Serum protease inhibitor capacity for elastase and the severity of pancreatitis. To clarify the relationship between the diminution of the serum protease inhibitor capacity and the severity of pancreatitis, the binding capacity of serum protease inhibitors for exogenous elastase 1 (E1) was investigated by gel filtration, the elastase activity of the alpha 2-macroglobulin (alpha 2-M)-elastase complex was measured, and the relationship between these findings and the severity of pancreatitis was studied in 13 patients with pancreatic disease and 6 healthy subjects. When 125I-labeled E1 was added to the sera of healthy subjects, it bound to alpha 2-M and alpha 1-protease inhibitor (alpha 1-PI) with a mean ratio of 72:28. In mild acute pancreatitis (n = 5), the binding capacity of alpha 2-M was less than that in healthy subjects. In severe pancreatitis (n = 4), most of the exogenous E1 bound to alpha 1-PI (alpha 2-M vs. alpha 1-PI, 13:87). This diminution in the binding capacity of alpha 2-M correlated well with the severity of acute pancreatitis. In the sera of patients (n = 4) with pancreatic cancer containing much immunoreactive E1, the proportion of exogenous E1 bound by alpha 2-M and alpha 1-PI (25:75) was similar to that seen in severe acute pancreatitis. A significant inverse relationship between the binding capacity of alpha 2-M and the activity of the endogenous elastase bound to alpha 2-M was seen in various pancreatic diseases.(ABSTRACT TRUNCATED AT 250 WORDS)


Modulation of pancreatic exocrine function in rodents by treatment with pancreatic polypeptide. The in vivo and in vitro treatment effects of pancreatic polypeptide (PP) were characterized by studying agonist-stimulated enzyme secretion in pancreatic acini prepared from 8-week-old mice treated for 2 days with PP (200 micrograms kg-1 day-1) and in pancreatic lobules from untreated male rats. In the mouse studies, enzyme secretion was evaluated on the basis of percentage total amylase released, amylase released per unit of DNA, and amylase released per unit of protein. When expressed as percentage total amylase released, the acini from mice treated with PP were significantly less responsive to pancreatic secretagogues than were acini from control animals. Chronic treatment with bovine PP lowered the maximal response to carbachol (12.3 +/- 0.3 vs. 9.0 +/- 0.3% total amylase release in control and PP treated, respectively), decreased the magnitude of the difference between basal and maximal amylase release (10.6 +/- 0.4 vs. 6.2 +/- 0.5% total amylase release in control and PP treated, respectively), and affected these changes without modifying the dose of carbachol producing half-maximal amylase release. Similarly, the percentage of total amylase released in response to all doses of cholecystokinin octapeptide (1-100 pM) was reduced by chronic treatment with PP. However, when amylase release was expressed relative to protein or DNA, no differences in enzyme release were detected between treatments with either secretagogue. Chronic treatment with PP increased the total amount of amylase in the acini (per unit DNA or protein), but the increased amylase appeared to be unavailable for release since the actual amount (per microgram DNA or milligram protein) released in response to agonists did not differ between treatments.(ABSTRACT TRUNCATED AT 250 WORDS)


Bioactivation of free-fat transfers: a potential new approach to improving graft survival. A new approach to free-fat autotransplantation resorption was evaluated experimentally in a rat animal model. Bioactive fat grafts were created by the addition of basic fibroblast growth factor delivered by dextran beads to the grafts and compared with free fat alone, free fat plus beads, and free fat plus beads and a control solution in the same animal. The grafts were assessed by weight and histology at 1 and 12 months postoperatively in 40 animals. A graded response in weight retention was observed at 1 and 12 months, with the growth factor-treated grafts exhibiting near complete weight maintenance after 1 year. All other bead-containing grafts had an intermediate response, with free fat alone averaging more than one-half graft weight loss after 1 year. Histologically, the bead-containing grafts had good fibroblastic ingrowth, but extensive intercellular collagen formation and the occurrence of small-sized adipocytes among the larger adipocytes were seen only in the growth factor-treated grafts. These findings indicate that graft manipulations that affect the preadipocyte cells of the graft or fibroblastic components of the recipient site, either through polypeptide stimulation or surface charge attraction, may offer a viable approach to postoperative fat-graft volume maintenance.


Study of functional and biochemical indicators of subclinical lung damage in bleomycin-treated patients. In 30 patients affected by testicular non-seminomatous cancer we evaluated pulmonary function tests before and after bleomycin-including combination chemotherapy. We paid particular attention to changes in diffusing lung capacity (DCO) and its two components, namely diffusing capacity of the alveolar-capillary membrane (Dm) and pulmonary capillary blood volume (Vc). In the same patients we also evaluated the behaviour of serum procollagen III aminopeptide (sP-III-P), assumed to be a biochemical equivalent to Dm, and of serum angiotensin converting enzyme (S-ACE), assumed to be a biochemical equivalent to Vc. We found that, after chemotherapy, patients showed a significant decline in several pulmonary function parameters, namely VC, TLC, and FEV1 (P < 0.0001, P = 0.0351, P = 0.0004, respectively), when compared to pre-treatment values. DCO was significantly impaired after chemotherapy (P < 0.0001), but with regard to its components, Vc values showed a significant decline (P = 0.0002), whereas Dm values were unchanged. Values of sP-III-P raised significantly after chemotherapy (P = 0.003), whereas S-ACE activity did not show any significant variation. When we looked at relationships between functional and biochemical parameter variations, the only significant correlation we found was between DCO and S-ACE (r2 = 0.112; P < 0.02). We conclude that in asymptomatic patients treated by bleomycin-including combination chemotherapy, DCO impairment is likely to occur because of a subclinical injury to pulmonary vessels, as suggested by Vc decline. Although the occurrence of pulmonary interstitial fibrosis after chemotherapy was excluded by chest X-ray examination and by stable values of Dm, sP-III-P elevation would suggest an accelerated type III collagen turnover in interstitial fibroblasts activated by bleomycin.


[Expression of adhesion molecules N.CAM, L1 and HNK1 epitope by medulloblastoma]. Twelve medulloblastomas were screened for their expression of adhesion molecules L1, N.CAM isoforms and HNK1 epitope by Western blotting and immunohistochemistry. Highly sialylated N.CAM isoforms were distinguished from total N.CAMs by using a monoclonal antibody (anti-MenB) specifically recognizing high polymers of 2-8 linked neuraminic acid. All tumors expressed HNK1 epitope, N.CAM and its highly sialylated isoforms on their surface membrane. L1 adhesion molecule was detected by immunohistochemistry in only one medulloblastoma. This spectrum of expression of cell surface adhesion molecules distinguishes medulloblastomas from other primitive neuroectodermal tumors. Medulloblastomas share some immunological features with post-mitotic cells forming the external granular layer of the cerebellum. Western blotting analysis of cerebrospinal fluid (CSF) samples with anti-MenB antibody enabled us to detect highly sialylated N.CAM in some samples. The presence of this antigen in CSF appears to correlate with meningeal spreading of medulloblastomas and could help monitoring chemotherapeutic treatment.


Pathogenesis of central nervous system paraneoplastic syndromes. The pathogenesis of central nervous system paraneoplastic syndromes has not been established. Increasing evidence supports the hypothesis that CNS paraneoplastic syndromes are immune disorders arising from an immune response to the underlying neoplasm. The hypothesis is that antigens or epitopes shared between the tumor and the nervous system are recognized as foreign by the host and elicit an immune response. The immune response is directed appropriately against the tumor leading to a more indolent course in those patients with paraneoplastic syndromes but is misdirected against the nervous system often leading to severe nervous system destruction. The evidence for this hypothesis consists of: 1) The finding of high titer autoantibodies that react with antigens in both tumor and central nervous system. 2) Intrathecal synthesis of the antibody. 3) The presence of the antibody in the central nervous system at autopsy. The development of animal models will be required to determine the exact nature of the pathogenesis of these disorders.


Amniocentesis--still important in the management of severe rhesus incompatibility. To determine the role of amniocentesis in the management of severe rhesus (Rh), incompatibility. A 10-year retrospective descriptive study. Department of Obstetrics and Gynaecology, Tygerberg Hospital, CP. 111 pregnancies complicated by severe Rh incompatibility where management had been based on results obtained from amniocentesis. Neonatal haemoglobin values, neonatal survival and short-term morbidity as compared with the position on the Liley chart after amniocentesis immediately before delivery. Average neonatal haemoglobin value decreased for the group in the lower zone through the midzone to the upper zone, but wide scattering of individual values between the various zones occurred. Perinatal mortality rate was 93/1,000 deliveries with Rh-related deaths occurring only in the upper midzone and upper zones. One death and 7 cases of morbidity occurred after acting on misleading information from the Liley curve. Amniotic fluid bilirubin values are at best an indirect evaluation of the state of disease. No serious complications are likely to occur when these values fall in the lower or lower mid-zones of the Liley chart. When bilirubin values fall in the upper midzone or upper zone, cordocentesis should be performed to prevent overreaction or to perform an intra-uterine transfusion.


Cytotoxic effects of cyclosporin A on the exocrine pancreas in rats. The current study was done to evaluate the subacute toxic effects of cyclosporin A (CS) on the exocrine pancreas and the protective effect of potent protease inhibitor camostate (FOY-305). CS administration (15 milligrams, twice a day) for 14 days caused a significant increase in serum amylase levels, pancreatic amylase and cathepsin B content and mild acinar cell vacuolization and interstitial edema. CS also caused the redistribution of cathepsin B activity from the lysosomal fraction to the zymogen fraction, indicating colocalization of lysosomal enzyme with pancreatic digestive enzymes. The administration of camostate (150 milligrams per kilogram, twice a day for 14 days) almost completely prevented the toxic changes induced by CS. These results indicate that CS induces exocrine pancreatic injury and that lysosomal enzymes play important roles in the pathogenesis of the injury. The results also suggest the usefulness of camostate in protecting the exocrine pancreas in patients treated with CS after organ transplantation, because it can inhibit some proteases that are closely involved in the fragility of the subcellular organelles.


Current management of hilar cholangiocarcinoma. The goal in the treatment of hilar cholangiocarcinoma is the relief of biliary obstruction with the quality survival. The choice of therapy for an individual patient requires complete preoperative staging to determine the resectability. This is best accomplished in a multidisciplinary setting with radiologists, gastroenterologists and surgeons participating in the diagnostic and therapeutic strategies. Operative removal of the tumor can be performed with low mortality and, when possible, provides the longest palliation and offers potential for cure. For tumors found at operation to be unresectable, biliary enteric bypass provides a durable method of palliation. The relative merits of the newer methods of radiologic stenting are encouraging, but further study will require prospective trials in comparison to operative biliary enteric bypass.


