Comparative assessment of SSR and SNP markers for inferring the population genetic structure of the common fungus Armillaria cepistipes.

During the last years, simple sequence repeats (SSRs, also known as microsatellites) and single-nucleotide polymorphisms (SNPs) have become the most popular molecular markers for describing neutral genetic variation in populations of a wide range of organisms. However, only a limited number of studies has focused on comparing the performance of these two types of markers for describing the underlying genetic structure of wild populations. Moreover, none of these studies targeted fungi, the group of organisms with one of the most complex reproductive strategies. We evaluated the utility of SSRs and SNPs for inferring the neutral genetic structure of Armillaria cepistipes (basidiomycetes) at different spatial scales. For that, 407 samples were collected across a small (150 km2) area in the Ukrainian Carpathians and a large (41 000 km2) area in the Swiss Alps. All isolates were analyzed at 17 SSR loci distributed throughout the whole genome and at 24 SNP loci located in different single-copy conserved genes. The two markers showed different patterns of structure within the two spatial scales studied. The multi-allelic SSR markers seemed to be best suited for detecting genetic structure in indigenous fungal populations at a rather small spatial scale (radius of ~50-100 km). The pattern observed at SNP markers rather reflected ancient divergence of distant (~1000 km) populations that in addition are separated by mountain ranges. Despite these differences, both marker types were suitable for detecting the weak genetic structure of the two A. cepistipes populations investigated.

The Tlg SNARE complex is required for TGN homotypic fusion.

Using a new assay for membrane fusion between late Golgi/endosomal compartments, we have reconstituted a rapid, robust homotypic fusion reaction between membranes containing Kex2p and Ste13p, two enzymes resident in the yeast trans-Golgi network (TGN). Fusion was temperature, ATP, and cytosol dependent. It was inhibited by dilution, Ca+2 chelation, N-ethylmaleimide, and detergent. Coimmunoisolation confirmed that the reaction resulted in cointegration of the two enzymes into the same bilayer. Antibody inhibition experiments coupled with antigen competition indicated a requirement for soluble NSF attachment protein receptor (SNARE) proteins Tlg1p, Tlg2p, and Vti1p in this reaction. Membrane fusion also required the rab protein Vps21p. Vps21p was sufficient if present on either the Kex2p or Ste13p membranes alone, indicative of an inherent symmetry in the reaction. These results identify roles for a Tlg SNARE complex composed of Tlg1p, Tlg2p, Vti1p, and the rab Vps21p in this previously uncharacterized homotypic TGN fusion reaction.

Lack of teratogenic effect of brief maternal insulin-induced hypoglycemia in rats during late neurulation.

We have previously shown that 1 h of maternal insulin-induced hypoglycemia is teratogenic to rat embryos during the initial stages of neurulation, when they are dependent on uninterrupted glycolysis (day 9.5-9.7 of development). To determine whether this vulnerability persists in later stages of neural tube and cardiac development, we infused insulin into 16 conscious pregnant rats for 1 h beginning after embryos had developed the capacity for aerobic glucose metabolism (day 10.6 of development). Half of the pregnant animals were allowed to become hypoglycemic (44 +/- 2 mg/dl) during the insulin infusions. The other half received glucose infusions to maintain normoglycemia (130 +/- 3 mg/dl). Normal plasma glucose levels were maintained in all animals after the insulin infusions. Embryos were examined on day 11.5 of development. At that time, 1 of 111 embryos from the normoglycemic group and 1 of 109 embryos from the hypoglycemic group were grossly malformed (P greater than .5). Means of embryo crown-rump length (4.15 +/- 0.03 vs. 4.14 +/- 0.03 mm), somite number (29.7 +/- 0.1 vs. 29.8 +/- 0.2), and total protein content (320 +/- 5 vs. 326 +/- 6 micrograms) were also similar in the two groups (P greater than .5). Thus, we could not detect an embryotoxic effect of 1 h of maternal insulin-induced hypoglycemia beginning at day 10.6 of development. This finding is in contrast to our prior demonstration that a similar period of hypoglycemia occurring earlier in neurulation (day 9.7) caused growth retardation and developmental anomalies in embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetic teratogenesis. In vitro evidence for a multifactorial etiology with little contribution from glucose per se.

