X-ray micro-analysis of cultured respiratory epithelial cells from patients with cystic fibrosis. X-ray micro-analysis was carried out on cultured respiratory cells from polyps removed from individuals with and without cystic fibrosis (CF). In a first set of experiments, proper experimental conditions were established. Washing the cells with 300 mmol l-1 mannitol in distilled water was found to give the best removal of the culture medium. The elemental concentrations stabilized in about 10 min after the start of the preincubation. Intracellular [Na] and [Cl] increased slightly with increasing passage number, whereas intracellular [K] decreased. Under resting conditions there were no significant differences in elemental content between CF and control cells, and there were no indications for abnormally high total [Ca] in CF cells. In normal cells, stimulation with a cAMP-analogue resulted in a decrease of cellular [Cl], whereas in CF cells an increase was measured. Exposure of both normal and CF cells to ouabain resulted in decreased [K] and increased [Na] and [Cl] level. The calcium ionophore A23187 had a similar effect on normal cells but did not affect CF cells markedly. Application of amiloride to the apical side of the cells resulted in a decrease of cellular [Na] in CF cells, whereas [Na] in control cells was not affected. The results correspond with what is known about the defective cAMP-regulated transepithelial Cl-transport in CF cells. The effect of the calcium ionophore on cellular electrolyte content is more complicated and may be the result of two separate effects: efflux of Cl- via a Ca(2+)-dependent mechanism and inhibition of the Na(+)-K(+)-ATPase by intracellular Ca2+ ions causing an influx of Na+ and Cl- ions.


Anti-C100-3 antibody status, viral genomic sequences, and clinical features in chronic hepatitic patients with hepatitis C virus RNA in sera. Since detection of hepatitis C virus RNA by the polymerase chain reaction (PCR) showed that there existed anti-C100-3 (anti-HCV) antibody negative patients infected with HCV, we attempted to find out whether there were any clinical or viral genomic differences between the anti-HCV antibody positive and negative groups. One hundred and fifty-nine patients with chronic liver diseases with hepatitis C virus RNA in their sera were selected. Anti-HCV antibody was tested for anti-C100-3 antibody by an enzyme linked immunosorbent assay. The incidence of anti-HCV antibody was 129/159. The concentration of serum gamma-globulin, the titier of ZTT, and the positive rate of the PCR with the primers of the NS3/4 region (NS3/4PCR) were significantly higher in the anti-HCV antibody positive group than in the negative group. However, the other data such as alanine aminotransferase activity or past history were not significantly different. Nucleotide sequence of the cDNA fragments of NS3/4 region amplified by the PCR did not differ significantly between isolates from anti-HCV antibody positive and negative sera. The sequences observed in the present study did not differ significantly from those reported previously. Although there remains the possibility that the variation of viral genomic sequences may cause the absence of anti-HCV antibody, these results suggested that the individual clinical backgrounds or immunoreactivity of the patients might influence the antibody development.


Localization of hepatitis C virus RNA in human liver biopsies by in situ hybridization using thymine-thymine dimerized oligo DNA probes: improved method. To establish the most proper method of in situ hybridization in detection of HCV-RNA in the liver, various detailed procedures were examined using frozen as well as paraffin-embedded sections of tissue derived from patients. In frozen sections of the liver from hepatitis C patients obtained at autopsy or surgery, HCV-RNA was detectable by in situ hybridization using thymine-thymine dimerized oligonucleotide DNA probes when the sections were treated with ethanol-acetic acid at first, then 0.2 N hydrochloric acid, proteinase K (0.02 u/ml) and DNase. When the paraffin-embedded liver sections were used, more intense proteinase K treatment (0.2-2 u/ml) was required to expose viral RNA and even after that, the positive HCV-RNA signals were less than those in frozen sections, because the cytoplasmic RNA in the routine paraffin-embedded sections was preserved unevenly and less than in frozen sections. These findings indicate that in situ hybridization of HCV-RNA is useful for diagnosing HCV infection and should be a potent tool for monitoring the state of virus activities during therapy. However, the liver biopsy method should be modified so that RNA is retained properly to utilize biopsies more effectively for the routine diagnosis of HCV infection.


A case of biliary cystadenocarcinoma arising in the liver with a congenital retention of indocyanine green. A case of biliary cystadenocarcinoma that occurred in a 45-year-old woman is reported. Ultrasonography and computed tomography clearly revealed papillary projections in the cyst of the liver. Percutaneous transhepatic cystography showed connection between the cyst and the common bile duct. The tumor was surgically resected and proved to be a mucinous papillary adenocarcinoma arising from a biliary cystadenoma. The patient is doing well 4 years after surgery. Interestingly, this is the first reported case of a biliary cystadenocarcinoma in the liver with markedly diminished excretion of indocyanine green.


Effect of oral contraceptives on plasma beta-endorphin and corticotropin at rest and during exercise. Concentrations of immunoreactive beta-endorphin and corticotropin in plasma were studied in 27 healthy physically active women at rest and after the exercise test on a treadmill requiring 60% and 90% of the maximal oxygen consumption. Eleven of the subjects were on a combination-type of oral contraceptive pills, and the remaining 16 did not use any pills. Plasma immunoreactive beta-endorphin levels at rest were higher in pill non-users than in pill users. Corticotropin levels at rest did not differ between the pill users and non-users. After the 60% exercise test a slight increase was found in the concentrations of corticotropin and beta-endorphin in the pill non-users but not in the pill users. In the 90% exercise test, plasma beta-endorphin and corticotropin levels increased significantly in both groups. We conclude that the use of oral contraceptives may elevate the threshold of the intensity of exercise required to increase beta-endorphin and corticotropin secretion. Decreased resting concentration of beta-endorphin in pill users can be explained by suppression of normal cyclic ovarian function.


