Lymphocyte activation and serine-esterase induction following recombinant interleukin-2 infusion for lymphomas and acute leukaemias

Summary C57BL mice inoculated with radiation leukemia virus (RadLV) develop preleukemic cells long before the onset of leukemia. These cells are potentially immunogenic but fail to elicit an immune response in the host because of the appearance of virus-specific suppressor T cells. We have studied the effect of polysaccharide K (PSK) on the generation of RadLV-specific cell-mediated immune responses in vitro. Long-term exposure to PSK in culture potentiated the ability of immunized T cells to respond to a RadLV-induced lymphoma. It also abrogated the suppressive activity of suppressor T cells and simultaneously boosted the ability of reactive T cells to respond. The dual immunostimulating activity of PSK resulted in the generation of T cytotoxic lymphocytes that could lyse lymphoma cells in vitro. The results suggest that PSK could be used as a prophylactic immune response modifier in preleukemia.     

Positive selection is a general phenomenon in the evolution of abalone sperm lysin

Lysin is a 16kDa acrosomal protein used by abalone sperm to create a hole in the egg vitelline envelope (VE). The interaction of lysin with the VE is species-selective and is one step in the multistep fertilization process that restricts heterospecific (cross-species) fertilization. For this reason, the evolution of lysin could play a role in establishing prezygotic reproductive isolation between species. Previously, we sequenced sperm lysin cDNAs from seven California abalone species and showed that positive Darwinian selection promotes their divergence. In this paper an additional 13 lysin sequences are presented representing species from Japan, Taiwan, Australia, New Zealand, South Africa, and Europe. The total of 20 sequences represents the most extensive analysis of a fertilization protein to date. The phylogenetic analysis divides the sequences into two major clades, one composed of species from the northern Pacific (California and Japan) and the other composed of species from other parts of the world. Analysis of nucleotide substitution demonstrates that positive selection is a general process in the evolution of this fertilization protein. Analysis of nucleotide and codon usage bias shows that neither parameter can account for the robust data supporting positive selection. The selection pressure responsible for the positive selection on lysin remains unknown.      

Open reading frame 1 of the Norwalk-like virus Camberwell: completion of sequence and expression in mammalian cells

The ORF1 sequence was determined for Camberwell virus, a genogroup 2 Norwalk-like virus, completing the full genome of 7,555 nucleotides. ORF1 cDNA was cloned into a simian virus 40-based expression vector, and the viral proteins synthesized following transfection into COS cells were analyzed. By using antisera directed against the helicase, protease, or polymerase regions, eight polypeptides ranging in size from 19 to 117 kDa were detected by radioimmunoprecipitation. The cleavage sites determining the amino and carboxy termini of the 3C-like protease were identified at E(1008)/A and E(1189)/G, respectively.     

Single amino acid substitution in Bacillus sphaericus phenylalanine dehydrogenase dramatically increases its discrimination between phenylalanine and tyrosine substrates

Homology-based modeling of phenylalanine dehydrogenases (PheDHs) from various sources, using the structures of homologous enzymes Clostridium symbiosum glutamate dehydrogenase and Bacillus sphaericus leucine dehydrogenase as a guide, revealed that an asparagine residue at position 145 of B. sphaericus PheDH was replaced by valine or alanine in PheDHs from other sources. This difference was proposed to be the basis for the poor discrimination by the B. sphaericus enzyme between the substrates L-phenylalanine and L-tyrosine. Residue 145 of this enzyme was altered, by site-specific mutagenesis, to hydrophobic residues alanine, valine, leucine, and isoleucine, respectively. The resultant mutants showed a high discrimination, above 50-fold, between L-phenylalanine and L-tyrosine. This higher specificity toward L-phenylalanine was due to K(m) values for L-phenylalanine lowered more than 20-fold compared to the values for L-tyrosine. The greater specificity for L-phenylalanine in the wild-type Bacillus badius enzyme, which has a valine residue in the corresponding position, was also found to be largely due to a lower K(m) for this substrate. Activities were also measured with a range of six amino acids with aliphatic, nonpolar side chains, and with the corresponding oxoacids, and in all cases the specificity constants for these substrates were increased in the mutant enzymes. As with phenylalanine, these increases are mainly attributable to large decreases in K(m) values.     

