Multiple HPV 16-related squamous cell carcinomas of the vulva, vagina, anus, skin and cervix in a 31-year-old woman. A 31-year-old woman is reported with in the genital region multiple squamous carcinomas of the skin (buttock), vulva, vagina, anus and cervix uteri. All these carcinomas were HPV 16 positive as tested by DNA in situ hybridization. The existence of areas with normal epithelium between all tumor localisations and the absence of distant metastases indicate multicentric development of these multiple carcinomas. The presence of HPV 16 DNA in all carcinoma cells, as detected by DNA in situ hybridisation, argues for an etiological role of HPV 16 in the development of these multiple tumors.


Effect of hormone replacement therapy and calcitonin on bone mass in postmenopausal women. A total of 104 postmenopausal women were randomly assigned to different therapeutic regimens: (a) calcitonin, (b) estrogen/progestogen (HRT) plus calcitonin, (c) estrogen/progestogen (HRT), (d) and the control group. The bone mass of the lumbar vertebrae of all patients was assessed with a dual beam photon absorptiometer (Norland GD 153). The 73 patients who completed the 1-yr study showed that postmenopausal bone loss could be prevented by either estrogen/progestogen (HRT) or calcitonin. In addition, the combination of hormonal replacement therapy and calcitonin not only prevented post-menopausal bone loss but resulted in a significant 10% gain in bone mass (P < 0.001).


Phosphotransferase activity associated with rat osseous plate alkaline phosphatase: a possible role in biomineralization. 1. Alkaline phosphatase from rat osseous plate catalyzed the transfer of phosphate from p-nitrophenylphosphate to glycerol, ethanolamines, Tris, glucose and 1-amino-1-methyl-2-propanol, in a wide range of pH. Serine did not stimulate phosphotransferase activity of the enzyme. 2. The best phosphotransferase acceptors were diethanolamine and glycerol while glucose was the poorest phosphotransferase acceptor used. 3. Diethanolamine and glycerol affected both VM and KM of p-nitrophenylphosphate hydrolysis with activation constants (KA) of 0.25 and 0.85 M, respectively. 4. A kinetic model was proposed for the phosphotransferase reaction observed with alkaline phosphatase from rat osseous plates.


Presence of a Vicia unijuga lectin-binding (Vgu) glycoprotein with Thomsen-Friedenreich (T) activity and Vgu glycoproteins in human primary hepatocellular carcinoma. 1. Twenty perchloric acid-soluble glycoprotein fractions (PASFs) were separated from carcinomatous and non-carcinomatous regions of the livers of 5 patients with primary hepatocellular carcinoma (HCC) and 5 patients with a complication of HCC and hepatocirrhosis (HC). 2. In autoradiography using 125I-labelled Vicia unijuga lectin (VUA) and 125I-labelled Arachis hypogaea anti-T lectin as probes, 10 PASFs from carcinomatous regions of the livers of 10 patients gave 1 Vgu glycoprotein with T activity and/or 1-4 Vgu glycoproteins, respectively, and among 10 PASFs from non-carcinomatous regions of the livers of 10 patients, 7 PASFs gave no glycoproteins, respectively. 3. These results show that a Vgu glycoprotein with T activity and Vgu glycoproteins occur in HCC and in a complication of HCC and HC as oncofetal antigens.


Carbohydrate-binding plasma proteins from the gastropod Biomphalaria glabrata: strain specificity and the effects of trematode infection. As lectins are believed to mediate non-self-recognition in molluscs, carbohydrate-binding proteins (CBP) from the circulating plasma of the gastropod Biomphalaria glabrata were harvested by affinity chromatography using six different monosaccharides as ligands. Pools of plasma were derived from B. glabrata of either the M line strain, which is susceptible to infection with the PR1 strain of the digenetic trematode Schistosoma mansoni, or from the 13-16-R1 strain, which is resistant to infection. For each strain, plasma was obtained from control snails and from snails exposed to infection 1 or 8 days previously with S. mansoni or the related digenean, Echinostoma paraensei, which is able to develop in either host strain. For control snails, only minor interstrain differences were noted. In M line snails exposed 8 days previously to either parasite, marked changes in CBP populations were observed. Only E. paraensei infections produced comparable alterations in 13-16-R1 snails. The most conspicuous changes noted were the increased production of 80-120 kDa CBP in both strains, 150-210 kDa in 13-16-R1 snails, and 190-210 kDa in M line snails. The results demonstrate 1) interstrain differences in CBP, particularly following exposure to trematodes; 2) that infection provokes increased production and diversity of CBP that bind with greater affinity to the columns; and 3) that snails of the same strain respond differently to the two parasites used.


Inhibitory action of orotate, 2-thioorotate and isoorotate on nucleotide metabolism and nucleic acid synthesis in hepatoma cells. 1. The specificity of the action of orotate on hepatoma cells was investigated. 2. Orotic acid and its methyl ester had similar inhibitory effects on the incorporation of [3H]thymidine into DNA of hepatoma cells. 3. In contrast to previous studies in vivo, incubation of rat kidney cells with orotate caused an increase in the ratio of UTP/ATP concentrations that was similar to effects on hepatic cells. 4. Inhibitory effects of 2-thioorotate and isoorotate on metabolism were found to be less selective and required higher concentrations than with orotate.


Cellular pathway(s) of antigen processing in fish APC: effect of varying in vitro temperatures on antigen catabolism. Studies were conducted to determine the effects of varying in vitro temperatures during cellular processing of T-dependent antigens by catfish antigen-presenting cells (APC). Strategy involved pulsing of long-term monocyte lines (used as APC) with antigen at different temperatures for various periods of time and then fixing and coculturing with autologous peripheral blood leukocytes (PBL) as responders at a permissive temperature (i.e., 27 degrees C). Results showed that APC incubated with antigen at low, nonpermissive, but physiologically relevant, temperatures (11 and 17 degrees C) elicited secondary proliferative responses by autologous PBL. However, responses elicited with APC pulsed at 11 and 17 degrees C required longer exposure to the antigen prior to fixation (i.e., greater than or equal to 8 h compared to 5 h, the optimal incubation time at 27 degrees C). Further, there was sufficient cell-associated antigen during a short-term pulsing period (1-2 h) at 11 and 17 degrees C to provide efficient presentation after subsequent incubation of the APC at 27 degrees C for an additional 10 h before fixation. In contrast, APC pulsed with antigen at an extremely low, physiologically irrelevant, temperature (4 degrees C) for extended periods of time did not elicit the proliferation of autologous responders unless antigen pulsing was carried out for 1-2 h and the APC subsequently shifted to 27 degrees C for an additional 10 hr. Antigen uptake assays revealed significant and similar levels of internalized antigen at 11, 17, and 27 degrees C, but not at 4 degrees C. However, intracellular degradation and formation of trichloroacetic acid-soluble antigen catabolites at 11 and 17 degrees C appeared to occur at a slower rate compared to APC at 27 degrees C. Significantly, primary anti-hapten plaque-forming cell responses were also observed with PBL cocultured with APC pulsed with hapten-carrier conjugates at 11, 17, and 27 degrees C. Consequently, the previously observed suppression of primary T-cell responses in fish at low, physiologically relevant, temperatures is not due to impaired antigen processing or presentation.


Interplay between proliferation and differentiation within the myogenic lineage. In muscle cells, as in a variety of cell types, proliferation and differentiation are mutually exclusive events controlled by a balance of opposing cellular signals. Members of the MyoD family of muscle-specific helix-loop-helix proteins which, in collaboration with ubiquitous factors, activate muscle differentiation and inhibit cell proliferation function at the nexus of the cellular circuits that control proliferation and differentiation of muscle cells. The activities of these myogenic regulators are negatively regulated by peptide growth factors and activated oncogenes whose products transmit growth signals from the membrane to the nucleus. Recent studies have revealed multiple mechanisms through which intracellular growth factor signals may interfere with the functions of the myogenic regulators. When expressed at high levels, members of the MyoD family can override mitogenic signals and can cause growth arrest independent of their effects on differentiation. The ability of these myogenic regulators to inhibit proliferation of normal as well as transformed cells from multiple lineages suggests that they interact with conserved components of the cellular machinery involved in cell cycle progression and that similar types of regulatory factors participate in differentiation and cell cycle control in diverse cell types.


Identification of a region of mouse zona pellucida glycoprotein mZP3 that possesses sperm receptor activity. The ability of mouse zona pellucida glycoprotein ZP3 (mZP3) to function as a sperm receptor is attributable to certain of its oligosaccharides, not to its polypeptide (P. M. Wassarman, 1990. Development 108, 1-17). Here, purified, radioiodinated mZP3 was digested by either papain or V8 protease, and the glycopeptides produced were fractionated by HPLC and assayed for sperm receptor activity in vitro. Each proteolytic digest of mZP3 contained a heavily glycosylated peptide, approximately 55,000 apparent M(r), that exhibited sperm receptor activity in vitro. To determine the region of mZP3 polypeptide from which the active glycopeptides were derived, Western gel immunoblotting, employing an antiserum directed against a specific mZP3 peptide epitope, and automated amino-terminal amino acid sequencing were employed. Results of these experiments strongly suggest that the active glycopeptides produced by digestion of mZP3 with either papain or V8 protease are derived from the same region of the carboxy-terminal half of the mZP3 polypeptide. These and other findings are discussed in terms of mZP3 structure and function.


Differential expression of inhibin subunits and follistatin, but not of activin receptor type II, during early murine embryonic development. Activins are known to be potentially important regulators of early developmental processes in amphibians, birds, and mammalians. In this study we report the expression of the inhibin subunits, including those that make up activin, the activin-binding protein follistatin, and activin receptor type II in several in vitro systems that model early murine embryonic development, namely embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and their differentiated derivatives. In addition, we examine the expression pattern of these factors in different stages of the mouse embryo itself. Expression of inhibin alpha and beta A subunits is restricted to certain differentiated cell types, while beta B subunits are expressed in both differentiated and undifferentiated cells. Our results further indicate a change in the expression pattern of inhibin subunits during early development from beta B at the blastocyst stage largely to beta A in postgastrulation embryos. This is similar to the expression pattern at equivalent stages of Xenopus and chick development. Expression of the activin-binding protein follistatin is altered by the induction of differentiation of P19 EC and ES cells by several factors, including retinoic acid. In contrast to the inhibin subunits and follistatin, activin receptor levels are not influenced by differentiation in these cell types. The results of this study demonstrate that the inhibin subunits and follistatin, but not the activin receptor type II, are differentially expressed during early murine development and suggest that the different forms of activin/inhibin are involved in the regulation of different developmental processes.


