Low Molecular Weight Bands Research Articles

Polymerization of normal and intact β2-microglobulin as the amyloidogenic protein in dialysis-amyloidosis

The primary structure of beta 2-microglobulin (beta 2m), the major constituent protein of beta 2-microglobulin amyloidosis (A beta 2m) or dialysis-amyloidosis, was initially shown to be identical to serum beta 2m, thereby strongly suggesting the polymerization of intact beta 2m in tissues. Recent biochemical data have been controversial, showing beta 2m acidic isoforms, fragmentation and amino acid sequence alteration of deposited beta 2m. The aim of this study was to reinvestigate beta 2m amyloid deposits for the presence of beta 2m fragments and/or amino acid sequence alteration. Four amyloid-laden tissues (3 femoral bone amyloid cysts and 1 heart tissue) from dialysis patients were used to isolate amyloidogenic beta 2m. Amyloid fibrils were isolated using the classic water extraction method, and purified in 6 M guanidine on a gel-filtration column. The protein was further purified on 17% SDS-PAGE gel, and transferred to a nitrocellulose membrane for immunostaining with antihuman beta 2m. beta 2m samples were microsequenced using the standard 03RPTH program on a 470A gas-phase sequencer, and HPLC was performed after digestion with trypsin. Two peaks were obtained with the gel filtration column, the second corresponding by molecular weight to beta 2m. SDS-PAGE analysis of this peak under reducing conditions, demonstrated one major band at 12,000 Da and a minor band at 25,000 Da (monomer and dimer), and no lower molecular weight bands were observed. The 12 kDa band was micro-sequenced and the amino acid sequence corresponded to that of normal beta 2m through the 40th residue. Amino acid sequence analysis showed no difference from normal beta 2m in any of the beta 2m proteins contained in the amyloid deposits isolated from the four studied tissues. Also, the HPLC profile of the four protein samples were strictly normal and identical to a commercial preparation of beta 2m. The present study demonstrates that beta 2m molecules polymerized in amyloid fibrils and deposits are intact and have a normal amino acid sequence, and produced by a specific and unique fibrillogenetic mechanism, which does not require proteolytic processing from the precursor protein to the amyloid fibrils.

Characterization and identification of a human dentin phosphophoryn

The present study further characterizes an extract from immature, human tooth apicies from which an intact dentin phosphoprotein has been identified. Third molar apicies from developing roots were decalcified in 10% EDTA until Ca2+ was undetectable in the decalcifying solution. The crude extract was run on 7.5% SDS-PAGE and stained with "Stains-All." Four distinct bands were found and the molecular weights were 140, 60, 50, and 34 k. When run on a SDS-PAGE under nonreducing conditions the 60, 50, and 34 k bands were absent. These results suggest that the lower molecular weight bands may be subunits of the larger protein. The extract was then further purified by adding CaCl2 and MgCl2 to precipitate the phosphoprotein. The precipitate was subjected to a DEAE-Sepharose CL6B column and eluted by 0-0.7 M NaCl gradient solution. The amino acid composition of the purified phosphoprotein was determined and the extract was found to be rich in serine and aspartic acid residues. The N-terminal peptide Asp-Asp-Pro was identified. The sequence of the three amino acids is identical to rat incisor phosphoprotein.

Cleavage of Human Corneal Type VI Collagen ??3 Chain by Matrix Metalloproteinase-2

The human cornea contains significant amounts of type VI collagen and matrix metalloproteinase-2 (MMP-2), but there has been no established relation between these two components. The objective of this study was to determine whether corneal type VI collagen was susceptible to digestion by MMP-2. Human corneas were frozen and then pulverized in liquid nitrogen. The type VI collagen was isolated by sequential extractions with sodium chloride buffer, guanidinium chloride solution, and guanidinium chloride/dithiothreitol solution. Visualization of type VI collagen alpha 3(VI) chain was made by using Western blots with specific monoclonal antibodies. The extracts were incubated up to 24 h with isolated, activated MMP-2. Within 4 h of incubation, two lower molecular weight bands (approximately 190 and 170 kDa) appeared. These bands increased in intensity with time but were not further digested into smaller fragments. This cleavage activity was inhibited by ethylenediaminetetraacetic acid (EDTA). Because type VI collagen represents approximately 25% of the corneal dry weight, its degradation properties may be important for the integrity of the stroma in scarring episodes and corneal diseases, such as keratoconus.

