Cellular kinetics of prednimustine versus chlorambucil plus prednisolone in vitro

Summary Intracellular concentrations of prednimustine (PM), chlorambucil (CLB), phenylacetic acid mustard (PAAM) and prednisolone (P) were measured in different experimental tumor cell lines that had been incubated with either PM or CLB+P. For intracellular analytical determination, we modified a high-pressure liquid chromatographic method for the detection of these substances in plasma. Intact PM could be detected in the intracellular compartment of the incubated tumor cells. PM-incubated cells from PM-injected rats exhibited a higher intracellular concentration-time integral (PAAM) and longer concentration-time profiles for drugs with alkylating capacity than did cells exposed to the CLB+P mixture or to CLB. PAAM was not detectable after incubation of cells with PM, whereas in CLB-incubated cells the AUC of PAAM exceeded that of the parent drug CLB. Our in vitro results therefore favour the concept of a facilitated intracellular uptake and an increased antiproliferative effect for PM versus CLB and CLB+P.Dedicated to Prof. Dr. H. J. Dengler on the occasion of his 65th birthday. This study was supported by the Ministry of Science and Research of Nordrhein-Westfalen     

Cultured rat mesangial cells contain smooth muscle alpha-actin not found in vivo.

A monoclonal antibody against smooth muscle alpha-actin (SM alpha-actin) was used to study the expression of SM alpha-actin in kidney sections and mesangial cell (MC) cultures. In the tissue sections, indirect immunofluorescence revealed intense labeling of vascular smooth muscle cells and precapillary pericytes for SM alpha-actin. Glomerular cells including MC were negative, with the exception of scattered smooth muscle cells in the wall of the intraglomerular segment of the efferent arteriole. In contrast, in MC cultures 50 to 95% of the cells displayed bright fluorescence. Immunoreactivity for SM alpha-actin first appeared 3 days after explanation of glomeruli and increased until the primary culture reached subconfluence. In each subculture (1 to 10) expression of SM alpha-actin was weak on day 1 and pronounced at subconfluence. Growth arrest of subconfluent cultures for 1 to 7 days in serum-free medium did not alter the percentage of cells positive for SM alpha-actin. However, exposure of MC to serum-free medium beginning on the first day of subculture curtailed expression of SM alpha-actin. Double-labeling with antibodies against proliferating cell nuclear antigen and SM alpha-actin revealed SM alpha-actin-positive filaments in both replicating and resting cells. In summary, our results demonstrate that some process or processes associated with cell proliferation and cell growth of MC are accompanied by de novo expression of SM alpha-actin. The relevance to the contractile behavior of the difference in SM alpha-actin expression under in vitro and in vivo conditions is unknown.     

Carried meningococci in the Czech Republic: a diverse recombining population

Population and evolutionary analyses of pathogenic bacteria are frequently hindered by sampling strategies that concentrate on isolates from patients with invasive disease. This is especially so for the gram-negative diplococcus Neisseria meningitidis, a cause of septicemia and meningitis worldwide. Meningococcal isolate collections almost exclusively comprise organisms originating from patients with invasive meningococcal disease, although this bacterium is a commensal inhabitant of the human nasopharynx and very rarely causes pathological effects. In the present study, molecular biology-based techniques were used to establish the genetic relationships of 156 meningococci isolated from healthy young adults in the Czech Republic during 1993. None of the individuals sampled had known links to patients with invasive disease. Multilocus sequence typing (MLST) showed that the bacterial population was highly diverse, comprising 71 different sequence types (STs) which were assigned to 34 distinct complexes or lineages. Three previously identified hyperinvasive lineages were present: 26 isolates (17%) belonged to the ST-41 complex (lineage 3); 4 (2.6%) belonged to the ST-11 (electrophoretic type [ET-37]) complex, and 1 (0.6%) belonged to the ST-32 (ET-5) complex. The data were consistent with the view that most nucleotide sequence diversity resulted from the reassortment of alleles by horizontal genetic exchange.     

