Genetic polymorphism of alpha 2HS-glycoprotein in a French population. Description of two new rare variants

A French population was investigated for genetic polymorphism of alpha 2HS-glycoprotein (A2HS; nomenclature according to Human Gene Mapping 7, Los Angeles, 1983) using isoelectric focusing and immunoblotting. Three variants were observed together with two common alleles A2HS 1 and A2HS 2, whose frequencies were significantly different from the data in Canadians and Egyptians. An anodal variant to A2HS 1 was identical to a variant with two different nomenclatures reported by three different groups, indicating that there is a confusion in the A2HS nomenclature. The others were new variants with cathodal isoelectric points to A2HS 2 in the native state.     

Effects of high-salt diet on CYP450-4A omega-hydroxylase expression and active tone in mesenteric resistance arteries

This study investigated the role of changes in the expression of the cytochrome P-450 4A (CYP450-4A) enzymes that produce 20-hydroxyeicosatetraenoic acid (20-HETE) in modulating the responses of rat mesenteric resistance arteries to norepinephrine (NE) and reduced Po(2) after short-term (3-day) changes in dietary salt intake. The CYP450-4A2, -4A3, and -4A8 isoforms were all detected by RT-PCR in arteries obtained from rats fed a high-salt (HS, 4% NaCl) diet, whereas only the CYP450-4A3 isoform was detected in vessels from rats fed a low-salt (LS, 0.4% NaCl) diet. Expression of the 51-kDa CYP450-4A protein was significantly increased by a HS diet. Inhibiting 20-HETE synthesis with 30 muM N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) reduced the vasoconstrictor response to NE in arteries obtained from rats fed either a LS or HS diet, but NE sensitivity after DDMS treatment was significantly lower in vessels from rats on a HS diet. DDMS treatment also restored the vasodilator response to reduced Po(2) that was impaired in arteries from rats on a HS diet. These findings suggest that 1) a HS diet increases the expression of CYP450-4A enzymes in the mesenteric vasculature, 2) 20-HETE contributes to the vasoconstrictor response to NE in mesenteric resistance arteries, 3) the contribution of 20-HETE to the vasoconstrictor response to NE is greater in rats fed a HS diet than in rats fed a LS diet, and 4) upregulation of the production of 20-HETE contributes to the impaired dilation of mesenteric resistance arteries in response to hypoxia in rats fed a HS diet.     

DNA homology comparison between American and European Borrelia burgdorferi strains

Seven strains of Borrelia burgdorferi isolated from ticks and from human beings in Europe and U.S.A. were analyzed for DNA restriction patterns with several enzymes and for DNA homology in Southern blot hybridizations. The restriction patterns showed a moderately high variability. In Southern blot hybridization, strain B31 (U.S.A.) DNA gave a strong signal with itself, strain Bsf (U.S.A.) and Alcaide (isolated in Italy but presumably contracted in Venezuela). Strain B45 (F.R.G.) hybridized to itself, strain BITS (Italy) and to strain D.A. (Italy). Strain Nancy (Italy) gave a signal only when hybridized to itself, although it was classified as Borrelia on the basis of the clinical manifestations, SDS-PAGE protein pattern and antigenic determinants. No hybridization differences were observed for strains isolated from different hosts in the same continental geographical area.     

Esterases A5-B5 in organophosphate-resistant Culex pipiens from Italy

Abstract. Culex pipiens mosquitoes from Lignano city, Udine province, northeast Italy, were found to carry over-produced non-specific esterases Al, A2-B2 and A4-B4 or A5-B5, detected by starch gel electrophoresis, giving multiple resistance to organophosphorus insecticides. In order to differentiate between A4-B4 and A5-B5 esterases, the latter known only from Cyprus whereas the former is widespread in Italy and elsewhere, restriction fragment length polymorphism (RFLP) analysis was performed at the esterase B locus. Both B4 and B5 haplotypes were found. This is the first record of A5-B5 esterase-mediated resistance in continental Europe.     

