Hydrogen-1 NMR evidence for three interconverting forms of staphylococcal nuclease: effects of mutations and solution conditions on their distribution

It has been known for several years that 1H NMR spectra of the enzyme staphylococcal nuclease contain resonances due to conformational heterogeneity [Markley, J. L., Williams, M. N., & Jardetzky, O. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 645-651]. One source of conformational heterogeneity has been attributed recently to cis/trans isomeriation of the Lys116-Pro117 peptide bond [Evans, P. A., Dobson, C. M., Kautz, R. A., Hatfull, G., & Fox, R. O. (1987) Nature (London) 329, 266-268]. In this paper we present evidence for three interconverting folded forms of nuclease. Forms N and N' are monomeric; form N" appears at higher nuclease concentrations and probably corresponds to dimerized enzyme. Saturation transfer was used to demonstrate that exchange occurs between the denatured state and N". The effects of temperature, pH, and Ca2+ and nucleotide binding on NMR spectra of nuclease were examined. When the temperature is increased or the pH is lowered, form N' is favored relative to N. Binding of a competitive inhibitor (thymidine 3',5'-bisphosphate plus calcium ion) strongly favors one form of nuclease. 1H NMR spectra of wild-type nuclease, the single-mutant nucleases L89F and H124L, and the double-mutant nuclease F76V+H124L were compared. In the unligated proteins, the equilibrium constant for the conformational equilibrium N in equilibrium with N' is approximately 0.1 in wild-type nuclease and nuclease H124L; by contrast, this equilibrium constant is about 0.7 in nuclease L89F and 1.2 in nuclease F76V+H124L under similar conditions.     

First detection of human metapneumovirus in children with respiratory infections in Romania

The human metapneumovirus (hMPV) was first isolated in 2001 in the Netherlands (Van der Hoogen and collaborators) from a nasopharyngeal aspirate sampled from an infant. Based on the morphological, biochemical and genetic characteristics, the hMPV was initially classified in the genus Metapneumovirus with the avian metapneumovirus (APV), the agent causing the respiratory infections of the upper tract in turkeys and other birds. Subsequently, together with the respiratory syncytial virus (RSV), it was classified in the Pneumovirus genus which is a part of the Pneumovirinae subfamily, the Paramyxoviridae family. The aim of the present study was to optimize hMPV molecular detection and to detect the virus in samples form children with respiratory infections in Romania. Two types of RTPCR commercial kits were evaluated for the detection of hMPV. Tests were performed on 28 pharyngeal exudates from children aged from 9 months to 6 years, which were negative for influenza viruses and for Respiratory Syncytial Virus (RSV). Among the tested samples 7 (25%) have been positive for hMPV by RT-PCR. These results document for the first time that hMPV is circulating in Romania and causes respiratory infections, especially in newborns and children under 6 years old.     

Os fossiles humains des grottes Muierii et Cioclovina, Roumanie

This paper presents the cultural and archaeological context of the human fossil bones from Muierii Cave, dated by us to the age of 30 150 ± 800 ¹⁴C years BP (Before Present) or 34 810 ± 927 cal years BP (calibrated years Before Present), and from Cioclovina Cave, dated to the age of 29 000 ± 700 ¹⁴C years BP or 33 540 ± 832 cal years BP, in the Southern Carpathians. These are among the most ancient dated human fossil remains from Central and South-Eastern Europe and are described in conjunction with other sites with Mousterian assemblages of the recent Neanderthal population, and sites with Aurignacian assemblage of early modern humans, from Romanian region, for the interval of time 34,000-26,000, the transitional period from the Middle Paleolithic to the Upper Paleolithic.     

Heterogeneous amylin fibril growth mechanisms imaged by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy has been used to visualize the fibrillization of amylin, a hormone which in aggregated forms plays a role in type 2 diabetes pathology. Data were obtained at acidic pH where fibrillization is hindered by the charging of histidine 18 and at slightly basic pH where the loss of charge on the histidine promotes aggregation. The experiments show three types of aggregate growth processes. In the earliest steps globular seeds are formed with some expanding radially during the course of the reaction. The dimensions of the globular seeds as well as their staining with the amyloid-specific dye thioflavin T indicate that they are plaques of short fibrils. The next species observed are fibrils that invariably grow from large globular seeds or smaller punctate granules. Fibril elongation appears to be unidirectional, although in some cases multiple fibrils radiate from a single seed or granule. After fibrils are formed, some show an increase in fluorescence intensity that we attribute to the growth of new fibrils alongside those previously formed. All three aggregation processes are suggestive of secondary (heterogeneous) nucleation mechanisms in which nucleation occurs on preformed fibrils. Consistently, electron micrographs show changes in fibril morphology well after fibrils are first formed, and the growth processes observed by fluorescence microscopy occur after the corresponding solution reactions have reached an initial apparent plateau. Taken together, the results highlight the importance of secondary nucleation in the fibrillization of amylin, as this could provide a pathway to continue fibril growth once an initial population of fibrils is established.     

