Interactions within the coiled-coil domain of RetGC-1 guanylyl cyclase are optimized for regulation rather than for high affinity

RetGC-1, a member of the membrane guanylyl cyclase family of proteins, is regulated in photoreceptor cells by a Ca(2+)-binding protein known as GCAP-1. Proper regulation of RetGC-1 is essential in photoreceptor cells for normal light adaptation and recovery to the dark state. In this study we show that cGMP synthesis by RetGC-1 requires dimerization, because critical functions in the catalytic site must be provided by each of the two polypeptide chains of the dimer. We also show that an intact alpha-helical coiled-coil structure is required to provide dimerization strength for the catalytic domain of RetGC-1. However, the dimerization strength of this domain must be precisely optimized for proper regulation by GCAP-1. We found that Arg(838) within the dimerization domain establishes the Ca(2+) sensitivity of RetGC-1 by determining the strength of the coiled-coil interaction. Arg(838) substitutions dominantly enhance cGMP synthesis even at the highest Ca(2+) concentrations that occur in normal dark-adapted photoreceptor cells. Molecular dynamics simulations suggest that Arg(838) substitutions disrupt a small network of salt bridges to allow an abnormal extension of coiled-coil structure. Substitutions at Arg(838) were first identified by linkage to the retinal degenerative disease, autosomal dominant cone rod dystrophy (adCORD). Consistent with the characteristics of this disease, the Arg(838)-substituted RetGC-1 mutants exhibit a dominant biochemical phenotype. We propose that accelerated cGMP synthesis in humans with adCORD is the primary cause of cone-rod degeneration.     

Pattern of skeletal malformations produced by Dominant hemimelia (Dh)

The hindlimb malformations in adult mice heterozygous for the dominant gene Dominant hemimelia (Dh) and +/+ littermates were characterized in skeletons that had been fixed, stained, and cleared. When the tibia was shortened, the deficiency was always an absence of the distal portion, and never the proximal portion. Although tibial hemimelia has been well documented in Dh mice, this study demonstrated a distinctive pattern of shortening of the tibia. Measurements of the length of the tibia (relative to the length of the humerus) showed only three patterns of shortening of the tibia (i.e., mild, moderate, and severe), rather than a continuous spectrum of shortening from mild to complete absence. The hindlimb malformation of Dh/+ mice occurred in association with a reduced number (five) of lumbar vertebrae. The interrelationship of the hindlimb malformations and the reduction in the vertebral number suggests a relationship between the development of the axial skeleton and the abnormal limb.     

Nucleotide binding activity of SecA homodimer is conformationally regulated by temperature and altered by prlD and azi mutations

SecA ATPase is critical for protein translocation across the Escherichia coli inner membrane. To understand this activity further, the high affinity nucleotide binding activity of SecA was characterized. We found that at 4 degrees C SecA homodimer binds one ADP molecule with high affinity. This nucleotide binding activity was conformationally regulated by temperature: at low temperature SecA affinity for ADP was high with a slow exchange rate, whereas at high temperature the converse was true. Azi- and PrlD-SecA proteins that confer azide-resistant and signal sequence suppressor phenotypes were found to have reduced affinity for ADP and accelerated exchange rates compared with wild type SecA. Consistent with this observation, fluorescence and proteolysis studies indicated that these proteins had a conformationally relaxed state at a reduced temperature compared with SecA. The level of Azi- and PrlD-SecA protein was also elevated in inverted membrane vesicles where it displayed higher membrane ATPase activity. These results provide the first direct evidence for conformational regulation of the SecA-dependent nucleotide cycle, its alteration in azi and prlD mutants, and its relevance to in vivo protein export.     

Putative Virulence Traits and Pathogenicity of Vibrio cholerae Non-O1, Non-O139 Isolates from Surface Waters in Kolkata, India

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (≥100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.     

Safety and Immunogenicity of a Live Oral Recombinant Cholera Vaccine VA1.4: A Randomized,Placebo Controlled Trial in Healthy Adults in a Cholera Endemic Area in Kolkata,India

Background

A live oral cholera vaccine VA 1.4 developed from a non-toxigenic Vibrio cholerae O1 El Tor strain using ctxB gene insertion was further developed into a clinical product following cGMP and was evaluated in a double-blind randomized placebo controlled parallel group two arm trial with allocation ratio of 1∶1 for safety and immunogenicity in men and women aged 18–60 years from Kolkata, India.

Method

A lyophilized dose of 1.9×10⁹ CFU (n = 44) or a placebo (n = 43) reconstituted with a diluent was administered within 5 minutes of drinking 100 ml of a buffer solution made of sodium bicarbonate and ascorbic acid and a second dose on day 14.

