Expression of LEKTI domains 6-9' in the baculovirus expression system: recombinant LEKTI domains 6-9' inhibit trypsin and subtilisin A

The precursor lympho-epithelial Kazal-type-related inhibitor (LEKTI), containing two Kazal-type and 13 nonKazal-type domains, is an efficient inhibitor of multiple serine proteinases, among them plasmin, subtilisin A, cathepsin G, elastase, and trypsin. To gain insight into the structure and function of some of these domains, a portion of the cDNA coding for LEKTI domains 6-9' was cloned and expressed in Sf9 cells using the baculovirus expression vector system (BEVS). Through a single purification step using a Co2+ column, 3-4 mg of purified recombinant LEKTI-domains 6-9' (rLEKTI6-9') with the predicted molecular mass of 34.6 kDa was obtained from the cell pellet of a 1-L culture. Unlike full-length LEKTI, rLEKTI6-9' inhibited trypsin and subtilisin A but not plasmin, cathepsin G, or elastase. The inhibition of trypsin and subtilisin A by rLEKTI6-9' occurred through a noncompetitive mechanism, with inhibitory constants (Ki) of 356 +/- 12 and 193 +/- 10 nM, respectively. On the basis of the Ki values, rLEKTI6-9' was determined to be a more potent trypsin inhibitor and a less potent subtilisin A inhibitor than the full-length LEKTI. In contrast to LEKTI domains 6-9', recombinant LEKTI domain 6 does not inhibit subtilisin A but competitively inhibited trypsin with a Ki of 200 +/- 10 nM. Taking LEKTI6-9' as an example, the BEVS should facilitate the structure-function analysis of naturally occurring processed LEKTI forms that have physiological relevance.     

Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome

Kindler syndrome is an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Linkage and homozygosity analysis in an isolated Panamanian cohort and in additional inbred families mapped the gene to 20p12.3. Loss-of-function mutations were identified in the FLJ20116 gene (renamed “KIND1” [encoding kindlin-1]). Kindlin-1 is a human homolog of the Caenorhabditis elegans protein UNC-112, a membrane-associated structural/signaling protein that has been implicated in linking the actin cytoskeleton to the extracellular matrix (ECM). Thus, Kindler syndrome is, to our knowledge, the first skin fragility disorder caused by a defect in actin-ECM linkage, rather than keratin-ECM linkage.     

Effect of human urotensin-II infusion on hemodynamics and cardiac function

Recent studies have shown that the vasoactive peptide urotensin-II (U-II) exerts a wide range of action on the cardiovascular system of various species. In the present study, we determined the in vivo effects of U-II on basal hemodynamics and cardiac function in the anesthetized intact rat. Intravenous bolus injection of human U-II resulted in a dose-dependent decrease in mean arterial pressure and left ventricular systolic pressure. Cardiac contractility represented by +/-dP/dt was decreased after injection of U-II. However, there was no significant change in heart rate or diastolic pressure. The present study suggests that upregulation of myocardial U-II may contribute to impaired myocardial function in disease conditions such as congestive heart failure.     

Synthesis and anti-HBV activity of thiouracils linked via S and N-1 to the 5-position of methyl beta-D-ribofuranoside

Reverse nucleoside derivatives of 2-(methylsulfanyl)uracils 6a-d were prepared by treating of the sodium salt of 2-(methylsulfanyl)uracils (5a-d) with methyl 2,3-O-isopropylidene-5-O-p-toluenesulfonyl-beta-D-ribofuranoside (2). The alkylation of 2-thiouracils 4a-d with methyl 5-deoxy-5-iodo-2,3-O-isopropylidene-D-ribofuranoside (3) afforded the corresponding S-ribofuranoside derivatives 8a-d. Deisopropylidenation of 6a-d and 8a-d afforded the corresponding deprotected derivatives 7a-d and 9a-d, respectively. The Anti-HBV activity of selected compounds was studied.     

Development and organization of polarity-specific segregation of primary vestibular afferent fibers in mice

A striking feature of vestibular hair cells is the polarized arrangement of their stereocilia as the basis for their directional sensitivity. In mammals, each of the vestibular end organs is characterized by a distinct distribution of these polarized cells. We utilized the technique of post-fixation transganglionic neuronal tracing with fluorescent lipid soluble dyes in embryonic and postnatal mice to investigate whether these polarity characteristics correlate with the pattern of connections between the endorgans and their central targets; the vestibular nuclei and cerebellum. We found that the cerebellar and brainstem projections develop independently from each other and have a non-overlapping distribution of neurons and afferents from E11.5 on. In addition, we show that the vestibular fibers projecting to the cerebellum originate preferentially from the lateral half of the utricular macula and the medial half of the saccular macula. In contrast, the brainstem vestibular afferents originate primarily from the medial half of the utricular macula and the lateral half of the saccular macula. This indicates that the line of hair cell polarity reversal within the striola region segregates almost mutually exclusive central projections. A possible interpretation of this feature is that this macular organization provides an inhibitory side-loop through the cerebellum to produce synergistic tuning effects in the vestibular nuclei. The canal cristae project to the brainstem vestibular nuclei and cerebellum, but the projection to the vestibulocerebellum originates preferentially from the superior half of each of the cristae. The reason for this pattern is not clear, but it may compensate for unequal activation of crista hair cells or may be an evolutionary atavism reflecting a different polarity organization in ancestral vertebrate ears.     