Is 1 mg of estradiol valerate or 0.625 mg of conjugated estrogens sufficient for all women to prevent menopausal bone loss? Bone mineral content was measured by dual photon absorptiometry in 35 women who needed estrogen replacement therapy but did not want the addition of progestogens because they did not want regular bleeding. A total of 23 women were treated with estradiol valerate 1 mg per day over a mean period of 3.7 years; 12 women received conjugated estrogens 0.625 mg per day over a mean period of 5.3 years. The mean values of bone mineral content in both groups did not change. In the women on estradiol valerate, 61% had a decrease, and in those on conjugated estrogens, 67% had a decrease in bone mineral content. However, the calculated decrease per year was within the limits of the intraindividual reproducibility of the measurements. A difference between two measurements with a decrease of > 1.0 g hydroxyapatite/year over a period of > 3 years is larger than the limits of the intraindividual reproducibility. A decrease in bone mineral content > 1.0 g hydroxyapatite/year over a mean period of 3.98 years, SD 0.35, was observed in six of 23 (26%) of the women on estradiol valerate with a mean decrease of 5.28 g hydroxyapatite, SD 0.97. Only one of 12 (8%) of the women on conjugated estrogens had a decrease of 6.1 g hydroxyapatite over a period of 5.2 years. Periodic measurement of bone mineral is recommended in women on estrogen replacement therapy with estradiol valerate 1 mg per day or conjugated estrogens 0.625 mg per day for prevention of postmenopausal bone loss.


Chemiluminescence of human bloodstream monocytes and neutrophils: an unusual oxidant(s) generated by monocytes during the respiratory burst. Maximal rates of O2- and H2O2 production by human bloodstream monocytes activated during the respiratory burst by phorbol ester were only about 10% of those of neutrophils. Furthermore, monocytes possess only about 5% of the myeloperoxidase activity of neutrophils and so can only produce low levels of HOCl and related compounds. These combined reductions in O2- generating ability and lower myeloperoxidase levels result in low levels of luminol chemiluminescence stimulated during the respiratory burst of monocytes. However, although monocytes generate much lower levels of O2- and H2O2 than neutrophils, these cells produce comparable rates of PMA-stimulated lucigenin chemiluminescence. Hence, this assay does not accurately reflect the production of either of these two oxidants by activated phagocytes, and further lucigenin must react with some other oxidant(s) via a process which leads to photon emission. This oxidant(s) is not O2-, H2O2, .OH, 1O2 or NO, but is derived from O2- generated during the respiratory burst and is generated in greater quantities by activated monocytes compared with neutrophils. Thus, lucigenin chemiluminescence is an indirect measure of superoxide release.


Parathyroid hormone-related protein stimulates prostaglandin E2 release from human osteoblast-like cells: modulating effect of peptide length. Parathyroid hormone-related protein (PTHrP) is a potent bone-resorbing protein that frequently mediates the humoral hypercalcemia of malignancy syndrome. Since prostaglandins may mediate the bone-resorptive action of certain hormones, we examined the effect of PTHrP on prostaglandin E2 (PGE2) secretion by human osteoblast-like cells. There was low-level basal secretion of PGE2 by Saos-2 cells (8.1 +/- 0.6 pg/ml). Using four different preparations of PTHrP, it was observed that with increasing peptide length, from 36 to 141 amino acids, a significant increase in efficacy for PGE2 release was seen in these cells. All forms of PTHrP were agonists for PGE2 release, with effects seen at concentrations as low as 10(-12) M in 48 h conditioned media. The amino terminus of the molecule appeared critical for this effect since the truncated derivative PTHrP-(7-34) did not induce significant PGE2 secretion. However, the influence of peptide length could not be explained by differential activation of adenylate cyclase since [Tyr36]PTHrP-(1-36)amide was equipotent to the longest peptide preparation, PTHrP-(1-141), in stimulating cyclic AMP accumulation in the Saos-2 cells. In contrast, PTHrP-(1-141) was significantly more effective than [Tyr35]PTHrP-(1-36)-amide in inducing a rise in cytosolic calcium. Further, this effect was noted at concentrations lower than those that caused significant cyclic AMP accumulation in the Saos-2 cells. PTHrP-(1-141) induced the release of PGE2 from primary human bone cell cultures to levels entirely comparable to those seen in the Saos-2 cells. PTHrP-(1-141) also induced PGE2 release by cultured fetal rat long bones at 72 h. We conclude that the carboxy-terminal region of PTHrP has important effects on cellular signal transduction pathways and on the release of a potent bone-active cytokine, PGE2.


[Calcified bladder oat cell associated with hypercalcemia and hypophosphoremia]. Description of a 69-year old male patient presenting calcified vesical tumour which, following a pathoanatomical study was shown to be an non-differentiated oat cell carcinoma associated to transitional carcinoma and adenocarcinoma. Biochemically it presented hypercalcemia and hypophosphoremia and the electron microscope study demonstrated the presence of neurosecretion granules. The immunohistochemical techniques were negative for protein S-100 and cytokeratins, but positive for the membrane epithelial antigen and the neuro-specific enolase. The patient was treated by transurethral resection and polychemotherapy, presenting an initially favourable response but dying 18 months later after widespread metastasis. These data are in agreement with those obtained from the literature review carried out with regard to clinical picture, pathoanatomical studies, aggressiveness and overall poor prognosis with this type of tumours. In our view, the tumour's mixed composition supports an origin of pluripotential cell (steam cell). Presence of neuroendocrine syndromes associated to oat cell vesical tumours is an unusual fact related to the tumour's hormonal secretion.


