1p36.22 region containing PGD gene is frequently gained in human cervical cancer.

AIM: To identify commonly occurring DNA copy number alterations in Korean cervical cancers. METHODS: DNA copy number alteration was screened by whole-genome array comparative genomic hybridization (CGH) analysis. For the array CGH discovery, genomic DNA from five cervical cancers and 10 normal cervical tissues were examined. For the independent validation of the most significant chromosomal alteration (1p36.22, PGD gene), 40 formalin-fixed paraffin-embedded cervical tissue samples were collected; 10 of them were used for quantitative polymerase chain reaction and the other 30 samples were used for immunohistochemical analysis. Chromosomal segments differently distributed between cancers and normal controls were determined to be recurrently altered regions (RAR). RESULTS: A total of 13 RAR (11 RAR losses and two RAR gains) were defined in this study. Of the 13 cervical cancer-specific RAR, RAR gain in the 1p36.22 locus where the PGD gene is located was the most commonly detected in cancers (P = 0.004). In the quantitative polymerase chain reaction replication, copy number gain of the PGD gene was consistently identified in cervical cancers but not in the normal tissues (P = 0.02). In immunohistochemical analysis, PGD expression was significantly higher in cervical cancers than normal tissues (P = 0.02). CONCLUSION: Our results will be helpful to understand cervical carcinogenesis, and the PGD gene can be a useful biomarker of cervical cancer.

Pentose phosphate pathway flux analysis for glycopeptide antibiotic vancomycin production during glucose-limited cultivation of Amycolatopsis orientalis.

In vivo pentose phosphate pathway (PPP) enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and transaldolase (TAL) activities as well as ATP- and ADP-level variations of Amycolatopsis orientalis were investigated with respect to glucose concentration and incubation period. G6PDH, 6PGDH, and TAL activities of A. orientalis reached maximum levels at 48 hr for all glucose concentrations used, after which the levels began to decline. G6PDH, 6PGDH, and TAL activities showed positive correlation with the glucose concentration up to 15 g/L, while further increases had an opposite effect. Intracellular ATP level showed a positive correlation with glucose concentrations, while ADP level increased up to 15 g/L. ATP concentration of A. orientalis increased rapidly at 48 hr of incubation, as was the case also for G6PDH, 6PGDH, and TAL activities, although the incubation period corresponding to maximum values of ADP shifted to 60 hr. Production of the glycopeptide antibiotic vancomycin increased with the increases in glucose concentrations up to 15 g/L, by showing coherence in the rates of oxidative and nonoxidative parts of the PPP.

Typing of four genetic loci discriminates among closely related species of New World Leishmania.

All New World Leishmania species can cause cutaneous lesions, while only Leishmania (Viannia) braziliensis has been associated with mucosal metastases. Multilocus enzyme electrophoresis (MLEE) is the optimal standard for species identification but is slow and costly. New methods for species identification are needed to ensure proper identification and therapy. The coding regions of four metabolic enzyme markers in the MLEE typing method: mannose phosphate isomerase (MPI), malate dehydrogenase (MDH), glucose-6-phosphate isomerase (GPI), and 6-phosphogluconate dehydrogenase (6PGD), were analysed from seven species of New World Leishmania isolated from patients with either cutaneous or mucosal lesions to identify specific genetic polymorphisms responsible for the phenotypic variations observed in the MLEE typing scheme. We identified species-specific polymorphisms and determined that a combination of sequencing of the mpi and 6pgd genes was sufficient to differentiate among seven closely related species of New World Leishmania and among isolates of L. braziliensis shown previously to have atypical MLEE patterns. When DNA isolated from 10 cutaneous lesion biopsies were evaluated, the sequence typing method was 100% concordant with the published MLEE/monoclonal antibody identification methods. The identification of species-specific polymorphisms can be used to design a DNA-based test with greater discriminatory power that requires shorter identification times. When the causative agent of the disease is L. braziliensis, this method ensures correct species identification, even when the agent is a genetic variant. Proper identification could facilitate adequate treatment, preventing the onset of the disfiguring mucosal form of the disease.

Polyporus s. str. in southern South America: isoenzyme analysis.

Forty-two dikaryotic and 42 monokaryotic isolates, and 34 pairings were examined by horizontal polyacrylamide gel electrophoresis (PAGE) for six enzymatic activities, viz. EST, 6PGD, IDH, MDH, SHDH and SOD. 44 bands were analysed. Numerical analysis of the isoenzymatic patterns was undertaken and compared with those from morphological characters. The analysis of six enzymatic systems showed the existence of four monomorphic systems (IDH, MDH, SHDH and SOD). The sterease system (EST) appears to be polymorphic in Polyporus ciliatus and in populations of P. tenuiculus from Argentina, being monomorphic in the remaining species studied. The 6PGD system is polymorphic in P. tucumanensis and monomorphic in the other species. Predominance of monomorphic enzymes and a clear distribution of the electromorphs among the species, indicates that isoenzymatic analysis is a good taxonomic tool within Polyporus. The low intraspecific variability allowed the use of interspecific differences to separate species. Numerical analysis showed a good correlation between morphological and molecular characters. In the isoenzymatic phenogram the similarity index is high only among very close species, showing a stressed separation of species.