Identification of H-specific determinants in flagellin of four Escherichia coli strains

The H serogroup of Escherichia coli is determined by the flagellar antigen, flagellin. Sequence analysis of the flagellin gene, fliC, reveals a central variable region and the highly conserved N- and C-termini. This variable region has been shown to encode both H-specific and cross-reactive epitopes. Using polyclonal antibodies, we mapped the linear H-specific determinants in flagellin from four E. coli serotypes O157:H10, 0138:H14, O157:H42 and O157:H43. The specificity of all potential fragments was verified with 52 ECRC (Escherichia coli Reference Center) H-specific antisera. Our results indicated that: (a) a specific determinant of H10 flagellin (1263 bp long) maps to the region covering amino acid residues 305-331; (b) a specific determinant of H14 flagellin (1653 bp long) maps to the region covering amino acid residues 430-461; (c) a specific determinant of H42 flagellin (1281 bp long) maps to a region covering amino acid residues 171-201; and (d) a specific determinant of H43 flagellin (1506 bp long) maps to a region covering amino acid residues 200-260.     

Home-based exercise rehabilitation in addition to specialist heart failure nurse care: design, rationale and recruitment to the Birmingham Rehabilitation Uptake Maximisation study for patients with congestive heart failure (BRUM-CHF): a randomised controlled trial

Background

We aimed to assess whether we could identify a graded association between increasing number of components of the metabolic syndrome and cardiac structural and functional abnormalities independently of predicted risk of coronary heart disease by the Framingham risk score.

Methods

We conducted a cross-sectional study on a random sample of the urban population of Porto aged 45 years or over. Six hundred and eighty-four participants were included. Data were collected by a structured clinical interview with a physician, ECG and a transthoracic M-mode and 2D echocardiogram. The metabolic syndrome was defined according to ATPIII-NCEP. The association between the number of features of the metabolic syndrome and the cardiac structural and functional abnormalities was assessed by 3 multivariate regression models: adjusting for age and gender, adjusting for the 10-year predicted risk of coronary heart disease by Framingham risk score and adjusting for age, gender and systolic blood pressure.

Results

There was a positive association between the number of features of metabolic syndrome and parameters of cardiac structure and function, with a consistent and statistically significant trend for all cardiac variables considered when adjusting for age and gender. Parameters of left ventricular geometry patterns, left atrial diameter and diastolic dysfunction maintained this trend when taking into account the 10-year predicted risk of coronary heart disease by the Framingham score as an independent variable, while left ventricular systolic dysfunction did not. The prevalence of left ventricular diastolic dysfunction, and the mean left ventricular mass, left ventricular diameter and left atrial diameter increased significantly with the number of features of the metabolic syndrome when additionally adjusting for systolic blood pressure as a continuous variable.

Conclusion

Increasing severity of metabolic syndrome was associated with increasingly compromised structure and function of the heart. This association was independent of Framingham risk score for indirect indices of diastolic dysfunction but not systolic dysfunction, and was not explained by blood pressure level.     

Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan^® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-10⁶ copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.     

Compartment‐specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition

Disruption of the functional protein balance in living cells activates protective quality control systems to repair damaged proteins or sequester potentially cytotoxic misfolded proteins into aggregates. The established model based on Saccharomyces cerevisiae indicates that aggregating proteins in the cytosol of eukaryotic cells partition between cytosolic juxtanuclear (JUNQ) and peripheral deposits. Substrate ubiquitination acts as the sorting principle determining JUNQ deposition and subsequent degradation. Here, we show that JUNQ unexpectedly resides inside the nucleus, defining a new intranuclear quality control compartment, INQ, for the deposition of both nuclear and cytosolic misfolded proteins, irrespective of ubiquitination. Deposition of misfolded cytosolic proteins at INQ involves chaperone‐assisted nuclear import via nuclear pores. The compartment‐specific aggregases, Btn2 (nuclear) and Hsp42 (cytosolic), direct protein deposition to nuclear INQ and cytosolic (CytoQ) sites, respectively. Intriguingly, Btn2 is transiently induced by both protein folding stress and DNA replication stress, with DNA surveillance proteins accumulating at INQ. Our data therefore reveal a bipartite, inter‐compartmental protein quality control system linked to DNA surveillance via INQ and Btn2.