Deinococcus as new chassis for industrial biotechnology: biology, physiology and tools.

Deinococcus spp are among the most radiation-resistant micro-organisms that have been discovered. They show remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation and oxidizing agents. Traditionally, Escherichia coli and Saccharomyces cerevisiae have been the two platforms of choice for engineering micro-organisms for biotechnological applications, because they are well understood and easy to work with. However, in recent years, researchers have begun using Deinococcus spp in biotechnologies and bioremediation due to their specific ability to grow and express novel engineered functions. More recently, the sequencing of several Deinococcus spp and comparative genomic analysis have provided new insight into the potential of this genus. Features such as the accumulation of genes encoding cell cleaning systems that eliminate organic and inorganic cell toxic components are widespread among Deinococcus spp. Other features such as the ability to degrade and metabolize sugars and polymeric sugars make Deinococcus spp. an attractive alternative for use in industrial biotechnology.

Repeated topical treatment, in contrast to single oral doses, with Vitamin A-containing preparations does not affect plasma concentrations of retinol, retinyl esters or retinoic acids in female subjects of child-bearing age.

BACKGROUND: Vitamin A is widely used in cosmetic preparations. Given that oral Vitamin A and its metabolites present a potential reproductive risk, the present study investigated the effect of topical Vitamin A on human endogenous plasma levels of Vitamin A and its metabolites. METHODS: Two groups of 14 female volunteers of child-bearing age were kept on a Vitamin A-poor diet and treated topically for 21 days with creams containing 0.30% retinol or 0.55% retinyl palmitate on approximately 3000 cm2 of their body surface area, amounting to a total of approximately 30,000 IU Vitamin A/subject/day. After a 12-day wash-out period, the study groups received single oral doses of 10,000 IU or 30,000 IU retinyl palmitate (RP), corresponding to the maximal EU allowance during pregnancy or three-times higher, respectively. Blood samples were collected over 24h on study days -3 (pre-study), 1, 21 (first and last days of topical treatment) and 34 (oral administration) at 0, 1, 2, 4, 6, 8, 12, 14-16 h and 24 h after treatment for determination of plasma concentrations of retinol (REL), retinyl palmitate (RP), oleate (RO) and stearate (RS), 9-cis-, 13-cis-, all-trans- (AT), 13-cis-4-oxo- or AT-4-oxo-retinoic acids (RAs). RESULTS: With the exception of transient mild (RP-group) to moderate (REL-group) local irritation on the treatment sites, no adverse local or systemic effects were noted. On days 1 or 21 of topical treatment, no changes were measured in individual or group mean plasma Cmax, AUC0-24 h or other pharmacokinetic parameters of REL, retinyl esters or RAs relative to pre-study data. In contrast, single oral doses of RP at 10,000 IU or 30,000 IU produced dose-related and sustained increases in Cmax and AUC0-24 h values of plasma RP, RO, RS, 13-cis- and 13-cis-4-oxo-RAs, as well as a transient increase in AT-RA. In conclusion, our results provide evidence that human topical exposure to retinol- or retinyl ester-containing cosmetic creams at 30,000 IU/day and maximal use concentrations do not affect plasma levels of retinol, retinyl esters or RAs, whereas single oral doses at 10,000 IU or 30,000 IU produce significant increases in plasma retinyl esters and RAs.

Regulation of the intracellular calcium level in human blood platelets: cyclic adenosine 3',5'-monophosphate dependent phosphorylation of a 22,000 dalton component in isolated Ca2+-accumulating vesicles.

Two protein kinase activities have been separated from the supernatants of homogenized human blood platelets by DEAE cellulose chromatography. One of them (peak I enzyme) is an efficient stimulator of the uptake of Ca2+ into isolated membrane vesicles in the presence of cyclic AMP and ATP. The second (peak II enzyme), although equally active towards histone, exerts only about one third of the activity of the peak I enzyme. The stimulation of Ca2+ uptake is accompanied by the phosphorylation of a membrane protein with an apparent molecular weight of 22 000, which appears to play an essential role in the regulation of the intracellular Ca2+ level and hence of platelet activity.

Radiotherapy of testicular intraepithelial neoplasia (TIN): a novel treatment regimen for a rare disease.