Effect of high and low sodium intake on urinary aquaporin-2 excretion in healthy humans

The degree of water transport via aquaporin-2 (AQP2) water channels in renal collecting duct principal cells is reflected by the level of the urinary excretion of AQP2 (u-AQP2). In rats, the AQP2 expression varies with sodium intake. In humans, the effect of sodium intake on u-AQP2 and the underlying mechanisms have not previously been studied. We measured the effect of 4 days of high sodium (HS) intake (300 mmol sodium/day; 17.5 g salt/day) and 4 days of low sodium (LS) intake (30 mmol sodium/day; 1.8 g salt/day) on u-AQP2, fractional sodium excretion (FE(Na)), free water clearance (C(H2O)), urinary excretion of PGE(2) (u-PGE(2)) and cAMP (u-cAMP), and plasma concentrations of vasopressin (AVP), renin (PRC), ANG II, aldosterone (Aldo), atrial natriuretic peptide (ANP), and brain natriuretic peptide (BNP) in a randomized, crossover study of 21 healthy subjects, during 24-h urine collection and after hypertonic saline infusion. The 24-h urinary sodium excretion was significantly higher during HS intake (213 vs. 41 mmol/24 h). ANP and BNP were significantly lower and PRC, ANG II, and Aldo were significantly higher during LS intake. AVP, u-cAMP, and u-PGE(2) were similar during HS and LS intake, but u-AQP2 was significantly higher during HS intake. The increases in AVP and u-AQP2 in response to hypertonic saline infusion were similar during HS and LS intake. In conclusion, u-AQP2 was increased during HS intake, indicating that water transport via AQP2 was increased. The effect was mediated by an unknown AVP-independent mechanism.     

Glucose-6-phosphate dehydrogenase deficiency and blood groups in northern Sardinia

Glucose-6-phosphate dehydrogenase deficiency, A1A2BO, Rhesus and Kell systems were investigated in a sample of 28,439 blood donors native of northern Sardinia. The frequency of deficient male individuals was 6.56%. Higher values of r, D, and CDe compared to the averages for continental Italy were observed. Variations within the island were found in the A1A2BO and Rhesus blood group systems.     

The functional VNTR of IGH enhancer HS1.2 associates with human longevity and interacts with TNFA promoter diplotype in a population of Central Italy

The dysregulation of both immune and inflammatory responses occurring with aging is believed to substantially contribute to morbidity and mortality in humans. We have already reported the association of the functional Variable Number of Tandem Repeat (VNTR) at the Immunoglobulin heavy chain (IGH) enhancer HS1.2 with Immunoglobulin levels and with several autoimmune diseases. Herein we tested the association of the VNTR at the HS1.2 enhancer with human longevity, also evaluating the possible modulatory effect of TNFA promoter diplotype (rs361525/rs1800629). HS1.2 enhancer genotypes have been determined for 193 unrelated healthy individuals from Central Italy divided into two groups: Group 1 (18–84 yrs, mean age 56.8 ± 19.4) and Group 2 (85–100 yrs, mean age 93.0 ± 3.5). Homozygous subjects for *2 allele were significantly disadvantaged in reaching higher life-expectancy (OR = 0.457, p = 0.021). A significant interaction between TNFA promoter diplotype status, HS1.2 2/2 genotype and the two Groups was found (p = 0.014). Of note, TNFA − 308A allele seems to exert a protective effect in HS1.2 2/2 carriers. These results support the hypothesis of an important role of HS1.2 VNTR in the puzzle of the immune-system regulation, evidenced also by the potential interaction with TNFA. Moreover, the previous results showing the association of HS1.2 *2 allele with inflammatory phenomena are consistent with the hypothesis that this allele is a detrimental factor in reaching advanced age.     

Recurrent heat shock impairs the proliferation and differentiation of C2C12 myoblasts

