Pleiotropic consequences of mutations towards antibiotic-hypersensitivity in Serratia marcescens

Various mutants (oxa ^s ) were isolated from Serratia marcescens SM-6 by selecting for hypersensitivity towards oxacillin. All mutants found are highly pleiotropic and able to yield spontaneous revertants which behave like the wild-type. Mutant W 1421 mostly studied shows the following phenotypic properties not found in the wild-type: (1) The growth is hypersensitive to various antibiotics, detergents and dyes which differ remarkably in their chemical structure and antibacterial action-mechanism, (2) the cells can be easily solubilized by 0.05% Sodium-dodecylsulfate, (3) the cells allow the adsorption of the roughmutant specific Salmonella phage 6SR, (4) strong cellular binding of crystal violet, (5) agglutination of the cells in 0.3% auramin solution and (6) reduced formation of red pigment. Strain W 1421 is assumed to be a lipopolysaccharide-defective mutant. The outer membrane of mutant W 1421 analyzed by Sodiumdodecylsulfate-polyacrylamide gel electrophoresis possesses a single protein less than that of the wild-type. Mutant W 1421 is further characterized by its low exolipase activity; exoprotease and exonuclease activities are as in the wild-type. This specific exoenzyme deficiency can be overcome either by backmutation to oxacillin-resistance or by growing mutant W 1421 in a medium supplemented with certain non-metabolizable polysaccharides, e.g. glycogen or pectin B. Both polysaccharides increase the exolipase activity of the wild-type too.List of Abbreviations amp ampicillin - LPS lipopolysaccharide - MIC minimal inhibitory concentration - NB nutrient broth - oxa oxacillin - str streptomycin - TBY tryptone broth with yeast extract - SDS sodium-dodecylsulfate - OD optical density This paper is dedicated to Prof. Dr. R. W. Kaplan, University of Frankfurt/M., on the occasion of his 65th birthday     

Mechanisms of chromosome banding

Photo-oxidation of mitotic human chromosomes has been used in conjunction with anti-cytosine and anti-adenosine antibodies to produce R-banding. To elucidate the mechanism of this banding procedure we have examined the effect of photo-oxidation alone on chromosomes and nuclei. With short exposures to light in the presence of dilute methylene blue, C-band areas on chromosomes 1, 9, 16 and the terminal segment of the Y stain poorly. We call this phenomena reverse C-banding. After 18 h of exposure to light the chromosomes are swollen and show very little staining with quinacrine or Giemsa. Quantitative autoradiography shows that their DNA is almost completely extracted. Cytophotometric measurements also confirm that nuclear DNA is progressively extracted according to the length of exposure to light. When chromosomes are exposed to dilute methylene blue alone, without light, G-banded chromosomes result. We suggest the following explanation for these observations. In dilute methylene blue, C-band regions take up the greatest amount of dye and after short periods of photo-oxidation the DNA of these regions is preferentially destroyed resulting in reverse C-banding. Autoradiography in photo-oxidized chromosomes suggested that this preferential destruction of C-segments occurred in our experiments. With more prolonged exposure the DNA of the G-bands regions is preferentially destroyed and staining the remaining DNA with sensitive fluorescent labeled anti-C antibodies results in R-banding.     

Predicting soils and environmental impacts associated with switchgrass for bioenergy production: a DAYCENT modeling approach

Switchgrass (Panicum virgatum L.) production has the potential to improve soils and the environment. However, little is known about the long‐term future assessment of soil and environmental impacts associated with switchgrass production. In this study, soil organic carbon (SOC), soil nitrate (), water‐filled pore space (WFPS), carbon dioxide (CO₂) and nitrous oxide (N₂O) fluxes, and biomass yield from switchgrass field were predicted using DAYCENT models for 2016 through 2050. Measured data for model calibration and validation at this study site managed with nitrogen fertilization rates (N rates) (low, 0 kg N ha^?1; medium, 56 kg N ha^?1; and high, 112 kg N ha^?1) and landscape positions (shoulder and footslope) for switchgrass production were collected from the previously published studies. Modeling results showed that the N fertilization can enhance SOC and soil NO₃^?, but increase soil N₂O and CO₂ fluxes. In this study, medium N fertilization was the optimum rate for enhancing switchgrass yield and reducing negative impact on the environment. Footslope position can be beneficial for improving SOC, , and yield, but contribute higher greenhouse gas (GHG) emissions compared to those of the shoulder. An increase in temperature and decrease in precipitation (climate scenarios) may reduce soil , WFPS, and N₂O flux. Switchgrass production can improve and maintain SOC and , and reduce N₂O and CO₂ fluxes over the predicted years. These findings indicate that switchgrass could be a sustainable bioenergy crop on marginally yielding lands for improving soils without significant negative impacts on the environment in the long run.     

dSTORM microscopy evidences in HeLa cells clustered and scattered γH2AX nanofoci sensitive to ATM,DNA-PK,and ATR kinase inhibitors

Molecular and Cellular Biochemistry - Discovered in the late 1980s as an extracellular vesicle of endosomal origin secreted from reticulocytes, exosomes recently gained scientific attention due to...     

