Factors affecting ciliary function in vitro: a preliminary study. Nasal ciliary function forms an important defence mechanism within the upper respiratory tract which has largely been ignored in recent years. The effects of various drugs used extensively in the treatment of diseases of the nose have not been fully established. Furthermore, the physiological control of ciliary activity is unclear. The aim of this study was therefore to investigate the effects of drugs on ciliary beat frequency in vitro using a photometric technique. A dose-dependent response to alpha and beta receptor drugs was found, cocaine hydrochloride achieved ciliostasis even at 40-fold dilution, and potassium ions, except at the limits of tolerance for excitable tissue, did not affect ciliary function. In conclusion, we suggest that alpha and beta receptors may be present on ciliated epithelium and be involved in the control of ciliary function in vivo. Ion fluxes across the ciliary cell membrane may also be important in ciliary activity akin to nervous tissue.


Zymosan induced 45Ca uptake by retinal pigment epithelial cells. 45Ca uptake was studied in isolated frog retinal pigment epithelial cells in response to the phagocytic stimuli, zymosan. 45Ca uptake was strongly stimulated immediately in the presence of zymosan particles. Calcium uptake was proportional to the zymosan concentrations. After 60 min in the presence of zymosan acid phosphatase and beta-glucuronidase activities showed a 25% and 50% increase, respectively. Rod outer segments induced a similar increase of these enzyme activities. The zymosan-induced lysosomal enzyme activities was inhibited by cytochalasin B and ruthenium red. The ionophore A23187 produced a remarkable increase in 45Ca uptake but did not affect the lysosomal enzyme activities. These results suggest that in vitro RPE cells are able to respond to zymosan as phagocytosable stimuli and that calcium mediate that response.


Inapparent ocular infection by Chlamydia trachomatis in experimental and human trachoma. There is substantial indirect evidence which suggests that Chlamydia trachomatis can generate inapparent, persistent infections in human. To confirm this directly, we examined ocular chlamydial infection in both the cynomolgus monkey model of trachoma and in patient samples from a trachoma-endemic area. In monkeys, ocular infection was studied over time using direct immunofluorescence cytology (DFA) and a molecular hybridization screening system which targets chlamydial ribosomal RNA. In eleven animals infected once with B serovar, DFA and probe screening of parallel conjunctival swabs gave congruent results through day 42 post-infection. Thereafter, DFA showed clearing of chlamydia and was negative by day 70, as in previous studies. In contrast, hybridization analysis indicated a continuing presence of chlamydial RNA in all samples from all animals through the end of the experiment at day 84 post-infection. Similarly, analysis of swabs from trachoma patients showed that a number of DFA-negative samples gave clear positive signal for chlamydial RNA. Taken together these data indicate that ocular chlamydial infection persists for longer periods than previously thought, judging solely on the basis of DFA, and they support the idea that inapparent ocular chlamydial infection occurs in vivo.


Insulin-like growth factor-1 and insulin-like growth factor binding protein-1 in early human pregnancy. Insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-1 (IGFBP-1) were measured in amniotic fluid, extraembryonic coelomic fluid and maternal serum from 23 women with apparently normal first trimester pregnancies prior to termination. The levels of IGF-1 and IGFBP-1 were significantly higher in coelomic fluid than amniotic fluid (IGF-1, P = 0.006; IGFBP-1, P = 0.0008 (paired t-test)). The levels of IGFBP-1 were lower in amniotic fluid than in maternal serum (P = 0.017), a finding in sharp contrast to the situation in the second and third trimesters of pregnancy. There was a significant relation between levels of IGF-1 and IGFBP-1 in amniotic fluid (r = 0.43; P = 0.04) and in coelomic fluid (r = 0.81; P less than 0.001) but not in maternal serum. The finding that both the absolute levels of IGFBP-1 and the ratio to IGF-1 were low in amniotic fluid implies that there is a very high level of unbound, biologically active IGF-1 in this compartment in the first trimester. Thus, the regulatory role of IGFBP-1 may change as pregnancy advances.


Protective effects of Ca2+, Mg2+, Cu2+, and Ni2+ on mercury and methylmercury toxicity to a cyanobacterium. Toxicological investigations of the impact of inorganic mercury (Hg2+) and methylmercury (CH3Hg+) in terms of growth, NH4+ uptake, in vivo glutamine synthetase (transferase) activity, and regulation of toxicity by Ca2+, Mg2+, Cu2+ and Ni2+ in the diazotrophic cyanobacterium Nostoc calcicola Bréb. have been completed. Photoautotrophic growth of the cyanobacterium was extremely sensitive to both mercury compounds, CH3Hg+ being 2.5 times more toxic than Hg2+. Although NH4+ uptake was 6 times more sensitive than in vivo GS activity against the two mercurials, both processes had a greater susceptibility toward CH3Hg+. On the basis of Km and Vmax, it is suggested that both mercury species inhibit such metabolic events noncompetitively. Ca2+, Mg2+, Cu2+, and Ni2+ did not change the nature of inhibition and effectively antagonized the Hg2+ and CH3Hg+ toxicities in the sequence Ca2+ greater than Mg2+ much greater than Cu2+ greater than Ni2+.


Milt characteristics, reproductive performance, and larval survival and development of white sucker exposed to bleached kraft mill effluent. White sucker from a Lake Superior bay which receives bleached kraft mill effluent (BKME) show increased hepatic mixed-function oxygenase (MFO) activity, reduced plasma sex steroid levels, decreased egg and gonad size, a decrease in the occurrence of secondary sexual characteristics, and an increased age to maturation. This study evaluated the reproductive performance of that white sucker population relative to a similar reference population. Spawning male white sucker from the BKME site had reduced spermatozoan motility but no significant differences in milt volume, spermatocrit levels, or seminal plasma constituents. BKME male and female fish had equal or greater fertilization potential compared to both male and female fish at the reference site. There was no difference either in the hatchability of the eggs or in larval size at hatch. BKME larvae did show reduced growth rates by 24 days posthatch but showed equal rates of yolk utilization. No difference in larval MFO activity was detected between sites at 21 days posthatch, indicating no parental transfer of induction to the progeny.


Joint action between binary mixtures of chlordimeform and insecticides. Chlordimeform (CDF) was tested for its ability to act jointly with an organophosphorus insecticide (parathion) and a series of carbamate insecticides. Three arthropod species were used as test subjects: the two-spotted spider mite, Tetranychus urticae; the German cockroach, Blatella germanica; and the flour beetle, Tenebrio castaneum. However, CDF antagonized the toxicity of parathion toward B. germanica while it acted in a greater than additive fashion toward T. castaneum. No combination of CDF and insecticide tested acted jointly toward T. urticae. Species-specific differences in sensitivity, absorption, metabolism, and mode of delivery account for the varying results. Metabolic studies of pairs of compounds using two radiolabeled carbamates showed that CDF altered the metabolic detoxification of both carbaryl and aldicarb. The results suggest that CDF may inhibit MFOs as its mode of action.


Coplanar PCBs in fish and mussels from marine and estuarine waters of New York State. Thirty samples of striped bass from marine and estuarine waters of New York State, six samples of mussels from Long Island Sound, and one composite sample of freshwater mussels from Troy were analyzed by a recently developed method which combines sulfuric acid cleanup, carbon chromatography, and high-resolution gas chromatography for the determination of non-ortho- and mono-ortho-substituted polychlorinated biphenyls (PCBs), which are biologically active congeners of PCBs and approximate isostereomers of 2,3,7,8-tetrachlorodibenzo-P-dioxin of (2,3,7,8-TCDD). Non-ortho coplanar PCBs ranged from 0.2 to 37.1 ppb in fish. The highest concentrations of 37.1 ng/g of 3,3',4,4'-tetrachlorobiphenyl (77) and 7.5 ng/g of 3,3',4,4',5-pentachlorobiphenyl (126) were detected in a fish caught near Troy/Albany, New York. Mono-ortho-substituted PCBs ranged from 0.4 to 790 ppb in fish, with the major components identified as 2,3',4,4',5-penta-(118) and 2,3,3',4,4'-pentachlorobiphenyls (105). When concentrations were converted to 2,3,7,8-TCDD picogram equivalents, 99% of the equivalents were derived from three congeners (77, 105, and 126), 3,3',4,4',5-pentachlorobiphenyl (126) accounting for approximately 80% of the activity. At Troy pg/g TCDD equivalents derived from PCB were approximately 3000, whereas the actual 2,3,7,8-TCDD concentration was only 20 pg/g; hence, accurate coplanar PCB measurement is important.


Impact of malathion and gamma-BHC on lipid metabolism in the freshwater female catfish, Heteropneustes fossilis. Female Heteropneustes fossilis were exposed to sublethal concentrations of malathion (5 and 20 microliters liter-1) and gamma-BHC (4 and 16 micrograms liter-1) for 4 weeks during different phases of their annual reproductive cycle. The impact of these pesticides on free fatty acids (FFA), monoglycerides (MG), diglycerides (DG), triglycerides (TG), phospholipids (PL), free cholesterol (CF), and esterified cholesterol (CE) in the liver, plasma, and ovary was assessed. During the preparatory phase both the pesticides reduced the levels of all the hepatic and ovarian lipids with elevated hepatic CE. During the prespawning phase these pesticides decreased all the lipids in the liver, plasma, and ovary with the elevation of hepatic FFA and CE. During the spawning phase a reduction of hepatic MG and CF with a decreased level of plasma and ovarian levels of FFA and PL was recorded, whereas ovarian levels of TG and CE were elevated in response to both the pesticides. During postspawning and resting phases all the hepatic lipids were reduced with the elevation of CE in response to the exposure. These pesticides also restricted their mobilization to the ovary. Cholesterol biosynthesis seemed unaffected but the hydrolysis of CE to CF was adversely affected during preparatory and prespawning phases which is a period of sex steroid hormone biosynthesis.


Accumulation of lead and effects on total lipids and lipid derivatives in the freshwater fish Anabas testudineus (Bloch). Exposure of a freshwater fish to several sublethal concentrations (1.25, 2.50, 5.00, 10.00, and 20.00 mg/liter) of lead for a period of 30 days showed significant accumulation of lead in the blood and tissues. Lead bioaccumulated in the study showed organ-specific distribution, with high levels in the blood followed by the kidney, gill, liver, and brain and comparatively lesser amounts in the ovary and muscle tissues. The lead accumulation in tissues was found to increase with lead in water up to a concentration of 5 mg/liter, and at concentrations of 10 and 20 mg/liter the lead accumulation in the tissues, although indicating an increase, was not proportional to the concentration in water. Exposure of the freshwater fish Anabas testudineus to a sublethal (5 ppm) concentration of lead nitrate for a period of 30 days during the preparatory phase of its annual reproductive cycle reduced the total lipids, phospholipids, and cholesterol levels in the liver and ovary tissues while the free fatty acid levels were increased and lipase activity was elevated. All the parameters in the blood were found to increase. These results suggest that lead nitrate affects the lipid metabolism of the fish and this may reduce the fecundity of the fish since lipids are known to play an important role in teleost reproduction as an energy source and a precursor of steroids.