The interaction of botrocetin with normal or variant von Willebrand factor (types IIA and IIB) and its inhibition by monoclonal antibodies that block receptor binding. We have recently shown the existence of two distinct forms of botrocetin (one-chain and two-chain), and demonstrated that the two-chain species is approximately 30 times more active than the one-chain in promoting von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib. The N-terminal sequence of two-chain botrocetin is highly homologous to sea-urchin Echinoidin and other Ca(2+)-dependent lectins (Fujimura et al., Biochemistry 1991; 30: 1957-64). Present data indicate that purified two-chain botrocetin binds to vWF from plasmas of patients with type IIA or IIB von Willebrand disease and its interaction is indistinguishable from that with vWF from normal individuals. However, an "activated complex" formed between botrocetin and IIB vWF expresses an enhanced biological activity for binding to GP Ib whereas the complex with IIA vWF has a decreased binding activity. Among several anti-vWF monoclonal antibodies (MoAbs) which inhibit ristocetin-induced platelet aggregation and/or vWF binding to GP Ib, only two MoAbs (NMC-4 and RFF-VIII RAG:1) abolished direct binding between purified botrocetin and vWF. This suggests that they recognize an epitope(s) on the vWF molecule in close proximity to the botrocetin binding site.


Enzyme-linked immunoassays for CRM positive hemophilia A using monoclonal antibodies with defined epitopes. Sensitive, monoclonal antibody-based, enzyme-linked immunosorbent assays (ELISAs) for plasma factor VIII (FVIII) antigen have been developed. The assays used five monoclonal anti-FVIII antibodies with known epitopes in four different pairwise combinations. The sensitivity of the assays was increased by use of the ELISA amplification method described by Bobrow et. al. (J. Immunol Methods 125:279, 1989). These assays can distinguish between CRM positive, CRM reduced and CRM negative hemophilia A. A total of 94 plasma samples from 78 different patients were screened and generally consistent results were obtained among the different assays. Such assays are potentially useful in the phenotypic and genotypic analysis of hemophilia A.


Tissue-type plasminogen activator, type 1 plasminogen activator inhibitor and their complex in plasma with disseminated intravascular coagulation. The levels of tissue-type plasminogen activator (t-PA), type 1 plasminogen activator inhibitor (PAI-1), and t-PA/PAI-1 complex antigens were analyzed in the plasma of disseminated intravascular coagulation (DIC) patients and healthy controls. Other fibrinolytic parameters such as the levels of plasminogen, alpha 2-antiplasmin (alpha 2-AP), plasmin/alpha 2-AP (PAP), and D-dimer were also estimated to clarify the fibrinolytic states in these plasmas. The antigens of t-PA, PAI-1, and t-PA/PAI-1 complex were found to increase from 8.5 +/- 4.3, 54.4 +/- 21.2, and 8.6 +/- 3.5 ng/ml in normal plasma to 36.4 +/- 25.1, 106.8 +/- 54.7, and 46.6 +/- 34.5 ng/ml in DIC plasma, respectively. The molar ratio of total t-PA to total PAI-1 was 1:6 and 1:3 in normal plasma and DIC plasma, respectively, indicating an enhanced fibrinolytic state in the DIC plasma. The DIC plasma revealed a significant consumption of plasminogen (62.1 +/- 27.8%), and alpha 2-AP (63.7 +/- 25.3%) and an increase in PAP (2.6 +/- 2.7 micrograms/ml) and D-dimer (3.9 +/- 10.7 micrograms/ml). These results suggest that the production and secretion of t-PA and PAI-1 from endothelial cells were enhanced in DIC, resulting in an increased t-PA/PAI-1 complex with dominant fibrinolytic activity.


Hydroxyethyl starch 200/0.5 reduces infarct volume after embolic stroke in rats. We evaluated isovolumic hemodilution with hydroxyethyl starch 200/0.5 in a rat model of focal cerebral ischemia. This compound avoids the unfavorable viscosity and erythrocyte aggregation abnormalities of low molecular weight dextran during administration over a period of several days. Sprague-Dawley rats, anesthetized with 0.5-1% halothane and 70% N2O, were subjected to silicon cylinder (treated and control groups) or sham (sham group) embolization of the cerebral circulation. Thirty minutes after embolization, the treated group (n = 5) was infused with 11 ml/kg of 10% hydroxyethyl starch 200/0.5, and the control (n = 5) and sham (n = 4) groups were infused with saline for 1 hour. In the treated group, 7.1 ml/kg of blood was withdrawn. After 24 hours, the animals were reanesthetized, and cerebral blood flow was determined with [14C]iodoantipyrine. Alternative brain slices were either incubated with 2,3,5-triphenyltetrazolium chloride for infarct volume determination or frozen for ischemic volume and cerebral blood flow determination using autoradiography. The hematocrit in the treated group was reduced from (mean +/- SEM) 46 +/- 1% to 35 +/- 2% at 1.5 hours (p < 0.01). Cortical blood flow was within the normal range of 115-185 ml/min/100 g, except for the ischemic cortex in the embolized groups, treated and control. The ischemic and infarct volume of the treated group was reduced by 74% (p < 0.05) and 89% (p < 0.05), respectively, from the control group. The treated and sham ischemic and infarct volumes were not statistically different. These data suggest that hydroxyethyl starch 200/0.5 could be an effective treatment for ischemic stroke when administered early, because it reduces infarct and ischemic volumes from control values to levels indistinguishable from those of the sham group.


Hematologic and bone marrow changes after short- and long-term administration of two recombinant bovine granulocyte colony-stimulating factors. Colony-stimulating factors are a category of glycoproteins that are instrumental in the regulation of hematopoiesis and inflammation. This investigation documented the clinical bone marrow and peripheral blood responses to short-term and long-term administration of a recombinant bovine granulocyte colony-stimulating factor (rb-GCSF) and an analog, where the cysteine at position 17 was substituted with a serine (rb-GCSF ser17). The colony-stimulating factors produced the expected changes in the hematologic findings of the bovine subjects in the study, and there was a cell-specific response to the compounds. The sustained neutrophilia in the long-term study indicates that the bovine species can tolerate the administration of recombinant forms of bovine GCSF for extended periods of time without detectable adverse side effects. The neutrophils from the short-term study revealed no apparent fluctuation, either as enhanced or reduced capability to reduce nitro blue tetrazolium as compared to pretreatment neutrophils. The administration of both recombinant forms of GCSF produced large increases in the bone marrow myeloid:erythroid (M:E) ratio concomitantly with the neutrophilias. This is the first preliminary report documenting the bone marrow response of cattle to the native and recombinant (rb-GCSF ser17) forms of bovine GCSF.


Characterization of the specificity and genetic restriction of human CD4+ cytotoxic T cell clones reactive to capsid antigen of rubella virus. Using 11 overlapping synthetic peptides covering more than 95% of the amino acid sequence of capsid protein of rubella virus, 7 CD4+ T cell clones (R10, R11, R18, A2, A10, A11, and A12) isolated from 2 rubella seropositive donors reacted strongly to rubella capsid peptides C6 (residues 119-152), C9 (residues 205-233), or C11 (residues 255-280), respectively, in both proliferation and cytotoxicity assay. Truncated peptides C6E (residues 125-139), C9B (residues 205-216), and C11E (residues 260-272) were shown to be involved directly to the T cell determinants of C6, C9, and C11, respectively. Genetic restriction of these T cell clones was analyzed by using human cell lines with various HLA-DR phenotypes as targets and/or antigen-presenting cells in cytotoxicity assay and/or proliferation assays. The results indicated that the recognition of peptide C6 by T cell clones (R11 and R18) was associated with DRw9 molecule, while the HLA restriction element of the responses of other T cell clones (A2 and A11, A10, and A12) that reacted with peptide C9 or C11 was DR4 molecule. However, there may be a cross-recognition by the T cell clone (A12) between DR1 and DR4 subtypes.


[Immunochemical analysis of the synthetic peptides incorporated into the protein antigenic determinant of the potato virus X coat]. Three peptides located in the N-terminal region of the potato virus X coat protein were synthesized by hand solid phase method for epitope mapping of this protein. One of these peptides (nanopeptide) interacted with monoclonal antibodies to native virus X. On the basis of these studies it was assumed, that amino acid sequence of the potato virus X coat protein, which included lysine residue in position 19, is located on the virion surface.


Synergistic interaction between ligands binding to the CD4 binding site and V3 domain of human immunodeficiency virus type I gp120. We demonstrate that soluble CD4 (sCD4) or a monoclonal antibody (mAb), 39.13g, binding to a conformational epitope of gp120 involved in CD4 binding, and mAbs binding to the V3 domain of gp120, can synergistically neutralize human immunodeficiency virus type I (HIV-1). In contrast, a neutralizing mAb binding to a linear epitope within the CD4 binding domain was unable to exert a synergistic effect in combination with V3 mAbs, suggesting that synergism is dependent on ligands binding to the critical, discontinuous, gp120 residues constituting the CD4 binding site. A number of V3 mAbs showed increased binding to virion gp120 in the presence of sCD4, suggesting a mechanism for the synergistic neutralization. This effect was not observed with recombinant or detergent solubilized viral gp120, suggesting that the oligomeric structure of gp120 on viral particles affects V3 epitope exposure. This hypothesis is supported by the ability of two new V3 mAbs, 8/38c and 8/64b, to only neutralize HIV-1 in the presence of sCD4 or mAb 39.13g; binding studies demonstrate that these mAbs only bind to virion gp120 in the presence of sCD4. Thus, V3 epitope exposure is modulated by the interaction of virion gp120 with ligands specific for the CD4 binding domain and results in enhanced antibody-mediated neutralization.


5' end-dependent translation initiation of hepatitis C viral RNA and the presence of putative positive and negative translational control elements within the 5' untranslated region. Hepatitis C virus (HCV) is a distant relative of pestiviruses and flaviviruses, but it has a 5' untranslated region (UTR) with some features structurally similar to that of picornaviruses. In order to test the role of the 5' UTR in controlling the expression of the HCV polyprotein, we fused full-length or deleted versions of the 5' UTR of HCV-1 RNA to chloramphenicol acetyl transferase (CAT) mRNA to monitor CAT activity in vivo. We found: (1) the full-length 5' UTR of HCV-1 RNA is translationally inactive while 5' deletions which mimic a 5' subgenomic RNA detected in vivo are active, (2) an efficient cis-acting element which represses translation is found at the 5' terminus, (3) a putative element which enhances translation is found near the 3' terminus of the 5' UTR, (4) additional cis-acting elements including small open reading frames (ORFs) upstream from the putative enhancer element downregulate translation. We did not find evidence supporting the existence of an internal ribosome entry site in the 5' UTR of HCV-1 RNA. These data suggest that HCV may employ a distinctive translation control strategy such as the generation of subgenomic viral mRNA in infected cells. Translational control of HCV might be responsible for some of the characteristic pathobiology seen in viral infection.


