Identification of subsidiary catalytic groups at the active site of beta-ketoacyl-CoA thiolase by covalent modification of the protein

beta-Ketoacyl-CoA thiolase (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) is known to possess sulfhydryl groups of cysteines at the active site that are essential for its catalytic activity. Other groups at the active site that participate in the catalytic process were identified by using anhydride reagents which covalently modify the protein by specifically reacting with any amino groups potentially present at the active site. Since these reagents may also react with thiol groups, the enzyme's amino groups were modified after masking the cysteine thiols present by an alkylalkane thiosulfonate-type reagent, methyl methanethiol-sulfonate (MMTS), that selectively formed a disulfide bridge, thus generating an inactive thiolmethylated enzyme. When this procedure was followed, the enzyme could be undoubtedly modified at its amino by the anhydride reagent, leading to a doubly modified protein. The thiomethyl group could then be removed by reduction with dithiothreitol, yielding an enzyme modified solely on the amino residues. The amino group could be unblocked in turn by exposure to acidic pH. The different anhydrides inactivated thiolase, but only acetoacetyl coenzyme A (AcAcCoA) provided any protection against inactivation. When thiolmethylcitraconyl thiolase was reduced with dithiothreitol the enzyme remained inactive, but when the doubly modified enzyme was exposed to pH 5 then the reduction led to formation of an active enzyme. These results are interpreted as demonstrating a role for an amino group at the enzyme active site. A catalytic mechanism is proposed for the enzyme which involves the amino group.     

The impact of COVID-19 on clinical research for Neglected Tropical Diseases (NTDs): A case study of bubonic plague

BackgroundAmong the many collaterals of the COVID-19 pandemic is the disruption of health services and vital clinical research. COVID-19 has magnified the challenges faced in research and threatens to slow research for urgently needed therapeutics for Neglected Tropical Diseases (NTDs) and diseases affecting the most vulnerable populations. Here we explore the impact of the pandemic on a clinical trial for plague therapeutics and strategies that have been considered to ensure research efforts continue.MethodsTo understand the impact of the COVID-19 pandemic on the trial accrual rate, we documented changes in patterns of all-cause consultations that took place before and during the pandemic at health centres in two districts of the Amoron’I Mania region of Madagascar where the trial is underway. We also considered trends in plague reporting and other external factors that may have contributed to slow recruitment.ResultsDuring the pandemic, we found a 27% decrease in consultations at the referral hospital, compared to an 11% increase at peripheral health centres, as well as an overall drop during the months of lockdown. We also found a nation-wide trend towards reduced number of reported plague cases.DiscussionCOVID-19 outbreaks are unlikely to dissipate in the near future. Declining NTD case numbers recorded during the pandemic period should not be viewed in isolation or taken as a marker of things to come. It is vitally important that researchers are prepared for a rebound in cases and, most importantly, that research continues to avoid NTDs becoming even more neglected.     

Egg-derived tyrosine phosphatase as a Potential Biomarker for Muscle Ageing and Degeneration in Drosophila melanogaster

Ageing is associated with declined activity of behaviors, physiology and metabolic processes （Arking, 2006）. In- vestigations in model organisms have indicated the exis- tence of ＂functional senescence＂, the progressive decline of biological functions with age and the decline in the activity may vary from tissue to tissue. Consequently, studies per- taining to the key organs/tissues whose functions deterio- rate/fail with age have led to the development of tissue specific ageing biomarkers （Grotewiel et al., 2005; Demontis et al., 2013）.     

Occurrence,conformational features and amino acid propensities for the pi-helix

The most abundant helix type in proteins is the alpha-helix, accounting for about 31% of amino acid secondary structure states, while the 3(10)-helix accounts for about 4%. The pi-helix appears to be extremely rare and is considered to be unstable. Existing secondary structure definition methods find very few within the Protein Data Bank. Using an improved pi-helix definition algorithm to search a non-redundant subset of high-resolution and well-refined protein structures, we found that almost every tenth protein contained a pi-helix. This enabled us to show for the first time that the pi-helix has structural parameters that are different from the hypothesized model values. It also has distinctive amino acid preferences and it is conserved within functionally related proteins. Features that may contribute to the stability of the pi-helical structure have also been identified. In addition to hydrogen bonds, several other factors contribute to the stability of pi-helices. The pi-helix may have some functional advantages over other helical structures. Thus, we describe cases where the side chains of functionally important residues at every fourth position within a pi-helix could be aligned and brought close together in a way that would not be allowed by any other helix type.     

Comparative efficiency of wheat straw and sugarcane bagasse biochar reduces the cadmium bioavailability to spinach and enhances the microbial activity in contaminated soil

Abstract

Biochar is considered a novel soil amendment for cadmium (Cd) stabilization in contaminated soils. A pot experiment was conducted to examine the efficiency of wheat straw and sugarcane bagasse induced biochar on Cd mobility in soil and its bioavailability to spinach in contaminated soil. Soil pH, Cd contents in plant tissues and microbial biomass were examined. Results showed that Cd was significantly decreased by 30.95% and 20.83% with wheat straw and sugarcane bagasse biochar at 2% application rate respectively, relative to the control. Similarly, Cd contents were decreased in plants shoots by 15.41 and 14.33%, while in roots by 48.3 and 35.54%, when wheat straw and sugarcane biochar were added at 2% application rate respectively. Moreover, soil microbial biomass was significantly increased with the application of all biochar types and their applications rates. Finally, wheat straw biochar at 2% application rate can be considered as an effective approach for Cd stabilization in contaminated soils.     