Evidence for the existence of juvenile hormones in ticks.

Juvenile hormones (JHs) comprise a family of sesquiterpenoid compounds identified primarily from insects. JHs are involved in preventing metamorphosis during moults in immature stages and regulating aspects of reproduction in adults[l]. The JH titre is strictly regulated by a number of different mechanisms at specific times during development. JHs are synthesized and released into the haemolymph, where they are bound to haemolymph binding-proteins which protect them from esterases[2]. When JHs are no longer needed, they are degraded by specific JH-esterases and JH-epoxidases to the inactive products, JH-acids and JH-diols[3]. Ticks (Acarina:lxodidea), like insects, pass through several immature stages before becoming reproductively competent. JHs have not been identified in ticks, but several reports suggest that JHs are present and have roles similar to those in insects[4,5]. This study presents evidence for the occurrence of haemolymph JH-binding proteins and of specific JH-esterase and epoxidase activities, thus, indirectly supporting the presence of JH-like compounds in the adult tick, Amblyomma hebraeum. Ticks were raised as previously described[6] and taken for assay at various times during the feeding cycle. For JHesterase and epoxidase studies, tick tissues were divided into three categories: haemolymph, gut tissue and 'remaining tissues' (included fat body, muscle, ovary, salivary glands, and synganglion). Total esterase and epoxidase activities were determined by homogenizing the tissue in phosphate buffered saline (0.1M phosphate buffer, 100 mM NaCI) and incubating an aliquot with 0.12 HCi [3H]JHIII (0.58 Ci/mol) for 4h at 26'C. The sample was then extracted into acetonitrile and radioactivity analyzed by HPLC connected to an on-line radioactivity detector. Separation was performed on a C I S column using a linear gradient of acetonitri1e:water (35:65. v/v) to acetonitrile over 45 min at a flow rate of 1 ml/min. Non-specific esterase activity was assessed according to Rotin et a1.[7]. Haemolymph binding studies using the photoaffinity label, [3H]epoxy-bis-farnesyI diazoacetate ([3H]EBDA), followed the procedure of King ef al.[8]. For this. diluted haemolymph was incubated with 1 pCi [3H]EBDH or I 1.11 [3H]EBDA + 10 nmol JHI in polyethylene glycol-treated quartz cuvettes and exposed to 254 nm light for 5 min in a Rayonete Photoreactor. Following SDS-PAGE, gels were prepared for fluorography according to Prestwich et al. [9]. Incubation of tissues with [3H]JHIII suggests that only the 'remaining tissues' group showed specific esterase activity (total esterase activity non-specific esterase activity). Epoxidase and JH-specific esterase activities in this group of tissues were low in small, partially fed females, peaked just prior to engorgement. then declined to initial levels at the time of engorgement. Of the two enzymes examined, the JHepoxidase showed the greatest activity. Gut tissue apparently could only non-specifically metabolize JHIIl to the inactive -acid, -diol, and -acid-diol derivatives. Haemolymph tissue showed no detectable esterase or hydrolase activity but this could be due to difficulties in obtaining a sufficient volume of haemolymph for the assay. Photoaffinity labelling experiments using the JHI photoaffinity analogue, [3H]EBDA, with haemolymph from partially fed females indicated the presence of two regions of [3H]EBDA binding on SDS-PAGE gels that could be displaced by excess JHI. These corresponded to a high molecular weight region (75-100 kDa) and a single, low molecular weight band (approximate molecular weight 44 kDa). Kulcsar et al.[10] identified a similar, high molecular weight binding region (66-97 kDa) using the JHIII-specific photoaffinity label [3H]epoxy-farnesyl diazoacetate but suggested that this labelling was due to non-specific binding of [3H]EBDA to lipoproteins. It is likely that the high molecular weight region we observe is also due to nonspecific binding of lipoproteins. The 44 kDa labelled protein observed on SDS-PAGE gels is more suggestive of a JHIdisplaceable binding protein, however, at present i t is not known whether this protein is specific for a JH-like compound or is developmentally regulated throughout the feeding cycle. Although direct identification of JHs in tick haemolymph/tissue is ultimately needed, a combination of indirect evidence using complementary techniques can be strongly suggestive of the presence of JH. The identification of a haemolymph JHI-displaceable binding protein using the photoaffinity label EBDA indicates that a mechanism for JH protection and distribution exists in ticks. However, binding displacement by JHI does not imply that endogenous JHI is present in ticks and may merely reflect cross-reactivity to another related compound. Furthermore. the enzymes responsible for JH degradation in insects are also present in tick tissues and these enzymes appear to be developmentally regulated. Given that ticks possess these basic mechanisms for strict JH titre regulation, it is compelling to suggest that JHs do have important functions in tick development.

