Morphological and biochemical properties ofStreptomyces hygroscopicus grown in chemostat

The basic regulation of macromoleoular synthesis in mycelium-forming bacteria is in principle the same as in unicellular bacteria, but modified by morphological peculiarities. The higher the specific growth rate the moreStreptomyces resembles unicellular bacteria.     

Effect of serine hydroxamate and methyl alpha-D-glucopyranoside treatment on nucleoside polyphosphate pools, RNA and protein accumulation in Streptomyces hygroscopicus

The accumulation of RNA and protein and the kinetics of nucleoside triphosphate and guanosine polyphosphate pools during amino acid starvation and carbon source downshift were investigated in Streptomyces hygroscopicus. RNA accumulation was controlled stringently during both amino acid starvation and carbon source downshift. The pool size of ppGpp increased dramatically under these conditions. However, the intracellular concentrations of nucleoside triphosphates were low and the concentration of guanosine polyphosphates was much lower than in Escherichia coli. The possible significance of this phenomenon in the regulation is discussed.     

Immunocytochemical double staining of cytokeratin and prostate specific antigen in individual prostatic tumour cells

Early dissemination of malignant cells is the main cause for metastatic relapse in patients with solid tumours. By use of monoclonal antibodies (mAbs) specific for cytokeratins, disseminated individual epithelial tumour cells can now be identified in mesenchymal organs such as bone marrow. Further to characterize such cells in patients with prostate cancer, an immunocytochemical procedure was developed for simultaneous labelling of cytokeratin component no. 18 (CK18) and prostate specific antigen (PSA). In a first step, cells were incubated with mAb ER-PR8 against PSA and secondary gold-conjugated goat anti-mouse antibodies. In a second step, biotinylated mAb CK2 to CK18 was applied as primary antibody and subsequently incubated with complexes of streptavidin-conjugated alkaline phosphatase, which were developed with the Newfuchsin substrate. The binding of gold-labelled antibodies was visualized by silver enhancement. The sensitivity and specificity of the technique was demonstrated on cryostat sections of hyperplastic prostatic tissue, and cytological preparations of LNCaP prostatic tumour cells. Double staining was restricted to cells derived from the secretory epithelium of the prostate. Cross-reactivity between both detection systems was excluded by several controls, including the use of unrelated antibodies of the same isotype and the staining of CK18⁺/PSA^– HT29 colon carcinoma cells. CK18⁺ cells co-expressing PSA were found in bone marrow aspirates from 5 out of 13 patients with carcinomas of the prostate, a finding that is consistent with the relative fraction of double-positive LNCaP cells. The specificity of CK18 for epithelial tumour cells in bone marrow was supported by negative staining of 12 control aspirates from patients with benign prostatic hypertrophy. In conclusion, the approach presented appears to be a reliable method to phenotype individual prostatic carcinoma cells.     

Expression of soluble, recombinant alphavbeta3 integrin fragments in Escherichia coli

No prokaryotic expression of integrin alphavbeta3 has been reported so far. We report here the expression of C-terminally truncated alphavbeta3 receptors in E. coli considering the known features required for dimerization and ligand binding. The expressed protein was insoluble despite of the addition of 'solubilizers' to the culture medium. Osmotic stress conditions combined with added exogenous solutes resulted in a small part of soluble receptor. The alphavbeta3 variants were purified from inclusion bodies or from soluble cytoplasmic maltose binding protein fusions. Heterodimerization of the subunits was proved by immunoprecipitation assays. Receptor-ligand binding was found to depend on the concentration. A competition assay with RGD peptides referred to unspecific receptor-ligand interaction. The latter fact was consistent with the finding that soluble receptors did not bind on RGD peptide-coupled sepharose (GRGDSPK sepharose).     

Establishment of a system suitable for analysis of balanced and unbalanced growth of Streptomyces hygroscopicus

A small-scale system was developed in which balanced growth of Streptomyces hygroscopicus occurred. Although the balanced growth, verified by corresponding increase of ATP, DNA, RNA, protein and mycelial length, was restricted to a relatively short period of the life cycle, it lasted for at least two doublings. The conditions for balanced growth could be altered by various treatments to induce imbalance. This system could be applied to study regulation in the mycelium under well-defined conditions.     

Development of an incubation system for an inverted microscopy for long-term observation of cell cultures using chamber slides]

Trifunctional bispecific antibodies open up new immunological possibilities in tumour treatment. Prior to clinical application, comprehensive investigations using animal models and in vitro examinations need to be done. To investigate long-term interactions between various immunologically active blood cells and individual tumour cells in the presence of antibodies, we developed an incubation system for experimental cell cultures on an inverted microscope. The system consists of a perspex box with a central moisture chamber with integrated water reservoir, external air circulation heating, and a CO2 supply. The sterile cell cultures are located in the wells of a slide positioned within a depression in the water reservoir. The newly developed incubation system enables continuous observation over the long term of experiments under optimal cell cultures conditions in combination with modern video techniques.     

Comparable Long-Term Efficacy of Lopinavir/Ritonavir and Similar Drug-Resistance Profiles in Different HIV-1 Subtypes

Background

Analysis of potentially different impact of Lopinavir/Ritonavir (LPV/r) on non-B subtypes is confounded by dissimilarities in the conditions existing in different countries. We retrospectively compared its impact on populations infected with subtypes B and C in Israel, where patients infected with different subtypes receive the same treatment.

