Effects of supplemental oxygen on urinary 8-hydroxy-2'-deoxyguanosine levels in extremely low birth weight infants.

As the effects of supplementary oxygen on urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) are poorly understood, urinary 8-OHdG levels (ng/mg creatinine) were determined longitudinally on the postnatal day (PND) 1, 3, and 30 in 16 neonates with birth weight < 1000 g. No supplementary oxygen was required in 9 neonates during the first 24 h of life. Urinary 8-OHdG level on PND 1 was inversely correlated with birth weight in these 9 neonates (P = 0.0323) and was higher in four with birth weight < 750 g than five with birth weight > 750 g (41.0 ± 6.9 vs. 5.6 ± 2.7, respectively, P = 0.0200). Median urinary 8-OHdG on PND 1 of these 9 neonates was significantly lower than that of 7 neonates with oxygen (9.3 vs. 60.2, respectively), although there were no significant differences in clinical background, such as birth weight, between the two groups. Five of the 9 did not require supplemental oxygen at all during the first 30 days of life. Median urinary 8-OHdG levels were consistently significantly lower in the 5 neonates than in 11 neonates with oxygen transiently or persistently (9.3 vs. 54.6, 19.1 vs. 61.4, and 28.3 vs. 145 on PND 1, 3, and 30, respectively), although there were no differences in clinical background, such as birth weight, between the two groups. Urinary 8-OHdG on PND 30 was significantly positively correlated with supplemental oxygen dose on PND 30 (P < 0.0001), but not with birth weight in the 16 neonates. These results suggest that higher supplemental oxygen tension caused higher urinary 8-OHdG in this population.

Nonsense mutation of feline beta-hexosaminidase beta-subunit (HEXB) gene causing Sandhoff disease in a family of Japanese domestic cats.

G(M2) gangliosidoses are inherited metabolic disorders and are caused by severely reduced enzymatic activity of lysosomal beta-hexosaminidase. In the present study, the open reading frame (ORF) of the HEXB gene in a family of Japanese domestic cats with G(M2) gangliosidosis variant 0 (Sandhoff disease) was determined. Two types of abnormal cDNA clones were obtained from the liver of an affected cat tissue. One showed a single nucleotide substitution from C to T at nucleotide position 667 of the HEXB ORF. In the deduced amino acid sequence, the codon of arginine was altered to a stop codon. The genotyping, using PCR-primer introduced restriction analysis confirmed that Sandhoff disease in this family is associated with this nonsense mutation. Discovery of the nonsense mutation will permit the confirmation of the clinical diagnosis of Sandhoff disease in conjugation with the already established enzyme-based test.

Anti-babesial ellagic acid rhamnosides from the bark of Elaeocarpus parvifolius.

Bioassay-guided investigation of the bark of Elaeocarpus parvifolius led to the isolation of three new ellagic acid derivatives, 4-O-methylellagic acid 3'-alpha-rhamnoside (2), 4-O-methylellagic acid 3'-(3''-O-acetyl)-alpha-rhamnoside (3), and 4-O-methylellagic acid 3'-(4''-O-acetyl)-alpha-rhamnoside (4) in addition to the known ellagic acid derivative, 4-O-methylellagic acid 3'-(2'',3''-di-O-acetyl)-alpha-rhamnoside (1). Their structures were elucidated on the basis of analysis of 1H NMR, 13C NMR, HMQC, HMBC and MS spectroscopic data. Compounds 1-4 were evaluated for their growth-inhibitory effect on Babesia gibsoni in vitro. Compounds 2 and 4 showed very weak activity, while compounds 1 and 3 showed moderate activity, with IC50 values of 28.5 and 52.1 microg/ml, respectively.

Modulatory influence of sodium 2-propenyl thiosulfate from garlic on cyclooxygenase activity in canine platelets: possible mechanism for the anti-aggregatory effect.