Evaluation of assay procedures measuring macrophage stimulation by immunomodulators in vitro. Several assay procedures for measuring the stimulatory effect on macrophages (møs) of bacterial-derived immunomodulators (OM-85, OM-89, OM-163, Laboratoires OM, Meyrin/Geneva, Switzerland) were evaluated with regard to their complexity, speed, and general convenience. To this effect, bone marrow-derived or peritoneal exudate macrophages were exposed to the immunomodulators in vitro, then tested for metabolic stimulation (glucose oxidation through the hexosemonophosphate shunt pathway, synthesis of type E prostaglandins, release of superoxide, and production of L-arginine-derived nitrogen oxidation products), as well as for enhancement of functional activities (production of tumor necrosis factor-alpha, extracellular cytolysis of P815 target cells, and intracellular parasite destruction). All these tests were found to provide adequate measurements of the mø response to the immunomodulators, with significant effects detectable using the compounds in the ng/ml to microgram/ml range. Concomitant incubation with crude macrophage activating factor or with recombinant murine interferon-gamma (IFN-gamma) dramatically increased the sensitivity of møs to the immunomodulators, and was an absolute requirement for induction of mø cytotoxic activities by the bacterial extracts. The measurement of nitrite production by møs exposed to the immunomodulators with or without treatment with 10 U/ml of IFN-gamma was found to be a highly convenient procedure, which correlated well with functional assays.


Disruption of the cytoskeleton-extracellular matrix linkage promotes the accumulation of plasminogen activators in F9 derived parietal endoderm. When F9 teratocarcinoma cells are treated with retinoic acid plus cyclic AMP (RACF9) they differentiate into parietal endoderm. Differentiation is accompanied by the acquisition of substrate adhesion sites and a change in the pattern of gene expression, including the synthesis of tissue-type plasminogen activator (tPA). We demonstrate here that dihydrocytochalasin B (DHCB) treatment of differentiating F9 cells prevents the assembly of a structured actin cytoskeleton and generates a more rounded and stellate cell morphology. This morphological change is accompanied by the accumulation of the usually visceral endoderm-specific marker urokinase-type plasminogen activator (uPA) and an increase in tPA levels in comparison to untreated RACF9 controls. The increase in tPA accumulation is preceded by an increase in tPA mRNA levels. These effects are reversible, with a lag, when DHCB is removed, and PA accumulation can be stimulated within 24 h when differentiated cells are exposed to DHCB. Exposure to the microtubule disrupting agent colchicine has no effect on uPA or tPA accumulation. In addition, antibody directed against the beta 1 integrin subunit can also specifically elicit increased PA production. Thus disturbing the cytoskeleton and cytoskeleton associated substrate adhesions promotes PA accumulation.


Studies of autonomic properties after healing of experimental myocardial infarction. The link between healed myocardial infarction, sympathetic activation, and sudden death has led to a series of studies in a specifically designed animal model. Transmural and non-transmural infarcts were produced in cat hearts and the studies were performed few months later. Using direct stimulation of cardiac sympathetic nerves and measuring local refractory periods it was found that there was an exaggerated response in the border zone. Competitive binding studies revealed that there was a modest reduction in the number of beta-adrenergic receptors in tissue adjacent to the scar. In similar preparations the norepinephrine content, in hearts with healed transmural infarcts, was significantly reduced in areas adjacent to scars. In contrast, in hearts with non-transmural infarcts, the norepinephrine content was not different from normal controls. Phenylephrine and isoproterenol induced afterdepolarizations, blocked respectively by phentolamine and propranolol, in 34% of Purkinje fibers of the preparations with a healed infarct. Thus, afterdepolarizations and triggered activity may be mediated by both alpha- and beta-adrenergic activity in hearts with healed myocardial infarction. These data suggest the persistence, after healing of myocardial infarction, of regional abnormalities in autonomic function at a myocardial level. These regional disparities are likely to influence propensity to arrhythmias.


[Syndromes of venous mesenteric ischemia: infarction and transient ischemia]. The reports of 8 patients with acute or subacute abdominal pain related to venous mesenteric ischemia were reviewed. None of the patients presented local or regional predisposing factors for venous thrombosis. In 4 patients, a localized segment of ischemic small bowel (median length 125 cm; range: 30-350) was resected without immediate anastomosis and postoperative anticoagulation therapy was given. Two of these patients developed recurrent ischemia involving the bowel adjacent to the stoma, treated successfully in 1 case by a repeat resection. The 4 other patients hospitalized with intestinal obstructive symptoms (1 case) or abdominal angina (3 cases) were treated by long term anticoagulation in 3 cases and artificial nutrition in 2 cases. None of them developed mesenteric infarction with a median follow up of 34 months. In 7 of the 8 patients, a coagulopathy was found: primary myeloproliferative disorder (1 case), hypercoagulation state (5 cases), autoimmune hemolytic anemia (1 case). These observations suggest that venous mesenteric ischemia included two different entities on the basis of clinical and morphological criteria: mesenteric infarction and subacute transient ischemia without bowel infarction. Most of apparently idiopathic cases of acute or subacute venous mesenteric ischemia are related to hypercoagulation states requiring a long term anticoagulation.


Calcified gastric carcinoma: CT findings. The computed tomographic (CT) findings of 13 cases of calcified gastric carcinoma were analyzed retrospectively. Eleven cases were confirmed as a mucinous adenocarcinoma by surgery (three cases), or endoscopic biopsy (eight cases). Two cases were diagnosed as adenocarcinoma by endoscopic biopsy. In all cases the calcifications were of the punctate or miliary shape and the size varied from 1-3 mm in diameter. The calcifications were located in the thickened gastric wall in all cases, and were seen in metastatic lesions such as lymph nodes and the liver in two cases. In 10 cases, some tumor portions showed lower attenuation number than that of the muscle on CT scans, and corresponded to mucin pool in tumor portions histologically. Twelve cases were in inoperable advanced stage.


MRI manifestations of peritoneal carcinomatosis. Three cases of proved peritoneal carcinomatosis were examined by magnetic resonance imaging (MRI). Air was used to distend the entire gastrointestinal tract via an antegrade method. The findings included seedings along the small intestine, transverse and sigmoid colon, stellate pattern in the mesentery, plaque-like and bulky tumor masses in the mesentery and greater omentum, and focal thickenings along the right subdiaphragmatic parietal peritoneum. Stenosis caused by tumor encasement at the duodenojejunal junction and ileocolic anastomosis were first detected by MRI and later confirmed by barium studies. Ascites was present in all cases. One case showed ascites located only along the left paracolic gutter. This report shows that MRI is also able to demonstrate peritoneal carcinomatosis by using air as a gastrointestinal contrast medium.


Hepatitis type C virus infection in patients with type B chronic liver disease. Anti-c100-3 (Ortho) was determined in the sera of 152 patients with HBs antigen-positive chronic liver diseases to assess coinfection of hepatitis B virus (HBV) and hepatitis C virus (HCV). Eleven patients (7.2%) were positive for anti-c100-3. Anti-CP-9 (Okamoto) and HCV-RNA (RT-PCR) were also examined in these 11 patients. Anti-CP-9 was detected in 7 patients and HCV-RNA was detected in all 11 patients. Four of the 11 anti-c100-3-positive patients were positive for HBe antigen (HBeAg) and others were negative. In 8 of the 11 patients, HCV was suspected to be superinfected by blood transfusion. In HBeAg-positive patients, serum glutamic pyruvic transaminase (SGPT) was elevated in relation to active replication of HBV shown by DNA-polymerase activity. The histological findings showed chronic active hepatitis, with or without cirrhosis. On the other hand, in HBeAg-negative patients, SGPT fluctuated without evidence of active replication of HBV. Active inflammation in the liver was observed in 3 of 5 HBeAg-negative patients by liver biopsy. These findings suggest that HBV might play an important role in chronic active inflammation in HBeAg-positive patients coinfected with HCV, and that HCV might be responsible for continuous inflammation in HBeAg-negative patients coinfected with HCV.


Arachidonate metabolism in D-galactosamine or carbon tetrachloride-induced acute and chronic liver injuries in rats. Arachidonate metabolism was examined in rats with experimentally induced acute and chronic liver injuries. Acute liver injury was induced by a single administration of D-galactosamine (D-Galn) and lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). Chronic liver injury was produced by several administrations of CCl4 for 5 weeks. Non-parenchymal liver cells from rats with D-Galn/LPS-induced acute liver injury produced prominently leukotriene B4 and 5-hydroxy-arachidonic acid which were hardly synthesized by the normal rat liver. No apparent changes were observed in the arachidonate metabolism of the non-parenchymal cells of the acute CCl4-injured liver. In chronic liver injury, the production of 6-ketoprostaglandin F1 alpha, a stable metabolite of prostaglandin I2, by the non-parenchymal cell fraction was significantly enhanced in contrast with the fixed amount of the other arachidonate metabolites. These results suggested the arachidonate metabolism by non-parenchymal liver cells might change according to the pathogenesis of the liver disease.


Alanine aminotransferase and HCV-RNA responses following interferon therapy of HCV-RNA positive chronic hepatitis. Interferon (IFN) has been shown to be effective for chronic hepatitis C. This study investigated changes of alanine aminotransferase (ALT) and HCV-RNA in chronic hepatitis C patients treated with alpha-IFN. IFN was given to 73 patients with HCV-RNA positive chronic hepatitis C. The pattern of changes in ALT activity after IFN administration was classified into five types. Type 1 was characterized by normalization of ALT (< or = 25 K.U) during IFN administration and sustained normalization after the IFN therapy. Type 2 involved a rebound of ALT after termination of IFN therapy and subsequent normalization. Type 3 had no ALT normalization during IFN administration, with normalization after the completion of the therapy. Type 4 involved transient normalization of ALT level during IFN therapy, with a subsequent reversion to abnormal levels after the termination of IFN therapy. Type 5 showed sustained abnormally high levels of ALT activity both during and after treatment. Twenty four patients (32.9%) had sustained normalization ALT (< or = 25 K.U) after the termination of IFN treatment. The HCV-RNA negative rate at 6 months after IFN therapy in patients with sustained normalization of ALT was 87.5% (21/24).