To determine the extent to which elevated glucose and 3-hydroxybutyrate (3OHB) concentrations contribute to the embryotoxic properties of diabetic serum, we tested the effects of serum from untreated or acutely insulin-treated diabetic rats on the development of mouse embryos during neurulation in vitro. Male Sprague-Dawley rats (n = 143) with streptozocin-induced diabetes for 1 week received infusions of insulin (n = 105) or saline (n = 38) for up to 120 min. The insulin-infused animals were exsanguinated when serum glucose concentrations fell to between 5.6 and 8.3 mM. Saline-infused animals were exsanguinated after a similar duration of infusion. Serum samples were tested for embryotoxic effects on 3-6 somite mouse embryos cultured in vitro for 24 h. Of embryos cultured in serum from untreated diabetic animals (glucose: 24 +/- 1 mM; 3OHB: 2.0 +/- 0.3 mM), 36% (31 of 87) exhibited gross malformations, mostly of the neural tube. Only 16% (10 of 62) of embryos grown in serum from acutely insulin-treated animals (glucose: 7.4 +/- 0.2 mM; 3OHB: 0.20 +/- 0.06 mM) were malformed. This rate that was less than half the rate caused by exposure to diabetic serum (P < 0.01), but a rate that remained much greater than the rate associated with culture in normal serum (0% in this study; < 2% historically). In vitro addition of glucose to serum from insulin-treated animals to re-establish hyperglycemia in the diabetic range (25 mM) resulted in a 17% (12 of 70) malformation rate, nearly identical to the 16% rate caused by normoglycemic serum from insulin-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced glucose tolerance in spontaneously hypertensive rats. Pancreatic beta-cell hyperfunction with normal insulin sensitivity.

We used intravenous glucose tolerance tests in vivo and 3-O-methylglucose transport into skeletal muscle in vitro to assess glucose tolerance, pancreatic beta-cell function, and insulin action in 9- to 11-wk-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar Kyoto rats (WKY). Body weight was slightly higher in the WKY (P less than 0.001), while blood pressure was elevated in the SHR (P less than 0.001). Insulin responses to intravenous glucose after 4 or 12 h of fasting in SHR were 2-3 times the responses of WKY rats (P less than 0.001). The greater insulin responses in SHR were associated with accelerated glucose disappearance P less than 0.001 vs. WKY rats). A direct correlation (r = 0.49, P less than 0.05) between the peak plasma insulin responses to glucose and Kg values in SHR suggested that the exaggerated insulin responses contributed to the accelerated glucose disappearance in that group. 3-O-methylglucose transport rates into epitrochlearis muscles in vitro did not differ significantly between SHR and WKY groups in the absence of insulin (P less than 0.2) or in the presence of insulin at physiological (600 pM, P greater than 0.4) or pharmacological (120,000 pM, P greater than 0.9) concentrations. Thus, compared with WKY rats, SHR had exaggerated insulin responses to glucose, similar insulin-mediated glucose transport into skeletal muscle, and enhanced glucose tolerance. Our findings indicate that young, hypertensive SHR have hyperfunction of pancreatic beta-cells that is unrelated to insulin resistance. The resultant nutrient-stimulated hyperinsulinemia could play a role in the development or maintenance of elevated blood pressure in SHR.

Glucose tolerance and insulin action in rats with renovascular hypertension.