A Novel Subepidermal Blistering Disease with Autoantibodies to a 200-kDa Antigen of the Basement Membrane Zone

Several components of the basement membrane zone (BMZ) have been identified as antigenic targets in autoimmune bullous diseases. We report a novel disease with autoantibodies to a BMZ antigen that is different from the targets described so far. The patient suffering from this disorder showed tense bullae and severe mucous membrane involvement rapidly responding to oral tetracyclines and colchicine. Histopathologic findings resembled those of dermatitis herpetiformis. Direct immunofluorescence microscopy showed linear deposits of IgG and C3 at the BMZ. By indirect immunofluorescence studies on split human skin, using both 1 M NaCl and suction blistering for dermal-epidermal separation, IgG antibodies localized exclusively to the dermal side of the split. The antibodies were mainly of the IgG4 sub-class. By Western blot analysis of epidermal and dermal extracts, the patient's serum unequivocally reacted with a dermal antigen of 200 kDa. It did not recognize bullous pemphigoid antigens (the autoantigen of epidermolysis bullosa acquisita), purified preparations of laminin-1 and laminin-5, or the recently described 105-kDa BMZ antigen. By immunoblotting of concentrated conditioned SCC-25 medium, the patient's antibodies reacted with a band of 200 kDa and several bands of lower molecular weight. No reactivity was seen with extracts of cultured human fibroblasts. By indirect immunogold electron microscopy, immunoreactants localized to the lower lamina lucida. After clearance of skin lesions, both indirect immunofluorescence and Western blot analysis became negative. This patient suffers from a novel autoimmune bullous disease with autoantibodies to a 200-kDa antigen of the BMZ.

Dynamic expression of a cell-surface determinant ofCandida albicans

Monoclonal antibodies against germ-tube specific antigens ofCandida albicans were produced by a selective immunization protocol. The monoclonal antibody (MAb) DC3:H10 recognized a determinant preferentially expressed by germ tubes of the fungus compared to yeast cells. The MAb DC3:H10 antigen was expressed by nearly all germ tubes of the strains 3153A and B311. It was also expressed by germ tubes of the white phenotype of strain WO-1, and by many exponentially growing yeast cells of the opaque phenotype of this strain. The determinant appeared very rapidly after induction of germ tubes of strain 3153A. The MAb DC3:H10 epitope was sensitive to proteolytic treatment but not to periodate treatment, indicating its protein nature. The reactive material could be extracted from intact germ tubes by treatment with β-mercaptoethanol. On elution from a G-200 Sephadex column it yielded an apparent molecular weight of 519 kDa. This fraction appeared heterogeneous since at least two major bands of lower molecular weight were detected by silver staining following electrophoretic separation under denaturing conditions.

Subspecies discrimination of staphylococci from revision arthroplasties by ribotyping

Sixty-six cultures of Staphylococcus spp. were obtained from bone and tissue samples collected from 37 patients during revision arthroplasties and were speciated and ribotyped to assess strain diversity in each species. There were 10 ribotypes among 51 isolates of S. epidermidis, three among three isolates of S. capitis, two among four isolates of S. aureus and two among two isolates of S. simulans. One ribotype was found among each of: two isolates of S. warneri; two isolates of S. haemolyticus and single isolates of S. cohnii and S. saprophyticus. Low molecular weight bands of ribotype patterns characterized one or two related species whereas high molecular weight bands were useful for distinguishing types within species. Specimens from 17 patients yielded more than one isolate of Staphylococcus spp. In 13 of these patients the isolates were representatives of a single species but in only eight did ribotyping show the isolates to be identical. The findings of multiple species and ribotypes from samples taken from the same patient may have implications for understanding the nature of infection in revision arthroplasty and for antibiotic therapy.