Cutaneous leishmaniasis: iso-enzyme characterisation of Leishmania tropica.

In order to identify and characterise the organisms responsible for Cutaneous Leishmaniasis, parasites were isolated from active lesions, grown in-vitro cultures and identified by iso-enzyme characterisation. Thirteen isolates from different patients were typed as L. tropica. Seven of these isolates were from Afghan refugees encamped in the suburbs of Islamabad, 3 were from patients in Multan, 1 was from a patient from Azad Jammu and Kashmir and 1 was from Besham (Swat, NWFP). The study confirms the presence of anthroponotic Cutaneous Leishamaniasis caused by L. Tropica in Pakistan.

Nickel deficiency alters liver lipid metabolism in rats.

The present investigation was designed to examine the effect of nickel deficiency on lipid metabolism in liver and serum lipoproteins of rats. Therefore, a study over two generations was conducted feeding a nickel-deficient diet containing 13 microg/kg nickel or a nickel-adequate diet supplemented with 1 mg/kg nickel. Male 7-wk-old pups from the second offspring were studied. Pups fed a diet poor in nickel tended to have lower weight gains (P < 0.15), nickel concentrations in liver (P < or = 0.1) and iron levels in serum (P < 0.1) than nickel-adequate rats. They were classified as nickel-deficient on the basis of significantly lower erythrocyte counts, hemoglobin concentrations, hematocrits and nickel concentrations in kidney compared with nickel-adequate rats. Nickel deficiency caused a significant triacylglycerol accumulation in liver, with greater concentrations of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids than nickel-adequate rats. Nickel deficiency had slight but significant effects on the fatty acid composition of liver total lipids and phosphatidylcholine and phosphatidylethanolamine. Moreover, nickel-deficient rats had significantly lower activities of the lipogenic enzymes glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malic enzyme and fatty acid synthase than nickel-adequate rats. Nickel-depleted pups had significantly higher concentrations of triacylglycerols and phospholipids in serum VLDL, and cholesterol in serum LDL than nickel-adequate pups. Most of these alterations in lipid metabolism are similar to those obtained in several iron-deficiency studies. Because nickel deficiency also slightly compromised iron status, it is possible that at least some of the observed alterations are due to the moderate iron deficiency.

Biochemical changes during regeneration of sunflower (Helianthus annuus L.).

The main developmental stages in Helianthus annuus organogenesis have been studied in the sunflower hybrid "Giove". Shoot regeneration was obtained with high efficiency from mature seed cotyledons. Two-dimensional electrophoresis of protein extracts as well as the isozyme patterns of acid phosphatase, alcohol dehydrogenase, esterase, gluconate-6-phosphate dehydrogenase and phosphoglucomutase were compared during growth, callusing and regeneration. Two-dimensional protein patterns were similar, although polypeptides specific for each developmental phase could be identified. Different 2,4-dichlorophenoxyacetic acid concentrations or the sampling of specific regions of the seed did not result in significant differences in protein patterns. The activity of alcohol dehydrogenase and phosphoglucomutase appeared very low. For gluconate-6-phosphate dehydrogenase no difference, related either to the genotype or to different morphological stages, could be observed; the expression of acid phosphatase varied in a nonsystematic fashion. The isozyme pattern of esterase was related to the genotype as well as to the morphogenic phase.

Biochemical characterisation of human isolates of Blastocystis hominis.

SDS-PAGE and iso-enzyme analysis of 11 human isolates of Blastocystis hominis revealed at least two variants with different polypeptide patterns and two zymodemes, respectively. This is the first iso-enzyme and the second protein analysis to indicate strain differences in B. hominis.

Electrophoretic conditions for high resolution citrus isozymes in polyacrylamide gel electrophoresis.

