Effects of naturally occurring G103D point mutation of AQP5 on its water permeability, trafficking and cellular localization in the submandibular gland of rats

Background information. AQPs (aquaporins) are water channel proteins that are expressed in almost all living things. In mammalians, 13 members of AQPs (AQP0–12) have been identified so far. AQP5 is known to be expressed mostly in the exocrine cells, including the salivary gland acinar cells. A naturally occurring point mutation (G308A, Gly¹⁰³ > Asp¹⁰³) was earlier found in the rat AQP5 gene [Murdiastuti, Purwanti, Karabasil, Li, Yao, Akamatsu, Kanamori and Hosoi (2006) Am. J. Physiol. 291 , G1081–G1088]; in this mutant, the rate of initial saliva secretion under stimulated and unstimulated conditions is less than that for the wt (wild‐type) animals. Results. Here the mutant molecule was characterized in detail. Using the Xenopus oocyte system, we demonstrated the mutant AQP5 to have water permeability almost the same as that of the wt molecule. Mutant and wt AQP5s, tagged with GFP (green fluorescent protein; GFP‐AQP5s) and expressed in polarized MDCK‐II (Madin—Darby canine kidney II) cells, first appeared in the vesicular structure(s) in the cytoplasm, and were translocated to the upper plasma membrane or apical membrane during cultivation, with the mutant GFP‐AQP5 being translocated less efficiently. Thapsigargin and H‐89 both induced translocation in vitro of either molecule, whereas colchicine inhibited this activity; the fraction of cells showing apical localization of mutant GFP‐AQP5 was less than that showing that of the wt molecule under any of the experimental conditions used. In the mutant SMG (submandibular gland) tissue, localization of AQP5 in the apical membrane of acinar cells was extremely reduced. Vesicular structures positive for AQP5 and present in the cytoplasm of the acinar cells were co‐localized with LAMP2 (lysosome‐associated membrane protein 2) or cathepsin D in the mutant gland, whereas such co‐localizations were very rare in the wt gland, suggesting that the mutant molecules largely entered lysosomes for degradation. Conclusion. Replacement of highly conserved hydrophobic Gly¹⁰³ with strongly hydrophilic Asp¹⁰³ in rat AQP5, though it did not affect water permeability, may possibly have resulted in less efficient membrane trafficking and increased lysosomal degradation, leading to its lower expression in the apical membrane of the acinar cells in the SMG.     

Interaction of calmodulin with Sec61α limits Ca2+ leakage from the endoplasmic reticulum

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, as formed by Sec61 complexes in the ER membrane, would seriously interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism for intracellular signalling. We identified a calmodulin (CaM)-binding motif in the cytosolic N-terminus of mammalian Sec61α that bound CaM but not Ca2+-free apocalmodulin with nanomolar affinity and sequence specificity. In single-channel measurements, CaM potently mediated Sec61-channel closure in Ca2+-dependent manner. At the cellular level, two different CaM antagonists stimulated calcium release from the ER through Sec61 channels. However, protein transport into microsomes was not modulated by Ca2+-CaM. Molecular modelling of the ribosome/Sec61/CaM complexes supports the view that simultaneous ribosome and CaM binding to the Sec61 complex may be possible. Overall, CaM is involved in limiting Ca2+ leakage from the ER.     

28-homobrassinolide improves growth and photosynthesis in <Emphasis Type="Italic">Cucumis sativus</Emphasis> L. through an enhanced antioxidant system in the presence of chilling stress

