Starvation-induced cross protection against osmotic challenge in Escherichia coli.

Stationary-phase Escherichia coli cultures showed enhanced osmotic resistance as compared with cultures in mid-logarithmic growth or preadapted to osmotic stress. The osmotolerance that developed during starvation or osmotic adaptation required de novo protein synthesis. Of the 22 polypeptides induced during osmotic shock, five were also starvation proteins.     

Enzymes of Carbohydrate Metabolism in Thiobacillus species

A study was made of enzymes of carbohydrate metabolism in representative thiobacilli grown with and without glucose. The data show that Thiobacillus perometabolis possesses an inducible Entner-Doudoroff pathway and is thus similar to T. intermedius and T. ferrooxidans. T. novellus lacks this pathway. Instead, a non-cyclic pentose phosphate pathway along with the Krebs cycle is apparently the major route of glucose dissimilation in this organism. Glucose does not support or stimulate the growth of strains of T. neapolitanus, T. thioparus, and T. thiooxidans examined, nor does its presence in the growth medium greatly influence their enzymatic constitution. These obligately chemolithotrophic thiobacilli do not possess the Entner-Doudoroff pathway. Their nicotinamide adenine dinucleotide (NAD)-linked isocitrate dehydrogenase activity predominates over their nicotinamide adenine dinucleotide phosphate (NADP)-linked activity; the converse is true for the other thiobacilli. The data suggest that NAD-linked isocitrate dehydrogenase activity in thiobacilli is involved in biosynthetic reactions.     

Murine antiestrogen-binding protein: characterization, solubilization and modulation by lipids

The properties of the antiestrogen-binding protein have been examined in mouse tissues, a species in which nonsteroidal antiestrogens are virtually pure agonists. As in other species studied, this protein was distributed in all tissues - highest levels being in the liver. Subcellular fractionation of mouse liver showed that 82% of the antiestrogen-binding protein was associated with the rough endoplasmic reticulum where it was confined to the membranous component. The antiestrogen-binding protein was also present in smooth endoplasmic reticulum, nuclei and cytosol. Its concentration in intact nuclei was at least 10-times higher than levels previously reported in intact rat liver nuclei. Binding of [3H]tamoxifen to the murine antiestrogen-binding protein was of high affinity (Kd = 1 nM) and was inhibited by unsaturated fatty acids and 7-ketocholesterol. In general, cis-isomers of unsaturated fatty acids were more effective binding inhibitors than trans-isomers. The antiestrogen-binding protein solubilized from rough endoplasmic reticulum membranes by the zwitterionic detergent CHAPS, had a molecular mass of approx. 700 kDa and a sedimentation coefficient of about 19 S. [3H]Tamoxifen binding capacity of the solubilized protein was abolished by trypsin and nonspecific proteinases but not by clostripain or Staphylococcus aureus V8 proteinase, suggesting that lysine residue(s) may be involved in [3H]tamoxifen binding.     

Susceptibility of chemostat-grown Yersinia enterocolitica and Klebsiella pneumoniae to chlorine dioxide.

The resistance of bacteria to antimicrobial agents could be influenced by growth environment. The susceptibility of two enteric bacteria, Yersinia enterocolitica and Klebsiella pneumoniae, to chlorine dioxide was investigated. These organisms were grown in a defined medium in a chemostat and the influence of growth rate, temperature, and cell density on the susceptibility was studied. All inactivation experiments were conducted with a dose of 0.25 mg of chlorine dioxide per liter in phosphate-buffered saline at pH 7.0 and 23 degrees C. The results indicated that populations grown under conditions that more closely approximate natural aquatic environments, e.g., low temperatures and growth at submaximal rates caused by nutrient limitation, were most resistant. The conclusion from this study is that antecedent growth conditions have a profound effect on the susceptibility of bacteria to disinfectants, and it is more appropriate to use the chemostat-grown bacteria as test organisms to evaluate the efficacy of a certain disinfectant.     

