Cytochemical and functional evaluation of ACTH responsive isolated rat adrenocortical cells. The maintenance of sex differences

The critical evaluation of isolation methods for obtaining the adrenocortical cell suspension due to trypsin or collagenase digestion was done. Some collagenase advantages were indicated by morphological observations on the staining smears as well as by ACTH stimulation test. The cytochemical reactions for enzyme activities had the limited applications for those purposes. It also appeared that commonly applied dye exclusion tests were inadequate for characterization of cell suspension. The possible role of the adrenocortical cell debris in the basal corticosterone production was pointed out. The maintenance of the sex dimorphism and the functional differences in the adrenocortical cells isolated from male and female rats have been observed.     

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.     

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.     

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.     

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.     

The influence of diet on the growth of Stenonema vicarium (Walker) (Ephemeroptera: Heptageniidae)

Laboratory studies compared the growth rate of Stenonema vicarium (Walker) nymphs on diets of detritus and natural stream periphyton. In three consecutive runs of the experiment, growth rates were consistently higher on periphyton (mean growth rate = 2.1% wet wt. d⁻¹) than detritus (mean = 1.8% wet wt. d⁻¹). The starting date of each run also significantly influenced growth rates. In each treatment growth rates generally decreased over the course of the 3 runs, and ca. one-half of the nymphs in the last run did not molt or grow. It appeared that growth of S. vicarium may be partially controlled by seasonal factors.