To test whether hypertension can cause hyperinsulinemia or insulin resistance, we performed intravenous glucose tolerance tests at 1 month and euglycemic clamps at 3 months after induction of two-kidney, one clip renovascular hypertension in rats. At 1 month, systolic pressure was higher in 21 clipped than in 12 control animals (161 +/- 5 mm Hg, range 134-187 mm Hg versus 119 +/- 3 mm Hg, range 108-146 mm Hg; p less than 0.001). Glucose tolerance, assessed as the glucose fractional disappearance rate between 3 and 11 minutes after the glucose injection, was similar in the clipped and sham groups (0.059 +/- 0.002 versus 0.056 +/- 0.002 min-1, respectively; p greater than 0.4). The total area under the insulin curve during glucose tolerance tests was also similar in the clipped and sham groups (926 +/- 95 versus 869 +/- 126 microunits/ml x min; p greater than 0.4). There was no significant relation between systolic blood pressure and insulin area during glucose tolerance tests in the clipped group, but there was a positive rectilinear relation in the control group (r = 0.66; p = 0.01). Fourteen animals had euglycemic clamps 2 months after glucose tolerance tests. At that time, systolic pressure (direct femoral measurement) was higher in the seven clipped animals (189 +/- 13 mm Hg versus 122 +/- 5 mm Hg in controls; p less than 0.001). Insulin infusions of 1 and 4 milliunits/min/kg body wt effected similar plasma insulin levels in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothermia is critical for survival during prolonged insulin-induced hypoglycemia in rats.

Hypothermia is a well-known concomitant of hypoglycemia in mammals. We tested the hypothesis that this hypothermia is an important adaptive response to hypoglycemia in 11 normal Sprague-Dawley rats. Twelve-hour fasted, conscious animals received primed, continuous insulin infusions for up to 8 hours. Plasma glucose was clamped between 30 and 40 mg/dL and core body temperature was monitored continuously during the insulin infusions. Five of the animals were maintained in a room temperature environment (22 to 24 degrees C) during the hypoglycemia; all became hypothermic (mean +/- SE nadir core temperature, 31 +/- 0.5 degrees C). Spontaneous activity was reduced in these animals, but they remained conscious and responsive to external stimuli. All five returned to normal behavior after euglycemia was restored at the end of the insulin infusions. In the remaining six animals, hypothermia was prevented during hypoglycemia by warming of the air in their cages (mean of hourly core temperatures, 37 +/- 0.1 degrees C). None of these animals survived more than 7 hours. The severity of the hypoglycemia was no greater in the euthermic than in the hypothermic group, as judged by the mean of individual nadir plasma glucose levels (25 +/- 1 v 24 +/- 1 mg/dl, respectively) and by the mean number of glucose values per animal that were less than 30 mg/dL (2 +/- 1 v 7 +/- 1). Plasma osmolality did not change significantly in either group during the period of hypoglycemia, suggesting that dehydration was not the cause of death in the euthermic animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroimmune link in the mucosa of chronic gastritis with Helicobacter pylori infection.

It is suggested that different neuropeptides regulate gastric mucosal integrity and participate in the development of chronic gastritis. The aim of this study was to examine the roles and changes of immunoreactive (IR) nerves and immunocompetent cells in human gastritis. Immunohistochemical, immunocytochemical, and confocal laser microscopic methods were used. All investigated nerve fibers were found in different quantities in the mucosa of both control and gastritis samples. The number of SP, NPY, and VIP IR nerve fibers increased significantly (P < 0.05) in gastritis. No IR immunocompetent cells (lymphocytes, plasma cells, mast cells) were found in the control, however, some showed NPY (16.8%) and SP (9.4%) immunoreactivity in chronic gastritis. The distance between nerve fibers and immunocompetent cells was 200 nm to 1 microm. In conclusion, the increased number of SP, NPY, and VIP IR nerves and IR immunocytes suggests that they participate in development of neurogenic inflammation, repairing processes of chronic gastritis.

[Pinching of the single sesamoid bone of the thumb into the metacarpo-phalangeal joint without luxation]. / A hüvelykujj egyetlen sesam-csontjának ficamodás nélküli becsípödése az alapízületbe.

Authors demonstrate the extremely rare case of articular pinching of the single and not well-developed sesam bone at the metacarpophalangeal joint on over extension without luxation and associated injuries. After reviewing the history and pathology it is described that this alteration can be treated with voluntary muscle function and without operation too.

