Haemolysis complicating acute viral hepatitis in patients with normal or deficient glucose-6-phosphate dehydrogenase activity

Haemolytic anaemia as a complication of acute hepatitis has been reported in up to 23% of patients. However, the incidence may rise up to 70-87% in patients who have glucose-6-phosphate dehydrogenase (G6PD) deficiency. Massive intravascular haemolysis with renal failure, hepatic encephalopathy and even death have been reported. In our retrospective study of patients with acute viral hepatitis, the overall incidence of acute haemolysis was 4% (17/434). Only 53% (9/17) of them had G6PD deficiency. Patients with acute haemolysis had a significantly higher peak bilirubin level and required more prolonged hospitalization. Since hepatitis A virus vaccination, unlike hepatitis B virus vaccination, is not yet recommended for routine immunization, we suggest subjects who are G6PD-deficient should be vaccinated against hepatitis A. In endemic areas of hepatitis A virus infection, universal immunization remains the definitive answer.     

Age-dependent tetrahydrothiophenium ion formation in young children and adults receiving high-dose busulfan

Busulfan, a bifunctional alkylating agent, is a mainstay of myeloablative preparative regimens before hematopoietic stem cell transplantation. The apparent oral clearance of busulfan expressed relative to body surface area is 2-3-fold higher in children 1-4 years old than it is in adults. The first step in busulfan elimination is the formation of a tetrahydrothiophenium ion (THT+) in a glutathione S-transferase-catalyzed reaction. We present computer simulations that demonstrate that the ratio of the AUC of THT+ to that of busulfan over 6 h [(AUC(THT+)/AUC(BU))(0-->6)] is highly correlated (r2 = 0.805) with the determinants of THT+ formation and is virtually independent of the determinants of its elimination (r2 = 0.0201). We compared (AUC(THT+)/AUC(BU))(0-->6) determined in 14 children (0.5-4 years) to that of 11 adults (12-54 years) and found a 1.5-fold elevation in the area ratio (P = 0.0098) and a similarly significant increase in busulfan apparent oral clearance expressed relative to body surface area (P = 0.042). The only common explanation for the elevated busulfan apparent oral clearance and (AUC(THT+)/AUC(BU))(0-->6) is an enhanced ability of children to metabolize busulfan through glutathione conjugation.     

Electron tomography of large, multicomponent biological structures

Genome-wide scans for linkage of chromosome regions to type 1 diabetes in affected sib pair families have revealed that the major susceptibility locus resides within the major histocompatibility complex (MHC) on chromosome 6p21 (lambda s = 2.5). It is recognised that the MHC contains multiple susceptibility loci (referred to collectively as IDDM1), including the class II antigen receptor genes, which control the major pathological feature of the disease: T lymphocyte-mediated autoimmune destruction of the insulin-producing pancreatic beta cells. However, the MHC genes, and a second locus, the insulin gene minisatellite on chromosome 11p15 (IDDM2; lambda s = 1.25), cannot account for all of the observed clustering of disease in families (lambda s = 15), and the scans suggested the presence of other susceptibility loci scattered throughout the genome. There are four additional loci for which there is currently sufficient evidence from linkage and association studies to justify fine mapping experiments: IDDM4 (FGF3/11q13), IDDM5 (ESR/6q22), IDDM8 (D6S281/6q27) and IDDM12 (CTLA-4/2q33), IDDM4, 5 and 8 were detected by genome scanning, and IDDM12 by a candidate gene strategy. The results suggest that the clustering of type 1 diabetes in families is due to the sharing of alleles at multiple loci, and that the as yet unidentified environmental factors are not causing clustering, but instead appear to influence the overall penetrance of genetically programmed susceptibility. The data are consistent with a polygenic threshold model for the inheritance of type 1 diabetes.     