The effect of pesticides on carp (Cyprinus carpio L). Acetylcholinesterase and its biochemical characterization. The activity and molecular forms of acetylcholinesterase (AChE) were characterized in tissues of the carp (Cyprinus carpio). Tissue AChE activity was determined in response to specific inhibitors (ethopropazine, BW 284 C51) or pesticides (CuSO4, paraquat (PQ), methidathion (MD)). The highest AChE activity was found in the serum (878 +/- 100 U/liter), followed by the brain (113 +/- 12 U/liter), heart (89 +/- 6 U/liter), and trunk muscle (35 +/- 5 U/liter). Experiments with specific choline esterase inhibitors revealed a very low amount of pseudocholinesterase in all tissues studied. The ratio of the membrane-bound to the cytoplasmic-free AChE molecular forms was increased in the order of brain, trunk muscle, and heart. In sera of fish treated with MD (2 ppm) there was an 80% inhibition of AChE lasting for 2 weeks. Treatment with CuSO4 or PQ (both 5 ppm) led to a 50% decrease in the serum AChE activity followed by a transient increase over the control level. After 2 weeks of chronic treatment, AChE activity in fish exposed to CuSO4 returned to the control level, whereas in fish treated with PQ an elevated level (130% when compared to the control level) of enzyme activity was found. Our present experimental data indicate that pesticides occurring in natural waters not only inhibit AChE activity in fish but may influence the resynthesis of the enzyme as well.


Integrated assessment of contaminated sediments in the lower Fox River and Green Bay, Wisconsin. Samples of sediment and biota were collected from sites in the lower Fox River and southern Green Bay to determine existing or potential impacts of sediment-associated contaminants on different ecosystem components of this Great Lakes area of concern. Evaluation of benthos revealed a relatively depauperate community, particularly at the lower Fox River sites. Sediment pore water and bulk sediments from several lower Fox River sites were toxic to a number of test species including Pimephales promelas, Ceriodaphnia dubia, Hexagenia limbata, Selenastrum capricornutum, and Photobacterium phosphorum. An important component of the observed toxicity appeared to be due to ammonia. Evaluation of three bullhead (Ictalurus) species from the lower Fox River revealed an absence of preneoplastic or neoplastic liver lesions, and the Salmonella typhimurium bioassay indicated relatively little mutagenicity in sediment extracts. Apparent adverse reproductive effects were noted in two species of birds nesting along the lower Fox River and on a confined disposal facility for sediments near the mouth of the river, and there were measurable concentrations of potentially toxic 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), and planar polychlorinated biphenyls (PCBs) both in the birds and in sediments from several of the study sites. Based on toxic equivalency factors and the results of an in vitro bioassay with H4IIE rat hepatoma cells, it appeared that the majority of potential toxicity of the PCB/PCDF/PCDD mixture in biota from the lower Fox River/Green Bay system was due to the planar PCBs. The results of these studies are discussed in terms of an integrated assessment focused on providing data for remedial action planning.


Bioconcentration kinetics of the organophosphorus insecticide chlorpyrifos in guppies (Poecilia reticulata). The bioconcentration kinetics of chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate) in guppies (Poecilia reticulata) were investigated. A static exposure was used to study the uptake of the compound. The amount absorbed was calculated from the difference in disappearance rates from the water phase in aquaria with and without fish. The uptake rate was found to be first-order with respect to the exposure concentration, and amounts to ca. 38 ml g-1 (fish) hr-1. The elimination rate was studied in separate experiments. Guppies were first given the opportunity to accumulate chlorpyrifos and, following transfer to clean water, the clearance of the compound was measured by determining the residual chlorpyrifos in the fish. It was found that the elimination rate was first-order with respect to the concentration in fish, but depended slightly on the length of the preexposure period. The half-life of tissue chlorpyrifos varied from 31 to 38 hr. Release of unchanged chlorpyrifos turned out to be negligible relative to the amount eliminated. This indicates that metabolic breakdown is the only pathway for elimination. From the kinetic rate parameters, a bioconcentration factor was calculated of about 1700. This value is very similar to that measured directly after an 8-day semistatic exposure. A two-compartment model is proposed to describe the kinetics of the bioconcentration process. The conditions limiting the applicability of the indirect method for measuring the toxicokinetics of chlorpyrifos are discussed.


Toxicity of lindane to freshwater insect larvae in compartments of an experimental pond. The acute and chronic toxicities of lindane to larvae of the freshwater insects Chironomus riparius Meigen, Chaoborus flavicans (Meigen), and Sigara striata (L.) were investigated in mesocosm compartments of an experimental pond. The following median lethal concentrations (LC50s) were determined: 240-hr LC50 of 2.0 micrograms lindane liter-1 for second instar C. riparius, 72-hr LC50 of 6.5 micrograms lindane liter-1 for fourth instar C. riparius, and 96-hr LC50s of 4.0 and 3.9 micrograms lindane liter-1 for fourth instar C. flavicans and fourth or fifth instar S. striata, respectively. Lindane significantly reduced the growth over 10 days of second instar C. riparius compared to that of the control at the treatment concentrations where larvae survived (1.0, 2.5, and 7.0 micrograms lindane liter-1). A significant increase in the median emergence time in comparison to that of the control was observed for C. riparius exposed to 0.8 and 2.0 micrograms lindane liter-1, with higher concentrations causing 100% mortality. The findings compare well with previously reported laboratory data on the toxicity of lindane to insects and support the methodology and results of a laboratory growth test for C. riparius.


Effects of hepatotoxicants on the induction of microsomal monooxygenase activity in sunfish liver by beta-naphthoflavone and benzo[a]pyrene. Effects of chemically induced hepatic injury on biotransformation enzymes in fish were studied. Sunfish hybrids (Lepomis macrochirus x L. cyanellus) were dosed per os with allyl formate (ALF) and carbon tetrachloride (CCl4), and the induction of liver EROD (7-ethoxyresorufin O-deethylase) activity was subsequently challenged by injections of beta-naphthoflavone (BNF) and benzo[a]pyrene (B[a]P). Hepatotoxicity of chemical treatments was assessed using blood enzymes (ASAT, ALAT, and LDH) along with other biochemical variables. Both hepatotoxicants partially abolished the induction of EROD (maximally by 76-89%), and the decrease in induction was dose related. The cytosolic activity of glutathione S-transferase (GST) in the liver decreased in parallel with the decrease in EROD induction. Fish receiving high doses of ALF exhibited significantly less microsomal and blood plasma proteins and, occasionally, were jaundiced. These symptoms, however, were less sensitive indicators of hepatotoxicity than alterations in liver EROD and GST. Both ALF and CCl4 increased the activities of hepatic enzymes in the blood plasma, indicating cytotoxicity. In addition B[a]P, unlike BNF, also increased plasma activities of LDH and ALAT at a dose inducing liver EROD, implying simultaneous hepatotoxicity at high sublethal levels of this xenobiotic. These data suggest that hepatotoxic chemicals absorbed by fish may act antagonistically by decreasing the degree of induction of the cytochrome P450 system relative to the inherent capacity of inducing xenobiotic chemicals present in the environment. Therefore, when assessing the toxicological status of water using fish health biomarkers, it is advisable to measure a concert of metabolic and biochemical variables instead of any single biomarker.


Endoscopic papillotomy in biliary tract pain and fluctuating cholestasis with common bile duct dilatation and small gallbladder stones. In patients suspected of having functional disorders of the papilla it is often difficult to establish the indications whether or not to perform endoscopic papillotomy (EP). We report on thirty-two consecutive patients referred for endoscopic retrograde cholangiopancreatography who all had longstanding biliary tract pain and episodes of liver enzyme elevation indicating cholestasis. Further features were: 1) a dilated common bile duct (CBD) after cholecystectomy (n = 11) or 2) a dilated CBD without or with larger (greater than cystic duct diameter) gallbladder stones (n = 6) or 3) multiple small gallbladder stones, with a normal or dilated CBD, in patients with signs of acute gallstone pancreatitis or in whom elective cholecystectomy was not indicated (n = 15). No CBD stones, organic obstruction or other disorders were found in these patients. Without further diagnostic procedures, EP was routinely performed. The laboratory (up to 3 months) and clinical findings (2 to 4 years follow up) showed improvement in all patients undergoing EP. We conclude that immediate EP appears justified in these selected patients.


Specific inhibition of Physarum polycephalum DNA-polymerase-alpha-primase by poly(L-malate) and related polyanions. Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-alpha-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-alpha-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(vinyl sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (N) interspaced between them. Poly(L-malate) (N = 5) and poly(D,L-malate) (N = 5) are the most efficient inhibitors, followed by poly(L-aspartate) (N = 6), poly(acrylate) (N = 3), poly(L-glutamate) (N = 8), poly(vinyl sulfate) (N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-alpha-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-alpha-primase in the G2 phase and M phase of the plasmodial cell cycle.


Dynamics of the exposure of epitopes on OmpF, an outer membrane protein of Escherichia coli. The OmpF protein is the major outer membrane trimeric porin of Escherichia coli B. The exposure of several cell-surface-exposed epitopes, that are recognized by various monoclonal antibodies directed against the protein, is investigated. Kinetic analyses show that two epitopes (E18 and E19) appear early during the in-vivo assembly on the folded monomer, just after the removal of the signal peptide, and are conserved in the native trimer. The trimerization that immediately follows or occurs in conjunction with the folding of monomers exposes another antigenic site (E21) at the surface of metastable forms. The binding of nascent lipopolysaccharide promotes the conversion of the heat-modifiable intermediate to a stable trimer and ensures the exposure of E20, E1, E3, E4 and E7. Late epitopes, E1, E3, E4 and E7 are only detected in the outer membrane fraction. These results suggest that different steps induce the sequential exposure of native antigenic sites. The detection of these epitopes depends on conformational changes occurring during the OmpF insertion into the outer membrane.