Heat-related illness and injury are becoming a growing safety concern for the farmers, construction workers, miners, firefighters, manufacturing workers, and other outdoor workforces who are exposed to heat stress in their routine lives. A primary response by a cell to an acute heat shock (HS) exposure is the induction of heat-shock proteins (HSPs), which chaperone and facilitate cellular protein folding and remodeling processes. While acute HS is well studied, the effect of repeated bouts of hyperthermia and the sustained production of HSPs in the myoblast-myotube model system of C2C12 cells are poorly characterized. In C2C12 myoblasts, we found that robust HS (43 °C, dose/time) significantly decreased the proliferation by 50% as early as on day 1 and maintained at the same level on days 2 and 3 of HS. This was accompanied by an accumulation of cells at G2 phase with reduced cell number in G1 phase indicating cell cycle arrest. FACS analysis indicates that there was no apparent change in apoptosis (markers) and cell death upon repeated HS. Immunoblot analysis and qPCR demonstrated a significant increase in the baseline expression of HSP25, 70, and 90 (among others) in cells after a single HS (43 °C) for 60 min as a typical HS response. Importantly, the repeated HS for 60 min each on days 2 and 3 maintained the elevated levels of HSPs compared to the control cells. Further, the continuous HS exposure resulted in significant inhibition of the differentiation of C2C12 myocytes to myotubes and only 1/10th of the cells underwent differentiation in HS relative to control. This was associated with significantly higher levels of HSPs and reduced expression of myogenin and Myh2 (P < 0.05), the genes involved in the differentiation process. Finally, the cell migration (scratch) assay indicated that the wound closure was significantly delayed in HS cells relative to the control cells. Overall, these results suggest that a repeated HS may perturb the active process of proliferation, motility, and differentiation processes in an in vitro murine myoblast-myotube model.     

Mast cell stabilization improves cardiac contractile function following hemorrhagic shock and resuscitation

Hemorrhagic shock (HS) is associated with cardiac contractile dysfunction. Mast cell (MC) degranulation is hypothesized to mediate the cardiodepressant effect. Cardiac function was assessed after HS and resuscitation (HS/R) with the administration of the MC stabilizers to prevent MC degranulation. Anesthetized male Sprague-Dawley rats were randomized to sham-operated control or HS/R groups and underwent 60 min of HS followed by 2 h of resuscitated reperfusion. Animals in the HS/R groups were randomized to receive cromolyn (5 mg/kg), ketotifen (1 mg/kg), or saline 15 min before shock. Hearts were excised following HS or 2 h of reperfusion, and function was assessed on a Langendorff apparatus. A second group of randomized animals had serial blood samples taken to assess MC degranulation by quantifying levels of serum beta-hexosaminidase. Hearts were excised at 0 min (before HS) and following 60 min of HS (before resuscitation) for a histological evaluation of MC density and degranulation. In vivo MC stabilization using ketotifen and cromolyn improved cardiac peak systolic pressure (P < 0.05), contractility (P < 0.05), and relaxation (P < 0.05) compared with that of HS controls. Serum beta-hexosaminidase increased during HS/R and was inhibited by MC stabilization (P < 0.05). Degranulation was inhibited when assessed by histochemistry and immune fluorescence. The inhibition of MC degranulation can significantly improve cardiac function following HS/R.     

Colonisation of hard substrata along a channel system in the deep Greenland Sea

The colonisation of hard substrata (HS) by epibenthic megafauna was studied by photographic surveys along the Ardencaple Canyon in the deep western Greenland Sea in 2000. Seven transects at 2,700–3,200 m water depth showed generally low densities of dropstones, sunken wood, and other substrata including anthropogenic material (range: 2–11 HS km⁻¹). Overall, 30 different taxa and morphotypes were found on or associated with HS. While the sea anemone Bathyphellia margaritacea and the pantopod Ascorhynchus abyssi dominated the fauna on the substrate surfaces, a ball-shaped morphotype of uncertain taxonomic origin characterised assemblages marginally associated with HS. Community analysis revealed differences in faunal patterns near the continental rise and towards the deep sea, but diversity and evenness did not differ significantly between the various regions. However, we conclude that dropstones and other hard substrata at the seafloor serve as colonisation islands and thereby generally increase small-scale habitat diversity in polar deep-sea environments.     

Immunoglobulin allotypes in sardinia

1218 individuals from Sardinia island (Italy) were tested for Gm and Km markers; 10 were not tested for Gm and only 401 were typed for Am markers. The peculiar genetic makeup of the Sardinian population is confirmed by their Gm allotypes. Their differences from those found in a control population of continental Italy (Ferrara), suggest ancient contacts with the Middle East and Africa. An indication for such contacts may also be found in the striking presence of the haplotype Gm f;n;bsc5, a haplotype not previously found in a human population. A significant difference of G2m(n) allotype was observed between highland and lowland regions. If confirmed, it may suggest an adaptive pressure related to the CH2 region of the gamma2 chain, possibly due to endemic malaria in the past.     

