Brush border enzymes as markers for ascending pyelonephritis: active immunization with pili

Activities of renal brush border membrane (BBM) enzymes, alkaline phosphatase, maltase and leucine aminopeptidase, were determined in control, pyelonephritic and immunized-infected rats. The activities of all enzymes decreased significantly (P < 0.05) in obstructed kidney while activities of alkaline phosphatase and leucine aminopeptidase increased significantly (P < 0.05) in unobstructed kidney in early stages of infection. The affinity constant (K_m) of all enzymes remained unaltered in control and experimental groups. Significant differences (P < 0.05) in the activities of BBM enzymes of infected and immunized-infected animals suggested a protective role of active immunization with pili.     

Isolation of the sodium-dependent d-glucose transport protein from brush-border membranes

Rabbit kidney brush-border membrane vesicles were exposed to bacterial protease which cleaves off a large number of externally oriented proteins. Na⁺-dependent d-glucose transport is left intact in the protease-treated vesicles. The protease-treated membrane was solubilized with deoxycholate and the deoxycholate-extracted proteins were further resolved by passage through Con A-Sepharose columns. Sodium-dependent d-glucose activity was found to reside in a fraction containing a single protein band of M_r ? 165000 which is apparently a dimer of M_r ? 85 000. When reconstituted and tested for transport, this protein showed Na⁺-dependent, stereo-specific and phlorizin-inhibitable glucose transport. Transport activity is completely recovered and is 20-fold increased in specific activity. A similar isolate was obtained from rabbit small intestinal brush-border membranes and kidneys from several other species of animals.     

Effect of inhibitors on glucose transport in malaria (Plasmodium berghei) infected erythrocytes

Effect of inhibitors on glucose transport in malaria (Plasmodium berghei) infected erythrocytes. International Journal for Parasitology16: 441–446. The effect of cytochalasin B and phloretin on transport of d-glucose and 2-deoxy-d-glucose into Plasmodium berghei-infected mouse erythrocytes was studied. Both the inhibitor-sensitive and insensitive fractions of transport in the infected erythrocytes were increased compared with normal erythrocytes. The i₅₀ values (concentrations of inhibitor producing 50% inhibition) were similar for both infected and normal erythrocytes, indicating that the binding affinities of the carrier were not substantially changed, but the turnover number, availability, or the number of the carrier may have increased in infection. There was a large increase in the transport of l-glucose into infected erythrocytes. Neither inhibitor showed any effect on transport of l-glucose into infected or normal erythrocytes. d-Galactose and d-fructose also showed a large transport increase mostly insensitive to cytochalasin B. The specificity of the transport increase raises the possibility of the presence of a new pathway other than simple diffusion, or the carrier-mediated pathway revealed by cytochalasin B or phloretin inhibition.     

Prevention of alterations in the transport of nutrients in pyelonephritic rats by immunization with pili

The uptake of D-glucose, L-aspartate, L-lysine and L-proline was studied in renal brush border membrane vesicles prepared from control, infected and actively immunized-infected rats. The uptake of D-glucose, L-lysine and L-proline was decreased significantly (p less than 0.05) during the course of infection in the infected animals. However, the uptake of L-aspartate was increased significantly (p less than 0.05) in early stages and decreased significantly (p less than 0.05) in later stages of infection in the infected animals. When the animals were actively immunized with pili, still there were changes in the uptake of D-glucose and L-aspartate, but the changes appeared later and less pronounced. No change in the uptake of L-lysine and L-proline was observed in the immunized-infected animals. The findings demonstrated that active immunization with pili prevents alterations in the uptake of nutrients in pyelonephritic rats.     

Action of 5-thio-D-glucose and its 1-phosphate with hexokinase and phosphoglucomutase.