Biosynthesis of the side chain of yeast glycosylphosphatidylinositol anchors is operated by novel mannosyltransferases located in the endoplasmic reticulum and the Golgi apparatus.

Glycosylphosphatidylinositol (GPI) anchors of the yeast Saccharomyces cerevisiae have been reported to contain three different types of side chains attached to contain three different types of side chains attached to the alpha 1,2-linked mannose of the conserved protein-ethanolamine-PO4-Man alpha 1,2Man alpha 1,6Man alpha 1,4GlcNH2-inositol glycan core. The possible side chains are Man alpha 1,2- or Man alpha 1,2Man alpha 1,2- or Man alpha 1,3Man alpha 1,2- (Fankhauser, C., Homan, S. W., Thomas Oates, J. E., McConville, M. J., Desponds, C., Conzelmann, A., and Ferguson, M. A. (1993) J. Biol. Chem. 268, 26365-26374). To determine in what subcellular compartment these side chains are made, we metabolically labeled GPI-anchored membrane proteins with myo-[2-3H]inositol in secretion mutants blocked at various stages of the secretory pathway and analyzed the anchor structure of the labeled glycoproteins. When the exit of vesicles from the endoplasmic reticulum or entry into the cis-Golgi were blocked in sec12 or sec18 cells, all anchors contained a side chain consisting of a single alpha 1,2-linked mannose. GPI proteins trapped in the cis-Golgi of sec7 contained Man alpha 1,3Man alpha 1,2- but no Man alpha 1,2Man alpha 1,2-side chains. Mutants blocked at later stages of the secretory pathway made increased amounts of side chains containing two mannoses. Man alpha 1,2Man alpha 1,2- and Man alpha 1,3Man alpha 1,2- side chains were preferentially associated with ceramide- and diacylglycerol-containing GPI anchors, respectively. Mnn1, mnn2, mnn3, mnn5, and mnt1(=kre2), i.e. mutants which lack or down-regulate 1,2- and 1,3- mannosyltransferases used in the elongation of N- and O-glycans in the Golgi, add the fifth mannose to GPI anchors normally. The same conclusion was reached through the analysis of deletion mutants in KTR1, KTR2, KTR3, KTR4, and YUR1 which all are open reading frames with high homology to MNT1. Mutants deficient in the Golgi elongation of N-glycans such as anp1, van1, mnn9 are deficient in the maturation of the N-glycans of GPI-anchored glycoproteins, but process the GPI anchor side chain normally. Data are consistent with the idea that the fourth mannose is added to proteins as part of the anchor precursor glycolipid in the endoplasmic reticulum, whereas the fifth mannose is added by not yet identified alpha 1,3- and alpha 1,2-mannosyltransferases located in the Golgi apparatus.

Glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae: absence of ceramides from complete precursor glycolipids.

Glycosylphosphatidylinositol (GPI) anchoring of membrane proteins occurs through two distinct steps, namely the assembly of a precursor glycolipid and its subsequent transfer onto newly synthesized proteins. To analyze the structure of the yeast precursor glycolipid we made use of the pmi40 mutant that incorporates very high amounts of [3H]mannose. Two very polar [3H]mannose-labeled glycolipids named CP1 and CP2 qualified as GPI precursor lipids since their carbohydrate head group, Man alpha 1,2(X-->PO4-->6)Man alpha 1,2Man alpha 1,6Man alpha-GlcN-inositol (with X most likely being ethanolamine) comprises the core structure which is common to all GPI anchors described so far. CP1 predominates in cells grown at 24 degrees C whereas CP2 is induced by stress conditions. The apparent structural identity of the head groups suggests that CP1 and CP2 contain different lipid moieties. The lipid moieties of both CP1 and CP2 can be removed by mild alkaline hydrolysis although the protein-bound GPI anchors made by the pmi40 cells under identical labeling conditions contain mild base resistant ceramides. These findings imply that the ceramide moiety found on the majority of yeast GPI anchored proteins is added through a lipid remodeling step that occurs after the addition of the GPI precursor glycolipids to proteins.