Structural analysis and mapping of DNase I hypersensitivity of HS5 of the beta-globin locus control region.

The beta-globin locus control region (LCR) is a cis regulatory element that is located in the 5' part of the locus and confers high-level erythroid lineage-specific and position-independent expression of the globin genes. The LCR is composed of five DNase I hypersensitive sites (HSs), four of which are formed in erythroid cells. The function of the 5'-most site, HS5, remains unknown. To gain insights into its function, mouse HS5 was cloned and sequenced. Comparison of the HS5 sequences of mouse, human, and galago revealed two extensively conserved regions, designated HS5A and HS5B. DNase I hypersensitivity mapping revealed that two hypersensitive sites are located within the HS5A region (designated HS5A(major) and HS5A(minor)), and two are located within the HS5B region (HS5B(major), HS5B(minor)). The positions of each of these HSs colocalize with either GATA-1 or Ap1/NF-E2 motifs, suggesting that these protein binding sites are implicated in the formation of HS5. Gel retardation assays indicated that the Ap1/NF-E2 motifs identified in murine HS5A and HS5B interact with NF-E2 or similar proteins. Studies of primary murine cells showed that HS5 is formed in all hemopoietic tissues tested (fetal liver, adult thymus, and spleen), indicating that this HS is not erythroid lineage specific. HS5 was detected in murine brain but not in murine kidney or adult liver, suggesting that this site is not ubiquitous. The presence of GATA-1 and NF-E2 motifs (which are common features of the DNase I hypersensitive sites of the LCR) suggests that the HS5 is organized in a manner similar to that of the other HSs. Taken together, our results suggest that HS5 is an inherent component of the beta-globin locus control region.     

The HLA system in Italy

4,902 Italians were typed for HLA-A antigens, 4,721 for HLA-B and 1,503 for HLA-C. The samples, which were composed of unrelated, healthy individuals born in Italy, were used for estimating HLA-A, HLA-B and HLA-C gene frequencies with the maximum-likelihood method. Different Italian regions showed significant differences in the HLA alleles, providing further evidence for the genetic heterogeneity of the Italian population. HLA gene frequencies place continental Italy and Sicily in a position which is similar to that of other Mediterranean populations, whereas the genetic isolation of Sardinia is quite evident. The most significant linkage disequilibrium values found in the Italian population (except for Sardinia) were in agreement with those observed in other Caucasian populations. The difference between Northern and Southern Italy and between continental Italy and Sardinia was emphasized by the linkage disequilibrium values and by the principal-component analysis as well.     

Humic substances and the water calcium content change the toxicity of malachite green

Laboratory experiments were conducted to test interactive effects of calcium (Ca²⁺) content and the presence of humic substance (HS) on malachite green (MAG)‐induced toxicity in fish embryos and larvae by means of a semistatic 144‐h‐embryo‐larval‐test with zebrafish (Danio rerio). Two kinds of reconstituted water samples were used to produce the test media by mixing salts into deionized water resulting in either hard water (↑Ca ? HS), or soft water (↓Ca ? HS). By adding HS two additional test media were produced (↑Ca + HS, ↓Ca + HS). MAG was tested in concentrations of 0.05, 0.10, 0.15, 0.20, 0.25 mg L^?1. The toxicity ranking of MAG (mg L^?1) to embryos based on 96‐h‐LC₅₀ in the different test water samples is: ↑Ca ? HS (0.061) > ↑Ca + HS (0.123) = ↓Ca ? HS (0.12) ≥ ↓Ca + HS (0.134) and on 144‐h‐LC₅₀ to larvae is: ↑Ca ? HS (0.038) > ↑Ca + HS (0.06) > ↓Ca ? HS (0.077) = ↓Ca + HS (0.077). Mortality of all the groups was significantly different (P < 0.05). Increased Ca²⁺ concentrations did not protect zebrafish embryos and larvae from MAG‐induced toxicity. At high Ca²⁺ conditions, the mortality of the embryos as well as of the larvae is reduced in the ↑Ca + HS group relative to the ↑Ca ? HS group. Thus, at high Ca²⁺ conditions the HS does affect the MAG‐induced mortality. The mechanism which causes the higher toxicity of MAG in the presence of higher Ca²⁺ concentrations is poorly understood. A probable explanation could be the stimulation of the calcium‐binding protein calmodulin as well as the calmodulin kinase II in cell membranes in the presence of high Ca²⁺ concentrations.     

