Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins

Tubulin is a major substrate for endogenous Ca2+-calmodulin-dependent phosphorylation in synaptic cytoplasm. The present study details the purification to apparent homogeneity and characterization of a brain cytosolic Ca2+-calmodulin-dependent kinase which phosphorylates tubulin and microtubule-associated proteins as major substrates. The cytosolic kinase system, purified by sequential chromatography on phosphocellulose resin, calmodulin-affinity resin, and Fractogel TSK HW-55, chromatographs as a homogeneous complex of approximately 600,000 Da on Sephacryl S-300. This calmodulin-dependent kinase possesses a group of properties which specifically characterize this enzyme system: 1) the enzyme contains two calmodulin-binding doublets, rho and sigma, of approximately 52,000 and 63,000 Da, respectively; 2) both the rho and the sigma subunits demonstrate isoelectric points between 6.7 and 7.2; 3) both the rho and sigma subunits demonstrate autophosphorylation; 4) both the rho and sigma subunits show significant homologies as assessed by tryptic peptide fingerprints; 5) in the absence of substrate, both the rho and sigma subunits manifest lower mobility autophosphorylated species; 6) the kinase phosphorylates beta-tubulin equally on threonine and serine residues. Substrate specificity, kinetic parameters, calmodulin-binding properties, subunit composition, and subunit isoelectric points clearly differentiate this enzyme from other previously reported calmodulin-dependent kinases.     

Iron-Chelating Compounds Produced by Soil Pseudomonads: Correlation with Fungal Growth Inhibition

Strains of Pseudomonas putida, Pseudomonas sp., and Pseudomonas aeruginosa were examined for their ability to grow in the presence of the iron chelator, ethylenediamine-di-(o-hydroxyphenylacetic acid). In vitro fungal inhibition assays showed that the isolates varied in their ability to inhibit the growth of representative fungal plant pathogens. Fungal inhibition in vitro was superior to that of previously reported Pseudomonas sp. Studies with Fusarium oxysporum forma sp. lycopersici and a susceptible tomato cultivar demonstrated that Pseudomonas putida PPU3.1 was able to significantly reduce wilt disease.     

The steroids and fatty acids of the basidiomycete Scleroderma polyrhizum

The fruit bodies of the Basidiomycete Scleroderma polyrhizum have been shown to contain the steroids ergosta-4,6,8(14) 22-tetraen-3-one and 5α,8α-epidoxyergosta-6,22-dien-3β-ol and also palmitic and oleic acids.     

Novel inhibitors of enkephalin-degrading enzymes. II: N5'-substituted-4-thioxohydantoic acids as aminopeptidase inhibitors

Some 2-substituted-(2'-aminophenyl)-4-thioxohydantoic acids (o-amino PTC-amino acids) have antinociceptive activity when administered (icv) alone (IC50 = 0.04-0.87 microM/animal) and show a striking prolongation of the antinociceptive action of (D-Ala-2 D-Leu5)-enkephalin (DADL) in combination. The effects are thought to be mediated via opioid receptors since they are naloxone-reversible. Although inhibitors of the enkephalin degrading puromycin-insensitive, bestatin-sensitive aminopeptidase (possibly aminopeptidase M) their action is weak (IC50 = 32 microM leucine, 536 microM, glycine) and they might be considered to have a direct antinociceptive effect on opioid receptors. The titled compounds constitute novel 'lead' compounds for the development of potent aminopeptidase M inhibitors.     