Quantitative expression patterns of multidrug-resistance P-glycoprotein (MDR1) and differentially spliced cystic-fibrosis transmembrane-conductance regulator mRNA transcripts in human epithelia. P-glycoprotein (MDR1), that confers multidrug resistance in cancer, and the cystic-fibrosis transmembrane-conductance regulator (CFTR), that is causative defective in cystic fibrosis, belong to the family of ATP-binding transport proteins. The expression of MDR1 and CFTR in human epithelial tissues and the cell lines T84 and HT29 was estimated by primer-directed reverse transcription (RT) and subsequent monitoring of the kinetics of cDNA product formation during the polymerase chain reaction (PCR). MDR1 mRNA was found in high levels, 15-50 amol mRNA/microgram RNA, in the intestine, kidney, liver and placenta, and in low levels, 0.2 amol/microgram RNA, in respiratory epithelium. Large amounts of CFTR mRNA were measured in the gastrointestinal tract, whereas the kidney, as the phenotypically normal organ, and the lung, as the most severely affected organ in cystic fibrosis, both contained low amounts, 3 amol CFTR/microgram RNA. CFTR transcript levels of 1-5 amol/microgram RNA were determined in lymphocytes and lymphoblast cell lines, suggesting that lymphoblasts are an accessible source for the study of the molecular pathogenesis of cystic fibrosis. When transcripts were scanned by overlapping RT/PCR analyses, only transcript of expected size was detected for MDR1 mRNA, where variable in-frame deletions of either exon 4, 9 or 12 were observed in CFTR mRNA. The complete loss of single exons was seen at proportions of 1-40% in all investigated tissues and cell lines with large donor-to-donor variation. Exons 9 and 12 of the CFTR gene encode parts of the evolutionarily well-conserved first nucleotide-binding fold including the two Walker motifs. Alternative splicing may give rise to various CFTR forms of different function and localization.


Treatment of hemorrhagic hypotension with hypertonic/hyperoncotic solutions: effects on regional cerebral blood flow and brain surface oxygen tension. Hypertonic/hyperoncotic solutions (e.g. HHS: 7.2% NaCl/10% dextran-60) are highly effective to normalize cardiovascular function in hemorrhagic shock due to rapid mobilization of fluid from the extravascular compartment. Since experiences are limited with regard to potential side effects of this treatment on the central nervous system, the present studies were carried out under particular consideration of the cerebral blood flow and O2 supply. HHS was administered in albino rabbits subjected to alpha-chloralose anesthesia and artificial ventilation with and without hemorrhagic hypovolemia. Hemorrhagic hypovolemia of 30 min duration was induced by withdrawal of approximately one third of the circulating blood volume resulting in a decrease in arterial blood pressure to 40 mm Hg. HHS was studied in addition to normovolemic animals. Cardiac output was rapidly normalized by infusion of HHS in animals with hypovolemia, while it increased intermittently in normovolemic animals. In animals with hemorrhagic shock arterial blood pressure recovered by treatment to approximately 70% of normal, whereas blood pressure remained unchanged after infusion of HHS in normovolemic controls. Cerebral blood flow, which was assessed by H2 clearance at the brain surface, had a range of 43.0-50.3 ml/100 g/min under control conditions. It remained virtually unchanged during hemorrhagic hypovolemia and also after infusion of HHS in normovolemic animals. Treatment of shock by HHS was followed 90 or 120 min later by a moderate increase in regional cerebral blood flow to 61 ml/100 g/min. Local tissue PO2 at the brain surface was obtained by an O2 multiwire electrode in the vicinity of the H2 clearance measurements using a weightless suspension system to avoid compression of the brain surface. Infusion of HHS in normovolemic animals did not affect the O2 supply of the brain. Hemorrhagic hypovolemia which led to a left shift of the cerebral PO2 histogram was followed by gradual normalization after fluid resuscitation. The current findings taken together do not indicate adverse side effects of this efficient method of fluid resuscitation with regard to the cerebral blood and O2 supply. The results make worthwhile further investigations on HHS in the presence of a focal brain lesion causing brain edema to find out whether the HHS are useful also for the treatment of intracranial hypertension.


An explanation of the achromatic bands produced by peroxidase isozymes in polyacrylamide electrophoresis gels stained for malate dehydrogenase. When plant tissue extracts are electrophoresed on polyacrylamide gels and the gels are stained for malate dehydrogenase by the standard NAD-dependent dehydrogenase reaction, terminating in the formation of reduced Nitroblue Tetrazolium (NBT), achromatic bands, in addition to the expected chromatic bands, are observed. The achromatic bands are seen when the staining conditions favor a generalized background staining of the gel and have been shown, in a previous study, to be caused by peroxidase isozymes [1]. The present study examined the mechanism by which peroxidase produced the achromatic bands using horseradish peroxidase (HRP). The generalized background staining resulted from the phenazine methosulfate (PMS)-mediated reduction of NBT. This reduction was enhanced by H2O2 and suppressed by HRP. Peroxidase apparently catalyzes the peroxidative oxidation of reduced PMS, which suppresses the generalized reduction of NBT in gel regions containing peroxidase isozymes producing the achromatic bands. In contrast, however, HRP also appears to catalyze the peroxidative oxidation of reduced NAD, but this reaction increases the reduction of NBT. The results are discussed in the context of the mechanisms proposed by others for the PMS-mediated reduction of NBT and for the peroxidase-catalyzed NADH-dependent formation of H2O2. This peroxidase-catalyzed reaction has been proposed for the plant peroxidases involved in lignification.


Histological and immunohistochemical investigations of hydroxyethyl-starch deposits in rat tissues. Tissue storage of hydroxyethyl starch (HES), a widely used artificial colloid, has been reported. In order to clarify whether storage of HES can be detected in tissues by immunohistochemical methods, use was made of a polyclonal rabbit anti-HES antiserum. Thirteen days after a single intravenous injection of HES rats were sacrificed and liver, spleen, lymph node, lung, kidney and skin were removed. On paraffin sections in all organs the anti-HES antiserum stained mainly cells which could be attributed to the mononuclear phagocyte system, as confirmed by the use of the antimacrophage monoclonal antibody ED1. The use of a polyclonal anti-HES antiserum may allow analysis of long-term storage and possible side effects in various tissues of man.


Differential sensitivity of CD34 epitopes to cleavage by Pasteurella haemolytica glycoprotease: implications for purification of CD34-positive progenitor cells. Our previous studies have shown that a unique glycoprotease from Pasteurella haemolytica specifically cleaves only proteins containing sialylated O-linked glycans. The hematopoietic progenitor cell antigen, CD34, which is heavily glycosylated with both N- and O-linked glycans, is readily cleaved by this protease. In this study, we demonstrate that the epitopes detected by five of the seven CD34 monoclonal antibodies are removed by the glycoprotease. The differential sensitivity of the CD34 epitopes to cleavage with either neuraminidase and/or glycoprotease establishes three classes of epitopes: 1) (class I) those identified by MY10, B1.3C5, 12.8, and ICH3 that are differentially affected by neuraminidase and removed by the glycoprotease; 2) (class II) the epitope detected by QBEND 10 that is removed only by the glycoprotease; and 3) (class III) those identified by TUK3 and 115.2 that are not removed by either enzyme. Cleavage of the 110-kd CD34 structure by the glycoprotease generates a major cell-bound fragment of about 75 kd, identified by the class III antibodies. We have also used the enzyme to improve the rapid recovery of CD34+ cells selected by immunomagnetic affinity techniques. In a preclinical model, we separated CD34+ KG1 cells with high yield (90%-95%) and high purity (94%-98%) from sham mixtures containing 50% CD34- cells. We also separated CD34+ blast cells from a patient in megakaryoblastic crisis of chronic myelogenous leukemia. In this case, the purity and yield were 93% and 94%, respectively. Enzyme treatment had no detrimental effect on cell viability, and the treated cells showed a normal quantitative expression and distribution of CD34 antigen as assessed with class III antibodies. We conclude that the P. haemolytica glycoprotease has potential to improve the isolation, from human bone marrow, of primitive hematopoietic cells that carry the CD34 antigen.


Purification and characterization of human macrophage-derived granulomonopoietic enhancing factor (GM-EF). A granulomonopoietic enhancing factor (GM-EF) capable of promoting the effect of colony-stimulating factors (CSFs) on myeloid progenitor cells has been purified to homogeneity from serum-free medium conditioned by fully mature human macrophages. GM-EF was a glycoprotein with an apparent molecular weight of 74 kd and an isoelectric point of 5.2-5.3. The purified protein was heat stable (75 degrees C for 30 min) and was sensitive to treatment with trypsin, papain, and bacterial protease but not to neuraminidase. The activity of GM-EF could be effectively neutralized by GM-EF-specific antiserum, and no antigenic cross-reactivity was observed using antisera against interleukin (IL)-1, IL-4, and IL-6. These results suggest that GM-EF is a unique cytokine that is different biochemically and antigenically from other hematopoietic enhancing factors such as IL-1, IL-4, and IL-6.


Long-term recombinant human granulocyte colony-stimulating factor (rhG-CSF) treatment severely depresses murine marrow erythropoiesis without causing an anemia. We hereby report profound effects of long-term granulocyte colony-stimulating factor (G-CSF) administration on murine erythropoiesis. Recombinant human (rh)G-CSF (150 micrograms/kg body weight/day) was administered over 24 days to female C57Bl mice. Marrow erythroid colony-forming units (CFU-E) and erythroblast numbers declined to less than 5% of normal, whereas splenic erythropoiesis simultaneously increased. Splenic erythropoiesis effectively compensated for the loss of marrow erythropoiesis as indicated by the maintenance of a normal hematocrit. In the marrow the numbers of spleen colony-forming units (CFU-S) and erythroid burst-forming units (BFU-E) declined as well. Simultaneously, however, these numbers increased both in the spleen and in the peripheral blood by a factor of 20 to 30. These findings suggest a continuous migration of stem cells and progenitor cells out of the marrow and an efficient seeding in the spleen, directly or indirectly induced by G-CSF. In addition the differentiation and/or amplification of BFU-E to CFU-E was impaired in the marrow but not in the spleen. The marrow and splenic microenvironment also behaved differently with respect to granulopoiesis. G-CSF did not lead to an enhanced granuloid amplification in the spleen but exerted its proliferation activity mainly in the marrow. These findings imply that prolonged G-CSF treatment might cause erythroid depression in animals and humans when spleen erythropoiesis is less efficient than in mice.


Ex vivo expansion of peripheral blood progenitor cells with recombinant cytokines. Peripheral blood mononuclear cells were isolated and cultured in the presence of Steel factor and/or PIXY321, a GM-CSF/IL-3 fusion protein. We compared the number of colony forming cells (CFC) per culture on day zero to the number of CFC after liquid culture in the presence of these cytokines. After a four day incubation with PIXY321 and Steel factor the number of CFU-GM increased 5.6-fold and the number of BFU-E increased 2.2-fold in four separate experiments. The expansion on day 8 post incubation was 16.1-fold for myeloid colony formation and 9.7-fold for erythroid colony formation. These studies demonstrate the potential to expand CFC from peripheral blood with a simple ex vivo culture procedure.