Role of the adenosine(2A) receptor-epoxyeicosatrienoic acid pathway in the development of salt-sensitive hypertension

Activation of rat adenosine(2A) receptors (A(2A) R) dilates preglomerular microvessels, an effect mediated by epoxyeicosatrienoic acids (EETs). High salt (HS) intake increases epoxygenase activity and adenosine levels. A greater vasodilator response to a stable adenosine analog, 2-chloroadenosine (2-CA), was seen in kidneys obtained from HS-fed rats which was mediated by increased EET release. Because this pathway is antipressor, we examined the role of the A(2A) R-EET pathway in a genetic model of salt-sensitive hypertension, the Dahl salt-sensitive (SS) rats. Dahl salt resistant (SR) rats fed a HS diet demonstrated a greater renal vasodilator response to 2-CA. In contrast, Dahl SS rats did not exhibit a difference in the vasodilator response to 2-CA whether fed normal salt (NS) or HS diet. In Dahl SR but not Dahl SS rats, HS intake significantly increased purine flux, augmented the protein expression of A(2A) R and cytochrome P450 2C23 and 2C11 epoxygenases, and elevated the renal efflux of EETs. Thus the Dahl SR rat is able to respond to HS intake by recruiting EET formation, whereas the Dahl SS rat appears to have exhausted its ability to increase EET synthesis above the levels observed on NS intake. In vivo inhibition of the A(2A) R-EET pathway in Dahl SR rats fed a HS diet results in reduced renal EETs levels, diminished natriuretic capacity and hypertension, thus supporting a role for the A(2A) R-EET pathway in the adaptive natriuretic response to modulate blood pressure during salt loading. An inability of Dahl SS rats to upregulate the A(2A) R-EET pathway in response to salt loading may contribute to the development of salt-sensitive hypertension.     

Identification of specific proteins synthesized by type II pneumocytes in primary culture

Proteins from primary cultures of type II granular pneumocytes have been examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to identify type II cell-specific proteins. The distribution of Coomassie Blue-stained bands in preparations of cellular proteins, culture medium, lavage and lamellar bodies have been compared. The most prominent stained band in the serum-free medium from type II cell cultures (HS1; Mr 39900) corresponds to a major protein in acellular sedimentable (20000 g for 30 min) crude surfactant obtained from rat lungs by saline (0.9% NaCl) lavage. A second protein (HS2; Mr 12000) is also found both in type II cell-conditioned medium and in lavage. Neither rat serum nor donor calf serum (used in the isolation of the type II cells) contains a protein co-migrating with HS1 or HS2 proteins. HS1 is also found in Coomassie Blue-stained gels of cellular proteins and of lamellar bodies isolated from whole lungs. Cultures of type II cells incorporate [14C]phenylalanine into HS1 and HS2 as shown by autoradiography of sodium dodecyl sulphate/polyacrylamide gels of culture medium. Rat lungs perfused in situ incorporate [35S]methionine into HS1 in the lamellar body fraction. A third protein (HS3; Mr 47000) is observed only in autoradiographs of cell culture medium; no corresponding Coomassie Blue-stained band can be identified in medium, in cells or in lung lavage. No protein bands corresponding to HS1, HS2 or HS3 are found in conditioned media from pulmonary alveolar macrophages, rat fibroblasts or bovine aorta endothelial cells. Two-dimensional gel electrophoresis of HS1 shows a single polypeptide with an isoelectric point of 6.3; HS3 appears as a chain of spots with a range of isoelectric points from 6.3 to 6.6. HS2 has not been identified on two-dimensional gels. The amino acid composition of HS1 does not differ significantly from that of surfactant apoproteins studied previously; however, HS1 is not detected by glycoprotein stains, nor does it appear to be a subunit of a thiol-linked multimer.     