We previously found that sodium 2-propenyl thiosulfate (2PTS) has an anti-aggregatory effect in vitro on both canine and human platelets at relatively low concentrations, but the extent of aggregation tends to return to the control level at high concentrations. To clarify the mechanism of this modulatory influence of 2PTS on the aggregation of platelets, we investigated the effects of 2PTS on cyclooxygenase (COX) activity and the reduced glutathione (GSH) concentration in canine platelets. Platelet COX activity was inhibited by 2PTS in a dose-dependent manner up to 0.1 mM, but tended to return to the control level at 1 mM. In contrast, the platelet GSH concentration decreased in a dose-dependent manner after treatment with 2PTS and a significant decrease was observed at 0.1 mM (P<0.05) and 1 mM (P<0.001). Furthermore, the activity of purified COX-1 was directly inhibited by addition of GSH in a dose-dependent manner. From these results, we conclude that the 2PTS-induced inhibition of platelet aggregation occurs as a result of inhibition of COX activity. Additionally, 2PTS may have a modulatory effect on platelet aggregation by affecting the platelet GSH concentration.

GM2-gangliosidosis variant 0 (Sandhoff-like disease) in a family of Japanese domestic cats.

A five-month-old, female Japanese domestic shorthair cat with proportionate dwarfism developed neurological disorders, including ataxia, decreased postural responses and generalised body and head tremors, at between two and five months of age. Leucocytosis due to lymphocytosis with abnormal cytoplasmic vacuolations was observed. The concentration of G(M2)-ganglioside in its cerebrospinal fluid was markedly higher than in normal cats, and the activities of beta-hexosaminidases A and B in its leucocytes were markedly reduced. On the basis of these biochemical data, the cat was diagnosed antemortem with G(M2)-gangliosidosis variant 0 (Sandhoff-like disease). The neurological signs became more severe and the cat died at 10 months of age. Histopathologically, neurons throughout the central nervous system were distended, and an ultrastructural study revealed membranous cytoplasmic bodies in these distended neurons. The compound which accumulated in the brain was identified as G(M2)-ganglioside, confirming G(M2)-gangliosidosis. A family study revealed that there were probable heterozygous carriers in which the activities of leucocyte beta-hexosaminidases A and B were less than half the normal value. The Sandhoff-like disease observed in this family of Japanese domestic cats is the first occurrence reported in Japan.

Acceleration of superoxide generation in polymorphonuclear leukocytes and inhibition of platelet aggregation by alk(en)yl thiosulfates derived from onion and garlic in dogs and humans.

We recently identified sodium n-propyl thiosulfate (NPTS) and sodium 2-propenyl thiosulfate (2PTS) from boiled onion and garlic, respectively, as causative agents of hemolytic anemia in dogs. We present here data concerning the effects of these alk(en)yl thiosulfates on superoxide (O(2)(-)) generation in peripheral polymorphonuclear leukocytes (PMNs) and on adenosine 5'-diphosphate (ADP)-induced platelet aggregation in dogs and humans in vitro. Both NPTS and 2PTS increased O(2)(-) generation significantly (P<0.05 at 1mM NPTS, P<0.005 at 0.1 and 1mM 2PTS) and reduced its reaction time significantly (P<0.05 between 0.01 and 1mM NPTS and at 1mM 2PTS) in canine PMNs stimulated by phorbol 12-myristate 13-acetate, compared with the control without alk(en)yl thiosulfates. However, a tendency to return to the control level was observed at 10mM of the alk(en)yl thiosulfates in both O(2)(-) generation and its reaction time. Although NPTS and 2PTS did not exert any significant effect on the O(2)(-) generation in human PMNs, 2PTS reduced its reaction time significantly (P<0.05) at 1 and 10mM compared with the control, showing that 2PTS accelerated O(2)(-) generation in human PMNs. The difference in effects on O(2)(-) generation may be due to that in susceptibility to alk(en)yl thiosulfates between canine and human PMNs. On the other hand, NPTS and 2PTS were shown to significantly inhibit ADP-induced platelet aggregation at 0.01mM (P<0.01) in canine platelets and at 0.001-0.1mM (P<0.05) in human platelets. In contrast, the maximal aggregation percentage returned to the control level at 1mM of alk(en)yl thiosulfates in both canine and human platelets. From these results, we conclude that NPTS and 2PTS have the potential to promote immune functions and prevent cardiovascular diseases.

Molecular cloning and phylogenetic analysis of Babesia gibsoni heat shock protein 70.