NMR assignments for the Sinorhizobium meliloti response regulator Sma0114

Response regulators are terminal ends of bacterial two-component systems that undergo extensive structural reorganization in response to phosphoryl transfer from their cognate histidine kinases. The response regulator encoded by the gene sma0114 of Sinorhizobium meliloti is a part of a unique class of two-component systems that employ HWE histidine kinases. The distinct features of Sma0114 include a PFxFATGY motif that houses the conserved threonine in the “Y–T coupling” conformational switch which mediates output response through downstream protein–protein interactions, and the replacement of the conserved phenylalanine/tyrosine in Y–T coupling by a leucine. Here we present ¹H, ¹⁵N, and ¹³C NMR assignments for Sma0114. We identify the secondary structure of the protein based on TALOS chemical shift analysis, ³J_HNHα coupling constants and hydrogen–deuterium exchange. The secondary structure determined by NMR is in good agreement with that predicted from the sequence. Both methods suggest that Sma0114 differs from standard CheY-like folds by missing the fourth α-helix. Our initial NMR characterization of Sma0114 paves the way to a full investigation of the structure and dynamics of this response regulator.     

Physiology engages with functional genomics - at last

A report on the XXXV International Congress of Physiological Sciences, held together with Experimental Biology 2005, San Diego, USA, 31 March - 6 April 2005.     

Thermodynamic and structural studies of carbohydrate binding by the agrin-G3 domain

Agrin is a key heparan sulfate proteoglycan involved in the development and maintenance of synaptic junctions between nerves and muscles. Agrin's important functions include clustering acetylcholine receptors on the postsynaptic membranes of muscles and binding to the muscle protein alpha-dystroglycan through its glycan chains. ITC and NMR were used to study the interactions of the C-terminal domain, agrin-G3, with carbohydrates implicated in agrin's functions. Sialic acid caps the glycan chains of alpha-dystroglycan and occurs as a posttranslational modification on the muscle-specific kinase component of the agrin receptor. We found that agrin-G3 binds sialic acid in a Ca2+-dependent manner. ITC data indicate that binding is exothermic and occurs with a 1:1 stoichiometry. NMR chemical shift changes map the sialic acid binding site to the loops that control the domain's acetylcholine receptor clustering activity. By contrast, the glycosaminoglycans heparin and heparan sulfate bind independently of Ca2+. Binding is endothermic, and the binding site spans about 12 saccharide units. The binding site for heparin occupies a similar location but is distinct from that for sialic acid. NMR translational diffusion experiments show that agrin-G3 binds heparin with a 2:1 stoichiometry. Comparisons between the muscle (B0) and neuronal (B8) isoforms of the agrin domain showed very similar Ca2+ and carbohydrate binding properties. Our work identifies agrin-G3 as a functional analogue of the concanavalin A-type lectins, highlights functional similarities between agrin and laminin G domains, and provides mechanistic clues about the roles of carbohydrates in agrin's functions.     

Microbiological quality of tomato (<i>Solanum lycopersicum</i> L.) produced under greenhouse conditions in five Municipalities of the State of Mexico

The aim of the current research was to determine tomato (Solanum lycopersicum L.) microbiological quality produced under greenhouse conditions in 5 municipalities of the State of Mexico. Studies were conducted during the 2013 production cycle to know the risks and apply prevention strategies prior to its consumption. A microbiological analysis of samples of irrigation water, soil and 100 tomato fruits variety cid was performed to determine Aerobic Mesophiles, Total Coliforms and Fecal Coliforms. The methodology used were those according to the Official Mexican Standards NOM- 109-SSA1-1994, NOM-110-SSA1-1994, NOM-092-SSA1-1994, NOM-113-SSA1-1994, and the Regulations of the National French Organization for Standardization (AFNOR) NF V08-60, and NOM-093-SSA1-1994, which establish the allowable limits for the study microorganisms. The results showed a zero level of pollution in water and soil samples. For fruits, levels of Aerobic Mesophilic were within the maximum limits permitted by the standards. The municipality of Texcaltitlan showed the highest average for these microorganisms (10083.80 CFU/mL). Huixquilucan showed 2266.84 CFU/mL for Total Coliforms. For Fecal Coliforms, municipalities of Coatepec and Texcaltitlan exceeded the allowed limit.