DNA divergence in and around the alcohol dehydrogenase locus in five closely related species of Hawaiian Drosophila

The alcohol dehydrogenase (Adh) region from five planitibia subgroup species of Hawaiian picture-wing Drosophila has been cloned. A total of 15 kb of DNA in and around the Adh gene has been compared among the five species. Genetic distances were calculated to determine evolutionary relationships. These distances agree with previous distances determined by protein polymorphism and DNA hybridization techniques and can be interpreted in terms of specific island colonization and speciation (founder) events over the past 5 Myr. Examination of the restriction maps of the cloned Adh region from the five species shows many instances of small deletions, insertion of a transposable element in D. heteroneura, and the existence of a highly variable region on the 3' side of the Adh gene. Clustering relationships and rates of DNA change are calculated and compared with the relationship found for other species of Drosophila.      

Recessive Mutations in COL25A1 Are a Cause of Congenital Cranial Dysinnervation Disorder

Abnormal ocular motility is a common clinical feature in congenital cranial dysinnervation disorder (CCDD). To date, eight genes related to neuronal development have been associated with different CCDD phenotypes. By using linkage analysis, candidate gene screening, and exome sequencing, we identified three mutations in collagen, type XXV, alpha 1 (COL25A1) in individuals with autosomal-recessive inheritance of CCDD ophthalmic phenotypes. These mutations affected either stability or levels of the protein. We further detected altered levels of sAPP (neuronal protein involved in axon guidance and synaptogenesis) and TUBB3 (encoded by TUBB3, which is mutated in CFEOM3) as a result of null mutations in COL25A1. Our data suggest that lack of COL25A1 might interfere with molecular pathways involved in oculomotor neuron development, leading to CCDD phenotypes.     

THE COMBINED USE OF WHOLE CUPHEA SEEDS CONTAINING MEDIUM CHAIN FATTY ACIDS AND AN EXOGENOUS LIPASE IN PIGLET NUTRITION

In search for an alternative for nutritional antimicrobials in piglet feeding, the effects of adding whole Cuphea seeds, as a natural source of medium chain fatty acids (MCFA), with known antimicrobial effects, and an exogenous lipase to a weaner diet were studied. The foregut flora, the gut morphology, some digestive parameters and the zootechnical performance of weaned piglets were investigated. Thirty newly weaned piglets, initial weight 7.0 ± 0.4 kg, were divided according to litter, sex and weight in two groups (control diet; Cuphea+lipase diet). The Cuphea seeds (lanceolata and ignea) (50 g kg^?1) were substituted for soybean oil (15 g kg^?1), Alphacell (25 g kg^?1) and soy protein isolate (10 g kg^?1) in the control diet. Also 500 mg kg^?1 microbial lipase was added to the Cuphea diet. The piglets were weighted individually on days 0, 3, 7, 14 and 16. Feed intake was recorded per pen during days 0 to 3, 3 to 7, 7 to 14 and 14 to 16. On day 7 five piglets of each experimental group were euthanized for counting the gastric and small intestinal gut flora and for gut morphology at two sites of the small intestine (proximal, distal). The results indicate a trend towards improved performances parameters by feeding Cuphea + lipase. The enzymic released MCFA (1.7 g kg^?1 fresh gastric contents) tended to decrease the number of Coliforms in the proximal small intestine, but increased the number in the stomach and distal small intestine. With Cuphea, the number of Streptococci was significantly lower in small intestine, but not in the stomach, while the number of Lactobacilli was significantly lower in the distal small intestine and tended to be lower in the stomach and proximal small intestine. No differences between the diets were noted for the total anaerobic microbial load in the stomach or in the gut. Feeding Cuphea+lipase resulted in a significantly greater villus height (distal small intestine) and a lesser crypt depth (proximal and distal small intestine) and greater villus/crypt ratio depth (proximal and distal small intestine). The intra-epithelial lymphocyte (IEL) counts per 100 enterocytes were significantly decreased in the proximal small intestine and tended to decrease in the distal small intestine by feeding the Cuphea+lipase diet. Both phenomena are indicative for a more healthy and better functional state of the mucosa. Present results are in line with foregoing research, showing that manipulation of the gut ecosystem by the enzymic in situ released MCFA in the stomach and foregut can result in improved performances of the piglets, which makes the concept a potential alternative for in-feed nutritional antibiotics.     

