Effects of micronutrient supplementation on performance and epigenetic status in dairy cows.

The postpartum period is crucial in dairy cows and is marked by major physiological and metabolic changes that affect milk production, immune response and fertility. Nutrition remains the most important lever for limiting the negative energy balance and its consequences on general health status in highly selected dairy cows. In order to analyze the effect of a commercial micronutrient on intrinsic parameters, performances and the epigenome of dairy cows, 2 groups of 12 Holstein cows were used: 1 fed a standard diet (mainly composed of corn silage, soybean meal and non-mineral supplement) and the other 1 fed the same diet supplemented with the commercial micronutrient (µ-nutrient supplementation) for 4 weeks before calving and 8 weeks thereafter. Milk production and composition, BW, body condition score (BCS), DM intake (DMI) and health (calving score, metritis and mastitis) were recorded over the study period. Milk samples were collected on D15 and D60 post-calving for analyses of casein, Na+ and K+ contents and metalloprotease activity. Milk leukocytes and milk mammary epithelial cells (mMECs) were purified and counted. The viability of mMECs was assessed, together with their activity, through an analysis of gene expression. At the same time points, peripheral blood mononuclear cells (PBMCs) were purified and counted. Using genomic DNA extracted from PBMCs, mMECs and milk leukocytes, we assessed global DNA methylation (Me-CCGG) to evaluate the epigenetic imprinting associated with the µ-nutrient-supplemented diet. The µ-nutrient supplementation increased BCS and BW without modifying DMI or milk yield and composition. It also improved calving condition, reducing the time interval between calving and first service. Each easily collectable cell type displayed a specific pattern of Me-CCGG with only subtle changes associated with lactation stages in PBMCs. In conclusion, the response to the µ-nutrient supplementation improved the body condition without alteration of global epigenetic status in dairy cows.

Silencing of essential genes by RNA interference in Haemonchus contortus.

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the 'essential' genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.

Using of high-resolution topsoil magnetic screening for assessment of dust deposition: comparison of forest and arable soil datasets.

Magnetic susceptibility (kappa) is an easily detectable geophysical parameter that can be used as a proxy or semi-quantitative tracer of atmospheric industrial and urban dusts deposited in topsoil. An enhanced kappa value of topsoil is in many cases also associated with high concentrations of soil pollutants (mostly heavy metals). High-resolution magnetic screening of topsoil in areas of high pollution influx is a useful tool for detection of pollution "hot spots". General and regional screening maps with a grid density of 10 or 5 km have been performed on the basis of forest topsoil measurement only. The purpose of this study was to perform high-resolution magnetic screening with different grid densities in both forested and agricultural areas (arable land). Our large study area (ca. 200 km(2)) was located in a relatively more polluted region of the central part of Upper Silesia, and a second (small) one (ca. 100 m(2)) was located in the western part of Upper Silesia, with considerably lower influx of pollution. In the framework of this study, we applied a statistical comparison of data obtained in forested areas and on arable land. The arable soil showed statistically significantly lower kappa values, the result of "physical dilution" of the arable layer caused by annual ploughing. Thus arable soils must be avoided during high-resolution field measurement. From semivariograms, it was clear that the spatial correlations in forest topsoil are much stronger than in arable soil, which suggests that a denser measurement grid is required in forested areas.

Regulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in Leishmania mexicana.

The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites.

Multisite comparison of methods for the quantitation of the surface expression of CD38 on CD8(+) T lymphocytes. The ACTG Advanced Flow Cytometry Focus Group.

We evaluated the effect of specimen processing variations and quantitation methods on quantitative determination of CD38 expression on CD8 T lymphocytes. Neither lysing reagent (ammonium chloride versus BD FACSlyse), fixation (paraformaldehyde versus no final fixation step), nor acquisition delay (acquisition within 6 h after fixation versus 24 h after fixation) had a significant effect on CD38 relative fluorescent intensity or CD38 quantitative estimates (RFI or antibodies bound per cell). The only significant difference in fluorescent intensity and CD38 antibodies bound per cell (ABC) was encountered when whole blood was held for 24 h prior to staining and fixation and then acquired after another 24-h hold. However, for all sample processing methods above, the CD4 biologic calibrator and QuantiBRITE bead methods gave significantly different estimates of CD38 intensity. In many cases, however, these differences are relatively small and were more pronounced in certain laboratories. We conclude that there is some flexibility in sample processing methods for quantitative CD38 determination; however, it is preferable for a laboratory to employ one method of fluorescence quantitation calculation consistently because small differences are detected between different methods. Cytometry (Comm. Clin. Cytometry) 42:174-179, 2000.

Leishmania mexicana mutants lacking glycosylphosphatidylinositol (GPI):protein transamidase provide insights into the biosynthesis and functions of GPI-anchored proteins.

The major surface proteins of the parasitic protozoon Leishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (DeltaGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein-anchor precursors. However, the synthesis and cellular levels of other non-protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the DeltaGPI8 mutant. Significantly, the DeltaGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.

Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana.

