Clustering proteins into families using artificial neural networks. An artificial neural network was used to cluster proteins into families. The network, composed of 7 x 7 neurons, was trained with the Kohonen unsupervised learning algorithm using, as inputs, matrix patterns derived from the bipeptide composition of 447 proteins, belonging to 13 different families. As a result of the training, and without any a priori indication of the number or composition of the expected families, the network self-organized the activation of its neurons into topologically ordered maps in which almost all the proteins (96.7%) were correctly clustered into the corresponding families. In a second computational experiment, a similar network was trained with one family of the previous learning set (76 cytochrome c sequences). The new neural map clustered these proteins into 25 different neurons (five in the first experiment), wherein phylogenetically related sequences were positioned close to each other. This result shows that the network can adapt the clustering resolution to the complexity of the learning set, a useful feature when working with an unknown number of clusters. Although the learning stage is time consuming, once the topological map is obtained, the classification of new proteins is very fast. Altogether, our results suggest that this novel approach may be a useful tool to organize the search for homologies in large macromolecular databases.


Common misconceptions in neurophysiology. An understanding of the mechanisms underlying nervous transmission is critical if we are to understand how anaesthetics work. In this review, I have concentrated on some areas of simple cellular neurophysiology that students often find puzzling: the relation between ionic concentration and membrane potential; how axonal and synaptic transmission differ; and presynaptic inhibition.


Biliary cystadenocarcinoma of the liver. A clinicopathologic and histochemical evaluation of nine cases. Nine cases of biliary cystadenocarcinoma of the liver were studied, with emphasis on its clinicopathologic features, mucin profiles, and immunohistochemical characteristics. In general, the cystic tumors had protrusions that consisted of well-differentiated papillary adenocarcinoma cells with or without benign-appearing epithelial elements. In invading or metastatic foci, the carcinoma cells tended to show distinctive anaplastic changes. Tumor growth was confined to the cystic lesions in five cases (noninvasive type), whereas in four cases it extended to the hepatic parenchyma or neighboring organs (invasive type). There was a considerable difference between the two groups in terms of prognosis. In fact, the patients included in the group with the noninvasive type had no sign of tumor recurrence after an appropriate surgical procedure. With mucin histochemical and immunohistochemical approaches, positive reactions with carcinoembryonic antigen, tissue polypeptide antigen, carbohydrate 19-9, and Dupan-2 and the predominance of sialomucin were observed in most cases of biliary cystadenocarcinoma, indicating a similar cellular nature of cholangiocarcinoma.


An analysis of proliferating cells in biopsy specimens from patients with small hepatocellular carcinoma. The proliferation of neoplastic and nonneoplastic hepatocytes is caused by various humoral growth factors with autocrine and paracrine mechanisms, and the proliferative activity of both hepatocytes and nonhepatocytic cells contributes to neoplastic growth. The authors attempted to detect various kinds of proliferating cells immunohistochemically in small hepatocellular carcinoma (HCC) using a monoclonal antibody against DNA polymerase alpha. Most of the HCC cells that stained for this enzyme were small, had basophilic cytoplasm with poorly developed organelles, and aggregated to form clusters distributed randomly within cancer nests. Nonhepatocytic cells also were stained, including some endothelial cells, Kupffer's cells, macrophages, and lymphocytes. Fat-storing cells were not stained. The number of stained sinusoidal (capillary) cells decreased in this order: Kupffer's cells and macrophages, endothelial cells, and fat-storing cells. Nonhepatocytic cells, including lymphocytes, proliferated more actively in areas with actively growing HCC cells than in those with quiescent cancer cells. The relationship between stained HCC cells and stained sinusoidal cells was clearly defined; the correlation coefficient was 0.97. These findings suggest the possibility of a relationship between the proliferative activity of neoplastic hepatocytes and that of sinusoidal cells, including lymphocytes.


Giant cell tumor-like cholangiocarcinoma associated with systemic cholelithiasis. A cholangiocarcinoma of the hepatic hilus with an element of giant cell tumor that occurred in a 59-year-old man is reported. His medical history included systemic cholelithiasis and repeated operations on the biliary passages. Four years after the last operation, which was a hepatic segmentectomy, he was readmitted because of persistent fever. A computed tomography scan showed a low-density area and stones in the hepatic hilus. He died of hepatic failure approximately 1 month later. At autopsy, a fist-sized tumor and gallstones were found at the hepatic hilus. Histologically, the tumor mainly showed sarcomatoid features and some tubular adenocarcinoma. An element of giant cell tumor consisting of many osteoclast-type giant cells also was noted. The results of immunohistochemical studies showed a positive reaction to cytokeratin and vimentin in some of the spindle-shaped sarcomatoid cells. Sarcomatoid bile duct carcinomas are rare, as are those with osteoclast-type giant cells. The authors also discuss the histogenesis of these giant cells.


Absence of correlation between nuclear morphometry and survival in stage I non-small cell lung carcinoma. To evaluate the utility of nuclear morphometry as a prognostic indicator in lung cancer, 5-year follow-up information was obtained in 46 cases of surgically resected Stage I non-small cell lung cancer (NSCLC). Nuclear area, perimeter, major diameter, minor diameter, and nuclear shape factor were determined from representative histologic sections of the tumors with a computer-assisted digitizing system. The morphometric parameters were compared between patients with favorable outcome (Group I: alive with no evidence of disease, n = 17) and those with poor outcome (Group II: dead of disease or with recurrence of disease, n = 29). No significant differences in any of the morphometric parameters were found between tumors in Groups I and II for individual tumor cell types or the combined cases. Failure to demonstrate a correlation between morphometric parameters and prognosis in Stage I NSCLC indicates that future efforts to determine objective prognostic factors should concentrate on other variables, such as specific genetic abnormalities.


Postoperative increase in soluble interleukin-2 receptor serum levels as predictor for early recurrence in non-small cell lung carcinoma. It is known that interleukin-2 (IL-2) plays an important role in the activation of host antitumor immune response. In addition to IL-2 cell surface receptor, a soluble form of IL-2 receptor (SIL-2R) may be released in the blood and potentially be involved in the regulation of IL-2 availability. High SIL-2R levels have been found in patients with lung cancer. The current study evaluated the influence of changes in SIL-2R serum levels during the perioperative period on early relapse rate in patients with operable non-small cell lung cancer. The study included 60 patients (epidermoid carcinoma, 33; adenocarcinoma, 27). Serum levels of SIL-2R were measured with an enzyme immunoassay before surgery and 7 and 30 days after surgery. A surgery-induced increase in SIL-2R levels was seen 7 days after surgery in 38 of 60 patients. On the 30th day after surgery, SIL-2R values were lower than the preoperative values in 32 patients (Group A) or still greater in the other 28 patients (Group B). After a median follow-up of 10 months, relapse occurred in 19 of 60 patients. The relapse rate was significantly higher in Group B than in Group A patients (16 of 28 versus 3 of 32, respectively; P less than 0.001). This difference also was significant in relation to histotype and node status. This study shows that the persistence of increased SIL-2R levels in the postoperative period is associated with a higher early relapse rate in patients with operable non-small cell lung cancer. The impact of SIL-2R levels on relapse suggests that host immune defenses may influence the clinical course of patients with lung cancer. Therefore, the evaluation of SIL-2R in the perioperative period may represent a new prognostic biologic factor in operable non-small cell lung cancer.


Prognostic importance of human papillomavirus type 16 DNA in cervical cancer. Human papillomavirus (HPV) type 16 DNA is frequent in invasive cervical cancers. Among 43 patients with invasive cervical cancer, HPV-16-positive tumors spread to the parametrial and pelvic lymph nodes significantly more often than did HPV-16-negative tumors (P less than 0.05). Demonstration of HPV-16 DNA in invasive cervical cancers may be an additional prognostic factor for this disease.


Heterogeneity among Epstein-Barr virus-seropositive donors in the generation of immunoblastic B-cell lymphomas in SCID mice receiving human peripheral blood leukocyte grafts. Epstein-Barr virus (EBV) is associated with B-cell malignancy in immunosuppressed humans and SCID mice receiving human peripheral blood leukocyte grafts (hu-PBL-SCID). We have further characterized the process of lymphoma development in hu-PBL-SCID mice. We report that EBV-seropositive donors differ markedly in the capacity of their PBL to give rise to immunoblastic lymphomas in SCID mice; some donors (high incidence) generated tumors rapidly in all hu-PBL-SCID mice, other donors (intermediate-low incidence) gave rise to sporadic tumors after a longer latent period (greater than 10 weeks), and some donors failed to produce tumors. B-cell lymphomas arising from high incidence donors were multiclonal in origin, and EBV replication was detected in all tumors. Tumors derived from intermediate-low incidence donors were monoclonal or oligoclonal and often had no evidence of viral replication. All tumors, regardless of the donor, resembled EBV-transformed lymphoblastoid cell lines in surface phenotype but differed from lymphoblastoid cell lines by having less Epstein-Barr nuclear antigen 2 and CD23 expression. The variable patterns of lymphomagenesis seen among different EBV-sero-positive donors may be explained by lower levels of specific immunity to EBV in high incidence donors, permitting activation of EBV replication and potential transformation of secondary B-cell targets. In addition, there may be differences in the transforming potential of EBV infecting different donors. The use of the hu-PBL-SCID model may help predict patients at high risk for posttransplant or acquired immunodeficiency syndrome-associated lymphomas.


Allelotype of non-small cell lung carcinoma--comparison between loss of heterozygosity in squamous cell carcinoma and adenocarcinoma. We examined loss of heterozygosity (LOH) on all autosomal chromosomes in 53 non-small cell lung carcinomas. Frequent LOH was observed on the long arms of chromosomes 1 (37%), 2 (31%), 5 (30%), 8 (31%), and 13 (32%), and the short arms of chromosomes 3 (54%) and 17 (62%). LOH on chromosomes 3p and 17p was observed in all informative cases of squamous cell carcinoma, but was significantly less frequent in adenocarcinomas (P = 0.003 and 0.001, respectively). Similarly, LOH on chromosome 13q was observed frequently in squamous cell carcinomas (5 of 9 informative cases, or 56%), but in only 5 of 26, or 19%, of adenocarcinomas. In contrast, LOH on chromosome 2q was observed only in adenocarcinomas. In addition, this chromosomal arm was lost more frequently in poorly differentiated, compared to well differentiated adenocarcinomas. Furthermore, a correlation between fractional allelic loss and pathohistological grade was identified. These results implicate the presence of several tumor suppressor genes associated with development and/or progression of non-small cell lung carcinomas.