C-banding and non-homologous associations

A chromosome complement formed by 16 autosomes and an Xy_p sex chromosome system was found in Epilachna paenulata Germar (Coleoptera: Coccinellidae). All autosomes were metacentric except pair 1 which was submetacentric. The X and the Y chromosomes were also submetacentric but the Y was minute. The whole chromosome set carried large paracentric heterochromatic C-segments representing about 15% of the haploid complement length. Heterochromatic segments associated progressively during early meiotic stages forming a large single chromocenter. After C-banding, chromocenters revealed an inner networklike filamentous structure. Starlike chromosome configurations resulted from the attachment of bivalents to the chromocenters. These associations were followed until early diakinesis. Thin remnant filaments were also observed connecting metaphase I chromosomes. Evidence is presented that, in this species, the Xy_p bivalent resulted from an end-to-end association of the long arms of the sex chromosomes. The parachute Xy_p bivalent appeared to be composed of three distinct segments: two intensely heterochromatic C-banded corpuscles formed the canopy and a V-shaped euchromatic filament connecting them represented the parachutist component. The triple constitution of the sex bivalent was interpreted as follows: each heterochromatic corpuscle corresponded to the paracentric C-segment of the X and Y chromosomes; the euchromatic filament represented mainly the long arm of the X chromosome terminally associated with the long arm of the Y chromosome. The complete sequence of the formation of the Xy_p bivalent starting from nonassociated sex chromosomes in early meiotic stages, and progressing through pairing of heterochromatic segments, coiling of the euchromatic filament, and movement of the heterochromatic corpuscles to opposite poles is described. These findings suggest that in E. paenulata the Xy_p sex bivalent formation is different than in other coleopteran species and that constitutive heterochromatic segments play an important role not only in chromosome associations but also in the Xy_p formation.     

Localization of chromosome breakpoints induced by DNase I in Chinese hamster ovary (CHO) cells

DNase I was electroporated into S-phase CHO cells and induced chromosome breakpoints were localized in G-banded metaphases. More than 75% of breakpoints mapped to Giemsa-light bands, 18% to Giemsa-dark bands and about 7% to band junctions. Chromosome breakpoint clusters produced by DNase I colocalized with chromosome breakpoints induced by the restriction endonucleases AluI and BamHI in the G1- and S-phases of the cell cycle in CHO cells. Digestion of metaphase spreads with AluI, BamHI and DNase I produced G-bands, indicating that G-light bands are more sensitive to endonuclease action. The possible role of nuclease-sensitive sites in active chromatin as selective targets for the induction of chromosome breakpoints by these endonucleases is discussed. Received: 15 January 1997; in revised form: 21 July 1997 / Accepted: 23 July 1997     

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses¹. Various strategies such as Staput^2,3, centrifugal elutriation¹, and flow cytometry (FC)^4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium^6,7 - and lack cell clumps¹ . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones¹ . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types^1,8 . The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting⁴, and ulterior gene expression studies⁹ .     

Assessment of different systems for the production of aldonic acids and sorbitol by calcium alginate-immobilized Zymomonas mobilis cells

Bioprocess and Biosystems Engineering - Equimolar amounts of lactobionic acid and sorbitol may be obtained in a reaction catalyzed by the enzymes glucose–fructose oxidoreductase and...     

Chromosomal aberrations: formation,identification and distribution

Chromosomal aberrations (CA) are the microscopically visible part of a wide spectrum of DNA changes generated by different repair mechanisms of DNA double strand breaks (DSB). The method of fluorescence in situ hybridisation (FISH) has uncovered unexpected complexities of CA and this will lead to changes in our thinking about the origin of CA. The inter- and intrachromosomal distribution of breakpoints is generally not random. CA breakpoints occur preferentially in active chromatin. Deviations from expected interchromosomal distributions of breakpoints may result from the arrangement of chromosomes in the interphase nucleus and/or from different sensitivities of chromosomes with respect to the formation of CA. Telomeres and interstitial telomere repeat like sequences play an important role in the formation of CA. Subtelomeric regions are hot spots for the formation of symmetrical exchanges between homologous chromatids and cryptic aberrations in these regions are associated with human congenital abnormalities.