The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. To study the role of the precursor to the membrane protein (prM) in flavivirus maturation, we inhibited the proteolytic processing of the Murray Valley encephalitis (MVE) virus prM to membrane protein in infected cells by adding the acidotropic agent ammonium chloride late in the virus replication cycle. Viruses purified from supernatants of ammonium chloride-treated cells contained prM protein and were unable to fuse C6/36 mosquito cells from without. When ammonium chloride was removed from the cells, both the processing of prM and the fusion activity of the purified viruses were partially restored. By using monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of MVE virus, we found that at least three epitopes were less accessible to their corresponding antibodies in the prM-containing MVE virus particles. Amino-terminal sequencing of proteolytic fragments of the E protein which were reactive with sequence-specific peptide antisera or MAb enabled us to estimate the site of the E protein interacting with the prM to be within amino acids 200 to 327. Since prM-containing viruses were up to 400-fold more resistant to a low pH environment, we conclude that the E-prM interaction might be necessary to protect the E protein from irreversible conformational changes caused by maturation into the acidic vesicles of the exocytic pathway.


CD4-independent inhibition of lymphocyte proliferation mediated by HIV-1 envelope glycoproteins. The cytopathic effects of HIV-1 produced by direct infection of human T cells do not account for the disproportionate loss of CD4-positive lymphocytes during the course of HIV infection. Previous studies have demonstrated the inhibition of uninfected human T cell activation and proliferation by the HIV-1 envelope glycoproteins, presumably due to gp120-CD4 interactions. To examine the ability of HIV-1 to inhibit T cell proliferation in the absence of both direct infection and gp120-CD4 interactions, we tested the effect of HIV-1 on mouse T cell proliferation. Culture media containing HIV-1 released from infected cells inhibited T lymphocyte proliferation in response to interleukin-2 (IL-2). Studies to explore the mechanism of this inhibition suggested that the decrease in proliferation resulted from interactions between HIV-1 and the mouse cells, but did not involve IL-2/IL-2 receptor interactions. We used monoclonal antibodies to demonstrate that the HIV-1 envelope glycoproteins were required for the inhibition of murine T cell proliferation. Anti-gp120 antibodies completely restored proliferation, indicating that the surface protein gp120 was primarily required for the inhibition of proliferation. However, antibodies directed against the transmembrane protein of HIV-1 (gp41) also partially restored lymphocyte proliferation. The functional significance of the HIV-1 envelope protein epitopes recognized by the monoclonal antibodies is discussed.


Identification of amino acids located in the antibody binding sites of human hepatitis A virus. Antigenic mutants of human hepatitis A virus (human-HAV) were isolated by their resistance to neutralizing monoclonal antibodies raised to human-HAV. The nucleotide sequence determined for the capsid regions of 12 mutants identified amino acid changes that clustered in three non-overlapping sites; one in VP3 and two in VP1. All mutants had a change at amino acid residue 70 in VP3, indicating its primary importance for antibody binding. Ten mutants had two amino acid changes occurring in the VP3 site as well as one in one of the two VP1 sites. These data suggest that both sites in VP1 interact with the single VP3 site to form the immunodominant epitope of HAV. The amino acid changes found in the antigenic mutants of human-HAV selected in this study were located in the same positions as changes found in strains of HAV isolated from Old World monkeys. These simian strains of HAV are not recognized by most monoclonal antibodies raised to human-HAV, suggesting that the observed amino acid changes are part of the antibody binding site.


[A histochemical analysis of the reactions of the lymphoid organs and liver in a radiation lesion (experimental research)]. A histochemical study is presented of the functional state of the lymphoid organs and liver of guinea pigs under the effect of small and large doses of roentgen rays. Changes were found in the quantitative content of nucleic acids and lipofuscin already several days after irradiation accompanied by structural-qualitative reorganization of cells.


[The factors influencing the incidence of heart rhythm disorders after the radical correction of the tetralogy of Fallot]. The frequency and factors favouring the development of cardiac rhythm disorders were studied in 306 patients operated on radically for Fallot's tetralogy (follow-up: 6 months--29 years). This complication was found in 99 patients (32.3%). Most patients showed ventricular arrhythmia--62 (62.6%). Paroxysmal tachycardia was noted in 11 patients, atrio-ventricular block in 15 patients, combination of different arrhythmias in 11 patients. Risk factors were: inadequate correction of the malformation, age over 15 at the time of operation, approach through the right ventricle with plastic repair of the drainage tract, five years after surgery.


Plasma levels of insulin-like growth factor binding protein-1, and growth hormone binding protein activity from birth to the third month of life. Serum levels of insulin-like growth factors (IGF-1 and IGF-2), insulin, insulin-like growth factor binding protein-1 (IGFBP-1), growth hormone (GH) and growth hormone-binding protein (GH-BP) activity were assessed in a group of healthy newborns and reevaluated at one and three months of life in six of them. A significant decrease in IGFBP-1 plasma levels was observed at one month (p < 0.002) and three months (p < 0.02) of life compared to cord blood values. IGF-1 plasma levels did not change during the first three months of life. In contrast, IGF-2 plasma levels increased significantly at three months of life compared to cord blood values (p < 0.002). GH plasma levels showed a significant decrease at three months of life (p < 0.03). GH-BP activity was low at birth and did not change significantly during the first three months of life. The low GH-BP activity may reflect the GH receptor status, indicating that GH receptors are poorly expressed in early infancy. The high IGFBP-1 plasma levels in newborns could be important in protecting them from hypoglycemia.


Phenotypic analysis of bronchoalveolar lavage lymphocytes from acquired immunodeficiency patients with and without Pneumocystis carinii pneumonia. A study was performed to reveal possible differences in lymphocyte subpopulations from bronchoalveolar lavage (BAL) of acquired immunodeficiency patients with and without Pneumocystis carinii pneumonia. Forty-one consecutive human immunodeficiency virus-seropositive patients were studied. Pneumocystis carinii infection was detected in the BAL fluid from 18 patients. The BAL lymphocyte subpopulations were determined by surface marker analysis with the immunoperoxidase slide assay. No significant differences in the percentage of CD4+ and CD8+ lymphocytes were found between the two groups. The percentage of CD57+ natural killer (NK) cells was significantly higher in the Pneumocystis carinii-negative group than in the -positive group. Since NK cells protect from microbial infections, it is conceivable that the loss of CD57+ NK cells may be one of the phenomena leading to the immunodeficiency state that underlies the pulmonary complications characteristic of the acquired immunodeficiency syndrome.


Serum parathyroid hormone-related protein concentrations in patients with hematologic malignancies or solid tumors. The clinical significance of parathyroid hormone-related protein in humoral hypercalcemia of malignancy was investigated by determining the serum parathyroid hormone-related protein concentrations in 167 normal subjects, 56 patients with hematologic malignancy and 144 patients with solid tumor. Serum parathyroid hormone-related protein was measured with a radioimmunoassay kit that recognizes the C-terminal portion of the molecule. The serum parathyroid hormone-related protein concentrations were 20.2-50.8 pmol/l (mean +/- 2 SD) in normal subjects, and were elevated in 80% of the patients with malignancies with hypercalcemia, including squamous cell carcinoma and adult T cell leukemia. Moreover, two cases of B cell non-Hodgkin's lymphoma with hypercalcemia had high serum parathyroid hormone-related protein concentrations, which varied in parallel with the tumor size during the clinical course. Of 136 patients with solid tumors with normocalcemia, the serum parathyroid hormone-related protein concentration was slightly elevated in only 5.1%, all of whom were at an advanced stage. These data indicate that determination of the serum parathyroid hormone-related protein concentration is useful for differential diagnosis of humoral hypercalcemia of malignancy and prediction of its development.


Growth hormone and insulin-like growth factor regulate insulin-like growth factor-binding protein-1 in Laron type dwarfism, growth hormone deficiency and constitutional short stature. Insulin-like growth factors (IGFs) mediate the effects of growth hormone (GH), and the insulin-like growth factor-binding proteins (IGFBPs) modulate the actions of IGFs in tissues. We studied the circulating levels of IGFBP-1 in 6 children and 9 adults with Laron type dwarfism (LTD), in 11 children and 21 adults with growth hormone deficiency (GHD), and in 8 children with constitutional short stature. Compared with the situation in healthy children, the basal serum IGFBP-1 concentration was 5.4-fold higher in LTD children, 4.1-fold higher in GHD children, and 3.8-fold higher in children with short stature (p < 0.02 vs controls in all groups). In adult patients with multiple pituitary hormone deficiency (MPHD), the IGFBP-1 concentration was 2-fold elevated, but it was normal in adult LTD patients. Intravenous (N = 10) or subcutaneous (N = 9) administration of IGF-I (75 micrograms.kg-1 and 150 micrograms.kg-1, respectively) in LTD children resulted in a rapid 50-60% fall in serum insulin (p < 0.02), a decline in blood glucose and a concomitant 40-60% rise of IGFBP-1 levels (p < 0.05). Treatment for seven days with IGF-I (150 micrograms.kg-1 x d-1) resulted in a decrease by 34% and 44% of serum IGFBP-1 level in two out of three children with LTD. After prolonged GH therapy, the IGFBP-1 level fell in GHD children by 29% (p < 0.05), in GHD adults by 52% (p < 0.02) and in children with constitutional short stature by 17% (p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)


Determination of insulin-like growth factor-2 in feto-maternal circulation during human pregnancy. In order to clarify the roles of insulin-like growth factors (IGFs) on the human maternal-fetal environment, IGF-2 and IGF-1 levels were investigated in human plasma and amniotic fluid during pregnancy. Initially, new radio-immunoassay (RIA) systems for human IGF-2 could be developed. The sensitivity of this assay was 17.5 pg/tube and the cross-reactivity with IGF-1 was 0.64%. The pattern of change of maternal plasma IGF-2 in early pregnancy differed from that of IGF-1, but both IGF levels increased progressively in the second half of gestation, and decreased to non-pregnancy levels in the puerperium. Maternal levels of IGF-2 were approximately seven times greater than those of IGF-1. The ratio of IGF-2 to IGF-1 was 3.2 in amniotic fluid. The IGF concentrations in amniotic fluid obtained in the second trimester were significantly greater than those of term specimens, and closely related to those of prolactin (PRL) in amniotic fluid. The highest IGF-2 to IGF-1 ratio (15.9) was found in umbilical vein plasma. On Sephadex G-150 gel-chromatography of maternal and fetal plasma at term, two apparent peaks of unsaturated IGF-2 binding protein (BP) could be detected in both 150 and 40 kilo dalton (kD) regions. One main peak of unsaturated IGF-2 BP could be determined in the 40 kD region in the amniotic fluid at term. High concentration of IGF-2 could be detected in feto-maternal circulation during human pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)