Unhydrolyzable analogues of adenosine 3':5'-monophosphate demonstrating growth inhibition and differentiation in human cancer cells. A set of adenosine 3':5'-monophosphate (cAMP) analogues that combine exocyclic sulfur substitutions in the equatorial (Rp) or the axial (Sp) position of the cyclophosphate ring with modifications in the adenine base of cAMP were tested for their effect on the growth of HL-60 human promyelocytic leukemia cells and LS-174T human colon carcinoma cells. Both diasteromeres of the phosphorothioate derivatives were growth inhibitory, exhibiting a concentration inhibiting 50% of cell proliferation of 3-100 microM. Among the analogues tested, Rp-8-Cl-cAMPS and Sp-8-Br-cAMPS were the two most potent. Rp-8-Cl-cAMPS was 5- to 10-fold less potent than 8-Cl-cAMP while Sp-8-Br-cAMPS was approximately 6-fold more potent than 8-Br-cAMP. The growth inhibition was not due to a block in a specific phase of the cell cycle or due to cytotoxicity. Rp-8-Cl-cAMPS enhanced its growth-inhibitory effect when added together with 8-Cl-cAMP and increased differentiation in combination with N6-benzyl-cAMP. The binding kinetics data showed that these Sp and Rp modifications brought about a greater decrease in affinity for Site B than for Site A of RI (the regulatory subunit of type I cAMP-dependent protein kinase) and a substantial decrease of affinity for Site A of RII (the regulatory subunit of type II protein kinase) but only a small decrease in affinity for Site B of RII, indicating the importance of the Site B binding of RII in the growth-inhibitory effect. These results show that the phosphorothioate analogues of cAMP are useful tools to investigate the mechanism of action of cAMP in growth control and differentiation and may have practical implication in the suppression of malignancy.


Selective loss of glucocorticoid-dependent responses in a variant of the DDT1MF-2 tumor cell line. Glucocorticoid treatment dramatically inhibits growth of the wild-type DDT1MF-2 hamster smooth muscle tumor cell line (DDT-WT) but not that of a glucocorticoid-selected clonal variant (DDT-GR). Our objective was to further define the level of glucocorticoid resistance in DDT-GR cells. Glucocorticoid receptors were confirmed to be less abundant in DDT-GR cells, but the immunoreactivity and molecular dimensions of the receptor and the ability of the receptor to undergo ligand-dependent nuclear accumulation was the same as that in DDT-WT cells. Glucocorticoid treatment also stimulated expression of the beta 2-adrenergic receptor gene to the same extent (approximately 2-fold at the mRNA and membrane protein level) in both cell lines. With the exception of the previously identified p29 protein, the pattern of detectably altered protein synthesis during glucocorticoid treatment was identical in both cell lines. All of the above responses that were shared by DDT-WT and DDT-GR cells as well as growth inhibition and p29 induction which are restricted to the DDT-WT cell could be blocked by the antiglucocorticoid, 17 beta-hydroxy-11 beta-[4-(dimethylamino)phenyl]-17 alpha-propynylestra- 4,9-dien-3-one. Together, these data indicate that DDT-GR cells contain enough functional glucocorticoid receptors to successfully regulate most of the normally responsive genes. Exploitation of this fact should greatly facilitate efforts to identify and study the function of those genes that are specifically involved in the antiproliferative action of glucocorticoid on the DDT-WT cell.


Interaction of an alpha-melanocyte-stimulating hormone-diphtheria toxin fusion protein with melanotropin receptors in human melanoma metastases. A hybrid toxin targeted to melanotropin receptors and selectively cytotoxic to melanoma cell lines in vitro has recently been developed. The toxin, a recombinant fusion protein (designated DAB389-MSH), contains the peptide sequences of alpha-melanocyte-stimulating hormone (alpha-MSH) and the catalytic (cytotoxic; Fragment A) and lipophilic (part of Fragment B) domains of diphtheria toxin. In the present study, binding of DAB389-MSH to melanotropin receptors in biopsy specimens of human and mouse melanoma metastases was assessed by measuring its ability to inhibit binding of a radiolabeled, superpotent analogue of alpha-MSH (125I-[Nle4,D-Phe7]-alpha-MSH; 125I-NDP-MSH) and comparing its potency in this system with those of the established ligands NDP-MSH and alpha-MSH. Radioligand binding to tissue sections in vitro was localized and quantified by autoradiography and image analysis. DAB389-MSH inhibited binding of 125I-NDP-MSH to experimental murine B16-F1C23 melanoma metastasis tissue and to melanoma metastases of three patients. In both mouse and human melanoma tissues, concentration-response relationships for DAB389-MSH-mediated inhibition of 125I-NDP-MSH binding were parallel, and its maximal effects were comparable in magnitude, to those of NDP-MSH and alpha-MSH. Half-maximal peptide concentrations for inhibition of 125I-NDP-MSH binding to mouse melanoma tissue sections were: NDP-MSH, 0.63 nM; alpha-MSH, 3.14 nM; and DAB389-MSH, 10.1 nM. In human melanoma tissues, the respective half-maximal peptide concentrations for inhibition of 125I-NDP-MSH binding to mouse melanoma tissue sections were: NDP-MSH, 1.80 nM; alpha-MSH, 2.43 nM; and DAB389-MSH, 11.9 nM. Taken together, these results suggest that NDP-MSH, alpha-MSH, and DAB389-MSH bind to a common melanotropin receptor in human metastatic melanoma cells. Since previous work has shown that melanotropin receptors are detectable in melanoma metastases of about 80% of human patients, malignant melanoma cells of many patients may be susceptible to killing by the melanotropin receptor-targeted cytotoxin DAB389-MSH.


Phosphatase inhibitors modulate the growth-regulatory effects of human tumor necrosis factor on tumor and normal cells. Tumor necrosis factor alpha (TNF-alpha) has been shown to inhibit the growth of tumor cells and stimulate the growth of certain normal cells in vitro. The mechanism by which TNF exerts its cell growth-regulatory effects is not known. In this report, we investigated the effects of phosphatase inhibitors on the cell growth-inhibitory effects of TNF on L-929, a highly sensitive murine connective tissue tumor cell line, and on the growth-stimulatory effects of TNF on normal human fibroblasts. The antiproliferative effects of TNF on L-929 cells were inhibited by orthovanadate, an inhibitor of phosphotyrosine phosphatases, in a dose-dependent manner. Okadaic acid, which is a specific inhibitor of phosphoserine- and phosphothreonine-specific phosphatases, also blocked the growth-inhibitory effects of TNF, suggesting that TNF may function through the activation of certain phosphatases. These inhibitors had no effect on TNF receptors. Addition of phosphatase inhibitor, even 12 h after the treatment of cells with TNF, was sufficient to block the antiproliferative effects of the cytokine, suggesting that the inhibitor is acting at a late event in the pathway of action of TNF. Cells were protected by orthovanadate from the cytotoxic effects of TNF even in the presence of actinomycin D or cycloheximide, thus indicating the lack of a requirement for de novo protein synthesis. Orthovanadate altered the cell morphology from flat spindle shapes to rounded ones. Besides anticellular effects, a phosphatase inhibitor also suppressed the proliferative effects of TNF on human fibroblasts. These results thus suggest that phosphatases may be needed for both proliferative and antiproliferative effects of this cytokine. This is the first report to suggest that phosphatases play a role in the growth-regulatory action of TNF.


Nephroblastomas in the Japanese eel, Anguilla japonica Temminck and Schlegel. Nephroblastomas were observed in 50 Japanese eel reared at a farm for 5 to 9 months from 1989 after collection in the wild. The tumors, arising from the trunk kidney near the anus, were noted externally as abdominal swellings and varied in size from 30 to 75 mm in maximum diameter. Most were elastic, solid, and well encapsulated. Histologically, the nephroblastomas were composed of combinations of three main tissue elements. Spindle- or oval-shaped cells resembling human blastema cells were observed in most tumors to some larger or smaller degree. Although variation was evident from tumor to tumor, and even within the same tumor, the most common histological type was epithelial with formation of alveolar nests, the cells sometimes being arranged in tubular structures simulating normal renal tubules. A muscle tissue element with distinct cross-striations was also observed. Liver metastases were found in one case. Histological examination of apparently normal kidneys from 100 eels revealed one early-stage nephroblastoma. The cause of these tumors is unclear, although they have been discovered with increasing incidence after the spread of indoor eel culture with raised water temperatures (26-27 degrees C) in Japan. Environmental factor(s) associated with the new aquaculture method may thus play a role in their genesis.


Frequent loss of expression of the potential tumor suppressor gene DCC in ductal pancreatic adenocarcinoma. The development of colon carcinomas is associated with allelic deletions on chromosomes 5q, 17p, and 18q. The DCC gene located on chromosome 18q21.3 codes for a potential tumor suppressor gene related to cellular adhesion receptors. We investigated the expression of this gene in several pancreatic carcinoma cell lines and in patients with ductal adenocarcinomas of the pancreas. In 8 of 11 cell lines and in 4 of 8 primary tumors a complete extinction of DCC gene expression was observed, whereas the c-Ki-ras gene was mutated at codon 12 in 7 of 8 tumors. A highly reduced or absent expression of DCC was found in all low or undifferentiated pancreatic tumor cell lines, whereas in the more differentiated ones DCC expression was conserved. These data suggest that loss of DCC gene expression is an important factor in the development or progress of pancreatic adenocarcinoma and may be linked to the differentiated phenotype of the pancreatic tumor cell.


Partial defluoridation of drinking water using fluorapatite precipitation. Ion adsorption and ion exchange are two methods commonly used in small home units to treat drinking water to bring the fluoride concentration to within acceptable limits. However, the necessary flowthrough system is often difficult to arrange where there is no piped supply and gradual exhaustion of the active agent is not easily detected. In an attempt to overcome these problems a defluoridation method based on the precipitation of a sparingly soluble fluoride salt, fluorapatite, has been studied. Samples of simulated high-fluoride drinking waters, approximately 10 ppm F, were saturated with brushite, resulting in a state of supersaturation with respect to fluorapatite. Subsequent seeding with hydroxyapatite caused a lowering of the calcium, phosphate, and fluoride concentrations in solution, indicative of fluorapatite precipitation. Repeating the process had an additive effect. Bone char was a less effective seed than hydroxyapatite with water containing fluoride only, but was a more effective seed with simulated Kenyan borehole water containing additional salts. Sixty-minute brushite saturation and apatite seeding steps were generally more effective than 10-min steps. The results suggest that apatite coprecipitation may be a convenient low-technology way to defluoridate drinking water, although prior testing might be useful to ensure adequate removal of fluoride.