The medullary relay from neck receptors to somatosensory thalamus in the rat: a neuroanatomical study. Experiments were performed on rats to determine the location of thalamic projecting neurones in the medulla which receive direct contacts from neck primary afferents. The medullary terminations of primary afferents from the cervical region were identified by silver staining their degenerating terminals, diffusely filling their axons with horseradish peroxidase (HRP), or reacting for transganglionically transported HRP applied to muscle or cutaneous nerves. Neurones projecting to the ventrobasal thalamus were identified in the same experimental animals by using retrograde transport of HRP or Fluoro-Gold. En passant swellings or terminals of neck primary afferents were found in the vicinity of neurones projecting to the thalamus in the dorsolateral part of the rostral cuneate nucleus, the ventral aspect of the external cuneate nucleus, and the border zone between the two. Terminals of neck afferents and retrogradely labelled cells also coincided in nucleus x. Putative synaptic contacts were found in the region between the dorsolateral part of the rostral cuneate nucleus and ventromedial external cuneate nucleus. Cutaneous afferents from the neck were associated with thalamic projecting cells located along the dorsolateral border of the rostral cuneate nucleus, and afferents from neck muscles were associated with thalamic projecting cells in the caudal third of the external cuneate nucleus and in nucleus x.


Isolation rearing of rats alters release of 5-hydroxytryptamine and dopamine in the frontal cortex: an in vivo electrochemical study. The effects of rearing hooded Lister rats either in groups of seven or singly on 5-hydroxytryptamine (5-HT) and dopamine (DA) release in the frontal cortex were investigated using in vivo voltammetry together with Nafion coated carbon fibre micro-electrodes. The selective detection of basal extracellular levels of 5-HT with this technique (Peak B) was confirmed with parallel experiments using intracranial microdialysis to measure 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels in vivo. The DA voltammetric signal (Peak A) was observed in vivo only following pharmacological or electrical stimulation of DA release. Enhanced efflux of cortical DA and 5-HT in response to local application of KCl and that of 5-HT following parentelar fenfluramine were selectively detected by the association: differential pulse voltammetry (DPV)-Nafion coated microbiosensors, supporting the capability of this electrochemical method to selectively monitor release of these amine neurotransmitters in vivo and in situ. The locomotor behaviour data indicated that isolation rearing resulted in augmented locomotor activity in a novel environment. In addition, the in vivo voltammetric results showed that following KCl or fenfluramine treatment cortical 5-HT release is prolonged while that of DA is increased in rats reared in isolation when compared with socially reared rats. This imbalance between extracellular levels of DA and 5-HT recorded in the frontal cortex of rats exposed to isolated housing conditions may contribute to the behavioural differences reported between isolation and group reared rats.


Descending projections of Forel's field H neurones to the brain stem and the upper cervical spinal cord in the cat. 1. Descending projections from Forel's field H (FFH) to the brain stem and upper cervical spinal cord were studied in cats. 2. Following implantation of HRP pellets into the spinal gray matter (C1-C3) or in the ponto-medullary reticular formation, the nucleus reticularis pontis caudalis (NRPC) or in the nucleus reticularis gigantocellularis (NRG), numerous neurones were retrogradely labelled in FFH on the ipsilateral side. In the former cases, the sizes of labelled neurones were medium-large (20-40 microns in diameter) while both small and medium-large neurones were labelled in the latter cases. 3. The lowest levels of spinal projection of single FFH neurones (n = 70) were assessed by antidromic spikes elicited by stimulating electrodes placed in C1, C3 and C7. The majority (59%) projected to C1 (but not to C3), about 27% to C3 (but not to C7), and only 14% to C7. 4. Axonal trajectories of single FFH neurones in C1-C3 segments were investigated by antidromic threshold mapping methods. The stem axons of spinal-projecting FFH neurones descended in the ventral or in the ventrolateral funiculi and the collaterals were projected to neck motor nuclei (lamina IX, Rexed 1954) and laminae V-VIII. The conduction velocities were estimated as 8-37 m/s from the antidromic latencies. 5. Axonal trajectories of 7 FFH neurones were investigated in the ponto-medullary reticular formation. All were antidromically activated from C1. In six neurones, the stem axons were located in the ventral part of the central tegmental tract and collaterals were projected to the NRPC and/or the NRG. Some of them projected to the inferior olive and the nucleus prepositus hypoglossi as well. The stem axon, in the remaining cell, was in the most dorso-medial part of the medial longitudinal fasciculus and collaterals were projected mainly to the dorsal part of the NRPC and the NRG, and also to the medial vestibular nucleus. 6. Anterograde transport of WGA-HRP injected into FFH revealed that in the upper cervical spinal cord, stem axons were found in the ventral funiculus and ventral part of the lateral funiculus. Collateral projections and presumed bouton-like deposits were observed in the laminae VI-IX, especially in their medial part. In the brain stem, dense bundles of the descending fibres were found in the central and the medial tegmental tracts and in the medial longitudinal fasciculus. FFH neurones projected densely to the caudal half of the NRPC and to the rostral half of the NRG.(ABSTRACT TRUNCATED AT 400 WORDS)


Mono- and disynaptic pathways from Forel's field H to dorsal neck motoneurones in the cat. 1. We analysed the synaptic actions produced by Forel's field H (FFH) neurones on dorsal neck motoneurones and the pathways mediating the effects. 2. Stimulation of ipsilateral FFH induced negative field potentials of several hundred microvolts with the latency of about 1.1 ms in the medial ponto-medullary reticular formation, being largest in the ventral part of the nucleus reticularis pontis caudalis (NRPC), and in the dorsal part of the nucleus reticularis gigantocellularis (NRG). 3. Stimulation of ipsilateral FFH induced excitatory postsynaptic potentials (EPSPs) in 90% (47/52) and inhibitory postsynaptic potentials (IPSPs) in 19% (10/52) of the reticulospinal neurones (RSNs) in the NRPC and the NRG. Latencies of the EPSPs and IPSPs were 0.7-3.0 ms, the majority of which were in the monosynaptic range. The monosynaptic connexions were confirmed by spike triggered averaging technique both in excitatory (n = 4) and inhibitory (n = 2) pathways. 4. Single stimulation of FFH induced EPSPs at the segmental latencies of 0.3-1.0 ms in neck motoneurones, which were clearly in the monosynaptic range. Repetitive stimulation of FFH produced marked temporal facilitation of EPSPs in neck motoneurones. The facilitated components of the EPSPs had a little longer latencies and their amplitude reached several times as large as that evoked by single stimulation in all the tested motoneurones. These facilitated excitations are assumed to be mediated by RSNs in the NRPC and NRG, since RSNs were mono- and polysynaptically fired by stimulation of FFH and they were previously shown to directly project to neck motoneurones. 5. EPSPs were induced in 91% (82/91) of motoneurones supplying m. biventer cervicis and complexus (BCC; head elevator), 10% (3/29) of motoneurones supplying m. splenius (SPL; lateral head flexor). Likewise, stimulation of FFH produced EMG responses in BCC muscles, while not in SPL muscle. Thus FFH neurones produce excitations preferentially in BCC motoneurones. 6. Systematic tracking in and around FFH revealed that the effective sites for evoking above effects were in FFH and extended caudally along their efferent axonal course. 7. These results suggested that FFH neurones connect with neck motoneurones (chiefly BCC, head elevator) mono-, di- and/or polysynaptically and are mainly concerned with the control of vertical head movements.


Identification of the axon pathways which mediate functional recovery of a paralyzed hemidiaphragm following spinal cord hemisection in the adult rat. Despite extensive neurophysiological work carried out to characterize the crossed phrenic phenomenon, relatively little is known about the morphological substrate of this reflex which restores function to a hemidiaphragm paralyzed by spinal cord injury. In the present study WGA-HRP was injected into normal and functionally recovered hemidiaphragm muscle in rats during the crossed phrenic phenomenon. The retrograde transynaptic transport characteristics of WGA-HRP was utilized to delineate the source of the neurons which mediate the crossed phrenic phenomenon. The results indicated that the neurons which drive phrenic motoneurons in spinal hemisected rats during the crossed phrenic phenomenon are located bilaterally in the rostral ventral respiratory group (rVRG) of the medulla. No transneuronal labeling of propriospinal neurons was noted in either normal or spinal-hemisected rats. Thus, propriospinal neurons do not relay respiratory drive to phrenic motoneurons. The neurons of the rVRG project monosynaptically to phrenic motoneurons. The present results suggest that both crossed and uncrossed bulbospinal pathways from the rVRG collateralize to both the left and right phrenic nucleic and functional recovery of a hemidiaphragm paralyzed by ipsilateral spinal cord hemisection is mediated by supraspinal neurons from both sides of the brain stem. These results are important to our complete understanding of the mechanisms which govern motor recovery in mammals following spinal cord injury.


Morphometric analysis of the effects of antineoplastic drugs on mucosa of normal ileum and ileal anastomoses in rats. Antineoplastic agents affect the healing of intestinal anastomoses. They often induce anorexia and diarrhea, possibly caused by morphological changes in the small intestinal mucosa. These changes were evaluated in the rat ileum. Animals in group I underwent only intestinal surgery while those in groups II and III underwent surgery on the third day of a 5-day course with cisplatin (in two different doses), bleomycin, and 5-fluorouracil. The parameters were: number of mitoses in crypts, crypt depth, villus height, width, and contour length, measured in the mucosa of primarily resected segments of the ileum and of the anastomotic area. Surgery yields an increased crypt depth and villus length in the anastomotic area without changing villus width. The changes in intestinal crypts precede those in villi. Antineoplastic drugs decrease crypt mitotic rate, villus height, width, and contour length. After cessation of antineoplastic chemotherapy mitotic activity increases. The shallower and shorter villi increase in width and length resulting in an increased villus contour length and area. A linear relation exists between villus contour length and villus height and width. Thus, antineoplastic polychemotherapy, dose-dependently, reduces and surgical trauma increases intestinal proliferative activity. However, the morphologic changes do not unequivocally explain possible metabolic disturbances causing retarded intestinal wound healing.


Level of expression controls modes of gating of a K+ channel. Several distinct subfamilies of K+ channel genes have been discovered by molecular cloning, however, in some cases the structural differences among them do not account for the diversity of K+ current types, ranging from transient A-type to slowly inactivating delayed rectifier-type, as members within each subfamily have been shown to code for K+ channels of different inactivation kinetics and pharmacological properties. We show that a single K+ channel cDNA of the Shaker subfamily (ShH4) can express in Xenopus oocytes not only a transient A-type K+ current but also, upon increased level of expression, slowly inactivating K+ currents with markedly reduced sensitivity to tetraethylammonium. In correlation with the macroscopic currents there are single-channel gating modes ranging from the fast-inactivation mode which underlies the transient A-type current, to slow-inactivation modes characterized by bursts of longer openings, and corresponding to the slowly inactivating macroscopic currents.


A large conductance ion channel in the nuclear envelope of a higher plant cell. To detect and characterize ion channel activity in the nuclear envelope of a higher plant cell, we performed patch clamp experiments on nuclei isolated from coconut endosperm cells and on giant liposomes containing nuclear envelope fragments prepared from the same cells. An ion channel exhibiting a number of conductance substates, with a maximum of ca. 1,000 pS, was observed. Above an applied potential of +/- 100 mV, the behavior of the channel was similar in isolated nuclei and liposomes, indicating that both patch clamp modes were detecting the same channel. That such a channel has now been identified in members of both the animal and plant kingdoms reinforces the notion that the nuclear pores are not always open to ions.