High dose aprotinin to reduce blood loss in patients undergoing redo open heart surgery. Recently, aprotinin has been demonstrated to be effective in reducing post-operative bleeding after open heart surgery. The aim of this study was to evaluate the effectiveness of high dose aprotinin in patients undergoing redo open heart surgery in whom perioperative bleeding is generally increased. Fifty-eight patients, mean age 55 years, underwent a second open heart surgery for single valve replacement in 37 patients (mitral or aortic), for double valve replacement in 16 patients and for coronary artery by-pass grafting in 4 patients. In 30 of the 58 patients (Group A) aprotinin was infused with the following scheme: 2 million KIU in 30 minutes during induction of anesthesia, 2 million KIU in the priming volume of extracorporeal circulation and 500,000 KIU every hour until the end of the surgical procedure. The remaining 28 patients served as control group (Group B). No adverse reactions to aprotinin were observed. There was no significant difference in cardiopulmonary by-pass time and minimum temperature achieved between the two groups of patients. During the operative day blood loss was significantly lower in group A than in group B (492 +/- 336 ml vs. 628 +/- 273 ml) (p < 0.01). This difference was not significant during the following days. However 3 days after the operation a still significant difference was reported in total blood loss between group A and group B (816 +/- 507 ml vs. 1018 +/- 373 ml) (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Blood loss and use of blood in internal mammary artery and saphenous vein bypass grafting with and without adding a single, low-dose of aprotinin (2 million units) to the pump prime. In comparison with saphenous vein bypass grafting (SVG) without a pleurotomy, internal mammary artery (IMA) bypass surgery might enhance more blood loss by the intrathoracic wound and a wide opened pleura. Low-dose aprotinin (Trasylol) reduces blood loss and consequently, the use of homologous blood. We studied the blood loss and blood requirements peri-operatively in 70 patients, who underwent either SVG-(n = 35) or IMA-surgery (n = 35) without aprotinin and another 70 patients who underwent SVG- (n = 35) or IMA-surgery (n = 35) with a single dose of aprotinin, added to the pump prime. We also determined the hemoglobin and total protein content of the shed blood. Without aprotinin administration, the mean intra-operative and post-operative blood loss was significantly less (p < 0.01) in SVG-patients, than in IMA-patients. We observed no statistical differences in the mean blood requirements between SVG- and IMA-surgery (1.2 units and 1.6 units). The use of any homologous blood product was similarly averted in 39% of the SVG-patients, and in 48% of the IMA-patients. Treatment with aprotinin significantly lessened (p < 0.01) the mean intra-operative blood loss only in IMA-patients. Post-operative blood loss was diminished with 45% in IMA-patients and 33% in SVG-patients, being significantly less (p < 0.01) after SVG, than after IMA-surgery. Blood requirements were significantly lowered (p < 0.01): a total mean of 0.2 units in SVG- and 0.6 units in IMA-patients (NS). Any homologous blood product was prevented in 78% of the IMA-patients and in 87% of the SVG-patients (NS). The mean loss of hemoglobin and total protein per 100 ml of shed blood was similar in IMA-, and SVG-patients with or without aprotinin, although aprotinin diminished the total amounts in both groups with 50% (p < 0.01).


Proliferative activity in normal salivary gland and pleomorphic adenoma. A study by argyrophilic nucleolar organizer region (AgNOR) staining. Proliferative activity of the structural components of normal salivary glands (27 cases) and characteristic histological areas in salivary pleomorphic adenoma (51 cases) was examined by visualization of argyrophilic nucleolar organizer regions (AgNORs). In the normal salivary gland, there were clusters of cells, each with two or three AgNOR dots, within the intercalated ducts. The mean number of AgNORs in this portion was 1.80, which was significantly higher than in the other epithelial parts. This indicates vigorous proliferative activity of the intercalated ducts, and thus the ducts may play an important role as reserve cells in neoplastic transformation. In pleomorphic adenoma, solid nests had a high AgNOR count (1.61), showing a significant difference from the tumor cells scattered in the "stroma". Thus the decrease in proliferative ability might occur concurrently with formation of the extracellular matrix.


Lipid-protein interactions in neurotropic Na-pump regulation. The role of lipid-protein interaction in the regulation of the brain Na-pump by different neurotropic agents (prostaglandin E2, middle molecules from blood plasma of uremic patients, neuropeptide galanin, the oligopeptide fraction from brain) was investigated. We established a definite correlation between the lipid status (the peroxidativity of the lipids, the phospholipids and cholesterol content) of the Na,K-ATPase enzyme preparation (plasma membrane fragments) and the influence of the neurotropic agents. Besides, after the treatment with delipidative agents (phospholipase A2, SDS) the inhibitory effects of these neurotropic agents, were diminished significantly. These facts do not contradict our previous suggestion that the lipid-protein interactions underlay the regulative action mechanism of the natural bioactive ligands.


Expression of insulin-like growth factors (IGFs) and their binding proteins (IGF BPs) during pancreatic development in rat, and modulation of IGF actions on rat islet DNA synthesis by IGF BPs. Growth of pancreatic beta-cells is an essential feature of development in order to maintain insulin availability. While glucose has been shown to increase beta-cell DNA synthetic rate in cultures of isolated islets of Langerhans, there is also evidence for a local control of beta-cell growth involving insulin-like growth factors (IGFs). IGF I and II and their specific binding proteins (IGF BPs) are expressed within the developing rat pancreas, and released by isolated fetal rat islets of Langerhans. Glucose-induced beta-cell growth is not mediated by IGF-I, since IGF release does not alter in response to changes in glucose availability. In contrast, IGF BP release is positively associated with glucose concentrations over the physiological range and is therefore a candidate. Exogenous hIGF BP1 or bIGF BP2 synergistically interacted with hIGF I or II to increase DNA synthesis within isolated fetal rat islets. These results suggest a role for IGF BPs in the regulation of beta-cell growth. They may act independently or by modulation of IGF bioavailability during pancreatic development.


Growth factor expression in normal and diabetic rats during peripheral nerve regeneration through silicone tubes. The silicone tube model of regeneration has proved to be an invaluable tool for experimental studies aimed at understanding expression of growth factors during normal and abnormal metabolic states of regeneration. Since the morphological parameters of nerve growth and myelination are well-defined and easily identified in this model, the expression of both diffusible and intracellular-acting growth factors can be readily correlated with the occurrence of these cellular events. These studies facilitate the study of the cellular and molecular events that accompany regeneration. Further, because the sciatic nerve can be traced up to its corresponding neurons, growth factor gene expression can also be studied by in situ hybridization and Northern blotting techniques. This is particularly important in defining the cell source of extracellularly released growth factors. Finally, and most importantly, the regeneration process in the normal or diseased metabolic state (such as diabetes) can be manipulated via the administration of adjuncts to the tube that either promote or inhibit regeneration. Further studies in this regard, and in the identification of growth factors involved and their role during regeneration should shed some light on the pathogenesis and possible means of mitigating or reversing diabetic neuropathy.


Interferon--a candidate mediator of cell growth. The IFNs are a class of compounds comprising at least three different entities alpha, beta, gamma, with a clearly defined capacity to enhance expression of MHC antigens, as well as modulate growth in a variety of tissues. They are thus candidates for autoimmune cellular destruction and growth modulation. IFN has now been shown to modulate important developmental and injury-related events in both the pancreas and CNS. Other cytokines may also be important players in these biological events and it remains to be elucidated how IFN and other cytokines exert their effects in these organs.


Response rate and cell-cycle changes due to intra-arterial infusion chemotherapy with cisplatin and bleomycin for locally recurrent uterine cervical cancer. Intra-arterial infusion with cisplatin (CDDP) and bleomycin (BLM) was carried out in 21 patients with locally recurrent uterine cervical cancer who were previously treated with irradiation alone. Patients were treated with a bolus infusion into both internal iliac arteries of 50 mg/m2 of CDDP and 30 mg/m2 of BLM. Two to four courses of the infusion therapy were given to each patient, and the response rate, the tumor and serum drug concentrations, and the cell kinetics in tumor tissue were evaluated. The response rate (CR+PR) was 71.4% according to the WHO criteria. There was no difference, in the tumor tissue concentrations of CDDP and BLM between responders and nonresponders. Although the DNA ploidy of tumor cells was not significantly different between the two groups before treatment, both the labeling index with BrdU and the proliferation index with flow cytometry significantly increased 24 hours after treatment in responding tumors but not in nonresponding tumors. These results show that intra-arterial infusion with CDDP and BLM improves the prognosis of recurrent cervical cancer and that labeling and proliferation indices may be useful for determining the response of cervical cancer to intra-arterial chemotherapy.


Conditions associated with normal levels of prostate-specific antigen and elevation of prostatic acid phosphatase. Thirteen cases are reported with normal levels of prostate-specific antigen and elevated prostatic acid phosphatase (PAP). Conditions that were associated with an elevated PAP were myeloproliferative syndromes (four cases), metastatic nonprostatic carcinoma (six cases), prostatic carcinoma (one case), tuberculosis with a concurrent lupus-like syndrome (one case), and osteomyelitis (one case). The inclusion of PAP in the initial investigation of cases with suspected prostatic disease results in a decreased specificity.


Hyperthermo-chemo-radiotherapy as a definitive treatment for patients with early esophageal carcinoma. Ten patients with early stage esophageal carcinoma were treated with hyperthermo-chemo-radiotherapy (HCR) without surgery. The reasons for the inoperability of these patients included medically inoperable unresectable cancers, advanced age, and/or refusal to undergo surgery. The diagnosis of early esophageal carcinoma was determined by esophagograms, endoscopy, and ultrasonography. Squamous cell carcinoma was histopathologically confirmed in each case. Each patient underwent four to nine sessions of hyperthermic treatment combined with external irradiation and chemotherapy using bleomycin; eight of these patients received additional radiation, and two terminated treatment after the HCR therapy. The tumors in all patients showed either a complete response (CR) or a partial response (PR) after HCR therapy; in two patients viable cancer cells remained, but later disappeared after additional radiation. Five patients experienced no local recurrence for 12 to 70 months and are now alive and doing well, three died of other medical conditions without any evidence of esophageal cancer, and two died of recurrent esophageal cancer 20 to 27 months after initial admission. All ten patients tolerated the HCR well without any systemic side effects. However, in two patients, esophageal erosion was recognized endoscopically. HCR therapy therefore deserves serious consideration when treating patients with small malignant lesions of the esophagus who, for various reasons, are unable to undergo surgery.