Epidermal growth factor activates calcium channels by phospholipase A2/5-lipoxygenase-mediated leukotriene C4 production. Epidermal growth factor (EGF) induces a Ca2+ influx in many cell types, but the underlying mechanisms are so far unresolved. We report that: EGF-induced Ca2+ channel activity is eliminated by lipoxygenase inhibition and is mimicked by artificial induction of lipoxygenase activity; addition of leukotriene C4 can fully mimic EGF in its ability to activate Ca2+ channels; and EGF induces a rapid accumulation of intracellular leukotriene C4. In addition, we show that EGF-induced, Ca(2+)-dependent membrane hyperpolarization and junB proto-oncogene expression are dependent on lipoxygenase activity, whereas EGF-induced cytoplasmic alkalinization is not. We conclude that PLA2/5-lipoxygenase-mediated leukotriene C4 production constitutes a novel and specific signal transduction pathway in growth factor action.


Disruption of the low affinity receptor-binding site in NGF allows neuronal survival and differentiation by binding to the trk gene product. Nerve growth factor (NGF), like many other growth factors and hormones, binds to two different receptor molecules on responsive cells. The product of the proto-oncogene trk, p140trk, is a tyrosine kinase receptor that has been identified as a signal-transducing receptor for NGF, while the role of the low affinity NGF receptor, p75NGFR, in signal transduction is less clear. The crystal structure of NGF has recently been determined, although structures involved in receptor binding and biological activity are unknown. Here we show that Lys-32, Lys-34, and Lys-95 form a positively charged interface involved in binding to p75NGFR. Simultaneous modification of Lys-32 with either of the two other lysines resulted in loss of binding to p75NGFR. Despite the lack of binding to p75NGFR, these mutants retained binding to p140trk and biological activity, demonstrating a functional dissociation between the two NGF receptors.


The identification of 18 nuclear genes required for the expression of the yeast mitochondrial gene encoding cytochrome c oxidase subunit 1. Eighteen nuclear mutants of the yeast Saccharomyces cerevisae, each disturbed in the biosynthesis of the mitochondrially encoded cytochrome c oxidase subunit 1 (cox1) and each representing a distinct complementation group, have been examined to identify the level at which COX1 expression is affected. RNA blotting revealed that most have a defect in the processing of COX1 precursor-mRNA; only a few are defective in COX1 transcription and/or pre-mRNA stability. In most RNA-processing mutants, the absence of the COX1 messenger results from a defect in the splicing of one or more COX1 introns. In turn, this defect can be ascribed to a mutation in a nuclear gene which is either directly involved in splicing or else acts indirectly by impairing COX1 translation.


The unusual reversion properties of a mitochondrial mutation in the structural gene of subunit I of cytochrome oxidase of Saccharomyces cerevisiae reveal a probable histidine ligand of the redox center. We have analyzed a mutation in the mitochondrial gene oxi3 coding for subunit I of cytochrome-oxidase in the yeast Saccharomyces cerevisiae. This mutation replaces one of the seven invariant histidines of the polypeptide (position 378) by a tyrosine, and leads to a respiratory deficient phenotype. A total of 157 revertants, which have recovered the ability to grow on a respiratory substrate, have been selected from this mutant (tyrosine 378). The nature of the reversion has been analysed by a rapid screening procedure and 32 of the revertants have been sequenced. They are all true back-mutations reintroducing the histidine in position 378. This very exceptional situation suggests that this histidine is a ligand of the redox center of cytochrome oxidase.


Linear, non-mitochondrial plasmids of Alternaria alternata. Three plasmids, with sizes of 7.0 kbp, 6.8 kbp, and 5.0 kbp and designated pAal-1, pAal-2 and pAal-3 respectively, have been found in a tentoxin-producing isolate of Alternaria alternata. Exonuclease digestions show these plasmids to be linear with blocked 5' ends. Plasmid pAal-1 does not hybridize to nuclear DNA, mitochondrial DNA, or double-stranded RNA from a mycovirus found in the isolate, but does hybridize weakly to a series of linear DNAs which are not visible on gels and may include pAal-2 and pAal-3. Cellular fractionation shows that, unlike other linear fungal plasmids, these plasmids are not localized in the mitochondria. Plasmids have not been found in other tentoxin-producing isolates and there is no evidence that these plasmids have any effect on the production of tentoxin.


Melittin-induced alterations in dynamic properties of human red blood cell membranes. The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10(-10) mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10(-10) mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10(-10) mol/mg (10(-4) mol melittin per mole of membrane phospholipid). The decrease in the ratio hw/hs of maleimide and the rise in relative rotational correlation time (tau c) of iodacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.


The present and future course of the AIDS epidemic in Côte d'Ivoire. An assessment of the current and future mortality and morbidity from acquired immunodeficiency syndrome (AIDS) in Côte d'Ivoire was made using the results of the 1989 national survey of the prevalence of human immunodeficiency (HIV) infection in the country and the AIDS projection model developed by WHO. For 1989 it was estimated that about 25,000 AIDS cases in adults and children had occurred, although the total number of cases reported for 1989 (up to 1 July 1991) was about 13% (1:6.9) of this estimated total. It is projected that by 1994 in Côte d'Ivoire the cumulative number of cases of AIDS in adults will be 89,000, and that for infants and children the corresponding number will be 41,000. It was also projected that about 371,000 uninfected children will have been born to HIV-infected mothers in Côte d'Ivoire by 1994 and that many of these children will have been orphaned by the deaths of their mothers from AIDS.


The Indian langur: preliminary report of a new nonhuman primate host for visceral leishmaniasis. Described are the susceptibility of the Indian langur (Presbytis entellus) to Leishmania donovani and the consequent haematological and serum biochemical changes. The host response to antileishmanial chemotherapy and the immunological profile were also examined. Each langur was inoculated intravenously with 1 x 10(8) amastigotes; a spleen biopsy carried out on day 35 post-infection (p.i.) revealed 10-13 L. donovani bodies per 500 cell nuclei, which reached a maximum of 130-195 at death (day 105-110 p.i.). The infected monkeys lost body weight, developed severe anaemia, lymphocytosis, hyperproteinaemia, hypergammaglobulinaemia, hypoalbuminaemia and an increase in the level of alkaline phosphatase and alanine aminotransferase (AAT). Treatment with sodium stibogluconate (60 mg Sb5+ per kg body weight intramuscularly for 10 days) reduced the number of spleen parasites (0-1 amastigotes per 500 cell nuclei) but after the therapy the parasites appeared in the skin, which had previously been free of infection. Relapse occurred on day 30 post-treatment (10-24 amastigotes per 500 cell nuclei) and the parasites were resistant to repeat intensive therapy (120 mg Sb5+ per kg per day x 30 days). The stibogluconate treatment caused a proportionate reduction in the haematological and biochemical parameters to normal values except for alkaline phosphatase and AAT, which remained elevated. The level of IgG antibodies, which rose during the infection, rapidly fell to the pretreatment value following the first therapeutic schedule and then increased a second time coinciding with relapse. Our findings suggest that langurs could serve as acceptable models for human visceral leishmaniasis.


Poliomyelitis case confirmation: characteristics for use by national eradication programmes. Highly sensitive case definitions were first introduced by national poliomyelitis eradication programmes to avoid missing true cases of the disease, though false-positive diagnostic errors could still occur owing to low specificity. Extensive data from all 1620 cases of acute, flaccid paralysis reported in Brazil during 1987-88 provided an opportunity to study the characteristics of confirmed poliomyelitis cases and epidemiologically to evaluate potential case definitions that maximized both sensitivity and specificity. Cases that had been confirmed by wild poliomyelitis virus isolation were compared with those that had been rejected (non-polio cases). To guarantee the consistency of clinical, epidemiological and laboratory investigations, only cases less than 10 years of age that had been investigated within 15 days of the onset and with complete laboratory specimens were included. No single practical case definition combining both high sensitivity and high specificity emerged from the study. However, the results showed that poliomyelitis endemic countries with limited resources should give priority to the investigation of cases in less than 5-year-olds, cases with prodromal fever, cases without involvement in all four limbs, cases without progression greater than 3 days after the onset, and cases occurring in areas where poliomyelitis had recently been confirmed. In countries without laboratory resources, cases of acute, flaccid paralysis with initial involvement in one or both lower limbs and residual neurological sequelae at 60 days should be confirmed. Countries that are close to eradication may selectively reject any cases lacking laboratory confirmation, despite adequate specimen collection, if they do not have initial involvement in one or both lower limbs and residual neurological sequelae at 60 days.


Early immunization of neonates with trivalent oral poliovirus vaccine. Described is the evaluation in Brazil of the immune response of early immunization with trivalent oral poliovirus vaccine (TOPV). A total of 85 normal neonates from São Paulo were assigned one of the following immunization schedules: group A--one dose of TOPV at birth and subsequent doses at 2, 4, and 9 months of age; or group B--one dose of TOPV at 2, 4 and 6 months of age. Blood samples were collected sequentially from the mother at delivery, from the umbilical cord, and from the child at 2, 4, 6, 9 and 12 months of age for assay of poliovirus neutralizing antibodies. Administration of TOPV at birth, in addition to establishing immunity against poliomyelitis at an earlier stage, produced a superior immune response to poliovirus type 3. At the end of the first year, the proportion of susceptible individuals was 3.7% in group A and 25.9% in group B. When immunization against poliomyelitis is started at birth, excellent seroconversion rates are obtained from the third dose onward.


Cisplatin, doxorubicin, cyclophosphamide, and etoposide combination chemotherapy for small-cell lung cancer. Because of potential synergistic interactions, we added 25 mg/m2 i.v. cisplatin (P) 25 given on days 1-5 to the combination of 45 mg/m2 i.v. doxorubicin (A) given on day 1, 800 mg/m2 i.v. cyclophosphamide (C) given on day 1, and 50 mg/m2 i.v. etoposide (E) given on days 1-5. The resulting PACE regimen was given every 21 days for the first three courses and then every 28 days for the next five courses. PACE was used in two trials: the first, for both limited and extensive disease, was conducted at the University of Maryland Cancer Center and North Shore University Hospital; and the second, for extensive disease, was carried out as a Cancer and Leukemia Group B pilot study. Chest irradiation was not used. Prophylactic cranial irradiation at a dose of 3,000 cGy was given to all patients achieving a complete response (CR). A total of 33 subjects were entered in the first study; 8 of the 15 (53%) presenting with limited disease and 7 of the 18 (39%) exhibiting extensive disease achieved a CR. A partial response (PR) was obtained in 27% and 33% of cases, respectively. Of the 34 patients entered in the second study, 25 were eligible; 8 (32%) achieved a CR and 6 (24%) showed a PR. Toxicity was severe in both studies, including greater than 90% severe or life-threatening leukopenia and thrombocytopenia. Serial creatinine-clearance evaluations in the first study indicated progressive deterioration, which required discontinuation of the cisplatin before the planned completion of treatment in most cases. Since the response rate was no higher than the historic data reported for the three-drug ACE combination and because the toxicity was severe, the studies were stopped and patients were followed for survival. After a follow-up period of greater than 6 years, the median survival was 24 months for limited disease, with 33% and 27% of the patients being alive at 3 and 6.5 years, respectively. The median survival for extensive disease was 15 and 11 months in the first and second studies, respectively. These pilot studies suggest that the addition of cisplatin may augment the activity of the ACE regimen, but at the cost of severe toxicity. Further studies seem warranted if the myelotoxicity can be better controlled.


