Flow cytometric detection of herpes simplex virus type 2 infected and transformed cells by immunoenzymatic and by indirect immunofluorescence staining

The glucose oxidase antiglucose oxidase (GAG) immunoenzymatic staining procedure has been used to detect herpes simplex virus (HSV) antigens microscopically. In this study, the GAG procedure was adapted to cells in suspension, and its potential usefulness in flow cytometry was examined. HSV-2 infected monkey kidney and HSV-2 transformed mouse cells were stained using antisera to HSV-2 or to an HSV-2 specific protein with a molecular weight of 38 Kd, respectively, with the GAG procedure. Flow cytometric analysis of the GAG stained cells was then performed by the measurement of scattered light intensity in the angular intervals 1 degree-2 degrees, 2.5 degrees-19 degrees, and 3 degrees-6 degrees. The greatest scattered light intensity decrement caused by staining occurred in the 3 degrees-6 degrees angular interval, as predicted by previous work. In infected cells, which stain intensely by immunofluorescence, the difference between positively and negatively stained cells was adequate for detecting infected cells using the GAG method; however, this was not the case for the lightly staining transformed cells. The indirect immunofluorescence method of analysis of the same populations was superior to the scattered light method of analysis of the GAG stained infected and transformed cells.     

Recipes for reconstituting skin

Reconstituted Living Skin Equivalent (LSE) is made up of a dermal equivalent (DE) on which keratinocytes are plated where they give rise to a multilayered differentiated epidermis. The dermal equivalent develops through interactions between fibroblasts and collagen fibrils that begin to form after the cell-matrix precursor is cast. The gel that forms as a result of collagen polymerization and fluid trapping is contracted uniformly in all dimensions. By securing it at ends and edges in the mold in which it is cast, the final dimensions, strength and morphology of the forming tissue are altered. The same phenomena are seen in casting tubular tissues for the fabrication of small caliber blood vessel equivalents. The cells of the dermal equivalent are biosynthetically active and enrich the matrix to different degrees with secretory products, depending on how the cells are stimulated and on the presence or absence of an epidermis. Collagen biosynthesis by dermal cells in the DE is sensitive to growth factors, ascorbate concentrations and amino acid pools. Both ascorbate and TGF beta 1 increase total collagen biosynthesis at least two-fold by one week after tissue formation. With TGF beta 1 present, the capacity of cells in the DE to synthesize collagen increases with time, over a two-week period. If ascorbate (200 micrograms/ml) is added just after the tissue is cast and daily thereafter, contraction lattice is blocked, and collagen biosynthesis is enhanced relative to contracted controls that had received 200 micrograms/ml ascorbate once. The increase was nearly an order of magnitude over that of controls and was coordinate with a comparable increase in hyaluronate and sulfated glycosaminoglycan (GAG) production as shown by TCA-precipitable glucosamine in the intercellular matrix of the DE. Both the LSE and the Living Dermal Equivalent (LDE) exhibit complex responses to UV radiation and to various chemicals that are greatly different from responses given by monolayered cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of AAC(3) and AAD(2″) genes fromAcinetobacter: differential expression in the host strain

The mechanism of resistance to gentamicin and tobramycin in a clinical isolate ofAcinetobacter baumannii, in which aminoglycoside-modifying enzymes were not detected, was investigated. For increase of the resistance gene product, DNA prepared from theA. baumannii isolate was cloned into pUC18 and introduced intoEscherichia coli by transformation. Gentamicin-resistant transformants were screened for aminoglycoside-modifying enzymes. This approach identified two genes encoding AAC(3) and AAD(2) activity, respectively. To determine whether both genes are expressed in the hostAcinetobacter strain, we extracted total cellular RNA from this strain, and Northern blots were hybridized with the cloned AAC(3) and AAD(2) structural genes. mRNA transcribed from the AAC(3) gene alone was detected. This shows that cloning a functional resistance gene is not sufficient in itself to investigate mechanisms of resistance in bacterial strains without detectable aminoglycoside-modifying activity. Furthermore, this study suggests a potential limitation of antibiotic resistance gene probes for studying mechanisms of resistance.     

Oestradiol is effective in stimulating 3H-thymidine incorporation but not on proliferation of breast cancer cultured cells

The growth of numerous human oestrogen target cell lines is said to have been stimulated by oestradiol. We studied the action of this hormone on the growth of two human cancer cell lines originating from endometrium (GUS), and from breast (FAM). Oestradiol was inactive on endometrial cell multiplication as well as on their tritiated thymidine uptake, but in FAM breast cancer cells, we noticed a discrepancy between tritiated thymidine uptake and actual cell proliferation: there was a 40% increase in DNA precursor uptake, but no change in either the number of cells or in their DNA content, both of which were verified by two different methods. Therefore, an actual increased nuclear (autoradiographic) uptake of thymidine did take place in oestrogenized cells, associated with an increase of incorporation into DNA (a rise of radioactivity in the acid-insoluble materials), but finally there was no greater total DNA increase in the whole treated population than in control cells. Then we examined the metabolism of tritiated thymidine in oestradiol-treated FAM cells. We extracted the radioactive thymine nucleotides and characterized them chromatographically: the oestradiol caused an increase in the labelling of deoxythymine monophosphate (TMP). How these results are consistent with both unmodified cell count and whole DNA content is discussed.     

Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.

We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of Bacillus subtilis for use in the positive selection and isolation of insertion sequence (IS) elements in gram-negative bacteria. Cells containing pUCD800 do not grow on medium containing 5% sucrose unless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NT1RE by selection for growth on sucrose medium. This putative IS element appears to be unique to Agrobacterium strains.     

Behavioral and biochemical assessment of time-related changes in globus pallidus and striatal dopamine induced by intranigrally administered neurotensin

Microinjection of neurotensin (NT; 2 and 5 μg) into the substantia nigra zona compacta caused an increase in dopamine (DA) and DA metabolites in the rodent globus pallidus and striatum which persisted for at least 20 hours after peptide administration. Similar NT treatments given unilaterally into the nigra caused circling away from the injected side in amphetamine-pretreated rats, but were without effect when microinjected into saline-pretreated animals. Circling also occurred when the animals were given amphetamine 20 hours after intranigral NT administration. Contralateral rotation was observed with unilateral intranigral injections of gamma-hydroxybutyric acid (GHB; 400 μg) or with lower intranigral GHB doses (250 μg) in amphetamine-pretreated animals. The effects of GHB and NT differed in the manner in which the animals rotated as well as in the profile of DA and DA metabolite changes induced by these drugs. These studies indicated that: (1) dopaminergic functions of the globus pallidus are influenced, like the striatum, by manipulations of the substantia nigra; (2) NT and GHB likely act via different mechanisms to effect nigral dopamine-containing cells; and (3) NT was capable of inducing changes in dopamine neurons which had long term consequences.     

Induction of acetoacetate decarboxylase in Clostridium acetobutylicum

Abstract Factors that may initiate the biosynthesis of acetoacetate decarboxylase were investigated in resting cells of Clostridium acetobutylicum . Linear acids from C₁ to C₄ were inducers, whereas branched acids and linear acids from C₅ to C₇ were not inducers of acetoacetate decarboxylase biosynthesis. Induction of acetoacetate decarboxylase was maximal at pH 4.8 in the presence of acid concentrations comparable with those found during fermentation. In growth conditions repression of acetoacetate decarboxylase biosynthesis was found. This fact explains that acetone production by Clostridium acetobutylicum occurs when growth slows down.     

Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli.

A clone bearing the structural gene sacB, coding for the exoenzyme levansucrase, was isolated from a library of Bacillus subtilis DNA that was cloned in phage lambda charon 4A on the basis of the transforming activity of the chimeric DNA. This lambda clone also was found to contain the sacR and smo loci. Subcloning the sacB-sacR region in plasmid pBR325 resulted in a clone which directed levansucrase synthesis in Escherichia coli. The nucleotide sequence coding for the secreted protein was localized on the physical map of the cloned DNA.     

Open-Face, Epoxy Embedding of Single Cells for Ultrathin Sections

Intact stamens of Tradescantia were fixed, dehydrated, and infiltrated with an epoxy resin. Each stamen was then put into a drop of resin on a microscope slide, which was transferred to the stage of a dissecting microscope so that individual hairs could be detached from the filament with fine tungsten needles. The detached hairs were transferred to drops of resin ca. 2 mm in diameter (6 or 7 in each of two rows) lying on a slide heavily coated with evaporated carbon. Polymerization was carried out in an oven until the resin attained a degree of viscosity that permitted orientation of the isolated hairs (by using a compound microscope) without their subsequent dislocation. When the small drops of resin had hardened after further polymerization, the positions of the hairs were marked by circumscribing the cells with India ink. The block was pried from the slide after rapid cooling with solid CO₂, and was then trimmed and sectioned. Cells suspended in culture medium were embedded in much the same way; they were centrifuged to obtain a pellet, which was fixed, dehydrated, and infiltrated. A small fragment of the pellet with a little resin was placed on a microscope slide, where the cells were dissociated under a dissecting microscope at ca. 100 × magnification. Individual cells were then picked up with tungsten needles and transferred to droplets of resin on a carbon-coated slide. The subsequent steps were similar to those described for the staminate hairs. Pieces of tissue in the 50-500 μ range were also handled by the foregoing technique. However, after infiltration they were put into large drops of resin on a slide coated with silicone mold-release rather than on a surface coated with carbon.