Previous work from this laboratory has shown that 5-thio-d-glucose is a competitive inhibitor for active transport of d-glucose. The present work indicates that the thiosugar analog and its 1-phosphate can also interfere with d-glucose 6-P formation.5-Thio-d-glucose serves as a substrate for yeast hexokinase with a K_m of 4 mm, and V of 8.8 nmol/min/μg of protein. The analog competitively inhibits d-glucose phosphorylation with a K_i of 20 mm.5-Thio-d-glucose 1-P can act as a substrate for rabbit skeletal muscle phosphoglucomutase with a K_m of 60 μm and V of 0.17 μmol/min/μg of protein. Thus, 5-thio-d-glucose 1-P behaves as a near metabolic analog of d-glucose 1-P. 5-Thio-d-glucose 1-P is a competitive inhibitor of d-glucose 1-P conversion to the 6-P with a K_i of 16.2 μm.5-Thio-d-glucose 6-P produced by phosphorylation of 5-thio-d-glucose and by conversion from 5-thio-d-glucose 1-P was identified by chromatographic mobility and by color reactions.     

Trichinella spiralis: peroxidase activity in isolated cells from the rat intestine

An understanding of physiologic events underlying resistance to parasitic worms depends on a knowledge of metabolic interactions between parasites and specific cells at the host-parasite interface. In the case of invasive intestinal parasites this interaction involves contact with epithelial cells and cells of the lamina propria. This investigation deals with the collection of epithelial cells and lamina propria cells from the small intestine of control rats and rats infected with the nematode, Trichinella spiralis, and measurement of peroxidase activity in these cells. Lamina propria cells were isolated by collagenase digestion of everted gut segments previously denuded of epithelium by treatment with hyaluronidase. Mean peroxidase activity in homogenates of lamina propria cells was equivalent to 40 nmoles H₂O₂ decomposed/min/mg of cell protein from control rats compared to 413 nmoles from infected animals. Epithelial cell peroxidase activity in homogenates of epithelial cells from both control and infected rats was less than 2 nmoles H₂O₂ decomposed/min/mg cell protein. The degree of contamination of lamina propria cells with epithelial cells was determined by measuring disaccharidase activity in both cell populations. The specific activity of maltase, sucrase, and trehalase in lamina propria cells was between 10 and 17% of that in epithelial cells. This work is a requisite for a study in which the role of intestinal cell peroxidase in resistance to Trichinella will be evaluated.     

Preparative production of hydroquinone from benzoquinone catalysed by immobilized d-glucose oxidase

Benzoquinone can replace O₂ as an electron acceptor in the oxidation of d-glucose catalysed by A. niger d-glucose oxidase. As a result, a useful chemical, hydroquinone, is formed in nearly 100% yield. A column packed with d-glucose oxidase immobilized onto alumina was operated for two weeks with no measurable decline in its catalytic efficiency and produced more than one hundred grams of hydroquinone.     

The effect of the strongly bound protein fraction on sugar transport in human erythrocyte ghosts

The protein fraction released from human erythrocyte membranes with 90% acetic acid enhanced the transport of several sugar species when enclosed in erythrocyte ghosts. Both the influx and the efflux of d-glucose were increased so that permeation rather than sugar binding was involved. The permeation increase was selective, being found with d-glucose, d-galactose and d-xylose but not with l-glucose or lactose. The protein-dependent sugar transport was saturable and the incorporation of proteins into the ghost membrane brought J_max to the level corresponding to intact erythrocytes, leaving K_m unchanged.     

Serological cross-reactivity between Strongyloides venezuelensis and Syphacia muris in Wistar rats (Rattus norvegicus)

One of the problems frequently faced in laboratory facilities is the possibility of the natural parasitic infection of lab animals, which can interfere with biomedical research results. The present study aimed to evaluate cross-reactivity among serum samples from Wistar rats (Rattus norvegicus) naturally infected with Syphacia muris and experimentally infected with Strongyloides venezuelensis. Forty rats were divided into four groups of ten animals each. Parasite load was evaluated by quantifying the adult worms from both helminthes species recovered from the intestines and the S. venezuelensis eggs eliminated in feces. Serological cross-reactivity by parasite-specific IgG detection was tested via enzyme linked immunosorbent assay (ELISA), immunofluorescence antibody test (IFAT) and immunoblotting. The results demonstrated that the quantity of S. venezuelensis eliminated eggs and parthenogenetic females decreased significantly in cases of co-infection with S. muris. ELISA revealed 100% cross-reactivity of serum samples from both species against the opposing antigen. IgG cross-reactivity was confirmed by IFAT using tissue sections of S. venezuelensis larvae and adult S. muris. Immunoblotting showed that IgG antibodies from the sera of animals infected with S. muris recognized eight antigenic bands from S. venezuelensis saline extract and that IgG antibodies from the sera of animals infected with S. venezuelensis recognized seven bands from S. muris saline extract. These results demonstrate the serological cross-reactivity between S. muris and S. venezuelensis in infected rats.     