The reversal of cisplatin-protein interactions by the modulating agent WR2721 and its metabolites WR1065 and WR33278. The reversibility of cisplatin-protein interactions by the modulating agent WR2721, its active thiol-metabolite WR1065, and the symmetrical disulfide WR33278 was studied using the model compounds (Pt(diethylenetriammine) monofunctionally bound to the sulfur in glutathione (Pt(dien)SG) and Pt(diethylenetriammine) monofunctionally bound to the sulfur in S-methylglutathione (Pt(dien)SMeG). Both model compounds could be quantified by high-performance liquid chromatography (HPLC) with UV detection. The Pt-cysteine-like bond in Pt(dien)SG could not be reversed by any of the WR compounds or by the strong nucleophiles thiosulfate (TS) and diethyldithiocarbamate (DDTC). However, the Pt-methionine-like bond in Pt(dien)SMeG could be reversed by WR1065, although the reversal was slow (k2 = 0.142 M-1 s-1) as compared with that obtained using the modulating agents TS (k2 = 10.1 M-1 s-1) and DDTC (k2 = 3.66 M-1 s-1). WR2721 was hardly able to reverse the Pt-S bond in Pt(dien)SMeG (k2 = 0.00529 M-1 s-1), and WR33278 showed no capacity to do so. The activity of cis-diamminedichloroplatinum(II) (CDDP)-inactivated fumarase was not appreciably restored by any of the WR compounds (16%, 7.7%, and 0 for 20 mM WR1065, WR2721, and WR33278, respectively) in contrast to the strong nucleophile DDTC (61% for 2 mM DDTC). These in vitro studies provide information at the molecular level that may explain why WR2721, in contrast to DDTC, does not provide protection against cisplatin-induced nephrotoxicity when it is given after platinum-containing chemotherapy. The results support the present clinical use of WR2721 prior to the administration of platinum compounds.


Specific receptor-guanine nucleotide binding protein interaction mediates the release of endothelium-derived relaxing factor. High affinity agonist-binding (HAB) sites are formed from specific receptor interaction with guanine nucleotide-binding (Gi) proteins. To determine whether the release of endothelium-derived relaxing factor (EDRF) is regulated by specific receptor-Gi protein coupling, we treated bovine aortic endothelial cells with 100 ng/ml pertussis toxin (PTX) for 16 hours to effect receptor-Gi protein uncoupling. The degree of receptor uncoupling as measured by the loss of HAB sites for the alpha 2-adrenergic receptor and bradykinin receptor was assessed by radioligand binding studies using partially purified bovine aortic endothelial cell membranes. The release of EDRF in response to UK14304 (an alpha 2-adrenergic receptor agonist) and bradykinin stimulation was measured with a bioassay apparatus. The Gi protein isoforms were characterized by Western blotting, and complete ADP-ribosylation of these proteins was confirmed by PTX-catalyzed [32P]NAD ribosylation. PTX produced a greater inhibition of EDRF release via the alpha 2-adrenergic receptor pathway compared with the bradykinin receptor pathway (80% versus 46%, p less than 0.01). This corresponded to the loss of HAB sites from the alpha 2-adrenergic receptor and bradykinin receptor pathway (72% versus 46%, p less than 0.01) as compared with complete loss of both HAB sites in the presence of GppNHp (0.1 mM). Since loss of HAB sites from PTX-mediated receptor uncoupling parallels the inhibition of EDRF release, these data suggest that Gi proteins contribute to a greater proportion of HAB sites derived from alpha 2-adrenergic receptor rather than bradykinin receptor interaction and that the inhibition of EDRF release by PTX is mainly due to the loss of these HAB sites.(ABSTRACT TRUNCATED AT 250 WORDS)


Heparin inhibits collagenase gene expression mediated by phorbol ester-responsive element in primate arterial smooth muscle cells. Heparin is a potent inhibitor of arterial smooth muscle cell (SMC) migration and proliferation in vivo and in vitro. We propose that heparin affects these SMC functions by interfering with either the expression or the activity of secreted proteases required for cell movement. We have reported that heparin selectively inhibits the expression of tissue-type plasminogen activator in SMCs during mitogenesis. In this study we show that the gene expression of another kind of protease, interstitial collagenase, is induced by fetal bovine serum and is also suppressed by heparin. The inhibitory effect on the induced collagenase mRNA is specific to heparin-like molecules and does not depend on the anticoagulant activity of heparin. The induction of the collagenase gene depends on the protein kinase C pathway, since it can be induced by phorbol esters such as phorbol 12-myristate 13-acetate and blocked by inhibitors such as H-7 and staurosporine. In transient transfection assays with chloramphenicol acetyltransferase constructs containing the phorbol ester-responsive element introduced into baboon SMCs, heparin inhibits transcription induced by serum or phorbol 12-myristate 13-acetate. These results support the conclusion that, in primate SMCs, interstitial collagenase gene transcription mediated by the phorbol ester-responsive element is blocked by heparin.


Exacerbation of left ventricular ischemic diastolic dysfunction by pressure-overload hypertrophy. Modification by specific inhibition of cardiac angiotensin converting enzyme. Hearts with compensatory pressure-overload hypertrophy show an increased intracardiac activation of angiotensin II that may contribute to ischemic diastolic dysfunction. We studied whether pressure-overload hypertrophy in response to aortic banding would result in exaggerated diastolic dysfunction during low-flow ischemia and whether the specific inhibition of the cardiac angiotensin converting enzyme by enalaprilat would modify systolic and diastolic function during ischemia and reperfusion in either hypertrophied or nonhypertrophied hearts. Isolated, red blood cell-perfused isovolumic nonhypertrophied and hypertrophied rat hearts were subjected to enalaprilat (2.5 x 10(-7) M final concentration) infusion during 20 minutes of baseline perfusion and during 30 minutes of low-flow ischemia and 30 minutes of reperfusion. Coronary flow per gram was similar in nonhypertrophied and hypertrophied hearts during baseline perfusion, ischemia, and reperfusion. At baseline, left ventricular developed pressure was higher in hypertrophied than nonhypertrophied hearts in untreated groups (224 +/- 8 versus 150 +/- 9 mm Hg; p less than 0.01) and in enalaprilat-treated groups (223 +/- 9 versus 145 +/- 8 mm Hg; p less than 0.01). During low-flow ischemia, left ventricular developed pressure was depressed but similar in all groups. All groups showed deterioration of diastolic function; however, left ventricular end-diastolic pressure increased to a significantly higher level in untreated hypertrophied than in nonhypertrophied hearts (65 +/- 7 versus 33 +/- 3 mm Hg; p less than 0.001). Enalaprilat had no effect in nonhypertrophied hearts, but it significantly attenuated the greater increase in left ventricular end-diastolic pressure in hypertrophied hearts treated with enalaprilat compared with no drug (65 +/- 7 versus 50 +/- 5 mm Hg; p less than 0.01). The beneficial effect could not be explained by differences in coronary blood flow per gram left ventricular weight, glycolytic flux as reported by lactate production, myocardial water content, oxygen consumption, and tissue levels of glycogen and high energy phosphate compounds. During reperfusion, all hearts showed a partial recovery of developed pressure to 70-74% of initial values. No effect of enalaprilat could be detected during reperfusion on systolic and diastolic function or restoration of tissue levels of high energy compounds. In conclusion, our experiments show that hypertrophied red blood cell-perfused hearts manifest a severe impairment of left ventricular diastolic relaxation in response to low-flow ischemia in comparison with control hearts. Further, our experiments support the hypothesis that the enhanced conversion of angiotensin I to angiotensin II in rats with pressure-overload hypertrophy contributes to the enhanced sensitivity of hypertrophied hearts to diastolic dysfunction during low-flow ischemia.


Role of peptidases in bradykinin-induced increase in vascular permeability in vivo. The purpose of this study was to examine whether neutral endopeptidase and angiotensin I-converting enzyme, two membrane-bound metalloenzymes that are widely distributed in the microcirculation, play a role in bradykinin-induced increase in vascular permeability in the hamster cheek pouch. Changes in vascular permeability were quantified by counting the number of leaky sites and by calculating the clearance of fluorescein isothiocyanate (FITC)-dextran (molecular mass, 70,000 d) during suffusion of the cheek pouch with bradykinin. Bradykinin produced a concentration- and time-dependent increase in the number of leaky sites and clearance of FITC-dextran. The selective, active site-directed neutral endopeptidase inhibitors phosphoramidon (1.0 microM) and thiorphan (10.0 microM) and the selective angiotensin I-converting enzyme inhibitor captopril (10.0 microM) each shifted the concentration-response curve to bradykinin significantly to the left. During suffusion with bradykinin (1.0 microM) and phosphoramidon, the number of leaky sites increased significantly from 17 +/- 2 to 27 +/- 4 sites per 0.11 cm2 (mean +/- SEM, p less than 0.05), and FITC-dextran clearance increased significantly from 1.0 +/- 0.2 to 2.1 +/- 0.3 ml/sex x 10(-6).(ABSTRACT TRUNCATED AT 250 WORDS)


Endogenous digitalis-like factors in human milk. We measured the concentration of endogenous digitalis-like factors (EDLFs) in milk or colostrum of women during nursing on different days after delivery. EDLF concentrations were assayed by a solid-phase RIA involving antidigoxin antibodies and by a radioreceptor assay (RRA) involving human placenta Na+/K(+)-ATPase. The mean (SD) EDLF concentrations as measured by RIA were 35.6 (19.4) ng of digoxin equivalents per liter in milk samples (n = 37) and 61.3 (12.5) ng/L in colostrum samples (n = 5); the mean EDLF concentration as measured by RRA in milk samples (n = 11) was 573 (717) ng/L (range 0-2098). EDLF concentration in milk is greater than circulating concentrations in healthy adults but is comparable with serum concentration in the third trimester of pregnancy. In milk and serum samples (n = 8) collected at the same time, heating and (or) extracting with Sep-Pak C18 cartridges before the RIA produced significantly different EDLF values from those in untreated serum (P less than 0.001) and milk (P = 0.035). EDLF in milk appeared to be not bound or weakly bound to milk protein, as indicated by the fact that boiling did not increase the digoxin-like immunoreactivity.