First report of Streptococcus agalactiae and Lactococcus garvieae from a wild bottlenose dolphin (Tursiops truncatus)

The isolation and characterization of two bacterial species, Streptococcus agalactiae and Lactococcus garvieae, previously unreported in wild marine mammals are described from a freshly dead bottlenose dolphin, Tursiops truncatus, from Kuwait Bay, Kuwait, in September 2001. Conventional and rapid identification systems were used to determine that isolates from muscle and kidney were S. agalactiae and L. garvieae, respectively. The isolates were gram-positive, catalase-negative, oxidase-negative, nonhemolytic cocci. The S. agalactiae was serotyped to group antigen B, whereas the L. garvieae could not be assigned to any serogroup. These Kuwait isolates displayed considerable homogeneity with corresponding American Type Culture Collection (ATCC) type isolates. Although the dolphin S. agalactiae isolate was nonhemolytic, it was biochemically similar to S. agalactiae isolated from mullet sampled in the concurrent Kuwait Bay fish kill. Some biochemical heterogeneity was observed between the dolphin isolates and corresponding mammalian ATCC type isolates, especially with Voges Proskauer, alanine-phenylanaline-proline arylamidase, and alpha-galactosidase tests. Nile tilapia, Oreochromis niloticus, experimentally infected with the dolphin S. agalactiae and L. garvieae isolates experienced 90% and 0% mortalities, respectively. This is the first isolation of S. agalactiae and L. garvieae from a wild marine mammal, and the microbial characteristics established here provide pertinent information for the future isolation of these bacteria.     

Control of post-translational modifications in antithrombin during murine post-natal development by miR-200a

Background

Developmental haemostatic studies may help identifying new elements involved in the control of key haemostatic proteins like antithrombin, the most relevant endogenous anticoagulant.

Results

In this study, we showed a significant reduction of sialic acid content in neonatal antithrombin compared with adult antithrombin in mice. mRNA levels of St3gal3 and St3gal4, two sialyltransferases potentially involved in antithrombin sialylation, were 85% lower in neonates in comparison with adults. In silico analysis of miRNAs overexpressed in neonates revealed that mir-200a might target these sialyltransferases. Moreover, in vitro studies in murine primary hepatocytes sustain this potential control.

Conclusions

These data suggest that in addition to the direct protein regulation, microRNAs may also modulate qualitative traits of selected proteins by an indirect control of post-translational processes.     

A novel locus (DFNA24) for prelingual nonprogressive autosomal dominant nonsyndromic hearing loss maps to 4q35-qter in a large Swiss German kindred

Nonsyndromic hearing loss is one of the most genetically heterogeneous traits known. A total of 30 autosomal dominant nonsyndromic hearing-loss loci have been mapped, and 11 genes have been isolated. In the majority of cases, autosomal dominant nonsyndromic hearing loss is postlingual and progressive, with the exception of hearing impairment in families in which the impairment is linked to DFNA3, DFNA8/12, and DFNA24, the novel locus described in this report. DFNA24 was identified in a large Swiss German kindred with a history of autosomal dominant hearing loss that dates back to the middle of the 19th century. The hearing-impaired individuals in this kindred have prelingual, nonprogressive, bilateral sensorineural hearing loss affecting mainly mid and high frequencies. The DFNA24 locus maps to 4q35-qter. A maximum multipoint LOD score of 11.6 was obtained at 208.1 cM at marker D4S1652. The 3.0-unit support interval for the map position of this locus ranges from 205.8 cM to 211.7 cM (5.9 cM).     

A decade of progress in understanding the structural basis of protein synthesis

The key reaction of protein synthesis, peptidyl transfer, is catalysed in all living organisms by the ribosome - an advanced and highly efficient molecular machine. During the last decade extensive X-ray crystallographic and NMR studies of the three-dimensional structure of ribosomal proteins, ribosomal RNA components and their complexes with ribosomal proteins, and of several translation factors in different functional states have taken us to a new level of understanding of the mechanism of function of the protein synthesis machinery. Among the new remarkable features revealed by structural studies, is the mimicry of the tRNA molecule by elongation factor G, ribosomal recycling factor and the eukaryotic release factor 1. Several other translation factors, for which three-dimensional structures are not yet known, are also expected to show some form of tRNA mimicry. The efforts of several crystallographic and biochemical groups have resulted in the determination by X-ray crystallography of the structures of the 30S and 50S subunits at moderate resolution, and of the structure of the 70S subunit both by X-ray crystallography and cryo-electron microscopy (EM). In addition, low resolution cryo-EM models of the ribosome with different translation factors and tRNA have been obtained. The new ribosomal models allowed for the first time a clear identification of the functional centres of the ribosome and of the binding sites for tRNA and ribosomal proteins with known three-dimensional structure. The new structural data have opened a way for the design of new experiments aimed at deeper understanding at an atomic level of the dynamics of the system.