Inhibition by activated neutrophils of the Ca2+ pump ATPase of intact red blood cells

Human neutrophils, activated by phorbol myristate acetate in the presence of intact red blood cells (RBCs), caused inhibition of the Ca2+ pump ATPase of the RBCs and fragmentation of the enzyme as well as other membrane proteins. Inhibition of the Ca2+ pump ATPase of intact RBCs was directly related to the neutrophil concentration and the time of incubation. Ca2+ pump ATPase activity was partially protected by the addition of exogenous glutathione-glutathione peroxidase, but not by superoxide dismutase. The addition of sodium azide, a potent inhibitor of endogenous RBC catalase, enhanced inhibition of the Ca2+ pump ATPase of intact RBCs. Examination by SDS-polyacrylamide gel electrophoresis of membrane proteins isolated from RBCs preincubated with activated neutrophils showed gross changes in banding patterns as compared to controls. Thus, a significant amount of methemoglobin appeared to be associated with the membrane proteins, and, in general, protein bands appeared to be more diffuse and less defined than proteins in control lanes. In addition, there was an increase in the low molecular weight protein bands. Using a monoclonal antibody to the Ca2+ pump ATPase, it was shown that the 140 kDa band representing the Ca2+ pump ATPase decreased, with concomitant appearance of two low molecular weight bands running at 8.2 and 6.8 kDa in the membrane proteins from RBCs preincubated with activated neutrophils. The data are interpreted to suggest that inhibition of the Ca2+ pump ATPase in intact RBCs under these conditions occurred as a result of: neutrophil-derived superoxide, dismutation of superoxide, to H2O2, diffusion of H2O2 into RBCs, a Fenton type reaction between oxyhemoglobin, and H2O2 producing hydroxyl radical and/or a ferryl radical capable of promoting protein fragmentation of RBC membrane proteins, including the plasma membrane Ca2+ pump ATPase.

Purification of a fraction of Giardia lamblia trophozoite extract associated with disaccharidase deficiencies in immune Mongolian gerbils (Meriones unguiculatus).

The effects on disaccharidase activities of challenging gerbils previously exposed to Giardia lamblia with fradions of the crude trophozoite extract were examined. Gel filtration of the soluble extract on a Sephacryl S-200 HR column resulted in 3 fradions: F1, F2 and F3. Only a challenge with fraction F1 (0.1 mg total dose) was found to induce disaccharidase deficiencies. Boiling F1 prior to challenge did not change this effect on the enzyme activities. However, the decreases were not obtained when the total F1 dose was reduced to 0.05 mg. Column chromatography of fraction F1 under dissociating and reducing conditions resulted in 2 further fractions: F1a and F1b. Challenging immune gerbils with F1b led to impairments of disaccharidose activity similar to those obtained with F1. Protein analysis of the crude extract, as well as the fractions of the extract, revealed several high and low molecular weight bonds. These findings indicate that a constituent(s) of fraction F1b is the portion of the parasite which induces disaccharidase deficiencies in immune gerbils. This fraction consists of proteins ranging in molecular weight from 32 to 200 kDa. In addition the G. lamblia fraction involved in the decreases in enzyme activity is heat-stable.