Modulation of mitogen-independent hepatocyte proliferation during the perinatal period in the rat

Summary Late gestation fetal rat hepatocytes can proliferate under defined in vitro conditions in the absence of added mitogens. However, this capacity declines with advancing gestational age of the fetus from which the hepatocytes are derived. The present studies were undertaken to investigate this change in fetal hepatocyte growth regulation. Examination of E19 fetal hepatocyte primary cultures using immunocytochemistry for 5-bromo-2′-deoxyuridine (BrdU) incorporation showed that approximately 80% of these cells traverse S-phase of the cell cycle over the first 48 h in culture. Similarly, 65% of E19 hepatocytes maintained in culture under defined mitogen-free conditions for 24 h showed nuclear expression of proliferating cell nuclear antigen (PCNA). These in vitro findings correlated with a high level of immunoreactive PCNA in immunofluorescent analyses of E19 liver. In contrast, E21 (term) liver showed little immunoreactive PCNA. The in vivo finding was recapitulated by in vitro studies showing that E21 hepatocytes had low levels of BrdU incorporation during the first day in culture and were PCNA negative shortly after isolation. However, within 12 h of plating, E21 hepatocytes showed cytoplasmic staining for PCNA. Although maintained under mitogen-free conditions, PCNA expression progressed synchronously to a nucleolar staining pattern at 24 to 48 h in culture followed by intense, diffuse nuclear staining at 60 h which disappeared by 72 h. This apparently synchronous cell cycle progression was confirmed by studies showing peak BrdU incorporation on the third day in culture. Whereas DNA synthesis by both E19 and E21 hepatocytes was potentiated by transforming growth factor α (TGFα), considerable mitogen-independent DNA synthesis was seen in hepatocytes from both gestational ages. These results may indicate that fetal hepatocytes come under the influence of an exogenous, in vivo growth inhibitory factor as term approaches and that this effect is relieved when term fetal hepatocytes are cultured.     

Comparative evaluation of D-glucosyl thiouronium, glucosylthio heterocycles, Daonil, and insulin as inhibitors for hepatic glycosidases

Comparison of the in vivo and in vitro effects of S-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)thiuronium bromide (1), 2-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosylthio-1,3,4-thiadiazolin-5-thione (2), and 2-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosylthio)-1,3-benzoxazole (3), as well as the antidiabetics Daonil and insulin on glycosidase enzymes has been investigated. Compound 1 inhibited both alpha- and beta-glucosidases, but the inhibition was more potent with the beta-enzyme. Compound 2 was found to be a weaker inhibitor of these enzymes, while compound 3 showed a slight apparent activation.     

Synthesis and In Vitro Antitumor Activity of New Substituted Thiopyrimidine Acyclic Nucleosides and Their Thioglycoside Analogs

Some new thiopyrimidine acyclic nucleosides and thioglycoside derivatives 3a-c, 4a-c, 6a,b, and 7a,b were synthesized. The cytotoxicity and antitumor evaluation of all prepared compounds have been tested in vitro against Ehrlich's ascites carcinoma cell line and their activity against glutathione peroxidase and catalase were reported. The role of the prepared compounds as free radical regulators and the therapeutic antitumor effect of a balanced generation of free radicals are discussed. Compounds 2, 3b, 3c, 4a, and 4c inhibited significantly in a dose dependent manner the growth of Ehrlich ascites carcinoma cells while the other compounds did not show any antitumor activity even at higher concentrations.     

ATM Mediates pRB Function To Control DNMT1 Protein Stability and DNA Methylation

The retinoblastoma tumor suppressor gene (RB) product has been implicated in epigenetic control of gene expression owing to its ability to physically bind to many chromatin modifiers. However, the biological and clinical significance of this activity was not well elucidated. To address this, we performed genetic and epigenetic analyses in an Rb-deficient mouse thyroid C cell tumor model. Here we report that the genetic interaction of Rb and ATM regulates DNMT1 protein stability and hence controls the DNA methylation status in the promoters of at least the Ink4a, Shc2, FoxO6, and Noggin genes. Furthermore, we demonstrate that inactivation of pRB promotes Tip60 (acetyltransferase)-dependent ATM activation; allows activated ATM to physically bind to DNMT1, forming a complex with Tip60 and UHRF1 (E3 ligase); and consequently accelerates DNMT1 ubiquitination driven by Tip60-dependent acetylation. Our results indicate that inactivation of the pRB pathway in coordination with aberration in the DNA damage response deregulates DNMT1 stability, leading to an abnormal DNA methylation pattern and malignant progression.     

Molecular mechanism for the inhibition of acetylcholinesterase enzyme by organophosphorothionates

The different mechanisms, whereby EPN and malathion inhibit the action of cholinesterase on acetylcholine, are described. Partially purified brain enzyme was used for the kinetic studies. The approach of the theory of Krupka and Laidler was followed. The ratio of [S]I opt/[S]opt = 1 + Ki [I] to the first power was found with malathion but to the square root of (1 + Ki [I]) 1/2 with EPN. The intercept on the slope axis of plots of slopes of (1/V not equal to [I]) against the reciprocal of substrate concentrations showed a non-zero value in the case of EPN and a zero value in the case of malathion. Accordingly, and based on the above theory, it seems that malathion acts as a competitive inhibitor of cholinesterase while EPN seems to be a mixed type inhibitor.