The murine IFN-gamma receptor does not transduce the activation signal to a murine promoter in human cells. MuIFN-gamma receptor cDNA has been stably transfected in the human WISH cell line. A stable transfectant, denominated WISH-C2, expressed 46;000 receptors/cell whose affinity was similar to that observed on murine cells (Kd = 2.6 x 10(-9) M). When WISH-C2 cells were treated with Hu- or MuIFN-gamma, antiviral and antiproliferative activities were observed with HuIFN-gamma only. These findings suggest that other species-specific components associated to the binding site are required for the signal transduction. To overcome the complexity of measuring biological functions that very likely involve more than one regulatory and structural gene, we set up for the first time a system where a reporter gene driven by a murine promoter was used to directly evaluate the interaction of MuIFN-gamma receptor with an inducible promoter through the missing transducer factor.


Using cue reactivity to screen medications for cocaine abuse: a test of amantadine hydrochloride. The use of responding to drug-related stimuli as a dependent measure for studies of anticraving medications was assessed. Cocaine-dependent subjects receiving either amantadine hydrochloride, a putative anticraving agent, or placebo were exposed to drug-related cues prior to and 7 days after the initiation of the medication. Measurements of heart rate, skin resistance, skin temperature, and self-reported craving were taken during each stimulus session. Amantadine increased physiological reactivity to the drug-related cues compared to the placebo while having no effect on craving. Although the results discourage the use of amantadine as an anticraving medication, they do suggest that responses elicited by drug-related stimuli provide a valuable set of dependent measures for use in future medication trials of anticraving agents.


Relationship between role-play measures of coping skills and alcoholism treatment outcome. Alcoholic clients were given role-play tests, involving various social and drinking-related scenarios, before and after two types of aftercare treatment. The assessments were used both to evaluate the effects of treatment and to determine whether any dimensions of pretreatment role-play performance interact with treatment type to predict treatment outcome. Eighty-nine patients were randomly assigned to aftercare group treatment involving either interactional therapy or coping skills training. Clients in both treatments experienced declines in their urge to drink during the role-play scenes from pre- to posttreatment, and these declines were related to reductions in heavy drinking. Three pretreatment role-play variables interacted with type of treatment to predict outcome: observer-rated skill, observer-rated anxiety, and self-reported urge to drink. In general, those patients who did better in the role plays had better drinking outcomes following interactional therapy. Those who experienced more difficulty in the role plays fared best with coping skills training. The results suggest that role-play measures could be used for patient-treatment matching, although it remains to be determined whether they will be superior to more easily assessed patient characteristics.


Development of an adolescent alcohol and other drug abuse screening scale: Personal Experience Screening Questionnaire. The development of a new adolescent alcohol and other drug abuse screening scale is summarized. The Personal Experience Screening Questionnaire (PESQ) is intended to meet the need for a quick, psychometrically adequate adolescent screening tool to measure the need for a comprehensive assessment. The development of the questionnaire's problem severity scale and evidence related to its reliability (internal consistency) and validity are described.


Structural analysis of periodate-oxidized heparin. Treatment of heparin with HONO at pH 1.5 cleaves the polymer at N-sulfated, but not at N-acetylated GlcN residues, and yields di- and tetrasaccharides. The GlcNSO3 residues at the sites of cleavage are converted into anhydromannose (AMan) residues. Reduction of heparin cleavage products with NaB3H4 yields mixtures of di- and tetrasaccharides with reducing terminal [3H]anhydromannitol residues. The identification and quantification of these oligosaccharides by HPLC procedures have been described. These procedures have been used to determine the rates of periodate oxidation of the susceptible unsulfated GlcA and IdoA residues in heparin by measuring the disappearance of the di- and tetrasaccharides that contain GlcA and IdoA. Complete oxidation with IO4- results in the total loss of the unsulfated uronic acid-containing oligosaccharides, but kinetic studies reported here show that IdoA is oxidized much more rapidly than the major fraction of the GlcA under all reaction conditions. As the pH is lowered from 7 down to 3, the overall rate of the oxidation slows markedly, but the relative rates of GlcA and IdoA oxidation do not change. The slow rate of oxidation of GlcA residues at all pH's yields oxidation products early in the reaction progress in which all of the unsulfated IdoA residues are oxidized while 70-80% of the pH 1.5 nitrous acid-releasable GlcA----AMan(3,6-(SO4)2) are retained. The anticoagulant activity (APTT) of the partially oxidized product is reduced from 170 IU/mg to 38 IU/mg. Further studies show that the GlcA residue in the antithrombin III binding pentasaccharide is oxidized much more rapidly than the bulk of the GlcA residues in heparin. The results suggest that heparin contains GlcA----AMan(3,6-(SO4)2) sequences that lie outside of the antithrombin-binding pentasaccharide.