The ameliorative role of 28-homobrassinolide under chilling stress in various growth, photosynthesis, enzymes and biochemical parameters of cucumber (Cucumis sativus L.) were investigated. Cucumber seedlings were sprayed with 0 (control), 10⁻⁸, or 10⁻⁶ M of 28-homobrassinolide at the 30-day stage. 48 h after treatment plants were exposed for 18 h to chilling temperature (10/8°C, 5/3°C). The most evident effect of chilling stress was the marked reduction in plant growth, chlorophyll (Chl) content, and net photosynthetic rate, efficiency of photosystem II and activities of nitrate reductase and carbonic anhydrase. Moreover, the activities of antioxidant enzymes; catalase (E.C. 1.11.1.6), peroxidase (E.C.1.11.1.7), superoxide dismutase (E.C. 1.15.1.1) along with the proline content in leaves of the cucumber seedlings increased in proportion to chilling temperature. The stressed seedlings of cucumber pretreated with 28-homobrassinolide maintained a higher value of antioxidant enzymes and proline content over the control suggesting the protective mechanism against the ill-effect caused by chilling stress might be operative through an improved antioxidant system. Furthermore, the protective role of 28-homobrassinolide was reflected in improved growth, water relations, photosynthesis and maximum quantum yield of photosystem II both in the presence and absence of chilling stress.     

Tetra primer ARMS-PCR relates folate/homocysteine pathway genes and ACE gene polymorphism with coronary artery disease

Cardiovascular disorders and coronary artery disease (CAD) are significant contributors to morbidity and mortality in heart patients. As genes of the folate/homocysteine pathway have been linked with the vascular disease, we investigated association of these gene polymorphisms with CAD/myocardial infarction (MI) using the novel approach of tetraprimer ARMS-PCR. A total of 230 participants (129 MI cases, 101 normal subjects) were recruited. We genotyped rs1801133 and rs1801131 SNPs in 5'10' methylenetetrahydrofolate reductase (MTHFR), rs1805087 SNP in 5' methyltetrahydrofolate homocysteine methyltransferase (MTR), rs662 SNP in paroxanse1 (PON1), and rs5742905 polymorphism in cystathionine beta synthase (CBS). Angiotensin converting enzyme (ACE) insertion/deletion polymorphism was detected through conventional PCR. Covariates included blood pressure, fasting blood sugar, serum cholesterol, and creatinine concentrations. Our results showed allele frequencies at rs1801133, rs1801131, rs1805087 and the ACE insertion/deletion (I/D) polymorphism varied between cases and controls. Logistic regression, after adjusting for covariates, demonstrated significant associations of rs1801133 and rs1805087 with CAD in the additive, dominant, and genotype model. In contrast, ACE I/D polymorphism was significantly related with CAD where recessive model was applied. Gene-gene interaction against the disease status revealed two polymorphism groups: rs1801133, rs662, and rs1805087; and rs1801131, rs662, and ACE I/D. Only the latter interaction maintained significance after adjusted for covariates. Our study concludes that folate pathway variants exert contributory influence on susceptibility to CAD. We further suggest that tetraprimer ARMS-PCR successfully resolves the genotypes in selected samples and might prove to be a superior technique compared to the conventional approach.     

Physiological changes in major soldiers of Macrotermes gilvus (Isoptera: Termitidae) induced by the endoparasitoid Misotermes mindeni (Diptera: Phoridae)

The majority of true parasitoids manipulate their host’s physiology for their own benefit. In this study, we documented the physiological changes that occurred in major soldiers of the subterranean termite Macrotermes gilvus (Hagen) (Isoptera: Termitidae) parasitized by the koinobiont larval endoparasitoid Misotermes mindeni Disney and Neoh (Diptera: Phoridae). We compared the metabolic rate, body water content, body water loss rate, cuticular permeability, and desiccation tolerance between parasitized and unparasitized major soldiers. The metabolic rate of parasitized hosts was significantly higher than that of unparasitized termites. Mean total body water content of parasitized major soldiers (64.73 ± 3.26%) was significantly lower than that of unparasitized termites (71.99 ± 2.23%). Parasitized hosts also had significantly lower total body water loss rates (5.72 ± 0.06%/h) and higher cuticular permeability (49.37 ± 11.26 μg/cm/h/mmHg) than unparasitized major soldiers (6.75 ± 0.16%/h and 60.76 ± 24.98 μg/cm/h/mmHg, respectively). Parasitized major soldiers survived almost twice as long as unparasitized termites (LT₅₀ = 6.66 h and LT₅₀ = 3.40 h, respectively) and they had significantly higher tolerance to water loss compared to unparasitized termites (45.28 ± 6.79% and 32.84 ± 7.69%, respectively). Body lipid content in parasitized hosts (19.84 ± 6.27%) was significantly higher than that of unparasitized termites (6.17 ± 7.87%). Finally, parasitized hosts had a significantly lower percentage of cuticular water content than unparasitized major soldiers (10.97 ± 1.84% and 13.17 ± 2.21%, respectively). Based on these data, we conclude that the parasitism-induced physiological changes in the host are beneficial to the parasitoids as the alterations can clearly increase the parasite’s chances of survival when exposed to extreme environmental conditions and ensure that the parasitoids are able to complete their larval development successfully before the host dies.     