Effects of aspirin on pathways of ion permeation in Necturus antrum: role of nutrient HCO3. Intracellular microelectrodes were used to evaluate electrical properties of the cell membranes in Necturus antral mucosa during exposure to luminal acid alone (pH 4) or to 5 mmol/L aspirin [acetylsalicylic acid (ASA)] in the presence of luminal acid. When nutrient solutions were buffered by HCO3- (pH 7.3), ASA moderately depolarized and increased the resistances of both cell membranes. When nutrient solutions were buffered by HEPES (pH 7.3), ASA induced even greater depolarizations of the cell membranes. In addition, resistance of the apical membrane did not increase and resistance of the basolateral membrane decreased. The changes in basolateral membrane resistance were observed when tissues were exposed to 5 mmol/L salicylate but not during exposure to luminal acid alone or to acidified luminal solutions containing 5 mmol/L acetate, a small and permeable organic acid. Electron microscopy confirmed that these initial electrophysiological changes precede alterations in cell morphology. The findings suggest that nutrient HCO3- attenuates changes in membrane potentials caused by ASA. Loss of nutrient HCO3- seems to accelerate alterations in basolateral membrane resistance caused by ASA and its salicylate moiety.


Intermittent jaundice by tumor emboli from intrahepatic cholangiocarcinoma. Free-floating tumor debris or mucobilia as a cause of intermittent obstruction has been described infrequently. A patient with intermittent jaundice caused by tumor emboli from an intrahepatic polypoid mucinous cholangiocarcinoma is presented. Symptoms of intermittent jaundice and midepigastric pain persisted over 5 years despite an initial cholecystectomy and common bile duct exploration before definitive diagnosis and treatment of an hepatic trisegmentectomy (segments II, III, and IV). Intraductal mucin was confirmed intraoperatively and pathologically as the cause of the obstructive jaundice. The patient remains asymptomatic and without evidence of disease more than 5 years postoperatively. This report of a predominantly mucin-producing intrahepatic cholangiocarcinoma details a rare protracted clinical course of intermittent biliary obstruction from mucus emboli and highlights the possibility of long-term survival after complete resection.


Slow off-rate of prazosin accounts for deviations from competitive antagonism in the isolated perfused kidney of rat. 1. The kinetics of the interaction of prazosin with noradrenaline, administered by bolus injection and infusion, was examined in the isolated perfused rat kidney. 2. Prazosin (1-30 nM) competitively antagonized vasoconstrictor responses to infusion of noradrenaline (pA2 = 9.51) whereas antagonism toward bolus injections of noradrenaline deviated from competition. 3. In the presence of prazosin, responses to bolus injection of noradrenaline became biphasic whereas the kinetics of the responses to infusions of noradrenaline were unchanged. 4. In contrast, phentolamine (30-300 nM) competitively antagonized responses to bolus injection of noradrenaline without altering response kinetics and in coperfusion studies with prazosin, prevented prazosin from inducing biphasic responses to bolus injection of noradrenaline. 5. It is concluded that prazosin acts as a pseudoirreversible antagonist toward vasoconstrictor responses to bolus injections noradrenaline (non-steady state conditions) whereas steady state conditions and competitive kinetics are observed with infused noradrenaline.


Effects of 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one on steroidogenesis in gonads of rat fetuses. 1. The effect of 5 alpha-reductase inhibitor 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA) on the fetal rat steroidogenesis was examined. 18- and 20-day-old fetal ovaries were incubated in the presence of dcAMP (1 mM) and spironolactone (10 mM) and progesterone production was assessed with and without 4-MA (0.01-50 microM). 2. High concentrations of 4-MA significantly decreased the production of progesterone. 3. Similar inhibition of the testosterone synthesis was observed in 16-day-old fetal testes explanted either in the control medium (M199) or in the presence of LH (100 ng/ml) when treated with 4-MA. The effect was rapidly reversible in both gonads.


Effects of nitrendipine and nisoldipine on biogenic amines in discrete areas of rat brain. 1. Biogenic amines and metabolites were determined in discrete brain sections obtained from rats once or repeatedly treated with nitrendipine or nisoldipine. 2. The increase in both the 5-HIAA levels and the 5-HIAA/5-HT ratio suggested that the drugs activated the serotonergic system in various brain areas. 3. Moreover, nisoldipine decreased the HVA content and the HVA/DA ratio in the striatum. 4. This suggested that nisoldipine, but not nitrendipine, inhibited dopaminergic neurotransmission in this brain area. 5. The effects induced by repeated treatment with nitrendipine or nisoldipine were reduced as compared to those caused by a single administration of these drugs. 6. All these facts could be explained on the basis of the pharmacokinetic properties of nitrendipine and nisoldipine and by the ability of these drugs to interact with the brain VSCC.


Stress response in the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata): interaction between CRF, ACTH, and biogenic amines. Previous studies reported that ACTH molecules influence chemotactic and phagocytic activities of hemocytes in the freshwater snail, Planorbarius corneus. The present study reveals that ACTH and CRF affect the release of biogenic amines. Hemocytes from P. corneus hemolymph incubated in vitro with ACTH for 15, 30, and 45 min released epinephrine, norepinephrine, and dopamine. The greatest release occurred after 15 min, while after 45 min the values were similar to those of the controls. Similar incubations with CRF also provoked a release of biogenic amines, this being mainly mediated by the release of endogenous ACTH. These data suggest that (i) ACTH and CRF provoke the release of biogenic amines; (ii) there is a direct relationship between CRF, ACTH, and biogenic amines, with the hemocytes as the target; (iii) exogenous ACTH can mimic an ancestral type of stress response; (iv) the major pathway of the stress response in P. corneus is mediated by a CRF-ACTH-biogenic amine axis. These data should help to unravel part of the complex molecular signaling mechanisms involved in the physiological/endocrinological reaction of invertebrate organisms to stress, and suggest that a stress response unexpectedly similar to that present in mammalian cells is detectable in invertebrates.


Alpha-melanocyte-stimulating hormone stimulates sodium excretion in the salt gland of the duck. Salt glands of ducks were induced to secrete sodium through the ingestion of salt water. In salt-adapted animals the administration of melanocyte-stimulating hormone (MSH) produced a rise in the sodium excreted by the salt gland, an effect which was not mimicked by adrenocorticotropin. Studies in vitro using incubations of gland slices and radioactive sodium ion showed that MSH increased sodium efflux, indicating that it acted directly upon the gland. We have previously observed that MSH has no effect on the pigmentary system of the duck. It is proposed that in the evolutionary process this hormone has acquired new target tissues in these birds.


Analysis of autofeedback mechanisms in the secretion of pro-opiomelanocortin-derived peptides by melanotrope cells of Xenopus laevis. The secretion of most pituitary hormones is under the control of feedback mechanisms. The feedback control of alpha-melanophore-stimulating hormone (alpha-MSH) from melanotrope cells is controversial. The possible existence of an autofeedback exerted by alpha-MSH or other POMC-derived peptides on melanotrope cells of the amphibian Xenopus laevis has been investigated. alpha-MSH or its potent agonist 4-norleucine,7-D-phenylalanine-alpha-MSH has no effect on the release of radiolabeled POMC-derived peptides or immunoreactive beta-endorphin from superfused neurointermediate pituitary lobes. Melanin concentrating hormone, previously reported to have an alpha-MSH-like effect on melanophores, did not affect alpha-MSH secretion. Neurointermediate lobe superfusate, which contains a mixture of POMC-derived peptides, failed to affect the secretory activity of melanotropes. It is concluded that in X. laevis the secretory activity of melanotropes is not under the control of short-term autofeedback mechanisms involving alpha-MSH or other POMC-derived peptides.


Nonlinear relationship between V+max and h infinity in frog skeletal muscle. The relationship between the maximum velocity of action potential upstroke (V+max) and steady-state Na+ channel inactivation (h infinity) was studied in frog skeletal muscle during repetitive discharges evoked in the presence of cevadine (1 mumol/l). Conventional microelectrodes and vaseline-gap voltage-clamp techniques were used. A severe degree of nonlinearity was found between (h infinity) and (V+max) especially when the Na+ conductance (gNa) was small. The observed nonlinearity could be explained as a property of the normal Na+ channel gating in skeletal muscle rather than that of cevadine-modified channels. Part of this work has been published in abstract form in Biophys. J. 57: 105A, 1990.


Study of activated oxygen production by some thiols using chemiluminescence. 2-3-dimercapto-1-propane sulfonic acid, D-penicillamine and meso-dimercapto succinic acid, drugs widely applied as antidota against metal poisoning, and cysteine and glutathione were studied with respect to their ability to generate and to scavenge superoxide anion radical. Superoxide production and scavenging were tested by means of luminol-dependent chemiluminescence. In presence of 1 mumol/l ADP-Fe3+ only cysteine and meso-dimercapto succinic acid induced chemiluminescence which could be inhibited by superoxide dismutase. 2,3-dimercapto-1-propane sulfonic acid, D-penicillamine and glutathione acted as O2- scavengers. These thiols inhibited O2(-)-dependent lipid peroxidation thus acting as antioxidants, whereas cysteine and meso-dimercapto succinic acid accelerated peroxidation. It is suggested that the toxic side effects of thiols may be due to their ability to generate or to scavenge free radicals.


Inhibitory effect of piperidinoethylesters of alkoxyphenylcarbamic acids on photosynthesis. Piperidinoethylesters of 2-, 3- and 4-alkoxysubstituted phenylcarbamic acids (alkoxy = methoxy - decyloxy) inhibit photosynthetic processes in algae and plant chloroplasts. The inhibitory activity is strongly dependent on the alkyl chain length of the alkoxy-substituent showing a typical quasi-parabolic dependence with maximum effect at 6-8 carbon atoms in the alkyl chain. The alkoxy-substitution in position 2 decreases the inhibitory activity of a compound when compared with its 3- and 4-substituted analogues. ESR studies of spinach chloroplasts confirm that the compounds studied cause destruction of PS II whereby, in the presence of the most effective of the derivatives tested, Mn2+ ions are released from the protein complex.


Absorption and Raman spectroscopy study of cyt c-thiol complexes in acidic solutions. Absorption UV-VIS and pre-resonance Raman spectra of acidic cyt c solutions with a series of thiols added (thiophenol, n-propanethiol, isopropanethiol, L-cysteine, dithiothreitol, 2-mercaptoethanol, N-acetyl-L-cysteine, p-acetamidothiophenol, 2-mercaptoethanamine, thioglycolic acid and mercaptopropionic acid), are presented. Interactions of cyt c molecule with the thiols were studied with the aim to identify binding of the thiols with the cyt c heme as its iron axial ligands. Absorption and Raman spectra showed some correlation between maxima of 700 nm region absorption band (typical for Fe-S axial bond in cyt c heme) and also wave numbers of spin state marker and axial ligand sensitive Raman bands on one, and pKa constant values of appropriate thiols on the other hand. These results imply thiol replacement of Met-80 from axial bond with heme iron and suggest that the force of Fe-L-cysteine axial bond is very close to the native axial bond (Fe-Met) for cyt c in neutral solution.