The admission serum lipase:amylase ratio differentiates alcoholic from nonalcoholic acute pancreatitis. To determine whether the lipase:amylase ratio differentiates alcoholic from nonalcoholic pancreatitis, we conducted a retrospective review of charts with the diagnosis of acute pancreatitis at the George Washington University Medical Center between January 1988 and July 1990. A total of 446 charts were reviewed. For a patient to be included in the subsequent analysis, the following criteria were met: 1) the patient had typical symptoms of pancreatitis, 2) serum amylase and lipase were analyzed on admission, and 3) a computerized tomographic (CT) scan or ultrasound of the abdomen was obtained within 72 h of admission. Forty-seven charts satisfied the requirements for inclusion in the study. Data collected from the charts included history of alcohol consumption, age, sex, race, admission serum amylase and serum lipase (from this the amylase:lipase ratio was calculated), peak serum amylase and serum lipase, and number of days of abdominal pain before admission. Patients with alcoholic pancreatitis had significantly lower serum amylase levels and significantly higher lipase:amylase ratios than those with nonalcoholic pancreatitis (p < 0.01). There was no difference in the serum lipase between the groups. The higher the lipase:amylase ratio, the greater the specificity of alcohol as the etiology of acute pancreatitis. Only patients with alcoholic acute pancreatitis had lipase:amylase ratios > 5.0 (sensitivity 31%, specificity 100%). Our data point to the clinical utility of the lipase:amylase ratio in differentiating alcoholic from nonalcoholic acute pancreatitis. Prospective studies will be needed to confirm the clinical utility of this ratio.


Des-gamma-carboxy prothrombin (PIVKA-II) and alpha-fetoprotein-producing IIc-type early gastric cancer. We describe the case of a 56-yr-old man with primary gastric adenocarcinoma, who had an extremely high plasma level of des-gamma-carboxy prothrombin (2.45 AU/ml) and of serum alpha-fetoprotein (2810 ng/ml). Histopathologically, the gastric cancer was a IIc type of early cancer which consisted of a combination of a poorly differentiated adenocarcinoma and a well-differentiated tubular adenocarcinoma. The association of a hepatic tumor including hepatocellular carcinoma or liver metastasis was ruled out by ultrasonography, computed tomography, radiocolloid liver scan, magnetic resonance imaging, and angiography. Foci strongly resembling hepatocellular carcinoma (hepatoid differentiation) were noted in the gastric tumor. Localization of des-gamma-carboxy prothrombin and alpha-fetoprotein within the tumor cells, especially within the hepatoid differentiated foci, was demonstrated by the immunohistochemical staining of tissue obtained at biopsy and the resected specimen. This case seems to be the first case reported in which des-gamma-carboxy prothrombin was produced by the gastric cancer. This finding supports the theory of hepatoid differentiation of a gastric cancer.


Audiologic profiles of children with Kawasaki disease. Kawasaki disease (KD) is an idiopathic vasculitis associated with systemic inflammation and profound immunoregulatory changes. Recent reports from Japan and the United States have documented the association of sensorineural hearing loss (SNHL) with acute KD. To further characterize the nature and prevalence of this complication, we prospectively evaluated the hearing of 40 consecutive patients with acute KD at a single institution. Standard audiometric procedures were used, including visual reinforcement audiometry and play audiometry. Auditory brainstem response (ABR) testing using clicks and tone pips (1000-4000 Hz) was performed in patients with abnormal or unreliable results on behavioral audiometry. Acoustic immittance measurements were obtained on all patients. Of the 23 males and 17 females (mean age 3.2 +/- 2.3 years, range 0.6-11.1 years), all but three were evaluated and treated with aspirin and intravenous gama globulin within 1 month of onset of fever. Seven children had test results suggesting sensorineural threshold shifts, 16 had normal hearing, and 14 had inconclusive hearing evaluations. Laboratory data in patients with hearing threshold shifts revealed significantly longer duration of fever (4.1 +/- 1.0 versus 1.9 +/- 0.5 days), and a tendency for higher temperatures and white blood cell counts at diagnosis compared to those with normal hearing. Results suggest that transient as well as persistent SNHL may be associated with the acute vasculitis of KD, and may be associated with laboratory markers indicating more severe systemic inflammation. Audiologic screening should be considered for all patients following KD.


Childhood peripheral neuropathy with autoantibodies to myelin glycoprotein P0. The serum of a 4-year-old child with severe hypertrophic peripheral neuropathy contained high-titer polyclonal antibodies, mainly of IgG class, reacting with the 30-kd P0 glycoprotein of peripheral nerve. This was demonstrated by indirect immunofluorescence, immunoblot analysis of myelin proteins, and enzyme-linked immunosorbent assay with purified P0 glycoprotein. The antibodies did not react with myelin-associated glycoprotein, sulfated glycuronic acid-containing paragloboside (SGPG and SGLPG), or other peripheral nerve glycolipids or gangliosides. To our knowledge this is the first report of strong antibody activity specifically directed against P0. This antibody may play a causative role in the pathogenesis of the peripheral neuropathy in this patient.


[Histopathology and ultrastructure of opportunistic infections of the digestive tract in acquired immunodeficiency syndrome]. Light and electron microscopy studies of digestive tract biopsy specimens from AIDS patients are very useful for the detection of opportunistic pathogens which may be located in: the lumen (bacteria, Candida albicans, Giardia lamblia), enterocyte brush border (Cryptosporidium sp), enterocyte cytoplasm (Enterocytozoon bieneusi), enterocyte nuclei (cytomegalovirus), or cytoplasm of lamina propria macrophages (Mycobacterium avium intracellulare). These studies may also be useful to detect combinations of morbid conditions and to evaluate the effectiveness (if any) of therapeutic agents.


Fontan procedure for hypoplastic left heart syndrome. Since 1985, 354 neonates have undergone palliative reconstruction for hypoplastic left heart syndrome with 109 early deaths and 12 late deaths. Of the survivors, before 1989, 77 patients underwent a subsequent modified Fontan operation, consisting of baffling the atrial septal defect to the tricuspid valve (initial 25 patients) or intraatrial baffling of the inferior vena cava to the pulmonary arteries and superior vena cava (52 patients). There were 17 early deaths and three late deaths. Major serous effusions developed in 42 patients (54%) after Fontan operation. Since 1989, a staged approach to Fontan's operation was undertaken in an effort to reduce the volume load of the right ventricle as early as possible, to minimize the impact of rapid changes in ventricular geometry and diastolic function that can accompany a primary Fontan operation, and to reduce effusive complications. Thus, at a mean age of 6 months, 121 patients have undergone closure of aortopulmonary shunt, augmentation of central pulmonary arteries, and association of the superior vena cava with the branch pulmonary arteries (hemi-Fontan procedure). Of these, 61 patients have already undergone completion of the Fontan procedure with six early deaths and three late deaths. Major serous effusions developed in 28 patients (46%) with the staged Fontan. For perspective, the contemporary experience since January 1991 consists of 58 neonates who have undergone initial palliation with 11 deaths (19%), 17 patients who have undergone the hemi-Fontan procedure with one death (6%), and 21 patients who have undergone completion of the Fontan operation with one death (5%).(ABSTRACT TRUNCATED AT 250 WORDS)


Aprotinin therapy for reoperative myocardial revascularization: a placebo-controlled study. We tested the efficacy and safety of aprotinin in 169 patients undergoing isolated reoperative myocardial revascularization. Patients were randomly assigned to high-dose aprotinin, low-dose aprotinin, or placebo treatment groups in a double-blind, placebo-controlled study. Treatment groups did not differ significantly with respect to age, sex, red cell mass, number of grafts, use of internal thoracic artery, or incidence of preoperative aspirin therapy. Patients treated with aprotinin had a significant reduction in postoperative chest tube drainage (720 +/- 753, 866 +/- 1,636, and 1,121 +/- 683 mL, respectively, for high-dose aprotinin, low-dose aprotinin, and placebo; p < 0.001). Transfusion requirements were reduced in aprotinin-treated patients (2.1 +/- 4.2, 4.8 +/- 11.8, and 4.1 +/- 6.2 units for high-dose, low-dose, and placebo, respectively; p < 0.001). A similar reduction in chest tube drainage and transfusion requirements was seen in patients using aspirin preoperatively. Q-wave myocardial infarctions were increased in the aprotinin subgroups (17.5%, 14.3%, and 8.9% for high-dose, low-dose, and placebo groups; not significant). Acute vein graft thrombosis was found in six of 12 vein grafts studied at postmortem examination in patients receiving aprotinin but not in any of five grafts in patients receiving placebo. We conclude that aprotinin is extremely effective in reducing bleeding and transfusion requirements and may increase the risk of graft thrombosis.


Low-dose aprotinin in internal mammary artery bypass operations contributes to important blood saving. The effect on postoperative blood loss and blood use of blood-saving treatment with or without 280 mg of low-dose aprotinin (2 million kallikrein inactivator units) was studied in 200 consecutive patients undergoing either unilateral or bilateral internal mammary artery bypass grafting. Postoperative blood loss and total units of homologous blood products were similar in patients having either bypass procedure without aprotinin treatment. In patients given aprotinin, postoperative blood loss and use of homologous blood products were significantly lower (p < 0.05). The use of any donor blood product was prevented in 78% of the patients given aprotinin versus only 45% of patients treated without aprotinin. None of the aprotinin-treated patients underwent repeat thoracotomy for excessive bleeding; repeat thoracotomy was indicated in 8% of the patients having bilateral internal mammary artery grafting without aprotinin treatment. These results demonstrate that low-dose aprotinin reduces blood loss and blood use significantly and prevents excessive bleeding.


[Treatment and staining of smears and sections for detection of microorganisms]. Detection of bacteria in smears and sections is necessary for a proper diagnosis of infectious diseases. Main methods of staining for a light- and luminescent microscopy and prescriptions of the dye preparation are presented.


