Hexokinase and phosphofructokinase activity and intracellular distribution correlate with aggressiveness and invasiveness of human breast carcinoma.

Glycolytic enzymes, such as hexokinase and phosphofructokinase, have been reported to be upregulated in many cancer types. Here, we evaluated these two enzymes in 54 breast cancer samples collected from volunteers subjected to mastectomy, and the results were correlated with the prognosis markers commonly used. We found that both enzymes positively correlate with the major markers for invasiveness and aggressiveness. For invasiveness, the enzymes activities increase in parallel to the tumor size. Moreover, we found augmented activities for both enzymes when the samples were extirpated from patients presenting lymph node involvement or occurrence of metastasis. For aggressiveness, we stained the samples for the estrogen and progesterone receptors, HER-2, p53 and Ki-67. The enzyme activities positively correlated with all markers but Ki-67. Finally, we conclude that these enzymes are good markers for breast cancer prognosis.

Kinetics of transport and phosphorylation of glucose in cancer cells.

Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS-30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D-[2-(3)H-glucose] as physiological substrate. Kinetic analysis revealed two components at low- and high-glucose concentrations in both tumor cells. At low glucose and 37 degrees C, the V(max) was 55 +/- 20 and 17.2 +/- 6 nmol (min x mg protein)(-1), whereas the K(m) was 0.52 +/- 0.7 and 9.3 +/- 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC(50) = 0.44 +/- 0.1; K(i) = 0.3 +/- 0.1 microM) and phloretin (IC(50) = 8.7 microM) in AS-30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS-30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS-30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS-30D carcinoma and HeLa cells. The much lower GLUT V(max) and catalytic efficiency (V(max)/K(m)) values in comparison to those of G6P-sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target.

Enhanced expression of hexokinase I in pancreatic islets induced by sucrose administration.

Administration of a sucrose-rich diet (SRD) to normal hamsters induces an insulin-resistant state and a significant increase of insulin secretion and beta-cell mass. Islets isolated from these animals had a marked increase in glucose metabolism and glucose-induced insulin secretion, at both low and high glucose concentrations. They also presented increased hexokinase (HK) activity, without measurable changes in glucokinase (GK) activity. In this study we measured HK and GK activity in homogenates of islets isolated from normal control and SRD-fed hamsters, as well as in their particulate and cytosolic fractions. We also measured transcription rate (mRNA by reverse transcriptase PCR) and expression levels (Western blotting) of both enzymes in these islets. We found an increase in HK activity and expression levels, without measurable changes in HK mRNA level in SRD-fed animals. Whereas a similar GK activity was measured in homogenates of islets isolated from both groups, such activity was significantly higher in the cytosolic fraction of SRD islets. On the other hand, GK transcription rate and expression level were similar in both experimental groups. Our results suggest that the increased beta-cell secretory response to low glucose can be partly ascribed to an increased activity of islet HK consecutive to an enhanced expression of the enzyme, while the enhanced response to high glucose could be due to changes in GK compartmentalization.

Hexose transporters GLUT1 and GLUT3 are colocalized with hexokinase I in caveolae microdomains of rat spermatogenic cells.

Postmeiotic spermatogenic cells, but not meiotic spermatogenic cells respond differentially with glucose-induced changes in [Ca2+]i indicating a differential transport of glucose via facilitative hexose transporters (GLUTs) specifically distributed in the plasma membrane. Several studies have indicated that plasma membrane in mammalian cells is not homogeneously organized, but contains specific microdomains known as detergent-resistant membrane domains (DRMDs), lipid rafts or caveolae. The association of these domains and GLUTs isoforms has not been characterized in spermatogenic cells. We analyzed the expression and function of GLUT1 and GLUT3 in isolated spermatocytes and spermatids. The results showed that spermatogenic cells express both glucose transporters, with spermatids exhibiting a higher affinity glucose transport system. In addition, spermatogenic cells express caveolin-1, and glucose transporters colocalize with caveolin-1 in caveolin-enriched membrane fractions. Experiments in which the integrity of caveolae was disrupted by pretreatment with methyl-beta-cyclodextrin, indicated that the involvement of cholesterol-enriched plasma membrane microdomains were involved in the localization of GLUTs and uptake of 2-deoxyglucose. We also observed cofractionation of GLUT3 and caveolin-1 in low-buoyant density membranes together with their shift to higher densities after methyl-beta-cyclodextrin treatment. GLUT1 was found in all fractions isolated. Immunofluorescent studies indicated that caveolin-1, GLUT1, and hexokinase I colocalize in spermatocytes while caveolin-1, GLUT3, and hexokinase I colocalize in spermatids. These findings suggest the presence of hexose transporters in DRMDs, and further support a role for intact caveolae or cholesterol-enriched membrane microdomains in relation to glucose uptake and glucose phosphorylation. The results would also explain the different glucose-induced changes in [Ca2+]i in both cells.

