Visualizing gene expression in time and space in the filamentous bacterium Streptomyces coelicolor.

Streptomycetes are prokaryotic microorganisms that exhibit a complex, mycelial fungus-like cycle of morphological differentiation. Development involves at least two spatially separated types of cells: the branching hyphae of the substrate mycelium, which penetrate the stratum upon which the colony feeds, and the upwardly protruding hyphae of the aerial mycelium, which undergo metamorphosis into spores. The luciferase-encoding luxA and luxB operon of the luminescent marine bacterium Vibrio harveyi was used as a promoter probe to visualize gene expression in differentiating colonies of Streptomyces coelicolor. Promoters for developmental genes of several kinds gave distinctive temporal and spatial patterns of light emission.

RNA polymerase heterogeneity in Streptomyces coelicolor.

Two forms of RNA polymerase holoenzyme have been identified in the filamentous differentiating bacterium Streptomyces coelicolor. They contain different species of sigma factor and are distinguishable by their ability to recognize different promoter classes. These and other holoenzyme forms may in part determine the selective expression of different gene sets in this morphologically-complex bacterium.

Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a "mini" mycobacterium-Escherichia coli shuttle vector.

Functional domains of pAL5000 were determined by gene disruption and deletion analysis. Of the five plasmid open reading frames (ORFs), ORF1 to ORF5, and a putative origin of replication previously identified (J. Rauzier, J. Moniz-Pereira, and B. Gicquel-Sanzey, Gene 71:315-321), two of the ORFs (ORF3 and ORF4) were deemed dispensable for plasmid replication. A "mini" mycobacterium-Escherichia coli shuttle plasmid applicable for general recombinant DNA studies in mycobacteria was constructed by using the gene for Kanr (Tn903) as a selective marker. Heterologous expression of the gene for Kanr was confirmed by Western blotting (immunoblotting) analysis.

Comparative analysis of CD8 expressed on mature CD4+ CD8+ T cell clones cultured with IL-4 and that on CD8+ T cell clones: implication for functional significance of CD8 beta.

Interleukin (IL-4) can induce CD8 expression on mature CD4+ T cells. To study this phenomenon in more detail, we characterized CD8 expressed on IL-4-induced CD4+ CD8+ (double positive) T cell clones in comparison with that on CD8+ T cell clones. Using 2ST8-5H7 mAb that detects CD8 beta expression, we found that double positive T cell clones isolated with IL-4 express CD8 alpha but not beta, in contrast to CD8+ CTL cell clones, which express both chains of CD8. Northern blot analysis revealed that these double positive clones expressed CD8 alpha but not beta mRNA, indicating that CD8 alpha and beta are independently regulated at the pre-translational level. Immunoprecipitation experiments showed that CD8 expressed on a representative IL-4-induced double positive T cell clone consists mainly of homodimers of a single 34 kd protein of CD8 alpha. The amount of multimers detected from this clone was much less than that from a CD8+ CTL clone. These results suggest that persistent expression of CD8 beta is specific for the CD8+ lineage and may be involved in polymerization and stabilization of CD8 which enhances the efficiency of class I-restricted antigen recognition.

Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model.

Diet and lifestyle produce major effects on tumour incidence, prevalence, and natural history. Moderate dietary restriction has long been recognised as a natural therapy that improves health, promotes longevity, and reduces both the incidence and growth of many tumour types. Dietary restriction differs from fasting or starvation by reducing total food and caloric intake without causing nutritional deficiencies. No prior studies have evaluated the responsiveness of malignant brain cancer to dietary restriction. We found that a moderate dietary restriction of 30-40% significantly inhibited the intracerebral growth of the CT-2A syngeneic malignant mouse astrocytoma by almost 80%. The total dietary intake for the ad libitum control group (n=9) and the dietary restriction experimental group (n=10) was about 20 and 13 Kcal x day(-1), respectively. Overall health and vitality was better in the dietary restriction-fed mice than in the ad libitum-fed mice. Tumour microvessel density (Factor VIII immunostaining) was two-fold less in the dietary restriction mice than in the ad libitum mice, whereas the tumour apoptotic index (TUNEL assay) was three-fold greater in the dietary restriction mice than in the ad libitum mice. CT-2A tumour cell-induced vascularity was also less in the dietary restriction mice than in the ad libitum mice in the in vivo Matrigel plug assay. These findings indicate that dietary restriction inhibited CT-2A growth by reducing angiogenesis and by enhancing apoptosis. Dietary restriction may shift the tumour microenvironment from a proangiogenic to an antiangiogenic state through multiple effects on the tumour cells and the tumour-associated host cells. Our data suggest that moderate dietary restriction may be an effective antiangiogenic therapy for recurrent malignant brain cancers.

N -butyldeoxynojirimycin reduces growth and ganglioside content of experimental mouse brain tumours.

Abnormalities in glycosphingolipid (GSL) biosynthesis have been implicated in the oncogenesis and malignancy of brain tumours. GSLs comprise the gangliosides and the neutral GSLs and are major components of the cell surface glycocalyx. N -butyldeoxynojirimycin (N B-DNJ) is an imino sugar that inhibits the glucosyltransferase catalysing the first step in GSL biosynthesis. The influence of N B-DNJ was studied on the growth and ganglioside composition of two 20-methylcholanthrene-induced experimental mouse brain tumours, EPEN and CT-2A, which were grown in vitro and in vivo. N B-DNJ (200 microM) inhibited the proliferation of the EPEN and CT-2A cells by 50%, but did not reduce cell viability. The drug, administered in the diet (2400 mg kg(-1)) to adult syngeneic C57BL/6 mice, reduced the growth and ganglioside content of subcutaneous and intracerebral EPEN and CT-2A tumours by at least 50% compared to the untreated controls. N B-DNJ treatment also shifted the relative distribution of tumour gangliosides in accordance with the depletion of metabolic substrates. Side effects of N B-DNJ treatment were generally mild and included reductions in body and spleen weights and intestinal distension. We conclude that N B-DNJ may inhibit tumour growth through an effect on ganglioside biosynthesis and may be useful as a new chemotherapy for brain tumours.