) beads had a specific surface area of 37.5 m(2)/g. Poly(MMA-MAH) beads were characterized by water uptake studies, FTIR, SEM and elemental analysis. Elemental analysis of MAH for nitrogen was estimated as 34.7 microM/g of polymer. Then, Cu(2+) ions were chelated on the beads. Cu(2+)-chelated beads with a swelling ratio of 38% were used in the adsorption of human-immunoglobulin G (HIgG) from both aqueous solutions and human plasma. The maximum adsorption capacities of the Cu(2+)-chelated beads were found to be 12.2 mg/g at pH 6.5 in phosphate buffer and 15.7 mg/g at pH 7.0 in MOPS. Higher adsorption value was obtained from human plasma (up to 54.3 mg/g) with a purity of 90.7%. The metal-chelate affinity beads allowed one-step separation of HIgG from human plasma. The adsorption-desorption cycle was repeated 10 times using the same beads without noticeable loss in their HIgG adsorption capacity.     

Degradation of fenitrothion byBacillus stearothermophilus adhering to silica

B. stearothermophilus strain AG-49, when cultivated in mineral medium in the presence of silica (SA), adhered to SA. Adhesion depended on age of culture, contact time and glucose concentration of the culture medium. Mid-exponential phase culture (5 h) required minimum contact time (30 min) for maximum adhesion. 0.6% glucose concentration was optimum. Quantitative variation in protein and saccharide extractable in sodium chloride and sodium dodecyl sulfate (SDS) was observed. Five % degradation of fenitrothion by adherentB. stearothermophilus could be achieved in 4 d.     

Phosphorylation of IP3R1 and the regulation of [Ca2+]i responses at fertilization: a role for the MAP kinase pathway.

A sperm-induced intracellular Ca2+ signal ([Ca2+]i) underlies the initiation of embryo development in most species studied to date. The inositol 1,4,5 trisphosphate receptor type 1 (IP3R1) in mammals, or its homologue in other species, is thought to mediate the majority of this Ca2+ release. IP3R1-mediated Ca2+ release is regulated during oocyte maturation such that it reaches maximal effectiveness at the time of fertilization, which, in mammalian eggs, occurs at the metaphase stage of the second meiosis (MII). Consistent with this, the [Ca2+]i oscillations associated with fertilization in these species occur most prominently during the MII stage. In this study, we have examined the molecular underpinnings of IP3R1 function in eggs. Using mouse and Xenopus eggs, we show that IP3R1 is phosphorylated during both maturation and the first cell cycle at a MPM2-detectable epitope(s), which is known to be a target of kinases controlling the cell cycle. In vitro phosphorylation studies reveal that MAPK/ERK2, one of the M-phase kinases, phosphorylates IP3R1 at at least one highly conserved site, and that its mutation abrogates IP3R1 phosphorylation in this domain. Our studies also found that activation of the MAPK/ERK pathway is required for the IP3R1 MPM2 reactivity observed in mouse eggs, and that eggs deprived of the MAPK/ERK pathway during maturation fail to mount normal [Ca2+]i oscillations in response to agonists and show compromised IP3R1 function. These findings identify IP3R1 phosphorylation by M-phase kinases as a regulatory mechanism of IP3R1 function in eggs that serves to optimize [Ca2+]i release at fertilization.     

The Role of Exoproteases in Governing Intraneuronal Metabolism of Botulinum Toxin

Botulinum toxin type A has a long duration of action, and thus it can block transmitter release for several weeks to several months. However, little is known about the precise mechanism that accounts for termination of toxin action. Therefore, experiments were done to gauge the effects of aminopeptidases and carboxypeptidases on the structure and function of the toxin. Exoproteases were added to the holotoxin, the native light chain, and a recombinant light chain. Treated toxin and light chain were examined for their effects on neuromuscular transmission and on isolated substrate. The data showed that aminopeptidase attack did not alter the N-terminus of the toxin/light chain, nor did it produce losses in biological activity. Carboxypeptidase attack did alter the C-terminus of the light chain, but not sufficiently to alter biological activity. The data suggest that the tertiary structure of the light chain confers upon the molecule substantial resistance to exoproteases.     

