Association between Knops blood group polymorphisms and susceptibility to malaria in an endemic area of the Brazilian Amazon

Complement receptor 1 (CR1) gene polymorphisms that are associated with Knops blood group antigens may influence the binding of Plasmodium parasites to erythrocytes, thereby affecting susceptibility to malaria. The aim of this study was to evaluate the genotype and allele and haplotype frequencies of single-nucleotide polymorphisms (SNPs) of Knops blood group antigens and examine their association with susceptibility to malaria in an endemic area of Brazil. One hundred and twenty-six individuals from the Brazilian Amazon were studied. The CR1-genomic fragment was amplified by PCR and six SNPs and haplotypes were identified after DNA sequence analysis. Allele and haplotype frequencies revealed that the Kn(b) allele and H8 haplotype were possibly associated with susceptibility to Plasmodium falciparum. The odds ratios were reasonably high, suggesting a potentially important association between two Knops blood antigens (Kn(b) and KAM(+)) that confer susceptibility to P. falciparum in individuals from the Brazilian Amazon.     

Estimating genomic instability mediated by Alu retroelements in breast cancer

Alu-PCR is a relatively simple technique that can be used to investigate genomic instability in cancer. This technique allows identification of the loss, gain or amplification of gene sequences based on the analysis of segments between two Alu elements coupled with quantitative and qualitative analyses of the profiles obtained from tumor samples, surgical margins and blood. In this work, we used Alu-PCR to identify gene alterations in ten patients with invasive ductal breast cancer. Several deletions and insertions were identified, indicating genomic instability in the tumor and adjacent normal tissue. Although not associated with specific genes, the alterations, which involved chromosomal bands 1p36.23, 1q41, 11q14.3, 13q14.2, occurred in areas of well-known genomic instability in breast and other types of cancer. These results indicate the potential usefulness of Alu-PCR in identifying altered gene sequences in breast cancer. However, caution is required in its application since the Alu primer can produce non-specific amplification.     

Determination of haemoglobin A(1c) by liquid chromatography using a new cation-exchange column

The use of a new cation-exchange column, ProPac SCX-10, for the determination of haemoglobin A(1c) (HbA(1c)) by high-performance liquid chromatography is described. After optimization of the analytical method for the separation of the various isoforms of haemoglobin with the ProPac SCX-10 column, the method was applied to the determination of HbA(1c) in blood from 59 volunteers. Three of the 59 had previously been diagnosed as diabetics. Interference studies for carbamylation, acetylation and pre-HbA(1c) were carried out via "in-vitro" experiments. No interference due to carbamylation was observed at the urea values normally found in uremic patients undergoing dialysis. No interference from pre-HbA(1c) was detected either. The method is able to separate haemoglobin A (alpha(2)beta(2)), haemoglobin S (haemoglobin from sickle cell anaemia patients) and haemoglobin A(2) (alpha(2)delta(2)) without interference. The method of Hampel was applied to detect outliers. A value of 3.29+/-0.44% (2sigma) for HbA(1c) was obtained in the analysis of 56 blood samples from non-diabetics. This average value is lower than that reported by most of the methods currently used in routine analyses.     

Characterization and location of Src-dependent tyrosine phosphorylation in rat brain mitochondria

Analysis of protein phosphorylation in highly purified rat brain mitochondria revealed the presence of several alkali-stable phosphoproteins whose phosphorylation markedly increases upon treatment with peroxovanadate and Mn(2+), a property indicating tyrosine phosphorylation. These include three prominent bands, with apparent sizes of 50, 60, and 75 kDa, which are detectable by anti-phosphotyrosine. Tyrosine phosphorylation disappears when mitochondria are treated with PP2, an inhibitor of the Src kinase family, suggesting the presence of members of this family in rat brain mitochondria. Immunoblotting and immunoprecipitation assays of mitochondrial lysates confirmed the presence of Fyn, Src and Lyn kinases, as well as Csk, a protein kinase which negatively controls the activity of the Src kinase family. Results show that tyrosine-phosphorylated proteins are membrane-bound and that they are located on the inner surface of the outer membrane and/or the external surface of the inner membrane. Instead, Src tyrosine kinases are mainly located in the intermembrane space - in particular, as revealed by immunogold experiments for Lyn kinase, in the cristal lumen. Rat brain mitochondria were also found to possess a marked level of tyrosine phosphatase activity, strongly inhibited by peroxovanadate.     

Phosphorylation of recombinant human spermidine/spermine N(1)-acetyltransferase by CK1 and modulation of its binding to mitochondria: a comparison with CK2.

Cytosolic spermidine/spermine acetyltransferase (SSAT) catalyzes the acetylation of the N(1)-propylamino groups of spermine and spermidine. The enzyme has a very short half-life and is rapidly induced by various stimuli. Once acetylated, these polyamines are subjected to the action of polyamine oxidase, which, besides initiating polyamine catabolism, may produce reactive oxygen species that in turn trigger modifications in subcellular compartments such as mitochondria. The present work evaluates the ability of the cAMP-independent Ser/Thr-protein kinase CK1 to phosphorylate SSAT. Results demonstrate that SSAT is phosphorylated by CK1, in sites distinct from those phosphorylated by CK2. Moreover, both phosphorylation processes are involved in the uptake of SSAT into rat liver mitochondria. Although CK2 is less effective than CK1 in phosphorylating SSAT, CK2 phosphorylation is much more powerful in preventing binding of SSAT to mitochondrial structures. These results suggest the involvement of CK1- and CK2-mediated SSAT phosphorylation in regulating the contents of polyamines and SSAT itself within subcellular compartments and implicate SSAT and polyamines as indirect modulators of progression through the cell cycle.     