Species-dependent differences in the effect of ionic strength on potassium transport of erythrocytes: the role of lipid composition. The Rb+(K+) efflux of erythrocytes from six mammalian species was investigated in solutions of physiological and low ionic strength. A species dependent increase of the Rb+(K+) efflux in low ionic strength solution could be observed. The rate constant of Rb+(K+) efflux of erythrocytes in physiological ionic strength solution correlates with the content of arachidonic acid of the membrane phospholipids. The same relation was observed in solution of low ionic strength with the exception of human erythrocytes. In addition, an age-dependent correlation of the rate constant of Rb+(K+) efflux from calf erythrocytes in low ionic strength solution with the content of arachidonic acid of the membrane phospholipids was found. The Rb+(K+) efflux of human erythrocytes, which is enhanced in low ionic strength solution, decreases with the decreasing temperature. The temperature-dependent ESR order parameter of a fatty acid spin label for human and cow erythrocytes in solution of physiological and low ionic strength media suggested that the effect of low ionic strength on Rb+(K+) efflux is not solely based on a change of membrane fluidity. The results are interpreted as being due to a specific influence of membrane phospholipids on the Rb+(K+) efflux.


The effect of arsenate and vanadate ions on the critical cell volume of bovine erythrocytes. Arsenate, used as an inhibitor of glycolysis, decreases the critical cell volume of erythrocytes, whereas orthovanadate, used as an accelerator of dephosphorylation of phosphatidyl-inositol-4,5-biphosphate, exerts an opposite effect. The ATP-dependent changes of the critical cell volume do not depend on the phosphorylation state of phosphatidylinositol. An interaction of the membrane skeleton with the lipid-protein matrix has been proposed as a regulation mechanism of the critical cell volume of erythrocytes.


A spin probe study of the effects of chlorpromazine and its derivatives on lipid-protein interactions in synaptosomal membranes. The electron spin resonance spectra of 16-doxyl stearic acid (16-SA) incorporated into synaptosomes mostly showed a fluid lipid component and a minor motionally-restricted component (MRC) of the molar fraction of 10-20%, measured at 0 degree C. At 10 mmol/l concentration, thioridazine (TRZ), chlorpromazine (CPZ), chlorprothixene (CPT), perphenazine (PFZ) and levopromazine (LPZ) raised the MRC molar fraction in the synaptosomes to 100, 92, 65, 41 and 39%, respectively (as detected by the spin probe at 0 degrees C). At 4% concentration, TRZ, CPZ, CPT, PFZ, and LPZ the respective MRC percentages were 100, 75, 41, 24 and 17%. In synaptosomal membranes, AMRC splitting values of MRC, induced by TRZ and CPZ, were similar to those of the probe in human serum albumin. MRC induced by CPZ and TRZ was constant (+/- 15%) within the temperature range from 0 to 30 degrees C. At drug/lipid ratios > or = 2 : 1, TRZ and CPZ formed rigid complexes with total lipids isolated from the rat brain. The complexes melted upon increasing the temperature of the samples over 10-20 degrees C. The drugs decreased the lipid concentrations in synaptosomes in the order of potency TRZ > CPZ > CPT > PFZ > or = LPZ; this was similar to their effect on MRC increase. The drugs tested increased the membrane dynamics/disordering, and their potency fairly correlated with their MRC increasing effects. It is supposed that the drug-induced 16-SA probe MRC increase in synaptosomes was a result of mainly decreased lipid/protein ratio in the synaptosomal membranes, which in turn probably is connected with perturbation of lipid-protein interactions and/or membrane proteins. The perturbation of lipid-protein interactions and/or membrane proteins may be connected with the drug perturbation effect on the bulk lipid membrane part.


Characterisation of the potassium influx in rat erythrocytes. In the rat erythrocyte membrane five different transport pathways for K+ are present. In addition to the well characterised K+ transport via the Na+ pump, the Na,K,Cl cotransport and the Ca(2+)-activated K+ channel, there are a K,Cl cotransport and a residual (leak) K+ transport. The K,Cl cotransport is already present under physiological conditions, and can be stimulated by N-ethylmaleimide treatment but not by a cell volume increase. A low ionic strength stimulated increase of the residual K+ influx can be demonstrated in rat erythrocytes after suppressing the K,Cl cotransport pathway. Between 11 and 19 weeks of age, rats show significant differences in all transport pathways of the erythrocyte potassium influx. Using influx data from individual rats a significant correlation between the total K+ influx and the ouabain-sensitive K+ influx has been found. Maintaining the rats on a diet poor in essential fatty acids leads to a significant change of the linoleic acid content of the erythrocyte membrane phospholipids. However, no significant effect on the various K+ transport pathways has been found. An analysis of the fatty acid composition of the erythrocyte membrane phospholipids showed significant correlations between the content of oleic acid, and arachidonic acid, and the ouabain-sensitive K+ influx (as well as the total K+ influx).


Maxi chloride channels in L6 myoblasts.

Maxi chloride channels in L6 myoblasts. The existence of a large conductance voltage sensitive chloride channel is documented in undifferentiated cells (myoblasts) of the L6 rat muscle cell line. At this stage of development the resting membrane conductance is dominated by potassium ions only (Kidokoro 1975). The conductance of the channel in symmetrical 120 mmol/l choline chloride is 331 +/- 4 pS. The probability of the channel being in the open state decreases with the increasing imposed voltage. Due to rapid inactivation at high membrane potential deviations (both negative and positive) from the equilibrium potential the channel can be resolved clearly by pulse technique protocols only. The incidence of the channel in successful patch trials was higher than usually reported. The channel was present after differentiation of the myoblasts into the myotubes. It showed at least one definite substate and pronounced flickerings between the substate and the main open state. The channel was observed in myoblast attached patches as well. It is supposed to belong to the category of maxi chloride channels, and to play probably a role in regulatory volume readjustment or in cell communication during myogenesis, respectively.


Sequence analysis and relationships between meningococcal class 3 serotype proteins and other porins from pathogenic and non-pathogenic Neisseria species. The presence of highly conserved regions within previously determined porin gene sequences from Neisseria meningitidis and Neisseria gonorrhoeae permitted the construction of oligonucleotide primers for PCR amplification of other neisserial porin genes. Although two separate porin genes (porA and porB) are present in N. meningitidis only a single fragment, corresponding to porB, could be amplified from this species. The amplified porB genes from four different meningococcal serotypes, which express the class 3 outer membrane protein, were sequenced. Amplified fragments corresponding to porin genes from N. lactamica and N. sicca were also sequenced. In common with the known neisserial porins, models of the organisation of the predicted proteins indicated trans-membrane structures with eight surface exposed loops. In the meningococcal class 3 proteins the main regions of sequence variation, which must be responsible for serotype specificity, were located on loops 5 and 7. A phylogenetic analysis of the family of porins from the Neisseria confirmed the close relationship of the meningococcal class 3 protein with the gonococcal PIA protein, while the gonococcal PIB protein was shown to be closely related to the N. lactamica porin. The close relationship seen between porins of the pathogenic and non-pathogenic Neisseriae identified no obvious virulence-associated regions in the proteins, but did suggest that the current nomenclature for neisserial porin genes may need reviewing.


Oligomerization-mediated activation of plasmid-borne genes in Escherichia coli. A plasmid carrying a weakly expressed neomycin phosphotransferase (neo) gene from the transposable element Tn5 was found to confer elevated levels of antibiotic resistance on its host cell when it existed in a non-monomeric state. This activation of the neo gene appeared to be a generalized effect which can be exerted on any plasmid-encoded gene, since specific sequences were not required for enhanced neo expression, and the activity of a plasmid-borne chloramphenicol acetyltransferase gene could be similarly induced by oligomerization. The potential role that multiple origins of replication present in such oligomeric plasmids play in these observed increases in gene expression is discussed.


Cloning and nucleotide sequence of the Pseudomonas aeruginosa nfxB gene, conferring resistance to new quinolones. The gene nfxB is one of the genes which affect the cell membrane permeability of quinolones in Pseudomonas aeruginosa PAO. Both wild-type nfxB and a mutant nfxB (nfx13E) were cloned and the DNA sequences were determined. The wild-type gene was dominant in PAO strains. The nfxB mutation was a point mutation (cytosine----guanine) which generates an amino acid exchange (arginine----glycine) in the putative nfxB product. The amino acid sequence of the wild-type NfxB protein revealed that it has a helix-turn-helix motif which may be responsible for the ability to bind in a sequence-specific manner to DNA. This finding indicated that the NfxB protein may regulate the expression of genes that are associated with cell permeability of drugs in P. aeruginosa. The position of the amino acid substitution between the NfxB protein and the Nfx13E protein was located within a possible DNA-binding domain, suggesting that the mutant protein (Nfx13E) may have lost DNA binding ability and regulator activity.


Reversibility of heat shock in Chlamydia trachomatis. The heat shock effect on chlamydia development was studied. We report here that the reversibility of the heat shock response did not depend on the stage of chlamydial morphogenesis at which transfer to high temperature occurred, and the infectivity of the particles produced was not affected significantly, so long as the heat shock exposure was not prolonged. Exposure to heat shock for more than 9 h resulted in stagnation of the growth cycle, appearance of aberrant reticulate body particles and loss of infectivity. SDS-PAGE analysis of proteins synthesized under prolonged heat shock showed increased relative abundance of heat shock proteins in common with other procaryotic organisms.


Purification and characterization of macrolide 2'-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics. The resistance mechanism of Escherichia coli BM2506 to macrolides was found to be due to inactivation. Inactivated oleandomycin was identified as oleandomycin 2'-phosphate by thin-layer chromatography. A new type of macrolide-phosphorylating enzyme, macrolide 2'-phosphotransferase type II (MPH(2')II), was detected, purified 95-fold and its enzymological properties investigated. MPH(2')II was a constitutive intracellular enzyme which showed high levels of activity with both 14-member-ring and 16-member-ring macrolides. The optimum pH for the inactivation of oleandomycin was 8.2 and the optimum temperature of the reaction was 40 degrees C. Enzyme activity was lost by heat treatment at 60 degrees C for 1 min. The isoelectric point and M(r) of the enzyme were 5.3 and 48,000, respectively. Purine nucleotides, such as ITP, GTP and ATP, were effective as cofactors in the inactivation of macrolides. An inhibitory effect of iodine, EDTA, or divalent cations on MPH(2')II activity was observed.