Physiological basis of the selective advantage of a Spirillum sp. in a carbon-limited environment

A Spirillum sp. and a Pseudomonas sp. possessing crossing substrate saturation curves for L-lactate were isolated from fresh water by chemostat enrichment. Their Ks and mumax values for L-lactate were: Spirillum sp., 23 micrometer and 0.35 h-1, respectively; Pseudomonas sp., 91 micrometer and 0.64 h-1, respectively. Under L-lactate limitation, pseudomonas sp. outgrew Spirillum s. at dilution rates (D) above 0.29 h-1, but the converse occurred at lower D values. The advantage of Spirillum sp. increased with decreasing D until, at D = 0.05 h-1 (i.e. L-lactate concentration of approximately 1 micrometer), Pseudomonas sp. was eliminated from the culture essentially as a non-growing population. In Spirillum sp. the Km for L-lactate transport (5.8 micrometer) was threefold lower than in Pseudomonas sp. (20 micrometer); Spirillum sp. also possessed a higher Vmax for the transport of this substrate. The surface to volume ratio was higher in Spirillum sp. and increased more markedly than in Pseudomonas sp. in response to decreasing D. Thus, a more efficient scavenging capacity contributes to the advantage of Spirillum sp. at low concentrations of the carbon source. Although most of the enzymes of L-lactate catabolism were more active in Pseudomonas sp., NADH oxidase activity was about twice as high in Spirillum sp.; and, unlike Pseudomonas sp., the cytochrome c content of this bacterium increased markedly with decreasing D. A more active and/or more efficient respiratory chain may therefore also play a role in the advantage of Spirillum sp. The other factors which appear to be involved include a lower energy of maintenance of Spirillum sp. [0.016 g L-lactate (g cell dry wt)-1 h-1 compared with 0.066 in Pseudomonas sp.] and a lower minimal growth rate.     

The CDK inhibitor p27 enhances neural differentiation in pluripotent NTERA2 human EC cells, but does not permit differentiation of 2102Ep nullipotent human EC cells

Embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, are the malignant counterparts of pluripotent embryonic stem (ES) cells, but commonly exhibit a reduced ability to differentiate, presumably because of continual selection for genetic changes that alter the balance between self-renewal, differentiation and apoptosis in favour of self-renewal. To explore the nature of the genetic changes that promote nullipotency, we have compared two human EC cell lines, a 'nullipotent' line, 2102Ep, and a 'pluripotent' line, NTERA2. A hybrid derived by fusion of these cells differentiates in response to retinoic acid but, unlike the parental NTERA2 line, does not form terminally differentiated neurons. This implies that the nullipotent EC cell line, 2102Ep, differs in expression of at least two functions in comparison with the NTERA2 pluripotent line, one affecting commitment to differentiation, and one affecting terminal neural differentiation. We have now investigated the possible role of the CDK inhibitor, p27kip1 (p27) in commitment and terminal differentiation. In NTERA2, but not in 2102Ep cells, retinoic acid induces up-regulation of p27 expression, suggesting that 2102Ep cells lack this capacity. However, constitutive expression of a p27 transgene does not overcome the block to differentiation in the 2102Ep parental cells; commitment to differentiation must be blocked elsewhere. On the other hand, constitutive over-expression of p27 from a transgene enhances the neural differentiation of NTERA2 cells. Our results suggest that p27 plays a role in terminal neuronal differentiation of human EC cells, but not in their initial commitment to differentiation, and that other factors, possibly Cyclin D2, specifically limit its ability to promote neural differentiation.     

Supportive properties of basement membrane layer of human amniotic membrane enable development of tissue engineering applications

Human amniotic membrane (HAM) has been widely used as a natural scaffold in tissue engineering due to many of its unique biological properties such as providing growth factors, cytokines and tissue inhibitors of metalloproteinases. This study aimed at finding the most suitable and supportive layer of HAM as a delivery system for autologous or allogeneic cell transplantation. Three different layers of HAM were examined including basement membrane, epithelial and stromal layers. In order to prepare the basement membrane, de-epithelialization was performed using 0.5 M NaOH and its efficiency was investigated by histological stainings, DNA quantification, biomechanical testing and electron microscopy. Adipose-derived stromal cells (ASCs) and a human immortalized keratinocyte cell line (HaCaT) were seeded on the three different layers of HAM and cultured for 3 weeks. The potential of the three different layers of HAM to support the attachment and viability of cells were then monitored by histology, electron microscopy and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, mechanical strengths of the basement membrane were assessed before and after cell culture. The results indicated that the integrity of extra cellular matrix (ECM) components was preserved after de-epithelialization and resulted in producing an intact basement amniotic membrane (BAM). Moreover, all three layers of HAM could support the attachment and proliferation of cells with no visible cytotoxic effects. However, the growth and viability of both cell types on the BAM were significantly higher than the other two layers. We conclude that growth stimulating effectors of BAM and its increased mechanical strength after culturing of ASCs, besides lack of immunogenicity make it an ideal model for delivering allogeneic cells and tissue engineering applications.     

