Chemical induction of the unfolded protein response in the liver increases glucose production and is activated during insulin-induced hypoglycaemia in rats.

AIMS/HYPOTHESIS: Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) can regulate insulin secretion, insulin action and in vitro hepatocyte glucose release. The aims of this study were to determine whether chemical agents that induce ER stress regulate glucose production in vivo and to identify a physiological setting in which this may be important. METHODS: A pancreatic clamp test was performed in anaesthetised rats, and insulin and glucagon were replaced at basal levels. [6,6-(2)H(2)]Glucose was infused in the absence (CON, n = 10) or presence of ER stress-inducing agents, namely, tunicamycin (Tun, n = 10) or thapsigargin (Thap, n = 10). RESULTS: Arterial insulin, glucagon, corticosterone and NEFA concentrations were constant throughout experiments and not different among groups. After 1 h, the glucose concentration was significantly increased in Tun and Thap rats (1.5 +/- 0.2 and 2.1 +/- 0.3 mmol/l, respectively; mean +/- SD), but did not change in CON rats. Glucose production increased (p < 0.05) by 11.0 +/- 1.6 and 13.2 +/- 2.2 micromol kg(-1) min(-1) in Tun and Thap rats, respectively, but did not change in CON rats. When glucose was infused in a fourth group (HYPER) to match the increase in glucose observed in the Tun and Thap rats, glucose production decreased by approximately 22 micromol kg(-1) min(-1). Liver phosphorylase activity was increased and glycogen decreased in the Tun and Thap groups compared with the CON and HYPER groups. Given that glucose deprivation induces ER stress in cells, we hypothesised that hypoglycaemia, a condition that elicits increased glucose production, would activate the UPR in the liver. Three hour hyperinsulinaemic (5 mU kg(-1) min(-1)) -euglycaemic (EUG, approximately 7.2 mmol/l, n = 6) or -hypoglycaemic (HYPO, approximately 2.8 mmol/l, n = 6) clamps were performed in conscious rats. Several biochemical markers of the UPR were significantly increased in the liver, but not in kidney or pancreas, in HYPO vs EUG rats. CONCLUSIONS/INTERPRETATION: Based on our findings that the chemical induction of the UPR increased glucose production and that prolonged hypoglycaemia activated the UPR in the liver, we propose that the UPR in the liver may contribute to the regulation of glucose production during prolonged hypoglycaemia.

Purification and characterization of beta-N-acetylhexosaminidase from bovine tick Boophilus microplus (Ixodide) larvae.

beta-N-Acetylhexosaminidase (HEX, E.C. 3.2.1.52) from larvae of the ixodid tick Boophilus microplus was purified to capillary zone electrophoresis homogeneity, and characterized. Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-200, p-aminobenzyl-N-acetyl-beta-D-thioglucosamine affinity, and Mono-Q FPLC columns. Purification was about 1600-fold, with a yield of 10%, as determined with p-nitrophenyl-N-acetylglucosaminide as substrate. The enzyme presented optimum pH 4.7, and optimum temperature 65 degrees C. The molecular weight of non-denatured enzyme was estimated as 127,000 by gel filtration chromatography, and 60,000 in SDS-PAGE. The tick hexosaminidase presented glycosyl residues, as evidenced by binding to Concanavalin-A. Among several p-nitrophenyl glycosides tested as substrate, HEX was active only on p-nitrophenyl-N-acetylglucosaminide and p-nitrophenyl-N-acetylgalactosaminide. The purified enzyme presented immunogenicity in rabbit, and the correspondent antibodies inhibited about 90% of its original, in vitro activity.

Effect of antibodies against beta-N-acetylhexosaminidase on reproductive efficiency of the bovine tick Boophilus microplus.

