Recent improvements in the optics and electronics of flow cytometry systems, as well as in staining techniques, permit the assay of such minute cellular constituents as the DNA and protein contents of micro-organisms. To assess the usefulness of this technique, DNA and protein content distributions were determined in Escherichia coli, Lactobacillus brevis, Lactobacillus casei, Chlorella kessleri 8k, Saccharomyces cerevisiae, Candida utilis, Schizosaccharomyces pombe and Euglena gracilis. Investigations of the DNA content distributions of polyploid strains of Saccharornyces cerevisiae indicated that the method can be used to determine ploidy. The rapidity of flow cytometry measurements allows accurate determinations in large populations.

Chemical screening of different Streptomycetes strains resulted in the detection, isolation, and structure elucidation of a number of novel carba-sugars. The constitution of these secondary metabolites, named gabosines A to K (1 to ll), was deduced from spectroscopic data as well as chemical transformation reactions. The gabosines exhibit a basic C7 skeleton and can be characterized as hydroxylated branched cyclohexanone derivatives, which show structural similarities to carbohydrates deriving from secondary metabolism. The configuration fo the gabosines (absolute stereochemistry for 1, 4, 5, and 6; relative configuration for the remaining metabolites) was determined by derivatization with chiral acids (Helmchen's method), NMR spectral analysis, as well as by a comparison of optical rotation values with those of the already known gabosines B (2) and C (3). The new term "ketocarbasugars" is used to characterize a typical ketone containing subgroup of carba-sugars originating from microbial sources. The well available natural gabosines can be used as suitable chiral building blocks.

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The structures of the novel 10-membered lactones, named decarestrictines A^A2(1/2), B (3), Ci/C2 (5/6) and D (7), are presented. The structures of these secondary metabolites, isolated from different Penicillium species, were established by spectroscopic analysis and confirmed by X-ray analysis of 7 and a derivative of 3 leading to the stereochemical information. The decarestrictines vary in the oxygenation pattern between C-3 and C-7 and show structural similarities to known lactones from other fungi.

The practical use of flow cytometry is shown in several microbial assays. Recent technical improvements in the optics and electronics of flow cytometric systems as well as in staining techniques permit the measurements of minute cellular components such as the cellular DNA and the protein content of bacteria, algae, moulds and yeasts. Single cell ingredients can be measured by this assay according to their specific stainability. The cell DNA was stained by propidium iodide while the cell protein was fluorochromed by fluorescein-iso-thiocyanate. The DNA synthesis of Saccharomyces cerevisiae and Saccharomyces pastorianus runs discontinuously while the protein content increases continuously during the vegetative growth. The different stages of DNA synthesis of yeast cells can be divided into two 'gap' phases, a synthesis and a mitosis period, corresponding to Howard and Pelc's model of DNA synthesis. Living and dead cells can be counted differentially after staining with Erythrosine B. The red fluorescence of the chlorophyll in algae can readily be used to determine the chlorophyll content of these cells.

Optical tweezers, based on a compact diode pumped Nd:YAG laser providing 350 mW at 1,064 nm coupled into a Zeiss IM 35 microscope, were used to sort CD4+ T cells into a capillary for further mechanical handling and to establish contact between single human natural killer (NK) cells and human erythroleukemia cells (K562) as targets. After contact and a lag phase of a few tens of seconds, the target cell starts to change its morphology and membrane blebbing occurs. The kinetics of the attack of the NK cell on K562 cells is not straightforward but governed by temporal oscillations in the shape of the target cell (zeosis).In a second application, the optical tweezers are combined with a UV laser microbeam based on a pulsed UV laser and with flow cytometry and sorting. With the pulsed laser, segments of sorted chromosome 1 of the Chinese hamster karyotype (CHV 79) can be easily microdissected and subsequently collected using the optical tweezers. This allows preparation of a few hundred chromosome segments per day without mechanical contact and in an absolutely sterile way and thus may provide an interesting basic technique in any type of genome sequencing project.

The glycolytic pathway inhibitor 2‐deoxyglucose (2‐DG) is capable of suppressing the transcription of the human pathogenic papillomavirus type 18 (HPV 18) in cervical carcinoma cells and derived non‐tumorigenic somatic cell hybrids at the level of transcription initiation. HPV down‐regulation is selective, since other reference genes are not affected or even up‐regulated under the same experimental conditions. Moreover, 2‐DG appears to restore the normal half‐life of the tumor suppressor gene product p53, because the protein is strongly up‐regulated after HPV18 E6/E7 suppression. The observed 2‐DG‐effect is not cytotoxic and is reversible after refeeding with fresh medium. HPV18 suppression by 2‐DG can be completely abrogated by simultaneous treatment with the intracellular Ca2+ antagonist TMB‐8, indicating that Ca2+, a known intracellular “second messenger”, is involved in this process. Elevated c‐myc and p53 expression appears to be responsible for the time‐dependent accumulation of apoptotic cells after prolonged 2‐DG treatment. The finding that 2‐DG acts selectively against the expression of a human pathogenic papillomavirus strongly suggests that an appropriate level of glycolysis is not only a pecularity of growing tumors, but even may be an essential prerequisite for the maintenance of virus‐specific E6/E7 gene expression. Our results may have substantial implications for the potential therapeutic application of 2‐DG or other glucose derivatives in the treatment of precancerous and malignant HPV‐associated lesions. Int. J. Cancer 76:000–000, 1998.© 1998 Wiley‐Liss, Inc.