Participation of C1q and its receptor in adherence of human diploid fibroblast

C1q binds through its collagen-like domain to specific surface receptors of fibroblasts and to adhesive elements of extracellular matrix including fibronectin, collagens, proteoglycans, and laminin. To determine whether C1q participates in fibroblast adhesion, cells in serum-free medium were plated on surfaces coated with purified C1q at physiologic ionic strength and pH. Surfaces coated with fibronectin or collagen type I served as positive controls, and those coated with BSA were negative controls. Substratum-adsorbed C1q promoted fibroblast adhesion to a maximum of 73% of available cells within 90 min at 37 degrees C. Adhesion was C1q concentration dependent, saturable, specific, and dependent on the collagen-like domain of the molecule. De novo protein synthesis plays a role in adhesion: pretreatment of fibroblasts with cycloheximide reduced adherence about 50% of controls. Addition of exogenous fibronectin, collagen type I, or C1q as soluble mediators did not affect adhesion of the cycloheximide-treated cells to C1q substrate. Adhesion could be accounted for primarily, although not completely, by the C1q receptors. Antibodies raised against the Raji cell C1q receptors (alpha C1qR Ab) specifically inhibited fibroblast adhesion to C1q substrates about 60% of controls. The binding of fibroblasts to C1q substrates could be inhibited about 24% of controls with the GRGDTP cell recognition peptide. GRGDTP and alpha C1q Ab had an additive effect on adhesion that was inhibited 77 to 80% of controls. We conclude from these data that aggregated rather than monomeric C1q may be the natural ligand of the fibroblast C1q receptor, and the biologic function of the receptor in cells of the connective tissue may be cell adhesion.     

Influence of metabolic genotype GSTM1 on levels of urinary mutagens in patients treated topically with coal tar

Fifteen hospitalized, non-smoking, dermatological patients were treated with ointment containing 2% coal tar (CT) in order to assess the influence of metabolic genotype GSTM1 on urinary mutagen levels. Urinary 1-pyrenol, the main metabolite of pyrene, was used to check the high exposure to PAH of this population. The mean levels of urinary 1-pyrenol found in the 24-h urine of our patients were 467. 8+/-211.0 nmoles-24 h (range 94.6-890.1 nmoles-24 h). Mutagenicity was assessed on urine samples collected over a period of 24 h, after three consecutive days of topical application, using the bacterial mutagenesis test on Salmonella typhimurium strains TA98 and YG1024 in the presence of microsomal enzymes. The latter strain turned out to be more sensitive than the former in revealing urinary mutagens in these patients (42 693+/-30 867 vs. 6877+/-6040 net revertants-24 h). The mutagenicity on YG1024 strain and 1-pyrenol levels of urine samples were correlated (Spearman's rank correlation coefficient=0. 6678, P<0.01, z=2.795). The influence of genotype GSTM1 on urinary mutagen levels was assessed on strain YG1024. The values of urinary mutagenicity of subjects with genotype GSTM1-null (n=6) were on average higher than those of GSTM1-positive subjects (n=9) (55 498+/-45 957 vs. 34 156+/-11 933 net rev.-24 h), a non-significant statistical difference. The mean total excretion of mutagens corrected for PAH exposure (net rev./nmoles of urinary 1-pyrenol) in GSTM1-null patients was double that of GSTM1-positive ones (136. 8+/-34.7 vs. 70.8+/-23.3 net rev./nmoles of urinary 1-pyrenol; one-tailed Mann-Whitney U-test, U=11.5, P<0.05). These results indicate a greater body burden of promutagens, resulting from skin application of CT, in GSTM1-null subjects.     

Recent developments in quantification methods for metallothionein.

The metallothioneins (MT), a family of proteins with relatively low molecular weight (6-7 kDa), are characterised by the intrinsic presence of 20 cysteinyl groups in their structure, which confers unique metal binding properties to the molecule. Since MT are involved in biological roles, quantification of MT remains an important task. To date, a large number of determination methods have been developed. In this paper recent developments, from 1995 to the present, in methodology employed in quantification studies of total MT and MT polymorphism are described. Different fields were taken into consideration, such as (i) separation techniques and hyphenated systems, (ii) electrochemical methods, (iii) immunological methods and (iv) quantification of MT mRNA. The data presented are based on our own and published results. A brief overview of the use of metallothionein as a biomarker is included as a relevant example of the importance of MT quantification. Finally, general problems associated with determination and evaluation of obtained results within the above four topics are mentioned.     

SARS‐CoV‐2 structural coverage map reveals viral protein assembly,mimicry, and hijacking mechanisms

We modeled 3D structures of all SARS‐CoV‐2 proteins, generating 2,060 models that span 69% of the viral proteome and provide details not available elsewhere. We found that ˜6% of the proteome mimicked human proteins, while ˜7% was implicated in hijacking mechanisms that reverse post‐translational modifications, block host translation, and disable host defenses; a further ˜29% self‐assembled into heteromeric states that provided insight into how the viral replication and translation complex forms. To make these 3D models more accessible, we devised a structural coverage map, a novel visualization method to show what is—and is not—known about the 3D structure of the viral proteome. We integrated the coverage map into an accompanying online resource (https://aquaria.ws/covid) that can be used to find and explore models corresponding to the 79 structural states identified in this work. The resulting Aquaria‐COVID resource helps scientists use emerging structural data to understand the mechanisms underlying coronavirus infection and draws attention to the 31% of the viral proteome that remains structurally unknown or dark.