Reconstitution of a minimal DNA replicase from Pseudomonas aeruginosa and stimulation by non-cognate auxiliary factors

DNA polymerase III holoenzyme is responsible for chromosomal replication in bacteria. The components and functions of Escherichia coli DNA polymerase III holoenzyme have been studied extensively. Here, we report the reconstitution of replicase activity by essential components of DNA polymerase holoenzyme from the pathogen Pseudomonas aeruginosa. We have expressed and purified the processivity factor (beta), single-stranded DNA-binding protein, a complex containing the polymerase (alpha) and exonuclease (epsilon) subunits, and the essential components of the DnaX complex (tau(3)deltadelta'). Efficient primer elongation requires the presence of alphaepsilon, beta, and tau(3)deltadelta'. Pseudomonas aeruginosa alphaepsilon can substitute completely for E. coli polymerase III in E. coli holoenzyme reconstitution assays. Pseudomonas beta and tau(3)deltadelta' exhibit a 10-fold lower activity relative to their E. coli counterparts in E. coli holoenzyme reconstitution assays. Although the Pseudomonas counterpart to the E. coli psi subunit was not apparent in sequence similarity searches, addition of purified E. coli chi and psi (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas tau(3)deltadelta' complex approximately 10-fold and enables the reconstituted enzyme to function better under physiological salt conditions.     

Pathogenicity of simian-human immunodeficiency virus SHIV-89.6P and SIVmac is attenuated in cynomolgus macaques and associated with early T-lymphocyte responses

Because most studies of AIDS pathogenesis in nonhuman primates have been performed in Indian-origin rhesus macaques (Macaca mulatta), little is known about lentiviral pathogenicity and control of virus replication following infection of alternative macaque species. Here, we report the consequences of simian-human immunodeficiency virus SHIV-89.6P and SIVmac251 infection in cynomolgus (Macaca fascicularis) and rhesus macaques of Chinese origin. Compared to the pathogenicity of the same viruses in Indian rhesus macaques, both cynomolgus and Chinese rhesus macaques showed lower levels of plasma virus. By 9 to 10 months after infection, both viruses became undetectable in plasma more frequently in cynomolgus than in either Chinese or Indian rhesus macaques. Furthermore, after SHIV-89.6P infection, CD4+ T-cell numbers declined less and survival was longer in cynomolgus and Chinese rhesus macaques than in Indian rhesus macaques. This attenuated pathogenicity was associated with gamma interferon ELISPOT responses to Gag and Env that were generated earlier and of higher frequency in cynomolgus than in Indian rhesus macaques. Cynomolgus macaques also developed higher titer neutralizing antibodies against SHIV-89.6 at 10 and 20 weeks postinoculation than Indian rhesus macaques. These studies demonstrate that the pathogenicity of nonhuman primate lentiviruses varies markedly based on the species or geographic origin of the macaques infected and suggest that the cellular immune responses may contribute to the control of pathogenicity in cynomolgus macaques. While cynomolgus and Chinese rhesus macaques provide alternative animal models of lentiviral infection, the lower levels of viremia in cynomolgus macaques limit the usefulness of infection of this species for vaccine trials that utilize viral load as an experimental endpoint.     

Deciphering the Role of EGL-3 for Neuropeptides Processing in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Using High-Resolution Quadrupole–Orbitrap Mass Spectrometry

Neuropeptides are derived from large and inactive proteins which require endoproteolytic processing for the biosynthesis of the bioactive peptides. The maturation of pro-neuropeptide to neuropeptide is believed to be performed by ortholog pro-protein convertase EGL-3 in Caenorhabditis elegans (C. elegans). Furthermore, ortholog of Cathepsin L, CPL-1 are found in C. elegans and can potentially cleave paired basic amino acids at the N-terminal suggesting the presence of both pathways. The objective of this study was to decipher the role of EGL-3 in the proteolysis of FMRF amide-related peptides (FLPs) or neuropeptide-like proteins (NLPs) using synthetic surrogate peptides based on a universal enzymatic cleavage pattern published by Schechter and Berger and used widely in enzymology. The results show evidence that proteolysis controls FLP-21 and NLP-8 related neuropeptide levels in C. elegans. Surrogate peptides were degraded rapidly when exposed to C. elegans S9 fractions leading to the formation of specific peptide fragments related to EGL-3 and CPL-1 pathway. The results suggest that CPL-1 pathway does not compensate for the loss of the EGL-3 pathway. Proteolysis of pro-neuropeptides associated to FLP-21 and NLP-8 in elg-3 mutants are severely hampered leading to a lack of mature bioactive neuropeptides.     

Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications

Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle. Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture. The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively. Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence. Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases. Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases. Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme. Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology. The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed.     

Catalytic activity of rabbit skeletal muscle aldolase in the crystalline state

The monoclinic crystalline form of aldolase from rabbit skeletal muscle grown at 29 degrees C is catalytically active in the direction of aldol cleavage. Activity was assayed for in a crystallization buffer containing 45% saturated ammonium sulfate using chemically unmodified single crystals cut to precise dimensions. Diffusion effects on velocities from assays employing aldolase crystals do not appear to be limiting when cut single crystals are crushed. Assays of crushed crystals are linear with respect to both time and enzyme concentration. Kinetic constants are reported for both substrates fructose 1-phosphate and fructose 1,6-phosphate. Maximal velocities and binding constants determined differ by no more than a factor of 2 between the crystalline and the soluble state of the enzyme. Analysis of the kinetic constants for fructose 1-phosphate as substrate shows that binding of substrate does not change in going to the crystalline state. Release of product is reduced roughly 2-fold in the crystalline state. A similar conclusion can be reached in the case of fructose 1,6-phosphate as substrate provided the "on" steps of substrate and product are only diffusion limited but independent of the physical state of the enzyme. It is not possible to distinguish between a more sluggish conformational change during catalysis or simply tighter product binding in the crystalline state as compared to the soluble enzyme state.     

Stability comparison between sample preparation procedures for mass spectrometry-based targeted or shotgun peptidomic analysis

The quantification of neuropeptides may play a significant role in future drug development targeting central nervous system functions. Adequate method precision and accuracy is essential, and sample stability is an important factor. This study compares three sample preparation protocols and assesses the stability of targeted neuropeptides under standard laboratory conditions. The results show that the concentrations of substance P, dynorphin A, and calcitonin gene-related peptide (CGRP) change significantly in time when spinal cord tissues are homogenized in phosphate-buffered saline (PBS) buffer or PBS buffer containing a mammalian protease inhibitor cocktail but is stabilized when tissues are homogenized in a 0.25% trifluoroacetic acid solution.     

Impaired EAT-4 Vesicular Glutamate Transporter Leads to Defective Nocifensive Response of Caenorhabditis elegans to Noxious Heat

Neurochemical Research - In mammals, glutamate is an important excitatory neurotransmitter. Glutamate and glutamate receptors are found in areas specifically involved in pain sensation,...     

Prevalence of mycobacterial sensitivity in Montreal children

The prevalence of sensitivity to atypical mycobacteria was found to be low in 2152 Montreal first-grade children tested with tuberculin and one of four atypical antigens randomly allocated. Forty-nine had positive reactions to an atypical antigen. PPD-G produced more reactions than PPD-A, -B or -K. In Greater Montreal there was a higher prevalence of sensitivity to atypical mycobacteria than in the two suburban areas studied. The range of this prevalence, standardized for the type of antigen, was from 0.86 to 2.92% according to region. The highest prevalence of sensitivity to any single atypical antigen, 5.3%, was found for PPD-G in Greater Montreal.Tuberculous infection was found in 1.35% of the children. Small tuberculin reactions (5 to 9 mm to stabilized PPD 5 TU) require clarification by differential tuberculin testing. More of them are caused by M. tuberculosis than by atypical mycobacteria in this particular age group and region.Routine BCG immunization is not indicated for the particular population studied; when given to individuals at risk, its effectiveness should not be impaired by atypical sensitivity at the low level found locally. Future BCG plans require more epidemiologic data.     

Protons and Mg2+ cations as probes in investigating the role of GTP in initiation complex formation.

fMet-tRNAfMet binding to both 30-S subunits and to 70-S particles is dependent on both pH AND Mg2+ concentration: for fMet-tRNAfMet binding to 70-S particles, variations of pH and Mg2+ concentration are tightly interdependent. This behavior can be interpreted by the polyelectrolyte theory as a direct consequence of the fact that the binding occurs in a polyanionic micro-environment. The pH-dependent binding to 70-S particles clearly shows the involvement of two prototropic groups which appear to be those carrying out GTP hydrolysis, therefore directly linked to initiation complex formation; in the presence of a non-hydrolyzable analogue to GTP, guanosine 5'-[beta, gamma-imido]triphosphate, the binding of fMet-tRNAfMet shows much less interdependence between variation of pH and Mg2+ concentration.