Influence of light,DMSO and glycerol on the hatchability ofThamnocephalus platyurus Packard cysts

Effects of two light intensities and different concentration of dimethyl sulfoxide (DMSO) and glycerol on hydrated cyst hatching inThamnocephalus platyurus were studied. A maximum of 65±6% hatching was recorded within seven days at 2500 lux continuous light regime. Hatching was at a minimum during the first two days, peaked between the third and fourth days, decreased thereafter. Hatching success was a function of duration of light exposure. Eight percent of cysts hatched in the dark, while cysts exposed to 24h light and subsequently incubated in the dark showed 27±2% hatching. Hatchability was significantly increased (23%) in 0.0375% DMSO and 0.0125% glycerol. Concentration above 0.05% DMSO and 0.025% glycerol had no or a negative influence on hatching. Since low concentrations of DMSO were non-toxic, apolar compounds like Ca²⁺ ionophore can be dissolved in DMSO to study the role of Calcium in cyst hatching.     

Persistent Enhancement of Sustained Calcium-Dependent Glutamate Release by Phorbol Esters: Role of Calmodulin-Independent Serine/Threonine Phosphorylation and Actin Disassembly

Abstract: The phorbol ester 4β-phorbol 12,13-dibutyrate increases the final extent of Ca²⁺-dependent glutamate release during the continuous depolarization of the synaptosomal plasma membrane. Based on this finding, we suggested that the sustained activation of protein kinase C has a positive influence on the efficiency of synaptic vesicle recycling in the presence of saturating concentrations of Ca²⁺. Previous work from our laboratory demonstrated that this 4β-phorbol 12,13-dibutyrate-dependent enhancement of synaptic vesicle recycling persists following the removal of 4β-phorbol 12,13-dibutyrate, requires localized Ca²⁺ entry through voltage-regulated channels, and is insensitive to the protein kinase inhibitor staurosporine. In the present study, we examined the possibility that the facilitation of glutamate release may be propagated through interactions between the protein kinase C- and multifunctional Ca²⁺/calmodulin-dependent protein kinase pathways. However, our data argue strongly against the involvement of such a mechanism in the persistent enhancement of sustained glutamate release. We observed that 4β-phorbol 12,13-dibutyrate did not increase the availability of cytosolic free calmodulin or the level of autonomous Ca²⁺/calmodulin-dependent protein kinase activity. In addition, we determined the effects of various serine/threonine kinase and phosphatase inhibitors on the phorbol ester-dependent enhancement of sustained glutamate release and found that protein kinase C increased the extent, but not the duration, of Ca²⁺-dependent glutamate release through a kinase-independent mechanism. Given our finding that the actin-depolymerizing agent cytochalasin D totally occluded the effect of 4β-phorbol 12,13-dibutyrate on release, we postulate that protein kinase C signals may be transduced through direct interactions between protein kinase C isoforms and cytoskeletal protein kinase C binding proteins.     

Expression and purification of recombinant cynomolgus monkey cholesteryl ester transfer protein from Chinese hamster ovary cells

Cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high- and low-density lipoproteins to triglyceride-rich lipoproteins, and reciprocally mediates triglyceride transfer. The gene for cynomolgus monkey CETP was expressed in serum-free CHO culture with 2g/ml insulin as its only exogenous protein supplement. Cell growth was facilitated by immobilizing the CHO cells in alginate beads. Recombinant CETP (rCETP) was purified 176-fold with a three-step protocol resulting in a 60% final yield as measured by a fluorescent CETP activity assay. Typically, 3.4 mg of rCETP was purified from 1700 ml of media by affinity-gel chromatography involving Reactive Red 120 (RR120) followed by concanavalin A Sepharose 4B and rechromatography on RR120. SDS-PAGE shows a single broad band ofM _r , ranging from 68,000 to 74,000 which immunoreacts in Western blot analysis. Amino acid analysis and protein sequencing of the purified protein agree with the theoretical amino acid composition and sequence of cynomolgus CETP.     