Solution structure of FK506 bound to FKBP-12. The complex of the immunosuppressant FK506 bound to FKBP-12 has been studied in solution using 1H and inverse-detected 13C NMR methods. The resonances of bound, 13C-labelled FK506 were assigned and a set of 66 intraligand NOE distance restraints were used to calculate the structure of the bound ligand by distance geometry and restrained molecular dynamics methods. The structure of bound FK506 in solution closely resembles that seen in the X-ray structure [17], except for the allyl region. The differences reflect the influence of intermolecular crystal contacts and have implications for interpretation of the interaction of the FK506/FKBP complex with its putative biological receptor.


Testosterone induction of poly(A)(+)-RNA synthesis and [35S]methionine incorporation into proteins of Rana esculenta Harderian gland. The role of androgens in the cyclic secretory activity of the Rana esculenta Harderian gland (HG) was studied. Total RNA showed a dramatic increase in October and May when the nuclear androgen receptors peak. During the resumption of the secretory activity a gradual increase of poly(A)(+)-RNA was detected; during the enhancement phase (May) a peak of the poly(A)(+)-RNA fraction was found. In in vitro experiments testosterone increased the incorporation of [3H]uridine into the poly(A)(+)-RNA fraction and also that of [35S]methionine into a newly synthesized protein fraction (100 kDa). The latter effect is prevented by the exposure of the cells to the antiandrogen, cyproterone acetate (CPA). These findings reveal that, besides hamsters, the HG is a target for androgens in the frog.


Manipulation of intracellular calcium affects in vitro juvenile hormone synthesis by larval corpora allata of Manduca sexta. The effect of altering intracellular free Ca2+ on juvenile hormone (JH) and acid synthesis by larval and pupally-committed corpora allata (CA) of fifth stadium Manduca sexta was investigated. Larval CA required extracellular Ca2+ greater than or equal to 0.1 mM for maximal JH synthesis, while JH acid synthesis by glands after pupal commitment was independent of extracellular Ca2+. Free Ca2+ in the hemolymph ranged from 1.4 to 2.1 mM during the fifth stadium. Both calcium ionophores and caffeine, which releases Ca2+ from intracellular stores, inhibited JH synthesis by larval CA but stimulated JH acid synthesis by post-commitment CA. These results suggest that intracellular stores may be the principal source of Ca2+ for the biosynthetic activity of the post-commitment gland. Calcium channel blockers (La3+, Cd2+) and antagonists (verapamil, isradipine and nitrendipine) decreased both JH and JH acid synthesis, indicating the existence of Ca2+ channels in the CA cell membrane. Calmodulin (CaM) antagonists inhibited the activity of both larval and post-commitment CA, suggesting an integral relationship of CaM to the effects of Ca2+ on gland activity. One of these effects is the demonstrated requirement of 0.1 mM extracellular Ca2+ for allatostatin inhibition of JH I synthesis by larval CA.


Inhibition of adrenocortical steroidogenesis by alpha 2-macroglobulin is caused by associated transforming growth factor beta. alpha 2-Macroglobulin (alpha 2M) is the major protein secreted by bovine adrenocortical cells in primary culture and its synthesis is stimulated by transforming growth factor beta (TGF beta). We investigated here the effects of alpha 2M on adrenocortical steroidogenesis. We observed that commercial preparations of bovine plasma alpha 2M were able to mimic the inhibitory action of TGF beta on adrenocortical cortisol production, with the same specificity of action directed at the steroid 17 alpha-hydroxylation step. This inhibition was time-dependent and dose-dependent (50% inhibition observed with 2 mg/ml alpha 2M). Acid/ethanol extracts of alpha 2M appeared to retain the full inhibitory activity of alpha 2M. Anti-TGF beta antibodies could reverse the inhibition caused by the acid/ethanol extract but not that caused by native alpha 2M. Taken together, these results indicate that the inhibition of adrenocortical steroidogenesis induced by alpha 2M is caused by associated TGF beta. We estimated that 2 mg of alpha 2M contained approximately 0.1 ng of TGF beta, corresponding to a molar ratio of 1/700,000 between TGF beta and alpha 2M. These results also clearly indicate that the alpha 2M-TGF beta complexes are biologically active on adrenocortical cells, suggesting that these cells possess the enzymatic equipment that can activate the latent alpha 2M-TGF beta complexes.


Stimulus-secretion coupling in beta-cells of transplantable human islets of Langerhans. Evidence for a critical role for Ca2+ entry. With human islets isolated for transplantation, we examined the applicability to humans of a metabolic fuel hypothesis of glucose transduction and a Ca2+ hypothesis of depolarization-secretion coupling, both previously proposed for rodent islet beta-cells. We report that several features of human beta-cell physiology are well accounted for by these hypotheses. With whole-islet perifusion, we demonstrated that insulin secretion induced by glucose, tolbutamide, or elevated K+ is dependent on extracellular Ca2+. Insulin release induced by these secretagogues is enhanced by the dihydropyridine Ca2+ channel agonist BAYk8644 and depressed by the dihydropyridine Ca(2+)-channel antagonist nifedipine. All of the aforementioned secretagogues provoke increases in cytosolic free Ca2+, which are dependent on extracellular Ca2+ and are altered by the dihydropyridine drugs. Individual beta-cells in the islet display diminished resting membrane conductance, graded depolarization, and complex electrical patterns, including bursts of action potentials in response to stimulatory concentrations of glucose or tolbutamide. Individual islet beta-cells display voltage-dependent Ca2+ currents that are activated at membrane potentials traversed during the excursion of the action potential. In most cells, the Ca2+ currents are enhanced by BAYk8644 and depressed by nifedipine at concentrations that have parallel effects on secretagogue-induced increases in cytosolic Ca2+ and insulin secretion. These survey studies should provide the basis for more detailed investigations of the relationship of voltage-dependent ionic currents to electrical activity patterns and of electrical activity patterns to granule exocytosis in single human beta-cells.


Developmental regulation of amylin and insulin-gene expression in lean (Fa/Fa) and obese (fa/fa) Zucker rats. Obese individuals are hyperinsulinemic and insulin resistant. Because amylin is cosecreted with insulin and may contribute to the insulin resistance of obesity, this study tested the hypothesis that insulin and amylin genes are coordinately regulated by obesity and carbohydrate feeding. Insulin and amylin gene expression were measured during the suckling/weaning transition in lean (Fa/Fa) and obese (fa/fa) Zucker rats, a period associated with marked changes in tissue insulin sensitivity. There was a decline in insulin mRNA (-90 +/- 15%, P less than 0.01) and amylin mRNA (-72 +/- 21%, P less than 0.01) content in pancreases of lean rats maintained on a high-fat diet from days 15 to 30, probably reflecting the relative increase in exocrine/endocrine development during this neonatal period and the effects of fat feeding. Weaning on high-carbohydrate versus high-fat diets resulted in enhanced expression of both insulin (P less than 0.05) and amylin (P less than 0.05) mRNAs. In contrast to the decline in pancreatic insulin and amylin mRNA content observed in lean rats, there was an increase in insulin mRNA (421.3 +/- 57.5%, P less than 0.05) and no change in amylin mRNA in obese rats maintained on a high-fat diet from days 15 to 30. There was no enhancement of insulin or amylin gene expression in obese rats with high carbohydrate relative to high-fat feeding, perhaps reflecting maximum rates of transcription in these obese insulin-resistant rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Histamine inhibits 5-hydroxytryptamine release from the porcine small intestine: involvement of H3 receptors. Strips of the porcine small intestine were incubated in vitro, and the release of 5-hydroxytryptamine (5-HT) was determined by high-pressure liquid chromatography with electrochemical detection. Removal of the mucosa resulted in a large reduction (95%) of tissue 5-HT, suggesting that enterochromaffin cells are the main source of 5-HT. The release of 5-HT was reduced by 70% after omission of calcium. Tetrodotoxin and hexamethonium reduced the release of 5-HT by 30%-40% in a nonadditive manner, indicating a spontaneous neuronal (nicotinic) excitatory input to the enterochromaffin cells. Histamine inhibited the release of 5-HT by about 50%. This effect was not affected by mepyramine or cimetidine but was effectively blocked by thioperamide, indicating the involvement of H3 receptors. The selective H3-receptor agonist R-alpha-methyl-histamine also inhibited 5-HT release. Because the effect of R-alpha-methyl-histamine was also observed in the presence of tetrodotoxin, an indirect, neuronally mediated action could be excluded. Therefore, the inhibitory H3 receptors may be localized directly at the enterochromaffin cells.


Increased levels of substance P in the myenteric plexus of Trichinella-infected rats. Changes in immunoreactive substance P concentrations were investigated in longitudinal muscle-myenteric plexus preparations from the inflamed jejunum of Trichinella spiralis-infected rats. The substance P concentration increased within 2 days of infection and increased fivefold by day 6; in contrast, there was no significant increase in substance P in the noninflamed ileum. In vitro exposure of preparations from infected rats to scorpion venom reduced substance P levels by 88%. In addition, no increase in substance P was observed in rats that had been treated with capsaicin as neonates or as adults before infection. Treatment of infected rats with betamethasone attenuated the inflammatory response to the infection and prevented the increase in substance P. Furthermore, no significant increase in substance P concentration was seen in congenitally athymic rats infected with T. spiralis. This study is consistent with the hypothesis that inflammation increases substance P in myenteric nerves by a process that involves T lymphocytes.


Truncated and native insulinlike growth factor I enhance mucosal adaptation after jejunoileal resection. It has been shown previously that insulinlike growth factors (IGFs) stimulate the proliferation of intestinal crypt cells in vitro. To examine the in vivo effects of IGF-I on mucosal adaptation, three groups of Sprague-Dawley rats underwent 80% jejunoileal resection. Miniosmotic pumps were then inserted under the skin immediately after resection to deliver vehicle (resected control), 1.5 mg/kg per day of IGF-I, or 1.5 mg/kg per day of des-(1-3)-IGF-I (des-IGF-I). Des-IGF-I is a truncated form of IGF-I that binds as well to type I IGF receptors but less tightly to several forms of IGF-binding proteins (IGFBPs) than IGF-I. Ad libitum food intake did not differ among the three resected groups. Body weight gains were greater in animals receiving des-IGF-I than in those receiving IGF-I, which were greater than resected controls. All animals were killed 7 days postoperatively, and the remaining small intestine was removed and divided at the anastomotic site. Both IGF-I and des-IGF-I induced hyperplasia (increased DNA and protein content) in the duodenojejunum but not in the ileum. IGF-I and des-IGF-I were equally active. In contrast, sucrase, maltase, and leucine aminopeptidase activities were greater only in the ileum of animals receiving IGF-I and des-IGF-I than in resected controls. Although more potent in stimulating overall body weight gain, des-IGF-I was not more potent than IGF-I when duodenal and ileal responses were determined. IGF infusion (IGF-I greater than des-IGF-I) increased the levels of circulating IGFBP-3 and IGFBP-2, which may act to modulate the biological effectiveness of the infused peptides. These results suggest that both IGF-I and des-IGF-I may have potential as therapeutic agents for short bowel patients.


