Characterization of the inhibitory effect of glucocorticoids on the DNA replication of adult rat hepatocytes growing at various cell densities

Dexamethasone inhibited the basal and EGF-stimulated DNA synthesis of adult rat hepatocytes in primary culture. The inhibition was glucocorticoid-specific: It was shown by dexamethasone and hydrocortisone, but not by progesterone, testosterone, or estradiol; and was counteracted by the glucocorticoid antagonist RU-38486 in a concentration-dependent manner. Dexamethasone acted by decreasing the rate of entry into S-phase (kG1/S), while cell cycle parameters were unaffected. The steroid was able to decrease the kG1/S severalfold even when added more than 20 hr after EGF, half-maximal effect occurring 11 hr after the addition of dexamethasone. Densely populated areas were much more sensitive to the inhibition by dexamethasone than sparsely populated areas within the same culture dish: A moderate (10 nM) concentration of dexamethasone nearly abolished the DNA synthesis in densely populated areas of hepatocyte cultures with only marginal effect on sparsely populated cells.     

The Salmonella typhimurium glycine cleavage enzyme system

Summary A glycine cleavage enzyme system, inducible by glycine, has been demonstrated in Salmonella typhimurium. The induced enzyme levels, however, are only about 20% of the induced levels found in Escherichia coli. Starting with a serine auxotroph, mutants were isolated that grow with a serine supplement, but not with a glycine supplement. Three independently isolated mutants have reduced or nondetectable glycine cleavage enzyme levels. The new mutations, designated gcv, were mapped between the serA and lys genes at 62.5 min on the S. typhimurium chromosome.Abbreviations C1 one-carbon - GCV glycine cleavage - GM glucose minimal - L agar Luria agar - LB Luria broth - Tc tetracycline     

Coexpression of cytokeratin and vimentin in Rathke's cysts of the human pituitary gland

Summary Immunohistochemistry with monoclonal and polyclonal antibodies revealed the presence of cytokeratins in epithelial cells of Rathke's cysts in the pars intermedia of the human pituitary gland. With monoclonal antibodies specific for individual cytokeratins, the expression of CK 18, CK 8, CK 7, and CK 19 could be shown in these cells. Within the hypophysis, CK 19 and CK 7 were restricted to Rathke's cysts and a few epithelial cell clusters in the pars tuberalis, whereas other cytokeratins were also present in endocrine cells of the pars distalis. Furthermore, vimentin and, focally, glial fibrillary acidic protein (GFAP) were detected in the cystic epithelia. By double labelling, coexpression of cytokeratin and vimentin, GFAP and cytokeratin, and GFAP and vimentin could be demonstrated. Compiled data of all known cases of coexpression of cytokeratin and vimentin in normal cells reveal physiological correlations and suggest a functional significance of this rare type of coexpression of intermediate filament proteins.     

Variations in the cytoplasmic region account for the heterogeneity of the chicken MHC class I (B-F) molecules

Molecular variation among major histocompatibility complex (MHC) class I (B-F) proteins from B-homozygous chickens is apparently caused by C-terminal variation. Analysis of the total B-F protein pool revealed substantial heterogeneity with two or three molecular mass constituents, each being comprised by several isoelectric focusing variants. This heterogeneity could not be reduced by enzymatic deglycosylation. By contrast, proteolytic removal of a small (M _r 1000–4000) fragment from the chain resulted in the generation of a M _r 36 000 fragment, common to all the molecular mass variants. Unlike the parent proteins, the M _r 36 000 fragment derived from isolated variants yielded identical, simple patterns in two-dimensional gel electrophoresis and identical finger prints in peptide mapping. This, together with N-terminal amino acid sequencing, as well as comparison of hydrophobicity properties of fragments obtained by gradual proteolytic digestion, indicated that the small peptide responsible for the major B-F heterogeneity was situated in the intracellular, C-terminal part.     

Statistic evaluation of the influence of species variation,culture conditions,surface wettability and fluid shear on attachment and biofilm development of marine bacteria

A budding coccoid bacterium, (CH1), a Vibrio sp. and a Pseudomonas sp. were investigated for factors governing their attachment to glass surfaces in static batch culture and laminar flow continuous culture systems. An analysis of variance showed that the three species exhibited very different responses. For CH1 attachment was dependent on cell density, incubation time and nutrient concentration. The Vibrio sp. was affected by nutrient concentration while the attachment of the Pseudomonas sp. was independent of cell density, incubation time and nutrient concentration. A comparison of attachment to hydrophilic and hydrophobic surfaces showed that attachment of the Vibrio sp. and CH1 to hydrophilic surfaces was 3 and 10 times greater respectively than to hydrophobic surfaces while Pseudomonas attached in equal numbers to both surfaces. The continuous culture system with defined flow hydrodynamics and growth conditions at steady state revealed a random sampling effect 3 times smaller than the batch culture system did. When the biofilm development of Pseudomonas sp. was followed during 46 h at different fluid shear under laminar and turbulent flow conditions, the former biofilm reached 3.3·10⁸ cells·cm^-2 and the latter 8.2·10⁷ cells·cm^-2.Non-common abbreviation NSS Nine salt solution     

Purification and in vitro phosphorylation of Myxococcus xanthus AsgA protein.

The deduced amino acid sequence of the Myxococcus xanthus AsgA protein contains an N-terminal domain that is homologous to the receiver of response regulators and a C-terminal domain that is homologous to the transmitter of histidine protein kinases. We overexpressed affinity-tagged AsgA in Escherichia coli, purified the recombinant protein, and showed that AsgA has autokinase activity in vitro. The results of chemical-stability assays suggest that AsgA is phosphorylated on a histidine and provide no evidence for transfer of the phosphoryl group to the conserved aspartate of the receiver domain.     

Loss of growth polarity and mislocalization of septa in a Neurospora mutant altered in the regulatory subunit of cAMP-dependent protein kinase.

In filamentous fungi, growth polarity (i.e. hyphal extension) and formation of septa require polarized deposition of new cell wall material. To explore this process, we analyzed a conditional Neurospora crassa mutant, mcb, which showed a complete loss of growth polarity when incubated at the restrictive temperature. Cloning and DNA sequence analysis of the mcb gene revealed that it encodes a regulatory subunit of cAMP-dependent protein kinase (PKA). Unexpectedly, the mcb mutant still formed septa when grown at the restrictive temperature, indicating that polarized deposition of wall material during septation is a process that is, at least in part, independent of polarized deposition during hyphal tip extension. However, septa formed in the mcb mutant growing at the restrictive temperature are mislocalized. Both polarized growth and septation are actin-dependent processes, and a concentration of actin patches is observed at growing hyphal tips and sites where septa are being formed. In the mcb mutant growing at the restrictive temperature, actin patches are uniformly distributed over the cell cortex; however, actin patches are still concentrated at sites of septation. Our results suggest that the PKA pathway regulates hyphal growth polarity, possibly through organizing actin patches at the cell cortex.