SPT4, SPT5 and SPT6 interactions: effects on transcription and viability in Saccharomyces cerevisiae. The SPT4, SPT5 and SPT6 genes of Saccharomyces cerevisiae were identified originally by mutations that suppress delta insertion mutations at HIS4 and LYS2. Subsequent analysis has demonstrated that spt4, spt5 and spt6 mutations confer similar pleiotropic phenotypes. They suppress delta insertion mutations by altering transcription and are believed to be required for normal transcription of several other loci. We have now analyzed interactions between SPT4, SPT5 and SPT6. First, the combination of mutations in any two of these three genes causes lethality in haploids. Second, some recessive mutations in different members of this set fail to complement each other. Third, mutations in all three genes alter transcription in similar ways. Finally, the results of coimmunoprecipitation experiments demonstrate that at least the SPT5 and SPT6 proteins interact physically. Taken together, these genetic and biochemical results indicate that SPT4, SPT5 and SPT6 function together in a transcriptional process that is essential for viability in yeast.


A mutant tRNA affects delta-mediated transcription in Saccharomyces cerevisiae. Mutations in the SPT3, SPT7, SPT8 and SPT15 genes define one class of trans-acting mutations that are strong suppressors of insertion mutations caused by Ty elements or by the Ty long terminal repeat sequence, delta. These SPT genes are required for normal transcription of Ty elements, and their gene products are believed to be involved in initiation of Ty transcription from delta sequences. We have isolated and analyzed extragenic suppressors of spt3 mutations. These new mutations, named rsp, partially suppress the requirement for SPT3, SPT7, SPT8 and SPT15 functions. In addition, rsp mutations cause changes in transcription of some delta insertions in an SPT+ genetic background. Interactions between mutations in the four identified RSP genes show a number of interesting genetic properties, including the failure of unlinked rsp mutations to complement for recessive phenotypes. Cloning and sequencing of one rsp mutant gene, rsp4-27, showed that it encodes a frameshift suppressor glycine tRNA. Our results indicate that the other three RSP genes also encode frameshift suppressor glycine tRNAs. In addition, other types of frameshift suppressor glycine tRNAs can confer some Rsp- phenotypes.


Somatic and germinal recombination of a direct repeat in Arabidopsis. Homologous recombination between a pair of directly repeated transgenes was studied in Arabidopsis. The test construct included two different internal, non-overlapping deletion alleles of npt (neomycin phosphotransferase) flanking an active HPT (hygromycin phosphotransferase) gene. This construct was introduced into Arabidopsis by agrobacterium-mediated transformation with selection for resistance to hygromycin, and two independent single-insert lines were analyzed. Selection for active NPT by resistance to kanamycin gave both fully and partly (chimeric) recombinant seedlings. Rates for one transgenic line were estimated at less than 2 x 10(-5) events per division for germinal and greater than 10(-6) events per division for somatic recombination, a much smaller difference than between meiotic and mitotic recombination in yeast. Southern analysis showed that recombinants could be formed by either crossing over or gene conversion. A surprisingly high fraction (at least 2/17) of the recombinants, however, appeared to result from the concerted action of two or more independent simple events. Some evolutionary implications are discussed.


[Isolation, characterization and mapping of the N15 phasmid insertion mutations]. Prophage of N15 temperate bacteriophage is stably maintained in Escherichia coli lysogens as a 46.33 kb linear plasmid. Using different transposons we obtained 18 insertion mutants of the N15 plasmid prophage. They were analysed for plaque formation ability, stability of the plasmid state and lysogenic conversion. Restriction mapping of the insertions allowed us to localize on the map the regions necessary for lytic growth and to map the lysogenic conversion gene. A recombinant phage encoding two antibiotic resistance genes was obtained. The phage contains an additional 4.77 kb DNA fragment (over 10% of the N15 genome).


Evidence that intergenic spacer repeats of Drosophila melanogaster rRNA genes function as X-Y pairing sites in male meiosis, and a general model for achiasmatic pairing. In Drosophila melanogaster males, X-Y meiotic chromosome pairing is mediated by the nucleolus organizers (NOs) which are located in the X heterochromatin (Xh) and near the Y centromere. Deficiencies for Xh disrupt X-Y meiotic pairing and cause high frequencies of X-Y nondisjunction. Insertion of cloned rRNA genes on an Xh- chromosome partially restores normal X-Y pairing and disjunction. To map the sequences within an inserted, X-linked rRNA gene responsible for stimulating X-Y pairing, partial deletions were generated by P element-mediated destabilization of the insert. Complete deletions of the rRNA transcription unit did not interfere with the ability to stimulate X-Y pairing as long as most of the intergenic spacer (IGS) remained. Within groups of deletions that lacked the entire transcription unit and differed only in length of residual IGS material, pairing ability was proportional to the dose of 240-bp intergenic spacer repeats. Deletions of the complete rRNA transcription unit or the 28S sequences alone blocked nucleolus formation, as determined by binding of an antinucleolar antibody, yet did not interfere with pairing ability, suggesting that X-Y pairing may not be mechanistically related to nucleolus formation. A model for achiasmatic pairing in Drosophila males based upon the combined action of topoisomerase I and a strand transferase is proposed.


The distribution of genes on human chromosomes as studied by in situ nick translation. We have studied the distribution of potentially active genes on human chromosomes, using two methods: DNAse I hypersensitivity and restriction enzyme--nick translation with enzymes sensitive to methylation of CpG doublets. DNAse hypersensitivity is known to be associated with potentially active genes, and, when the reaction is detected by "in situ" nick translation, produces an R-banding pattern. Digestion of chromosomes with HpaII or CfoI, both of which should preferentially cut unmethylated sequences in the CpG islands associated with the majority of genes, also produces R-banding patterns. Deviations are attributable to overdigestion of the chromosomes, leading to extraction of DNA and loss of the specific sites that were to be detected. Contrary to the results of a number of previous workers, we have failed to demonstrate any differences between the DNAse I hypersensitivity or the degree of methylation of the active and inactive X chromosomes in metaphases from females.


DNA-promoted assembly of the active tetramer of the Mu transposase. A stable tetramer of the Mu transposase (MuA) bound to the ends of the Mu DNA promotes recombination. Assembly of this active protein-DNA complex from monomers of MuA requires an intricate array of MuA protein-binding sites on supercoiled DNA, divalent metal ions, and the Escherichia coli HU protein. Under altered reaction conditions, many of these factors stimulate assembly of the MuA tetramer but are not essential, allowing their role in formation of the complex to be analyzed. End-type MuA-binding sites and divalent metal ions are most critical and probably promote a conformational change in MuA that is necessary for multimerization. Multiple MuA-binding sites on the DNA contribute synergistically to tetramer formation. DNA superhelicity assists cooperativity between the sites on the two Mu DNA ends if they are properly oriented. HU specifically promotes assembly involving the left end of the Mu DNA. In addition to dissecting the assembly pathway, these data demonstrate that the tetrameric conformation is intrinsic to MuA and constitutes the form of the protein active in catalysis.


The aryldialkylphosphatase-encoding gene adpB from Nocardia sp. strain B-1: cloning, sequencing and expression in Escherichia coli. Using degenerate oligodeoxyribonucleotides (oligos) derived from the N-terminal sequence of an aryldialkylphosphatase (ADPase) from Nocardia sp. strain B-1, an amplification reaction was used to isolate a DNA segment containing a 57-bp fragment from the adpB gene. Based on the nucleotide (nt) sequence of this fragment, a nondegenerate oligo was synthesized and used to screen a subgenomic library of strain B-1 DNA for fragments containing adpB. A 3.55-kb PstI fragment containing adpB was cloned into Escherichia coli, and the nt sequence of a 1600-bp region containing adpB was determined. Under control of the lac promoter of pUC19, adpB expression in E. coli cultures was approx. 15-fold higher than in strain B-1 under the native adpB promoter. Comparison of adpB with the Flavobacterium ADPase-encoding gene, opd, revealed no significant homology at the nt or aa levels.


Transcriptional regulation of the Tn5276-located Lactococcus lactis sucrose operon and characterization of the sacA gene encoding sucrose-6-phosphate hydrolase. The Lactococcus lactis sucrose operon was located on the conjugative transposon Tn5276 and the nucleotide sequence of the sacA gene, encoding sucrose-6-phosphate hydrolase, and its surrounding regions was determined. Northern blot analysis showed that the sucrose operon contains two divergent transcriptional units of 3.2 and 3.6 kb, the expression of which is considerably higher in cells grown on sucrose than in cells grown on glucose. This was confirmed by primer extension studies which demonstrated that transcription is initiated at two sucrose-inducible promoters with a back-to-back organization. The 3.2-kb transcriptional unit includes the sacB gene which most probably encodes the sucrose-specific enzyme II of the phosphotransferase system, and may contain the gene encoding fructokinase. The 3.6-kb transcriptional unit includes genes sacA and sacR. The protein encoded by the sacR gene is likely to be involved in the regulation of the sac operon expression, since its deduced N terminus is homologous to helix-turn-helix DNA-binding domains found in several regulatory proteins.


Anti-hepatitis C virus in the elderly: a seroepidemiological study in a home for the aged. A seroepidemiological survey of anti-hepatitis C virus (anti-HCV) was carried out in 315 institutionalized elderly people. HBV serum markers were tested in the same sera. Clinical details were also studied in the anti-HCV-positive subjects. The overall prevalence of anti-HCV was 2.2%, while the prevalence of HBV serum markers was 36.8% (the HBsAg prevalence was 0.6%). In 1 subject anti-HCV was found in association with HBsAg positivity. Serum transaminase levels were found within the normal range in all 315 subjects (either anti-HCV+ve and anti-HCV-ve), except in the subject who was found to be HBsAg-positive and anti-HCV+ve. In conclusion we found in the institutionalized elderly people a similar prevalence of anti-HCV compared to blood donors of the same geographical area; homes for the aged appear to bring together subjects with previously acquired infections.


[The hygienic validation of the regulation of the use of phosphate slag in agriculture as a mineral fertilizer]. Phosphate slag MAC in the soil was accepted at the level of 500 mg/kg by the common sanitary and translocation (from the soil to plants) indexes. The phosphate slag had some embryotoxic effects, its threshold dose was at the level of 100 mg/kg. Maximum noneffective dose was 10 mg/kg in experiments on animals.


[Presence of human Herpes simplex virus-2 (HSV-2) in women attending a clinic for cervico-uterine cancer control]. For this prospective study, 284 women were recruited who attended a cancer detection (Papanicolaou testing) program and appeared to be healthy. The objective was to obtain cervicovaginal samples from this group and culture them on human foreskin fibroblasts to isolate and identify Herpes simplex virus by immunofluorescence with monoclonal antibodies. A total of 12 women (4.2%) were positive for the virus; upon comparison of various sociodemographic factors in positive and negative culture patients, it was possible to establish certain associations as follows: The presence of Herpes simplex virus type 2 was related to age, start of sexual life, number of sex partners, lower education and minimum income. Other markers observed in infected women were mucopurulent cervicitis, typical and non-typical confluent ulcerations and minor disruptions of the epithelium. The knowledge of this subclinical or somewhat asymptomatic Herpes prevalence should be considered to prevent further spread among couples and also to avoid the risk of reactivation in pregnant women.