Heparan sulfate glycosaminoglycans as primary cell surface receptors for herpes simplex virus. Our current incomplete picture of the earliest events in HSV infection may be summarized as follows. The initial interaction of virus with cells is the binding of virion gC to heparan sulfate moieties of cell surface proteoglycans. Stable binding of virus to cells may require the interaction of other virion glycoproteins with other cell surface receptors as well (including the interaction of gB with heparan sulfate). Penetration of virus into the cell is mediated by fusion of the virion envelope with the cell plasma membrane. Events leading up to this fusion require the action of at least three viral glycoproteins (gB, gD and gH), one or more of which may interact with specific cell surface components. It seems likely that binding of gB to cell surface heparan sulfate may occur and may be important in the activation of some event required for virus penetration. Heparan sulfate is present not only as a constituent of cell surface proteoglycans but also as a component of the extracellular matrix and basement membranes in organized tissues. In addition, body fluids contain both heparin and heparin-binding proteins, either of which can prevent the binding of HSV to cells (WuDunn and Spear, 1989). As a consequence, the spread of HSV infection is probably influenced, not only by immune responses to the virus, but also by the probability that virus will be entrapped or inhibited from binding to cells by extracellular forms of heparin or heparan sulfate.


Calories, fat, fibers, and cellular proliferation in Swiss Webster mice. Increased cellular proliferation has been associated with the enhanced expression of several key stages in carcinogenesis. A standard protocol was used to investigate the effect of specific dietary regimens on cellular proliferation. Young adult Swiss Webster mice were fed for 30 days with modified AIN-76A semi-purified diets designed to illustrate the effects of the levels of dietary or calorie restriction, different fibers and bulking agents, and different fats on cellular proliferation. Female mice were used for the restriction and fat studies, males for the fiber and bulking agent studies. Vaginal smears were taken from females from treatment day 15, and the mice killed 2 days following the first estrus following 30 days feeding; males were killed on the 30th day. One hour before death, mice were injected ip with 0.25 micro Ci/g 3[H]-thymidine. Slides were prepared for radioautography and histopathology. Both dietary and calorie restriction led to reduced 3[H]-thymidine labeling indices in each of the seven tissues studied, the mammary gland being the most severely affected. Different fibers and bulking agents, in specific cases, reduced labeling in the duodenum but not to a consistent statistically significant extent in the colon or colo-rectal region. In the duodenum, oat bran and oat gum were the most effective while wood cellulose (alphacel) had no effect. Investigations on the effects of different fats is continuing. High levels of lard, menhaden oil, or cod liver oil as the fat component of the AIN-76A diet, led to much higher levels of labeled cells in the mammary gland or colo-rectal region than did fat components rich in vegetable oils. The labeling indices appeared to be inversely correlated with the level of linoleic acid in the diet, a presumption that has been confirmed by investigating a series of diets containing different levels of this acid. Anti-oxidants were not used in any of these fat-modified diets. The overall results obtained in these studies clearly indicate the utility of cellular proliferation studies in investigating the effects of dietary modifications.


Human papillomavirus infections.

Human papillomavirus infections. Though the existence of disease associated with HPV has been documented for centuries, it has been only within the past 2 decades that we have recognized the clinical diversity and significant morbidity and mortality associated with HPV infections. The original lack of interest and nonavailability of in vitro culture systems has hampered research. However, with the advent of molecular diagnostic techniques, strong evidence suggests that HPV plays a major role in the development of specific anogenital cancers, including cervical, vaginal, vulvar, penile, and anal. It is this association between anogenital cancers and HPV that may result in treatment guidelines that eventually will eliminate these cancers. Moreover, to the extent that this association has resulted in overdiagnosis and unnecessarily aggressive treatment, new and ongoing research may create more appropriate treatment options. The principles for diagnosis and treatment should be based on adequate evaluation, including colposcopic examination and histologic confirmation for suspected neoplastic lesions. The majority of HPV-induced disease should be treated conservatively (often best with observation). The role of HPV DNA testing in clinical outcome is yet to be defined, with the exception of high-grade neoplastic disease, which should be treated aggressively.


Measurement of MPO activity as model for detection of granulocyte infiltration in different tissues. Activity of myeloperoxidase (MPO) was determined in different tissues to detect granulocyte infiltration. MPO was measured in the mouse ear after injection of interleukin-1 beta, in the rat paw after carrageenan-induced edema and in the lung of sensitized guinea pigs after ovalbumin inhalation. Pretreatment of the animals with antiinflammatory drugs abolished the increase of MPO activity in tissues induced by this different stimuli.


The effect of lidocaine on oxygen free radical production by polymorphonuclear neutrophils. During myocardial ischemia and subsequent reperfusion, activation of polymorphonuclear neutrophils occurs and results in an increased production of oxygen free radicals that participate in the generation of reperfusion arrhythmias. We investigated the effect of antiarrhythmic drug lidocaine on oxygen free radicals production by activated neutrophils in vitro with a chemiluminescence assay. Lidocaine reduced lucigenin-amplified chemiluminescence related to the superoxide anion production but failed to affect hydrogen-peroxide dependent luminol chemiluminescence.


Inhibition of butyric acid-induced colitis in mice by 16,16-dimethyl prostaglandin E2. A standard colitic lesion was induced in male BKA mice by intrarectal administration of butyric acid (7.5%, 0.1 ml, 10 sec contact). Animals were killed after 5 h and the 'colitic score', increase in colonic tissue water ('oedema') and colonic tissue content of myeloperoxidase (MPO, a marker for neutrophils) were determined. Drug was administered intrarectally in 0.2 ml saline 20 min before colitis induction. In colitic animals given vehicle alone, all these parameters increased (P less than 0.05) compared to the non-colitic controls. In colitic animals given 16,16-dimethyl PGE2 (0.2-20000 micrograms/kg), colitic score was reduced (P less than 0.05) at all dose levels when compared with vehicle-treated colitic animals. The oedema and MPO showed a dose-related reduction (r = -0.895 and -0.904 respectively). In mouse colon 16,16-dimethyl PGE2 showed a protective action against butyric acid-induced colitic damage.