Electrophoretic conditions including electrode and gel buffers, acrylamide concentration, use of stacking gels, voltage, current, and run time were investigated in order to produce isozyme bands of high resolution which would facilitate densitometric quantification of enzyme activity following polyacrylamide gel electrophoresis (PAGE). Electrode buffers which provided optimal conditions for gels stained for the isozymes of malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6-PGD), phosphoglucose isomerase (PGI), and shikimate dehydrogenase (SkDH) were 0.02 M Tris-glycine, pH 8.5, 0.1 M sodium borate, pH 6.0, 0.1 M sodium borate, pH 8.7, and 0.07 M sodium borate, pH 7.0, respectively. A 0.5 M Tris-HCl, pH 7.5, gel buffer was optimal for gels stained for the isozymes of 6-PGD, PGI and SkDH. A 0.5 M Tris-HCl, pH 8.5, gel buffer was best for gels stained for MDH. Stacking gels were found to be detrimental to enzyme activity and showed no improvement in resolution for any of the enzymes. Acrylamide concentration for gels stained for MDH were 8.7%, gels stained for 6-PGD and PGI were 7.5%, while gels stained for SkDH had an acrylamide concentration of 5.0%. Higher concentrations above these levels caused a reduction and in some cases loss of band activity, while below this concentration there was a decrease in band resolution.(ABSTRACT TRUNCATED AT 250 WORDS)

A randomized clinical trial to investigate the usefulness of the addition of prednisolone to tamoxifen as adjuvants to mastectomy in primary breast cancer patients with a high risk of recurrence: a preliminary report.

The efficacy of the addition of prednisolone to tamoxifen as adjuvants to mastectomy in patients with primary breast cancer who were at a high risk of recurrence was investigated in a randomized trial. Primary carcinomas were collected from a series of 169 patients with loco-regional disease, undergoing mastectomy. The activities of alpha-glycerolphosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the carcinomas were estimated biochemically and the ratio of the two enzymes was used to as the parameter to determine the risk of recurrence. 116 patients with a high risk of recurrence within five years of mastectomy were then randomized to either tamoxifen (2x20 mg/day) or tamoxifen+prednisolone (3x2.5 mg/per day) until recurrence. The patients are currently followed quarterly. The data were analysed at a median follow-up of 26 months (range 7-62 months). The probabilities of both disease-free and overall survival were not significantly different in either arm of the trial, indicating that there is no advantage in combining prednisolone with the antioestrogen. Recently, similar findings in terms of response have been reported for patients with metastatic disease treated with the same combination, raising doubts over the role of prednisolone in the management of patients with endocrine treatments.

Histochemistry of human urethral glands.

Human urethral glands were reacted histochemically and immunohistochemically to identify glycoproteins, some androgen metabolic enzymes, and VIP-like immunoreactivity. Neutral/acid mucosubstances were detected mainly in the apical cytoplasm of the principal cells. 3 beta-, 17 beta-, and 3 alpha-hydroxysteroid dehydrogenase, G6PD, and 6PGD reactivity were intense in all the glandular epithelium. Small amounts of VIP-positive fibers were noted around the secretory elements.

Cord blood red-cell enzymes and reduced glutathione in Indian neonates, normal and with pathologic jaundice.

Red-cell enzymes and reduced glutathione (GSH) were assayed on cord blood in 307 Indian neonates. On follow-up, 20 of them developed pathologic jaundice. Of these, six had red-cell enzyme/GSH deficiency, seven had associated non-enzymatic causes of jaundice, and in the remaining seven the cause could not be diagnosed. In 41 other neonates, there was enzyme/GSH deficiency without pathologic jaundice. The degree of enzyme deficiency had no relation with jaundice. Red-cell enzyme/GSH deficiency state in neonates was associated with pathologic jaundice more frequently (six of 47; 12.8%) than in the absence of such deficiency (14 of 260; 5.4%). None of the jaundiced patients had very high levels of bilirubin nor needed exchange blood transfusion. There was no reduction in haemoglobin level in the enzyme/GSH deficient group with jaundice in comparison with non-deficient jaundiced neonates or normal subjects. The incidence of red-cell enzyme/GSH deficiency appears to be high in Indian neonates; however, the majority of them do not precipitate pathologic jaundice.

Isolation of motoneuron cell bodies from spinal cord stored at -70 degrees C in ethylene glycol.

Pretreatment of spinal cord with ethylene glycol permits long-term storage of the tissue at -70 degrees C prior to isolation and biochemical analysis of the cell bodies of spinal motoneurons. The method is useful for storing spinal tissue from laboratory animals, as well as from human post mortem specimens, where aliquots of tissue may then be used for motoneuron isolation over an indefinitely long period. In addition to inhibiting the loss of soluble proteins from the neurons during freezing and thawing, cryoprotection increases the yield and improves the appearance of the isolated cell bodies. The method should aid biochemical studies of many kinds of neuronal subpopulations isolated from small amounts of starting material.

Biochemical tissue markers of human colorectal carcinoma.

