The extended arms of DNA-binding domains: a tale of tails

DNA-binding domains (DBDs) frequently have N- or C-terminal tails, rich in lysine and/or arginine and disordered in free solution, that bind the DNA separately from and in the opposite groove to the folded domain. Is their role simply to increase affinity for DNA or do they have a role in specificity, that is, sequence recognition? One approach to answering this question is to analyze the contribution of such tails to the overall energetics of binding. It turns out that, despite similarities of amino acid sequence, three distinct categories of DBD extension exist: (i) those that are purely electrostatic and lack specificity, (ii) those that are largely non-electrostatic with a high contribution to specificity and (iii) those of mixed character that show sequence preference. Because in all cases the tails also increase the affinity for target DNA, they represent a crucial component of the machinery for selective gene activation or repression.     

Irreversible global DNA hypomethylation as a key step in hepatocarcinogenesis induced by dietary methyl deficiency

Dietary methyl group deprivation is now well recognized as a model of hepatocarcinogenesis in rodents. In the present study, we examined the effects of feeding a methyl-deficient diet followed by a methyl-adequate diet on the extent of methylation of liver DNA and on the formation and evolution of altered hepatic foci. Male F344 rats were fed a methyl-deficient diet for 9, 18, 24, and 36 weeks, followed by re-feeding a methyl-adequate diet for a total of 54 weeks. Similar to previous findings, the methyl-deficient diet resulted in decreased levels of S-adenosylmethionine (SAM), SAM/SAH ratios, and global DNA hypomethylation. Feeding the methyl-adequate diet restored the liver SAM levels and SAM/SAH ratios to control levels in all experimental groups. In contrast, re-feeding the complete diet restored DNA methylation to normal level only in the group that had been fed the methyl-deficient diet for 9 weeks; in animals exposed to methyl deprivation longer, the methyl-adequate diet failed to reverse the hypomethylation of DNA. Liver tissue of rats exposed to methyl deficiency for 9, 18, 24, or 36 weeks was characterized by the persistent presence of placental isoform of glutathione-S-transferase (GSTpi)-positive lesions despite re-feeding the methyl-adequate diet. The persistence of altered hepatic foci in liver after withdrawal of methyl-deficient diet serves as an indication of the carcinogenic potential of a methyl-deficient diet. Substitution of the methyl-deficient diet with complete diet failed to prevent the expansion of initiated foci and restore DNA methylation in animals exposed to deficiency for 18, 24, or 36 weeks. The association between DNA hypomethylation and expansion of foci suggests that stable DNA hypomethylation is a promoting factor for clonal expansion of initiated cells. These results provide an experimental evidence and a mechanistic basis by which epigenetic alterations may contribute to the initiation and promotion steps of carcinogenesis.     

SUMOylation participates in induction of ischemic tolerance

Ground squirrels in hibernation torpor have been shown to have striking increases in global SUMOylation on tissue immunoblots. Here, we find evidence that global SUMOylation is also involved in ischemic tolerance in primary cortical neuronal cultures (from rats and mice) and SHSY5Y human neuroblastoma cells. Cultured cortical neurons preconditioned by sublethal oxygen/glucose deprivation (OGD) were less vulnerable to severe OGD than non-preconditioned neurons. Preconditioned neurons maintained elevated SUMO-1 conjugation levels (and, to a lesser extent those of SUMO-2/3) on western blots in contrast to non-preconditioned cells. Further, cortical neurons and SHSY5Y cells in which transfected SUMO-1 or SUMO-2 were over-expressed showed increased survival after severe OGD. In contrast, cell cultures subjected to depletion of endogenous SUMO-1 protein by RNAi had reduced survival after exposure to this form of in vitro ischemia and an attenuated protective response to preconditioning. These findings suggest that maintenance of a globally elevated SUMO-1 (and maybe SUMO-2/3) conjugation level as revealed by immunoblot assays is a component of ischemic tolerance.     

Behavioral and electroencephalographic manifestations of thioacetamide-induced encephalopathy in rats

The aim of our study was to investigate the behavioral and electroencephalographic manifestations of thioacetamide-induced encephalopathy in rats. Male Wistar rats were divided among (i) control, saline-treated, and (ii) thioacetamide-treated groups (TAA(300) (300?mg/kg body mass); TAA(600) (600?mg/kg); and TAA(900) (900?mg/kg)). The daily dose of thioacetamide (300?mg/kg) was administered intraperitoneally once (TAA(300)), twice (TAA(600)), or 3 times (TAA(900)), on subsequent days. Behavioral manifestations were determined at 0, 2, 4, 6, and 24?h, while electroencephalographic changes were recorded 22-24?h after the last dose. General motor activity and exploratory behavior, as well as head shake, auditory startle reflex, placement, and equlibrium tests were diminished in the TAA(600) and TAA(900) groups compared with the control, and were absent in the TAA(900) group 24?h after treatment. Corneal, withdrawal, grasping, and righting reflexes were significantly diminished in the TAA(900) group compared with the control. Mean electroencephalographic power spectra density was significantly higher in TAA(300) and TAA(600) and lower in the TAA(900) group by comparison with the control. Only a score of 3 (mean dominant frequency?≤ 7.3?Hz and δ relative power?≥ 45%) was observed in the TAA(900) group. Thioacetamide induces encephalopathy in rats in a dose-dependent manner. A dose of 900?mg/kg TAA may be used as a suitable model of all stages of hepatic encephalopathy.     