Synthesis and characterization of rat interleukin-10 (IL-10) cDNA clones from the RNA of cultured OX8- OX22- thoracic duct T cells. A cDNA of the complete coding region of rat IL-10 was cloned and sequenced using RNA isolated from a cultured population of thoracic duct T-lymphocytes obtained from Trichinella spiralis infected animals. The OX8- OX22-T-helper cells were stimulated in vitro with Concanavalin A for 24 hours prior to harvest. Reverse transcription of cellular RNA was primed with oligo-dT followed by amplification of IL-10 specific cDNA by polymerase chain reaction with synthetic oligo nucleotide primers chosen from two highly conserved regions of mouse and human IL-10. The sequence of the coding region of the amplified, cloned rat IL-10 cDNA is 90% identical to the mouse and 82% identical to the human IL-10 cDNA coding regions.


Functional study of the germinal angiotensin I-converting enzyme promoter. Polymerase chain amplification experiments indicate that the germinal specific promoter of the angiotensin I-converting enzyme (ACE) is completely extinguished in somatic tissues. Despite this very strict specificity of expression, the germinal ACE promoter is active in transient transfection experiments in two somatic cell lines and one cell line of germinal origin. The analysis of the promoter shows the existence two regulatory elements within the first 350 bp: a proximal positive element and a distal negative element.


Loss of collagenase gene expression in immortalized clones of SV40 T antigen-transformed human diploid fibroblasts. We isolated a cDNA clone whose expression was lost during immortalization. The subtractive hybridization was performed between a genetically matched pair of mortal and immortal lines of SV40 T antigen-transformed MRC-5. The clone was found to code human interstitial collagenase. The expression of collagenase gene was almost completely shut off in seven out of eight independent immortalized clones. In addition, the levels of collagenase expression were dramatically increased toward crisis in the T antigen-transformed but mortal cells. These findings suggest the possibility that the regulatory mechanism of collagenase expression is related to both processes of in vitro aging and immortalization.


cDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of the silkworm, Bombyx mori. We have identified the cDNAs encoding pheromone biosynthesis activating neuropeptide (PBAN) using PCR technique. The nucleotide sequence showed that the PBAN gene encodes, besides PBAN, diapause hormone and three putative amidated peptides. These four peptides share with PBAN the C-terminal pentapeptide amide which is corresponding to the shortest fragment with pheromonotropic activity. The organization of the PBAN gene is characteristic of several short neuropeptides and has some degree of similarity to that of the gene for the insect neuropeptide FMRFamide. Thus, the PBAN gene products construct a family of structurally related peptides and have various biological functions.


Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from L-arginine. The objective of this study was to determine whether constitutive nitric oxide (NO) synthase from rat cerebellum could be regulated by the two products of the reaction, NO and L-citrulline, utilizing L-arginine as substrate. NO synthase activity was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine in the presence of added cofactors. The rate of citrulline formation in enzyme reaction mixtures was non-linear. Addition of superoxide dismutase (SOD; 100 units) inhibited NO synthase activity and made the rate of product formation more non-linear, whereas addition of oxyhemoglobin (HbO2; 30 microM) increased NO synthase activity, made the rate of product formation linear and also abolished the effect of SOD. Added NO (10 microM) inhibited NO synthase activity and this inhibition was potentiated by SOD and abolished by HbO2. Added L-citrulline (1 mM) did not alter NO synthase activity. The two NO donors, S-nitroso-N-acetylpenicillamine (200 microM) and N-methyl-N'-nitro-N-nitrosoguanidine (200 microM) mimicked the inhibitory effect of NO and inhibition of NO synthase activity by NO was reversible. These observations indicate clearly that NO formed during the NO synthase reaction or added to the enzyme reaction mixture causes a reversible inhibition of NO synthase activity. Thus, NO may function as a negative feedback modulator of its own synthesis.


Molecular cloning of the human brain and gastric cholecystokinin receptor: structure, functional expression and chromosomal localization. The receptors for the brain and gastrointestinal peptide, cholecystokinin, can be classified into CCKA and CCKB subtypes. Having recently cloned the rat CCKB receptor, we used it's cDNA to isolate the human CCKB receptor homologue from brain and stomach which encodes a 447 amino acid protein with 90% identity to both rat CCKB and canine gastrin receptors. Northern hybridization identifies transcripts from stomach, pancreas, brain and gallbladder. The CCKB receptor gene maps to chromosome 11. Expression of the receptor cDNA in COS-7 cells was characteristic of a CCKB receptor subtype pharmacology. These data confirm that we have cloned a novel gene for the human brain and stomach CCKB receptor.


Cancer cell binding to E-selectin transfected human endothelia. Human endothelial cells were transiently transfected with E-Selectin which enabled us to study tumor cell/endothelial interactions following engagement of E-Selectin without the added complications of metabolic stimulation, morphological changes, and/or up regulation of other adhesion molecules due to cytokine induction. Similar results were received from in vitro binding studies and FACS analyses on both Tumor Necrosis Factor-alpha activated and E-Selectin transfected endothelial cells. These data suggest that this methodology is appropriate for dissecting the individual activities of E-selectin while minimizing the participation of other adhesion molecules, thereby allowing us to develop a better understanding of the role of E-Selectin and endothelia in metastatic disease.


Expression of DRG during murine embryonic development. We had previously characterised a cDNA which encodes a novel GTP-binding protein DRG. The expression of drg gene is down-regulated during the embryonic development of murine central nervous system. Further analysis of drg mRNA and protein in adult mouse tissues and various cell lines of different origins indicated that it is expressed widely, albeit at low and variable levels. In situ hybridisation analysis of mRNA expression in sections of mouse embryos indicated that drg is expressed strongly in various embryonic tissues. The expression of drg mRNA is greatly reduced in newborn animals. At cellular level, DRG protein can be detected in the cytoplasm. These observations suggest that DRG may play multiple roles in development and normal cell metabolism.


Hepatocyte growth factor down-regulates the alpha-fetoprotein gene expression in PLC/PRF/5 human hepatoma cells. Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes; however, in certain human hepatoma cell lines, the growth is inhibited by HGF. In the present study, the effect of HGF on the alpha-fetoprotein (AFP) gene expression was analyzed in PLC/PRF/5 human hepatoma cells. HGF did not inhibit cell proliferation, but dose-dependently suppressed AFP secretion at the concentrations of 10 ng/ml or less. By Northern blot analysis, the levels of AFP mRNA were suppressed by HGF, whereas the levels of beta-actin mRNA used as a control did not show any significant changes. In the transient chloramphenicol acetyltransferase plasmid transfection assays, the AFP promoter activity was repressed by HGF, in contrast, the AFP enhancer activity was not affected by HGF. These results suggest that the AFP gene expression is down-regulated by HGF through the suppression of its promoter activity in human hepatoma cells.


Possible activity of acidic fibroblast growth factor as a progression factor rather than a transforming factor. Acidic and basic fibroblast growth factors (aFGF and bFGF) are mitogens for mesoderm- and neuroectoderm-derived cells. The facts that FGF-related proteins are oncogenic and that FGFs are expressed in a variety of tumor cell lines or tumor tissues suggest the transforming activities of FGFs. To examine such an activity of aFGF, we introduced a human aFGF expression vector into two populations of Rat-1 cells; one was non-transformed (nRat-1), the other was partially-transformed (tRat-1). tRat-1 cells transfected with aFGF cDNA formed larger colonies in soft agar and produced larger and more malignant tumors in nude mice than those of parental cells. In contrast, nRat-1 cells transfected with aFGF cDNA neither formed colonies in soft agar nor produced tumors in nude mice. Our results suggest that high expression of aFGF may enhance a tumorigenic potential of Rat-1 cells rather than confer such a potential de novo.


Purification of a mammary-derived growth inhibitor (MDGI) related polypeptide expressed during pregnancy. The present study was undertaken to screen immunochemically for MDGI-related proteins in the mammary gland. A new form, MDGI 2, not present in lactation could be detected in the bovine gland during pregnancy. It was further distinguished from MDGI by its lower molecular weight, its association with a complex binding to WGA, and by lacking immunoreactivity to an anti-MDGI antibody directed against the C-terminus of MDGI. MDGI 2 was purified by chromatography over DEAE-Sepharose, Bio-Gel P-30 in 1% acetic acid, Sephacryl S-200 in 6 M urea and Mono Q. Final purification included HPLC on TSK G-3000 SW and electroelution from SDS-gels. Cell-free translation of poly (A+)mRNA from glands of pregnant animal yielded one form identical with MDGI. We assume that posttranslational processing of MDGI is involved in its activities.


Increased mitochondrial activities in pigmented (melanized) fish cells and nucleotide sequence of mitochondrial large rRNA. Using the subtracted probe approach, we had previously isolated eight cDNAs whose corresponding RNAs are more abundant in pigmented (melanized) than in closely related unpigmented goldfish cell lines. We report here that two of these are of mitochondrial origin, suggesting that pigmentation is accompanied by higher content (activity) of mitochondria. We also present the complete nucleotide sequence of the full length cDNA for the large mitochondrial rRNA, showing the presence of a polyA tail, two polyadenylation signals and a long open reading frame potentially encoding for a polypeptide of 166 amino acids and with no known protein homologue. It is however unknown whether such a polypeptide is actually produced.


Methylation-sensitive protein-DNA interaction at the cell cycle regulatory domain of human thymidine kinase promoter. We have investigated the DNA-protein complex formation in nuclear extracts of human cells using the sequence of cell-cycle regulatory unit (CCRU) of human thymidine kinase (TK) promoter. It appeared that a distinct DNA-protein complex was present in three human tumor cell lines and that the CCAAT box within the sequence of CCRU was a necessary element for complex formation. Upon 4 days of serum deprivation, this DNA-protein complex remained unchanged in HeLa cells, but the expression of TK mRNA was decreased. Furthermore, DNA methylation of the Hhal site of the CCRU sequence of the TK promoter greatly reduced the binding activity of nuclear proteins from different human tumor cell lines. On the basis of these data, we proposed a possible role for DNA methylation in the regulation of TK transcription during late G1/S phase progression of the cell cycle.


Molecular cloning of hippocalcin, a novel calcium-binding protein of the recoverin family exclusively expressed in hippocampus. We have isolated a cDNA clone encoding a novel calcium-binding protein of the recoverin family from rat brain cDNA library. This clone (PCB11) has 588 nucleotides in the open reading frame including the termination codon, 174 nucleotides of the 5' leader and 800 nucleotides of the 3' noncoding region. The complete amino acid sequence deduced from the cDNA is composed of 195 residues, has a calculated molecular mass of 22,574 Daltons, and contains three putative calcium-binding domains of the EF-hand structure. The deduced amino acid sequence has a striking sequence homology to those of the retinal recoverin family (recoverin, visinin, P26, 23kD protein, S-modulin) and the brain-derived recoverin family (P23k, 21-kDa CaBP and neurocalcin). Northern blot, in situ hybridization, immunoblot and immunohistochemical analyses revealed that the protein is exclusively expressed in pyramidal layer of the hippocampus. The protein was therefore designated hippocalcin.