Distinguishing Cowper''s glands from neoplastic and pseudoneoplastic lesions of prostate: immunohistochemical and ultrastructural studies

beta-Glucosidase of indigo plant (Polygonum tinctorium) has a high substrate specificity for indican (indoxyl beta-D-glucoside). To examine the localization of this beta-glucosidase, we fractionated the cells of the leaves and analysed them immunocytochemically. Immunoelectron micrographs with specific antibodies against the beta-glucosidase clearly showed that the beta-glucosidase was localized in the stroma of the chloroplasts in mesophyll cells, but not in the thylakoid membrane. Chloroplasts were isolated from the crude homogenate of the fresh leaves by Percoll density gradient centrifugation and then subjected to suborganellar fractionation. beta-Glucosidase activity was specifically detected in the stromal fraction, but not in the thylakoid membrane. This was also supported by the result of an immunoblot of the fraction with anti-beta-glucosidase antibodies. The beta-glucosidase was immunocytochemically localized in the chloroplasts of mesophyll cells, but not in any chloroplasts in marginal cells of the vascular bundle or epidermal cells; ribulose 1,5-bisphosphate carboxylase (Rubisco), a typical stromal protein, was observed in all chloroplasts in these cells. These results suggest that beta-glucosidase is tissue specific in its expression in the leaves of the indigo plant.     

99Tcm-MDP scintigraphy in high-voltage electrical burn patients

In high-voltage electrical burn injuries (> 1000 V), it is difficult to identify the site and extent of non-viable deep tissue damage for debridement to avoid further tissue injury from wound infection and the risk of sepsis. This prospective study was designed to evaluate the usefulness of 99Tcm-methylene di-phosphonate (99Tcm-MDP) scintigraphy in detecting the extent of tissue injury and determining the level of amputation required for electrical burn patients. Over a 5 year period, 33 high-voltage electrical burn patients were studied. Blood flow and blood pool studies revealed absent perfusion in 37 limbs, all of which eventually were amputated. In addition to a routine three-phase bone scan, images were obtained at 30-60 min (early images) to evaluate whether soft tissue injury could be detected better at that time. For comparison of the detection rate from the early images and bone (delayed) images, 164 corresponding spot views of both images were reviewed. Eighty-three and 125 tissue necrotic lesions were demonstrated by the early images and bone images respectively. All of the 83 lesions found by the early images were more clearly identified by the bone images. All but one of the 125 lesions underwent surgical debridement or amputation. We concluded that the blood flow and blood pool images correlated well with the level of amputation required. The site and extent of tissue necrotic lesions can be clearly identified on 99Tcm-MDP bone scans. Because the early images were less sensitive in detecting tissue necrosis, we suggest that early imaging is not necessary.     

Immunological detection of membrane-associated human luteinizing hormone correlates with gene expression in cultured human cancer and fetal cells

We have demonstrated the expression of membrane-associated hCG and its subunits and fragments by cells from 78 human cancer cell lines of different types and origins, indicating that such expression is a common phenotypic characteristic of cultured human malignant cells. Because human (h) LH beta has 80% homology with hCG beta and is coded by one of the seven genes in the gene cluster located in chromosome 19, it was important to determine whether hLH and its beta-subunit are also expressed as membrane-associated proteins by cells from human cancer cell lines. Thus, 11 cancer cell lines of different types and origins were adapted to grow in serumless medium, with Nutridoma-HU or SP as serum substitute, and analyzed by flow cytometry using two monoclonal antibodies directed to different conformational epitopes of intact hLH and a monoclonal antibody reacting with an epitope of hLH beta-free. The cells were also analyzed simultaneously for the expression of hCG and its subunits and fragments. Determination of translatable levels of hLH beta and hCG beta messenger RNAs (mRNAs) was performed in cells from some of the cancer cell lines, including the JEG-3 choriocarcinoma cell line, and in cells from a human fetal lung cell line. The analytical flow cytometry studies showed that in addition to the expression of membrane-associated hCG in all of its forms, expression of membrane-associated intact (holo) hLH and its free beta-subunit occurred in every case. These findings were corroborated by the presence of translatable levels of hLH beta and hCG beta mRNAs in all of the cancer cell lines analyzed, indicating that the expression of these membrane-associated glycoproteins is a phenotypic characteristic of human cancer cells and that the activation of the hCG beta-hLH beta gene cluster is nonselective. The presence of translatable levels of hCG beta-hLH beta mRNAs in the cultured human fetal lung cells punctuates once more the in vivo and in vitro biochemical similarities between fetal and cancer cells.     

