Kinamycin acetyltransferase I from Streptomyces murayamaensis, an apparently large, membrane-associated enzyme.

Identification and initial characterization of an apparently large, membrane-associated multifunctional enzyme, kinamycin acetyltransferase I (KAT I), is described. KAT I activity was enriched 29-fold over the level in cell-free extracts of Streptomyces murayamaensis. Two acetyltransferase activities catalyzing acetyl coenzyme A dependent conversion of kinamycin F and E to kinamycin E and D, respectively, were inseparable in the course of the partial purification. Partial purification involved separation of KAT I from cytosolic proteins by differential ultracentrifugation, solubilization with 0.5% CHAPS zwitterionic detergent followed by ultracentrifugation, and Sephacryl S400 gel filtration chromatography of the resulting supernatant.

Mechanics of tensile and shear stress generation in fixed partial denture retainers.

Movements caused by the application of chewing loads to a fixed partial denture (FPD) are predictable. There are translational and rotational movements of the FPD and flexure of the structure. The location and magnitude of tensile and shear stresses affecting cement within retainers during mastication is related to the type of movement and determined by differences in mobility of abutments at each end of the FPD, length of span, and point of chewing load. The incidence of cement failure could be reduced with improved strategic stress resistance.

A threshold effect of the major isoforms of NCAM on neurite outgrowth.

Interactions between recognition molecules on the surface of neuronal growth cones and guidance cues present in the local cellular environment are thought to account for the growth of neurites in the highly stereospecific manner that contributes to correct target cell innervation. In vitro assays have been used to identify candidate molecular components of this system, either directly by demonstrating their ability to promote neurite outgrowth, or indirectly by the ability of specific antibodies to inhibit neurite outgrowth. The role of the neural cell adhesion molecule (NCAM) in pathway finding is not fully understood. Some immunological studies support a positive role; others do not, and it has been reported that purified NCAM does not support neurite outgrowth. We have previously shown that an arbitrary biochemical index of neurite outgrowth, the relative level of immunoreactive neurofilament protein, is increased when human and rat dorsal root ganglion neurons are cultured on monolayers of cells expressing transfected human NCAM. But, the complexity of growth precluded a simple morphological analysis and we did not determine the 'dose-response' relationship between NCAM expression and neuronal response. Here, we report on the morphology of rat cerebellar neurons cultured on monolayers of 3T3 cells transfected with complementary DNAs encoding all of the main NCAM isoforms found in cells such as astrocytes, Schwann cells and skeletal muscle. The data indicate that both transmembrane and glycosyl-phosphatidylinositol linked NCAM isoforms are potent substrates for neurite extension. A critical threshold value of NCAM expression is required for increased neurite outgrowth. Above this threshold, small increases in NCAM induce substantial increases in neurite outgrowth.

Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms.

Full length cDNAs for a variety of human N-CAM isoforms have been transfected into mouse L-cells and/or 3T3 cells. Three independent clones of each cell line that were shown to express human N-CAM were tested for their ability to support the morphological differentiation of sensory neurons. The cell surface expression of N-CAM isoforms, linked to the membrane directly by an integral transmembrane spanning domain or indirectly via covalent attachment to a glycosyl-phosphatidylinositol moiety, were consistently found to be associated with a significant increase in the morphological differentiation of both human and rat dorsal root ganglion neurons. Modification of the extracellular structure of both classes of N-CAM, consequent to the expression of a glycosylated 37-amino acid sequence normally found expressed exclusively in muscle N-CAM isoforms did not obviously affect the ability of transfected cells to support increased neuronal differentiation. 3T3 cells that were transfected with a full length cDNA encoding a secreted N-CAM isoform, and that have previously been shown to secrete N-CAM into the growth media rather than link it to the membrane did not significantly differ from control cells in their ability to support neuronal differentiation. These data provide direct evidence for both transmembrane and lipid-linked N-CAM isoforms being components of the regulatory machinery that determines neuronal morphology and process outgrowth.

Obstetric practice. A decade under review.

As part of a prospective study concerning the cause of neonatal jaundice, information relating to obstetric practice over a 10 year period (1977 to 1987) in the New England area of Australia was collected. The data reveal certain trends over the decade and provide baseline data for obstetricians practising in a provincial setting.

New products related to kinamycin from Streptomyces murayamaensis. I. Taxonomy, production, isolation and biological properties.

Six new products of Streptomyces murayamaensis sp. nov. Hata et Ohtani, the producer of the kinamycins, were isolated by silica gel column chromatography. The antibacterial activities of the new products, as well as that of dehydrorabelomycin and murayaquinone, previously isolated products of the same organism, were compared to the kinamycins. Three of the products had antibacterial activities similar to the kinamycins, while two others had activity only against Gram-positive bacteria. Dehydrorabelomycin and one other metabolite had no detectable antibacterial activity. The organism was found to be capable of aerial mycelium formation, with sporophores branched at regular intervals bearing square-ended spores with smooth surfaces. The culture contains L,L-diaminopimelic acid in the cell wall (Type I), is highly resistant to lysozyme, and lecithinase- and melanin-positive, suggesting a relationship with the genus Streptoverticillium and the lavendulae group of the genus Streptomyces.