Methods

Clinical and demographic data were reported by physicians. Resistance was tested after treatment failure. Statistical analyses were conducted using SPSS.

Results

607 LPV/r treated patients (365 male) were included. 139 had HIV subtype B, 391 C, and 77 other subtypes. At study end 429 (71%) were receiving LPV/r. No significant differences in PI treatment history and in median viral-load (VL) at treatment initiation and termination existed between subtypes. MSM discontinued LPV/r more often than others even when the virologic outcome was good (p = 0.001). VL was below detection level in 81% of patients for whom LPV/r was first PI and in 67% when it was second (P = 0.001). Median VL decrease from baseline was 1.9±0.1 logs and was not significantly associated with subtype. Median CD4 increase was: 162 and 92cells/µl, respectively, for patients receiving LPV/r as first and second PI (P = 0.001), and 175 and 98, respectively, for subtypes B and C (P<0.001). Only 52 (22%) of 237 patients genotyped while under LPV/r were fully resistant to the drug; 12(5%) were partially resistant. In48%, population sequencing did not reveal resistance to any drug notwithstanding the virologic failure. No difference was found in the rates of resistance development between B and C (p = 0.16).

Conclusions

Treatment with LPV/r appeared efficient and tolerable in both subtypes, B and C, but CD4 recovery was significantly better in virologically suppressed subtype-B patients. In both subtypes, LPV/r was more beneficial when given as first PI. Mostly, reasons other than resistance development caused discontinuation of treatment.     

Dependence of macromolecular composition and morphology of Streptomyces hygroscopicus on specific growth rate.

The dependence of macromolecular composition and morphology of Streptomyces hygroscopicus on specific growth rate micron was investigated. The percentage of DNA on dry weight (%DNA) is constant, % protein is also nearly independent of micron whereas %RNA rises considerably with increasing micron, regarding mycelia grown in glucose-limited and ammonium-limited continuous cultures as well as in discontinuous cultures with various carbon sources. It is probable that the overall synthesis of DNA, RNA and protein is regulated in the mycelium-forming bacterium S. hygroscopicus by the same mechanisms found in unicellular bacteria like Escherichia coli because of the qualitatively similar dependence of %DNA, %RNA and %protein on micron. But differences exist in quantitative regard whereby %DNA, %RNA and %protein of S. hygroscopicus are much smaller at low micron and, with increasing micron, approach those of unicellular bacteria. The hypothesis about the increase of the hyphal regions showing high synthesis activity in S. hygroscopicus mycelia grown in glucose-limited continuous cultures with increasing micron -- derived from comparison of macromolecular composition of S. hygroscopicus and unicellular bacteria -- was confirmed autoradiographically with respect to protein synthesis. The increase of the part of mycelial regions showing high cytoplasmic activity results in an increase of mean hyphal diameter, of mean relative apical growth rate alpha and/or mean relative branching rate beta. Beta depends sigmoidally and alpha inverses sigmoidally on micron. Therefore, the morphology of the mycelium determined by alpha and beta also depends on micron. The hyphal growth unit L/N, the distance from apex to first branch Lp and the mean distance between neighbouring branches Ln decline with increasing micron and reach a minimum at micron = 0.32 (1/h). A further rise of micron is accompanied with an increase of L/N, Lp and Ln. This means that mycelia growing slowly or very quickly have a loose form whereas quickly growing mycelia are characterized by a more compact form. The complicated dependence of alpha, beta, L/N, Lp and Ln on micron indicates that the morphology is regulated by different mechanisms depending on the specific growth rate.     

Maximizing the expression of recombinant Kringle 1 (Streptokinase) synthesized in Escherichia coli. Influence of culture and induction conditions

Summary We examined the influence of culture conditions on the production of the recombinant fused protein Kringle 1 (Streptokinase) (K1(SK)) expressed in E. coli under the control of the Ipp-lac promoter. The growth and the protein production were severely inhibited at glucose concentrations higher than 5 g/L. The highest yield of K1(SK) from the soluble extract was obtained using synthetic medium. Concentrations of inducer between 0.1 and 1 mM of IPTG did not significantly affect the production level of K1(SK) or the final cell density. Under optimal conditions the K1(SK) protein was expressed in an intracellular soluble form at about 3–4% of the total cellular protein.     

Influence of stringent and relaxed response on excretion of recombinant proteins and fatty acid composition in Escherichia coli

In contrast to stringent (relA⁺) cells of Escherichia coli, relaxed (relA) cells excreted recombinant proteins (-lactamase, interferon 1) into the culture medium during amino acid limitation. Comparative analyses of overall fatty acid composition in relA⁺ cells and relA cells were performed and revealed that, in wild-type cells, drastic alterations occurred during the stringent response. The portion of saturated fatty acids (C16:0) and the fractions of cyclopropane fatty acids (C17_cyc and C19_cyc) increased whereas the portions of unsaturated fatty acids (C16:1 and C18:1) decreased. In cells of the relaxed mutant, no significant changes in the overall composition of the fatty acids were observed after the onset amino acid limitation. These results indicate that a change in fatty acid composition of membrane lipids after starvation of cells may be responsible for the prevention of loss of cellular proteins into the culture medium in stringent controlled cells of Escherichia coli.