Calbindin-D28k and calretinin in the rat posterior pituitary; Light and electron microscopic localization and upregulation with dehydration

Ca²⁺ binding proteins (CaBPs), calbindin-D_28k (calbindin) and calretinin, are thought to contribute to the regulation of intracellular Ca²⁺ in many neuronal populations and perhaps more importantly, signal functional modulation in neuronal activity. In the present experiments, light microscopic immunohistochemistry revealed that the immunoreactivity of calbindin and calretinin was contained in varicose axons in the posterior pituitary. The dual labeling study with confocal microscopy demonstrated that calbindin immunoreactivity was present in the terminals of both oxytocin (OXT) and arginine-vasopressin (AVP) neurons. However, calretinin immunoreactivity was exclusively seen in the OXT terminals. Moreover, the dual labeling study showed that most calretinin-positive terminals contained calbindin immunoreactivity, demonstrating the colocalization of calbindin and calretinin in the same OXT nerve terminals. By electron microscopy, calbindin and calretinin immunoreactivities were seen in the neurosecretory axons and nerve terminals. These immunoreactive nerve terminals were seen to contain more clear microvesicles than dense-core neurosecretory granules. This immunoelectron microscopic observation suggests that both calbindin and calretinin localize preferentially in the active zone of the nerve terminals, which usually face the perivascular space around fenestrated capillaries. In spite of similar localization of calbindin and calretinin within the posterior pituitary, Western blot analysis showed some differences between the two CaBPs. Calbindin was present mostly in the soluble fraction with little in the insoluble fraction, but a substantial portion of calretinin was present in both the insoluble and soluble fractions. Moreover, dehydration induced by drinking 2% NaCl solution and deprivation of drinking water increased calretinin levels in the posterior pituitary as compared with control, but the calbindin level was not changed. The present findings demonstrate that calbindin and calretinin colocalize in the active zones of OXT nerve terminals, but only calretinin is upregulated with dehydration, suggesting different physiological role of calbindin and calretinin in the nerve terminals.     

A strategy to make constitutively active MAP kinase by fusing with constitutively active MAP kinase kinase.

Classical mitogen-activated protein kinases (MAPKs) play a pivotal role in a variety of cellular signal transduction pathways. MAPKs are activated by phosphorylation at specific threonine and tyrosine residues catalyzed by upstream MAPK kinases (MAPKKs). Mutations of these two activation phosphorylation sites into acidic amino acids, however, do not convert MAPKs into constitutively active forms. Here, we report an approach to make a molecule with constitutive MAPK activity. The nuclear export signal-disrupted, constitutively active MAPKK was fused to the N-terminal end of wild-type MAPK. When the resulting fusion protein was expressed in Escherichia coli, the MAPK moiety became phosphorylated and the fusion protein was constitutively active as MAPK. Moreover, when expressed in mammalian cultured cells, the fusion protein was also activated as MAPK and was able to induce marked morphological changes in NIH-3T3 cells. These results suggest that the fusion protein can work as constitutively active MAPK and that this approach may be applicable to other members of the MAPK family to make constitutively active forms.     

Localization of germination-specific spore-lytic enzymes in Clostridium perfringens S40 spores detected by immunoelectron microscopy

The localization of germination-specific spore-lytic enzymes, an amidase and a muramidase, in Clostridium perfringens S40 spores was examined by immunoelectron microscopy with respective antisera raised against the enzymes and a colloidal gold-immunoglobulin G complex. For both antisera, immunogold particles were visualized on the outside of the cortex of dormant spores, and they were not detected in germinated spores and decoated spores.     

Validation and application of a 96-well format solid-phase extraction and liquid chromatography-tandem mass spectrometry method for the quantitation of digoxin in human plasma

To evaluate the pharmacokinetics of digoxin in humans, a sensitive and specific LC/MS/MS method was developed and validated for the determination of digoxin concentrations in human plasma. The method was shown to be more sensitive, specific, accurate, and reproducible than common techniques such as RIA. For detection, a LC/MS/MS system with electro spray ionization tandem mass spectrometry in the positive ion-multiple reaction-monitoring (MRM) mode was used to monitor precursor to product ions of m/z 798.5-51.5 for digoxin and m/z 782.5-35.5 for the internal standard, digitoxin. The method was validated over a concentration range of 0.02-5 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 80%. Imidafenacin, coadministered in a drug-drug interaction study, had no detectable influence on the determination of digoxin in human plasma. The novel method was applied to a drug-drug interaction study of digoxin and imidafenacin and the characterization of steady-state pharmacokinetics of digoxin in humans after oral administration at a dose of 0.25 mg on days 1 and 2 followed by 0.125 mg daily doses on days 3 through 8.     

Mechanisms of resistance to spinosad in the western flower thrip, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae)

Cross‐resistance, resistance mechanisms, and mode of inheritance of spinosad resistance were studied in the western flower thrip, Frankliniella occidentalis (Pergande). Spinosad (naturalyte insecticide) showed low cross‐resistance to prothiophos (organophosphorus insecticide) and chlorphenapyr (respiratory inhibitor) showed some cross‐resistance to thiocyclam (nereistoxin). The synergists PBO (piperonyl butoxide), DEM (diethyl maleate), and DEF (s, s, s‐tributyl phosphorotrithioate) did not show any synergism on the toxicity of spinosad in the resistant strain (ICS), indicating that metabolic‐mediated detoxification was not responsible for the spinosad resistance, suggesting that spinosad may reduce sensitivity of the target site: the nicotinic acetylcholine receptor and GABA receptor. Following reciprocal crosses, dose‐response lines and dominance ratios indicated that spinosad resistance was incompletely dominant and there were no maternal effects. The results of backcross showed that spinosad resistance did not fit a single‐gene hypothesis, suggesting that resistance was influenced by several genes.     