[Relevance of Entamoeba dispar in amebiasis]. / Relevancia del reconocimiento de Entamoeba dispar en la amibiasis.

Entamoeba histolytica was redefined to recognize the existence of two morphologically indistinguishable species but genetically distinct: E. histolytica and E. dispar. The former is a pathogen responsible for amebiasis while the latter is a commensal. This redefinition has dramatically changed the understanding of amebiasis and its clinical management.

The effect of Walker-256 tumour development upon Kupffer cell metabolism.

The liver plays a central role in the establishment and maintenance of the cachectic state in rats bearing extra-hepatic tumours. Kupffer cells, which as macrophages, show a strong relationship between metabolism and function could be involved in the alterations observed in the disruption of many functions of the organ as a whole. To assess whether the metabolic/functional pattern of Kupffer cells was altered by cachexia we have investigated the utilization of glucose, glutamine and palmitate by the cells from tumour-bearing and control rats. We have found an enhanced utilization of the three substrates by the cells from tumour-bearing rats as compared with controls, which was related to greater energy production through the Krebs cycle and enhanced production of precursors for the synthesis of the many substances the cells secrete when activated. The use of palmitate as substrate was also augmented in these cells, in the opposition to the observation in stimulated peritoneal macrophages. The availability of palmitate however, was not associated with a reduction of glucose or glutamine consumption. The cycle of interconversion, free fatty acids/triacyglycerol in Kupffer cells from tumour-bearing rats was also found to be increased, as was hydrogen peroxide production. Taken together the results suggest an increased utilization of substrates for both energy production and for synthetic processes (e.g. NADPH for hydrogen peroxide production).

[Isoenzymatic characterization of the hexokinase in the development of Plebeia droryana (Hymenoptera, Apidae) and its relation with flight activity]. / Caracterização isoenzimática da hexoquinase no desenvolvimento de Plebeia droryana (Hymenoptera, Apidae) e sua relação com a atividade de vôo.

Hexokinase enzyme was studied by means of starch-agarose gel electrophoresis in Plebeia droryana. Three anodal bands with enzyme activity were observed during the development: hexokinase-1 (HK-1), the intensity of which increases from larvae to adult, probably related with energy supply to thoracic flight muscles and, therefore, with flight activity; hexokinase-2 (HK-2), that reaches maximum intensity in clear brown eyed pupae and hexokinase-3 (HK-3), the intensity of which reaches maximum peak in winged pupae and is not observed in the adult phase. This isozyme should have important functions in the bee metamorphosis.

Caracterizaçäo isoenzimática da hexoquinase no desenvolvimento de Plebeia droryana (Hymenoptera, Apidae) e sua relaçäo com a atividade de vôo / Isoenzymatic characterization of hexokinase in the development of Plebeia droryana (Hymenoptera, Apidae) and its relation with flight activity

A enzima Hexoquinase foi estudada em P. droryana por meio de eletroforese em gel de amido agarose. Três regiöes anódicas com atividade enzimática foram observadas durante o desenvolvimento. A hexoquinase-1 (HK-1), cuja intensidade de coloraçäo aumenta de larva até adulto, provavelmente relacionada ao fornecimento de energia para os músculos torácicos de vôo e, conseqüentemente, com a atividade de vôo nesta espécie. A hexoquinase-2 (HK-2), que alcança intensidade máxima em pupa de olho marrom claro e hexoquinase-3 (HK-3), que alcança seu pico máximo de intensidade em imago e näo é observada na fase adulta. Esta isoenzima deve ter funçäo importante na metamorfose desta espécie.

Analysis of dissociation and unfolding of oligomeric proteins using a flat bed gel electrophoresis at high pressure.

A slab gel electrophoresis apparatus with the ability to operate over a pressure range of 10(-3) to 2 kbar is described. The system presented here is an improvement of a previous apparatus (A. A. Paladini, J. L. Silva, and G. Weber, Anal. Biochem. 161, 358-364, 1987). It consists of a flat bed gel, with a significantly enlarged buffer reservoir, which eliminates the requirement of high concentrations of running buffers, and at the same time allows shorter runs, leading to enhanced resolution and reproducibility. The application of the method to the dissociation of the tetramer glycogen phosphorylase a as a function of hydrostatic pressure is described. The flat geometry of the apparatus allows for the first time the analysis of the stability of oligomers and their constituent subunits to chemical denaturation by urea gradient electrophoresis gels at high pressure. Dimeric hexokinase shows a reversible cooperative unfolding transition with a midpoint at 3.8 M urea. In contrast, the monomers unfold at very low urea concentration (< 1.0 M). The observed differences in stability validates oligomerization as an important stabilizing element of the protein structure.

