Resolution of myocardial phospholipase C into several forms with distinct properties.

Phospholipase C activity capable of hydrolysing phosphatidylinositol in bovine heart was resolved into four forms (I-IV) by ion-exchange chromatography. Some of these forms could only be detected if the assay was performed at acidic pH (I and IV) or in the presence of deoxycholate (II). Gel-filtration chromatography indicated that the four forms had different molecular weights in the range 40000-120000. I, II and III all had pH optima in the range 4.5-5.5. However, the major form (III) also had substantial activity at pH 7.0 and above. The activities of I, II and III at pH 7.0 were stimulated by deoxycholate; this effect was most marked with I and II, which had very low activity at this pH. All forms of the enzyme were inhibited by EGTA and required 2-5 mM-CaCl2 for maximal activity. When the fractions eluted from the ion-exchange and gel-filtration columns were assayed with polyphosphoinositides as substrates there was a close correspondence to the elution profile obtained with phosphatidylinositol as substrate; there was no evidence for the existence in heart of phospholipase C activities specific for individual phosphoinositides.     

Effects of insulin in vitro on protein turnover in rat epitrochlearis muscle.

The hexapeptide Arg-Asn-Gly-epoxyethylglycine-Ala-Val-OMe specifically inactivates membrane-bound N-glycosyltransferases. The specificity is demonstrated by the inability of peptides containing 2,3-epoxypropyl-, allyl- and vinyl-glycine in the epoxyethylglycine position to function as inhibitors. The inhibition is concentration-dependent and follows first-order kinetics, but requires disruption of the membrane vesicles by detergents to achieve accessibility to the transferase. The enzyme can be protected partially against inactivation by the addition of the acceptor peptide Arg-Asn-Gly-Thr-Ala-Val-OMe, pointing to an active-site-directed reaction. Exhaustion of the endogenous pool of glycosyl donor molecules by preincubation of the membrane vesicles with the acceptor peptide before inhibitor application is accompanied by an additional decrease in the inhibition rate. This suggests that inactivation occurs only under conditions where glycosyl transfer is catalysed. A mechanism of inactivation is proposed in which the transferase catalyses its own inactivation by a kind of 'suicide' mechanism.     

Relation of Catalase to Substrate Utilization by Mycoplasma pneumoniae

No catalase activity was detected in four strains of glucose-grown Mycoplasma pneumoniae at any time during the replication of the organism. Exogenous catalase dramatically increased the O(2) uptake with glycerol, presumably by releasing inhibition caused by hydrogen peroxide. The effect of added catalase on the O(2) uptake of washed organisms with glucose as substrate was moderate and variable in degree. The production of hydrogen peroxide was demonstrated by the quantitative enzymatic assay for inorganic peroxide and by the fact that added pyruvate, which is non-enzymatically oxidized by H(2)O(2) to acetic acid and CO(2) could mimic the action of catalase.     

Inversion of Transfer Modes and Sex Factor-Chromosome Interactions in Conjugation in Escherichia coli

A study was made of the mating properties of an unusual system of interconvertible donor strains of Escherichia coli K-12: Ra-1, Ra-2, and RaF⁺. The Ra-1 and Ra-2 strains are Hfr strains whose origins are widely separated on the chromosome and whose transfer modes proceed in the opposite direction from one another. When Ra-1 cells were mated with females, a small fraction of the donors transferred markers via the Ra-2 mode. This effect was enhanced by preconjugal ultraviolet (UV) treatment of the Ra-1 cells. Among the survivors of UV-treated Ra-1 cells, a few stable Ra-2 cells were found. When Ra-2 cells were used as the donors, some of them were found to mate via the Ra-1 mode, in analogy with the Ra-1 to Ra-2 alteration with inversion of F mentioned above. Related experiments suggested that the inversion occurs by detachment of the F factor from one Hfr origin locus, followed by reassociation of the F factor with the other Hfr origin locus. Both the Ra-1 and Ra-2 strains reverted spontaneously to an F⁺ strain, called RaF⁺. Cultures of RaF⁺ cells were found to mate primarily according to the Ra-1 and Ra-2 transfer modes, with smaller contributions also coming from transfer modes with origins elsewhere on the chromosome in a way which is similar to the transfer of markers from a normal F⁺ strain. The RaF⁺ sex factor was found to be wild type, whereas the chromosome was found to carry irregularities (sex factor affinity loci) at the locations of the Ra-1 and Ra-2 origins. Only about 10% of the donor capacity of the RaF⁺ strain was due to stable spontaneous Hfr cells in cultures of RaF⁺ cells.     

POLYESTER-METHACRYLATE EMBEDMENTS FOR ELECTRON MICROSCOPY

It has been found that tissues fixed for electron microscopy and dehydrated in acetone can be embedded in mixtures of n-butyl methacrylate and polyester resin. Activation with 1 per cent tert-butyl hydroperoxide followed by 12 to 48 hours at 60°C produces blocks that section well with glass knives. The ribbons are cleared of methacrylate by heat (200–250°C for 1 hour) and/or immersion in organic solvents (CCL₄, acetone-ether). After removal of the methacrylate the residual polyester matrix provides thermostable and insoluble support for the tissue. Its insolubility permits staining by immersion of cleared preparations in organic solvents carrying heavy metal compounds in solution. Clearing by heat stabilizes section-grid relationships. The removal of volatile materials by clearing substantially reduces contamination of both specimen and microscope. Tissue fine structure is well preserved in these preparations.     