The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all mannose additions in the biosynthesis of both the anchor and free GPIs, except for a alpha1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol-phosphate-mannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.

Characterization of a novel GDP-mannose:Serine-protein mannose-1-phosphotransferase from Leishmania mexicana.

Protozoan parasites of the genus Leishmania secrete a number of glycoproteins and mucin-like proteoglycans that appear to be important parasite virulence factors. We have previously proposed that the polypeptide backbones of these molecules are extensively modified with a complex array of phosphoglycan chains that are linked to Ser/Thr-rich domains via a common Manalpha1-PO4-Ser linkage (Ilg, T., Overath, P., Ferguson, M. A. J., Rutherford, T., Campbell, D. G., and McConville, M. J. (1994) J. Biol. Chem. 269, 24073-24081). In this study, we show that Leishmania mexicana promastigotes contain a peptide-specific mannose-1-phosphotransferase (pep-MPT) activity that adds Manalpha1-P to serine residues in a range of defined peptides. The presence and location of the Manalpha1-PO4-Ser linkage in these peptides were determined by electrospray ionization mass spectrometry and chemical and enzymatic treatments. The pep-MPT activity was solubilized in non-ionic detergents, was dependent on Mn2+, utilized GDP-Man as the mannose donor, and was expressed in all developmental stages of the parasite. The pep-MPT activity was maximal against peptides containing Ser/Thr-rich domains of the endogenous acceptors and, based on competition assays with oligosaccharide acceptors, was distinct from other leishmanial MPTs involved in the initiation and elongation of lipid-linked phosphoglycan chains. In subcellular fractionation experiments, pep-MPT was resolved from the endoplasmic reticulum marker BiP, but had an overlapping distribution with the cis-Golgi marker Rab1. Although Man-PO4 residues in the mature secreted glycoproteins are extensively modified with mannose oligosaccharides and phosphoglycan chains, similar modifications were not added to peptide-linked Man-PO4 residues in the in vitro assays. Similarly, Man-PO4 residues on endogenous polypeptide acceptors were also poorly extended, although the elongating enzymes were still active, suggesting that the pep-MPT activity and elongating enzymes may be present in separate subcellular compartments.

Quantitation of CD38 expression using QuantiBRITE beads.

The QuantiBRITE bead method was compared with the CD4 biological calibration method for quantitation of CD38 expression on CD8+ T-lymphocytes of Multicenter AIDS Cohort Study participants. Results were expressed as CD38 antibodies bound per cell (ABCs) and were the same with the two methods provided two conditions were met. These were the use of repurified (> 95% of the monoclonal antibodies [mAbs] have 1 phycoerythrin [PE] molecule per mAb) CD38-PE for both methods and use of repurified CD4-PE to calculate the relative fluorescence intensity multiplier for the CD4 biological calibration method. Our results indicate that the prognostic significance of CD38 values obtained using the QuantiBRITE method can be interpreted using previously published reports (Liu et al.: J Acquir Immune Defic Syndr Hum Retrovirol 16:83-92, 1997 and 18:332-340, 1998). Sample preparation using NH4Cl and FACS lysing solution gave similar results for CD38 relative fluorescence intensity. Dilution into either phosphate-buffered saline with 2% fetal calf serum and 0.1% sodium azide or fixation in 1% paraformaldehyde for 1 or 24 h also gave similar results. In experiments using Raji cells, which express high levels of CD38, the valence of binding of the intact Leu 17 antibody was approximately 68% bivalent and approximately 32% monovalent. This emphasizes the complexity of determining antigen density from ABCs. We conclude that repurified PE conjugates of CD38, which can be consistently made, together with QuantiBRITE PE beads, provide a convenient and reliable method for quantitation of CD38 expression as ABCs.

The glycoinositolphospholipids from Leishmania panamensis contain unusual glycan and lipid moieties.

The cell surface of Leishmania parasites is coated by glycosylphosphatidylinositol (GPI)-anchored macromolecules (glycoproteins and a lipophosphoglycan) and a polymorphic family of free GPI glycolipids or glycoinositolphospholipids (GIPLs). Here we show that GIPLs with unusual glycan and lipid moieties are likely to be major cell surface components of L. panamensis (subgenus Viannia) promastigotes. These glycolipids were purified by high performance thin layer chromatography and their structures determined by gas-liquid chromatography-mass spectrometry, fast-atom bombardment mass spectrometry, methylation analysis and chemical and enzymatic sequencing of the glycan headgroups. The major GIPLs contained two glycan core sequences, Manalpha1-3Manalpha1-4GlcN-phosphatidylinositol (type-2 series) or Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1- 4GlcN-phosphatidylinosit ol (hybrid series), which were elaborated with Galalpha1-2Galbeta1- or Galalpha1-2/3Galalpha1-2Galbeta1- extensions that were attached to the 3-position of the alpha1-3 linked mannose. The phosphatidylinositol moiety contained exclusively diacylglycerol with palmitoyl, stearoyl and heptadecanoyl chains. Non-galactosylated GIPL species with the same core structures were also found. The galactose extensions and the presence of diacylglycerol in the lipid moieties are novel features for the GIPLs of Leishmania spp. The implications of these structures for the biosynthesis of leishmanial GIPLs and their putative function in the mammalian host are discussed.