Inhibition of leukotriene B4(LTB4) by recombinant interleukin-1 receptor antagonist (IL-1RA) on human monocytes. Macrophages are a primary source of interleukin-1 (IL-1), a glycoprotein which plays an important and essential role in the immune response and inflammation. Cytokines stimulate many different cells to produce increasing amounts of arachidonic acid metabolites such as prostaglandins and leukotrienes. Recently, interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 released by macrophages, has been reported to inhibit PGE2. In accordance with these data our results show that the pretreatment, for 60 min, of purified human peripheral monocytes with IL-1ra at different concentrations (0.25-250 ng/ml) inhibits, in a dose-dependent manner, the generation of LTB4 released after 10 min treatment with calcium ionophore A23187 (5 microM). The inhibition of LTB4 synthesis by hrIL-1ra suggests the possibility that this new glycoprotein plays a modulatory role in immunity and inflammation.


Eicosanoid production by density-defined human peritoneal macrophages during inflammation. Density-defined macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B4 (LTB4, 16%) and 5-hydroxy-eicosatetraenoic acid (5-HETE, 24%) and the cyclooxygenase products 12-hydroxyheptadecatrienoic acid (HHT, 22%) and thromboxane B2 (TXB2, 18%). The synthesis of eicosanoids was linear with the maturity of the macrophage subpopulations, suggesting that eicosanoid production is increased in in-vivo activated macrophages.


[The dynamics of melanin-affinitive and non-affinitive antibacterial agents in the iris-ciliary body of rabbit eyes--comparative studies in pigmented and albino rabbits]. The differences between the drug penetration levels in the iris-ciliary bodies of sparfloxacin (SPFX) and cefmenoxime (CMX), which respectively have high and low affinity to melanin, were examined using pigmented and albino rabbit eyes. Each drug was mixed with a homogenate of the iris-ciliary bodies of pigmented and albino rabbit eye, respectively. All CMX was distributed in a water soluble protein of the above homogenate of both pigmented and albino eyes, while all SPFX was detected from the water soluble protein of the tissue homogenate of the albino eye. However, in homogenate of the pigmented eyes, 60% of the drug was detected from water soluble protein and 20% of that was detected from water non-soluble protein. In the in vivo study, each drug was topically administered to pigmented and albino rabbit eyes. The SPFX concentration in the iris-ciliary body was significantly higher in the pigmented than in the albino eyes. The results indicated that the intraocular dynamics of the drug which has a high affinity to melanin showed significant differences between pigmented and albino rabbit eyes. This should be considered in studies of ocular pharmacology as an important factor which influences intraocular drug dynamics.


Color flow Doppler characterization of focal hepatic lesions. The purpose of this study was to determine the sensitivity and specificity of color flow Doppler sonography for the specific diagnosis of focal hepatic lesions. Color flow Doppler images of 118 focal hepatic lesions in 108 patients were analyzed prospectively. In most patients, liver disease was suspected or known to be present before the Doppler images were obtained. Experienced sonologists obtained and interpreted all sonograms. The lesions were classified, according to their color flow pattern, into two main categories: lesions with internal vascularity and lesions with no internal vascularity. The color flow Doppler pattern of each lesion was correlated with the diagnosis of the lesion on a lesion-by-lesion basis. One hundred two lesions were proved by biopsy and 16 lesions were confirmed by evaluation with other imaging techniques. Lesions included 29 hepatocellular carcinomas, 64 metastases, one cholangiocarcinoma, and 24 benign lesions. The sensitivity and specificity of vascularity as shown by color Doppler imaging in the diagnosis of hepatocellular carcinoma were determined. The majority of hepatocellular carcinoma lesions (76%) had internal vascularity. Most of the metastases (67%) and benign lesions (75%) had no internal vascularity. When the presence of internal vascularity was used as the discriminating criterion, the sensitivity of color flow Doppler findings for the diagnosis of hepatocellular carcinoma was 0.76. The specificity of internal vascularity for the diagnosis of hepatocellular carcinoma vs other focal lesions was 0.69; for hepatocellular carcinoma vs metastases it was 0.67. Although most hepatocellular carcinomas have internal vascularity on color flow Doppler images, a significant number of metastases also have internal vascularity. This overlap limits the usefulness of color flow Doppler imaging for distinguishing hepatocellular carcinoma from metastatic tumors.


Dietary prevention and treatment of disease. Diet is a major factor in five of the 10 leading causes of death in the United States and is a contributing factor in many other diseases and health conditions. Certain diseases can be prevented and general health can be maintained or improved if patients are willing to follow advice about nutrition. Hence, nutrition-related consideration of fiber, cholesterol, carbohydrates and energy should be integral to the management of health conditions.