Intrahepatic bile duct loss in biliary atresia despite portoenterostomy: a consequence of ongoing obstruction? The histological and immunohistochemical characteristics of the liver in 44 children (28 boys, 16 girls) with extrahepatic biliary atresia at different stages of the clinical course were studied. Thirty-four wedge liver biopsy specimens taken during Kasai operations (25 specimens) and relaparotomy (9 specimens) and 20 hepatectomy explants taken at the time of transplantation were examined. Routine histological stains and monoclonal antibodies against different molecular weight cytokeratins and HLA-DR were used. The histopathological changes and the pattern of cytokeratin expression observed during the course of the disease were suggestive of persistent or recurrent extrahepatic biliary obstruction that occurred despite the Kasai operation and eventually led to cirrhosis and liver failure. Quantitative studies showed a progressive loss of intrahepatic bile ducts over the time course of the disease. This destruction of bile ducts had a geographic anatomical distribution in hepatectomy specimens, and in two livers it occurred predominantly in only one lobe. This geographic distribution of the vanishing bile ducts probably indicates an unpredictable and uneven obliteration of bile ducts in the porta hepatis during portoenterostomy wound healing and scarring.


Construction and characterization of an Escherichia coli mutant deficient in the metY gene encoding tRNA(f2Met): either tRNA(f1Met) or tRNA(f2Met) is required for cell growth. The Escherichia coli metY gene, encoding tRNA(f2Met), was split by the kanamycin-resistance-encoding gene. The resulting mutant exhibited the same growth rate as the wild type, indicating that tRNA(f2Met) is not indispensable as is the case with the metZ gene encoding tRNA(f1Met) [Kenri et al., Gene 103 (191) 31-36]. beta-Galactosidase was produced efficiently from the start codon AUG of the intact lacZ gene or a trpA'::lac'Z fusion gene, in the metY mutant. The lac repressor from the lacI gene and the chimeric protein from a hupB'::lac'Z fusion gene, whose start codons are GUG, were also synthesized efficiently in the insertion mutant. These results provide evidence that tRNA(f2Met) is not essential for growth of E. coli and that the start codons, AUG and GUG, are both recognized by tRNA(f1Met), a major N-formyl methionine-specific tRNA, in the tRNA(f2Met)-depleted cells. We were unable to construct mutants deficient in both tRNA(f1Met) and tRNA(f2Met) by P1 phage-mediated transduction with the metY and metZ mutations. Moreover, the ampicillin-resistance marker of the pUC9 plasmid carrying metZ+ was not cured at 42 degrees C in host cells with the polAts and metY-metZ double mutations. These results indicate that either tRNA(f1Met) or tRNA(f2Met) is required for the growth of E. coli.


Corrected nucleotide sequence of M13mp18 gene III. There are seven differences between the actual nucleotide (nt) sequence of bacteriophage M13mp18 gene III and the previously reported nt sequence (which had been compiled based on the nt sequence of wild-type bacteriophage M13 gene III).


Interactions between parents and pediatric primary care physicians about children's mental health. Interaction patterns between parents and pediatricians were examined during 1,378 well-child visits to four public and private pediatric clinics. During 327 visits, parents listed at least one psychosocial concern related to their child's mental health. At 37 percent of these visits, parents said they did not wish to discuss the concern with the physician. Physicians failed to address concerns during approximately 35 percent of visits at which parents were willing to discuss them. Successful parent-physician interactions were three times more frequent in private practices than in a public clinic; they were more likely when fewer concerns were stated and less likely when behavior problems were the concern. Parents concerned about the parent-infant relationship were four times more likely to be referred to outside mental health services, although these cases were relatively rare. Older children and families receiving Medicaid were also more likely to be referred to such services.


Variation in gene copy number and polymorphism of the human salivary amylase isoenzyme system in Caucasians. The polymorphic patterns of human salivary amylase of a large number of individuals of Caucasian origin were determined by using isoelectric focusing and polyacrylamide gel electrophoresis. Nine different salivary amylase protein variants were found; three of them are recorded for the first time and their heredity is shown. Some of the variants are encoded by haplotypes expressing three allozymes. Most variants display low frequencies. Analysis of the relative intensities of variant-specific isozyme bands, combined with segregation analysis, show that extensive quantitative variation is present in the population. The numbers of salivary amylase genes in some families showing quantitative variation at the protein level have been estimated by the polymerase chain reaction. We present evidence that quantitative variations in amylase protein patterns do not always reflect variations in gene copy number but that other mechanisms are also involved.


Localization of the human insulin-like growth-factor-binding protein 4 gene to chromosomal region 17q12-21.1. Insulin-like growth-factor-binding proteins (IGFBPs) constitute a family of structurally related proteins that specifically bind insulin-like growth factors and modulate their functions. In this study, the chromosomal localization was determined for the gene encoding IGFBP4, i.e. inhibitory-IGFBP. A polymerase chain reaction (PCR) fragment corresponding to the previously published cDNA sequence was used to isolate overlapping cosmid clones. By fluorescent in situ hybridization to metaphase chromosomes, the IGFBP4 gene was then localized to chromosomal region 17q21-21.1. This result was in agreement with PCR analysis of a panel of somatic cell hybrids.


The occurrence of various non-delta F508 CFTR gene mutations among Hungarian cystic fibrosis patients. Cystic fibrosis (CF) is an autosomal recessive disease caused by different mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The frequency of the major mutation (delta F508) in the Hungarian population is 64%. To identify other common mutations in CF families from Hungary, 30 non-delta F508 CF chromosomes were analyzed for selected mutations in exon 11 (G551D, R553X, G542X), intron 4 (621 + 1G----T), intron 10 (1717-1G----A), exon 20 (W1282X), and in exon 21 (N1303K) of the CFTR gene. In 6 of the 30 non-delta F508 CF chromosomes the following mutations were detected: R553X, G542X, 1717-1G----A, W1282X, and N1303K. After analysis of the above eight mutations, 30% of CF chromosomes are as yet undefined and further analysis is planned.


"Self" to cytotoxic T cells has to be 1,000 or less high affinity nonapeptides per MHC antigen. In order to serve as the effective target of a relevant cytotoxic T-cell receptor, the same peptide fragment has to occupy at least 0.1% of the class I major histocompatibility complex (MHC) antigen sites on the plasma membrane. Because of this need, I content that the thymic educator cell of "self" to cytotoxic T cells can suppress autoreactive T-cell clones only with regard to at the most, 1,000 self nonapeptides per a given allelic form of class I MHC antigens; e.g., HLA-A2. Each allelic form of class I MHC antigen apparently developed the preferential binding affinity toward a specific set of nonapeptides. The requirement for preferential binding can either be permissive or stringent. In the case of human HLA-A2, those nonapeptides having either Leu or Met at the second position and mainly Val, but occasionally Leu at the ninth position are preferred. Since both Leu and Val are very common residues, the typical somatic cell type readily supplies nearly 3000 high affinity host nonapeptides preferred by HLA-A2. Of those, the tolerance can be induced, at the most, to only 1,000 nonapeptides. In view of this, permissive class I MHC antigens such as HLA-A2 carefully avoid high affinity nonapeptides in viral proteins, for their status as to self or nonself is uncertain, and they choose second choice nonapeptides as T epitopes. In sharp contrast to human HLA-A2, mouse H-2Db represents the stringent class I MHC antigens. In order to show the high binding affinity toward H-2Db, nonapeptides are required to carry Asn at position 5 and Met or Ile at the equally critical position 9. Inasmuch as Asn and Met are rare residues and Ile, too, is not a common residue, the typical somatic cell type can supply only several hundred host nonapeptides having the high binding affinity toward H-2Db. Under the circumstance, there is no problem in memorizing the selfness of all of them. Accordingly, T epitopes are almost invariably chosen from the high affinity nonapeptides that are present in their viral proteins.


Immunoreactivity of recombinant carcinoembryonic antigen proteins expressed in Escherichia coli. Immunoreactivities of recombinant carcinoembryonic antigen (CEA) proteins expressed in Escherichia coli (E. coli) were analyzed in relation to the CEA domain structure [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M]. We reconstructed in a prokaryotic expression vector, pUCPL-cI, the cDNAs fro CEA-N, CEA-I, CEA-II, and CEA-III-M. The latter three were expressed as fusion products with bacterial beta-galactosidase. The recombinant proteins were solubilized by sonication in 1% sodium dodecyl sulfate (SDS) and purified by preparative SDS-polyacrylamide gel electrophoresis followed by electroelution. Their molecular weights judged from Western blotting coincided with those calculated from their cDNA sequences, respectively. By solid-phase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 21 distinct anti-CEA monoclonal antibodies (MAbs) which had been found to recognize the peptide epitopes of the CEA molecule and to be reactive with the recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells. Fourteen of the 21 MAbs reacted with the recombinant CEA proteins expressed in E. coli and confirmed the localization of the epitopes identified by using the recombinant CEA proteins expressed in CHO cells. The reactivities of 5 MAbs with the recombinant proteins expressed in E. coli were remarkably low when compared with those of the proteins expressed in CHO cells but also confirmed the localization of the epitopes identified with the recombinant CEA proteins expressed in CHO cells. The remaining 2 MAbs did not react with any recombinant protein expressed in E. coli. These results indicate that the fusion CEA-proteins expressed in E. coli are useful in the localization of the epitopes on the polypeptide chains when they reacted with the MAbs tested. However, one third of the epitopes of CEA peptides may be profoundly affected by the presence of disulfide bonds and/or sugar chains which do not seem to be formed well in E. coli.


FKBP-12 is not an inhibitor of protein kinase C. It was recently noted that the amino acid sequence of FK506 binding protein (FKBP-12) is nearly identical to that of an endogenous inhibitor of protein kinase C, PKCI-2. To follow up on this observation, we have tested the hypothesis that FKBP-12 is an inhibitor of PKC. The kinase activity of rat brain protein kinase C (PKC) was not inhibited by the presence of up to 700 micrograms recombinant human FKBP-12 per ml, in either the presence or absence of FK506. FKBP-12 also did not affect PMA-induced phosphorylation of an endogenous PKC substrate, an 80 kDa protein, in permeabilized cells. To test whether FKBP-12 could account for endogenous PKC inhibitory activity in cells, Jurkat cell lysate was chromatographed on an anion exchange column. A peak of PKC inhibitory activity was eluted at approximately 200 mM NaCl. As shown by both Western blots and FK506 binding activity, FKBP-12 was eluted only in the flow-through and wash fractions. These results demonstrate that FKBP-12 is clearly distinct from endogenous PKC inhibitory activity.