Permeability defect with bicarbonate leak as a mechanism of immune-related distal renal tubular acidosis.

We present a 15-year-old girl with distal renal tubular acidosis (dRTA) appearing in what is probably a very early stage of primary Sjögren's syndrome. On the basis of tests evaluating renal handling of H+, we attempt to explain the mechanism of the urine acidification disorder. The inability to decrease urinary pH during systemic acidosis, together with the normal increase of urinary carbon dioxide partial pressure (pCO2) values after sodium bicarbonate and neutral phosphate loading, suggest a gradient-type dRTA. The inability to lower urinary pH in response to furosemide, accompanied by markedly increased urinary excretion of NH4, HCO3, Na, and K, points to a collecting tubule permeability disorder with bicarbonate leak to the tubular lumen. This patient had never been exposed to amphotericin B. To our knowledge, immune-related dRTA as a result of a gradient defect with bicarbonate leak into the tubular lumen has not been described.

Mechanism of hypouricemia in a child with the normotensive form of Gordon's syndrome.

The mechanism of renal tubular urate transport disorder was studied by the pyrazinamide and probenecid tests in a 12-year-old hypouricemic boy suffering from the normotensive form of Gordon's syndrome, with increased distal tubular reabsorption of NaCl (confirmed by the hypotonic saline diuresis test). The aim of the study was to determine the impact of oral hydration during the tests on the phases of renal tubular urate transport: before (I), and during long-term hydrochlorothiazide therapy (0.5 mg/kg BW/day) (II). In both periods (I,II), presecretory reabsorption of urate was within normal limits. Hypouricemia in our patient was caused by decreased postsecretory reabsorption, with or without simultaneous increase of tubular urate secretion. The degree of overhydration determines which of these mechanisms is responsible for increased renal urate clearance.

Primary malignant melanoma of the esophagus. A case report.

A case of primary malignant melanoma of the esophagus has been reported. This rare entity accounts for less than 0.2% of all esophageal neoplasms. A polypoid, deep blue and tough tumour was present on endoscopic examination during which a few samples were taken. Microscopically melanotic tissue occupied the whole submucosa and scanty deposits of melanin pigment were dispersed. Positive immunohistochemical stain with HMB-45 antibody confirmed the diagnosis of malignant melanoma. The patient was admitted to the Oncological Center for resection of the tumour, which is the treatment of choice in such cases.

Endoscopic obliteration for bleeding peptic ulcer.

A group of 133 patients treated for bleeding peptic ulcer in our Department, is reviewed. Within several hours of admission, all patients underwent upper gastrointestinal tract gastroscopy and obliteration of the bleeding ulcer. Bleeding gastric ulcers were found in 41 patients, and duodenal ulcers in 92 patients. Patients were classified according to the Forrest scale: IA - 11 patients, IB - 49 patients, IIA - 35 patients, lIB - 40 patients. In 126 (94.7%) patients the bleeding was stopped, and 7 required urgent surgery: 3 patients with gastric ulcer underwent gastrectomy, and 4 with duodenal ulcer - truncal vagotomy with pyloroplasty and had the bleeding site underpinned. Fifty-five patients underwent elective surgery: gastrectomy and vagotomy (18 patients with gastric ulcer), highly selective vagotomy (25 patients with duodenal ulcer) and truncal vagotomy and pyloroplasty (12 patients with duodenal ulcer). None of the patients was observed to have recurrent bleeding.

Disorders of magnesium homeostasis in the course of liver disease in children.

Magnesium deficiency can develop in patients with acute or chronic liver disease as a result of low dietary magnesium intake, low intestinal absorption or renal magnesium loss caused by natriuretic drugs. The aim of this study was to evaluate magnesium homeostasis in 39 children: 10 with acute liver failure due to Amanita phalloides poisoning. 14 with chronic liver diseases without cholestasis, and 15 with chronic liver diseases with cholestasis. Serum magnesium and fractional and 24 h urinary magnesium excretion were measured in all the children. Magnesium retention after intravenous infusion was also evaluated. Tissue magnesium deficit was found in 30 per cent of children with acute or chronic liver disease.

[Uric acid metabolism abnormalities in IgA nephropathies]. / Zaburzenia metabolizmu kwasu moczowego w nefropatii IgA (doniesienie wstepne).

The possibility of uric acid tract stones forming was analysed in 12 children with IgA nephropathy (group I) and 10 children with other hematuric glomerulopathies (group II). Elevated serum uric acid level and higher urinary excretion was found only in children with IgAN during exacerbation of the disease. Uric acid nephrolithiasis was found only in children with IgA nephropathy. The prophylactic treatment (proper diet, high fluid intake, adjusting urinary pH to 6.5-6.8) was effective in decreasing the number of exacerbations, urinary tract infections and formation of uric acid stones.