Characterization of a caveolin‐1 mutation associated with both pulmonary arterial hypertension and congenital generalized lipodystrophy

Congenital generalized lipodystrophy (CGL) and pulmonary arterial hypertension (PAH) have recently been associated with mutations in the caveolin‐1 ( CAV1 ) gene, which encodes the primary structural protein of caveolae. However, little is currently known about how these CAV1 mutations impact caveolae formation or contribute to the development of disease. Here, we identify a heterozygous F160X CAV1 mutation predicted to generate a C‐terminally truncated mutant protein in a patient with both PAH and CGL using whole exome sequencing, and characterize the properties of CAV1 , caveolae‐associated proteins and caveolae in skin fibroblasts isolated from the patient. We show that morphologically defined caveolae are present in patient fibroblasts and that they function in mechanoprotection. However, they exhibited several notable defects, including enhanced accessibility of the C‐terminus of wild‐type CAV1 in caveolae, reduced colocalization of cavin‐1 with CAV1 and decreased stability of both 8S and 70S oligomeric CAV1 complexes that are necessary for caveolae formation. These results were verified independently in reconstituted CAV1 ^?/? mouse embryonic fibroblasts. These findings identify defects in caveolae that may serve as contributing factors to the development of PAH and CGL and broaden our knowledge of CAV1 mutations associated with human disease.      

Knowledge‐guided laboratory evolution of protein thermolability

In rare but nevertheless important cases it is of practical interest to decrease the thermostability of an enzyme, that is, to increase thermolability in a controlled manner. In the present model study, this unconventional goal has been reached by applying directed evolution to the lipase from Pseudomonas aeruginosa (PAL). By utilizing the B‐factor iterative test (B‐FIT), previously developed to increase the thermostability of enzymes, it was possible to reduce the value from 71.6°C in the case of wild type (WT‐PAL) to 35.6°C (best mutant) without affecting the catalytic profile in terms of substrate acceptance or enantioselectivity at room temperature. Accordingly, saturation mutagenesis was performed at sites in PAL, which on the basis of its X‐ray structure, have the lowest B‐factors indicative of high rigidity. Focused mutations were introduced which can be expected to decrease rigidity, the ensuing increased flexibility leading to higher thermolability without changing the actual catalytic profile. Biotechnol. Bioeng. 2009;102: 1712–1717. © 2008 Wiley Periodicals, Inc.     

The prevalence of factor V Leiden,prothrombin G20210A and methylenetetrahydrofolate reductase polymorphism C677T among G6PD deficient individuals from Western Iran

It has been suggested that the allele frequency of thrombophilic mutations is affected by glucose-6-phosphate dehydrogenase (G6PD) deficiency. The prevalence of thrombophilic mutations were studied in sixty G6PD deficient individuals including 57 males and three females with the mean age of 15 ± 3.08 and 110 age and sex matched healthy individuals consisted of 95 males and 15 females with the mean age of 16.19 ± 2.17 from the Kermanshah Province of Iran. Using a combination of PCR-RFLP technique, single strand conformation polymorphism (SSCP) analysis and DNA sequencing polymorphic G6PD mutations were identified. The factor V Leiden, prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR) C677T were detected by PCR-RFLP method using MnlI, HindIII and HinfI restriction enzymes, respectively. Three mutations, G6PD Mediterranean, G6PD Chatham and G6PD Cosenza were identified in 60 G6PD deficient individuals with highest prevalence of G6PD Mediterranean (91.6%). In G6PD deficient individuals the prevalence of factor V Leiden tended to be higher (5%) compared to healthy individuals (2.7%). The prevalence of prothrombin G20210A mutation in G6PD deficient individuals was 1.7%. However, in normal subjects the prevalence of this mutation was 2.7%. The frequency of T allele in G6PD deficient individuals were insignificantly higher (29.16%) than those in healthy individuals (26.8%). Our finding indicates that the prevalence of factor V Leiden, prothrombin G20210A and MTHFR C677T in G6PD deficient individuals is not statistically different compared to normal subjects and G6PD deficiency is not associated with these thrombophilic mutations in Western Iran.     

Inter-sexual differences in Antarctic fur seal pup growth rates: evidence of environmental regulation?

We investigated the variation of Antarctic fur seal (Arctocephalus gazella) pup growth rates in response to sex, breeding season and duration of both maternal foraging trip and attendance bouts. Data were collected during five consecutive rearing seasons at Cape Shirreff, the most important breeding colony in the South Shetland Islands, Antarctica. Our results showed significant interannual and sexual variations in pup growth rates. Male pups grew significantly faster than female pups during 2000, 2001, 2002 and 2004 seasons, whilst during 2003 no difference was found. The interannual variation in pup growth rates was correlated with the interannual fluctuation in maternal foraging trip and attendance bouts. There was a significant effect of pup sex and maternal foraging trip duration on pup growth rates, which varied between years having foraging trip duration a major effect during 2003, when females spent more time at sea and interestingly on that year there were no sexual differences in pup growth rates. The effect of attendance bout on pup growth rates was not significant. Diet analysis showed that Antarctic krill (Euphausia superba) was the most frequent prey item during the study period. Analysis of krill size distribution showed a significant difference in krill length, during 2003, when A. gazella preyed upon the smallest sizes of krill. In this study, sex was the most important factor on pup growth rates, but when prey availability seemed more limited, there are longer foraging trips and shorter attendance bouts the sex factor became less significant.     

