Two calcium‐binding chaperones from the fat body of the Colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) involved in diapause

Molecular chaperones are crucial for the correct folding of newly synthesized polypeptides, in particular, under stress conditions. Various studies have revealed the involvement of molecular chaperones, such as heat shock proteins, in diapause maintenance and starvation; however, the role of other chaperones in diapause and starvation relatively is unknown. In the current study, we identified two lectin‐type chaperones with calcium affinity, a calreticulin (LdCrT) and a calnexin (LdCnX), that were present in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) during diapause. Both proteins possessed an N‐globular domain, a P‐arm domain, and a highly charged C‐terminal domain, while an additional transmembrane domain was present in LdCnX. Phylogenetic analysis revealed distinction at the order level. Both genes were expressed in multiple tissues in larval and adult stages, and constitutively throughout development, though a starvation response was detected only for LdCrT. In females, diapause‐related expression analysis in the whole body revealed an upregulation of both genes by post‐diapause, but a downregulation by diapause only for LdCrT. By contrast, males revealed no alteration in their diapause‐related expression pattern in the entire body for both genes. Fat body‐specific expression analysis of both genes in relation to diapause revealed the same expression pattern with no alteration in females and downregulation in males by post‐diapause. This study suggests that calcium‐binding chaperones play similar and possibly gender‐specific roles during diapause.     

Osmotic characteristics of mouse spermatozoa in the presence of extenders and sugars

Successful cryopreservation requires cells to tolerate volume excursions experienced during permeating cryoprotectant equilibration and during cooling and warming. However, prior studies have demonstrated that mouse spermatozoa are extremely sensitive to osmotically induced volume changes. A series of three experiments were conducted 1) to test the efficacy of two commonly used extender media components, egg yolk (EY) and skim milk (SM), in broadening the osmotic tolerance limits (OTL) of ICR and B6C3F1 murine spermatozoa; 2) to determine if the extender components affected sperm plasma membrane permeability coefficients for water and cryoprotective agent (CPA) characteristics; and 3) to test the effects of permeating and nonpermeating CPA on mouse sperm morphology. In experiment 1, sperm samples were added to 150, 225, 300, 450, or 600 mOsm NaCl, EY, SM, sucrose, or choline chloride at 22 degrees C and then returned to isosmotic conditions. In experiment 2, epididymal sperm were preequilibrated in 1 M glycerol (Gly) or 2 M ethylene glycol (EG) prepared in SM extender, abruptly exposed to isosmotic conditions at 22, 15, or 2 degrees C, and the corresponding volume excursions were measured and analyzed. In experiment 3, the effects of permeating CPA (0.3 M EG or dimethyl sulfoxide) or nonpermeating CPA (12% sucrose or 18% raffinose) on sperm morphology (i.e., principle midpiece folding and putative membrane fusion) were evaluated. Experiment 1 showed that spermatozoa from ICR and B6C3F1 mice have effectively broader OTL when exposed to EY or SM extenders. The results of experiment 2 indicated that, for ICR sperm, the activation energy (E(a)) for the hydraulic conductivity (L(p)) was unchanged in SM extender. However, for B6C3F1 sperm, there were significant differences in E(a) of L(p) in the presence of Gly and EG. The result of experiment 3 indicated that permeating CPAs damage sperm membrane integrity, causing a high frequency of head-to-tail or tail-to-tail membrane fusion, whereas this occurrence in the presence of nonpermeating CPA was less than 3%. Finally, the results of experiments 1 and 2 were combined in a mathematical model to predict Gly and EG addition and removal in the presence of SM extender, which would prevent mouse sperm membrane damage. These predictions indicated that, for ICR sperm, both Gly and EG may be added and removed in a single step. However, for B6C3F1 spermatozoa, Gly required a two-step addition while EG only required a single step. For removal from B6C3F1 sperm, Gly required a three-step removal process while EG required a two-step removal.     

Mutations in CSPP1 Cause Primary Cilia Abnormalities and Joubert Syndrome with or without Jeune Asphyxiating Thoracic Dystrophy

Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain ∼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.     