Synthesis and characterization of acrylate-terminated unsaturated copolyesters

The reaction of the potassium salt of acrylic acid with the potassium salt of a dicarboxylic acid and 1,4-dibromobutane was applied to the synthesis of three series of acrylate-terminated copolyesters: poly(tetramethylene maleate-co-tetramethylene phthalate) (BMPA), poly(tetramethylene fumarate-co-tetramethylenephthalate) (BFPA) and poly(tetramethylene phthalate-co-tetramethylene succinate) (BPSA). The monofunctional monomer, the potassium salt of acrylic acid, was used for the introduction of double bonds at the end of the chains and for the regulation of the molecular weight of the polyesters. The telechelic copolyesters were characterized by ¹H and ¹³C NMR spectroscopy, in almost all cases providing an acrylate functionality of two.     

Shb null allele is inherited with a transmission ratio distortion and causes reduced viability in utero.

SHB is an Src homology 2 domain-containing adapter protein that has been found to be involved in numerous cellular responses. We have generated an Shb knockout mouse. No Shb-/- pups or embryos were obtained on the C57Bl6 background, indicating an early defect as a consequence of Shb- gene inactivation on this genetic background. Breeding heterozygotes for Shb gene inactivation (Shb+/-) on a mixed genetic background (FVB/C57Bl6/129Sv) reveals a distorted transmission ratio of the null allele with reduced numbers of Shb+/+ and Shb-/- animals, but increased number of Shb+/- animals. The Shb- allele is associated with various forms of malformations, explaining the relative reduction in the number of Shb-/- offspring. Shb-/- animals that were born were viable, fertile, and showed no obvious defects. However, Shb+/- female mice ovulated preferentially Shb- oocytes explaining the reduced frequency of Shb+/+ mice. Our study suggests a role of SHB during reproduction and development.     

emm typing and validation of provisional M types for group A streptococci

This report discusses the following issues related to typing of group A streptococci (GAS): The development and use of the 5' emm variable region sequencing (emm typing) in relation to the existing serologic typing system; the designation of emm types in relation to M types; a system for validation of new emm types; criteria for validation of provisional M types to new M-types; a list of reference type cultures for each of the M-type or emm-type strains of GAS; the results of the first culture exchange program for a quality control testing system among the national and World Health Organization collaborating centers for streptococci; and dissemination of new approaches to typing of GAS to the international streptococcal community.     

Role of tyrosine kinase signaling for beta-cell replication and survival

Diabetes mellitus is commonly considered as a disease of a scant beta-cell mass that fails to respond adequately to the functional demand. Tyrosine kinases may play a role for beta-cell replication, differentiation (neoformation) and survival. Transfection of beta-cells with DNA constructs coding for tyrosine kinase receptors yields a ligand-dependent increase of DNA synthesis in beta-cells. A PCR-based technique was adopted to assess the repertoire of tyrosine kinases expressed in fetal islet-like structures, adult islets or RINm5F cells. Several tyrosine kinase receptors, such as the VEGFR-2 (vascular endothelial growth factor receptor 2) and c-Kit, were found to be present in pancreatic duct cells. Because ducts are thought to harbor beta-cell precursor cells, these receptors may play a role for the neoformation of beta-cells. The Src-like tyrosine kinase mouse Gtk (previously named Bsk/Iyk) is expressed in islet cells, and was found to inhibit cell proliferation. Furthermore, it conferred decreased viability in response to cytokine exposure. Shb is a Src homology 2 domain adaptor protein which participates in tyrosine kinase signaling. Transgenic mice overexpressing Shb in beta-cells exhibit an increase in the neonatal beta-cell mass, an improved glucose homeostasis, but also decreased survival in response to cytokines and streptozotocin. It is concluded that tyrosine kinase signaling may generate multiple responses in beta-cells, involving proliferation, survival and differentiation.