Inhibition of low pH evoked activation of airway sensory nerves by capsazepine, a novel capsaicin-receptor antagonist. Low pH is a well known sensory irritant in pathological conditions such as inflammation. The mechanisms underlying this low pH effect were therefore studied in the guinea pig. Acid exposure caused marked nasal irritation via a specific subset of sensory nerves sensitive to capsaicin. Furthermore, acid caused bronchoconstriction via release of neuropeptides from capsaicin sensitive afferents. Interestingly, capsazepine, a recently developed competitive capsaicin receptor antagonist, selectively inhibited these responses to low pH. Ruthenium red, which blocks the cation channel associated with the capsaicin receptor, had effects similar to those of capsazepine. Therefore, acid irritation of the airway mucosa may involve capsaicin-receptor mechanisms and capsazepine represents a novel protective agent.


Co-purification of 130 kD nitric oxide synthase and a 22 kD link protein from human neutrophils. The synthesis of nitric oxide (NO) from L-arginine has been demonstrated in several cell types. Both constitutive and inducible forms of NO synthase have been described in different cells. We purified the constitutive form of NO synthase enzyme in human neutrophils using a two-column procedure. Crude 100,000g supernatant of human neutrophils was passed through a 2'-5'-ADP-agarose column followed by a DEAE-Bio-Gel A anion exchange column. NO synthase enzyme migrated as a single band (MW approximately 130,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its activity was dependent upon nicotinamide adenine dinucleotide phosphate (NADPH) and (6R)-tetrahydro-L-biopterin (BH4). In addition, flavin adenine dinucleotide (FAD) was also found to be essential for its maximal activity. A second NADPH, FAD-dependent component (MW approximately 22kD) was also found consistently on the SDS-PAGE gel. These observations suggest co-regulation between NO synthase enzyme and this NADPH, FAD-dependent component, which may be associated with the superoxide radical generating system.


Detection of antibody to hepatitis C E2/NS1 protein in patients with type C hepatitis. Putative E2/NS1 sequence of hepatitis C virus was expressed in E. coli as a fusion protein with maltose binding protein. Approximately 80 kDa protein was obtained containing 38 kDa E2/NS1 protein. The antibody to this protein was detectable in the same serum from which the sequence was amplified. It was also detectable in none of 7 acute hepatitis, in 2 of 12 chronic persistent hepatitis, in 3 of 25 chronic active hepatitis, and in 2 of 4 cirrhosis. It was detectable in none of 10 normal subjects. In 3 cases who were positive for the antibody before the interferon treatment, it became undetectable after the treatment. Thus, it seems that the antibody is not a neutralizing antibody and is related to active viral replication.


Protein tyrosine phosphorylation as an indicator of 2,3,7,8-tetrachloro-p-dioxin exposure in vivo and in vitro. A dose-dependent increase in tyrosine phosphorylation of five hepatic intracellular proteins with approximate molecular weights of 17, 21, 27, 29, and 34 kDa was seen 24 h after administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to C57BL/6J female mice. The ED50 values for tyrosylphosphorylation of these five proteins, respectively, were 0.26, 0.21, 0.26, 0.31, and 0.38 micrograms TCDD/Kg. TCDD induction of 7-ethoxyresorufin O-deethylase activity (EROD) was characterized by an ED50 of 2.5 micrograms/Kg. An eighteen h exposure of a human lymphoblastoma cell line (X3) to TCDD increased tyrosylphosphorylation status of ten proteins with approximate molecular weights of 16, 17, 24, 26, 27, 32, 33, 34, 35, and 36 kDa in a dose-dependent manner. The EC50 values for these TCDD-dependent tyrosylphosphorylation ranged from 0.01 to 0.07 nM TCDD. EROD induction by TCDD in X3 cells exhibited an EC50 of 0.14 nM. These data indicate that TCDD alters intracellular protein tyrosine phosphorylation and these changes are more sensitive biological indicators of TCDD exposure than induction of EROD.


Mouse dopamine beta-hydroxylase: primary structure deduced from the cDNA sequence and exon/intron organization of the gene. Genomic clones for mouse dopamine beta-hydroxylase (DBH) were isolated from two genomic libraries derived from DBA/2J and 129/SV mouse strains, by plaque hybridization with the human DBH cDNA probe. Subsequently, cDNA encoding mouse DBH was amplified with reverse transcription-polymerase chain reaction (RT-PCR) method using primers corresponding to 5'- and 3'-portions of the mouse DBH mRNA, subcloned into a plasmid vector, and subjected to nucleotide sequence analysis. The clone encoded a protein of 621 amino acids with a calculated molecular mass of 70,186 daltons. The predicted amino acid sequence of mouse DBH showed 87%, 80% and 79% identities with the rat, bovine and human enzymes, respectively. Several potential amino acid sequences that are involved in the posttranslational modification and catalytic function of DBH were identified in mouse DBH protein. Nucleotide sequence analysis of the overlapping genomic clones showed that the mouse DBH gene was composed of 12 exons about 17 kb in length. Typical TATA and CCAAT boxes were observed in the 5'-upstream region of the gene. Northern blot analysis of adrenal gland RNA detected a single size species of the mouse DBH mRNA.


Platelet-derived growth factor gene expression in cultured human retinal pigment epithelial cells. Gene expression of platelet-derived growth factor (PDGF) and its receptors in cultured human retinal pigment epithelial (RPE) cells was studied by using semiquantitative polymerase chain reaction. The RPE cells were found to express PDGF A- and B-chain genes as well as alpha- and beta-receptor genes with dominant expression of B-chain and beta-receptor isoforms. Phorbol myristate acetate (PMA) and thrombin increased the expression of PDGF B-chain gene to 19.8 +/- 1.75 and 15.9 +/- 1.84 fold (n = 3) of the control without affecting beta-receptor gene expression. PDGF produced by the RPE cells may play an important role in the pathogenesis of some ocular proliferative diseases.


Inactivation of substance P by granulation tissue-derived gelatinase. An active gelatinase has been purified from the conditioned medium of granulation tissue culture formed by carrageenin injection in rats. The purified gelatinase gave a single band corresponding to a M(r) of 57 kDa on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-gelatin PAGE. The granulation tissue-derived gelatinase selectively cleaved the Gln6-Phe7 bond of substance P (SP) with a Km of 0.17 mM and a Vmax of 0.027 nmol SP7-11/min/micrograms protein, resulting in the generation of biologically inactive fragments, SP1-6 and SP7-11. Our data suggest that the gelatinase produced by granulation tissue participates in the inactivation of SP in the inflammatory site.


Inhibition of nicotinic acetylcholine receptor-mediated secretion and synthesis of catecholamines by sea urchin toxin in cultured bovine adrenal medullary cells. We previously reported the partial purification and characterization of a toxic substance (sea urchin toxin) isolated from the pedicellariae of the sea urchin Toxopneustes pileolus (Nakagawa and Kimura, Jpn J Pharmacol 32: 966-968, 1982). In the present study, we examined the effect of sea urchin toxin on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. Sea urchin toxin inhibited the secretion of catecholamines stimulated by carbachol and nicotine but not by veratridine or a high concentration of K+. The toxin inhibited the carbachol-evoked influx of 22Na+ and 45Ca2+ at concentrations similar to those for catecholamine secretion. The inhibition of catecholamine secretion by sea urchin toxin was not overcome by increasing the concentration of carbachol. Preincubation of cells with the toxin caused a time-dependent inhibition in the secretion stimulated by carbachol even when the toxin was removed from the incubation medium. The toxin suppressed catecholamine synthesis and tyrosine hydroxylase activity in carbachol-stimulated cells. In addition, sea urchin toxin inhibited [3H]phencyclidine binding to adrenal medullary cells whereas it did not alter cyclic GMP accumulation caused by muscarine. Further purified fractions from sea urchin toxin by concanavalin A affinity column chromatography also inhibited carbachol-evoked secretion of catecholamines. These results suggest that sea urchin toxin inhibits carbachol-enhanced secretion and synthesis of catecholamines by suppression of nicotinic acetylcholine receptor-mediated Na+ influx and subsequent Ca2+ influx in cultured adrenal medullary cells.


Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 94-100 (antigenic site 3) of myoglobin. Amino acid substitutions outside protein antigenic sites are very frequently assumed to exert no effect on binding to antiprotein antibodies, especially if these are monoclonal antibodies (mAbs). In fact, a very popular method for localization of residues in protein antigenic sites is based on the interpretation that whenever a replacement causes a change in binding to antibody, then that residue will be located in the antigenic site. To test this assumption, mAbs of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any carrier) synthetic peptide representing region 94-100 of sperm whale myoglobin (Mb). The cross-reactivities and relative affinities of three mAbs with eight Mb variants were studied. Five Mb variants which had no substitutions within the boundaries of the designed antigenic site exhibited remarkable, and in two cases almost complete, loss in cross-reactivity relative to the reference antigen, sperm whale Mb. Two myoglobins, each of which had one substitution within region 94-100, showed little or no reactivity with the three mAbs. It is concluded that substitutions outside an antigenic site can exert drastic effects on the reactivity of a protein with mAbs against the site and that caution should be exercised in interpreting cross-reactivity data of proteins to implicate residues directly in an antigenic site.


Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 15-22 (antigenic site 1) of myoglobin. Monoclonal antibodies (mAbs) of predetermined specificity were prepared by immunizing with a free (i.e., not conjugated to any carrier) synthetic peptide representing region 15-22 (site 1) of sperm whale myoglobin (SpMb). The cross-reactions of Mb variants with three mAbs were studied in order to determine whether such interactions are influenced by substitutions outside the site. Finback whale Mb, which has no substitutions within region 15-22, showed lower cross-reactivity and relative binding affinity than the reference antigen, SpMb. Bottle-nose Atlantic dolphin myoglobin (BdMb) and badger myoglobin (BgMb), although they have identical substitutions within region 15-22 (Ala-15 to Gly and Val-21 to Leu), showed very different binding properties. The cross-reaction of BdMb was quite comparable to that of SpMb, while that of BgMb was much lower. Since the two proteins have identical structures in regions 15-22, the differences in their cross-reactivities are readily attributed to the effects of substitutions outside this region. Another pair of myoglobins, horse myoglobins (HsMb) and chicken myoglobin (ChMb), also have two identical substitutions (Ala-15 to Gly and Val-21 to Ile) within region 15-22, but possessed different cross-reactivity. The results indicate that the reaction of mAbs, whose specificity is precisely known and predetermined by the immunizing free peptide, can be markedly affected by substitutions outside the indicated binding region on the protein.


Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 56-62 (antigenic site 2) of myoglobin. This work was carried out in order to study the effects of substitutions outside antigenic site 2 of sperm whale myoglobin (SpMb) on the reactivity of protein variants with antisite 2 monoclonal antibodies (mAbs). A synthetic peptide corresponding to region 56-62 (site 2) of SpMb was used as an immunogen in mice in its free form (i.e., without coupling to any carrier) to prepare a panel of mAbs whose predetermined specificity is directed, by design, against this region. The binding of three of these mAbs to eight Mbs from different species was studied. Myoglobins of Pacific common dolphin, finback whale, and horse, which have no substitutions within region 56-62 relative to SpMb, showed remarkable differences in their cross-reactivities and relative affinities with each of the mAbs. Myoglobins of badger, chicken, and dog, although they have an identical substitution within the site (Ala-57 to Gly), exhibited cross-reactivities with a given mAb that were affected differently. Echidna Mb, which has one replacement (Glu-59 to Ala) within region 56-62, displayed greatly reduced cross-reactivities and relative binding affinities. The results, especially those from Mbs that have no substitutions within the boundaries of site 2, clearly indicate that substitutions outside site 2 of Mb can exert drastic effects on the binding of the Mb variants with mAbs whose specificity was predesigned to be against the site. These indirect effects and their impact on site reactivity will completely explain previous findings on cross-reactivities of Mb variants with mAbs of unknown specificity and will rule out the postulations of discontinuous sites in Mb, which were based on the assumption that every substitution affecting reactivity is directly involved in binding to antibody.


Covalent attachment of ribonucleic acids to proteins. As a prerequisite for the synthesis of affinity labels, we describe methods to couple histones to ribonucleic acids. For the synthesis of these covalent hybrid molecules, we used a population of histones H1, H2A, H2B, H3, and H4 from calf thymus and polyadenylic acid with an average chain length of up to 260-280 bases, representing the size of poly(A)-tails from mature mRNAs. Three methods were investigated. (a) Poly(A) containing an 8-N3-A residue was cross-linked to histones by ultraviolet irradiation. (b) The 3'-end of the polynucleotide was connected to a mononucleotide containing an aliphatic amino group, and the resulting poly(A)-derivative was coupled to histones via derivation with a bromoacetyl group. (c) The 3'-end of the polynucleotide was oxidized with sodium periodate and bound covalently to an amino group of the polypeptide. To demonstrate the RNA content of the hybrid molecule, the poly(A) was removed with RNase T2.


Effect of alcohol on urinary and blood dolichols. Alcohol appears to affect dolichol metabolism, as both serum and urinary dolichol concentrations were found to be significantly higher in alcoholics than in social drinkers. Furthermore, acute heavy drinking (5.5 g alcohol/kg body weight during 42 h) increased urinary dolichol excretion significantly, whereas moderate drinking (60 g/day for 10 days) had no effect. Increased urinary dolichol concentrations in alcoholics returned rapidly to normal with a half-life decay of 3 days, whereas increased serum dolichol concentrations did not change during a 7-day observation period. The mechanism behind alcohol-induced alterations in dolichol metabolism remains unclear, but based on our results, it seems likely that serum and urinary dolichols are regulated independently from each other.


Development of T7 phage and T7 phage containing apurinic sites in an exonuclease III, endonuclease IV double mutant of Escherichia coli. The development of bacteriophage T7 was examined in an Escherichia coli double mutant defective for the two major apurinic, apyrimidinic endonucleases (exonuclease III and endonuclease IV, xth nfo). In cells infected with phages containing apurinic sites, the defect in repair enzymes led to a decrease of phage survival and a total absence of bacterial DNA degradation and of phage DNA synthesis. These results directly demonstrate the toxic action of apurinic sites on bacteriophage T7 at the intracellular level and its alleviation by DNA repair. In addition, untreated T7 phage unexpectedly displayed reduced plating efficiency and decreased DNA synthesis in the xth nfo double mutant.


Therapeutic applications of oligonucleotides. This review focuses strictly on the pharmacodynamic considerations of the use of oligonucleotides designed to interact with nucleic acids as therapeutics. The objectives are to place oligonucleotide therapeutics in the context of modern drug discovery and development and to summarize recent progress.


The internal representation of arithmetical word problem sentences: frontal and posterior-injured patients compared. Patients with frontal and left posterior brain lesions have severe difficulties in arithmetical word problem solving. In the present study the origin of these difficulties is investigated from an information-processing perspective. Following this perspective the first stage in word problem solving consists of a translation of individual sentences to an internal representation. This translation process is examined in 30 frontal patients, 10 left posterior-injured patients, and 10 healthy controls with a recognition and a sentence-picture matching task. In addition, the relationship between sentence representation and arithmetical word problem solving is studied. The results suggest that error rates in the translation of different types of arithmetical word problem propositions differ substantially in our three groups. A relationship between translation skills and arithmetical word problem solving ability is also found.


Treatment of subfoveal choroidal neovascular membranes with systemic interferon-alpha 2a. Systemic interferon-alpha has recently been reported to be useful in the treatment of choroidal neovascular membranes (CNVM) in age-related macular degeneration. To further investigate its effect we treated eight patients with subfoveal choroidal neovascular membranes of various aetiologies including idiopathic, age-related macular degeneration and myopic chorioretinal degeneration. Only one of the eight patients responded with an angiographically demonstrated decrease in the size of the CNVM; this was in a patient from the idiopathic group. Considerable side-effects were reported with marked lethargy and malaise limiting compliance with some patients. Although this is an initial report with limited patient numbers and follow-up, it does suggest a role for interferon-alpha in patients with idiopathic neovascular membranes where the retina is otherwise normal, but raises some doubt as to its efficacy in the treatment of age-related macular degeneration.


Corneal storage at 4 degrees Celsius in a chondroitin sulphate and dextran containing medium. Corneas were stored at 4 degrees C in a chondroitin sulphate and dextran containing medium from one to ten days. The viability of the corneal endothelium was assessed after storage with the vital stain, trypan blue.


Identification of a mouse B-type cyclin which exhibits developmentally regulated expression in the germ line. To begin to examine the function of cyclins in mammalian germ cells, we have screened an adult mouse testis cDNA library for the presence of B-type cyclins. We have isolated cDNAs that encode a murine B-type cyclin, which has been designated cycB1. cycB1 was shown to be expressed in several adult tissues and in the midgestation mouse embryo. In the adult tissues, the highest levels of cycB1 transcripts were seen in the testis and ovary, which contain germ cells at various stages of differentiation. The major transcripts corresponding to cycB1 are 1.7 and 2.5 kb, with the 1.7 kb species being the predominant testicular transcript and the 2.5 kb species more abundant in the ovary. Examination of cDNAs corresponding to the 2.5 kb and 1.7 kb mRNAs revealed that these transcripts encode identical proteins, differing only in the polyadenylation signal used and therefore in the length of their 3' untranslated regions. Northern blot and in situ hybridization analyses revealed that the predominant sites of cycB1 expression in the testis and ovary were in the germinal compartment, particularly in early round spermatids in the testis and growing oocytes in the ovary. Thus cycB1 is expressed in both meiotic and postmeiotic cells. This pattern of cycB1 expression further suggests that cycB1 may have different functions in the two cell types, only one of which correlates with progression of the cell cycle.


Role of tyrosine kinases in IgE-mediated signal transduction in human lung mast cells and basophils. Recent evidence suggests that tyrosine kinases play an important role in signal transduction mechanisms utilized by a range of different agonists in many cell types. We have investigated the effects of four different inhibitors of tyrosine kinases on IgE-dependent histamine release from human lung mast cells and basophils. Genistein inhibited the anti-IgE-induced histamine release from human basophils (at 10 microM genistein, inhibition = 55 +/- 5%, n = 17, P < 0.005) with an IC50 of 8 microM, but was much less effective in the human lung mast cell (at 10 microM, inhibition = 18 +/- 6%, n = 11, P < 0.05). Two inactive analogs of genistein, genistin and diadzein, failed to affect anti-IgE-induced histamine release significantly in either mast cells or basophils. A second inhibitor of tyrosine kinases, tyrphostin 25, inhibited IgE-dependent release from basophils (at 10 microM, inhibition = 25 +/- 7%, n = 6, P < 0.05) though it was less effective than genistein and failed to affect IgE-induced histamine release from human lung mast cells (at 10 microM, inhibition = 22 +/- 16%, n = 5, P = NS). In contrast, methyl 2,5 dihydroxycinnamate (MDC) failed to inhibit anti-IgE-dependent histamine release in human basophils (at 10 microM, inhibition = 3 +/- 3%, n = 5, P = NS) but proved to be an effective inhibitor of anti-IgE-induced degranulation in human lung mast cells (at 10 microM, inhibition = 53 +/- 16%, n = 5, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Murine strain differences in acute lung injury and activation of poly(ADP-ribose) polymerase by in vitro exposure of lung slices to bleomycin. The DNA-cleaving, antitumor antibiotic bleomycin (BLM) causes pulmonary fibrosis, but the essential early events initiating the fibrotic state have not been well characterized. Thus, we have directly examined BLM-mediated pulmonary cell injury by monitoring lactate dehydrogenase (LDH) release and nuclear poly(ADP-ribose) polymerase (PAP) activity, which is stimulated by DNA breakage, using lung slices isolated from BLM-sensitive (C57B1/6) and BLM-resistant (BALB/c) mice. Lung slices were incubated continuously with or without the PAP inhibitor, 3-aminobenzamide (3-AB), and exposed to BLM for 45 min. LDH release from C57B1/6 lung slices increased 2-fold by 8.5 h after treatment with BLM. In contrast, BLM failed to enhance cumulative LDH release by BALB/c mouse lung slices. Co-incubation of C57B1/6 lung slices with 3-AB prevented BLM-induced LDH release. Nuclear PAP was activated 3- to 4-fold 1.25 h after exposure of C57B1/6 lung slices to BLM but returned to control levels by 3.75 h. Nuclear PAP was only marginally affected at these times in BALB/c lung slices. Co-incubation of C57B1/6 slices with 3-AB prevented the early increases in PAP activity. These results demonstrate that murine strain sensitivity to acute cell injury and early PAP activation by BLM in lung slices parallels the in vivo sensitivity of lungs. In addition, 3-AB suppresses PAP activation and acute cell injury in lung slices. Differential activation of PAP appears to govern murine strain variation in response to BLM and is consistent with the hypothesis that activation of PAP participates in acute pneumocyte injury, initiating the process of BLM-induced fibrosis.