Allosteric regulation of glycogen synthase controls glycogen synthesis in muscle

Glycogen synthase (GS), a key enzyme in glycogen synthesis, is activated by the allosteric stimulator glucose-6-phosphate (G6P) and by dephosphorylation through inactivation of GS kinase-3 with insulin. The relative importance of these two regulatory mechanisms in controlling GS is not established, mainly due to the complex interplay between multiple phosphorylation sites and allosteric effectors. Here we identify a residue that plays an important role in the allosteric activation of GS by G6P. We generated knockin mice in which wild-type muscle GS was replaced by a mutant that could not be activated by G6P but could still be activated normally by dephosphorylation. We demonstrate that knockin mice expressing the G6P-insensitive mutant display an ～80% reduced muscle glycogen synthesis by insulin and markedly reduced glycogen levels. Our study provides genetic evidence that allosteric activation of GS is the primary mechanism by which insulin promotes muscle glycogen accumulation in?vivo.     

Morphological and microsatellite diversity associated with ecological factors in natural populations of Medicago laciniata Mill. (Fabaceae)

Genetic variability in 10 natural Tunisian populations of Medicago laciniata were analysed using 19 quantitative traits and 12 polymorphic microsatellite loci. A large degree of genetic variability within-populations and among-populations was detected for both quantitative characters and molecular markers. High genetic differentiation among populations for quantitative traits was seen, with Q _ST = 0.47, and F _ST = 0.47 for microsatellite markers. Several quantitative traits displayed no statistical difference in the levels of Q _ST and F _ST . Further, significant correlations between quantitative traits and eco-geographical factors suggest that divergence in the traits among populations may track environmental differences. There was no significant correlation between genetic variability at quantitative traits and microsatellite markers within populations. The site-of-origin of eco-geographical factors explain between 18.13% and 23.40% of genetic variance among populations at quantitative traits and microsatellite markers, respectively. The environmental factors that most influence variation in measured traits among populations are assimilated phosphorus (P₂0₅) and mean annual rainfall, followed by climate and soil texture, altitude and organic matter. Significant associations between eco-geographical factors and gene diversity, H _e , were established in five-microsatellite loci suggesting that these simple sequence repeats (SSRs) are not necessarily biologically neutral.     

Increased DNA damage in children caused by passive smoking as assessed by comet assay and oxidative stress

The present study aimed to evaluate the association between the environmental tobacco smoke (ETS) and DNA damage in relation to oxidative stress (OS) in children. Sixty-four children of age 1-8 years, selected from the outpatient clinic of Mansoura University Children Hospital were divided into two groups (23 children/group) based on high (>20 cigarettes/day) or low (<20 cigarettes/day) exposure to ETS at home. Twenty symptom-free children with normal cotinine level and with no exposure to ETS were recruited as controls. The comet assay was used to quantify the level of DNA damage in lymphocytes isolated from all children. Spectrophotometric methods were used to assess the serum level of malondialdehyde (MDA) and activity of glutathione peroxidase (GSH-Px) in erythrocytes. Also, serum level of tocopherol fractions (alpha, gamma, delta) was assessed by high performance liquid chromatography (HPLC). Children exposed to ETS exhibited retarded growth, more chest problems, and gastroenteritis than the control. A significant increase in mean comet tail length indicating DNA damage was observed in ETS-exposed children (P<0.001) compared to controls. ETS-exposed children had significantly (P<0.001) higher MDA level paralleled with significant (P<0.001) decrease in the level of GSH-Px and tocopherol fractions compared with controls. The GSH-Px activity and tocopherol levels were inversely correlated with the increase of ETS exposure. These results show that inhalation of ETS is associated with an increase in the level of oxidants and a simultaneous decrease in the level of antioxidants in the children's blood. This status of oxidant-antioxidant imbalance (OS) may be one of the mechanisms leading to DNA damage detected in lymphocytes of ETS-exposed children. In conclusion, the present study gives an indication of an association between DNA damage and ETS exposure in children.     

Synthesis, structure and magnetic properties of a new family of chiral metal(II) citramalates

A new series of chiral carboxylate-bridged complexes of Mn(II), Co(II), and Ni(II) has been synthesized by reaction of M(II) salts with (S)-2-hydroxy-2-methyl-butanedioic acid ((S)-citramalic acid) under solvothermal conditions. The Mn(II) compound 1 is obtained as a crystalline powder, whereas the Co(II) and Ni(II) compounds (2 and 3 respectively) are obtained as single crystals. All the compounds crystallize in orthorhombic chiral space group P2₁2₁2₁. Compounds 2 and 3 are isostructural, and their structure consists in helicoïdal chains of M(II) centres linked by carboxylate bridges. The magnetic data indicate a rather weak coupling interaction between paramagnetic centres. The Mn(II) compound 1 exhibits antiferromagnetic ordering at T_N = 2.64 K. The Co(II) and Ni(II) compounds show ferromagnetic interactions within the chains. For 3, the chains couple antiferromagnetically, which leads to a metamagnetic behaviour with T_N = 1.69 K.