Differential Expression and Subcellular Localization of Protein Kinase C Isoforms in Neuronal and Cardiac Tissues of Limulus polyphemus

The presence and subcellular distribution of specific protein kinase C (PKC) isoforms in Limulus neuronal and cardiac tissue extracts were determined using polyclonal antibodies to individual PKC isoforms. Western blot analysis revealed that the brain, abdominal ganglia, cardiac ganglia and heart all contained PKC′s α, β, γ, ϵ and ζ. PKC δ was detected in all neuronal tissues. PKC θ was found only in the cardiac ganglia and heart. PKC′s γ and δ were predominantly localized in membrane fractions while the α, β, ϵ, ζ and θ isoforms were found in both fractions. Antibodies to PKC′s ϵ and ζ detected relatively high molecular weight bands in membrane fractions and lower molecular weight bands in the cytosolic fractions. The results demonstrate that Limulus neuronal and cardiac tissues differentially express the conventional, novel and atypical isoforms of protein kinase C.

β-endorphin binding in cultured adrenal cortical cells

The polypeptide β-endorphin binds to cultured bovine adrenal cortical cells in a naloxone insensitive manner, β-endorphin and N-Acetyl-β-endorphin are equipotent in inhibiting binding. The amino terminal 27 amino acid fragment referred to as β-endorphin[1-27] shows no ability to inhibit binding, whereas the carboxy-terminal tetrapeptide Lys-Lys-Gly-Glu partially inhibits binding. ACTH, angiotensin II and met-enkephalin show little or no ability to inhibit β-endorphin binding. Competition bin-ding reveals an apparently single affinity class with Kd of 33 nM. Molecular cross linking experiments reveal putative receptor subunits of 85 kD, 64 kD, 54 kD and 44 kD. The lower molecular weight bands are preferentially cross-linked by a hydrophobic cross linking reagent, in contrast to the two higher molecular weight bands, which are cross linked equally by hydrophobic and water soluble cross linking reagents. The β-endorphin binding characteristics of adrenal cortical cells revealed here are quite similar to those of a class of non-opioid β-endorphin receptors previously shown to exist in cells of the immune system.

713-3 β-Blocker Agents Prevent Oxygen Radical-induced Human Apolipoprotein-B100Oxidation

Oxygen radicals may induce both peroxidation of polyunsaturated lipids and protein alterations in low density lipoprotein (LDL). Oxidized LDL taken up by macrophages via the “scavenger” receptor may contribute to formation of foam cells. Previous studies have shown that β -blocker agents reduce experimental atherosclerosis in primates. In addition, we have previously shown that β -blocker agents inhibit oxygen radical-induced lipid peroxidation. Thus, in this study we have investigated whether β -blockers may prevent oxidative modifications induced by oxygen radicals on apolipoprotein-B 100 (apo-B 100 ) of LDL. Purified human LDL was exposed to oxygen radicals generated by CuSO 4 (15 μ M for 20 hs at 37° C) under control conditioincubation with propranolol (P: 1–10μmM). The index of peroxidation malonildihaldehyde, by thiobarbiturate method, was 2.1 ± 0.5* nmoles/mg of protein in native LDL, 34.5 ± 4.1 in control LDL and decreased to 6.5 ± 1.8* in LDL incubated with P (*p < 0.01 vs controls). When oxidized LDL was run on SDS-polyacrylamide electrophoresis (SDS-PAGE: 5 to 10% gradient) extensive apo-B 100 fragmentation was observed. Addition of P prevented the apo-B 100 fragmentation (mean reduction from 12 to 3 bands of low molecular weight). Moreover, oxidized LDL had increased mobility on 0.8% agarose gel electrophoresis (2.8 ± 0.4 time than controls). Similarly, incubation with P reduced LDL mobility to 0.8 ± 0.2 fold in respect to controls (p < 0.05). These data demostrate protection by propranonol on apo-B 100 oxidation in vitro, at clinically relevant concentrations. Although β -blockers are known to adversely affect lipid metabolism, inhibition of both LDL peroxidation and apo-B 100 fragmentation may reduce uptake of oxidized LDL by macrophages and hence foam cell formation.

Apoptosis (programmed cell death) in arteries of the neonatal lamb.