New chromogen for assay of glucose in serum. We describe a colorimetric assay for glucose determination in human serum, with use of the chromogen 2-amino-4-hydroxybenzenesulfonic acid (AHBS), glucose oxidase, and peroxidase. With this assay, glucose concentrations less than or equal to 27.8 mmol/L can be measured in serum, with a sample/reagent volume ratio as low as 0.0025. The chromogen itself is easily soluble in water and does not require other components for the color change, making the reagent composition less complex. A single working reagent is used, and the reaction is completed within 10 min at 37 degrees C. The absorbance of the yellow reaction product is measured at 415 nm, and a blank sample measurement is not needed. The average analytical recovery of glucose in different human sera was 97.6%, with no significant interference of reducing compounds in serum. The results of the recommended procedure correlated well with those of the phenol/4-aminoantipyrine method of Trinder.


Advanced glycosylation end products in the mesenteric artery. We measured advanced glycosylation end products in the mesenteric artery of 37 patients (ages 29-82 years), 34 of whom were nondiabetic. Samples of arterial tissue were obtained during bowel resectioning. Advanced glycosylation end products were measured as collagen-linked fluorescence (excitation wavelength 370 nm, emission wavelength 440 nm) after collagenase digestion of tissue samples. Mean fluorescence of the arterial samples was 15 U/mg (range 5.3-27). Collagen fluorescence correlated with patients' age (r = 0.57; P less than 0.001). No difference in the collagen-linked fluorescence was observed between men and women (P = 0.63), hypertensive and normotensive patients (P = 0.44), smokers and nonsmokers (P = -0.52), and patients with and without symptomatic coronary heart disease (P = 0.7). This study demonstrates, for the first time, the relationship between collagen-linked fluorescence and patients' age in human arterial tissue ex vivo.


Role for cyclic adenosine monophosphate in the synergistic interaction between oxytocin and corticotrophin-releasing factor in isolated human gestational myometrium. We wished to investigate the role of cAMP in the synergistic effect of corticotrophin-releasing factor and oxytocin on human myometrial contractility. Isolated human gestational myometrium obtained from Caesarean sections at term was studied in vitro. Static incubation techniques as well as tension recordings were applied to the tissue obtained. The subjects were healthy pregnant women undergoing lower segment Caesarean section at term, prior to labour. Specimens obtained were immediately dissected into small strips and either incubated in multi-well trays (strip weight 2.75 mg) or superfused and used for tension recordings (strip weight 2.00 mg). cAMP accumulation was measured after incubation with oxytocin (0.1-10 nM), corticotrophin-releasing factor (1 nM) or a combination of both peptides. Tension generated by the muscle strips was recorded isometrically and response to oxytocin (0.01-10 nM), corticotrophin-releasing factor (1 nM) and forskolin (10 nM) expressed in force per gram wet tissue (N/g). Oxytocin (0.1 nM) causes a statistically significant dose-related decrease in cAMP when combined with 1 nM corticotrophin-releasing factor (P less than 0.001), as compared with cAMP stimulation by corticotrophin-releasing factor alone. Time course studies suggest a maximal effect at 1 minute. The hypothesis that an intracellular reduction of cAMP is a prerequisite for the synergistic response in contraction force was tested with tension recordings. Prevention of a decrease in cAMP in the tissue by addition of 10 nM forskolin to the superfusate abolished the potentiation between oxytocin and corticotrophin-releasing factor. These results indicate that a fall in cAMP concentration plays a vital mediating role in the synergistic interaction between oxytocin and corticotrophin-releasing factor.


Endocrine function in patients with Cushing's disease before and after treatment. To estimate the value of some commonly used tests in diagnosing Cushing's disease and to assess the outcome after treatment. Follow-up of a consecutive group of patients for 4.1 to 109.6 months, median 34.4. Forty-six patients assumed to have ACTH dependent hypercorticism (of 50 patients with Cushing's syndrome) were included. Forty-five underwent transsphenoidal neurosurgery. Ten were treated preoperatively with radiotherapy. Pituitary, adrenal, thyroid and gonadal function, radiology and pituitary histology were evaluated. One main finding was a significant correlation between the urinary excretion of cortisol before and during administration of dexamethasone. Thus patients with modestly elevated urinary cortisol excretion had an apparently normal suppression. The urinary cortisol values during the dexamethasone test were significantly related to the peak plasma cortisol concentrations at the 30-minute ACTH tests. Computed tomography failed to identify an adenoma in 10 of the 19 patients who were histologically proved to harbour a corticotroph adenoma. At 6 months after radiotherapy, clinical and biochemical improvement was noted in none. Cure was achieved in 36 after neurosurgery. Eventually, adrenalectomy was needed in eight patients. Sixteen patients developed persisting adrenal insufficiency after neurosurgery so that the total number of patients on permanent steroid substitution was 24. Post-operative thyroid and gonadal insufficiency (in men and women of fertile age) was found in 36 and 49%, respectively. The diagnostic value of measuring the cortisol excretion during dexamethasone administration appears doubtful. The outcome after neurosurgical treatment for Cushing's disease is not entirely satisfactory. Further studies are needed to decide whether adrenalectomy as the first line of therapy should be considered relevant in some patients with Cushing's disease.


Virus infections complicating bone marrow transplantation. Virus infections account for considerable morbidity in bone marrow transplant (BMT) recipients. In all ages, members of the herpes virus group are the predominant pathogens. Of these, cytomegalovirus is pre-eminent in being the most frequent cause of death due to infection associated with the transplant procedure. Considerable effort is being invested in the development of preventative and therapeutic strategies to control this virus. Other potentially life-threatening virus infections may be acquired on the transplant unit as a result of cross-infection and can be caused by enteroviruses, rotaviruses and adenoviruses. Prevention of these infections is best achieved by implementation of strict infection-control measures.


[Histiocyte activation syndrome associated with EBV in children]. VAHS (virus associated hemophagocytic syndrome) is a severe clinical and biological condition associated with certain menacing viral infections. It has been observed in patients with manifest immunodeficiency but also in previously apparently healthy children. Epstein-Barr virus (EBV) infection, although it is sometimes unrecognized and insidious, is one of the etiologies to be considered. Differential diagnosis with other causes of macrophage activation is a delicate task in pediatrics. Better understanding of the pathophysiology of the syndrome offers interesting therapeutic prospects.


[New therapeutic trends in AIDS].

[New therapeutic trends in AIDS]. In this paper we describe the current status of the chemotherapy of HIV-related disease, and the newly emerging approaches to this problem. Azidothymidine, the first anti-HIV drug, is now used by thousands of patients with AIDS, and is able to induce a substantial improvement of their clinical status. However, due to its toxicity and its very limited activity against HIV replication in chronically infected cells (the natural reservoir of the virus in the body), it is crucial that new drugs be developed. A number of compounds belonging to the dideoxynucleoside family (the same of AZT) have been synthesized and used in HIV-infected patients, with promising results. Nevertheless, new compounds with different mechanisms of action, and with excellent anti-HIV efficacy need to be developed, particularly those that can inhibit the late stages of HIV replication. This will permit a polychemotherapeutic approach against HIV infection that, as in the case of anticancer chemotherapy, has conceivably better chances to be effective in patients with HIV-related disease.


Hereditary flat adenoma syndrome: a variant of familial adenomatous polyposis? We describe the clinical and pathologic features in four extended kindreds that are consistent with the hereditary flat adenoma syndrome (HFAS). This colon cancer susceptibility disorder is believed to be inherited as an autosomal dominant. The principal phenotypic marker is multiple colonic adenomas (usually less than 100), with a tendency for proximal location. The majority of these adenomas are flat or slightly raised and plaquelike, as opposed to polypoid. Colon cancers have typically developed in middle age and show no unusual histologic features. There are a variety of extracolonic manifestations, including adenomas and carcinomas of the small bowel and fundic gland polyps. The HFAS is contrasted with hereditary nonpolyposis colorectal cancer and familial adenomatous polyposis (FAP) and shown to be distinct from both in the numbers and distribution of colonic adenomas and the typical age of cancer diagnosis. The clinical implications of these findings are discussed. Given its linkage to the FAP locus on 5q and the phenotypic parallels between HFAS and FAP, we conclude that HFAS is a variant of FAP.


The utility of ancillary studies in pediatric FNA cytology. We evaluated the diagnostic contribution of adjunct studies performed on aspirated material in the work-up of pediatric fine-needle aspiration (FNA) biopsies. Ancillary studies were performed on 54 of 136 (39.7%) pediatric FNA biopsies during a 5-year period. In 23 (16.9%) cases, immunocytochemical (ICC) studies, consisting of immunoperoxidase staining of direct smears and/or cell blocks or flow cytometric immunophenotyping, were performed. The studies were adequate in 14 cases (60.9%), suboptimal in five cases (21.7%), and inadequate in four cases (17.4%). Of the adequate and suboptimal cases, the ICC data helped to narrow the differential diagnosis or classify the disease process in eight cases (42.1%), confirmed cytologic impression in nine cases (47.4%), and gave contradictory results in two cases (10.5%). Adequate material for electron microscopy (EM) was obtained in 14/19 cases (73.7%). Ultrastructural studies were diagnostic, or helped classify the disease process in five cases (35.7%), confirmed the cytologic impression in four cases (28.6%), helped exclude diagnostic considerations in three cases (21.4%), and were judged to be non-contributory in two cases (14.3%). Cytogenetic studies revealed six of seven cases (all neoplasms) to have abnormal karyotypes. Special stains for organisms performed on smears from 25 cases including Ziehl-Neelsen, Gomori methenamine silver (GMS), Gram, and Warthin-Starry (WS) were negative except for 1/16 GMS and 4/9 Gram stains. In summary, we found that with appropriate case selection, ancillary studies performed on aspirated material can provide useful information in pediatric FNA cytology.


Postoperative spindle-cell nodule of urinary bladder with unusual intracytoplasmic inclusions. Postoperative spindle-cell nodule of the urinary bladder (or in the absence of previous surgical procedure, inflammatory pseudotumor) is a reactive process of unknown etiology mimicking a sarcoma. Intracytoplasmic inclusion bodies were noted inside the atypical spindle cells on Diff-Quik stained smears in a recent case of this entity. Although fluorescence was noted in the inclusion bodies with monoclonal antibodies against the major outer membrane protein of the Chlamydia species, the exact nature of these inclusion bodies remains unknown.