HIV risks among female sex workers in Croatia and Montenegro

The study analyzed the prevalence and determinants of HIV-risks among female sex workers (FSWs) in Croatia and Montenegro. Face-to-face interviews were carried out in Zagreb, Split, and Podgorica during the 2006-2008 period. Croatian participants (n = 154) reported fewer clients, more consistent condom use, higher rates of HIV testing, and greater HIV knowledge. The participants interviewed in Montenegro (n = 119) were more likely to have injected drugs and to have experienced sexual abuse in the previous year. Although Montenegrin FSWs were more exposed to HIV-risks than Croatian FSWs, they reported lower HIV-risk self-assessment. Consistent condom use was significantly associated with education and HIV-risk self-assessment in the Croatian and the experience of physical/sexual abuse in the Montenegrin sample. In spite of a number of methodological limitations, the empirical insights provided by this study may assist in improving the existing HIVV prevention initiatives.     

The benefits of regular kinesiotherapy once a week for postmenopausal women: an aged-matched study

Regular exercise training improves overall physical fitness and quality of life in postmenopausal women. The exigent training frequency depends on a user-specified training aim. The aim of this study was to confirm the benefits of regular once a week exercise training for the maintenance of fitness in postmenopausal women. The test group included 20 postmenopausal women (65 +/- 3.1 years) who have been attending the exercise training program conducted by the physiotherapist once a week for three years. The age-matched control group included 20 healthy women (65.5 +/- 2.4 years) who did not regularly attend the training program. The outcomes were: right and left lateral trunk flexion, right and left shoulder flexion, right and left grip strength, endurance capacity of the trunk extensors, lower limb muscle strength (1' chair stand test), and balance (one-leg standing duration time with eyes open and closed). Women from the test group achieved statistically significant better results in the following outcomes: right lateral trunk flexion (15.4 cm: 12.6 cm, p < 0.001), left lateral trunk flexion (15.4 cm: 12.6 cm, p = 0.001), trunk extension muscle endurance (53.4 s: 40.5 s, p < 0.001), lower limb muscle strength (28.4 x: 25 x, p < 0.001), and one-leg standing duration time with open eyes (33.5 s: 19.7 s, p < 0.001). The results suggest that a regular once a week exercise training program designed and conducted by the physiotherapist, may be helpful in the improvement or maintenance of flexibility, muscle strength and capacity, and balance in postmenopausal women. The better fitness proved by our study could be a result of other causes and not solely that of the designed training program.     

β-Arrestin2 Regulates Lysophosphatidic Acid-Induced Human Breast Tumor Cell Migration and Invasion via Rap1 and IQGAP1

β-arrestins play critical roles in chemotaxis and cytoskeletal reorganization downstream of several receptor types, including G protein-coupled receptors (GPCRs), which are targets for greater than 50% of all pharmaceuticals. Among them, receptors for lysophosphatidic acid (LPA), namely LPA₁ are overexpressed in breast cancer and promote metastatic spread. We have recently reported that β-arrestin2 regulates LPA₁-mediated breast cancer cell migration and invasion, although the underlying molecular mechanisms are not clearly understood. We show here that LPA induces activity of the small G protein, Rap1 in breast cancer cells in a β-arrestin2-dependent manner, but fails to activate Rap1 in non-malignant mammary epithelial cells. We found that Rap1A mRNA levels are higher in human breast tumors compared to healthy patient samples and Rap1A is robustly expressed in human ductal carcinoma in situ and invasive tumors, in contrast to the normal mammary ducts. Rap1A protein expression is also higher in aggressive breast cancer cells (MDA-MB-231 and Hs578t) relative to the weakly invasive MCF-7 cells or non-malignant MCF10A mammary cells. Depletion of Rap1A expression significantly impaired LPA-stimulated migration of breast cancer cells and invasiveness in three-dimensional Matrigel cultures. Furthermore, we found that β-arrestin2 associates with the actin binding protein IQGAP1 in breast cancer cells, and is necessary for the recruitment of IQGAP1 to the leading edge of migratory cells. Depletion of IQGAP1 blocked LPA-stimulated breast cancer cell invasion. Finally, we have identified that LPA enhances the binding of endogenous Rap1A to β-arrestin2, and also stimulates Rap1A and IQGAP1 to associate with LPA₁. Thus our data establish novel roles for Rap1A and IQGAP1 as critical regulators of LPA-induced breast cancer cell migration and invasion.     

Une nouvelle espèce de Pseudamaurops Jeannel, 1948 du Monténégro,et remarques sur les genres voisins (Coleoptera: Pselaphinae: Amauropini)

Une nouvelle espèce du genre Pseudamaurops Jeannel, 1948, récoltée dans une grotte à proximité de Virpazar, au Monténégro, et décrite et illustrée. Il s’agit là du troisième représentant du genre, deux autres espèces ayant été décrites d’Albanie. La position taxonomique de la nouvelle espèce est discutée, quelques remarques sur le genre et deux genres apparentés, Bergrothia Reitter, 1884 et Zoufalia Reitter, 1918, sont fournies.     

Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8 mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5 %, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10 months (ten cycles). A total of 20 % of the mature SSEs from cabbage and 55 % from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1 mg l^?1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56 % in cabbage and 79 % in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.