We have examined whether cell death contributes to postnatal remodeling of arteries in lambs. First, abdominal aortic smooth muscle cell proliferation rates fell from 2.87 +/- 0.08% per day at 3 days of age to 1.75 +/- 0.15% per day at 21 days. These proliferation rates would yield a 50% increase in DNA content in the absence of cell death. No increase in DNA content was observed (P < .05 for predicted versus measured accumulation); therefore, significant cell death was inferred. The same analysis did not indicate high cell-death rates in the carotid, renal, or iliac arteries; however, cell death was detected in situ by end-labeling partially degraded DNA with terminal deoxynucleotidyl transferase or by nuclear labeling with propidium iodide, a fluorescent dye that permeates only nonviable cells. Nuclei were labeled in all arteries, although labeling was most frequent in the abdominal aorta, a vessel that regresses substantially after birth. Cell death was apoptotic because DNA extracted from arteries and end-labeled with [32P]dCTP produced a series of low molecular weight bands (DNA ladder) on an agarose gel, a hallmark of apoptosis. The ladder was strong for neonatal abdominal aorta but weak for other arteries. Only weak laddering was observed for fetal abdominal aortas in late gestation, confirming that high apoptosis rates in this vessel were initiated after birth. Intense DNA ladders and frequent in situ labeling indicated high rates of apoptosis in the postnatal intra-abdominal umbilical artery, another vessel that regresses after birth. We conclude that apoptosis contributes to postpartum arterial remodeling. This contribution is greatest in arteries that regress after birth.

Amaranth seed proteins: effect of defatting on extraction yield and on electrophoretic patterns.

Acetone and hexane were used to know the effect of defatting amaranth flour on the extraction yield of protein fractions and on the electrophoretic patterns. It was found that albumins (33%) and globulins (20%) did not present yield changes when using these two solvents, but it was noted that with hexane compared to acetone, prolamins extraction was reduced by half (3.0 to 1.6%) whereas glutelins increased from 26.5 to 30%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of prolamins extracted with acetone and hexane showed one band of low molecular weight (22 KDa) and five bands between 52 to 22 KDa, respectively. No electrophoretic changes were observed in the remaining fractions.

Membrane glycoprotein modifications of G6PD deficient red blood cells.

In this study, the composition and the role of membrane glycoproteins in phagocytosis were determined in G6PD deficient RBCs. G6PD deficient RBCs were recognized and significantly phagocytosed by murine macrophages, without pre-exposure to oxidants in vivo. Phagocytosis was partially (60%) inhibited by incubating macrophages with either galactose or mannose, or by incubating RBCs with beta-galactosidase, indicating the involvement of lectin-like receptors in the recognition of G6PD deficient RBCs. Membrane glycoproteins on G6PD deficient cells were detected by binding of Con A to both intact RBCs and to purified membrane proteins. The results demonstrated modifications in the glycoprotein pattern of G6PD deficient RBCs compared to untreated controls. These included reduction in the amounts of several high molecular weight glycoproteins and appearance of lower molecular weight bands. These results suggest that G6PD deficient RBCs undergo glycoprotein modifications, which may lead to premature removal from circulation, even in non-acute hemolysis.

Lactoferrin interaction with salmonellae potentiates antibiotic susceptibility in vitro

Interaction of lactoferrin (Lf) with the cell envelope (CE) and outer membrane (OM) of Salmonella typhimurium- type strain ATCC13311 was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western-blot analyses. The peroxidase-labeled bovine Lf (BLf) and human Lf both recognized a heat-modifiable protein with an estimated molecular mass of 38 kD in the OM. Simultaneous immunoblotting with an antiporin monoclonal antibody specific for a conserved porin domain in members of enterobacteriaceae confirmed that the Lf-binding protein is a porin. Such Lf-binding porin proteins (37–39 kD range) were readily detected in nine other common Salmonella species: S. dublin, S. panama, S. rostock, S. abony, S. hartford, S, kentucky, S. pullorum, S. thompson , and S. virchow . The latter six species also demonstrated one to three weak Lf-reactive bands of low molecular weight in their CE. The antibiotic susceptibility of Salmonella in the presence of Lf was examined. A mixture containing sub-minimum inhibitory concentration (MIC) levels of Lf (MIC/4) and cefuroxime (MIC/2) inhibited the bacterial growth. Lf strongly potentiated the action of erythromycin (eightfold), whereas it increased the activity only by two-fold for ampicillin, ciprofloxacin, chloramphenicol, and rifampicin; similarly, these antibiotics also reduced the MIC of BLf by twofold in S. typhimurium . Such antimicrobial potentiation was not observed with BLf mixtures containing cefalexin, gentamycin, or polymyxin B against strain ATCC13311. BLf and cefuroxime also demonstrated potentiation of varying degrees (two to 16-fold) with nine other Salmonella species. These data established the binding of Lf to porins in salmonellae and a potentiation effect of Lf with certain antibiotics.