Peritoneal cytology of uncommon ovarian tumors. Peritoneal cytology has been well established as a diagnostic and staging tool in the management of the common epithelial tumors of ovary. Germ cell, mesenchymal, and sex-cord stromal tumors are much less frequently encountered in peritoneal specimens, often with cytologic features that may pose problems in differential diagnosis. This report presents the cytomorphology of the ascitic fluid in cases of endodermal sinus tumor, dysgerminoma, and Sertoli-Leydig-cell tumor, and peritoneal washings in a case of ovarian malignant mixed mullerian tumor. The cytologic features of Sertoli-Leydig-cell tumors have not been well described. Careful correlation of peritoneal cytologic findings, cell-block preparations, and immunocytochemistry with the cytohistologic features of these tumors is crucial for correct tumor classification.


Induction of chromosomal aberrations in bone marrow cells of asbestotic rats. In the present study, cytogenetic effects of Indian chrysotile asbestos in rat bone marrow cells after 290 days of intratracheal inoculation (5 mg dust/0.5 ml normal saline), when it develops massive pulmonary fibrosis, were investigated. The pulmonary fibrosis was confirmed by both histopathological studies and increased collagen content in the lung of the treated animals. In the asbestotic rats a significant increase in chromosomal aberrations was recorded and a decrease in mitotic index of bone marrow cells. The types of chromosomal aberrations in these cells were chromatid gaps and breaks. The results indicate the significant cytogenetic changes in the bone marrow cells of asbestotic rats and also suggest that these changes directly or indirectly may be one of the biological events involved in eliciting the asbestos-mediated toxic responses.


A structurally abnormal erythropoietin receptor gene in a human erythroleukemia cell line. Restriction endonuclease mapping demonstrates a 3' end deletion of one erythropoietin receptor (EpoR) gene in TF-1 cells, a human erythroleukemia cell line that overexpresses the EpoR and proliferates in response to erythropoietin (Epo). EpoR mRNA transcripts are highly abundant and normal in size. These findings raise interesting questions about the possible role of this EpoR gene abnormality in the pathogenesis of the erythroleukemia from which this cell line was derived. This is the first report of an abnormal human erythropoietin receptor gene.


Sodium vanadate, a tyrosine phosphatase inhibitor, affects expression of hematopoietic growth factors and extracellular matrix RNAs in SV40-transformed human marrow stromal cells. Protein tyrosine kinases represent a subset of proteins that mediate signal transduction between the extracellular environment and the nucleus. We have previously described a coordinated upregulation between RNA transcripts of a tyrosine kinase, c-abl, and those of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL-6) in human marrow stromal cells (SVMSC). Moreover, an inverse relationship exists between expression of c-abl transcripts and those of extracellular matrix proteins such as type collagen I transcripts. In the present study, these inverse relationships were again seen in SVMSC when tyrosine kinase effects were enhanced by treatment of the cells with the tyrosine phosphatase inhibitor sodium orthovanadate. This suggests that tyrosine kinases are involved in the coordinate regulation of these genes.


Protein kinases and phosphatases are involved in erythropoietin-mediated signal transduction. To study the role of protein phosphorylation in erythropoietin (EPO)-mediated signal transduction, we examined the effects of tyrosine phosphatase and tyrosine and serine-threonine kinase inhibitors as well as activators of serine kinases on DNA synthesis and cell proliferation in the murine EPO-dependent cell line HCD-57. HCD-57 cells were obtained synchronized in G0 by centrifugal elutriation, and DNA synthesis was measured by incorporation of labeled thymidine into DNA. Half-maximal DNA synthesis was stimulated by 0.001 U/ml of EPO. Sodium orthovanadate (Na3VO4), a tyrosine phosphatase inhibitor, at 5 microM potentiated a subsaturating concentration of EPO. Na3VO4 alone stimulated HCD-57 DNA synthesis at concentrations of 0.1-20 microM. Zinc chloride, another tyrosine phosphatase inhibitor, also stimulated HCD-57 DNA synthesis at concentrations of 50-100 microM. Genistein, a tyrosine kinase inhibitor, blocked the effect of EPO at a concentration of 5 micrograms/ml. Bryostatin, a protein kinase C (PKC) activator, stimulated DNA synthesis in HCD-57 cells at concentrations of 10(-9)-10(-10) M, whereas the phorbol ester, phorbol 12,13-dibutyrate (PDBu), was stimulatory only at a concentration of 10(-11) M. Staurosporine, a PKC inhibitor, blocked the effect of EPO at a concentration of 10(-7) M, and H-7, a nonspecific protein kinase inhibitor, was not inhibitory. These agents also had similar effects on the in vitro proliferation of HCD-57 cells. Taken together, the data indicate that the EPO-mediated transition from G0 to S phase in HCD-57 cells involves the activation of both tyrosine and serine-threonine kinases and is modulated by tyrosine phosphatase activity.


Stoichiometric association of extrinsic cytochrome c550 and 12 kDa protein with a highly purified oxygen-evolving photosystem II core complex from Synechococcus vulcanus. A highly purified, native photosystem II (PS II) core complex was isolated from thylakoids of Synechococcus vulcanus, a thermophilic cyanobacterium by lauryldimethylamine N-oxide (LDAO) and dodecyl beta-D-maltoside solubilization. This native PS II core complex contained, in addition to the proteins that have been well characterized in the core complex previously purified by LDAO and Triton X-100, two more extrinsic proteins with apparent molecular weights of 17 and 12 kDa. These two proteins were associated with the core complex in stoichiometric amounts and could be released by treatment with 1 M CaCl2 or 1 M alkaline Tris but not by 2 M NaCl or low-glycerol treatment, indicating that they are the real components of PS II of this cyanobacterium. N-Terminal sequencing revealed that the 17 and 12 kDa proteins correspond to the apoprotein of cytochrome c550, a low potential c-type cytochrome, and the 9 kDa extrinsic protein previously found in a partially purified PS II preparation from Phormidium laminosum, respectively. In spite of retention of these two extrinsic proteins, no homologues of higher plant 23 and 17 kDa extrinsic proteins could be detected in this cyanobacterial PS II core complex.


Induction of vascular smooth muscle cell growth by selective activation of the thrombin receptor. Effect of heparin. The synthetic peptide, SFLLRNPNDKYEPF, has been recently described as a peptide mimicking the new amino-terminus created by cleavage of the thrombin receptor, therefore acting as an agonist of the thrombin receptor. This peptide was a potent mitogen for rabbit arterial smooth muscle cells (SMC) and exhibited the same activity as that of native alpha-thrombin. Both compounds stimulated the proliferation of growth-arrested SMCs with half-maximum mitogenic responses at 1 nM. NAPAP, a synthetic inhibitor of the enzymatic activity of thrombin, specifically inhibited thrombin-induced SMC growth (IC50 = 0.35 +/- 0.04 microM) but was without effect on the mitogenic effect of the agonist peptide. These results therefore demonstrate that the mitogenic effect of alpha-thrombin for SMCs is intimately linked to its esterolytic activity. Heparin, which inhibited fetal calf serum-induced SMC growth, was without effect on thrombin-induced SMC growth but strongly reduced the mitogenic effect of the agonist peptide (IC50 = 32 +/- 5 micrograms/ml). This effect was not related to the anti-coagulant activity of heparin but was highly dependent on molecular mass and on the global charge of the molecule and was also observed for other sulphated polysaccharides such as pentosan polysulphate.


Atomic force microscopy of DNA molecules. DNA-cytochrome c complexes adsorbed on carbon-coated mica surfaces were directly imaged by atomic force microscopy in air using commercially available cantilevers, with a routine resolution of 6 nm. Images of M13 phage DNA and M13-DNA polymerase complex are also shown.


Curcumin inhibits nitrite-induced methemoglobin formation. Curcumin protects hemoglobin from nitrite-induced oxidation to methemoglobin. The protection is not observed when curcumin is added after the autocatalytic stage of the oxidation of hemoglobin by nitrite. The ability of curcumin to scavenge superoxide may be responsible since superoxide is implicated in promoting the autocatalytic stage of oxidation of hemoglobin by nitrite.


Ubiquitin, a central component of selective cytoplasmic proteolysis, is linked to proteins residing at the locus of non-selective proteolysis, the vacuole. Ubiquitin, an evolutionary highly conserved protein, is known to be involved in selective proteolysis in the cytoplasm. Here we show that ubiquitin-protein conjugates are also found in the yeast vacuole. Mutants defective in the major vacuolar endopeptidases, proteinase yscA and yscB, lead to accumulation of ubiquitin-protein conjugates in this cellular organelle.


The corticotrophin releasing factor test: a valuable and rapid procedure for the differential diagnosis of hirsutism. Plasma concentrations of adrenocorticotrophic hormone, cortisol, 17-hydroxyprogesterone, testosterone, delta 4-androstenedione and dehydroepiandrosterone sulphate were determined in 10 regularly menstruating hirsute women 0, 15, 30, 60, 90 and 120 min after intravenous injection of 100 micrograms corticotrophin releasing factor. The baseline concentrations of adrenocorticotrophic hormone, cortisol, progesterone and dehydroepiandrosterone sulphate were within the normal range, whereas the mean delta 4-androstenedione and testosterone concentrations were mildly elevated. The administration of corticotrophin releasing factor induced a rapid and significant increase in plasma concentrations of adrenocorticotrophic hormone, cortisol, 17-hydroxyprogesterone and delta 4-androstenedione after 60 min, whereas dehydroepiandrosterone sulphate and testosterone concentrations were not significantly elevated. These findings suggest that the corticotrophin releasing factor test can readily be used as an out-patient procedure to exclude adrenal hirsutism.


[Changes in levels of blood coagulation inhibitors in cirrhosis. Prospective study in 33 patients]. The variations of the main plasma inhibitors of coagulation were prospectively studied in 33 cirrhotic patients, of which 9 presented with hepatocellular carcinoma, 5 of those associated with portal vein thrombosis. The mean prothrombin index was 49 +/- 16 percent. All plasma values of inhibitors were diminished, but to varied degrees: the mean values were: protein C (PC): 33 +/- 15 percent, antithrombin III (AT III): 50 +/- 23 percent, total protein S (PST): 67 +/- 20 percent. The more severe the cirrhosis, the more decreased were the values of antithrombin II and protein C. According to Child classes A, B, and C, antithrombin III plasma values were 64 +/- 20, 50 +/- 21 and 26 +/- 11 percent and protein C values were 43 +/- 16, 32 +/- 8 and 19 +/- 9 percent, respectively. We were able to define expected plasma values of the plasma inhibitors as a function of coagulation factors during cirrhosis; AT III (percent) = 1.16 x factor II (percent) - 7.85; PC (percent) = 0.49 x AT III (percent) + 8.96; PC (percent) = 0.55 x factor II (percent) + 5.55; PST (percent) = 0.76 x factor II (percent) + 28.74. However those equations cannot be extrapolated to patients presenting with cirrhosis complicated with portal thrombosis.