Rumpshaker: an X-linked mutation causing hypomyelination: developmental differences in myelination and glial cells between the optic nerve and spinal cord. The X-linked mutation rumpshaker (rsh), which is probably an allele of jimpy (jp), causes hypomyelination in the CNS of mice. This study examines the developmental expression of the morphology, glial cells, and immunostaining of myelin proteins in the optic nerve and spinal cord. The optic nerve contains varying numbers of amyelinated and myelinated fibres. The majority of such sheaths are of normal thickness whereas in the spinal cord most axons are associated with a disproportionately thin sheath which changes little in thickness during development. In the optic nerve glial cell numbers are elevated in mutants during early and peak myelination but then fall slightly below normal in adults. In contrast, the number of glial cells is consistently elevated after 16 days of age in the spinal cord. The majority of the alterations to total glial cells are due to corresponding changes in the oligodendrocyte population. Immunostaining intensity is somewhat reduced for myelin basic protein (MBP) and the C-terminal common to proteolipid protein (PLP) and DM-20 and profoundly decreased for the PLP-specific peptide. Glial fibrillary acidic protein (GFAP) is increased in rsh. It is probable that some of the variation in myelination between optic nerve and cord in rsh is related to the difference in axon diameter in the two locations, as there are adequate numbers of oligodendrocytes at the time of myelination. However, the effect of the mutation on cell development in the brain and the spinal cord may be different. The immunostaining indicates a marked deficiency in PLP in myelin but suggests that DM-20 levels may be relatively normal. rsh shows several major differences from jp and other X-linked myelin mutants, particularly in relation to oligodendrocyte numbers, and will be useful to elucidate the role of the PLP gene in influencing oligodendrocyte differentiation and survival.


Developmental appearance, antigenic profile, and proliferation of glial cells of the human embryonic spinal cord: an immunocytochemical study using dissociated cultured cells. We have investigated the time of appearance of the earliest differentiating glial cell types of human spinal cord using a panel of antigenic markers to identify them in cultures from 6- to 9-week-old human embryos. Immunolabeling performed at 14 h in vitro with the O4 mAb, an early oligodendrocyte marker, showed the presence of oligodendrocytes during the 7th week of age. At 8 weeks only a few of the O4+ cells expressed galactocerebroside (GalC), a marker of more differentiated oligodendrocytes. All the O4+ and GalC+ cells were vimentin+ and some of the GalC+ cells were A2B5+, GD3+ and SSEA-1+. During the first week in vitro many of the O4+ cells exhibiting a more immature, bi- or tri-polar morphology incorporated [3H]thymidine into their nuclei. Cells expressing the astrocyte-specific marker GFAP could be first observed at 8 weeks; almost all of these GFAP+ cells, which should correspond to radial glia on the basis of the current literature, were vimentin+, A2B5+, GD3+, and SSEA-1+. At 2 days in vitro incorporation of [3H]thymidine could be shown in a small fraction of these cells. The finding that radial glia and oligodendrocytes expressed similar antigenic features and the additional observation that a small, but consistent fraction of the cells were simultaneously labeled by O4 and anti-GFAP antibodies support the hypothesis that, in the human spinal cord, radial glial cells can give rise to both oligodendrocytes and astrocytes; in this respect, radial glial cells may be similar to the A2B5+, GD3+, vimentin+ bipotential glial progenitors previously identified in cultures from developing rat CNS, which also express A2B5, GD3, and vimentin.


Heme oxygenase is a heat shock protein and PEST protein in rat astroglial cells. Cultured rat forebrain astrocytes contained significant amounts of immunostainable heme oxygenase-1 (HO-1) isozyme, whereas HO-1 was undetectable in spontaneously transformed rat astroglial cells (ATs). HO-1 was inducible in both cell types by heat shock and by submicromolar amounts of H2O2. Inhibition of RNA synthesis with actinomycin D or protein synthesis with cycloheximide resulted in the rapid loss of immunostainable heme oxygenase in astrocytes. Analysis of the primary structure of heme oxygenase suggests that it is a PEST protein, i.e., targeted for rapid turnover.


Analysis of Escherichia coli colonization factor antigen I linear B-cell epitopes, as determined by primate responses, following protein sequence verification. Colonization factor antigen I (CFA/I)-bearing strains of enterotoxigenic Escherichia coli (ETEC) are responsible for a significant percentage of ETEC diarrheal disease worldwide whether the disease presents as infant diarrhea with high mortality or as traveler's diarrhea. CFA/I pili (fimbriae) are virulence determinants that consist of repeating protein subunits (pilin), are found in several ETEC serogroups, and promote attachment to human intestinal mucosa. While CFA/I pili are highly immunogenic, the antigenic determinants of CFA/I have not been defined. We wished to identify the linear B-cell epitopes within the CFA/I molecule as determined by primate response to the immunizing protein. To do this, we (i) resolved the discrepancies in the literature on the complete amino acid sequence of CFA/I by N-terminal and internal protein sequencing of purified and selected proteolytic fragments of CFA/I, (ii) utilized this sequence to synthesize 140 overlapping octapeptides covalently attached to polyethylene pins which represented the entire CFA/I protein, (iii) immunized three rhesus monkeys with multiple intramuscular injections of purified CFA/I subunit in Freund's adjuvant, and (iv) tested serum from each monkey for its ability to recognize the octapeptides in a capture enzyme-linked immunosorbent assay. Eight linear B-cell epitopes were identified; the region containing an epitope at amino acids 11 to 21 was strongly recognized by all three individual rhesus monkeys, while the amino acid stretches 22 to 29, 66 to 74, 93 to 101, and 124 to 136 each contained an epitope that was recognized by two of the three rhesus monkeys. The three other regions containing epitopes were recognized by one of the three individuals. The monkey antiserum to pilus subunits recognized native intact pili by immunogold labeling of CFA/I pili present on whole H10407 cells. Therefore, immunization with pilus subunits induces antibody that clearly recognizes both synthetic linear epitopes and intact pili. We are currently studying the importance of these defined epitope-containing regions as vaccine candidates.


Characterization of the epitope specificity of murine monoclonal antibodies directed against lipid A. A series of monoclonal antibodies directed against lipid A was characterized by using synthetic lipid A analogs and partial structures. These compounds vary in phosphate substitution, acylation pattern (type, number, and distribution of fatty acids), and, in the case of monosaccharides, in their backbone glycosyl residue. The monoclonal antibodies tested could be subdivided into five groups according to their reactivity patterns. One group reacted exclusively with 1,4'-bisphosphoryl lipid A, and a second also reacted with 4'-monophosphoryl lipid A. Two further groups recognized either 4-phosphoryl or 1-phosphoryl monosaccharide partial structures of lipid A. The fifth group reacted with 4-phosphoryl monosaccharide structures and with phosphate-free compounds. Antibodies reactive with monosaccharide structures also recognized their epitopes in corresponding phosphorylated disaccharide compounds. Both groups of monosaccharide and monophosphoryl lipid A-recognizing antibodies have access to their epitopes in bisphosphoryl compounds as well. Because of this unidirectional reactivity with more complex structures, the various specificities cannot be distinguished by using bisphosphoryl lipid A (e.g., Escherichia coli lipid A) as a test antigen. The epitopes recognized by the various monoclonal antibodies all reside in the hydrophilic backbone of lipid A, and there was no indication that fatty acids were part of the epitopes recognized. Nevertheless, the reactivities of compounds in the different test systems are strongly influenced by their acylation patterns; i.e., acyl groups may modulate the exposure of lipid A epitopes.


Rat monoclonal antibodies against Aspergillus galactomannan. Monoclonal antibodies (MAbs) against Aspergillus fumigatus galactomannan were produced in rats. Seven of them, EB-A1 through EB-A7, were characterized in more detail. They were all immunoglobulin M antibodies, reacting in an indirect enzyme-linked immunosorbent assay with purified A. fumigatus galactomannan, with avidity constants of between 2 x 10(9) and 5 x 10(9)/M. Enzyme-linked immunosorbent assay inhibition experiments with modified galactomannan and synthetic oligomers of beta (1----5)galactofuranose demonstrated that the MAbs bound to an epitope located on the beta(1----5)galactofuranose-containing side chains of the galactomannan molecule. An identical or similar epitope also seemed to be present in other fungi. Immunofluorescence and immunoelectron microscopy experiments with EB-A2 revealed the presence of the antigen in the fungal wall and inside the cell. Immunoblotting experiments demonstrated that the epitope recognized by the MAbs was a common oligosaccharide moiety of a wide range of intracellular and extracellular glycoproteins in A. fumigatus. The characteristics of the MAbs justify their use in the diagnosis of invasive aspergillosis by antigen detection.


Monoclonal antibodies define genus-specific, species-specific, and cross-reactive epitopes of the chlamydial 60-kilodalton heat shock protein (hsp60): specific immunodetection and purification of chlamydial hsp60. Ocular and urogenital tract infections with Chlamydia trachomatis can progress to chronic inflammatory diseases that produce blindness and tubal infertility. The pathophysiology of these chronic disease conditions is thought to be immunologically mediated, and the chlamydial 60-kDa heat shock protein (hsp60) has been implicated as a major target antigen that stimulates the immunopathological response. The lack of chlamydial hsp60 antibodies and purified hsp60 has severely restricted studies to define more thoroughly the role of this protein in the immunopathogenesis of chlamydial disease. We produced a panel of antichlamydial hsp60 monoclonal antibodies (MAbs) and defined their specificities by immunoblotting against lysates of C. trachomatis, C. psittaci, and six other genera of bacteria. Three patterns of anti-hsp60 immunoreactivity were observed: chlamydial species specific, chlamydial genus specific, and cross-reactive. The epitopes recognized by these MAbs were localized within the primary amino acid sequence of hsp60 by immunoblotting against recombinant amino-terminal truncated hsp60 fusion polypeptides and then precisely mapped by use of overlapping synthetic peptides. The majority of the MAbs mapped to either the amino or the carboxyl termini of hsp60. Epitopes defining all three MAb reactivities mapped within amino-terminal residues 6 to 16. Genus-specific hsp60 MAbs mapped to epitopes located within this region and to residues 17 to 28 and 177 to 189. Antichlamydial hsp60 MAbs stained inclusions as effectively as MAbs specific for the major outer membrane protein. Homogeneous preparations of full-length recombinant chlamydial hsp60 and amino-terminal truncated recombinant hsp60 polypeptides were obtained by immunoabsorption chromatography with an hsp60 MAb reactive to the carboxyl terminus of the protein. Thus, the antichlamydial MAbs described here should be extremely useful for the specific immunodetection of hsp60 in tissues from individuals having different disease manifestations and for the purification of hsp60 or truncated hsp60 polypeptides for use in serologic and lymphocyte proliferation assays. The availability of these MAbs will facilitate studies to define more precisely the role of hsp60 in the immunopathogenesis of chlamydial disease.