Effects of guava intake on serum total and high-density lipoprotein cholesterol levels and on systemic blood pressure. There is evidence that inclusion of high fiber foods such as oats, fruits and vegetables in the diet can decrease fat intake and modulate blood lipids. To test this hypothesis, 61 group A and 59 group B patients with essential hypertension were administered guava fruit preferably before meals in a foods-to-eat approach rather than foods-to-restrict, in a randomized and single-blind fashion for 12 weeks. At entry into the study, mean age, male sex, mean body mass index, percentages of risk factors and mean levels of blood lipids were comparable between groups A and B. Adherence to guava consumption was assessed by questionnaires and weighing of guava intake by 24-hour recall after 12 weeks of follow-up. Nutrient intakes including saturated and total fat were significantly decreased; carbohydrates, total and soluble fiber and vitamins and mineral intakes were significantly higher in group A than in group B at 12 weeks. There was a significant net decrease in serum total cholesterol (9.9%), triglycerides (7.7%) and blood pressures (9.0/8.0 mm Hg) with a significant net increase in high-density lipoprotein cholesterol (8.0%) after 12 weeks of guava fruit substitution in group A than in group B. By adding moderate amounts of guava fruit in the usual diet, changes in dietary fatty acids and carbohydrates may occur, providing significant amounts of soluble dietary fiber and antioxidant vitamins and minerals without any adverse effects. There is a greater decrease in lipoprotein metabolism and blood pressures.


MR of the brain using fluid-attenuated inversion recovery (FLAIR) pulse sequences. Results from conventional T2-weighted spin-echo sequences were compared with those obtained using fluid attenuated inversion recovery (FLAIR) pulse sequences in order to assess their relative merits in detecting disease. Forty adult patients with suspected disease of the brain were examined with spin-echo sequences (TE = 20 and TE = 80), and results were compared with FLAIR sequences of several types with inversion times of 1800-3000 msec and echo times of 130-240 msec. Scans were assessed by two radiologists for lesion number, conspicuity, and extent. A total of 48 lesions or groups of lesions were recognized with both sequences. In 22 instances, more lesions were seen with FLAIR sequences, and, in the remaining 26, equal numbers were seen. In 42 lesions, conspicuity was better with FLAIR sequences, equal in five and worse in one cystic lesion. Lesion extent was better assessed in 28 of the 48 cases with FLAIR sequences and equally well seen in the remainder. By virtue of their long echo time and relative freedom from cerebrospinal fluid artifact FLAIR sequences provide high sensitivity to a wide range of disease. The basic sequence is easy to implement but is relatively time consuming.


Molecular and cellular aspects of calcium channel antagonism. Calcium fluxes play a key role in controlling many physiologic processes and responses in the body. The past few years have witnessed major advances in understanding of L-type calcium channels and their blockade with calcium channel antagonists. The L-type calcium channels comprise 5 subunits termed alpha 1, alpha 2, beta, gamma, and delta. Elucidation of the mechanisms of action of the calcium channel antagonists has been advanced by cloning and genetic manipulation of these subunits. The alpha 1 subunit appears to be responsible for channel opening and voltage dependency, and it contains receptors for calcium channel antagonists; these geographically distinct receptors correspond to each of the 3 different chemical classes of antagonists, exemplified by diltiazem, nifedipine, and verapamil. Diltiazem appears to have an inhibitory effect on mitochondrial sodium-calcium exchange that is unique among calcium channel antagonists. Preliminary data suggest that a diltiazem-specific receptor also exists in the endothelium. It appears, therefore, that despite advances in the understanding of L-type calcium channels, much remains to be learned about other possible receptors for the calcium channel antagonists.


Peripheral neuromuscular dysfunction and falls in an elderly cohort. In a prospective study of 169 tenants of senior citizen housing in New Jersey in 1986-1987, the relations between tests of peripheral sensory and motor functions in the lower extremities and the rate of first falls were evaluated. The mean age of the cohort was 79.8 years. Fifty-seven persons fell at least once during the follow-up period (mean, 5.6 months). After adjustment for history of stroke, heart failure, emphysema, and use of a walker or cane, rate ratios for first falls were elevated in subjects with reduced toe joint position sense (rate ratio (RR) = 2.2) and sharp-dull discrimination (RR = 2.0), but to a lesser extent for reduced ankle strength (RR = 1.5). Presence of one or more of these three deficits was defined as a peripheral neuromuscular dysfunction and was associated with first falls after adjustment for multiple covariates (RR = 2.4, 95% confidence interval 1.3-4.5). Having two or all three sensory or motor deficits increased the rate of falling 3.9 times (95% confidence interval 2.1-7.0) compared with persons without these deficits. These data suggest that impaired sensory and motor function of the lower extremities plays an important role in falls in the elderly.


