Effect of tubulozole, a new synthetic microtubule inhibitor, on the induction of casein gene expression by prolactin

Colchicine and related drugs are known to inhibit milk secretion. They are also able to prevent stimulation of casein and DNA synthesis by prolactin in the mammary gland. The present report reports data obtained with tubulozole, a new antimitotic drug. Tubulozole C added to culture medium of isolated rabbit epithelial mammary cells strongly inhibited their multiplication. Simultaneously, at a concentration of 1 microM, it prevented almost completely the induction of beta-casein mRNA. Induced cells were rapidly deinduced by addition of the drug to the medium. A similar inhibition was observed when the induction was obtained with prolactin alone or with its two stimulators insulin and glucocorticoids. Tubulozole T, an isomer of tubulozole C which is known to be ineffective in disrupting microtubules, did not alter prolactin actions. These data and those obtained with other tubulin-binding drugs strongly suggest that the integrity of microtubules is required for prolactin to deliver its message to the mammary cell.     

Autoradiographic, ultrastructural and interferometric analyses of the temperature effect on the synthesis and migration of ribosomes in root meristem cells of Helianthus annuus L. relation to S phase initiation

Experimental model consisted in blocking cells in G1 phase by cold treatment (12 h, 10 degrees C); following 3 h of postincubation at 20 degrees C, cells initiated S phase. In the present studies it has been shown that 2 h postincubation at 20 degrees C of cold-treated young seedlings of Helianthus annuus L. results in transformation of inactive meristematic nucleoli, characterized by small sizes, reduced amount of dry mass and granular component and by the presence of few and large fibrillar centres into large active nucleoli displaying high dry mass and granular component contents, numerous and small fibrillar centres. After 3 h of postincubation at 20 degrees C, nucleoli lose their granular component, decrease in size and dry mass content. At this moment cytoplasm enriches in ribosomes and its dry mass increases. Maximum of nucleolar activity is preceded by an accumulation of proteins in nucleoli. It is concluded that an enhanced transport of ribosomes is one of the conditions of S phase initiation.     

Effects of isotretinoin on the behavior of neural crest cells in vitro

Isotretinoin (13-cis-retinoic acid), an anti-acne medication, has been found to cause severe birth defects which affect the craniofacial elements, ear, heart, thymus, and central nervous system. Many of these structures receive contributions from the cranial neural crest. Here, we examine the possibility that these teratogenic effects are due to disturbances in neural crest development. Cranial and trunk neural crest explant cultures were exposed to different concentrations of isotretinoin and the cell morphology was monitored at daily intervals. Treated neural crest cells often became rounded or spindle shaped, separated from their neighbors, and frequently detached from the substrate or clumped together. In contrast, neural tube cells and cardiac fibroblasts were relatively unaffected by the drug. These results suggest that isotretinoin selectively affects neural crest cells by decreasing their cell-substratum adhesion.     

Subcellular location of enzymes involved in the N-glycosylation and processing of asparagine-linked oligosaccharides in Saccharomyces cerevisiae

A particulate translation system isolated from the yeast Saccharomyces cerevisiae was shown to translate faithfully in-vitro-transcribed mRNA coding for a mating hormone precursor (prepro-alpha-factor mRNA) and to N-glycosylate the primary translation product after its translocation into the lumen of the microsomal vesicles. Glycosylation of its three potential sugar attachment sites was found to be competitively inhibited by acceptor peptides containing the consensus sequence Asn-Xaa-Thr, supporting the view that the glycan chains are N-glycosidically attached to the prepro-alpha-factor polypeptide. The accumulation in the presence of acceptor peptides of a membrane-specific, unglycosylated translation product (pp-alpha-F0) differing in molecular mass from a cytosolically located, protease-K-sensitive alpha-factor polypeptide (pp-alpha-Fcyt) by about 1.3 kDa, suggests that, in contrast to previous reports, a signal sequence is cleaved from the mating hormone precursor on/after translocation. This conclusion is supported by the observation that the multiply glycosylated alpha-factor precursor is cleaved by endoglucosaminidase H to a product with a molecular mass smaller than the primary translation product pp-alpha-Fcyt but larger than the membrane-specific pp-alpha-F0. Translation and glycosylation experiments carried out in the presence of various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-1-deoxynojirimyin and 1-deoxymannojirimycin) indicate that the N-linked oligosaccharide chains of the glycosylated prepro-alpha-factor species are extensively processed under the in vitro conditions of translation. From the specificity of the glycosidase inhibitors applied and the differences in the molecular mass of the glycosylated translation products generated in their presence, we conclude that the glycosylation-competent microsomes contain trimming enzymes, most likely glucosidase I, glucosidase II and a trimming mannosidase, which process the prepro-alpha-factor glycans down to the (Man)8(GlcNAc)2 stage. Furthermore, several arguments strongly suggest that these three enzymes, which apparently represent the full array of trimming activities in yeast, are exclusively located in the lumen of microsomal vesicles derived from endoplasmic reticulum membranes.     