Result

The vaccine did not elicit any diarrhea related adverse events. Other adverse events were rare, mild and similar in two groups. One subject in the vaccine group excreted the vaccine strain on the second day after first dose. The proportion of participants who seroconverted (i.e. had 4-folds or higher rise in reciprocal titre) in the vaccine group were 65.9% (95% CI: 50.1%–79.5%) at both 7 days (i.e. after 1^st dose) and 21 days (i.e. after 2^nd dose). None of the placebo recipients seroconverted. Anti-cholera toxin antibody was detected in very few recipients of the vaccine.

Conclusion

This study demonstrates that VA 1.4 at a single dose of 1.9×10⁹ is safe and immunogenic in adults from a cholera endemic region. No additional benefit after two doses was seen.

Trial Registration

Clinical Trials Registry-India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2012/04/002582     

Parental Age Affects Somatic Mutation Rates in the Progeny of Flowering Plants

In humans, it is well known that the parental reproductive age has a strong influence on mutations transmitted to their progeny. Meiotic nondisjunction is known to increase in older mothers, and base substitutions tend to go up with paternal reproductive age. Hence, it is clear that the germinal mutation rates are a function of both maternal and paternal ages in humans. In contrast, it is unknown whether the parental reproductive age has an effect on somatic mutation rates in the progeny, because these are rare and difficult to detect. To address this question, we took advantage of the plant model system Arabidopsis (Arabidopsis thaliana), where mutation detector lines allow for an easy quantitation of somatic mutations, to test the effect of parental age on somatic mutation rates in the progeny. Although we found no significant effect of parental age on base substitutions, we found that frameshift mutations and transposition events increased in the progeny of older parents, an effect that is stronger through the maternal line. In contrast, intrachromosomal recombination events in the progeny decrease with the age of the parents in a parent-of-origin-dependent manner. Our results clearly show that parental reproductive age affects somatic mutation rates in the progeny and, thus, that some form of age-dependent information, which affects the frequency of double-strand breaks and possibly other processes involved in maintaining genome integrity, is transmitted through the gametes.In humans, it has long been recognized that the reproductive age of the parents has an influence on the health of their progeny. An older reproductive age of the mother is known to increase the fraction of aneuploid gamete formation (Hurles, 2012). For instance, the risk for a trisomy increases from 2% to 3% for mothers in their 20s to more than 30% for mothers in their 40s (Hassold and Hunt, 2009). The age of the father also has an effect on the frequency of spontaneous congenital disorders and common complex diseases, such as autism and some cancers (Goriely and Wilkie, 2012). Indeed, sperm from 36- to 57-year-old men have more double-strand breaks (DSBs) than those of 20- to 35-year-old individuals (Singh et al., 2003). Similarly, the efficiency of DSB repair was reported to decrease with age in vegetative tissues of the plant model system Arabidopsis (Arabidopsis thaliana; Boyko et al., 2006).Owing to the continuous divisions of spermatogonial stem cells, the male germline of humans is thought to be more mutagenic than the female germline. Indeed, it was shown that the paternal germline is more mutagenic than the maternal one with respect to base substitutions (Kong et al., 2012) and replication slippage errors at microsatellites (Sun et al., 2012). It is also known that carriers of germline mutations in mismatch repair (MMR) genes in humans are prone to get colorectal cancer and that the risk depends on the parent-of-origin of the mutation (van Vliet et al., 2011). The molecular basis of these parental effects is not entirely clear but is likely to involve higher rates of nondisjunction during female meiosis, higher mutation rates during spermatogenesis, and probably additional effects of aging.In contrast to the effect of parental age on germline mutations, not much is known about potential effects of parental reproductive age on somatic mutation rates in the offspring. However, it has been shown in animal studies that radiation of males can lead to somatic mutations in their progeny—and subsequent generations—that cannot be attributed to mutations in the paternal germline (for review, see Little et al., 2013). Moreover, several recent studies have illustrated the existence of complex parental and transgenerational effects in humans, although their molecular basis is not clear (Grossniklaus et al., 2013). These effects can be of either genetic nature (but the effect is seen even in offspring that did not inherit the genetic variant from their parents; for review, see Nadeau, 2009) or epigenetic nature (where environmental influences can possibly exert effects on subsequent generations; for review, see Pembrey et al., 2006; Pembrey, 2010; Curley et al., 2011). It is currently not known whether such parental effects affect the somatic mutation rates in the offspring or whether the effects are modulated by parental age.Taking advantage of the plant model system Arabidopsis, in which various somatic mutation rates can readily be assessed (Bashir et al., 2014), we investigated the effects of parental reproductive age on somatic mutation rates in the progeny. We report that there is a pronounced effect of parental age on somatic mutation rates in their offspring in a parent-of-origin-dependent fashion. Thus, some form of parental information, which is inherited through the gametes to the next generation, seems to alter the somatic mutation rates in the progeny and changes with parental reproductive age.