PURPOSE: Testicular intraepithelial neoplasia (TIN) is a consistent precursor of most invasive germ cell tumors, currently treated by radiotherapy with 20 Gy, which destroys TIN but preserves Leydig cells. Nevertheless, analysis has shown dose-dependent dysfunction even with low therapeutic doses of 20 Gy in some cases. Therefore, we tested a dose reduction regimen by delivering smaller fractional doses to enhance the tolerance of Leydig cells. METHODS AND MATERIALS: Between 1993 and 1999, 9 patients were treated for TIN in a prospective multicenter trial. A total dose of 13 Gy was administered in 10 fractions of 1.3 Gy. Hormonal levels of follicle-stimulating hormone, luteinizing hormone, and testosterone were assayed serially. RESULTS: During a median follow-up time of 36 months, no patient showed evidence of local disease. A first postradiation biopsy was obtained 3-12 months after radiotherapy; 5 patients underwent a second biopsy 2-3 years after treatment. All biopsies showed a Sertoli cell-only pattern. Follicle-stimulating hormone levels continued to increase 1 year after radiotherapy, signaling eradicated spermiogenesis. Luteinizing hormone and testosterone remained within the normal range 2 years after radiotherapy. CONCLUSIONS: In the treatment of TIN, there seems to be a dose reduction potential to 13 Gy by lowering single fractional doses, which enhances the therapeutic ratio in favor of the Leydig cells.

Ortho-phenanthroline modulates enzymes of cellular energy metabolism.

The lipophilic iron chelator 1,10-phenanthroline has been used in mechanistic studies on intracellular oxidant damage because iron is assumed to play a role in the endogenous formation of highly reactive oxygen species. This study shows that 1,10-phenanthroline has enzyme-modulatory properties in addition to its antioxidant activity. In rat hepatocytes, 1,10-phenanthroline caused inhibition of respiration and enhancement of cellular ATP content, pyruvate release and CO2 formation from glycerol resulting from a modulatory action of 1,10-phenanthroline on various enzymes involved in cellular energy metabolism. In intact mitochondria and in submitochondrial particles, oxygen consumption, complex I activity, and ATPase degradation are inhibited by 1,10-phenanthroline. In submitochondrial particles, complex II activity can also be suppressed by 1,10-phenanthroline. The purified cytosolic enzymes lactate dehydrogenase and glycerol-3-phosphate dehydrogenase are inhibited while purified glyceraldehyde-3-phosphate dehydrogenase is activated by 1,10-phenanthroline. The results suggest that 1,10-phenanthroline modulates various enzyme activities linked to cellular energy metabolism and that this property must be taken into account when using 1,10-phenanthroline as a tool in experiments on oxidant effects in cells.

Influence of acetaminophen treatment and hydrogen peroxide treatment on the release of a CINC-related protein and TNF-alpha from rat hepatocyte cultures.

Western blot analysis of conditioned media from hepatocytes exposed to H2O2 revealed that a 28 kDa protein was released dose-dependently in response to 1-10 mM H2O2. The 28 kDa protein was present in freshly isolated hepatocytes and exhibited cross-reactivity towards an antibody against CINC/gro. The intracellular amount of the protein decreased in parallel to the H2O2-induced release into the medium. The CINC-related protein was absent in media harvested after 1 h of treatment. The delivery of CINC-related protein correlated with the extent of cell damage as judged from lactate dehydrogenase leakage. Likewise, exposure of hepatocytes to 10-50 mM acetaminophen resulted in a dose-dependent release of the CINC-related protein after 24 h of culture. In contrast, monomeric CINC (molecular weight approximately 6.5 kDa) but not the 28 kDa CINC-related protein was released by lipopolysaccharide (LPS)-stimulated Kupffer cells. The amount of monomeric CINC liberated by Kupffer cells was diminished upon acetaminophen-treatment. Also, the release of tumor necrosis factor-alpha by hepatocytes was reduced after exposure to high acetaminophen doses (40-50 mM). In contrast to this finding, TNF-alpha release from hepatocyte cultures was not affected after H2O2 treatment. These data suggest that damaged hepatocytes release proinflammatory cytokines which may aggravate liver injury through activation of neutrophils and monocytes. The results indicate that the appearance of the CINC-related protein is due to impairment of plasma membrane integrity as the consequence of massive cell damage. In addition, APAP inhibited the release of monomeric CINC from LPS-activated Kupffer cells and of TNF-alpha from hepatocytes even at concentrations that were not sufficient to affect cell viability.

Impairment of TNF-alpha expression and secretion in primary rat liver cell cultures by acetaminophen treatment.

Tumor necrosis factor-alpha (TNF-alpha) is assumed to act as a mediator in toxic liver injury, aggravating the primary damage to the parenchymal liver cell, but also stimulating liver regeneration. Reports on the effect of acetaminophen in vivo on TNF-alpha transcript concentrations and serum TNF-alpha concentrations, under different experimental, or clinical conditions have yielded controversial results. We used primary rat hepatocyte and Kupffer cell cultures to test the direct action of subtoxic and toxic concentrations of acetaminophen on TNF-alpha expression and release. We observed a dose-dependent decrease of TNF-alpha mRNA in the hepatocytes, and of TNF-alpha release into the medium of hepatocyte cultures. The data also indicate an impairment of TNF-alpha release in Kupffer cell cultures after treatment with nontoxic, as well as with toxic, acetaminophen concentrations. The results suggest that inhibition of TNF-alpha expression and release in the liver is a consequence of acetaminophen exposure. It is at present unknown how this effect modulates the course of acetaminophen intoxication.