A polyclonal antibody (anti-HEX) was developed against a soluble N-acetylhexosaminidase (HEX) isolated from larval extracts of Boophilus microplus. Purified hexosaminidase was strongly inhibited by the IgG fraction of this antibody. The antibody inhibited the hexosaminidase activity of other sources, such as haemolymph and larval membranes. The antibody reacted with different antigens in the tick haemolymph, but did not recognize any antigen in saliva, as seen by immunoblot analysis. The anti-HEX was inoculated into fully engorged B. microplus females, resulting in a decrease in oviposition of approximately 26%, relative to the effect of pre-immune IgG. These data show the potential of the use of this tick enzyme as an antigen in vaccine development.

Isolation of an aspartic proteinase precursor from the egg of a hard tick, Boophilus microplus.

An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.

Immunization of bovines with an aspartic proteinase precursor isolated from Boophilus microplus eggs.

The capacity of the Boophilus Yolk pro-Cathepsin (BYC) to induce a protective immune response in cattle against Boophilus microplus infestation was tested by vaccination experiments and by inoculation of monoclonal antibody (MAb) against BYC into fully engorged tick females. In immunization experiments the measurement of various biological parameters demonstrated a partial protection against B. microplus. A continuous decrease in the levels of specific antibodies was observed over 11 months when six bovines were maintained in field conditions. The inoculation of the MAb into tick females produced a dose-dependent decrease in oviposition and survival of the ectoparasite compared to the control.

Monoclonal antibodies against Boophilus microplus and their effects on tick reproductive efficiency.

Four monoclonal antibodies (mAbs) against extracts of embryo and gut tissue obtained from fully engorged Boophilus microplus were produced. The mAb BrBml reacted with different instars and tissues, the BrBm2 recognized only antigens present in gut extract and the mAbs BrBm3 and BrBm4 recognized vitellin. The effect of inoculation of these mAbs into fully engorged Boophilus microplus females was also evaluated. The mAbs BrBm1 and BrBm2 caused a decrease in oviposition of approximately 50% and 70%, respectively, and the mAbs BrBm3 and BrBm4 did not affect reproductive efficiency. This assay may be useful as a low-cost test to provide preliminary information on the possible effects of anti-tick antibodies in damaging ticks before attempting cattle vaccination experiments.

Functional bovine immunoglobulins in Boophilus microplus hemolymph.

The aim of the present work was to quantify the passage of bovine immunoglobulins into the hemolymph of the tick Boophilus microplus during the feeding process and to determine their antibody activity. The knowledge is of paramount importance when vector control or blocking of disease transmission is attempted by vaccination of cattle. Approximately 2% of bovine immunoglobulin present in the serum as determined by competitive ELISA was demonstrated in hemolymph of B. microplus and antibody activity against an antigen of B. microplus in the hemolymph of ticks fed on bovine immunized with the antigen purified from tick eggs was detected by Western blot assay. The antibody reactivity detected against the B. microplus antigen showed that functional antibodies are present in the hemolymph of fully engorged ticks for at least 48 h after completing the parasitic life cycle.

Changing patterns of vitellin-related peptides during development of the cattle tick Boophilus microplus.

The major components of protein extracts from the cattle tick Boophilus microplus eggs and larvae of various ages were characterized by molecular sieving chromatography, ion exchange chromatography and SDS-PAGE. The fractions analysed showed a changing chromatographic pattern development. A serum raised against the components of a fraction showing characteristics of vitellin strongly reacted in Western blots with the major peptides of extracts from eggs, larvae, gut and ovary. Comparison of patterns obtained by electrophoresis in non-denaturing PAGE, stained with Coomassie blue or with benzidine/hydrogen peroxide, revealed that the major proteins of these extracts are haemoproteins, possibly in different aggregation states or heterogeneous in composition.

Genetic analyses of tattered, an X-linked dominant, developmental mouse mutation.