Alternative lipid remodelling pathways for glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins of Saccharomyces cerevisiae exist with two types of lipid moiety--diacylglycerol or ceramide--both of which contain 26:0 fatty acids. To understand at which stage of biosynthesis these long-chain fatty acids become incorporated into diacylglycerol anchors, we compared the phosphatidylinositol moieties isolated from myo-[2-(3)H]inositol-labelled protein anchors and from GPI intermediates. There is no evidence for the presence of long-chain fatty acids in any intermediate of GPI biosynthesis. However, GPI-anchored proteins contain either the phosphatidylinositol moiety characteristic of the precursor lipids or a version with a long-chain fatty acid in the sn-2 position of glycerol. The introduction of long-chain fatty acids into sn-2 occurs in the endoplasmic reticulum (ER) and is independent of the sn-2-specific acyltransferase SLC1. Analysis of ceramide anchors revealed the presence of two types of ceramide, one added in the ER and another more polar molecule which is found only on proteins which have reached the mid Golgi. In summary, the lipid of GPI-anchored proteins can be exchanged by at least three different remodelling pathways: (i) remodelling from diacylglycerol to ceramide in the ER as proposed previously; (ii) remodelling from diacylglycerol to a more hydrophobic diacylglycerol with a long-chain fatty acid in sn-2 in the ER; and (iii) remodelling to a more polar ceramide in the Golgi.

Whole body insulin sensitivity in Osborne-Mendel and S 5B/Pl rats eating a low- or high-fat diet.

To determine whether whole body insulin sensitivity differs between a rat strain that does not (S 5B/Pl) and a strain that does [Osborne-Mendel (OM)] become obese when eating a high-fat diet, we performed euglycemic clamp studies in animals from each strain during low- and high-fat feeding. Clamps were performed after 2 days ("initial clamp") and 9 days ("final clamp") on each diet. Plasma glucose and insulin levels during the final 60 min of initial and final clamps were similar in S 5B/Pl and OM rats regardless of diet. Insulin sensitivity, measured as the glucose clearance rate during the final 60 min of the clamp, averaged 35 +/- 3 ml.kg-1.min-1 in S 5B/Pl rats after 2 days on a low-fat diet. This did not change significantly during an additional 7 days on the low-fat diet. The high-fat diet was associated with a 13% reduction in insulin sensitivity after 2 days and a 30% reduction after 9 days in S 5B/Pl rats. OM rats exhibited similar patterns of insulin sensitivity during low- and high-fat diets, albeit at lower insulin sensitivity overall (P < 0.0005 vs. S 5B/Pl). Mean glucose clearance after 2 days on the low-fat diet was 27 +/- 2 mg.kg-1.min-1 and did not change significantly during seven more days of low-fat feeding. The high-fat diet was associated with a 19% reduction in glucose clearance after 2 days and a 38% reduction after 9 days in OM rats. The magnitude of reduction in insulin sensitivity during high-fat diets did not differ significantly between strains.(ABSTRACT TRUNCATED AT 250 WORDS)

The collagen-binding site of type-II units of bovine seminal fluid protein PDC-109 and fibronectin.

A single type-II domain has been isolated by limited proteolysis of the collagen-binding bovine seminal fluid protein, PDC-109. The 45-residue fragment corresponding to the second type-II domain of the parent molecule was found to have retained affinity for immobilized collagen, indicating that this minidomain carries critical regions of the collagen-binding site. Studies on various fragments of fibronectin have also implicated the two type-II units of this molecule in collagen-binding. In the present work we have found that type-II domains of human fibronectin, expressed in Escherichia coli as beta-galactosidase fusion proteins, bind specifically to immobilized collagen.

Diffuse plasmacytosis in a child with brainstem glioma following multiagent chemotherapy and intensive growth factor support.