Stage-specific expression and intracellular shedding of the cell surface trans-sialidase of Trypanosoma cruzi. We have used antibodies to the Trypanosoma cruzi trans-sialidase and to its product, the host cell invasion-related Ssp-3 epitope, to study the expression of the corresponding antigens during the intracellular development of the parasite and in the extracellular trypomastigotes. As soon as 2 h after host cell invasion, trans-sialidase was no longer detected, whereas the Ssp-3 epitope was still present on intracellular parasites. The amastigotes which subsequently developed remained nonreactive with the antibodies. Expression of enzymatically active T. cruzi trans-sialidase started again only after transformation of the amastigotes into trypomastigotes 72 h after host cell invasion. trans-Sialidase was shed from the trypanosomes into the host cell cytoplasm, where the enzyme accumulated until release of the parasites. All released trypomastigotes expressed trans-sialidase on their surfaces and in the flagellar pockets, but stumpy trypomastigotes were stained more intensely than slender trypomastigotes. Ssp-3, the sialylated reaction product of trans-sialidase, was assembled only after rupture of the host cell membrane and was detected on the plasma membranes and in the flagellar pockets of all trypomastigotes.


Prolonged elevation of interleukin-8 and interleukin-6 concentrations in plasma and of leukocyte interleukin-8 mRNA levels during septicemic and localized Pseudomonas pseudomallei infection. Patients suffering from serious bacterial infection present to the hospital after early inflammatory events, such as release of tumor necrosis factor (TNF), have been initiated. The role of other cytokines, such as interleukin-8 (IL-8), a neutrophil chemoattractant and activator, in the pathophysiology of human sepsis is not well characterized, and there are only limited data on IL-6. We studied serial concentrations of TNF, IL-6 (involved in the acute-phase response), and IL-8 in plasma and leukocyte levels of mRNA for these cytokines in patients with localized and septicemic Pseudomonas pseudomallei infection on admission to the hospital and during a prolonged recovery phase (up to 30 days). Of 18 patients, 8 had detectable plasma IL-8 and all had raised plasma IL-6 concentrations. In patients who died median initial concentration of IL-8 (167 pg/ml; range, 97 to 362 pg/ml) and IL-6 (4,800 pg/ml; range, 60 to 9,245 pg/ml) in plasma were higher than those in survivors (P less than 0.008 and P = 0.007, respectively). Septic patients who survived and patients with localized disease had similar cytokine levels. Plasma IL-8 and IL-6 concentrations were elevated throughout the inpatient period of recovery. Circulating leukocytes contained mRNA for IL-8 but not for IL-6 and TNF, and they may secrete IL-8. An elevated plasma IL-6 concentration (greater than 1,000 pg/ml) had 75% mortality) was the best predictor of mortality in P. pseudomallei sepsis. Fifty percent of patients with detectable plasma IL-8 concentrations died. In contrast, plasma TNF bioactivity did not relate to outcome; 75% of patients who did never had detectable plasma TNF activity.


Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A. The toxin A gene of Clostridium difficile contains a 2.5-kb region encoding a series of contiguous repeating units located at the COOH terminus of the molecule. We previously showed that the monoclonal antibody (MAb) PCG-4, which neutralizes the enterotoxic activity of toxin A, binds to epitopes located within these repeating units. In the present study, we subcloned a series of fragments from this portion of the gene. The recombinant peptides expressed from the gene fragments were examined for reactivity with MAb PCG-4 to identify the epitopes involved in binding. Our results showed that MAb PCG-4 recognizes epitopes in amino acid residues 2097 through 2141 and amino acid residues 2355 through 2398.


Role of specific determinants in mannan of Candida albicans serotype A in adherence to human buccal epithelial cells. Candida albicans serotype A (C. albicans A) possesses a specific antigen, designated antigen 6, which resides in mannans on the cell surface. To determine the role of the mannan moiety of the C. albicans cell wall in adherence to buccal epithelial cells, we used antigen 6-deficient mutants which had been isolated by screening with an agglutinating monoclonal antibody against antigen 6 (MAb-6). 1H nuclear magnetic resonance spectral analysis of the purified mannans from the mutants showed a loss of the signals related to that beta-linkage of the side chains. Moreover, acetolyzed fragments of the mutant mannans showed a decreased amount of mannohexaose and mannopentaose. The mutant yeast cells exhibited significantly reduced ability to adhere both to exfoliated buccal epithelial cells and to a human buccal cell line. A number of strains of C. albicans A, C. tropicalis, and C. glabrata, all of which bear antigen 6, showed significantly higher adherence to the cell line than did those of C. albicans serotype B, which lack antigen 6. The whole mannan from the C. albicans A parent inhibited the adherence of C. albicans A to epithelial cells dose dependently, whereas mannan from a mutant strains did not. Moreover, C. albicans A treated with MAb-6 or polyclonal factor 6 serum showed reduced adherence. A close correlation was found between adhesive ability and agglutinability with MAb-6 in the C. albicans A parent, the antigenic mutants, and their spontaneous revertants. These results suggest that so far as mannan adhesion is concerned, serotype A-specific determinants are largely involved in the mechanisms of adherence of C. albicans A to human buccal epithelial cells.


Effect of adenylate cyclase activators on C5a-induced human neutrophil aggregation, enzyme release and superoxide production. The effect of adenylate cyclase activators on C5a- and f-Met-Leu-Phe-induced human neutrophil aggregation, enzyme release and superoxide production was investigated. C5a-stimulated superoxide production was markedly inhibited by adenylate cyclase activators, and the order of potency was PGE1 greater than isoproterenol greater than epinephrine greater than PGF2 alpha, which correlated with intracellular cAMP levels. However, neutrophil aggregation was inhibited by PGE1, PGE2, isoproterenol and epinephrine only at concentrations greater than 10(-6) M. Lysozyme release was inhibited only via PGEs in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine. These results suggest that in the human neutrophil: (1) C5a-induced superoxide production is more sensitive to regulation by cAMP than neutrophil aggregation or enzyme release, and (2) the type of receptor occupied as well as the threshold level of cAMP are important in the regulation of neutrophil aggregation and enzyme release stimulated by C5a.


Antigenic differences between subsets of peripheral blood lymphocytes differing in their right angle light scatter in flow cytometric analysis. The expression of various cell surface markers on peripheral blood lymphocytes (PBL) of young children and of adults was determined by flow cytometry among cells with either high or low light side scatter (SSC). In adults and in children, CD4+ lymphocytes (helper T cells) were more abundant among PBL with low SSC than among PBL with high SSC. The proportion of CD57+ (Leu-7+) cells (NK cells and a subset of CD8+ T cells) was significantly elevated among high SSC PBL, while that of CD8+ PBL (cytotoxic suppressor T cells) was only slightly elevated. CD4+ and CD8+ lymphocytes which coexpressed the CD29 marker, characteristic for activated or memory T cells, were significantly more abundant among lymphocytes with high SSC. In adults, the proportion of CD4+ CD45RA+ lymphocytes, naive T cells, was significantly higher among low SSC cells. The present study indicates that determination of the SSC of lymphocyte subsets by flow cytometry can improve the discrimination among lymphocyte subpopulations and contribute to assessment of their state of activation.


Comparison of the modulatory effect of ketotifen, sodium cromoglycate, procaterol and salbutamol in human skin, lung and tonsil mast cells. To assess the influence of mast cell heterogeneity on the inhibition of mediator release by drugs, the effects of ketotifen, sodium cromoglycate and beta-adrenoceptor agonists were examined against IgE-dependent histamine and prostaglandin D2 release from enzymatically dispersed human skin, lung and tonsillar mast cells. At high concentrations, ketotifen was an inhibitor of histamine and prostaglandin D2 release from lung and tonsillar mast cells. No cross-tachyphylaxis with sodium cromoglycate was seen. In skin mast cells no inhibition of mediator release was observed with 1.0 microM ketotifen, above which histamine release was induced. Sodium cromoglycate was a weak inhibitor of histamine and prostaglandin D2 release from lung and tonsillar mast cells and showed tachyphylaxis. Sodium cromoglycate did not inhibit histamine and prostaglandin D2 release from skin mast cells. On the other hand, no heterogeneity was observed with the beta-adrenoceptor agonists, procaterol and salbutamol. beta-Adrenoceptor stimulants were significantly more effective in inhibiting prostaglandin D2 than histamine release. No tachyphylaxis was seen with prolongation of the incubation time before challenge. Our results suggest that human mast cells are heterogeneous with respect to the modulation of mediator release by ketotifen and sodium cromoglycate but not beta-adrenoceptor agonists.


Effect of phospholipase A2 inhibitor ONO-RS-082 on substance P-induced histamine release from rat peritoneal mast cells. Rat peritoneal mast cells purified on a Percoll gradient were challenged with substance P (SP) and the effect of phospholipase A2 inhibitor ONO-RS-082 on SP-induced histamine release from the cells was investigated. 10(-5) mol/l SP caused a significant histamine release and the amount of histamine release reached maximum at 1 min after the challenge. ONO-RS-082 inhibited the SP-induced histamine release in a concentration-dependent manner at the concentration from 10(-6) to 10(-4) mol/l, suggesting possible involvement of phospholipase A2 in SP-induced histamine release from rat peritoneal mast cells.


Comparison of substance P-induced and compound 48/80-induced neutrophil infiltrations in mouse skin. It has recently been shown that substance P induces neutrophil infiltration in the skin, which is mediated through mast cell degranulation. Since substance P activates both skin mast cells and vascular endothelial cells, we compared the potencies of substance P and a mast cell-degranulating agent, compound 48/80, which is inactive for vascular endothelial cells, in inducing neutrophil infiltration in mouse skin. We also examined the effect of the C-terminal peptide of substance P, SP6-11, which is active for vascular endothelial cells, on compound 48/80-induced neutrophil infiltration in the skin. Subcutaneous administrations of substance P (10(-7) to 10(-5) M; 0.1 ml) and compound 48/80 (0.5-50 micrograms/ml) induced neutrophil infiltrations and mast cell degranulations in mouse skin in a concentration-dependent fashion. Moreover, substance P induced more neutrophil infiltrations than compound 48/80 in terms of the magnitude of mast cell degranulations. SP6-11 (10(-6) to 10(-4) M) induced no significant neutrophil infiltration or mast cell degranulation, but increased the vascular permeability of endothelial cells in the skin. Furthermore, SP6-11 enhanced compound 48/80-induced neutrophil infiltration without any increase in mast cell degranulation. Our results indicate that, in addition to mast cell degranulation, the activation of vascular endothelial cells is involved in substance P-induced neutrophil infiltration in the skin.