On possible different mechanisms subserving the release of arachidonic and di-homo-gamma-linolenic acids for the formation of monoenoic and bisenoic prostaglandins in uteri from ovariectomized rats

The uptake of arachidonic acid (AA) and of di-homo-gamma-linolenic acid (DGLA) and their incorporations into phospholipids (PLs) and into neutral lipids (NLs) of uteri isolated from spayed rats and the effect of inhibiting triglyceride (TG) metabolism with 4-pentenoic acid (4-PEA) on tissue TG levels and the output of prostaglandins (PGs), were explored. Attempts were also made to determine whether the acylation of labelled AA and of labelled DGLA into PLs and TGs is different and to confirm possible correlations between the synthesis of PGE1 and the degradation of TGs. Uterine PLs incorporated significantly less DGLA than AA (P less than 0.05). AA was acylated mainly into the phosphatidylinositol (PI) and into phosphatidylcholine (PC) subfractions of rat uteri, whereas the incorporation of DGLA into these two subfractions was significantly smaller than that of AA. The acylation of labelled DGLA into NL fractions, mainly into triacylglycerol, almost doubled that of labelled AA. The levels of TGs in isolated rat uteri suspended in glucose-free medium during a period of 60 minutes were significantly less than immediately after isolation (P less than 0.001). PGE1 released from uteri into the incubating solution, was significantly higher than that of PGE2. Moreover, the presence of 4-PEA (1.0 mM), added after tissue isolation, prevented the decrement of TGs observed following 60 minutes of incubation and simultaneously diminished significantly (P less than 0.001) the enhanced output of PGE1, without altering that of PGE2. Results presented herein suggest that PLs are not normal precursors for the synthesis of PGE1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opsonizing properties of natural antibodies of rainbow trout, Oncorhynchus mykiss (Walbaum)

In order to confirm previous observations in which a protective effect of rainbow trout natural antibodies against furunculosis was suspected, phagocytosis studies wereconducted in vitro , using combinations of rainbow trout sera with high or low levels of natural antibodies and active or inactivated complement as opsonizing factors. Opsonization was observed in all the cases where complement was present, and to a lesser degree with sera containing only natural antibodies. The results confirm the prime importance of the complement system and provide additional evidence for a possible role of natural antibodies in antimicrobial defences.     

Pharmacological Characterization of Somatostatin Receptors in the Rat Cerebellum During Development

Abstract: Somatostatin (SRIF) receptors (SRIF-Rs) are transiently expressed in a germinative lamina of the rat cerebellum, the external granule cell layer. The appearance of SRIF-Rs coincides with the expression of SRIF-like immunoreactivity in the cerebellum. However, the cellular location of SRIF-Rs does not overlap with the distribution of SRIF-like immunoreactivity, with the latter being restricted to ascending fibers arising from the brainstem, to perikarya within the white matter, and to some Purkinje cells. The characterization of SRIF-Rs in the immature (13–day-old) rat cerebellum was conducted by means of binding experiments in membraneenriched preparations and autoradiography, using two radioligands, [¹²⁵I-Tyr⁰,D-Trp⁸]SRIF-14 ([¹²⁵I-Tyr⁰,d -Trp⁸]S14) and ^I25I-SMS 204–090. The pharmacological profile of cerebellar SRIF-Rs was compared with that of adult cortical SRIF-Rs. Saturation studies performed in 13–day-old rat cerebellum showed that the A'_D values for [¹²⁵I-Tyr⁰,D-Trp⁸]S14 and ¹²⁵I-SMS 204–090 binding were 0.35 ± 0.04 and 0.39 ± 0.01 nM, respectively. The corresponding B_max values were 52.7 ± 4.8 and 49.9 ± 5.3 fmol/mg of protein, a result indicating that radioligands with high specific radioactivity (2,000 Ci/mmol) bind to a single class of high-affinity sites (SSI). Competition studies showed that different D-Trp-sub-stituted analogs displaced [¹²⁵I-Tyr⁰,d -Trp⁸]S14 binding with Hill coefficients >1, a finding indicating the existence of different subtypes of binding sites. When [Tyr⁰,d -Trp⁸]S14 was used as a competitor, two sites were resolved by Scatchard analysis in both 13–day-old cerebellum and adult cerebral cortex. The higher-affinity sites correspond to the SSI subtype identified in saturation experiments, whereas the lower-affinity sites most likely correspond to the SS2 subtype. Ionic supplementation studies showed that divalent cations were required to obtain maximal specific binding on the SSI sites. In particular, Mn²⁺ was the most efficient cation for promoting binding of [¹²⁵I-Tyr⁰,d -Trp⁸]S14. Addition of GTP to the incubation buffer induced a marked reduction of specific binding. The results obtained by membrane binding assays were similar to those obtained by quantitative autoradiography, a result indicating that the microenvironment of SRIF-Rs was preserved in both types of tissue preparations. Receptors expressed in the developing rat cerebellum exhibited the same K_D and similar pharmacological profile as those observed in the adult rat cortex. These results show that SRIF-binding sites transiently expressed in the external granule cell layer of the cerebellum of young rats are indistinguishable from adult rat brain SRIF-Rs. The extremely high density of SRIF-Rs found in the external granule cell layer in 13–day-old rats suggests that SRIF may play a pivotal role in the proliferation and/or differentiation of these germinative cells.     