Hematological and serum biochemical analyses in experimental caprine besnoitiosis

This study was undertaken to investigate the hematological and biochemical changes in experimentally infected goats with Besnoitia caprae from the time of infection till 360 days post-infection (PI). Six male goats were inoculated subcutaneously with 13 × 10(7) bradyzoites of B. caprae, and blood samples were collected from the jugular vein. The total erythrocyte and total leukocyte counts, hematocrit value, and differential leukocyte counts were determined. Serum biochemical analysis, including the total protein, albumin, total globulin, cholesterol, triglyceride, chloride, testosterone, calcium (Ca(2+)), inorganic phosphorus, sodium (Na(+)), potassium (K(+)), iron (Fe(2+)), glucose, serum amyloid A (SAA), haptoglobin (Hp), fibrinogen, ceruloplasmin, aspartate aminotransferase, alanine aminotransferase, creatine kinase, lactate dehydrogenase, and alkaline phosphatase, was undertaken. Skin biopsy from the limbs were collected at weekly intervals and histologically examined for Besnoitia cysts. Cysts were present in the skin biopsies of the leg of the infected goats from day 28 PI. There were variations in hematological analyses, but no significant difference was seen. From day 30 to 360 PI, results showed that SAA, Hp, fibrinogen, and ceruloplasmin concentrations increased, whereas testosterone concentrations decreased. Infected goats exhibited decrease of albumin and increase of serum total protein and globulin concentrations. By contrast, there were no significant differences in the remained analyses concentrations.     

Protein and DNA destabilization by osmolytes: the other side of the coin

Osmolytes are naturally occurring small molecules accumulated intracellularly to protect organisms from various denaturing stresses. Similar to the two faces of a coin, several of these osmolytes are stabilizing and destabilizing proteins depending on the concentrations and/or solvent conditions. For example, the well known stabilizing osmolyte, trehalose destabilizes some proteins at high concentration and/or high pH. In spite of the fact that destabilizing aspects of osmolytes can modulate many cellular processes including regulation of protein homeostasis (proteostasis), protein-protein interaction, and protein-DNA interaction, researchers have mostly focused on the stabilizing aspects of osmolytes. Thus, it is important to look into both aspects of osmolytes to determine their precise role under physiological conditions. In this article, we have discussed both stabilizing and destabilizing/denaturant aspects of osmolytes to uncover both sides of the coin.     

Genotypic characterization of Iranian camel (Camelus dromedarius) isolates of Echinoccocus granulosus

In the present study, Echinoccocus granulosus isolates collected from camels (Camelus dromedarius) in eastern Iran were characterized based on the nucleotide sequences of mitochondrial CO1 and NDI genes. The molecular results for camel isolates demonstrated that at least 2 different genotypes are present, i.e., a buffalo genotype (G3) and the camel genotype (G6). Although the sequences of the Iranian camel genotype (G6) are completely homologous to the reference sequence of G6 (M84666) of E. granulosus , a nucleotide mutation (C to T at position 168) was detected in the CO1 sequences of the Iranian G3 isolates (HM626405) when compared with the reference G3 genotype (M84663). The findings of the present study represent the demonstration of the buffalo strain in camels. As previously reported, humans can be infected by this genotype; accordingly, the epidemiological importance of this genotype in the camel population should be considered in further studies.