Lamprey <Emphasis Type="Italic">snail</Emphasis> highlights conserved and novel patterning roles in vertebrate embryos

snail genes mark presumptive mesoderm across bilaterian animals. In gnathostome vertebrates, snail genes are a multimember family that are also markers of premigratory neural crest (pnc) and some postmigratory neural crest derivatives in the pharyngeal arches. Previous studies of nonvertebrate chordates indicate that they have single snail genes that retain ancestral functions in mesoderm development and perhaps in specification of a pnc-like cell population. Lampreys are the most basal extant vertebrates, with well-defined developmental morphology. Here, we identify a single snail gene from the lamprey Petromyzon marinus that is the phylogenetic outgroup of all gnathostome snail genes. This single lamprey snail gene retains ancestral snail patterning domains present in nonvertebrate chordates. Lamprey snail is also expressed in tissues that are broadly equivalent to the combined sites of expression of all three gnathostome snail paralogy groups, excepting in embryonic tissues that are unique to gnathostomes. Importantly, while snail does not appear to demarcate an early neural crest population in lampreys as it does in gnathostomes, it may be involved in later neural crest development. Together, our results indicate that significant cis-regulatory innovation occurred in a single snail gene before the vertebrate radiation, and significant subfunctionalization occurred after snail gene duplications in the gnathostome lineages.     

Analysis of beta-hemolysis in human blood agars by Streptococcus pyogenes

The aim of the study was to assess the reliability of human blood agar media (HuBA) in identifying Streptococcus pyogenes by hemolysis analysis. We analyze several factors that might affect the accuracy of HuBA media for microbial analysis, including incubation time, blood group, Rh factor and presence of antistreptolysin-o.     

Escitalopram or Novel Herbal Mixture Treatments during or following Exposure to Stress Reduce Anxiety-Like Behavior through Corticosterone and BDNF Modifications

Anxiety disorders are a major public health concern worldwide. Studies indicate that repeated exposure to adverse experiences early in life can lead to anxiety disorders in adulthood. Current treatments for anxiety disorders are characterized by a low success rate and are associated with a wide variety of side effects. The aim of the present study was to evaluate the anxiolytic effects of a novel herbal treatment, in comparison to treatment with the selective serotonin reuptake inhibitor escitalopram. We recently demonstrated the anxiolytic effects of these treatments in BALB mice previously exposed to one week of stress. In the present study, ICR mice were exposed to post natal maternal separation and to 4 weeks of unpredictable chronic mild stress in adolescence, and treated during or following exposure to stress with the novel herbal treatment or with escitalopram. Anxiety-like behavior was evaluated in the elevated plus maze. Blood corticosterone levels were evaluated using radioimmunoassay. Brain derived neurotrophic factor levels in the hippocampus were evaluated using enzyme-linked immunosorbent assay. We found that (1) exposure to stress in childhood and adolescence increased anxiety-like behavior in adulthood; (2) the herbal treatment reduced anxiety-like behavior, both when treated during or following exposure to stress; (3) blood corticosterone levels were reduced following treatment with the herbal treatment or escitalopram, when treated during or following exposure to stress; (4) brain derived neurotrophic factor levels in the hippocampus of mice treated with the herbal treatment or escitalopram were increased, when treated either during or following exposure to stress. This study expands our previous findings and further points to the proposed herbal compound''s potential to be highly efficacious in treating anxiety disorders in humans.     

Comparative Evaluation of Cardiac Markers in Differentiated Cells from Menstrual Blood and Bone Marrow-Derived Stem Cells In Vitro

In recent years, menstrual blood-derived stem cells (MenSCs) have been introduced as easily accessible and refreshing stem cell source without ethical considerations in the field of regenerative medicine. The aim of this study was to investigate in vitro cardiac differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs) under two protocols using 5-aza-2′-deoxycytidine (5-aza) and basic fibroblast growth factor (bFGF). Our data revealed that differentiated MenSCs and BMSCs acquired some features of cardiomyocytes; however, degree of differentiation was dependent on the protocol. In a similar manner with BMSCs, differentiated MenSCs showed upper levels of mRNA/protein of late-stage cardiac markers under 5-aza stimulation and continuous treatment with bFGF (protocol 2) compared to those induced by 5-aza alone (protocol 1) evidencing the key role of bFGF in cardiac development of stem cells. Compared to corresponding undifferentiated cells differentiated MenSCs under protocol 2 showed remarkable expression of connexin-43 and TNNT2 at both gene and protein levels, whereas developed BMSCs under the same condition only expressed connextin-43 at the higher level. Superiority of protocol 2 over protocol 1 was confirmed by assessment of LDH and cTnI production by differentiated cells. Based on the accumulative data, our study provided convincing evidence that MenSCs have relatively higher capability to be differentiated toward cardiomyocyte compared with BMSCs. Furthermore, usage of bFGF and 5-aza to induce in vitro cardiac differentiation of MenSCs is highly recommended.