Lectin binding patterns to terminal sugars of rat lung alveolar epithelial cells.

We used post-embedding cytochemical techniques to investigate the lectin binding profiles of rat lung alveolar epithelial cells. Sections from rat lung embedded in the hydrophilic resin Lowicryl K4M were incubated either directly with a lectin-gold complex or with an unlabeled lectin followed by a specific glycoprotein-gold complex. The binding patterns of the five lectins used could be divided into three categories according to their reactivity with alveolar epithelial cells: (a) the Limax flavus lectin and Ricinus communis I lectin bound to both type I and type II cell plasma membranes; (b) the Helix pomatia lectin and Sambucus nigra L. lectin bound to type II but not type I cells; and (c) the Erythrina cristagalli lectin reacted with type I cells but was unreactive with type II cells. The specificity of staining was assessed by control experiments, including pre-absorption of the lectins with various oligosaccharides and enzymatic pre-treatment of sections with highly purified glycosidases to remove specific sugar residues. The results demonstrate that these lectins can be used to distinguish between type I and type II cells and would therefore be useful probes for investigating cell dynamics during lung development and remodeling.     

Expression of major histocompatibility complex antigens on the bovine placenta

Immunohistochemistry was utilized to determine expression of the major histocompatibility complex (MHC) antigens on Day 8-9 hatched blastocysts and fetal membranes of mid- to late gestation cows and to examine the pattern of leucocytic infiltration into the gravid uterus. Hatched blastocysts were weakly positive for MHC class I antigens. In the mature placenta, chorioallantoic membranes in the interplacentomal area showed positive immunostaining for class I antigens on the chorionic epithelium but had no staining for class II antigens. There was an accumulation of lymphoid cells expressing class II antigens directly beneath the luminal epithelium of the endometrium. In addition, cells staining for leucocyte common antigen were present both within and beneath the luminal epithelium. Some cells positive for class II and leucocyte common antigen (CD45) were also associated with uterine glands. In the placentomes, class I antigens were expressed only on maternal caruncular septa. Fetal cotyledonary villi had no detectable immunostaining for class I and II antigens. No distinct pattern of leucocyte infiltration in the maternal caruncular tissue was observed; the caruncular septa contained some cells that were labelled for CD45 and a few class II-positive cells around blood vessels. The results indicate that the fetal placenta of the cow expresses MHC class I antigens in a regionally defined manner and there is a differential accumulation of lymphoid cells in the uterus.     

Differential adhesion,activity, and carbohydrate: Protein ratios ofPseudomonas atlantica monocultures attaching to stainless steel in a linear shear gradient

Biofilm formation on metallic surfaces in marine and freshwater environments often precedes corrosion and other biofouling conditions. Attachment is mediated by such environmental factors as the presence of surface conditioning films, fluid dynamics, bulk-phase nutrient levels, and surface chemistry. In this study, we utilized a Fowler Cell Adhesion Measurement Module to demonstrate that the changes in cellular concentration and composition of a monoculture ofPseudomonas atlantica biofilms on stainless steel were a function of the applied shear force. At shear forces in the range of 3–10 dynes cm^–2 (1.0 liter min^–1), attachment as measured by acridine orange direct microscopic counts was greatest at the higher shear forces.¹⁴C-Acetate uptake activity on the stainless steel surfaces increased with shear stress. Acetate incorporation ranged from 1×10^–5 to 19×10^–5 mol cm^–2 between 0.15 and 30 dynes cm^–2 for 30 min uptake periods. On a per cell basis, however, activity decreased with shear, indicating a shift in metabolism. Fourier transform infrared spectroscopy revealed that protein and carbohydrate concentrations also increased with the applied shear. Increased biofilm CN ratios and total fatty acids were associated with the higher shear stresses. Neither radius of interaction nor biofilm age appeared to significantly influence the relationship between fluid shear and attachment and cellular composition ofP. atlantica biofilms in the range of 1–10 dynes cm^–2.     

The regulation of proenkephalin expression in a distinct population of glial cells.

The expression of opioid genes was examined in isolated populations of glial cells in primary culture. Northern blot analysis of purified type I astrocytes, oligodendrocytes and mixed oligodendrocyte-type-2-astrocyte lineage cells derived from cerebral cortex demonstrated robust expression of proenkephalin mRNA exclusively in type I astrocytes. The expression of proenkephalin mRNA was stimulated by the beta-adrenergic agonist isoproterenol, and 8-(4-chlorophenyl thio)adenosine 3'-5'-cyclic monophosphate (cpt-cAMP). Both of these compounds regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into type I astrocytes. HPLC and immunoassay of the cell culture media revealed significant levels of unprocessed proenkephalin secreted by the cell and this secretion was stimulated by isoproterenol and cpt-cAMP. The relatively high levels of proenkephalin expressed suggest that enhanced expression in astrocytes may be important during neural development, in trauma-induced gliosis and in neuroimmune interactions.