Effects on properties of a thiol protease from Xenopus embryos of changes in substrate and assay conditions.

A protease was purified from Xenopus embryos. Proteolytic activity of the protease against BSA had an optimum pH of 3.8 in acetate buffer and was not detectable at neutral pH. However, when embryonic proteins were used as substrates and digested in phosphate buffer, proteolysis of embryonic proteins was enhanced and was detectable from pH 5.0 to pH 7.0. Digestion of three proteins were mainly detected in digestion of total embryonic proteins. The proteins digested had the same mobilities (on SDS polyacrylamide gel) as yolk proteins. The protease was present in the cytoplasm and around yolk granules. We propose that this protease mainly cleaves a certain yolk proteins in the cytoplasm of Xenopus embryos.     

New insights into dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p in shuttling of PTS1-receptor Pex5p during peroxisome biogenesis

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders such as Zellweger syndrome. Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for PBDs of complementation groups 1 and 4, respectively. PEX26 responsible for peroxisome biogenesis disorders of complementation group 8 codes for C-tail-anchored type-II membrane peroxin Pex26p, the recruiter of Pex1p-Pex6p complexes to peroxisomes. Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by ATPase cycle. Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome targeting signal type-1 and shuttles between the cytosol and peroxisomes. AAA peroxins are involved in the export from peroxisomes of Pex5p. Pex5p is ubiquitinated at the conserved cysteine11 in a form associated with peroxisomes. Pex5p with a mutation of the cysteine11 to alanine, termed Pex5p-C11A, abrogates peroxisomal import of proteins harboring peroxisome targeting signals 1 and 2 in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, hence suggesting an essential role of the cysteine residue in the export of Pex5p.     

Prevention and reversible solubilization of advanced glycation and products (AGE) by organic germanium compounds as derivatives of amino acids

Summary The amino-carbonyl reaction (The Maillard reaction) of bovine lens crystallin, serum albumin or skin collagen with glucose was investigated to find effective means to prevent the formation of Advanced Glycation End Products (AGE) and induce the reversible solubilization of polymerized glycated proteins. The organic germanium compounds (Ge-132, 373, 385), derivatives of amino acids containing germanium as the linker of framework, were combined by the box titration method to determine the dose that would be most effective, compared with Aminoguanidine-HCl (AMG),-tocopherol (VE), and pirenoxine (Catalin-K, CK). Although AMG suppressed the formation of AGE, effective concentrations were higher than 20 mM. Ge-385, when administered by itself at a low dose, induced the reversible solubilization of AGE made from crystallin, and albumin. The addition of any two reagents such as AMG, VE, CK and Ge-132 or 385 together to proteins lessened the effective range, and the peaks of smaller molecules in the profiles of HPLC and PAGE were quite remarkable. Examination was made of the effects of Ge-132 on the eyes of SAM mice, which show senescence accelerated cataracts at a relatively young age. The prevention of cataract-genesis and induction of reversible transparency of turbid lenses became evident following the administration of Ge-132 to the eyes 4 times a day. The mode of action of organic germanium compounds was demonstrated quite capable of disconnecting the sugar-parts from AGE by decarbonylation, resulting in the formation of glucosone and amino residues, and further leading subsequently to fewer AGE.Abbreviations used in this paper: BLC bovine lens crystallin; BSA bovine serum albumin; AsCol acid soluble bovine skin collagen type III; AGE advanced glycation end products; Ge-132 2-Carboxyethylgermanium sesquioxide, Ge-373 2-Carboxy-2-amino-6-phenyl germanium sesquioxide; Ge-385 2-Carboxy-ethyl-2-aminogermanium sesquioxide; AMG or AG aminoguanidine-HCl; V. E. vitamin E or-tocopherol; CK 1-Hydroxy-5-oxo-5H-pyrido [3, 2-a] phenoxazine-3-carboxylic acid or catalin-K or pirenoxine; PACE polyacrylamide gel electrophoresis; SAM senescence accelerated mouse; HPLC high pressure liquid chromatography; SDS sodium laurylsulfate; FT fructose-p-toluidine.     

温度对杀扑磷抗性奥氏钝绥螨Amblyseius womersleyi(Acari:Phytoseiidae)繁殖适合度的影响

研究了奥氏钝绥螨杀扑磷抗性种群在20℃、25℃与30℃温度下的繁殖适合度．结果表明,在20—30℃温度范围培养下,随着温度的升高,奥氏钝绥螨杀扑磷抗性种群的各发育历期缩短,雌性比降低,每雌日产卵量升高,但在25℃温度下每雌总产卵量均明显高于在20℃与30℃的每雌总产卵量．25℃温度是室内人工繁育奥氏钝绥螨杀扑磷抗性种群的最适温度。