Phorbol ester binding and protein kinase C activity in normal and transformed human keratinocytes

Normal keratinocytes, SV40-transformed keratinocytes (SVK14), and various squamous carcinoma cell (SCC) lines have been used as an in vitro model system to study the properties of phorbol ester receptor and protein kinase C expression during keratinocyte differentiation. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-4; moreover, all cell lines respond to a low external Ca2+ concentration by a decreased capacity to differentiate. Normal keratinocytes exhibited the highest number of phorbol ester receptors as compared to the other cell lines, while each individual cell line exhibited a higher number of phorbol ester receptors during growth under normal Ca2+ conditions as compared to cells grown under low Ca2+ conditions. The apparent dissociation constant (Kd) demonstrated only small variations in the various cell lines. In contrast, the cytoplasmic protein kinase C activity, was found to be higher in cells grown under low Ca2+ conditions than in cells grown under normal Ca2+ conditions, indicating the absence of a causal relationship between cytoplasmic protein kinase C activity and phorbol ester receptor expression. Therefore the properties of protein kinase C have been determined in more detail in normal keratinocytes and SCC-15 cells. These studies revealed differences between protein kinase C properties from the two cell lines grown under normal and low Ca2+ conditions. The differences included the effect of phorbol 12-myristate 13-acetate (PMA) on the redistribution pattern of protein kinase C between the cytoplasmic and particulate fractions as well as the activating effect of diolein in vitro on protein kinase C activity, partly purified from particulate or cytoplasmic fractions. These observations demonstrate that the functional protein kinase C activity of keratinocytes is determined by various endogenous and exogenous activators and that these activators are modulated differently in various cell lines, under various growth conditions (low Ca2+ versus normal Ca2+).     

Current approaches to the synthesis and reconstruction of genetic material

Advanced approaches to the synthesis and reconstruction of genetic material developed in the Institutes of Molecular Biology and Genetics during the past years are summarized. The evolution of methods for oligonucleotide synthesis and scopes for their use in gene production are discussed. The principles of localised mutagenesis methods developed in the Institute are described, such as: a) mutagenesis directed to the regulatory gene regions; b) segment-localized mutagenesis; c) mutagenesis directed by phosphotriester analogues of oligonucleotides. Examples of employing these methods for induction of regulatory mutants of phage lambda, production of fused genes, mutant interferon genes, construction of new DNA vectors, construction of hybrid H1-H3 subtype haemagglutinine gene of influenza virus etc. are presented. The approach to in vivo site-directed mutagenesis is experimentally substantiated.     

Microflora associated with the internal surfaces of rubber and stainless steel milk transfer pipeline

Sterile sections of rubber and stainless steel milk transfer pipeline were inserted sequentially into a milking installation and soiled with fresh raw milk over a period of 5 d. The resultant adherent microbial population was removed and the generic composition of mesophilic and psychotropic types was determined. In all cases Acinetobacter spp. were found to predominate (59.5-75.6%). The generic composition of the raw milk used to soil the milking unit (with inserted pipe section) was determined once during each 5-d soiling period. In general the milk was found to contain a mixed flora in which Gram-positive organisms predominated.     

The physiology of Clostridium sporogenes NCIB 8053 growing in defined media

The physiology of Clostridium sporogenes was investigated in defined, minimal media. In batch culture, the major end products of glucose dissimilation were acetate, ethanol and formate. When L-proline was present as an electron acceptor, acetate production was strongly enhanced at the expense of ethanol. As judged by assay of the relevant enzymes, glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The growth energetics of Cl. sporogenes were investigated in glucose- or L-valine-limited chemostat cultures. In the former case, the addition of L-proline to the medium caused a significant increase in the molar growth yield (as calculated by extrapolation to infinite dilution rate). This finding adds weight to the view that the reduction of L-proline by Cl. sporogenes is coupled to the conservation of free energy.     

Radiation cell survival and growth delay studies in multicellular spheroids of small-cell lung carcinoma

The radiation sensitivity of two small-cell lung carcinoma cell lines growing as multicellular spheroids in static culture was determined using clonogenic cell survival and growth delay as endpoints. Growth delay determination suggested that clonogenic cell kill was less than was obtained by direct assay of cell survival. Recovery from potentially lethal damage was assayed in one line (HC12) but was not demonstrable, and clonogenic cell survival decreased with time in treated spheroids with diameters greater than 300 microns which contained a hypoxic cell population. Microscopic examination of the treated spheroids showed the emergence of an abnormal giant-cell population, and the progressive clonogenic cell loss that occurred after treatment was thought to be due to oxygen and nutrient deprivation of the remaining viable cells by this doomed cell population. Correction of the growth delay measurements for changes in cell size and clonogenic cell population allowed correlation of the growth delay and cell survival data.