Tattered (Td) is an X-linked dominant mouse mutation that causes prenatal lethality in affected males. To map the locus, we analyzed 199 normal male and affected female progeny from a backcross of Td and Mus castaneus. Pedigree analysis of these animals suggests a gene order of cen-DXWas70-(Td, DXMit26, Gata1, Tcfe3)-(Cybb, Otc)-tel, where Tcfe3 is a transcription factor homologous to a gene involved in the murine microphthalmia (mi) mutation [Hodgkinson et al. Cell 74, 395-404, 1993]. To evaluate Tcfe3 as a candidate for Td, heterozygous tattered females were crossed to xid males to obtain females in which > 95% of B cells expressed genes solely from the Td X Chromosome (Chr). Fluorescent activated cell sorting (FACS) analysis and Western blotting of isolated splenocytes from Td/xid double heterozygotes rule out Tcfe3 as a likely candidate for the Td mutation.

Therapeutic and persistent efficacy of doramectin against Boophilus microplus in cattle.

One therapeutic and one persistent efficacy study were conducted in Brazil to evaluate doramectin at a dose rate of 200 micrograms kg-1 against induced infestations of the single host tick, Boophilus microplus. Doramectin was highly effective in eliminating established tick populations from cattle and also in preventing infestation by the parasite. In the therapeutic study, 12 calves were infested three times a week along the dorsal line with 2500 recently hatched larvae, for a total of 11 times before treatment. Animals were allocated to two groups on the basis of uniformity of established engorged tick burdens. Six calves were treated with doramectin and six received saline solution. From Day -3 to Day 21 post-treatment, individual collections of detached engorged female ticks were made from each calf. In the persistent efficacy study, 12 calves were allocated to two groups of six animals. Six calves were treated with doramectin and six received saline solution. From Day 1 to Day 17 post-treatment, each animal was infested three times a week along the dorsal line with 2500 recently hatched Boophilus microplus larvae, for a total of nine times. From Day 18 to Day 42 post-treatment, daily collections of detached engorged female ticks were made from individual animals. In the therapeutic study, efficacy (reduction of collected engorged female ticks) progressed from 51% at 24 h post-treatment (p.t.) to at least 99% at 4 days p.t., and reached 100% at 8 days p.t. With the exception of one tick that did not lay eggs, recovered from one animal at 11 days p.t., no more ticks were recovered from doramectin-treated calves for the duration of the experiment. For the first 6 days after treatment, only a few detached engorged ticks were collected from treated animals, and their oviposition and hatchability declined rapidly. In the persistent efficacy study, doramectin treatment was highly efficacious in preventing the establishment of Boophilus microplus populations for 20 days after the first ticks completed their cycle in the non-treated group. The oviposition and hatchability of the few ticks that completed their life cycle in the doramectin group were severely reduced.

Comparative mapping on the mouse and human X chromosomes of a human cDNA clone encoding the vasopressin renal-type receptor (AVP2R).

Mutations in the gene for the human renal-type vasopressin receptor (V2R) have recently been identified in patients with nephrogenic diabetes insipidus (NDI). Both V2R and NDI have been independently mapped to Xq28. Using a combination of genetic and physical mapping, we have localized the murine V2r locus to within 100 kb of L1Cam on the mouse X chromosome in a region syntenic with human Xq28. Based on conserved gene order of mouse and human loci in this region, physical mapping using DNA derived from human lymphoblasts has established that the corresponding human loci V2R and L1CAM are linked within 210 kb. The efficiency and precision of genetic mapping of V2r and other loci in the mouse suggest that it might be easier to map additional human genes in the mouse first and infer the corresponding human location. More precise physical mapping in man could then be performed using pulsed-field gel electrophoresis and/or yeast artificial chromosomes.

Genetic mapping of the X-linked dominant mutations striated (Str) and bare patches (Bpa) to a 600-kb region of the mouse X chromosome: implications for mapping human disorders in Xq28.