The hexosamine biosynthetic pathway and glucose-induced down regulation of glucose transport in L 6 myotubes

Based on experiments in cultured adipocytes, it has been proposed that glucose-induced down regulation of glucose transport is mediated by the conversion of fructose-6-phosphate to glucosamine-6-phosphate via the first and rate-determining enzyme of the hexasamine biosynthetic pathway, glutamine: fructose-6-phosphate amidotransferase (glutamine hexosephosphate aminotransferase). Evidence for this assertion was: (a) l-glutamine, the provider group for the aminotransferase was essential; (b) two inhibitors of glutamine hexosephosphate aminotransferase, 6-diazo-5-oxonorleucine ( l form) and azaserine, blocked glucose-induced down regulation of glucose transport; (c) azaserine inhibited the activity of the aminotransferase, (d) glucosamine, which enters the hexosamine pathway distal to this enzyme was 40-times more potent than glucose; and (e) azaserine was unable to block the effect of glucosmaine. Since muscle is quantitatively much more important than adipose tissue for whole body glucose utilization, we sought to determine if the hexosamine pathway was involved in glucose-induced down regulation of glucose transport in L 6 myotubes. Glucose was effective, both in the presence and absence of glutamine in the incubation media. Glucosamine was also effective but was as equipotent as glucose. Small amounts of glutamine hexosephosphate aminotransferase were present in the L 6 myotubes and although the leucine derivative (20 μM)_ inhibited the enzyme, it did not impair glucose-induced down regulation of glucose transport. Total GLUT-1 levels were similar when the cells were incubated in the absence or presence of 5 mM glucose or glucosamine although glucosamine was associated with a marked increase in a lower molecular weight band. These results do not suggest that the hexosamine biosynthetic pathway is involved in glucose-induced down regulation of glucose transport in L 6 myotubes. Thus, this phenomenon is regulated differently in muscle and fat.

Sequence analysis of the 5′ flanking sequence of ovine Lipoprotein Lipase (LPL)

Lipoprotein lipase is found in many species and Ussue types 11) and is the pivotal enzyme in controlling lipid flux between plasma triacylglycerol rich lipoprotein particles and the tissues. Because tissue LPL activities alter with the physiological state of an organism, it is of great commercial and economic importance, and an understanding of the way factors interact to regulate its expression may, eventually, allow the remote manipulation of deposition of tissue lipids in v i m The 5 flanking sequence of Human I2.31. Mouse 141 and Chicken [5] LPL have already been determined and although chicken, in general, shares a lower level of sequence homology with human and mouse there is a remarkable level of conservation of cisacting elements across the species. Therefore this study was devised to investigate the 5 flanking sequence of ovine LPL to investigate whether these control elements were also conserved in sheep, a ruminant, with a very different metabolism to the species already investigated. High molecular weight ovine genomic DNA was isolated, from liver, and used to construct a sheep genomic library. This library consisted of 1.2 million independent clones with an average insert size of 14Kbk4Kb. A 1.7% ovine LPL cDNA (R1660) had already been isolated and sequenced previously[6] and was used to screen approx. 200.000 plaques of the sheep genomic library. From this screen one putative plaque was isolated, purified to homogeneity and designated LPLl. During the sequencing of R1660 a fragment corresponding to 225bp of the 5 end of R1660 had been generated using Pst I. This fragment was cloned into pGEM 3Zf and was designated LPL225c. LPLl was characterised by single and double digests with a range of restriction enzymes (HindIII. Sacl. Kpnl. BamHI, EcoFU and PstI). I t was determined from these digests that LPLl contained an insert of approx. 18Kb. The above digests were transferred to a nylon membrane and probed with 32P-labelled LPL225c. A single low molecular weight band of approx. 700bp was observed in the Pst I digest. This fragment was then subcloned into a vector suitable for sequencing. The vector chosen was pGEM 3Zf. Ligations were performed, transformed into JM109 and recombinants selected by blue/white screening. Plasmids were extracted from recombinant colonies and digested with Pst I. Digests were separated on an agarose gel, the DNA was transferred to a nylon membrane and probed again with 32P labelled LPL225c. A positive clone was identified and designated LPL7OOg. LPL7OOg was used as the template for sequencing reactions using the M 1 3 universal forward and reverse sequencing primers. Using the sequence already obtained, oligonucleotides were synthesised to internal sites in LPL7OOg in an attempt to walk through the clone. The sequence obtained from this Pst I sub-clone of LPLl included part of exon 1 and in addition 430bp of the 5 flanking sequence of LPL. This sequence was searched for known cis-elements using FINDPA'ITERNS from the Genetics Computer Group (GCG) suite of programs using the transcription factors database as the dictionary of ciselements and additionally by REGULON the authors own programme. The results of this analysis is shown in Table 1.