Trichinella spiralis: Rapid,immunologically influenced reduction of intestinal,sodium-coupled sugar transport in the rat

Epithelium of isolated small intestinal segments were studied in Ussing-type chambers to detect physiological changes associated with rapid, immune rejection of Trichinella spiralis infective larvae. Electrophysiological parameters associated with Na⁺-coupled hexose transport were measured. Changes in transepithelial electrical potential difference (PD), resistance, and short circuit current (I_sc) due to the addition of actively absorbed β-methyl-d-glucoside (BMG) to the mucosal solution were determined. Measurements were made prior to and 30 min after primary and secondary infections. Animals were infected by intraduodenal inoculation. As the infective larval dose in primarily infected (nonimmunized) rats increased from 50 to 2000 larvae the magnitude of the rise in I_sc elicited by BMG decreased in a dose-dependent fashion, with 50 larvae per rat having no effect. In previously infected (immunized) rats challenged with a secondary inoculum, all doses, ranging from 50 to 2000 larvae per rat, decreased the BMG-stimulated change in I_sc by approximately 50%. The effect of 50 worms per rat in immunized hosts was equivalent to that produced by ~1600 worms in nonimmunized animals. Measurements of ¹⁴C-BMG mucosa-to-serosa flux confirmed that Na⁺-BMG cotransport was responsible for observed changes in I_sc. Results support the conclusion that changes in intestinal epithelial function are associated with larval challenge of immune rats.     

Hymenolepis diminuta: lack of pathogenicity in the healthy rat host.

Whether Hymenolepis diminuta (Cestoda: Cyclophyllidea) might affect adversely the growth of its host under normal conditions was studied. Rats were divided into four experimental groups: (1) rats infected with the tapeworm and fed ad libitum, (2) uninfected rats fed ad libitum, (3) and infected rats fed isocalorically with (4) uninfected rats. Growth rates of infected rats did not differ from infected animals. Infected, meal fed rats limited to 15 g synthetic diet/day grew as rapidly as their uninfected counterparts, and infected rats fed ad libitum did not consume more food than the comparable infected group. There were no significant differences in consumption or in excrement produced between groups (1) and (2) and groups (3) and (4). Weights attained by the worms were not affected by mode of host feeding (ad libitum or meal fed), whether expressed as wet or dry weight. Since H. diminuta appears not to affect nutrient utilization or consumption in a healthy, unstressed host, at least on a gross level, it probably should be considered an endocommensal.     

Action of porcine-pancreatic amylase on oxidized-reduced amylose of low degree of modification

Amylose was oxidized with 0.1–0.2 mol of periodate per glucose residue (G), and then reduced with sodium borohydride or borotritide to give an oxidized-reduced amylose of low degree of modification. Mild acid hydrolysis gave erythritol, 2-O-α-d-glucosyl-l-erythritol, higher homologs, and other products. Extensive action of porcine-pancreatic amylase on the polymer gave, besides d-glucose and maltose, oligosaccharides containing one or more oxidized-reduced (modified, M), acyclic residues. The enzymic products containing only one oxidized-reduced residue were identified as a modified tetrasaccharide (MG₃) and a modified pentasaccharide (MG₄). Structures of MG₃ and MG₄ were identified by a combination of enzymic and chemical approaches. With glucoamylase, MG₄ was converted into MG plus d-glucose, whereas MG₃ was totally resistant. On mild acidic hydrolysis, MG₃ was converted into 2-O-α-d-glucosyl-d-erythritol plus maltose. These results indicate that MG₃ is G-M-G-G and that MG₄ is G-G-M-G-G. In principle, MG₄ could occupy the five d-glucose residue, substrate-binding site of porcine-pancreatic amylase in such a way that M, the acyclic structure replacing a d-glucose residue, is placed just to the “left” of the catalytic site. The modified structure, being very vulnerable to acidic hydrolysis, might then be expected to be very readily attacked by the amylase, but in fact, it is not.     

Echinococcus multilocularis in the cotton rat: the in vitro protoscolicidal activity of peritoneal cells.