The potential therapeutic effects of differentiating agents on leukemic and solid tumor cells are being evaluated worldwide. These effects can be followed by morphologic as well as biochemical parameters. The enzymatic profile of four enzymes and the level of carcinoembryonic antigen were studied in 24 human colorectal carcinoma specimens and their adjacent uninvolved mucosa. The enzymes studied were thymidine kinase and 6-phosphogluconate dehydrogenase as markers of proliferation, and alkaline phosphatase and gamma-glutamyl transpeptidase as markers of differentiation. A consistent finding was a marked increase in the activities of thymidine kinase and 6-phosphogluconate dehydrogenase in the tumor cells as compared with the adjacent normal mucosa. The activity of gamma-glutamyl transpeptidase was not significantly different between tumor and uninvolved colon tissue. Alkaline phosphatase activity was markedly reduced in the tumor specimens. A relationship between the degree of differentiation and the degree of penetration and CEA expression was demonstrated in the tumor specimens as well as in their surrounding uninvolved mucosa.

Hexose monophosphate shunt enzymes in lung tumors from normal and glucose-6-phosphate-dehydrogenase-deficient subjects.

Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate, dehydrogenase the key enzymes of the hexose monophosphate shunt pathway, were measured in both surrounding and tumoral lung tissues from normal and G6PD-deficient subjects. A significant increase of these enzymatic activities in tumoral tissue was found not only in G6PD-normal patients, but also in G6PD-deficient patients with very low or nonmeasurable G6PD activity in both erythrocytes and normal lung tissue.

Enzymatic characterization of Babesia bovis.

Agarose gel electrophoresis was used to identify metabolic enzymes in Babesia bovis and B. bigemina. Glutamate dehydrogenase, lactate dehydrogenase, glucose phosphate isomerase, and hexokinase were identified in B. bovis- and B. bigemina-infected erythrocytes and B. bovis merozoite preparations. A specific electrophoretic mobility was observed for each enzyme. Malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and adenylate kinase were only detected in normal erythrocyte preparations. Inter-species, but not intra-species, variation was noted when comparing electrophoretograms of both species. Kinin-activating activity was not detected in B. bovis-infected erythrocyte or merozoite preparations at pH 4.2 or 7.6.

Metabolic reduction of chromium by alveolar macrophages and its relationships to cigarette smoke.

Pulmonary alveolar macrophages (PAM), obtained by bronchoalveolar lavage from 47 individuals, reduced hexavalent chromium [Cr(VI)] and decreased its mutagenicity. Their specific activity--mostly mediated by cytosolic, enzyme-catalyzed mechanisms--was significantly higher than in corresponding preparations of mixed-cell populations from human peripheral lung parenchyma or bronchial tree, or from rat lung or liver. At equivalent number of PAM, Cr(VI) reduction, total protein, and some oxidoreductase activities were significantly increased in smokers. No appreciable variation could be detected between lung cancer and noncancer patients. In rats, the Cr(VI)-reducing activity of PAM preparations was induced by Aroclor 1254. Thus, alveolar macrophages provide crucial defense mechanisms not only by phagocytizing metals, but also by metabolically reducing Cr(VI). The epithelial-lining fluid (ELF) also displayed some Cr(VI) reduction. Together with already investigated metabolic processes occurring inside lung cells, these mechanisms are expected to determine thresholds in the pulmonary carcinogenicity of chromium.

The value of 6-phosphogluconate dehydrogenase (6-PGDH) activity as a marker of tumour cellularity and prognostic indicator in primary breast cancer.

The value of 6-phosphogluconate dehydrogenase (6-PGDH) activity as a cytosolic marker of tumour cellularity has been assessed, together with its use as a prognostic indicator for primary breast cancer in 87 patients over 4 years. 6-PGDH activity shows a good correlation with histologically-assessed tumour cellularity in a sample of 114 patients (correlation coefficient = 0.83). Patients whose primary breast tumour had a high 6-PGDH activity showed poor relapse-free survival times when compared to those with low 6-PGDH activities (Log Rank chi 2 = 6.87, P less than 0.01). This compared with a Log Rank chi 2 of 2.22, P less than 0.20 for oestrogen positive and negative patients. These results suggest that 6-PGDH activity is a better prognostic indicator in primary breast cancer than oestrogen receptor status.

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.

Glycolytic enzymes in human breast carcinoma cytosols: the influence of commonly used reference parameters.

The activities of selected glycolytic enzymes in breast tumor tissues were examined based on earlier studies showing a relationship between therapy and/or prognosis of breast cancer and tissue enzyme. In the present study, five glycolytic enzymatic activities (pyruvate kinase [PK], glucose-6-phosphate dehydrogenase [G6PD], phosphohexose-isomerase [PHI], lactate dehydrogenase [LDH], and 6-phospho-glucocate dehydrogenase [6PGD]) were measured in the cytosol (105,000 g) of 57 breast carcinomas and 22 benign breast lesions. Nucleic acids and DNA were also determined. The results were related to the wet weight of the tissue, to total and tissue cytosol proteins, and to DNA. The various means of expressing the results were compared. The correlations were satisfactory except for PHI and LDH. In these cases, this might have been due to blood contamination. The enzyme activities content was lower in the benign breast lesions than in the breast carcinomas irrespective of the way the results were expressed.