Work-related mesothelioma in Québec, 1967-1990. Prior surveys of malignant mesothelioma in Québec have noted that almost all the excess in occupational exposure related mesothelioma was in the manufacture and industrial application of asbestos rather than in the mining and milling operations. To evaluate the current status of malignant pleural mesothelioma in the Québec workforce, we reviewed all cases of pleural mesothelioma seen and accepted by the Québec Workman's Compensation Board (CSST) for work related compensation of industrial disease. We identified 120 cases, 7 of whom were females. They were of an average age of 59 +/- 8.5 yrs (sd) (range 42-84); they were exposed to asbestos dust in the workplace for an average of 26 +/- 14.3 yrs (range 0.5-50). The cases were subdivided into 3 groups according to workplace asbestos exposures. There were 49 cases originating in the mines and mills of the Québec Eastern Township region (primary industry, group 1), 50 cases from the manufacture and industrial application sector (secondary industry, group 2), and 21 cases from industries where asbestos was not a major work material, often an "incidental" material (tertiary industry, group 3). Group 1 was of an average age of 62 +/- 8 years, exposed to asbestos dust 31 +/- 14 years and the distribution of exposure time was as follows: 15% cases with < or = 10 year-exposure and 77% > or = 25 year-exposure. In group 2, the age was significantly lower at 57 +/- 9 years; the exposure time was also significantly lower at 22 +/- 14 years, and the distribution of exposure time differed from the above (29% cases with < or = 10 year-exposure and 48% > or = 25 year-exposure). In group 3, the average age was 58 +/- 7 years, the exposure time was also significantly lower at 28 +/- 12 years and the distribution of exposure time differed from the above (33% cases with < or = 10 year-exposure and 62% > or = 25 year-exposure). Analyses of the yearly incidence of new cases in each group documented the general incremental trend in all groups, with the sharpest rises in group 3. In the mining towns of Thetford and Asbestos, the incidence of mesothelioma was proportional to the workforce, thus suggesting that the tremolite air contamination, which is 7.5 x higher in Thetford, may not be a significant determinant of the disease in these workers.(ABSTRACT TRUNCATED AT 400 WORDS)


An environmental survey in chrysotile asbestos milling processes in India. Environmental monitoring to determine airborne asbestos fiber levels has been carried out in four different mills processing chrysotile asbestos in the Cuddapah District (Andhra Pradesh) of India. The "membrane filter method" comprising standard asbestos sampling techniques, acetone-triacetin method for sample preparation, fiber counting, and sizing using the phase contrast optical microscope were adopted in the study. Fiber concentrations both with respect to personal exposures and processing areas were found in most of the cases to be much higher than the prescribed Threshold Limit Value (TLVs) of the developed and developing countries for chrysotile asbestos. By optical microscopy, fiber length distribution showed 70% of fibers in the milling processes were in size range > 5-10 microns, whereas in > 10-20 and > 20 microns, 20% and 8%, respectively. Fiber identification for major elemental content, also done by using scanning electron microscope equipped with an energy dispersive X-ray analyzer, indicated the presence of tremolite along with chrysotile. The study stresses the urgent need to adopt suitable engineering controls at the dust generating sources to reduce the fiber level in the mill environment below the threshold limit.


Preparation and storage of single-dose portions of exametazime: effects on radiochemical purity after labeling. The effect of exametazime concentration, storage time, and the volume of the radiolabeling compound on the radiochemical purity of labeled exametazime doses was studied. Exametazime cold unit doses (CUDs) of 0.50, 0.33, 0.25, 0.17, and 0.13 mg/mL were prepared by reconstituting exametazime kits with 0.9% sodium chloride injection. After either one or two days of storage at -10 degrees C, four CUDs of each concentration were labeled with 0.2-0.3 mL of sodium pertechnetate Tc 99m (10-20 mCi). The radiochemical purity of CUDs was evaluated 15 minutes later by instant thin-layer chromatography. In a second experiment, exametazime CUDs of 0.5 mg/mL were prepared. After 0-19 days of storage at -10 degrees C, four CUDs were each labeled with 0.2 mL of sodium pertechnetate Tc 99m (10-20 mCi), and radiochemical purity was measured after 15 minutes. In a third experiment, exametazime CUDs of 0.5 mg/mL were labeled with 2.0 mL of sodium pertechnetate Tc 99m (10-20 mCi) after zero to five days of storage at -10 degrees C. The mean radiochemical purity was unacceptably low (less than 80%) for exametazime CUDs of 0.33, 0.25, 0.17, and 0.13 mg/mL; the 0.5-mg/mL CUDs were acceptably stable. Purity was less than 80% for CUDs stored for more than two days. The radiochemical purity of CUDs labeled with 2.0 mL of sodium pertechnetate Tc 99m was significantly greater than the purity of CUDs labeled with 0.2 mL for storage times exceeding two days.(ABSTRACT TRUNCATED AT 250 WORDS)


Itraconazole therapy for blastomycosis and histoplasmosis. NIAID Mycoses Study Group. To assess the efficacy and toxicity of orally administered itraconazole in the treatment of nonmeningeal, nonlife-threatening forms of blastomycosis and histoplasmosis. Prospective, nonrandomized, open trial. Multicenter trial at 14 university referral centers. Eighty-five patients with culture or histopathologic evidence of blastomycosis (48 patients) or histoplasmosis (37 patients). Patients receiving other systemic antifungal therapy were excluded. Itraconazole was administered orally at doses of 200 to 400 mg/d. Patients in whom treatment was considered a success were treated for a median duration of 6.2 months (blastomycosis) and 9.0 months (histoplasmosis). Disease activity was assessed at baseline; drug efficacy and toxicity were evaluated at monthly intervals during therapy, and efficacy was evaluated at regular follow-up visits after completion of therapy. The median duration of posttreatment evaluation for successfully treated patients was 11.9 months (blastomycosis) and 12.1 months (histoplasmosis). Among the 48 patients with blastomycosis, success was documented in 43 (90%). The success rate for patients treated for more than 2 months was 95% (38 of 40). Among the 37 patients with histoplasmosis, success was documented in 30 (81%). The success rate for patients treated for more than 2 months was 86% (30 of 35). All patients with histoplasmosis in whom treatment failed had chronic cavitary pulmonary disease. Toxicity was minor; only 25 (29%) patients experienced any side effects, and itraconazole toxicity necessitated stopping therapy in only 1 patient. Itraconazole is a highly effective therapy for nonmeningeal, nonlife-threatening blastomycosis and histoplasmosis. The drug is associated with minimal toxicity.