[Behavior of nucleolar organizers in the normal cervix, human papilloma virus infection and cervico-uterine cancer]. Thirty five cervical biopsies were divided into four groups: 5 cases normal cervix, 10 cases with papillomavirus without atypias (HPVIO), 10 cases with human papillomavirus with atypias (HPVIA) and 10 cases with squamous carcinoma. With the colloidal silver technique, the proteins associated with nucleolar organizers were stained; they were found to increase significantly in number in the groups studied being lowest in the normal cervix and highest in the biopsies with carcinoma. The number of nucleolar organizers in HPVIO in our study was found to be similar to that in cervical intraepithelial neoplasia I (CIN I), according to Egan and the number in HPVIA similar to that in CIN II, suggesting that HPVIO is a low risk lesion and HPVIA a high risk lesion for cervical carcinoma.


[Results of treatment of condyloma in the male and its effect on the couple in the presence of intraepithelial cervical neoplasia]. Genital papillomavirus is an infection sexually transmitted, besides it is frequently associated with cancer of the uterine cervix. It is controversial if it is useful to treat the sexual partner. The present study analyzes in a comparative way patients with CIN with treated partner and untreated partner. From 1986 to 1991 251 women treated for CIN, were prospectively studied; they had completed a one year follow up and during this time they presented, or not, treatment failure. Fifty six were examined and their sexual partners were treated for genital papillomavirus, and the control were 195 women, similar to the study group as to age, race, socio-economical level, CIN histological degree, distribution of lesions and therapeutical methods, in whom the male was not examined nor treated. Statistical methods were Umann-Whitney, homogeneity test, and the fractions difference test to compare the groups. It were obtained the persistence and recidive of the treated couple and untreated couple (41% vs 32.8% with P greater than 0.05) not significant, which shows that papillomavirus treatment of the male partner has no effect on CIN treatment failure in the woman. These statistical results should be considered with caution as it is known that the male is a virus reservoir that together with other cofactors may explain the greater risk for the woman to develop CIN.


[Premorbid states of the bronchopulmonary system in workers in nitrogen fertilizer manufacture]. Prophylactic medical examination of workers engaged in the nitrous mineral fertilizers production (NMFP) revealed a group with the premorbid conditions of nonspecific pulmonary diseases. Advanced examination of subjects with premorbid forms of chronic bronchitis showed disorders in immunologic status and markedly changed external respiration parameters in restrictive and obstructive types. The disorders depended on the length of service. Immunologic tests combined with bronchial patency examination were recommended form early diagnosis of nonspecific pulmonary diseases in NMFP workers. Autoimmune reactions to the tracheal and pulmonary antigens play an important role in diagnosis of nonspecific pulmonary diseases.


[Morbidity of the peripheral nervous system among the crew of fishing vessels and their treatment under shipboard conditions]. The incidence of diseases of the peripheral nervous system was studied during the voyage in different groups of sea sailors. The diseases due to overcooling and overexercise (cervico-thoracic and lumbosacral radiculosis) appeared to predominate. Methods of electroneurostimulation of acupuncture points and reflexogenic zones, lymphotropic therapy were used in the treatment administered on board the ship.


Comparison of the effects of ispaghula and wheat bran on rat caecal and colonic fermentation. The effects of ispaghula and wheat bran on the contents of the caecum and proximal and distal colon of the rat were investigated to identify any differences that might account for their effects on colonic motility. Rats fed diets supplemented with 5% ispaghula and 10% wheat bran for 28 days were killed and the contents of the gut collected. Caecal and colonic content wet and dry weight and short chain fatty acid (SCFA) content were measured. In additional in vitro fermentations in batch cultures of mixed rat caecal bacteria with ispaghula and bran, SCFA production was monitored over 24 hours. Both ispaghula and wheat bran increased faecal weight but ispaghula was more effective. Ispaghula resulted in greater and more liquid contents, with a characteristic pattern of SCFA production (higher propionic acid) maintained throughout the colon. In contrast, wheat bran affected only the caecum and faeces. SCFA content and wet and dry weight in the proximal and distal colon were unaffected by wheat bran. Caecal butyrate was characteristically higher in wheat bran fed rats but ispaghula produced higher butyrate in the distal colon. In contrast, ispaghula seemed to be fermented more quickly in vitro than wheat bran. Thus, wheat bran has a portion that is rapidly fermented and an inert residue that may stimulate motility. Ispaghula seems to be fermented throughout the colon but maintains a high water content which dilutes the luminal contents.


Serum autoantibodies and the diagnosis of type-1 autoimmune hepatitis in Italy: a reappraisal at the light of hepatitis C virus infection. Antinuclear antibodies with the homogeneous pattern (ANA-H) and smooth muscle antibodies with antiactin specificity (SMA-AA) are regarded as the serum markers of type-1 autoimmune chronic hepatitis. Their diagnostic relevance, however, has been questioned recently after the detection of signs of hepatitis C virus infection in autoimmune chronic hepatitis patients. To further evaluate this point, antihepatitis C virus antibodies were sought by two second generation assays (ELISA 2 and RIBA 2) in 100 Italian patients with chronic liver disease of unknown aetiology, including 46 with (autoimmune chronic hepatitis) and 54 without the above antibodies (cryptogenic). By ELISA 2, antihepatitis C virus, although significantly prevalent in cryptogenic (83%), were found also in a substantial proportion of autoimmune chronic hepatitis patients (46%) (p < 0.0001), their occurrence was confirmed by RIBA 2 in almost all cases (96% and 86%, respectively). Autoimmune patients with either ANA-H or SMA-AA exhibited similar antihepatitis C virus prevalences (59% and 52%, respectively); by contrast, the eight cases positive for both the autoantibodies were consistently antihepatitis C virus negative. These findings confirm that in countries with high hepatitis C virus circulation (like Italy) an overlap between autoimmune chronic hepatitis and hepatitis C virus infection, reflected by 'true' antihepatitis C virus antibodies, does occur. The detection of ANA-H or SMA-AA, in fact, identifies chronic liver disease patients with a relatively low prevalence of antihepatitis C virus, but does not exclude hepatitis C virus infection. Positive findings for both ANA-H and SMA-AA, however, is an appropriate marker for hepatitis C virus free 'primary' autoimmune chronic hepatitis.


Malignant mixed Müllerian tumor of the fallopian tube: report of two cases and review of literature. Malignant mixed Müllerian tumors are usually found in the endometrium, vagina, cervix, and ovary. It is extremely rare for this tumor to arise in the fallopian tube, and to date only 37 tubal cases have been reported. We recently experienced 2 such cases. The clinical features, pathologic findings, diagnosis, therapy, and outcome of these 39 cases were reviewed. The clinical features and diagnosis were similar to those of primary carcinoma of the fallopian tube. Correct preoperative diagnosis was difficult. Histologically, 18 patients had homologous elements and 21 had heterologous elements in the sarcomatous components. The most common type of heterologous element was cartilage, followed by striated muscle and bone. The clinical stage (FIGO staging of ovarian carcinoma) was stage I in 15 cases, stage II in 11 cases, stage III in 8 cases, stage IV in 3 cases, and unknown in 2 cases. In all the patients except 1, the tumor was surgically removed. Postoperatively, radiotherapy was given to 9 patients, chemotherapy to 9 patients, and both to 2 patients. Sixteen patients died of the disease, after a mean period of 16.1 months. Of the 15 stage I patients, 10 survived more than 12 months. The most important prognostic factor was spread of the tumor at diagnosis.


Treatment of small cell carcinoma of the cervix with cisplatin, doxorubicin, and etoposide. Between July 1986 and February 1991, 10 patients with small cell carcinoma of the cervix were prospectively treated with combination chemotherapy using cisplatin (50 mg/m2) and doxorubicin (50 mg/m2) on Day 1 and etoposide (75 mg/m2) on Days 1-3. All patients underwent an extensive pretreatment metastatic survey and had histologic confirmation of small cell carcinoma prior to entry in the study. Seven patients had stage Ib, 1 stage IIa, and 2 stage IIb. Nine patients received chemotherapy at primary presentation and 1 was treated for recurrent disease. In 6 cases, chemotherapy was given and then followed by radiation therapy. Three patients received chemotherapy following radical hysterectomy and 1 was treated for persistent disease after radiation therapy. Patients received a median of four courses of chemotherapy (range 2-6). Neutropenia was the dose-limiting toxicity with 9 of 10 patients requiring a dose reduction. There was no instance of neutropenic sepsis or other major toxicity. Seven patients had measurable disease at the start of therapy. Three of these patients had a complete clinical response, 1 had a partial response, 2 had stable disease, and 1 had progressive disease (response rate = 57%). The median survival was 28 months. At the time of this report, 4 of 6 patients with stage Ib cancers given primary treatment on this regimen remained free of disease (with 28 months the median follow-up). Our pilot study indicates that this chemotherapy regimen has activity in small cell carcinoma of the cervix and should be further evaluated as an adjuvant to surgery or radiation in patients with early stage disease.


Acute stress stimulates 3H-inositol phosphates accumulation in rat cerebral cortex. An in vivo determination. Using an in vivo method, we examined the effects of acute stress on rat brain inositol phosphates (IPs) levels. Cerebral phosphoinositides were prelabeled with 3H-myoinositol injected i.c.v. 24 h prior to a 15 min forced swimming stress. The stressful condition resulted in a 35% increase in whole brain 3H-IPs content, mainly due to an enhancement in cerebral cortical 3H-IPs accumulation. Cerebellar and brain minus cortex levels of 3H-IPs showed no alteration in stressed animals. Among the IPs, stress induced a 102% and 92% increase in the cerebral cortical content of 3H-IP and 3H-IP2 respectively. Pretreatment of animals with diazepam (2 mg/kg, i.p.) prevented the stress-induced 3H-IPs increase, in both the whole brain and the cerebral cortex. Diazepam administration alone did not produce changes in 3H-IPs. These results suggest that an acute stress condition stimulates in vivo the brain IPs turnover, probably because of an enhanced receptor-linked hydrolysis of phosphoinositides.