Taxonomy and function of C1 protein kinase C homology domains.

C1 domains are compact alpha/beta structural units of about 50 amino acids which tightly bind two zinc ions. These domains were first discovered as the loci of phorbol ester and diacylglycerol binding to conventional protein kinase C isozymes, which contain 2 C1 domains (C1A and C1B) in their N-terminal regulatory regions. We present a comprehensive list of 54 C1 domains occurring singly or doubly in 34 different proteins. Many C1 domains and C1 domain-containing proteins bind phorbol esters, but many others do not. By combining analysis of 54 C1 domain sequences with information from previously reported solution and crystal structure determinations and site-directed mutagenesis, profiles are derived and used to classify C1 domains. Twenty-six C1 domains fit the profile for phorbol-ester binding and are termed "typical." Twenty-eight other domains fit the profile for the overall C1 domain fold but do not fit the profile for phorbol ester binding, and are termed "atypical." Proteins containing typical C1 domains are predicted to be regulated by diacylglycerol, whereas those containing only atypical domains are not.     

N-Acetyl-aspartylglutamate (NAAG) in human cerebrospinal fluid: Determination by high performance liquid chromatography,and influence of biological variables

Summary NAAG is one of the neuropeptides found in highest concentrations in the CNS. The presence of micromolar concentrations of NAAG in human CSF was demonstrated by using two different and complementary analytical approaches: 1) isocratic separation of endogenous NAAG by reverse-phase high performance liquid chromatography (HPLC) with dual wavelength detection and 2) derivatization of endogenous NAAG with acidic methanol and subsequent HPLC analysis of the derivative NAAG-trimethyl ester. The NAAG concentration was between 0.44µmol/l and 7.16µmol/l (mean of 2.19 ± 1.53µmol/l) in CSF samples from forty neuropsychiatric patients. Endogenous NAAG or [³H]NAAG added to CSF samples were not significantly degraded when the CSF was incubated at 37°C during one hour, suggesting that the peptide is a highly stable metabolite in the subarachnoid space. In addition, evidence is provided that NAAG does not present a concentration gradient along the lower subarachnoid space.     

长臂生物素对硝基苯酯的合成

以羰基二咪唑为催化剂,生物素与6－氨基己酸甲酯反应生成生物素－6－氨基己酸甲酯,该化合物通过酸碱萃取与原料分离,皂化后生成生物素氨基己酸．生物素氨基己酸在吡啶存在下与三氟乙酸对硝基苯酯进行转酯反应即得生物素对硝基苯酯．最后经光谱、色谱及核酸杂交证实了长臂生物素对硝基苯酯的化学结构和生物学活性．     

Degradation of soluble collagen by ozone or hydroxyl radicals

Collagen exposed to ozone or hydroxyl radicals was degraded in a time- and dose-dependent manner. This degradation was inhibited by free radical scavengers. Furthermore, lower levels of these oxidants did not degrade the molecule, but caused it to become susceptible to proteolytic degradation. We suggest an alternative mechanism by which oxygen-derived free radicals participate in the destruction of extracellular matrix observed during acute lung injury by oxidant gas, in addition to the commonly accepted proteinase-antiproteinase theory of lung injury.     

Ultraviolet-crosslinking reveals specific affinity of eukaryotic initiation factors for Semliki Forest virus mRNA

Eukaryotic initiation factors (eIF) associate readily with ³²P-labeled Semliki Forest virus (SFV) mRNA in vitro, forming complexes which can be crosslinked by 254 nm ultraviolet irradiation. After ribonuclease digestion, the initiation factors were released and analysed by gel electrophoresis. Autoradiography revealed proteins by virtue of crosslinked ³²P-labeled mRNA fragments. eIF-4A, -4B and -4C as well as three subunits of eIF-3 could be crosslinked with SFV mRNA. None of these proteins bound to ribosomal RNAs.