Biochemical characterization of a novel beta-1--3, 1--4 glucan 4-glucanohydrolase from Thermomonospora sp. having a single active site for lichenan and xylan

A bifunctional high molecular weight (Mr, 64,500 Da) beta-1-3, 1-4 glucan 4-glucanohydrolase was purified to homogeneity from Thermomonospora sp., exhibiting activity towards lichenan and xylan. A kinetic method was used to analyze the active site that hydrolyzes lichenan and xylan. The experimental data was in agreement with the theoretical values calculated for a single active site. Probing the conformation and microenvironment at active site of the enzyme by fluorescent chemo-affinity label, OPTA resulted in the formation of an isoindole derivative with complete inactivation of the enzyme to hydrolyse both lichenan and xylan confirmed the results of kinetic method. OPTA forms an isoindole derivative by cross-linking the proximal thiol and amino groups. The modification of cysteine and lysine residues by DTNB and TNBS respectively abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating the participation of cysteine and lysine in the formation of isoindole complex.     

History of click-speaking populations of Africa inferred from mtDNA and Y chromosome genetic variation

Little is known about the history of click-speaking populations in Africa. Prior genetic studies revealed that the click-speaking Hadza of eastern Africa are as distantly related to click speakers of southern Africa as are most other African populations. The Sandawe, who currently live within 150 km of the Hadza, are the only other population in eastern Africa whose language has been classified as part of the Khoisan language family. Linguists disagree on whether there is any detectable relationship between the Hadza and Sandawe click languages. We characterized both mtDNA and Y chromosome variation of the Sandawe, Hadza, and neighboring Tanzanian populations. New genetic data show that the Sandawe and southern African click speakers share rare mtDNA and Y chromosome haplogroups; however, common ancestry of the 2 populations dates back >35,000 years. These data also indicate that common ancestry of the Hadza and Sandawe populations dates back >15,000 years. These findings suggest that at the time of the spread of agriculture and pastoralism, the click-speaking populations were already isolated from one another and are consistent with relatively deep linguistic divergence among the respective click languages.     

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.     

Engineering of a linear inactive analog of human β‐defensin 4 to generate peptides with potent antimicrobial activity

Human β‐defensins (HBDs) are cationic antimicrobial peptides constrained by three disulfide bridges. They have diverse range of functions in the innate immune response. It is of interest to investigate whether linear analogs of defensins can be generated, which possess antimicrobial activity. In this study, we have designed linear peptides with potent antimicrobial activity from an inactive peptide spanning the N‐terminus of HBD4. Our results show that l ‐arginine to d ‐arginine substitution imparts considerable antimicrobial activity against both bacteria and Candida albicans. Increase in hydrophobicity by fatty acylation of the peptides with myristic acid further enhances their potency. In the presence of high concentrations of salt, antimicrobial activity of the myristoylated peptide with l ‐arginine is attenuated relatively to a lesser extent as compared with the linear active peptide with d ‐arginine. Substitution of cysteine with the hydrophobic helix‐promoting amino acid α‐aminoisobutyric acid favors candidacidal activity but not antibacterial activity. The mechanism of killing by d ‐arginine substituted unacylated analog involves transient interaction with the bacterial membrane followed by translocation into the cytoplasm without membrane permeabilization. Accumulation of peptides in the cytoplasm can affect various cellular processes that lead to cell death. However, the peptide causes membrane permeabilization in case of C. albicans. Myristoylation results in greater interaction of the peptide chain with the microbial cell surface and causes membrane permeabilization. Results described in the study demonstrate that it is possible to generate highly active linear analogs of defensins by selective introduction of d ‐amino acids and fatty acids, which could be attractive candidates for development as therapeutic agents. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

N-terminal myristoylation alters the calcium binding pathways in neuronal calcium sensor-1