Effect of dietary fiber on colonic bacterial enzymes and bile acids in relation to colon cancer. Because of the potential significance of colonic bacteria and secondary bile acids in the pathogenesis of colon cancer, the present study investigated the effect of different types of dietary fiber on fecal bacterial enzymes, namely, beta-glucuronidase, 7 alpha-dehydroxylase, nitroreductase, and azoreductase, and on bile acids and neutral sterols in premenopausal women. The subjects consumed 13-15 g of wheat, oat, or corn bran daily for 8 weeks in addition to their normal diet. Stools collected during the normal and fiber diet periods were analyzed for the above constituents. Dietary wheat bran decreased the concentrations of fecal deoxycholic acid, lithocholic acid, 12-ketolithocholic acid, and neutral sterols and the activities of all bacterial enzymes. Oat bran had no effect on secondary bile acids and 7 alpha-dehydroxylase but decreased beta-glucuronidase, nitroreductase, and azoreductase levels. Dietary corn bran increased 7 alpha-dehydroxylase, lithocholic acid, and cholesterol levels and decreased deoxycholic acid coprostanol, cholestenone, nitroreductase, and azoreductase levels. These results show that the modifying effect of dietary fiber on secondary bile acids and bacterial enzymes that may play a role in carcinogenesis depends on the type of fiber consumed.


Increased jejunal intraepithelial lymphocytes bearing gamma/delta T-cell receptor in dermatitis herpetiformis. T-cell receptor 1 (gamma/delta) expression was studied in 19 jejunal or duodenal specimens from patients with dermatitis herpetiformis and in 16 jejunal or duodenal specimens showing normal histology. In normal specimens, gamma/delta+ cells represented 10.8% of intraepithelial CD3+ lymphocytes. Around 50% of these cells were recognized by the A13 monoclonal antibody, which detects products of the V gamma 1/V delta 1 gene rearrangement and the non-disulfide-linked form of T-cell receptor 1. The remaining 50% reacted with the BB3 monoclonal antibody, which recognizes products of the V gamma 9/V delta 2 rearrangement and the disulfide-linked form of receptor. Very few gamma/delta+ cells were observed in the lamina propria. In jejunal specimens from patients with dermatitis herpetiformis, a significant increase in the prevalence of gamma/delta+ intraepithelial lymphocytes was observed (P less than 0.001). This finding was largely accounted for by an increase in those cells recognized by the A13 monoclonal antibody, thus possibly expressing the V gamma 1/V delta 1 rearrangement and the nondisulfide-linked form of receptor. These data suggest that similar pathogenetic mechanisms may be active in determining the jejunal damage in celiac disease and dermatitis herpetiformis.


A comparative analysis of two models of colitis in rats. Two models of colitis produced in rats that have received significant attention over the past few years are the acetic acid and trinitrobenzene sulfonic acid (TNBS) models. The objective of this study was to quantify and compare the temporal relationship among mucosal permeability, epithelial injury, and inflammation induced by acetic acid, ethanol (vehicle), ethanol plus TNBS (unbuffered, pH 1.0), and ethanol plus TNBS (pH 7.4). Data obtained show that the inflammation induced by these four irritants results from caustic injury to the colonic epithelium and interstitium as measured by the rapid and dramatic increases in mucosal permeability and tissue water content as well as by histological analysis. The injurious nature of TNBS was confirmed in a separate series of studies showing that buffered TNBS (pH 7.4), in the absence of ethanol, is toxic to cultured rat intestinal epithelial cell monolayers. Only after 1-2 days of the initial insult, were signs of classical inflammation observed, including increases in colonic myeloperoxidase activity (neutrophil infiltration) and colon weight as well as hyperemia and mucosal ulcerations. Although ethanol plus TNBS (pH 1.0 or 7.4) tended to produce higher mucosal permeabilities (epithelial cell injury) at 1-2 weeks after the enemas than acetic acid or ethanol groups, only the ethanol plus TNBS (pH 7.4) permeabilities were found to be significantly enhanced. In addition, all four groups showed significant elevations in colonic myeloperoxidase activity and colon weight at 1-2 weeks after enema. It is suggested that these models of colitis are useful to study events that occur at the time of inflammation and repair. However, these models may have significant limitations in understanding events that initiate inflammation of the intestine in human inflammatory bowel disease.


Effect of cell culture on rabbit colonic smooth muscle bradykinin receptors. The aim of this study was to assess the effect of cell culture on the bradykinin receptor of rabbit colon myocytes. In longitudinal muscle strips prepared from distal colon, bradykinin stimulated dose-dependent contraction that was 62% of the maximal response to bethanecol. At 4 degrees C, [3H]bradykinin binding to fresh muscle homogenates from the distal colon was time dependent, saturable, and linearly related to tissue concentration. Specific binding of 0.6 nmol/L [3H]bradykinin was 80% +/- 2% of total binding. In competitive binding studies, Hill coefficients approached unity, suggesting the presence of a single class of receptors. The order of potency was bradykinin greater than [D-Phe7]bradykinin much greater than des-Arg9, [Leu8]bradykinin, which is consistent with results of a B2 receptor subclass. Colon myocytes from the longitudinal muscle layer achieved confluence and were harvested for studies after 12-14 days in culture. Bradykinin receptors were of high affinity [disassociation constant (Kd) = 672 pmol/L] and numbered 10,217 +/- 2567/cell. To show that the receptors on cultured myocytes were functional, the effect of bradykinin was measured (a) on intracellular calcium concentration using Fura 2 and (b) on prostaglandin E2 concentration in the culture media using radioimmunoassay. In cells grown to confluence on cover slips and preloaded with Fura 2, bradykinin stimulated the threshold response at 1 nmol/L and maximal response (increased intracellular calcium concentration from 229 to 633 nmol/L) at 1 mumol/L. Bradykinin, 100 nmol/L, increased Prostaglandin E2 in the culture media threefold. In summary, colon myocytes express functioning bradykinin receptors, which, unlike muscarinic receptors, persist in culture. Bradykinin appears to be a suitable agonist for studies of receptor-mediated intracellular events in cultured colon myocytes.


Duration and specificity of antibodies to hepatitis C virus in chronic active hepatitis. To determine the duration and specificity of antibodies to hepatitis C virus in hepatitis B surface antigen-negative chronic active hepatitis, sera from 19 patients seropositive by enzyme immunoassay were assessed by recombinant immunoblot assay. Only 12 of the 19 patients were reactive by immunoblot assay (63%). Patients nonreactive by immunoblot assay had lower signal-cutoff ratios by enzyme immunoassay (1.3 +/- 0.2 vs. 6.5 +/- 0.1; P less than 0.05), higher serum immunoglobulin G levels (4082 +/- 301 vs. 1760 +/- 143 mg/dL; P less than 0.05), and higher serum gamma globulin levels (3.3 +/- 0.5 vs. 2.04 +/- 0.1 g/dL; P less than 0.05) than reactive patients. Twelve of 14 patients with serial studies remained seropositive after 39 +/- 11 months of follow-up (range, 7-113 months). Only patients nonreactive by immunoblot assay became seronegative by enzyme immunoassay during corticosteroid therapy (2/3 vs. 0/6 patients). It is concluded that seropositivity by enzyme immunoassay may not be documented by immunoblot assay. Patients nonreactive by immunoblot assay have lower signal-cutoff ratios and higher gamma globulin levels than reactive patients, and their seropositivity may be nonspecific. Patients nonreactive by immunoblot assay may lose seropositivity by enzyme immunoassay during corticosteroid therapy.


Depression of plasma gelsolin level during acute liver injury. Human plasma contains two actin-binding proteins, plasma gelsolin and vitamin D-binding protein. These proteins are considered to play an important role in the disposition of actin derived from injured tissue. To evaluate this actin-scavenger system, gelsolin concentrations were measured in serial plasma samples obtained from patients with acute liver injury using an enzyme-linked immunosorbent assay. Plasma gelsolin levels in 43 healthy persons were 226 +/- 52 micrograms/mL. They were markedly reduced to 80 +/- 40 micrograms/mL in 14 patients with an early stage of acute hepatitis and returned to normal levels of 232 +/- 38 micrograms/mL as the disease resolved. Moreover, they showed a significant negative correlation with serum aminotransferase and bilirubin levels. In 7 patients with hepatocellular carcinoma, plasma gelsolin levels rapidly decreased from 182 +/- 42 to 87 +/- 41 micrograms/mL after transcatheter arterial embolization therapy. Because plasma gelsolin is not a hepatic protein, the decreased levels are considered to depend exclusively on the extent of actin leakage from the injured liver.


[Intrauterine pregnancy with uterine myoma after downregulation with GnRH analogs]. GnRH analogues are widely used for the reduction of uterine fibroids. This case report describes the therapy of a 27-year-old woman, whose uterus had a diameter of about 13 cm. After 7 injections of Zoladex Depot (ICI Pharma, Heidelberg, Germany), the uterus was reduced to normal size carrying dorsally a myoma of the same size. After only 7 weeks of medroxyprogesterone acetate (5 mg twice daily, Clinofem, Upjohn, Heppenheim, Germany) the uterus had grown again, so that therapy was changed to Zoladex Depot for another 3 months. On the 27th day of the first spontaneous cycle, the patient ovulated and conceived. Up to the 29th week of gestation (Aug '91), the foetal growth and development was normal, the uterus was normal in size, a childhead sized myoma being situated unproblematically behind the foetus.


Copper-ligand interactions and physiological free radical processes. pH-dependent influence of Cu2+ ions on Fe2(+)-driven OH. generation. Prior to comparative studies on the reactivity of various copper complexes with respect to OH. radicals, the influence of free Cu2+ ions on the superoxide-independent generation of OH. radicals through Fenton assays and water gamma radiolysis has been tested in the present work. Cu2+ ions have been shown to behave in a distinct manner towards each of these two production systems. As was logically expected from the noninvolvement of copper in OH. radical production through gamma radiolysis, no influence of Cu2+ ions has been observed on the amount of radicals detected in that case. In contrast, Cu2+ ions do influence OH. radical generation through iron-driven Fenton reactions, but differently depending on copper concentration. When present in high concentrations, Cu2+ ions significantly contribute to OH. radical production, which confirms previous observations on the reactivity of these in the presence of hydrogen peroxide. At lower levels corresponding to copper/iron ratios below unity on the contrary, Cu2+ ions behave as inhibitors of the OH. production in a pH-dependent manner over the 1-6 range investigated: the lower the pH, the greater the inhibition. The possible origin of this previously unreported inhibitory effect is discussed.