Use of starvation promoters to limit growth and selectively enrich expression of trichloroethylene- and phenol-transforming activity in recombinant Escherichia coli [corrected]

The expression of much useful bacterial activity is facilitated by rapid growth. This coupling can create problems in bacterial fermentations and in situ bioremediation. In the latter process, for example, it necessitates addition of large amounts of nutrients to contaminated environments, such as aquifers. This approach, termed biostimulation, can be technically difficult. Moreover, the resulting in situ bacterial biomass production can have undesirable consequences. In an attempt to minimize coupling between expression of biodegradative activity and growth, we used Escherichia coli starvation promoters to control toluene monooxygenase synthesis. This enzyme complex can degrade the environmental contaminants trichloroethylene (TCE) and phenol. Totally starving cell suspensions of such strains degraded phenol and TCE. Furthermore, rapid conversions occurred in the postexponential batch or very slow growth (dilution) rate chemostat cultures, and the nutrient demand and biomass formation for transforming a given amount of TCE or phenol were reduced by 60 to 90%. Strong starvation promoters have recently been clones and characterized in environmentally relevant bacteria like Pseudomonas species; thus, starvation promoter-driven degradative systems can now be constructed in such bacteria and tested for in situ efficacy.     

Analysis of Novel Soluble Chromate and Uranyl Reductases and Generation of an Improved Enzyme by Directed Evolution

Most polluted sites contain mixed waste. This is especially true of the U.S. Department of Energy (DOE) waste sites which hold a complex mixture of heavy metals, radionuclides, and organic solvents. In such environments enzymes that can remediate multiple pollutants are advantageous. We report here evolution of an enzyme, ChrR6 (formerly referred to as Y6), which shows a markedly enhanced capacity for remediating two of the most serious and prevalent DOE contaminants, chromate and uranyl. ChrR6 is a soluble enzyme and reduces chromate and uranyl intracellularly. Thus, the reduced product is at least partially sequestered and nucleated, minimizing the chances of reoxidation. Only one amino acid change, ^Tyr128^Asn, was responsible for the observed improvement. We show here that ChrR6 makes Pseudomonas putida and Escherichia coli more efficient agents for bioremediation if the cellular permeability barrier to the metals is decreased.     

Blastema cells derived from New Zealand white rabbit’s pinna carry stemness properties as shown by differentiation into insulin producing,neural, and osteogenic lineages representing three embryonic germ layers

Stem cells (SCs) are known as undifferentiated cells with self-renewal and differentiation capacities. Regeneration is a phenomenon that occurs in a limited number of animals after injury, during which blastema tissue is formed. It has been hypothesized that upon injury, the dedifferentiation of surrounding tissues leads into the appearance of cells with SC characteristics. In present study, stem-like cells (SLCs) were obtained from regenerating tissue of New Zealand white rabbit’s pinna and their stemness properties were examined by their capacity to differentiate toward insulin producing cells (IPCs), as well as neural and osteogenic lineages. Differentiation was induced by culture of SLCs in defined medium, and cell fates were monitored by specific staining, RT-PCR and flow cytometry assays. Our results revealed that dithizone positive cells, which represent IPCs, and islet-like structures appeared 1 week after induction of SLCs, and this observation was confirmed by the elevated expression of Ins, Pax6 and Glut4 at mRNA level. Furthermore, SLCs were able to express neural markers as early as 1 week after retinoic acid treatment. Finally, SLCs were able to differentiate into osteogenic lineage, as confirmed by Alizarin Red S staining and RT-PCR studies. In conclusion, SLCs, which could successfully differentiate into cells derived from all three germ layers, can be considered as a valuable model to study developmental biology and regenerative medicine.