Differences in low pH tolerance among closely related sunfish of the genus Enneacanthus

Synopsis Three closely related sunfish in the genus Enneacanthus were examined to determine if differences existed in their tolerance to low pH that could explain their contrasting distributions. Na fluxes of E. obesus, E. gloriosus, and E. chaetodon were measured during 12 h exposure to pH 4.0 and 3.5 (all species), and 3.25 (former 2 species only). All experienced ionic disturbances upon acid exposure resulting from inhibition of active Na influx and stimulation of passive Na efflux, but E. gloriosus and E. chaetodon experienced greater disturbances than E. obesus at all pH's tested. Body and plasma Na concentrations of E. gloriosus were measured after one week of exposure to a range of pH's for comparison with previously published data from E. obesus. Exposure to pH 4.0 and below caused a depression in body and plasma Na concentration of E. gloriosus, and only two of 10 fish survived the one week test period at pH 3.5; none survived at pH 3.25. In contrast, exposure to pH 4.0 for five weeks had no effect on body Na concentration of E. obesus, all 10 fish survived exposure to pH 3.5 for two weeks. Growth of E. gloriosus and E. obesus were measured separately during 12 weeks of exposure to a range of pH's. E. gloriosus exposed to pH 4.25 and 4.0 grew at a lower rate than those at higher pH's (4.5, 5.0, and 5.8), and body Na concentrations of fish at pH's 4.25 and 4.0 were significantly less than the others. With declining pH E. obesus did not exhibit reduced growth until pH 3.75 was reached; no depression in body Na concentration occurred at this pH. These results show that there are marked differences in low pH tolerance among closely related species of Enneacanthus which could affect their distributions and competitive interactions.     

Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization

The N-nitrosodimethylamine demethylase (P450I-IE1) is induced severalfold in liver by giving rats ethanol, acetone, pyrazole, and other related small molecular weight compounds. This induction is not the result of an increase in IIE1 mRNA, but could be due to either an increase in translation rate or a decrease in protein degradation. To determine the mechanism of induction, we measured IIE1 synthesis and degradation rates in untreated and acetone-treated rats. This was accomplished by immunopurification of radiolabeled IIE1 protein using a specific monoclonal antibody subsequent to in vivo labeling of total cellular protein with either NaH14CO3 or [3H]leucine. We found that in rats fed acetone, the rate of IIE1 synthesis was not changed; however, IIE1 degradation was markedly altered. In untreated rats, IIE1 protein was degraded via a biphasic pathway consisting of both a rapid and slow component with approximate half-lives of 7 and 37 h, respectively. However, in acetone-treated rats, only a monophasic curve with a half-life of 37 h was observed. The abolition of the rapid degradation component of the IIE1 turnover cycle indicates that induction of IIE1 by acetone is primarily due to specific stabilization of IIE1 protein. Since acetone is also metabolized by IIE1, we believe that this may be a substrate-induced enzyme stabilization.