Striated (Str) and bare patches (Bpa) are X-irradiation-induced, X-linked dominant mouse mutations that are lethal prenatally in hemizygous males. To map the Str mutation, we generated a backcross involving Mus castaneus. Pedigree analysis of 193 affected female and normal male progeny from the cross places Str extremely close to DXMIT1 and favors a gene order of (Cf-9)-Ids-Gabra3-DXS1104h-(Str, DXMIT1)-F8a-DXPas8-DXBay6-DXMIT6 for the loci studied. This region of the mouse X Chromosome (Chr) is syntenic with proximal human Xq28. Based on the mode of inheritance and clinical phenotype, Str may be a homolog of human familial incontinentia pigmenti (IP2). Further refinement of our genetic mapping of bare patches positions that locus between DXS1104h and DXPas8 in the same region as Str, raising the possibility that Bpa and Str may be allelic or are due to mutations in overlapping contiguous genes.

Major histocompatibility complex class II alleles in Kawasaki syndrome--lack of consistent correlation with disease or cardiac involvement.

Recent refinements in molecular genetic typing have allowed the precise determination of the extensive polymorphism now recognized in class II major histocompatibility complex (MHC) genotypes. This could have important applications in Kawasaki syndrome (KS), where the relative contribution of genetic factors is now well known. Accordingly, 44 Caucasian, 13 Asian, and 5 American black patients, as well as 221 Caucasian controls were typed for HLA-DRB1, DRB3, DRB4, DQA1, DQB1, and DPB1 alleles by oligonucleotide probe hybridization of polymerase chain reaction amplified genomic DNA using probes and primers supplied by the 11th International Histocompatibility Testing Workshop. Among the 15 HLA-DRB1, 3 DRB3, 9 DQA1, 15 DQB1, and 19 DPB1 alleles examined, none were found to be significantly associated with KS, except for an increased frequency of HLA-DRB3*0301 in Houston Caucasian patients when compared to Houston Caucasian controls (38 vs 11%, pc = 0.012, RR = 5.0). Twelve patients developed coronary artery involvement of whom 7 had aneurysms and 5 had dilatation (8 Caucasians, 2 blacks, 2 Asians). No specific HLA class II allele was associated with this disease complication. Despite a regional association, our data fail to support a consistent role for MHC class II alleles in the pathogenesis of KS.

DNA analysis of HLA-DR, DQ, and DP alleles in children with polyarticular juvenile rheumatoid arthritis.

HLA-DR, DQ and DP alleles were determined by restriction fragment length polymorphism analysis and oligonucleotide probe hybridization of polymerase chain reaction amplified genomic DNA in 94 Caucasian children with polyarticular juvenile rheumatoid arthritis (JRA) [13 rheumatoid factor (RF)+ and 81 RF-] and 100 healthy controls. HLA-DRw8, DQw4, DQA1*0401, DQB1*0402 were increased in frequency in those patients with RF seronegative disease, with highest frequencies seen in patients with young age at onset (< 5 years of age). These findings were similar to what we observed in children with pauciarticular JRA, especially those with young age at onset. DPB1*0301 was also found in increased frequency in the RF- group, and in particular those seronegative for antinuclear antibody. In contrast to what is observed in patients with pauciarticular JRA, the frequency of DPB1*0201 was not increased in any polyarticular JRA patient group. These data suggest that polyarticular JRA shares many genetic features with pauciarticular JRA.

Extrachromosomal nucleic acids in bovine Babesia.

Two kinds of small extrachromosomal nucleic acid elements were found in the bovine babesias, Babesia bovis and B. bigemina. One element with an apparent size of 5.5 kilobase pairs (kbp) is a double stranded RNA related to virus like particles. Another molecule is a double stranded DNA with a molecular size of about 6.2 kbp. Southern blot comparison of restriction DNA fragments of the latter molecule, which is present in both B. bovis and B. bigemina is described.

DNA analysis of HLA-DR, DQ and DP genes in pauciarticular juvenile rheumatoid arthritis.

