Differentiation of the myoepithelial cells of the rat submandibular gland in vivo and in vitro: an ultrastructural study

Cytodifferentiation of the myoepithelial cells (MEC) of the rat submandibular gland (SMG) was observed by studying the prenatal and postnatal development of the gland in vivo and in vitro by light and electron microscopy. The anlage of the SMG first appeared on the fourteenth day of gestation and, from its earliest inception, was surrounded by an intact basal lamina. Presumptive myoepithelial cells were first seen at 18 days of gestation coinciding with the onset of secretion in the rudiment. These cells were flattened, peripherally located and subjacent to the epithelial basal lamina. Initial deposition of cytofilaments in the MEC's was observed during the first three days following birth and fully matured cells were seen as early as one week after birth. Presumptive and immature MEC's were observed undergoing mitosis, but once cytofilament deposition had begun in the cells they did not divide. Myoepithelium developed in relation to embryonic secretory structures and were only observed surounding acini and intercalated ducts in the adult gland. New myoepithelial cells were formed as long as new acinar-intercalated duct units were formed. Myoepithelial cells did not produce secretory type granules at any time during their development or in their mature state. Development of the MEC's in vitro paralleled that in vivo and supported the above observations.     

The effects of fatty acids on phosphoinositide synthesis and myo-inositol accumulation in exocrine pancreas

The effects of arachidonic acid (20:4) on phosphoinositide turnover were examined in rat pancreatic acinar cells prelabeled with myo-[3H]inositol. Arachidonic acid (50 microM) increased the accumulation of myo-[3H]inositol, but not that of [3H]inositol monophosphate, [3H]inositol bisphosphate, or [3H]inositol trisphosphate. By contrast, 10 microM carbamoylcholine increased the accumulation of all four compounds. A combination of arachidonic acid plus carbamoylcholine caused a selective and marked accumulation of myo-[3H]inositol, which was abolished by 10 mM LiCl. Arachidonic acid (10-100 microM) produced a concentration-dependent inhibition of myo-[3H]inositol incorporation into phosphoinositides and markedly depressed carbamoylcholine-induced increases in myo-[3H]inositol incorporation into inositol phospholipids. Several other unsaturated and saturated fatty acids failed to elicit a synergistic response with carbamoylcholine in stimulating myo-[3H]inositol accumulation and did not retard the incorporation of myo-[3H]inositol into phosphoinositides. The fact that eicosapentaenoic acid (20:5), but not arachidic acid (20:0), mimicked the depressant effect of arachidonate on phosphoinositide labeling suggests that the degree of unsaturation of the fatty acid, rather than chain length, is important for inhibition of phosphoinositide synthesis. The arachidonate-induced decrease in myo-[3H]inositol incorporation was accompanied by a reduction in the steady state level of [32P]phosphatidylinositol 4,5-bisphosphate. The mass of arachidonic acid liberated in response to carbamoylcholine was measured by gas chromatography-mass spectrometry, and the time course of stimulated arachidonate accumulation paralleled that of inositol phosphate accumulation and amylase release. These observations suggest that in exocrine pancreas, endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover.     

Neisseria meningitidis: heterogenicity in the outer membrane proteins released into the growth medium

Eight strains of Neisseria meningitidis belonging to different serogroups were analysed for their virulence in mice and their release of outer membrane proteins into the medium during growth. All strains released proteins. No detectable lipopolysaccharide was observed. However, SDS-PAGE showed a heterogenicity in the protein number and profile among the different strains of N. meningitidis tested.     

Casein kinase II activity and polyamine-stimulated protein phosphorylation of cytosolic and plasma membrane proteins in bovine sperm

A highly purified preparation of sperm cytosolic protein kinase was obtained by repeated chromatography with phosphocellulose. The preferred substrate of the enzyme was casein and the activity was not stimulated by added Ca2+, calmodulin, or cAMP. With casein as substrate, both ATP and GTP served as phosphate donors and the activity was inhibited by low micromolar heparin and stimulated by low millimolar spermine and spermidine. These properties are characteristic of casein kinase II from other cells. Endogenous protein substrates of the enzyme in sperm cytosolic fractions and in plasma membranes were demonstrated by incubating the preparations with [gamma-32P]GTP, under conditions unfavorable to other protein kinases, and analyzing the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Spermine greatly enhanced the phosphorylation of three (55, 92, and 106 kDa) proteins in both cytosolic and plasma membrane preparations. Our results indicate that polyamines play a role in modulating the phosphorylation state of proteins in sperm and may further regulate sperm function through this mechanism.     