The Babesia gibsoni heat shock protein 70 gene (BGHsp70) was cloned by polymerase chain reaction (PCR) and sequenced. The length of the gene was 1938 bp and the predicted polypeptide was 646 amino acids long with a calculated molecular weight of 70,627. The amino acid sequences of BGHsp70 from 17 isolates were identical, though there were six types of polymorphisms among the corresponding nucleotide sequences. There was no intron in the BGHsp70 gene. Phylogenetic analysis of the amino acid sequence of Hsp70 showed that B. gibsoni was most closely related to B. bovis and lies within a phylogenetic cluster with Theileria. These results suggest that Hsp70 was well conserved among intraerythrocytic protozoa.

Sandhoff disease in a golden retriever dog.

A golden retriever dog is described with total hexosaminidase deficiency and raised GM2-ganglioside in CSF. The animal represents a model for human Sandhoff disease.

A novel mutation in the gene for canine acid beta-galactosidase that causes GM1-gangliosidosis in Shiba dogs.

A homozygous recessive mutation, causing GM1-gangliosidosis in Shiba dogs, was identified as a deletion of C nucleotide 1668 in the gene for canine acid beta-galactosidase, which was a novel mutation in canine GM1-gangliosidosis.

Increased generation of superoxide in erythrocytes infected with Babesia gibsoni.

The present study was conducted to clarify the mechanism underlying the oxidative process in erythrocytes infected with Babesia gibsoni. The parasite B. gibsoni was cultured together with erythrocytes from normal dogs for 7 days. When parasitemia reached 12.0-13.4% at Day 7. the production of superoxide in erythrocytes was significantly higher in the parasitized culture than in the control culture (p<0.005). The concentration of thiobarbituric acid reactive substances (TBARS) in erythrocytes in parasitized culture was also significantly increased compared with the control culture (p<0.005), indicating that lipid peroxidation was greater in infected erythrocytes than in non-infected cells. In addition, the rates of superoxide generation in the blood of B. gibsoni-infected dogs were also significantly higher than in non-infected dogs (p<0.001). These results indicate that superoxide anions are increased in erythrocytes parasitized with B. gibsoni. and suggest that oxidative damage, due to lipid peroxidation, might be caused in host erythrocytes by the parasite.

Prevalence of genotypes 1 and 2 of bovine viral diarrhea virus in Lower Saxony, Germany.

The aim of this study was to find whether an antigenic drift had occurred in Lower Saxony in the past 40 years. For this, the genetic diversity of bovine viral diarrhea virus (BVDV) isolates mainly from Lower Saxony was estimated by RT-PCR and sequencing of a 420 bp fragment of the E2 glycoprotein gene. Sixty-one field virus isolates collected during routine diagnostics between 1960 and 2000 in Lower Saxony, Northern Germany, were analyzed. Phylogenetic analysis allowed discrimination of genotypes BVDV 1 and 2. Excepting two isolates, which were of BVDV type 2, most of the isolates were classified as BVDV type 1. This group could be further subdivided into four subgroups and one disparate isolate. Independent of the year of isolation and geographical localization, 54 isolates clustered in two subtypes (BVDV subtypes 1b and 1d). Only one isolate was classified as BVDV type 1a, thus being similar to the North American NADL strain, and to the vaccine strain Oregon C24V, which was extensively used for vaccination in Germany. The remaining isolates belonged to new clusters tentatively designated as BVDV subtypes 1g and 1f. To compare the cluster designation with that of other studies, phylogenetic analysis of representatives of each of the subgroups based on the 5' untranslated region (5'UTR) was performed. It grouped the viruses similarly. The results indicate that the BVDV population seems to be relatively stable over 40 years in Lower Saxony.

Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni.