The use of granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) in order to abrogate chemotherapy-induced neutropenia has become a routine part of many cancer treatment regimes. However, there are still very few data available about possible complications related to repeated or prolonged use of these agents in patients with malignant solid tumors. The authors report a child with brainstem glioma who received repeated cycles of multiagent chemotherapy with G- or GM-CSF support. During this period of 10 months, no clinical side effects were observed that could have been attributed to growth factor administration. However, postmortem histological examination revealed the presence of diffuse plasmacytosis, a rare hematological disorder in childhood. Undifferentiated plasma cells of nonmonoclonal origin could be demonstrated infiltrating bone marrow, lungs, and lymph nodes of the patient. Based on previously published in vitro and in vivo evidence on the interleukin-6 (IL-6)-mediated stimulatory effect of G- and GM-CSF on myeloma cell proliferation, the authors suggest a possible link between extensive growth factor support and the development of plasmacytosis in this patient.

Hypertension without peripheral insulin resistance in spontaneously hypertensive rats.

We performed euglycemic clamp studies with labeled glucose to measure insulin's effect on hepatic glucose output (HGO) and peripheral glucose clearance in eight conscious mobile spontaneously hypertensive rats (SHR) and eleven normotensive Wistar-Kyoto (WKY) rats age 9-10 wk. Systolic blood pressure was elevated in the SHR (P less than 0.001), whereas means of 12-h-fasted plasma insulin (P greater than 0.4), glucose (P greater than 0.07), HGO (P greater than 0.25), and glucose clearance (P greater than 0.2) did not differ significantly between groups. Infusions of human insulin into SHR and WKY rats (1 and 1.5 mU.min-1.kg-1, respectively) during concomitant somatostatin administration reestablished basal insulinemia in both groups. Neither HGO (P greater than 0.15) nor glucose clearance (P greater than 0.3) differed significantly between SHR and WKY rats under those conditions. Somatostatin plus higher-dose insulin infusions (4 mU.min-1.kg-1 in SHR and 3 or 6 mU.min-1.kg-1 in WKY rats) resulted in physiological hyperinsulinemia in all rats. Insulin sensitivity, calculated as the increase in glucose clearance effected by an increase in circulating insulin during higher-dose insulin infusions, did not differ significantly between SHR and WKY groups (P greater than 0.3). HGO was completely suppressed in SHR and WKY rats during the higher-dose insulin infusions. Our data indicate that hypertension is present in SHR at an age when insulin-mediated glucose disposal is not different from age-matched WKY rats. These findings do not support a role for peripheral insulin resistance in the genesis of hypertension in SHR.

Deletion of GPI7, a yeast gene required for addition of a side chain to the glycosylphosphatidylinositol (GPI) core structure, affects GPI protein transport, remodeling, and cell wall integrity.

Gpi7 was isolated by screening for mutants defective in the surface expression of glycosylphosphatidylinositol (GPI) proteins. Gpi7 mutants are deficient in YJL062w, herein named GPI7. GPI7 is not essential, but its deletion renders cells hypersensitive to Calcofluor White, indicating cell wall fragility. Several aspects of GPI biosynthesis are disturbed in Deltagpi7. The extent of anchor remodeling, i.e. replacement of the primary lipid moiety of GPI anchors by ceramide, is significantly reduced, and the transport of GPI proteins to the Golgi is delayed. Gpi7p is a highly glycosylated integral membrane protein with 9-11 predicted transmembrane domains in the C-terminal part and a large, hydrophilic N-terminal ectodomain. The bulk of Gpi7p is located at the plasma membrane, but a small amount is found in the endoplasmic reticulum. GPI7 has homologues in Saccharomyces cerevisiae, Caenorhabditis elegans, and man, but the precise biochemical function of this protein family is unknown. Based on the analysis of M4, an abnormal GPI lipid accumulating in gpi7, we propose that Gpi7p adds a side chain onto the GPI core structure. Indeed, when compared with complete GPI lipids, M4 lacks a previously unrecognized phosphodiester-linked side chain, possibly an ethanolamine phosphate. Gpi7p contains significant homology with phosphodiesterases suggesting that Gpi7p itself is the transferase adding a side chain to the alpha1,6-linked mannose of the GPI core structure.