Impact of tannic acid on blood pressure,oxidative stress and urinary parameters in L-NNA-induced hypertensive rats

Hypertension is a major health problem with increasing prevalence around the world. Tannic acid is water-soluble polyphenol that is present in tea, green tea, coffee, red wine, nuts, fruits and many plant foods. It has been reported to serve as an antioxidant or a pro-oxidant depending on the type of cells and its concentration. The purpose of our study was to evaluate the effect of tannic acid on systolic blood pressure, oxidative stress and some urinary parameters in the rat model of essential hypertension. Blood pressures of all rats were measured using the tail-cuff method. The nitric oxide synthase inhibitor N (omega)-nitro-L-arginine was administered orally at a dose of 0.5 g/l/day for 15 days to rats in order to create an animal model of hypertension. Tannic acid was intraperitoneally injected at a dose of 50 mg/kg for 15 days. Superoxide dismutase, catalase activity and the concentration of malondialdehyde (MDA) were determined in blood plasma and homogenates of heart, liver and kidney. In order to evaluate renal functions, urine pH, urine volume, urine creatine, uric acid, and urea nitrogen values were measured. Compared with the hypertension group, a decrease in MDA concentrations of heart tissue (p < 0.01), urea nitrogen values (p < 0.01) and urine volumes (p < 0.001) were established in hypertension + tannic acid group. There was also a decrease in blood pressure values (20th and 30th days) of this group, but there was no a statistical difference according to hypertension group. The findings of our research show the effect of tannic acid in lowering blood pressure in hypertensive rats.     

Improved felid embryo development by group culture is maintained with heterospecific companions

Domestic cat embryos of excellent quality appear to improve development of conspecific embryos when cultured together, providing an avenue for improving development of embryos from valuable species or individuals. To have relevance to rare species, it would be useful to understand if this advantage could be conferred by heterospecific companions because there usually are severely limited numbers of conspecific embryos available from wildlife donors. In the first study, we incubated single test cat embryos alone (controls) or with 10 cat embryos or 10 or 20 mouse embryos under similar regimented conditions (each group shared 20 microl medium). In the second study, single test cat embryos were cultured alone, with 10 conspecific or 20 mouse embryos or 10 cattle embryos (each group shared 20 microl medium). Single test embryos in all treatment groups achieved similar (P>0.05) stages of compaction and blastocyst development. In the first study, only the test embryos incubated with 10 cat or 20 mouse companion embryos achieved blastocyst expansion. The average total cell number within test embryos incubated with 10 cat or 20 mouse companions was greater (P<0.05) than controls or those placed with 10 mouse embryos. In the second study, test embryos in all groups achieved blastocyst expansion and had more (P<0.05) total cells per embryo than the solitary controls. Inner cell mass to trophoblast cell ratio did not differ among treatments in either study. Thus, companion mouse and cattle embryos selected for excellent quality confer a benefit to singleton cat embryos, although the number of companions necessary to grant an advantage may be species dependent. If this phenomenon can be extrapolated across species, this may be an avenue for 'common animal embryos' to improve developmental potential of embryos from rare, unrelated taxa.     

Mutations in RIPK4 cause the autosomal-recessive form of popliteal pterygium syndrome

The autosomal-recessive form of popliteal pterygium syndrome, also known as Bartsocas-Papas syndrome, is a rare, but frequently lethal disorder characterized by marked popliteal pterygium associated with multiple congenital malformations. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this malformation syndrome to chromosomal region 21q22.3. Direct sequencing of RIPK4 (receptor-interacting serine/threonine kinase protein 4) showed a homozygous transversion (c.362T>A) that causes substitution of a conserved isoleucine with asparagine at amino acid position 121 (p.Ile121Asn) in the serine/threonine kinase domain of the protein. Additional pathogenic mutations-a homozygous transition (c.551C>T) that leads to a missense substitution (p.Thr184Ile) at a conserved position and a homozygous one base-pair insertion mutation (c.777_778insA) predicted to lead to a premature stop codon (p.Arg260ThrfsX14) within the kinase domain-were observed in two families. Molecular modeling of the kinase domain showed that both the Ile121 and Thr184 positions are critical for the protein's stability and kinase activity. Luciferase reporter assays also demonstrated that these mutations are critical for the catalytic activity of RIPK4. RIPK4 mediates activation of the nuclear factor-κB (NF-κB) signaling pathway and is required for keratinocyte differentiation and craniofacial and limb development. The phenotype of Ripk4(-/-) mice is consistent with the human phenotype presented herein. Additionally, the spectrum of malformations observed in the presented families is similar, but less severe than the conserved helix-loop-helix ubiquitous kinase (CHUK)-deficient human fetus phenotype; known as Cocoon syndrome; this similarity indicates that RIPK4 and CHUK might function via closely related pathways to promote keratinocyte differentiation and epithelial growth.