Effects of CDP-choline administration on receptor-stimulated inositol phospholipid hydrolysis in brain slices and neuronal cultures. Repeated addition of CDP-choline (100 microM, once a day since the 2nd day of maturation in culture) to corticostriatal neurons led to an increased basal hydrolysis of inositol phospholipids, as revealed by an enhanced formation of [3H]inositolmonophosphate ([3H]InsP) in the presence of 10 mM Li+. This increase was prevented by the muscarinic receptor antagonist, atropine, or by tetrodotoxin, but not by other receptor antagonists, such as L-2-amino-4-phosphonobutanoate (L-AP4), prazosin or ketanserin. The increase in inositol phospholipid hydrolysis induced by repeated addition of CDP-choline was obliterated when cultures were incubated in the presence of the muscarinic receptor agonist, carbamylcholine. CDP-choline had no effect on inositol phospholipid hydrolysis in cultured cerebellar neurons, which are devoid of cholinergic cells. The basal hydrolysis of inositol phospholipids was also increased in hippocampal slices prepared from rats repeatedly injected with CDP-choline (200 mg/kg, i.p. for 15 days). As observed in cultured cortico-striatal neurons, this increase was prevented by atropine and was masked in the presence of carabamylcholine. Taken collectively, these data indicate that repeated exposure to exogenous CDP-choline increases polyphosphoinositide turnover, an effect that results from an increased availability of acetylcholine acting on muscarinic receptors.


Rubidium shows effects different from lithium on phosphatidylinositol metabolism in a cell line of human neuroblastoma. In a SK-N-BE human neuroblastoma cell line the incubation of rubidium (1 and 10 mM) for 24 h significantly increased IP2 formation, whereas it apparently did not affect other inositol phosphates. In comparison to lithium (10 mM), which significantly enhanced inositolmonophosphate and IP2 accumulation following carbamoylcholine (1 mM) stimulation, rubidium at the same concentration, was unable to affect inositol phosphate accumulation. In conclusion, the present experiments show that rubidium, compared with lithium, shows a different profile on phosphoinositide metabolism since its main action is an increase in phosphatidylinositol turnover. These results may have some relevance to the use of rubidium as antidepressant in man.


A follow-up study of 49 adult patients with idiopathic thrombocytopenic purpura treated with high-dose immunoglobulins and anti-D immunoglobulins. We analyze here short- and long-term results in 49 patients with idiopathic thrombocytopenic purpura (ITP), consecutively treated with high-dose (h.d.) immunoglobulins (Ig) or anti-D Ig. The major aims of this study were to assess the prognostic power of some patient characteristics and to verify the possibility that repeated courses of treatment can induce a stable remission. Moreover, the relative efficacy and safety of these two treatments were compared. Group A included 28 patients with chronic ITP and 17 with ITP of recent onset who were receiving h.d. Ig; Group B included 5 patients with ITP of recent onset and 7 with chronic ITP treated with anti-D Ig. Eight cases, receiving both treatment, were included in both groups. Response to treatment was defined as any increase in platelet count above 30 x 10(9)/l, when the platelet count was less than 10 x 10(9)/l, or any doubling of the basal platelet count otherwise. Remission was defined as any platelet count higher than 100 x 10(9)/l lasting for 3 months or longer without therapy. Cumulative response and remission rate was not statistically different in the two groups. Multivariate logistic regression analysis showed no influence of sex, previous therapy or duration of disease. Patients older than 60 years were definitely less responsive to h.d. Ig (58% vs 92%, p = 0.03). In Group A, two patients obtained remission after the first course of h.d. Ig. No additional remissions were obtained by repeated courses of h.d. Ig, apart from a single case of ITP of recent onset.(ABSTRACT TRUNCATED AT 250 WORDS)


Intraduodenal absorption in the rabbit of a novel heparin salt. The intraduodenal absorption of a new low-molecular-weight heparin (LMWH) diamine salt (ITF 1331) was compared with the parent compound ITF 1060 and with sodium LMWH, in anaesthetized rabbits. The administration of either salt, but not of sodium LMWH, resulted in a dose-related increase in plasma anti-Xa activity. In this respect ITF 1331 was slightly superior to ITF 1060, and in acute-toxicity studies the counterion itself (ITF 258) was less toxic than that in ITF 1060 (counterion No. 4). These data confirm that a tertiary diamine within the counterion is an important structural requirement for the bioavailability of heparin by the intraduodenal route, and suggest that ITF 1331 may represent an important advance in the search for an oral heparin.


Effective pre-emptive therapy with Ganciclovir and specific immunoglobulins for CMV infection in a bone marrow transplanted patient. A 29-year-old man, who underwent allogeneic bone marrow transplantation (BMT) for acute non lymphoid leukemia (ANLL), presented with blood pancytopenia and mild hypoxemia on day +39, without signs of acute graft versus host disease (GVHD). Cytomegalovirus (CMV) antigens were detected on WBCs and cells from bronchoalveolar lavage by immunofluorescence (IF) with specific murine monoclonal antibodies. Prompt treatment with Ganciclovir and high titer CMV immunoglobulins was followed by disappearance of the laboratory findings of CMV infection. Therapy was well tolerated and no side effects were recorded except for hematological depression that did not reverse after withdrawal of the therapy. The patient is relatively well on day + 210. Detection of CMV antigenemia appears to be a valuable tool for deciding whether to start CMV therapy with potentially toxic antiviral drugs in BMT patients in early engraftment, with or without overt signs of CMV infection.


[Effects of bufadienolides and some kinds of cardiotonics on guinea pig hearts]. Effects of bufadienolides such as bufalin (BF) and cinobufagin (CB), the main components of Senso (Ch'an Su), on myocardial Na+,K(+)-ATPase activity, the cardiotonic activity in vivo and the action potential of isolated guinea pig papillary muscle cells were compared with those of other cardiotonic drugs. 1) The rank order of potency for inhibition of myocardial Na+,K(+)-ATPase activity was BF greater than digoxin (DG) greater than digitoxin (DT) greater than telocinobufagin greater than gamabufotalin greater than cinobufotalin greater than CB greater than g-strophanthin (GS) greater than digitoxigenin (DTG) greater than resibufogenin (RB) when compared at the 50% inhibitory concentration. 2) In isolated papillary muscle cells, CB shortened the action potential duration (APD) dose-dependently. The order of potency for shortening of APD was GS greater than CB greater than DTG much greater than DT. 3) In open-chest guinea pigs, intraduodenal administration of BF or CB increased the myocardial contractile force (MCF), but did not affect the heart rate. The order of potency for increase in MCF was as follows: methyldigoxin, proscillaridin greater than BF greater than CB greater than DG greater than Senso much greater than DT, DTG, RB. These results indicate that CB has a shortening effect on APD and an inhibitory effect on Na+,K(+)-ATPase activity along with its cardiotonic effect, like GS.


[High affinity binding of Y-25130 for serotonin 3 receptor]. The binding of Y-25130 on serotonin 3 (5-HT3) receptors was evaluated by examining its effect on 3H-BRL 43694 (3H-granisetron) binding to rat cerebral cortex membranes in comparison with those of granisetron, ondansetron and metoclopramide. Y-25130, granisetron, ondansetron, metoclopramide, 5-HT and 2-methyl-5-HT displaced the specific binding of 3H-granisetron to the synaptosomal membranes in a concentration-dependent manner. The rank order of potency for inhibition of 3H-granisetron binding by the test compounds was Y-25130 not equal to granisetron greater than ondansetron much greater than 2-methyl-5-HT not equal to 5-HT not equal to metoclopramide. To determine the manner of interaction between Y-25130 and 5-HT3 receptors, Scatchard analysis of 3H-granisetron specific binding to the membranes of the cerebral cortex in the absence or presence of various concentrations of Y-25130 was performed. It was indicated that Y-25130 exerted a typical competitive-type inhibition of 3H-granisetron binding. The present study indicates that Y-25130 binds competitively to 5-HT3 receptors with high affinity.


[23Na-NMR measurements of the sodium concentration in guinea pig erythrocytes: the effects of cardiac glycoside and asebotoxin III]. Intra- and extracellular sodium in guinea pig erythrocytes was evaluated with sodium-23 nuclear magnetic resonance (23Na-NMR) by the use of a shift reagent, Dy(TTHA)3- or Dy(PPPi)2(7-). The test medium contained erythrocytes at 40% hematocrit level and NMR buffer (145 mM NaCl, 10 mM Dy(TTHA)3-, 10% D2O, adjusted to pH 7.4 with tris, at 35 degrees C). NMR spectra were obtained with a JEOL GSX 400 spectrometer operating at the Fourier transform mode of resonance signals, and the accumulated signals provoked by radio-frequency pulses of 90 degrees were recorded on paper. Quantitative Na determination was performed by measuring the area under the peak of intracellular sodium (Nai) NMR signals. Ouabain (Oua: 0.3 mM) and asebotoxin-III (ATX-III: 0.3 mM) produced an increase in Nai-NMR signals to a level of 188.1% and 138.1% of the control, respectively. Combined use of Oua (0.15 mM) and ATX-III (0.15 mM) produced an elevation of Nai concentration to a high level of 219.0% of the control in a superadditive manner. Mechanisms of the Nai elevation with Oua and ATX-III can be interpreted by assuming two different actions: ATX-III increases net Na(+)-influx via Na+ channels, while Oua inhibits the pumping out of Na+ from the cell.


[Therapy of neuropathies with a vitamin B combination. Symptomatic treatment of painful diseases of the peripheral nervous system with a combination preparation of thiamine, pyridoxine and cyanocobalamin]. In an open, multicentric observational study involving 234 doctors in private practice, the evolution of symptoms and the tolerability of a vitamin B preparation (Neurotrat forte) used as treatment in 1,149 patients with polyneuropathy, neuralgia, radiculopathy and neuritis associated with pain and paresthesias, were observed. The form of administration (ampoules, dragées), dosage and duration of treatment were left to the individual care-providing physician. The target symptoms evaluated were intensity of pain, muscle weakness affecting the legs, and paresthesia. Under treatment, there was a clear improvement in these symptoms. At a second examination approximately three weeks after initiation of treatment, a positive effect on pain in particular was observed in 69% of the cases. Similar observations were also made for paresthesias and muscular weakness in the legs.