Microbiological and biotechnological aspects of metabolism of carbamates and organophosphates.

Several carbamate and organophosphate compounds are used to control a wide variety of insect pests, weeds, and disease-transmitting vectors. These chemicals were introduced to replace the recalcitrant and hazardous chlorinated pesticides. Although newly introduced pesticides were considered to be biodegradable, some of them are highly toxic and their residues are found in certain environments. In addition, degradation of some of the carbamates generates metabolites that are also toxic. In general, hydrolysis of the carbamate and organophosphates yields less toxic metabolites compared with the metabolites produced from oxidation. Although microorganisms capable of degrading many of these pesticides have been isolated, knowledge about the biochemical pathways and respective genes involved in the degradation is sparse. Recently, a great deal of interest in the mechanisms of biodegradation of carbamate and organophosphate compounds has been shown because (1) an efficient mineralization of the pesticides used for insect control could eliminate the problems of environmental pollution, (2) a balance between degradation and efficacy of pesticides could result in safer application and effective insect control, and (3) knowledge about the mechanisms of biodegradation could help to deal with situations leading to the generation of toxic metabolites and bioremediation of polluted environments. In addition, advances in genetic engineering and biotechnology offer great potential to exploit the degradative properties of microorganisms in order to develop bioremediation strategies and novel applications such as development of economic plants tolerant to herbicides. In this review, recent advances in the biochemical and genetic aspects of microbial degradation of carbamate and organophosphates are discussed and areas in need of further investigation identified.     

Cytochrome b from Escherichia coli nitrate reductase. Its properties and association with the enzyme complex

Membrane-bound nitrate reductase purified from Escherichia coli was resolved into two separate forms. The majority of the enzyme complex had a subunit composition of 2A:2B:4C, exhibited cytochrome b spectra, and was found to be stable after purification. A second form of nitrate reductase activity was a modified complex with a subunit composition of 2A:2B and lacked cytochrome. The subunit B from this complex was altered in its mobility on sodium dodecyl sulfate-polyacrylamide gels. The cytochrome-containing enzyme had 28 +/- 2 atoms of iron and 1.35 atoms of molybdenum whereas iron and molybdenum in cytochromeless enzyme were 24 +/- 2 atoms and 1.18 atoms/molecule, respectively. Besides cytochrome-containing nitrate reductase, two other cytochrome b-containing fractions were also resolved. These were cytochrome b associated with formate dehydrogenase and a novel cytochrome b with reduced absorption maxima at 430, 529.5, and 560 nm. Nitrate reductase cytochrome b (subunit C) was isolated from subunits A and B as a partially denatured form and its renaturation was accomplished by dialyzing against hemin. The renatured cytochrome yielded absorption spectra similar to the holoenzyme. The pure cytochrome aggregated upon heating, even in the presence of sodium dodecyl sulfate. It had a high isoelectric point (pH greater than 9.5) and had 45% hydrophobic amino acids.     

Estradiol regulation of secretory component: Expression by rat uterine epithelial cells

Sex hormones are known to play an important role in the regulation of mucosal immunity in the female reproductive tract. The purpose of this study was to examine the effect of estradiol (E₂) on secretory component (SC) expression by epithelial cells in the rat uterus and to determine whether SC mRNA is present in uterine tissues and is under hormonal control. When ovariectomized rats treated with E₂ for 3 days and sacrificed 12 h after the last injection, expression of SC on luminal and glandular epithelial cells, as determined by immunohistochemistry, was elevated when compared to control animals. To determine whether E₂ regulation of SC involves mRNA synthesis, uterine RNA was extracted and analyzed by Northern blot. These experiments demonstrated that SC RNA is present in uteri from intact rats and markedly increased when ovariectomized animals are treated with E₂. In other studies, uterine epithelial cells from adult rats were isolated and grown on permeable membranes for 5 to 10 days. Under these conditions, isolated epithelial cells grow to confluence, form tight junctions, and preferentially secrete SC into the apical medium. These studies identify epithelial cells as a key target cell in the uterus for the regulation of mucosal immunity by E₂, which we postulate will play an important role in studies to prevent and/or control the spread of sexually transmitted diseases.