Generation of active oxygen species in blood platelets--spin trapping analysis. Generation of active oxygen species by bovine blood platelets was examined by the electron spin resonance (ESR) spin trapping technique with 5,5-dimethyl-1-pyroline-1-oxide (DMPO). The hydroxyl spin-trapped adduct 5,5-dimethyl-2-hydroxyl-1-pyrolidinyloxy (DMPO-OH) was formed in the presence of platelets, indicating the generation of hydroxyl radicals (.OH) by the platelets. Generation of .OH was observed even with platelets in the resting state, but was markedly enhanced when the platelets were activated with stimulants. Stronger stimulants such as the calcium ionophore ionomycin, induced greater radical generation than the weaker stimulant ADP. When the platelets were stimulated by thrombin, generation of .OH was greatest after 1.5 min, and depended on the dose of the stimulant. It was inhibited by inhibitors of platelet activation such as forskolin and phenolic antioxidants.


Hydroxyl radicals are generated by hepatic microsomes during NADPH oxidation: relationship to ethanol metabolism. Ethanol is metabolized to acetaldehyde by hepatic microsomes in a reaction that requires cytochrome P-450, and a role for hydroxyl radicals has been implicated in this process. However, previous spin trapping experiments have failed to demonstrate the production of hydroxyl radicals by liver microsomes unless iron or other metal catalysts have been added. The spin trapping experiments described in this report provide unambiguous evidence that liver microsomes form hydroxyl radicals during oxidation of NADPH, that the addition of exogenous iron is unnecessary for this process, and that hydroxyl radicals participate in the metabolism of ethanol. Liver microsomes are known to metabolize ethanol to the 1-hydroxyethyl radical, and our experimental data support the conclusion that a significant part of the production of the 1-hydroxethyl radical occurs as a consequence of hydroxyl radical attack on ethanol. Lack of previous observation of microsomal hydroxyl radical production in spin trapping experiments is shown to be related to the contamination of the microsomes with catalase.


Calmodulin content and activity in normal and coeliac duodenum. Calmodulin is an important modulator of intracellular calcium processes and may be implicated in the calcium malabsorption of coeliac disease. The calmodulin content in extracts of duodenal biopsy specimens from 48 normal control subjects and 28 patients with coeliac disease was determined. Radioimmunoassay was used to measure immunoreactive calmodulin while a cyclic adenosine 3',5'-monophosphate phosphodiesterase activity assay was used to measure biologically active calmodulin. Calmodulin values measured by both assays were similar for control and disease groups. Mean (SEM) immunoreactive calmodulin values were 1.68 (0.09) micrograms/mg protein for controls and 1.67 (0.15) and 1.45 (0.15) micrograms/mg protein for partial and total villous atrophy respectively. These values were not significantly different. Biologically active calmodulin values were 2.77 (0.21), 1.82 (0.34), and 3.24 (0.33) micrograms/mg protein for control, partial, and total villous atrophy subjects respectively. The biologically active calmodulin values in the partial villous atrophy group were significantly lower than in controls and total villous atrophy subjects. In the phosphodiesterase assay, the calmodulin antagonist trifluoperazine inhibited the activity stimulated by purified calmodulin and by the extracts to the same extent. These results show that calmodulin values are normal in coeliac disease and provide no evidence that changes in calmodulin account for the abnormal calcium absorption in these patients.


Intestinal smooth muscle dysfunction after intraperitoneal injection of zymosan in the rat: are oxygen radicals involved? Zymosan is frequently used as an activator of granulocytes to study inflammatory responses. We used zymosan as a model to understand the mechanisms involved in intestinal inflammatory diseases, and our special interest was focused on the smooth muscle function. Moreover, we investigated the role of oxidative stress in intestinal pathology after inflammatory processes. Intraperitoneal injection of zymosan induces a peritoneal inflammation, characterised by exudate in the peritoneum and peritoneal fibrosis. Three days after injection of zymosan (25-40 mg/100 g) we measured a decreased beta adrenergic smooth muscle response, while the muscarinic receptor-mediated contraction was not significantly affected. Efforts were made to correlate these observations with the development of oxidative stress; however, the intestinal glutathione balance remained undisturbed and no increase in lipid peroxidation products in the intestine was observed. Our conclusion is the peritoneal inflammation will lead to a release of various mediators, which may destroy receptor systems, among which are beta adrenoceptors. There was no evidence of an important role for reactive oxygen metabolites in this effect.


Epidemiology of familial adenomatous polyposis in Finland: impact of family screening on the colorectal cancer rate and survival. The incidence and prevalence rates of familial adenomatous polyposis (FAP) in Finland between 1961 and 1990 were estimated from Finnish polyposis registry data comprising 81 FAP families, including 251 affected patients. In addition, the effect of family screening on the occurrence of colorectal carcinoma was evaluated by comparing the call up and proband groups and calculating the proportion of FAP among all patients with colorectal carcinoma. The incidence of FAP was 0.62 to 2.38 per million and the prevalence increased steadily from 0.88 to 26.3 million during the study period suggesting improving prognosis. Altogether 76 of 116 probands (65.5%) had colorectal carcinoma compared with only five of 76 call up patients (6.6%). Consequently, the life expectancy of the call up patients was significantly better than that of the probands from the age of 31 years and above. However, at most, 0.53% of all colorectal carcinomas were associated with FAP in 1966-70, and the diminishing frequency of this proportion was more a result of an increase in sporadic colorectal carcinomas in Finland than of family screening for FAP. Family screening is very effective in FAP and must always be undertaken when a new proband is diagnosed.


The ring constriction syndrome.

The ring constriction syndrome. This series of 37 cases treated by the author over a twenty-seven year period, covers the full spectrum of deformity produced by this condition. These include simple ring constrictions, those with a distal atrophy or oedema, digit fusions, and amputations.


Clinicopathological study on neural invasion to the extrapancreatic nerve plexus in pancreatic cancer. Thirty-four cases that underwent resection of cancer of the pancreatic head were examined clinicopathologically to elucidate neural invasion of cancer of the pancreatic head into the extrapancreatic nerve plexus. Invasion of cancer into the retropancreatic tissue (rpe) was seen in 29 (86 per cent) of the 34 cases and neural invasion of the extra-pancreatic nerve plexus in 21 (72 per cent) of the 29 rpe cases. The incidence of invasion of the IInd portion of the nerve plexus of the pancreatic head was high (14 = 67 per cent). The degree of neural invasion tended to increase with the extent of lymph vessel invasion. Observation of serial sections revealed continuity of the neural invasion into the nerve plexus. From these findings we conclude that neural invasion of the extrapancreatic nerve plexus is mainly the result of continuous spread, and that en bloc resection of the retropancreatic tissue involving the nerve plexus and fat tissue is necessary in the surgical treatment of cancer of the pancreatic head.


Effect of synthetic protease inhibitors on superoxide (O2-), hydrogen peroxide (H2O2) and hydroxyl radical production by human polymorphonuclear leukocytes. The effects of clinically used protease inhibitors (aprotinin, nafamostat mesilate, gabexate mesilate) on the production of oxygen-derived free radicals (O2-, H2O2, .OH) by human polymorphonuclear leukocytes were examined. Nafamostat mesilate and gabexate mesilate markedly and dose-dependently inhibited zymosan-stimulated O2- production by human polymorphonuclear leukocytes. However, aprotinin had a slight scavenging effect on O2- produced by the xanthine-xanthine oxidase system. All the protease inhibitors inhibited H2O2 production, but had no significant scavenging effect on H2O2. Nafamostat mesilate and gabexate mesilate slightly inhibited .OH production. These results indicate that the synthetic protease inhibitors nafamostat mesilate and gabexate mesilate inhibit the production of various activated oxygen radicals by human polymorphonuclear leukocytes, and the differences in their inhibitory effects suggest that each synthetic protease inhibitor is specific for a particular oxygen-derived free radical.


An unusual growth pattern of a mucin-producing intrahepatic cholangiocellular carcinoma involving the hepatic hilus. The case of a mucin-producing intrahepatic cholangiocellular carcinoma in a 73 year-old-man is presented. A tumor originating in the right posterior inferior segment of the liver was found to be invading the right posterior and anterior bile ducts, and the hepatic hilus. Extensive superficial spread was observed in the entire posterior segmental bile duct extending to the hepatic hilus. Mucin produced and excreted by the tumor was retained in the common hepatic and common bile duct. The diagnosis in this case was suggested by percutaneous transhepatic aspiration of mucinous bile, and was confirmed by utilizing the techniques of ultrasonography, percutaneous transhepatic cholangiography, computed tomography and angiography. Curative surgery, which included right hepatic lobectomy with total caudate lobectomy and bile duct resection, was performed. Biliary continuity was maintained by left hepaticojejunostomy using a Roux-en-Y jejunal loop. The histological diagnosis was mucin-producing papillary adenocarcinoma originating in the right posterior inferior segment of the liver. Postoperative recovery was very good and the patient has now been enjoying a good active social life for the last 20 months with no signs of tumor recurrence. This case report discusses the unusual growth pattern of a mucin-producing intrahepatic cholangiocellular carcinoma involving the hepatic hilus, and suggests rational surgical treatment.


Non-A, non-B chronic hepatitis is chronic hepatitis C: a sensitive assay for detection of hepatitis C virus RNA in the liver. To study the role of hepatitis C virus in non-A, non-B chronic hepatitis, 49 liver biopsy samples from 40 patients with non-A, non-B chronic hepatitis and 9 control patients were analyzed by complementary DNA/polymerase chain reaction. Two segments of the HCV genome, one in the nonstructural region and the other in the noncoding region, were amplified by two sets of primer pairs. With use of the nonstructural region primers, hepatitis C virus RNA was detected in 24 (60%) of 40 patients with non-A, non-B chronic hepatitis. Of these 40 patients, RNA was detected in 19 (70%) of 27 patients positive for antibody to hepatitis C virus and in 5 (38%) of 13 patients negative for antibody to hepatitis C virus. However, with the noncoding region primers, hepatitis C virus RNA was detected in 38 (95%) of 40 patients with non-A, non-B chronic hepatitis. Of these patients, the RNA was detected in 26 (96%) of 27 patients positive for antibody to hepatitis C virus and also in 12 (92%) of 13 patients positive for antibody to hepatitis C virus. Hepatitis C virus RNA was not detected in any of the control patients. Sequence analysis showed homology between our samples and the prototype to be only 66% to 77% in the nonstructural region but 99% to 100% in the noncoding region. We conclude that almost all patients with non-A, non-B chronic hepatitis in Japan are currently infected with hepatitis C virus, regardless of the presence or absence of antibody to hepatitis C virus.(ABSTRACT TRUNCATED AT 250 WORDS)