Lactobacillus divergens sp. nov., a New Heterofermentative Lactobacillus Species Producing L(+)-Lactate.

A group of 8 heterofermentative Lactobacillus strains were isolated from raw vacuum-packaged, as well as SO(2)-treated, minced beef, in the course of shelf life studies on this product. These strains were found to differ from all other described heterofermentative Lactobacillus species in their production of virtually pure L( + )-lactic acid from hexoses and pentoses, and also in their low mol% G + C in the DNA (34 ± 0.8% G + C). The m-Dpm type of peptidoglycan was typical of all strains. Their pattern of sugar metabolism seems to differ from the traditional HMP-pathway typical of the heterofermentative lactobacilli. The incorporation of these strains in a new species, Lactobacillus divergens sp. nov., is suggested.

Grammatical morphology and the lexicon in children with specific language impairment.

We examined the use of grammatical morphology by preschool-age English-speaking children with specific language impairment (SLI) as a function of their lexical diversity. Relative to a group of normally developing (ND) preschoolers, these children's use of finite-verb morphology logged behind expectations based on the number of different verbs they used. Noun-related morphology fell below expectations based on overall lexical diversity. Differences between the ND children and children with SLI were also seen for the slope of the increases in finite-verb morphology as a function of lexical diversity, with shallower slopes in the SLI data. The findings of this study add to existing evidence suggesting that a measure of finite grammatical-morphology use has promise as a clinical marker of SLI in English.

Some metabolic and biochemical alterations during the development of stress ulcers in rats forced to swim.

Swimming of the rats in the water below body temperature (23 degrees C) for a period of 5 hours resulted in a number of acute haemorrhagic lesions, principally erosions, in the glandular part of the stomach. The objective of this study was to establish the possible roles of some hormonal, biochemical and metabolic factors in the pathogenesis of stress ulcer produced in rats forced to swim. It was found that (i) prior ligation of the pylorus caused a considerable decrease of both the incidence and the severity of the ulcers resulting from the swim-stress, (ii) a significant decrease of the gastric secretion and rectal temperature resulted during the swim-stress condition, (iii) metabolic acidosis developed during the forced swimming period, (iv) considerable increases of the plasma corticosterone and blood glucose concentration also developed, (v) the gastric mucosal level of cAMP also increased, and (vi) a prior period of starvation increased the incidence and severity of the acute ulcers resulting from the swim-stress. It was concluded that various humoral, biochemical and metabolic factors play important roles in the development of stress ulceration in rats forced to swim.

[Determination of therapy management in rectal carcinoma by staging with 18-FDG-PET]. / Bestimmung des Therapiemanagements beim Rectumcarcinom durch Staging mittels 18-FDG-PET.

The curative treatment of carcinoma of the rectum in the early stage of the disease is radical local surgery. If there is a solitary liver metastasis, resection is also a curative treatment. This report describes a female patient with rectal carcinoma, in whom a solitary liver metastasis in the left lobe was diagnosed only by FDG-PET and verified at surgery. This case report demonstrates the potential role of FDG-PET even for primary staging in detecting occult hepatic and extrahepatic metastases, thus significantly influencing the therapeutic management and prognosis of these patients.

Special aspects of cosmetic spray safety evaluations: principles on inhalation risk assessment.

The consumer exposure to the vast majority of cosmetic products is limited to dermal contact. Even spray applications tend to be topically exposed to skin or hair. Besides this skin contact, spray products require additional considerations in regard to potential inhalation for building a robust and reliable safety assessment. Over the years, cosmetic industry developed prediction models for the best estimate of inhalation exposure combining data from computer simulation programs available in the market, individual real measured data and last but not least the experience from the market. Such attempt is driven by the toxicological profile of individual used ingredients. The focus of this review is on the determination of inhalation exposure, and the derivation of safe exposure levels for cosmetic spray products. Many of the methods employed to ensure product safety of cosmetic sprays in accordance with the general requirements of the EC Cosmetics Directive are based on industry experience which are not necessarily consistent across companies. This paper presents an approach to compile common principles for risk assessment and thus contribute to standardisation of safety assessment methodologies utilized for spray product evaluation without interfering with the flexibility of the individual safety assessor. It is based on the experience within the author's companies and may be useful as a support document as well for SME (Small and Medium Enterprises) companies safety assessors. In this respect it can be seen as one fundamental step in a tiered approach of cosmetic spray safety evaluation.

Mutations in fibrillin-1 cause congenital scleroderma: stiff skin syndrome.

The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence-encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor-beta (TGFbeta). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFbeta concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.