HLA-DR, DQ, and DP alleles were determined by restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization analysis in 50 Caucasian children with pauciarticular juvenile rheumatoid arthritis (PaJRA) and 82 controls. There was an increased frequency of DR5, DRw8, and DQw4, as well as individual DQ alpha and beta chains, DQA*0401 and DQB1*0402, respectively, in this group of patients. There was an absolute association between DRw8, DQw4, DQA1*0401, and DQB1*0402 in the patient population. HLA-DPw2.1 was also increased in frequency. There was little evidence of linkage disequilibrium found between DPw2.1 and DR5, DRw8, or DQw4. These MHC Class II associations were more characteristic of those patients with young age of onset (less than 5 years), rather than those with onset greater than or equal to 5 years of age. Our data confirmed the previous associations of HLA-DR5, DRw8, and DPw2.1 with PaJRA and suggested a new association for DQ alpha and beta genes in the clinical expression of this disease.

A putative RNA virus in Babesia bovis.

Babesia bovis is an intraerythrocytic protozoan that causes bovine babesiosis. Agarose gel electrophoresis of nucleic acids extracted from two isolates of B. bovis reveals, besides bulk DNA, an ethidium bromide-stainable band at about 5.5 kb. Further characterization of the latter with DNase I, RNase and mung bean nuclease suggested it to be a double-stranded RNA. Sonicated parasites were fractionated in a CsCl buoyant density gradient. A sample containing the 5.5-kb RNA was analysed under an electron microscope and a virus-like particle was observed.

Ocorrência de Boophilus microplus livre de Babesia spp em regiäo indene (Santa Vitória do Palmar, RS, Brasil) / Occorrence of Boophilus microplus free from Babesia spp in undamaged region (Santa Vitória do Palmar, RS, Brazil)

A entrada de animais infestados por carrapatos nas regiöes livres, nos meses de veräo, provoca surtos de tristeza parasitária nos animais nativos, que normalmente se encontram sem proteçäo para esta enfermidade. Nos veröes de 1987 e 1988, ocorreram vários focos isolados de carrapatos em Santa Vitória do Palmar, regiäo indene do extremo sul do Brasil. O presente trabalho teve como objetivo colaborar no esclarecimento do assunto, chegando os autores à conclusäo de que o carrapato ali existente é o Boophilus microplus, naturalmente livre de Babesia spp

Total disconnection of the right ventricular free wall: surgical treatment of right ventricular tachycardia associated with right ventricular dysplasia.

Arrhythmogenic right ventricular dysplasia is a myopathy that affects the right ventricular free wall (RVFW) and gives rise to recurrent reentrant ventricular tachycardia (VT). Because the entire right ventricle is potentially arrhythmogenic, ablating a single site of VT may not eliminate the arrhythmia. We developed an operation to confine any arrhythmic activity arising from the right ventricle to that chamber: total disconnection of the RVFW from the left ventricle. We performed RVFW disconnection in two patients with refractory VT associated with arrhythmogenic right ventricular dysplasia. At least two sites or origin of morphologically distinct VT were identified in the RVFW in each patient. RVFW disconnection was carried out under normothermic cardiopulmonary bypass. An encircling incision was made along the attachment of the RVFW to the aortoventricular unit and the tricuspid annulus; the right coronary artery and its RVFW branches were left intact. Electrical activity of the two chambers became dissociated, and VT arising from the RVFW was confined to that chamber. Postoperatively, there was no clinical evidence of hemodynamic impairment (follow-up 4 months and 3 months). Left ventricular function was unchanged and right ventricular flow was maintained by atrial contraction and motion of the septum toward the RVFW during left ventricular systole. One patient had incessant right ventricular tachycardia confined to the RVFW for 3 weeks. We conclude that RVFW disconnection is feasible and applicable to patients with refractory VT originating in the diffusely diseased RVFW.

Varices colonicas. Presentacion de dos casos y revision de la literatura / Colonic varices. Report of 2 cases and review of the literature

Se presentan 2 pacientes con varices colonicas. Se hace una breve revision de la literatura, la cual incluye un total de otros 33 casos. La mayoria de los pacientes tienen hipertension portal. El motivo de consulta mas frecuente es el sangramiento rectal. La endoscopia es el metodo mas util para el diagnostico. El tratamiento que mejores resultados parece dar en los casos de hemorragia masiva es la anatomosis porto-cava