Antibodies to proteins from Pityrosporum ovale in the sera from patients with psoriasis.

In order to analyse the humoral immune response to the commensal yeast Pityrosporum ovale, we developed a western immunoblot technique with a salt soluble extract of P. ovale cytoplasm. In the present study, we tested sera from patients with psoriasis (n = 15), seborrhoeic dermatitis (n = 10), pityriasis versicolor (n = 8), and normal controls (n = 10). Seventy-three per cent (11/15) of the patients with psoriasis showed specific reactivity with a protein derived from P. ovale of estimated molecular mass 120 kDa, and 46% (7/15) of the cases recognized a 100-kDa protein. Sera from pityriasis versicolor and normal donors showed nonspecific reactivity with several bands of lower molecular weight. To characterize the location of the 100 and 120-kDa proteins, we performed a lyticase digestion of the cell wall, and analysed the soluble digested products by western blotting. The sera from psoriasis patients detected several bands in the range 100-120 kDa. The finding of the immunoreactive 120-kDa protein in this fraction suggests its location at the space between cell wall and membrane (periplasmic space). As a control, we performed an extraction of the cytoplasmic proteins of the dimorphic yeast Candida albicans. C. albicans showed a different pattern of banding in SDS-PAGE. Immunoblots with C. albicans did not allow the detection of any related band. A smear was observed in the high molecular weight range consistent with the presence of lipopolysaccharides. The role of the immune response in infection by P. ovale has not yet been fully explored.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and regulatory properties of a single hexokinase from the citric acid accumulating fungus Aspergillus niger

A single glucose-phosphorylating enzyme has been detected and purified from the citric acid accumulating fungus Aspergillus niger. The enzyme was formed constitutively, and high activities were formed on glucose and sucrose as carbon sources. Highest activities were formed during growth on high concentrations of glucose or sucrose. The enzyme, purified about 600-fold from cell-free extracts prepared from glucose-grown mycelia, gave a double band in denaturing (SDS)-polyacrylamide gel electrophoresis. Tryptic peptide patterns suggest that the lower molecular weight band was the product of either C- or N-terminal truncation. The specific activity of the enzyme was about 40 and 35 μmol/min and mg protein with glucose and fructose as substrates, respectively. The affinity for glucose was about 10 3-fold higher than for fructose. The subunit molecular weight was 50 000 and the molecular weight of the native protein was 100 000 by gel permeation chromatography. Of the reaction products ADP, but not glucose 6-phosphate, inhibited hexokinase activity. Citrate inhibited ( K I 0.15 mM) non-competitively with respect to both glucose and ATP, which was not due to Mg 2+ -chelation. 2-Deoxyglucose resistant mutant strains of A. niger were isolated which showed decreased growth rate and activity of hexokinase during growth on glucose, while their growth on fructose and hexokinase activities were comparable to the parent strain. They displayed a reduced rate of citric acid accumulation. It is concluded that the synthesis of very high hexokinase activities may counteract citrate inhibition, thereby guaranteeing a high glycolytic flux during citric acid accumulation.