Neuronal calcium sensor-1 (NCS-1) interacts with many membranes and cytosolic proteins, both in a Ca²⁺-dependent and in a Ca₂₊-independent manner, and its physiological role is governed by its N-terminal myristoylation. To understand the role of myristoylation in altering Ca²⁺ response and other basic biophysical properties, we have characterized the Ca²⁺ filling pathways in both myristoylated (myr) and non-myristoylated (non-myr) forms of NCS-1. We have observed that Ca²⁺ binds simultaneously to all three active EF-hands in non-myr NCS-1, whereas in the case of myr NCS-1, the process is sequential, where the second EF-hand is filled first, followed by the third and fourth EF-hands. In the case of myr NCS-1, the observed sequential Ca²⁺ binding process becomes more prominent in the presence of Mg²⁺. Besides, the analysis of ¹⁵N-relaxation data reveals that non-myr NCS-1 is more dynamic than myr NCS-1. The overall molecular tumbling correlation time increases by approximately 20% upon myristoylation. Comparing the apo forms of non-myr NCS-1 and myr NCS-1, we found the possibility of existence of some substates, which are structurally closer to the holo form of the protein. There are more such substates in the case of non-myr NCS-1 than in the case of the myr NCS-1, suggesting that the former accesses larger volumes of conformational substates compared with the latter. Further, the study reveals that the possibility of Ca²⁺ binding simultaneously to different parts of the protein is more favourable in non-myr NCS-1 than in myr NCS-1.     

Conformation and activity of delta-lysin and its analogs

Delta-Lysin is a 26-residue hemolytic peptide secreted by Staphylococcus aureus. Unlike the bee venom peptide melittin, delta-lysin does not exhibit antibacterial activity. We have synthesized delta-lysin and several analogs wherein the N-terminal residues of the toxin were sequentially deleted. The toxin has three aspartic acids, four lysines and no prolines. Analogs were also generated in which all the aspartic acids were replaced with lysines. A proline residue was introduced in the native sequences as well as in the analogs where aspartic acids were replaced with lysines. We observed that 20- and 22-residue peptides corresponding to residues 7-26 and 5-26 of delta-lysin, respectively, had greater hemolytic activity than the parent peptide. These shorter peptides, unlike delta-lysin, did not self-associate to adopt alpha-helical conformation in water, at lytic concentrations. Introduction of proline or substitution of aspartic acids by lysines resulted in loss in propensity to adopt helical conformation in water. When proline was introduced in the peptides corresponding to the native toxin sequence, loss of hemolytic activity was observed. Substitution of all the aspartic acids with lysines resulted in enhanced hemolytic activity in all the analogs. However, when both proline and aspartic acid to lysine changes were made, only antibacterial activity was observed in the shorter peptides. Our investigations on delta-lysin and its analogs provide insights into the positioning of anionic, cationic residues and proline in determining hemolytic and antibacterial activities.     

Recognition of partially concealed leopards by wild bonnet macaques (Macaca radiata). The role of the spotted coat

Wild bonnet macaques (Macaca radiata) have been shown to recognize models of leopards (Panthera pardus), based on their configuration and spotted yellow coat. This study examined whether bonnet macaques could recognize the spotted and dark melanic morph when partially concealed by vegetation. Seven troops were studied at two sites in southern India, the Mudumalai Wildlife Sanctuary and the Kalakad-Mundanthurai Tiger Reserve. The forequarters and hindquarters of the two leopard morphs were presented from behind thick vegetation to individuals at feeding stations 25 m away. Flight reaction times and frequency of flight were obtained from video for only those individuals who oriented towards the models prior to hearing alarm calls. Bonnet macaques exhibited faster reaction times and greater frequency of flight after looking at the spotted morph's forequarter than after looking at either its spotted hindquarter or the dark morph's forequarter. The hindquarter of the dark morph was ignored completely. Artificial neural network modeling examined the perceptual aspects of leopard face recognition and the role of spots as camouflage. When spots were integrated into the pattern recognition process via network training, these spots contributed to leopard face recognition. When networks were not trained with spots, spots did not act as camouflage by disrupting facial features.