In vivo relationship between morphonuclear parameters and hormone sensitivity of the murine MXT mammary tumor

The initially hormonosensitive (HS) MXT mouse mammary tumor spontaneously evolved to hormonoin-dependence (HI). Using a SAMBA 200 cell image processor, we compared the DNA content and the chromatin structure of HS and HI tumor cells squashed onto histologic slides; the nuclei were colored by the Feulgen reaction. We compared HI and HS nuclei by a discriminant analysis using the 15 parameters obtained on each nucleus. We show that the percentage of well-classified granulocytes (2n DNA content control) versus HS or HI nuclei exceeded 99. On the other hand, this percentage did not reach 70 when we compared HS and HI. The cell cycle analysis revealed that the percentage of cells in S and G2 + M phases were significantly higher in HI tumors than in the HS. Hence, HI and HS MXT tumor nuclei seem to be morphologically identical, but are significantly different if we refer to cell proliferation rates.     

Hypertonic saline enhances neutrophil elastase release through activation of P2 and A3 receptors

Hypertonic saline (HS) holds promise as a novel resuscitation fluid for the treatment of trauma patients because HS inhibits polymorphonuclear neutrophil (PMN) activation and thereby prevents host tissue damage and associated posttraumatic complications. However, depending on conditions of cell activation, HS can increase PMN degranulation, which could exacerbate tissue damage in trauma victims. The cellular mechanism by which HS increases degranulation is unknown. In the present study, we tested whether HS-induced ATP release from PMN and feedback via P1 and/or P2 receptors may be involved in the enhancement of degranulation by HS. We found that HS enhances elastase release and ERK and p38 MAPK activation when HS is added after activation of PMN with formyl peptide (fMLP) or phorbol ester (PMA). Agonists of P2 nucleotide and A3 adenosine receptors mimicked these enhancing effects of HS, whereas antagonists of A3 receptors or removal of extracellular ATP with apyrase diminished the response to HS. A1 adenosine receptor antagonists increased the enhancing effect of HS, whereas A1 receptor agonists inhibited elastase release. These data suggest that HS upregulates degranulation via ATP release and positive feedback through P2 and A3 receptors. We propose that these feedback mechanisms can serve as potential pharmacological targets to fine-tune the clinical effectiveness of HS resuscitation. resuscitation; inflammation; osmotic stimulation; nucleotide receptor signaling     

Dietary vitamin A can improve immune function in heat-stressed broilers

This experiment was undertaken to evaluate the effect of dietary vitamin A on the performance and immune competence of broilers under heat stress (HS). A total of 180 birds, at 22 days of age, were randomly assigned to be reared either at 24°C (thermoneutral, TN, 24°C, constant) or 24°C to 38°C (heat stress, HS, cycling) until the age of 42 days. Birds were then supplemented with vitamin A at 750, 1500, 15 000 IU/kg. Each of the 2 × 3 factorially arranged treatments were replicated in six cages, each containing five birds. Humoral immunity was assessed by intravenous injection of 7% sheep red blood cells (SRBC) followed by evaluation of serum for antibody titers in primary and secondary responses. Cell-mediated immunity was assessed by using a Sephadax stimulation method to recruit abdominal exudate cells (AEC) to evaluate macrophage phagocytic ability. Body weight (BW) and feed conversion were significantly affected by dietary vitamin A (P < 0.05). HS significantly reduced BW, feed intake and feed conversion (P < 0.05). Numbers of AEC, percentage of macrophages in AEC, phagocytic macrophages, internalized opsonized and unopsonized SRBC were increased by dietary vitamin A (P < 0.05). Both primary and secondary antibody responses were characterized by increasing titers of antibody to SRBC by dietary vitamin A when birds were exposed to HS (P < 0.05). Lymphoid organ weights, antibody responses, incidence of macrophages in AEC and phagocytic ability of macrophages were all significantly reduced under HS. These results indicated that HS severely reduced performance and immunocompetence of broilers, whereas the immune response of broilers improved by dietary vitamin A supplementation under HS.