OBJECTIVE: To determine the usefulness of canine RBC with high concentrations of potassium, reduced glutathione (GSH), and amino acid(i.e., HK cells) for in vitro cultivation of Babesia gibsoni. ANIMALS: RBC were obtained from 3 dogs that had inherited HK cells and from 3 genetically unaffected dogs that, therefore, had RBC with lower potassium (LK) concentrations (i.e., LK cells). PROCEDURES: First, B. gibsoni were cultivated using HK or LK cells in alpha-modification of Eagle medium, consisting of Earle salts with glutamine and without ribosides, deoxyribosides, and sodium bicarbonate under a humidified atmosphere containing 5% CO2 at 37 C. Second, parasites were cultivated with LK- or HK-cell lysates. Finally, HK cells were separated into 3 fractions (bottom, middle, top layers) by density gradient centrifugation, and B. gibsoni were cultivated with each of the HK-cell fractions. In addition, the concentrations of free amino acids and reduced glutathione (GSH) in each HK-cell fraction were measured. RESULTS: B. gibsoni preferentially multiplied in HK-cell cultures rather than in LK-cell cultures. Furthermore, the addition of HK-cell lysate to the culture medium resulted in enhanced multiplication of the parasites. Higher multiplication of the parasites was observed in HK cells from the top layer, compared with HK cells from the middle and bottom layers. The HK cells from the top layer had higher concentrations of glutamate, aspartate, and GSH, compared with HK cells from the middle and bottom layer. CONCLUSIONS: Canine HK cells are useful host cells for in vitro cultivation of B. gibsoni, and the high concentrations of glutamate, aspartate, and GSH may result in enhancement of multiplication of the parasites in HK cells.

Hematologic changes associated with the appearance of eccentrocytes after intragastric administration of garlic extract to dogs.

OBJECTIVE: To determine whether dogs given garlic extract developed hemolytic anemia and to establish the hematologic characteristics induced experimentally by intragastric administration of garlic extract. ANIMALS: 8 healthy adult mixed-breed dogs. PROCEDURE: 4 dogs were given 1.25 ml of garlic extract/kg of body weight (5 g of whole garlic/kg) intragastrically once a day for 7 days. The remaining 4 control dogs received water instead of garlic extract. Complete blood counts were performed, and methemoglobin and erythrocyte-reduced glutathione concentrations, percentage of erythrocytes with Heinz bodies, and percentage of eccentrocytes were determined before and for 30 days after administration of the first dose of garlic extract. Ultrastructural analysis of eccentrocytes was performed. RESULTS: Compared with initial values, erythrocyte count, Hct, and hemoglobin concentration decreased to a minimum value on days 9 to 11 in dogs given garlic extract. Heinz body formation, an increase in erythrocyte-reduced glutathione concentration, and eccentrocytes were also detected in these dogs. However, no dog developed hemolytic anemia. CONCLUSIONS AND CLINICAL RELEVANCE: The constituents of garlic have the potential to oxidize erythrocyte membranes and hemoglobin, inducing hemolysis associated with the appearance of eccentrocytes in dogs. Thus, foods containing garlic should not be fed to dogs. Eccentrocytosis appears to be a major diagnostic feature of garlic-induced hemolysis in dogs.

The cause of the predilection of Babesia gibsoni for reticulocytes.

This study was conducted to determine why Babesia gibsoni replicates well in reticulocytes. First, B. gibsoni was cultivated in resealed erythrocyte ghosts loaded with either erythrocyte or reticulocyte lysate, and in reticulocyte ghosts loaded with either erythrocyte or reticulocyte lysate. The parasites multiplied well in the erythrocyte or reticulocyte ghosts loaded with reticulocyte lysate compared to the other resealed cells loaded with erythrocyte lysate. Second, the parasites were cultivated in erythrocytes in culture medium supplemented with either erythrocyte or reticulocyte lysate. The parasites multiplied better in reticulocyte lysate-containing cultures than in erythrocyte lysate-containing cultures. Finally, the parasites were cultivated in erythrocytes in culture medium supplemented with glutamate, aspartate, asparagine, glycine, isoleucine, proline, taurine or GSH, which were present in higher concentrations in reticulocytes than in erythrocytes. Supplementation of the culture medium with glutamate and GSH resulted in enhancement of the multiplication of the parasites, while the other amino acids did not enhance the multiplication. These results indicated that the high levels of the multiplication of B. gibsoni in reticulocytes are partly due to the high concentrations of glutamate and GSH in reticulocytes.

GM1 gangliosidosis in shiba dogs.