Protoscolices of Echinococcus multilocularis were incubated in vitro with peritoneal cells and with sera from normal and infected cotton rats. The cells from rats bearing massive subcutaneous and intraperitoneal hydatid cysts rapidly killed protoscolices; normal cells and cells from lightly infected animals did not. Only sera from rats bearing large, subcutaneous cysts displayed significant protoscolicidal activity. These data suggest that the phenomenon whereby large, established cysts of E. multilocularis effectively suppress the establishment, growth, and metastasis of distant foci of infection has an immunological component.     

Prostaglandin E2 and muscle protein turnover inPseudomonas aeruginosa sepsis

Rats were injected intraperitoneally withPseudomonas aeruginosa (septic group) or sterile 0.9% NaCl (controls). Soleus muscles were excised 7 h later, and muscle prostaglandin E₂ release and tyrosine release were measured in vitro. Muscles of septic rats exhibited 226–326% higher release of prostaglandin E₂ and 54–84% higher net proteolysis than muscles of controls. Inclusion of aspirin or indomethacin in the incubation medium almost completely inhibited prostaglandin E₂ production, but had no effect on net proteolysis in muscles from either group. Inclusion of cycloheximide, a protein synthesis inhibitor, increased tyrosine release of control muscles by 42%, whereas no statistically significant increase was observed in muscles from infected rats. However, total proteolytic rate, indexed by tyrosine release in the presence of cycloheximide, was 22% higher in muscles of septic rats compared to that of control animals. Concomitantly, inclusion of cycloheximide inhibited prostaglandin E₂ release by muscles of infected rats by 91% and that of controls by 65%. It is concluded that (a) muscles of septic animals exhibit a pronounced stimulation of prostaglandin E₂ release and net proteolysis, combined with a small increase in total proteolytic rate, (b) the stimulation of net proteolysis is mainly due to inhibition of protein synthesis, (c) the increases in net and total proteolysis appear to be independent of prostaglandin E₂ production, (d) cycloheximide has a previously unrecognized inhibitory effect on muscle prostaglandin E₂ production.     

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes

Human placental microsomes exhibit uptake of d-[³H]glucose which is sensitive to inhibition by cytochalasin B (apparent K_i = 0.78 /gm M). Characterization of [³H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED₅₀ = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a K_d = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [³H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED₅₀ = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [³H]cytochalasin B incorporation into soluble protein (M_r range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [³H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.     

Oxidative Stress in the Heart of Rats Infected with Trypanosoma evansi

This study was conducted to investigate the occurrence of oxidative stress in the heart tissue of rats infected with Trypanosoma evansi. Rats were divided into 2 groups (A and B) with 12 animals each, and further subdivided into 4 subgroups (A1 and A2, 6 animals/each; and B1 and B2, 6 animals/each). Animals in the groups B1 and B2 were subcutaneously inoculated with T. evansi. Thiobarbituric acid reactive substances (TBARS), superoxide dismutase activity (SOD), glutathione S-transferase activity (GST), reduced glutathione activity (GSH), and non-protein thiols (NPSH) in the heart tissue were evaluated. At day 5 and 15 post-infection (PI), an increase in the TBARS levels and a decrease in the SOD activity (P<0.05) were observed. GSH and GST activities were decreased in infected animals at day 15 PI (P<0.05). Considering the proper functioning of the heart, it is possible that the changes in the activity of these enzymes involved in the oxidative stress may be related, at least in part, in the pathophysiology of rats infected with T. evansi.     

Involvement of ectonucleotidases and purinergic receptor expression during acute Chagas disease in the cortex of mice treated with resveratrol and benznidazole