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P

For PCR-based genotyping using polymorphic microsatellite markers, DNA from decomposed postmortem human tissues was fractionated into six groups according to molecular size. The minimum required amounts of this degraded DNA, for detecting alleles at five microsatellite loci (ACTBP2, CMAG, HUMTH01, CYP19, and LPL) and one minisatellite locus (MCT118) were investigated respectively. The allele patterns were detected by electrophoresis of the PCR products on a 6%-denaturing polyacrylamide gel following silver staining. The detection of alleles for the loci with large allele size required more template DNA with higher molecular size than for that with small allele size. Amounts from 0.3 ng to 5 ng were needed for allele detection on genomic DNA from fresh blood. When the decomposed DNA mixture was used as the template, approximately ten times the amount of genomic DNA was required to detect alleles at the three loci of LPL, CYP19 and HUMTH01, while 24 to 67 times was required for the loci, CMAG, ACTBP2 and MCT118. It was demonstrated that a minimum molecular, size and amount of template DNA was needed for amplifying alleles of the six loci, and degraded DNA less than minimum size in the samples would prevent the detection of the loci which have large allele size.     

Pharmacological and biochemical evidence for the regulation of osteocalcin secretion by potassium channels in human osteoblast-like MG-63 cells

Previous reports have suggested the involvement of voltage-activated calcium (Ca2+) channels in bone metabolism and in particular on the secretion of osteocalcin by osteoblast-like cells. We now report that potassium (K+) channels can also modulate the secretion of osteocalcin by MG-63 cells, a human osteosarcoma cell line. When 1,25-dihydroxyvitamin D3(1,25(OH)2D3)-treated MG-63 cells were depolarized by step increases of the extracellular K+ concentration ([K+]out) from 5-30 mM, osteocalcin (OC) secretion increased from a control value of 218 +/- 13 to 369 +/- 18 ng/mg of protein/48 h (p < 0.005 by analysis of variance). In contrast, in the absence of 1,25(OH)2D3, there is no osteocalcin secretion nor any effect of cell depolarization on this activity. The depolarization-induced increase in 1,25(OH)2D3-dependent osteocalcin secretion was totally inhibited in the presence of 10 microM Nitrendipine (a Ca2+ channel blocker, p < 0.005) without affecting cellular alkaline phosphatase nor cell growth. Charybdotoxin, a selective blocker of Ca2+-dependent K+ channels (maxi-K) present in MG-63 cells, stimulated 1,25(OH)2D3-induced osteocalcin synthesis about 2-fold (p < 0.005) after either 30, 60, or 120 minutes of treatment. However, Charybdotoxin was without effect on basal release of osteocalcin in the absence of 1,25(OH)2D3 pretreatment. Using patch clamp technique, we occasionally observed the presence of a small conductance K+ channel, compatible with an ATP-dependent K+ channel (GK[ATP]) in nonstimulated cells, whereas multiple channel openings were observed when cells were treated with Diazoxide, a sulfonamide derivative which opens GK(ATP). Western blot analysis revealed the presence of the N-terminal peptide of GK(ATP) in MG-63 cells, and its expression was regulated with the proliferation rate of these cells, maximal detection by Western blots being observed during the logarithmic phase of the cycle. Glipizide and Glybenclamide, selective sulfonylureas which can block GK(ATP), dose-dependently enhanced 1,25(OH)2D3-induced OC secretion (p < 0.005). Reducing the extracellular calcium concentration with EGTA (microM range) totally inhibited the effect of Glipizide and Glybenclamide on osteocalcin secretion (p < 0.005), which remained at the same levels as controls. Diazoxide totally prevented the effect of these sulfonylureas. These results suggest that voltage-activated Ca2+ channels triggered via cell depolarization can enhance 1,25(OH)2D3-induced OC release by MG-63 cells. In addition, OC secretion is increased by blocking two types of K+ channels: maxi-K channels, which normally hyperpolarize cells and close Ca2+ channels, and GK(ATP) channels. The role of these channels is closely linked to the extracellular Ca2+ concentration.