A six-month-old shiba dog with a one-month history of progressive motor dysfunction showed clinical signs of a cerebellar disorder, including ataxia, dysmetria and intention tremor of the head. Histopathological and ultrastructural studies revealed distended neurons packed with membranous cytoplasmic bodies throughout the central nervous system. The activities of lysosomal acid beta-galactosidase in its leucocytes and liver were less than 2 per cent of the control levels, and the compound accumulated in the brain was identified as GM1 ganglioside. A sibling which died immediately after birth was shown to have a beta-galactosidase deficiency in the brain and visceral organs. A family study revealed that the sire and dam of the probands were heterozygotes with approximately half of the normal level of beta-galactosidase activity, suggesting an autosomal recessive pattern of inheritance.

Vascular endothelial growth factor expression in progression of cervical cancer: correlation with thymidine phosphorylase expression, angiogenesis, tumor cell proliferation, and apoptosis.

BACKGROUND: Vascular endothelial growth factor (VEGF) has been linked not only to angiogenic activity but also to thymidine phosphorylase (TP), rapid tumor growth, and inhibition of apoptotic cell death. Our purpose was to examine how VEGF expression affect these factors in cervical cancer at varying stages of progression. METHODS: VEGF expression, TP expression, the microvessel count (reflected by factor VIII-related antigen), and proliferating cell nuclear antigen (PCNA) were assessed immunohistochemically in 19 specimens of normal cervical epithelium, 35 of carcinoma in situ (CIS), 34 of microinvasive carcinoma (MIC), and 34 of invasive cervical squamous cell carcinoma (SCC). Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. RESULTS: VEGF expression progressively increased along a continuum from normal epithelium to invasive SCC (P < 0.0001). VEGF expression significantly correlated with TP expression and PCNA index (P < 0.01 and P < 0.0001, respectively). In analyses within histological stages, VEGF expression significantly correlated with the PCNA index in CIS and MIC (P < 0.01 and P < 0.05, respectively), but not in invasive SCC. The PCNA index for combined analysis of VEGF and TP expression was similar to that for VEGF expression alone. VEGF expression tended to correlate with microvessel count, however, the difference was not significant (P = 0.09). No significant correlation was observed between VEGF expression and the apoptotic index. CONCLUSIONS: VEGF expression may stimulate tumor cell proliferation in the early stages of cervical cancer, and may be responsible for cervical tumorigenesis.

'Babesia gibsoni' of dogs from North America and Asia belong to different species.

18S rDNA sequences from 4 isolates of Babesia gibsoni originating from Japan, Malaysia and Sri Lanka were compared with a previously published, 0.5 kb portion of the 18S rDNA from a B. gibsoni isolate from California, USA, and with the corresponding 18S rDNA sequences of other Babesia spp. Distance, parsimony and maximum likelihood analyses showed almost identical genotypes among the small canine Babesia from Asia, but an unexpectedly distant genetic relationship to that from the USA. While the American isolate segregated together with B. equi, the Asian isolates showed a close relationship to B. divergens and B. odocoilei. These results indicate that small Babesia of dogs originating from North America and Asia belong to different, genetically distantly related species.

Expression of thymidine phosphorylase in epithelial ovarian cancer: correlation with angiogenesis, apoptosis, and ultrasound-derived peak systolic velocity.

OBJECTIVE: The objective of this study was to determine whether the expression of thymidine phosphorylase (TP) by epithelial ovarian cancer cells correlates with the density of microvessels within the tumor, apoptotic index (AI) within the tumor cells, and ultrasound-derived blood flow. METHODS: Color Doppler imaging and pulsed Doppler spectral analysis ultrasonography were used to scan 44 patients with an overt ovarian mass immediately before laparotomy. Sections of malignant tumors were analyzed for the cellular expression of TP and the intratumoral density of microvessels by immunohistochemistry using antibodies to TP and factor-VIII-related antigen, respectively. Moreover, AI was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling methods. RESULTS: Forty-four epithelial ovarian cancers were studied (6 low malignant potential, 15 serous cystadenocarcinoma, 9 mucinous cystadenocarcinoma, 8 endometrioid adenocarcinoma, 4 clear cell carcinoma, and 2 malignant Brenner tumors); 19 were Stage I, 6 Stage II, 15 Stage III, and 4 Stage IV. Fourteen tumors (32%) were classified as being TP positive. The proportion of Stage I tumors that was TP positive (16%) was significantly lower (P = 0.022) that the corresponding value for Stages II-IV (44%), although the values for microvessel count, AI, and peak systolic velocity (PSV) were similar. AI was significantly lower in TP-positive tumors than in TP-negative tumors (P = 0.015). The PSV was significantly higher in TP-positive tumors (P = 0.02). There was a significant correlation between the microvessel count and the PSV (r = 0.34, P = 0.024). Moreover, AI was significantly inversely related to the PSV (r = 0. 35, P = 0.023). The PSV in a subgroup with a high microvessel count and low AI was significantly higher than that in a subgroup with a low microvessel count and high AI (P = 0.0006). These findings significantly associated with TP expression (P = 0.024). CONCLUSIONS: The intratumoral PSV, as determined by color Doppler imaging and spectral analysis, might reflect the coordination of angiogenesis and apoptosis associated with TP expression in epithelial ovarian cancer.