Virulence of nonpathogenic zymodemes of Entamoeba histolytica isolates from asymptomatic subjects in Calcutta, India.

1. The zymodeme and virulence of 31 Entamoeba histolytica isolates obtained from asymptomatic human subjects in Calcutta, India are described. 2. Virulence was measured by the extent of lesion diameter (mm) induced by each isolate in the liver of golden hamsters and resistance of isolates to non-immune hamster sera in vitro. 3. Two nonpathogenic zymodemes, III (N = 17) and IV (N = 14), were detected among 31 isolates by isoenzyme electrophoresis. Most of the zymodeme III isolates were moderately to highly virulent while a quarter of the zymodeme IV were invasive although with low virulence. 4. The virulence of the isolates was found to have a significant positive correlation (r = 0.96, P < 0.001) with their greater resistance to complement-mediated lysis. 5. The data suggest that the virulence of E. histolytica is probably not related to its zymodeme.

Giardia lamblia: isoenzyme analysis of 19 axenic strains isolated from symptomatic and asymptomatic patients in Mexico.

Infection of the small intestine of humans with the parasitic protozoon Giardia lamblia may have an asymptomatic course, or it may produce acute or chronic diarrhoea. In order to establish if the different clinical outcome of giardiasis in children could be due, at least partially, to strain differences, 19 isolates from asymptomatic and symptomatic cases studied in Mexico City were cultured under axenic conditions and the isoenzyme electrophoretic patterns of 10 different enzymes were compared. Strains from carriers and from symptomatic cases of giardiasis were equally amenable to isolation and axenization. Isoenzyme electrophoresis demonstrated remarkable homogeneity in 7 enzyme patterns for all 19 isolates, except for phosphoglucomutase, for which 3 different zymodemes were found. Therefore, these isolates of G. lamblia, obtained from a single geographical location, tended to be genetically homogeneous. In addition, there were no consistent zymodeme differences between isolates from symptomatic and asymptomatic human infections.

Isozyme profiles of Trypanosoma cruzi stocks from Colombia and Ecuador.

A total of 74 of 82 domestic Rhodnius prolixus from the same locality in eastern Colombia were found to be infected with Trypanosoma cruzi or T. rangeli. One of three domestic Triatoma dimidiata from Ecuador also showed T. cruzi infection. A total of 59 T. cruzi stocks from these and five other localities in Colombia were isolated from man, marsupials and triatomine bugs. Cellulose-acetate electrophoresis of nine or ten enzymes characterized all T. cruzi stocks as zymodeme 1 (reference clone Silvio X10/1). Differences in electrophoretic patterns between the newly isolated stocks and the zymodeme 1 standard were seen with the enzymes G6PD and HK. These results are in agreement with the previously described geographical distribution of T. cruzi zymodemes. Stocks were isolated from both low and high altitudes and there was no evidence of adaptative significance of T. cruzi enzyme polymorphism.

The use of isoelectrofocusing in thin layer polyacrylamide and agarose gels as a method for the characterization of Venezuelan Trypanosoma cruzi stocks.

The technique of isoelectrofocusing has been used to compare culture forms of 12 stocks of T. cruzi isolated in different regions of Venezuela. The following seven enzymes have been used for the characterization: unspecific esterase (E.C.3.1.1), malate dehydrogenase (E.C.1.1.1.37), "malic enzyme" (E.C.1.1.1.40), hexokinase (E.C.2.7.1.1), phosphoglucomutase (E.C.2.7.5.1), glucosephosphate isomerase (E.C.5.3.1.9) and glucose-6-phosphate dehydrogenase (E.C.1.1.1.49). The isoelectrofocusing method allows to determine reproducible enzyme patterns of high selectivity and with a number of bands. This permits to recognize possible differences within the T. cruzi-complex much easier than previous methods. The Venezuelan T. cruzi stocks showed a remarkable homogenous behaviour concerning the enzyme profiles. Most of them were identical. Different types seen for "malic enzyme", phosphoglucomutase, and glucose-6-phosphate dehydrogenase were observed in only three stocks, It was not possible to find a clear relationship between the types and the histories of stocks.

Isozyme frequency patterns in Drosophila pavani associated with geographical and seasonal variables.

Fourteen population samples of Drosophila pavani were obtained from a number of localities in Chile. The populations sampled were dispersed over 7 degrees of latitude and 1800 meters of elevation, and were drawn at three different times. Sixteen electrophoretic loci were assayed for each population; eight of the loci were analyzed statistically for geographic variation; the other eight were essentially monomorphic. For all eight variable loci, variation in allelic frequencies among populations was highly significant. In all cases, a significant portion of the variation among populations was associated with variation in gross environmental variables (latitude, elevation, month of collection). The implications of the evidence were discussed, and the authors concluded that there was suggestive evidence for selection.