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi. CD affects people worldwide, primarily in tropical areas. The central nervous system (CNS) is an essential site for T. cruzi persistence during infection. The protozoan may pass through the blood–brain barrier and may cause motor and cognitive neuronal damage. Once in the CNS, T. cruzi triggers immune responses that the purinergic system can regulate. Treatment for CD is based on benznidazole (BNZ); however, this agent has negative side-effects and is toxic to the host. For this reason, we investigated whether resveratrol (RSV), a potent antioxidant and neuroprotective molecule, would modulate purinergic signaling and RSV alone or in combination with BNZ would prevent changes in purinergic signaling and oxidative damage caused by T. cruzi. We infected mice with T. cruzi and treated them with RSV or BNZ for 8 days. Increases in ATP and ADP hydrolysis by NTPDase in the total cortex of infected animals were observed. The treatment with RSV in infected group diminished ATP, ADP, and AMP hydrolysis compared to infected group. The combination of RSV + BNZ decreased AMP hydrolysis in infected animals compared to the INF group, exerting an anti-inflammatory effect. RSV acted as a neuroprotector, decreasing adenosine levels. Infected animals presented an increase of P2X₇ and A_2A density of purine receptors. RSV reduced P2X₇ and A_2A and increased A1 density receptors in infected animals. In addition, infected animals showed higher TBARS and reactive oxygen species (ROS) levels than control. RSV diminished ROS levels in infected mice, possibly due to antioxidant properties. In short, we conclude that resveratrol could act as a neuroprotective molecule, probably preventing inflammatory changes caused by infection by T. cruzi, even though the mice experienced high levels of parasitemia.     

The utilization of bathocuproinedisulfonic acid as a reagent for determining D-glucose and D-galactose levels in glycoconjugates

A sensitive, rapid, and reliable method for measuring d-glucose and d-galactose levels in glycoconjugates has been developed. In this method, the NAD(P)H produced from the enzymatic oxidation of the monosaccharides is reacted with a CuSO₄-bathocuproinedisulfonic acid reagent (Cu-BCS) to produce a color complex absorbing maximally at 486 nm. With galactose dehydrogenase and glucose dehydrogenase serving as the model enzymes, graphs of absorbance versus varying d-glucose or d-galactose concentrations yielded a linear plot from 2.5 to 250 nmol of sugar. Using this procedure, sugar released by acid hydrolysis from lactose, porcine submaxillary mucin and raffinose was quantified. When p-nitrophenyl-α-d-glucopyranoside and p-nitrophenyl-β-d-galactopyranoside were acid hydrolyzed and assayed with the Cu-BCS reagent, the amount of sugar released from each of the p-nitrophenyl compounds was found to be equal to the levels of p-nitrophenol in solution. This method is easy to use and with minor modifications can be employed for the quantification of d-glucose and d-galactose in other glycoconjugates.     

A simple apparatus for performing short-time (1–2 seconds) uptake measurements in small volumes; its application to d-glucose transport studies in brush border vesicles from rabbit jejunum and ileum

An automated procedure allows uptake measurements with incubation times as short as 0.5 s and with volumes of 10–20 μl. Using this technique the kinetic parameters K_m and V of d-glucose transport in brush border vesicles from rabbit small intestine could be determined from unidirectional fluxes. A comparison of the data obtained from jejunum and from ileum shows that the K_m for d-glucose is the same in both parts of the intestine, whereas the maximum flux is significantly larger in the jejunum.     

Imaging of systemic Candida albicans infections with a radioiodinated monoclonal antibody: Experimental study in the guinea pig

Guinea pigs intravenously infected with Candida albicans were scanned to evaluate the use of radioiodinated monoclonal antibodies (MAb) to fungal antigens for detecting tissue infection sites. A total of 18 infected and 8 uninfected animals were used. MAb and F(ab′)₂ fragments directed against cell wall glycoproteins of C. albicans were labeled with ¹³¹I. Another MAb directed against a Schistosoma mansoni glycoprotein was labeled with ¹²⁵I and used as a nonspecific control. Radiolabeled MAbs were injected at a dose of 12.5 μg (500 kBq) per animal. Images were acquired 24 h later. Animals were then killed and the dissected organs were separately gamma-counted. The number of C. albicans colony forming units (cfu) per gram was determined in each organ. A clear relationship was found between the anatomic distributions of C. albicans and ¹³¹I. The biodistribution of ¹³¹I radioactivity associated with anti-Candida MAb was greater in infected animals than in healthy animals and increased with the number of cfu per g in each organ. The distribution was highly specific in animals with Candida endophthalmitis, a pathognomic feature of organ involvement during hematogenous dissemination. In contrast, the distribution of ¹²⁵I radioactivity associated with the nonspecific MAb was similar in healthy and infected animals. In infected animals, it was totally independent of the intensity of fungal infection.