Expression of thymidine phosphorylase and vascular endothelial growth factor in epithelial ovarian cancer: correlation with angiogenesis and progression of the tumor.

OBJECTIVE: The aim was first, to determine whether the expression of thymidine phosphorylase (TP) and vascular endothelial growth factor (VEGF) by epithelial ovarian cancer cells is correlated with the density of microvessels within the tumor and with ultrasonographic findings (B-mode classification and pulsed Doppler blood flow) and second, to speculate how these two angiogenesis factors participate in the tumorigenesis and tumor progression of epithelial ovarian cancer. METHODS: B-mode ultrasonography and color Doppler imaging and pulsed Doppler spectral analysis were used to scan patients with an overt ovarian mass immediately before laparotomy. Sections of malignant tumors were analyzed for the cellular expression of TP and VEGF and the intratumoral density of microvessels by immunohistochemistry using antibodies to TP, VEGF and Factor VIII related antigen, respectively. The main outcome measures were the histological classification of the tumor, the stage of the disease, whether or not the tumor cells were TP and VEGF positive or negative, the microvessel count and B-mode classification and the peak systolic velocity (PSV). RESULTS: Forty-four epithelial ovarian cancers were studied (6 of low malignant potential, 15 serous cystadenocarcinoma, 9 mucinous cystadenocarcinoma, 8 endometrioid adenocarcinoma, 4 clear cell carcinoma and 2 malignant Brenner tumor); 19 were Stage I, 6 Stage II, 15 Stage III and 4 Stage IV. Fourteen tumors (32%) were classified as TP positive and 21 (48%) as VEGF positive. The proportion of Stage I tumors that were TP positive (16%) was significantly lower (p = 0.022) than the corresponding value for Stages II-IV (44%), but the proportion with VEGF positive, the values for microvessel count and PSV were similar. Microvessel count did not significantly related to TP nor VEGF expression. The PSV was significantly higher in TP-positive tumors (p = 0.02) and VEGF-positive tumors (p = 0.006), respectively. There was a significant correlation between the microvessel count and the PSV (r = 0.34, p = 0.024). Moreover, specific B-mode classification significantly associated with disease stage and with TP expression, but not with VEGF expression. CONCLUSIONS: Angiogenesis is an early, critical step in the tumorigenesis of epithelial ovarian cancer, however, it does not provide significant information about tumor aggressiveness. When TP expression is superimposed upon the VEGF expression, the tumor might acquire the aggressive tumor phenotype.

Relation between erythrocyte reduced glutathione and glutamate concentrations in Korean Jindo dogs with erythrocytes possessing hereditary high activity of Na-K-ATPase and a high concentration of potassium.

The concentrations of sodium, potassium, reduced glutathione (GSH) and free amino acids and Na-K-ATPase activity in erythrocytes were examined in 35 purebred Jindo dogs in Korea. The incidence of Jindo dogs with a high potassium concentration and high activity of Na-K-ATPase in erythrocytes (HK phenotype) was 25.7%. The erythrocyte GSH concentration in HK Jindo dogs varied widely, from 2.45 to 12.38 mmol/l of RBCs, and was positively correlated with the erythrocyte glutamate concentration. These results indicate that HK Jindo dogs have normal to very high levels of erythrocyte GSH, which might result from the varying quantity of Na-dependent glutamate influx in the erythrocytes.