Protective effects of topical alpha-tocopherol acetate on UVB irradiation in guinea pigs: importance of free radicals

Reactive oxygen species can be generated by daily exposure of the skin to ultraviolet light and may cause some subchronic and chronic skin disorders. The aim of this study was to investigate a possible preventive role of alpha-tocopherol acetate (ATA) on ultraviolet B (UVB) induced peroxidation by assessing lipid peroxide (LPO) levels and activity of reactive oxygen scavenging enzymes including glutathione peroxidase and superoxide dismutase (SOD) in guinea pigs. ATA was topically applied to the skin for three weeks before a single dose of 0.9 J/cm2 UVB irradiation on the skin and lipid peroxide levels and antioxidants in plasma, skin and liver and erythrocytes were determined after decapitation. Topical application of ATA prevented the UVB irradiation-induced reduction of scavenging enzyme activities in skin and erythrocytes. In conclusion, we suggest that topical applications of ATA before UVB irradiation is effective in protecting the skin from unwanted effects of UVB irradiation.     

The mitogenic activity of endothelin-1 on megakaryocytopoiesis in vitro

Endothelin (ET)-1 induces proliferation of various cells including smooth muscle cells, fibroblasts, glomerular mesangial cells, endothelial cells and osteoblasts. ET-1 also stimulates synthesis of interleukin (IL)-6 in endothelial and bone marrow stromal cells of rat. It is well known that IL-6 modulates megakaryocytopoiesis. Some studies have indicated that megakaryocytes express both ET receptors and they are targets for ET. Therefore we planned to examine the effects of ET-1 on the growth of normal megakaryocytic cells in rat bone marrow primary cell culture. Bone marrow cells were cultured at 37 degrees C, in an incubator atmosphere of 5% CO2 in air and 95% relative humidity for nine days. ET-1 at 10(-7), 10(-8 ) and 10(-11) M, and control with saline were added at the beginning of the experiment protocol. At each day, plasma clots were stained using direct-coloring thiocholin method for acetylcholinesterase activity. Although 10(-7) M ET-1 did not change the proliferation of megakaryocytic cells, this could be due to the presence of over crowded fibroblasts in the same environment. 10(-8) M ET-1 stimulated megakaryocytic cell growth to 234% over the control on the fifth day. ET-1 at a concentration of 10(-11) M also rised the megakaryocytic cell number significantly reaching up to 86% at the sixth day. Our results indicate that ET-1 may modulate the growth of megakaryocytic cells by an autocrine and/or paracrine action.     

Two new dihydrochalcones from Myrica gale

From the fruits of Myrica gale the isolation of 2′,6′dihydroxy-4′-methoxy-3′,5′-dimethyldihydrochalcone and 4,4,6-trimethyl-2-(3-phenylpropionyl)-cyclohexane-1,3,5-trione is described. The constitutions have been deduced from spectroscopic data and confirmed by synthesis.     

Optimization and enhanced production of α-amylase and protease by a newly isolated Bacillus licheniformis ZB-05 under solid-state fermentation

Eight different agro-residues were tested for α-amylase and protease production by using Bacillus licheniformis ZB-05. Among them, rice husk (RH) was proved as the best substrate for two enzymes (α-amylase 443 U/g and protease 469,000 U/g). Maximum enzyme production was observed to be 30 % initial moisture, with a growth period of 36 h in 20 and 30 % inoculum volumes for α-amylase and protease, respectively. The best enzyme recovery from solid mass was obtained when extracted with tap water. Among the tested various nitrogen sources, 1 % ammonium sulphate followed by 2 % Bacto liver, 2 % ammonium sulphate and 1 % Bacto casaminoacid served as the best inorganic and organic nitrogen sources for α-amylase and protease production, respectively. As additional carbon sources, 2 % soluble starch enhanced α-amylase production, while 1 % maltose enhanced protease production.