[Studies on lymphokine-activated killer (LAK) cell: accumulation in tumor tissue and the therapeutic effects of adoptive immunotherapy]. We studied the therapeutic effects of adoptive immunotherapy with lymphokine-activated killer (LAK) cells combined with chemotherapy on BMT-11 fibrosarcoma in C57BL/6 mice. Compared with the untreated group, no significant therapeutic effect was brought about by CY therapy alone or LAK.rIL-2 alone and all mice belonging to these three groups died with a mean survival time (MST) of 45.3, 51.8 and 45.9 days respectively. CY plus LAK.rIL-2 brought about complete cures in 3 out of 8 mice (37.5%) and a significant prolongation of MST of mice which died (64.4 days) and the accumulation of LAK cells (% Dose/g) at tumor sites was enhanced more than 7-fold by combination with CY. On the other hand, the therapeutic effects of cytotoxic T lymphocytes (CTLs) was sufficiently high even in the CTL.rIL-2 alone and were only slightly enhanced by combination with CY compared with LAK cells. Also, we detected LAK-attractant activity in the conditioned medium (CM) of CY-treated tumor tissues but not in that of untreated tumor tissues, and peak activity was reached 5 days after CY-treatment. This attractant activity was located in two major 10,000-50,000 M. W. fractions of CM. We then observed that LAK-attractant was produced in CM of host reactive cell enriched fractions from CY-treated tumor tissues, but not in that of tumor cell enriched fractions. The above findings imply that the effects of adoptive immunotherapy depend upon the accumulation of transferred effector cells at tumor sites, and we believe that the production of LAK-attractant by tumor tissue, facilitated by chemotherapy, is one of the mechanisms responsible for enhanced LAK-cell-accumulation at tumor sites. We performed a preliminary clinical trial with adriamycin, autologous spleen-LAK cells and rIL-2 on 30 hepatocellular carcinoma patients after radical resection on the basis of the experimental results above. There were no significant therapeutic effects after adoptive immunotherapy during the postoperative course but tendency for temporary inhibition of recurrence. Thus it is shown that this method has probable value as effective adjuvant postoperative therapy.


[Characterization of glycolipid sulfotransferase fractionated from rat kidney and involvement of the enzyme in human cancer]. 3'-Phosphoadenosine 5'-phosphosulphate: galactosylceramide sulfotransferase which catalyzes the sulfation of galactosylceramide (GalCer) was partially purified from a rat kidney light membrane fraction, and the properties were studied with special reference to substrate specificity. In order to study minimum molecular requirement of the acceptor substrate for the sulfotransferase, analogues of galactosylceramide where omega-amiocaproic or omega-aminododecanoic acid is substituted for the acyl moiety of the native glycolipid were chemically synthesized by improved procedures. The artificial glycolipids were sulfated effectively by the kidney enzyme, suggesting that the synthetic compounds will serve for affinity ligands for the purification of the enzyme. The extent of sulfation in the synthetic compounds was comparable with that of galactosylceramide containing normal acids and higher than that of galactosylsphingonine in which one of hydrocarbon chains is deleted from the native glycolipid. Through substrate specificity experiments, the sulfotransferase has relatively broad specificity acting on beta-linked galactosides at nonreducing ends of mono- and disaccharides which bind, at least, one hydrocarbon chain. Enzyme kinetic analysis by competition assay using mixed acceptors demonstrated that the same, single sulfotransferase catalyzes sulfation of galactosylceramide, lactosylceramide and galactosylsphingosine. As to human cancer, the sulfotransferase activity was hardly detectable in Wilms' tumor tissues that contrasts with renal cell carcinoma tissues where the markedly elevated level was previously demonstrated. When sera from patients with various cancers were examined for the enzyme level, many cases of hepatocellular carcinoma showed significantly increased activity, whereas the hepatoma tissues had hardly detectable level of the enzyme. These observations suggest that a humoral factor derived from the hepatoma induces the sulfotransferase.


Gonadotropin receptors in human ovarian follicles and corpora lutea throughout the menstrual cycle. To study changes in the binding of gonadotropins to human follicles and corpora lutea, we have examined the binding of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the human ovary throughout the menstrual cycle, by both quantitative binding assays and surface binding autoradiography. The specific high-affinity low-capacity receptors for hLH were demonstrated in both human follicles and corpora lutea. Binding of 125I-hFSH was identified in the granulosa cells, but not in the thecal cells of the preantral and antral follicles at various stages of follicular development. The binding of 125I-hLH to the thecal cells increased during follicular development, and a dramatic increase was preferentially observed in the granulosa cells of the preovulatory follicles. In the corpora lutea, the binding of 125I-hLH increased from the early luteal phase to the midluteal phase and decreased towards the late luteal phase. The results of the present study suggest that changes in the binding of gonadotropins in the ovarian target cells during the menstrual cycle might play an important role in the regulation of follicular and luteal function.


Steroid hormones: effect on brain development and function. Hormones secreted by the adrenals, gonads and thyroid play an important role in mediating how the environment shapes the structure and function of the brain during early development, adult life and senescence. Many of these hormone effects occur at the level of gene transcription, via the actions of intracellular hormone receptors which are DNA-binding proteins. Other effects occur at the membrane level via receptors on the cell surface that produce rapid effects on bioelectrical activity and secondary messenger systems. Hormone effects on the brain are classified as organizational, occurring during development; cyclical, occurring during maturity; experiential, depending on the individual experiences; and disorganizational, leading to damage and destruction of neural tissue. Organizational effects, such as occur as a result of testosterone action during sexual differentiation, give rise to group differences; whereas experiential effects, in which hormone secretion is evoked on an individual basis according to personal life events, are responsible for individual differences even between identical twins having the same genetic constitution. Experiential effects, often involving stress and possibly thyroid hormones, may result in adaptation or may lead to disorganization and damage under extreme and deleterious conditions.


M4 and M9 antibodies in the overlap syndrome of primary biliary cirrhosis and chronic active hepatitis: epitopes or epiphenomena? Before the identification of the major mitochondrial antigens of primary biliary cirrhosis as components of the 2-oxo-acid dehydrogenase enzyme family, mitochondrial autoantigens were believed to be extremely heterogeneous and were divided into nine subtypes termed M1 to M9. This classification was based on the data derived from the relatively nonspecific biochemical and immunological techniques that were available. After the cloning and definition of the major autoantigens, more than 95% of the sera of patients with primary biliary cirrhosis were found to react with components of the 2-oxo-dehydrogenase enzymes; these enzymes correspond to the old M2 classification. Two other "M" species, dubbed M4 and M9, have attracted significant attention because they have been postulated to be prognostic indicators and more recently have been tentatively identified respectively as sulfite oxidase (EC 1.8.3.1) and glycogen phosphorylase (EC 2.4.1.1). Indeed, patients with the "overlap syndrome" are reported to have antibodies to M4 and a poor prognosis, whereas patients with antibodies to M9 have a favorable prognosis. To address the significance and definition of M4 and M9, we performed in-depth studies of sera from 11 patients with the overlap syndrome, 75 patients with primary biliary cirrhosis, 19 chronic active hepatitis patients, 13 patients with primary sclerosing cholangitis, 10 patients with cholangiocarcinoma, 20 patients with systemic lupus erythematosus, 20 patients with alcoholic cirrhosis, 17 patients with scleroderma and 30 normal individuals, using techniques of ELISA, complement fixation, immunoblotting and enzyme inhibition. We report herein that we were unable to show any disease-specific reactivity toward the proposed M4 and M9 antigens.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatitis C virus reinfection in allografts after orthotopic liver transplantation. From September 1988 to May 1991, 160 orthotopic liver transplantations were performed in our hospital. Twenty-four patients had end-stage cirrhosis caused by chronic non-A, non-B hepatitis. Antibodies against hepatitis C virus were documented before and after orthotopic liver transplantation in 13 patients. Studies using the polymerase chain reaction demonstrated hepatitis C virus RNA in the serum and liver tissue of 17 patients (10 of whom tested positive for hepatitis C virus antibodies) before orthotopic liver transplantation. Tissue samples taken from liver grafts during the operation were hepatitis C virus RNA negative in every case. Ten of these 17 patients had positive hepatitis C virus RNA findings in serum and liver biopsy specimens within the first month after surgery. One patient died of Mucor sepsis 2 mo after orthotopic liver transplantation. Another patient died of multi-organ failure 3 mo after a retransplantation. Two patients underwent retransplantation for graft rejection at 2 and 3 mo, respectively. One year after orthotopic liver transplantation, hepatitis C virus RNA was demonstrated in allograft biopsy specimens in 13 of 15 patients. Two patients remained hepatitis C virus RNA negative in repeated biopsies up to 12 mo. Mild portal and lobular hepatitis developed within 6 months of orthotopic liver transplantation in four patients and within 1 yr in five additional patients. The data suggest that persistent hepatitis C virus reinfects the allograft in most cases, but the risk of acute organ damage caused by hepatitis C virus reinfection is low.


Oxidative metabolism in cirrhotic patients with and without hepatocellular carcinoma: effects of malnutrition. Progressive degrees of metabolic alterations are frequent in cirrhosis impairing peripheral tissue and body composition. Hepatocellular carcinoma worsens protein wasting and malnutrition. A normal energy production rate and an abnormal substrate oxidation rate are well-known findings in cirrhosis; however, no data are available on cirrhotic patients with hepatocellular carcinoma. The aim of this study was to measure oxidative metabolism in cirrhotic patients with and without hepatocellular carcinoma and to investigate the correlation between energy production rate, respiratory quotient and nutritional state. Thirteen male cirrhotic patients with hepatocellular carcinoma (8 well-nourished and 5 malnourished) were compared with 17 cirrhotic patients without hepatocellular carcinoma (11 well-nourished and 6 malnourished) and six controls who were age and sex matched. A diagnosis of malnutrition was made if the fat mass percentage was reduced to less than 20% of the patient's body weight. Indirect calorimetry was performed between 8 and 10 AM, after a 12-hr fast, for 30 min (with a 10-min steady-state period), and measured energy production rate was calculated according to Weir's formula. Body composition was assessed by means of the Durnin and Womersley formula. Anthropometry and bioelectric impedance analysis showed no variations in kilograms of fat-free mass in our malnourished patients. Our data show that, when the energy production rate is measured while the patient is at rest and corrected for fat-free mass, the energy requirements of cirrhotic patients and cirrhotic patients with hepatocellular carcinoma matched that of the controls, regardless of nutritional state.(ABSTRACT TRUNCATED AT 250 WORDS)


Overexpression of p53: a rare event in a large series of white patients with hepatocellular carcinoma. Mutant p53 has been found in a wide variety of human malignancies including carcinomas of the lung, breast and colon. Because of the controversial mutational rate of the p53 gene in hepatocellular carcinoma, a large series of liver tumors from white patients with different risk factors was examined immunohistochemically for expression of the p53 mutant to assess its prevalence and the relationships between p53 overexpression and clinicopathological data. Nine of 58 specimens were found to have detectable evidence of p53 gene mutation by virtue of the immunohistochemical detection of mutant p53 protein. The p53 mutation was more frequent in patients with serological hepatitis B and C markers than in patients without these markers (p = 0.046). The prevalence of p53-positive tumors was also significantly higher in the group of tumors with invaded portal branches than in the group without (p = 0.02). Our results showed that p53-positive hepatocellular carcinoma is a rare finding in patients exposed to a low dietary aflatoxin intake and that p53 mutation seems to occur at a late stage of the tumoral process and could contribute to an aggressive tumoral phenotype.