New products related to kinamycin from Streptomyces murayamaensis. II. Structures of pre-kinamycin, keto-anhydrokinamycin, and kinamycins E and F.

Four new colored components of Streptomyces murayamaensis sp. nov. Hata et Ohtani, the producer of the kinamycins, have been isolated and their structures determined by a combination of mass spectral, high field NMR and biosynthetic techniques. The first compound with the benz[b]carbazole skeleton in the biosynthetic pathway of kinamycin D has been named "pre-kinamycin" (7). A keto-epoxide kinamycin intermediate has been labeled "keto-anhydrokinamycin" (9), and the 1'-monoacetate of kinamycin has been called kinamycin E (12). Natural production of deacetylkinamycin (13, now labeled kinamycin F) by S. murayamaensis has been confirmed by an isotope trapping experiment. The role of these new intermediates in kinamycin biosynthesis is discussed.

Factors controlling the expression of the NGF receptor in PC12 cells.

An enzyme-linked immunoadsorbent assay (ELISA) has been used to study the relative expression of the nerve growth factor receptor (NGF-R) in PC12 cells. Both nerve growth factor (NGF) and fibroblast growth factor (FGF) were found to induce time-dependent increases in receptor expression. The former response was half-maximal at approximately 2 ng/ml and maximal at 25-50 ng/ml. The induction of NGF-R expression by NGF was associated with an increase in the relative level of a 3.8 kb mRNA species encoding this protein. Whereas the induction of the NGF-R by NGF and FGF were both fully inhibited by cholera toxin and cordycepin, the responses were differentially inhibited by the kinase inhibitor K-252a.

A novel way of removing quiescent astrocytes in a culture of subcortical neurons grown in a chemically defined medium.

A new method has been described for removing a very small number of contaminating astrocytes in neuronal cultures (derived from the septal-diagonal band region of 17-day-old embryonic rat brain) grown in a chemically defined medium. The proportion of these glial fibrillary acidic protein (GFAP)-positive cells was usually less than 1.5% up to 10 days, but thereafter their number increased rapidly reaching 10-15% by 22 days in vitro. A prolonged exposure to normally used concentration of cytosine arabinoside (Ara-C; 10 microM) was toxic to both astroglial and neuronal cells, while a brief treatment (48 h) with a low level (4 microM) of Ara-C failed to eliminate these astrocytes, as judged by glutamine synthetase activity and GFAP-positive cell count. However, these quiescent astroglial cells could be easily eliminated if they were induced to proliferate by epidermal growth factor before exposure to Ara-C. The combined treatment with these agents had no effect on the number of acetylcholinesterase-positive cells, and on the development of cholinergic and GABA-ergic neurons, as measured in terms of choline acetyltransferase and glutamate decarboxylase activity, respectively.

The mechanism of cytosine arabinoside toxicity on quiescent astrocytes in vitro appears to be analogous to in vivo brain injury.

Neuronal cultures derived from the septal diagonal band region of the embryonic rat brain and grown in a chemically defined medium contained a very small number of contaminating astroglial cells. During the first week in culture, these cells were well dispersed in the form of a single isolated cell with fine fibrous branched processes. Treatment with 4 microM cytosine arabinoside for 24 h failed to kill these astrocytes (most probably present in quiescent form), as judged by glutamine synthetase activity and glial fibrillary acidic protein-positive cell count. On the other hand, the exposure of cultures to cytosine arabinoside resulted in a marked increase in choline acetyltransferase enzyme activity. The overall results, together with our previous findings, are consistent with the proposal that a brief exposure to a relatively low concentration of cytosine arabinoside induces quiescent astrocytes to produce a large quantity of a neurotrophic factor that is involved in the regulation of cholinergic cells.

Birth weight, and use of oxytocin and analgesic agents in labour in relation to neonatal jaundice.

A prospective study of 1977 babies, delivered in two district hospitals in the Northern Tablelands area of New South Wales, revealed a highly significant relationship between neonatal jaundice and birth weight in all cases of neonatal jaundice (P = 0.0001). The use of oxytocin was associated with a significant increase in the incidence of jaundice (all cases, P = 0.003; severely jaundiced babies, P = 0.05). This effect was related to the total dose of oxytocin used (P = 0.003 for all cases; P = 0.056 for severely jaundiced babies), but not to its rate of administration. No significant difference was apparent whether oxytocin was administered to initiate or to accelerate labour. The suggestion that oxytocin use is associated with delivery of immature babies is refuted. The use of analgesic agents during labour was not associated with an increased incidence of neonatal jaundice.