Evaluation of thyroid function in patients with isolated adrenocorticotropin deficiency. Thyroid hormone and thyrotropin (TSH) levels were evaluated before and after adrenal replacement in eight patients (six men and two women, 35-62 years old) with isolated adrenocorticotropin (ACTH) deficiency. Six patients (cases 1-6) showed TSH excess before treatment. Four patients (cases 1-4), who initially had subnormal thyroid hormone levels, showed resolution of biochemical features of primary hypothyroidism after treatment, although TSH excess has persisted in two patients (cases 1 and 2). Case 1 had an extremely high titer of antimicrosomal antibody (MCHA), and cases 2 and 3 showed histologically and cytologically chronic thyroiditis, despite negative results for MCHA and antithyroglobulin antibody, respectively. Two patients (cases 5 and 6), who had had normal thyroid hormone levels and did not show the significant rise in serum T3 in TSH releasing hormone testing, showed TSH normalization without changes in serum thyroid hormone levels after treatment. The other two patients (cases 7 and 8), who initially had normal TSH and thyroid hormone levels, did not show the significant changes in serum TSH and thyroid hormone levels after treatment. The prevalence of chronic thyroiditis coexistence in isolated ACTH deficiency may be higher than predicted. Therefore, TSH excess before adrenal replacement may be attributed to not only direct enhancement of TSH release due to chronic cortisol deficiency but also to thyroid dysfunction due to chronic thyroiditis. It is possible that hypothyroidism due to chronic thyroiditis can be improved only by adrenal supplementation.


Effects of calcium channel blockade with verapamil on the prolactin responses to TRH, L-dopa, and bromocriptine. To determine the mechanisms by which calcium channel blockade with verapamil causes hyperprolactinemia, the authors investigated the effects of this blockade on the prolactin (PRL) responses to stimulation by thyrotropin releasing hormone (TRH) and inhibition by dopamine, using L-dopa and bromocriptine. Verapamil, given for 1 week at a dosage of 240 mg orally to eight healthy volunteers, induced a significant elevation of basal PRL levels (17.3 +/- 1.8 ng/ml to 30.9 +/- 4.3 ng/ml, p < 0.005). Verapamil also caused an increase in the PRL response to a TRH (100 micrograms). However, when the increased basal level was considered by calculating the area under the THR response curve and subtracting the basal values, this increase (1763.4 +/- 202.6 ng/ml.min to 2260.6 +/- 223.9 ng/ml.min) was not found to be statistically significant (p > 0.05). Verapamil had no effect on the basal or TRH-stimulated thyroid stimulating hormone levels. In these same volunteers, PRL levels decreased from 13.2 +/- 2.5 ng/ml to a nadir of 5.5 +/- 1.6 ng/ml in response to L-dopa. After 1 week of verapamil 240 mg, basal PRL levels were elevated to 21.5 +/- 3.1 ng/ml, then decreased to 8.2 +/- 1.8 ng/ml with L-dopa. The percentage decreased in PRL in response to L-dopa (60 +/- 5% versus 62 +/- 3%) were not significantly different (p > 0.05). Verapamil had no effect on the basal or L-dopa-stimulated growth hormone levels. Bromocriptine 2.5 mg given to five volunteers twice daily caused PRL levels to fall from 13.3 +/- 1.6 ng/ml to 5.0 +/- 0.9 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)


Self-administered home cervicovaginal lavage: a novel tool for the clinical-epidemiologic investigation of genital human papillomavirus infections. Our objective was to evaluate a self-administered cervicovaginal irrigation apparatus (MY-PAP, Medtech, Bohemia, N.Y.) for the detection of cervicovaginal human papillomavirus infection. Twenty-five women referred to a colposcopy clinic because of a recent abnormal Papanicolaou smear were studied. Human papillomavirus infection test results detected by Southern blot hybridization and polymerase chain reaction from physician-administered and MY-PAP self-administered cervicovaginal lavage were paired for comparison. Seventeen (68%) patients returned home samples by mail a mean of 13 days after the clinic visit. All clinic samples and 16 of 17 (94%) home samples yielded adequate deoxyribonucleic acid for human papillomavirus infection analyses. Human papillomavirus infection was detected by polymerase chain reaction in 14 of 16 (88%) samples with complete concordance of viral detection in paired samples from the clinic and home. Southern blot detected human papillomavirus infection in 13 of 25 clinic samples (52%) and 7 of 16 home samples (44%), with concordance in 12 of 16 paired samples (75%). Specific viral type was the same in all positive paired samples (6/6). Concordance was high when the initial sample had a strong (2+ to 4+) human papillomavirus infection signal (5/6, 83%) or a negative result (5/6) or when a dysplastic (cervical intraepithelial neoplasia grade 1 to 3) lesion (9/11, 82%) was seen on cervical biopsy. Self-administered lavage represents an extremely promising technique for obtaining cervicovaginal lavage samples for human papillomavirus infection analyses.


Mechanisms involved in the pituitary desensitization induced by gonadotropin-releasing hormone agonists. We investigated the mechanisms of desensitization induced by gonadotropin-releasing hormone agonist in the pituitary. Effects of gonadotropin-releasing hormone agonist on the pituitary were studied in vitro and in vivo in the rat. In the clinical study serum luteinizing hormone was measured by radioimmunoassay with a polyclonal luteinizing hormone antibody (luteinizing hormone-radioimmunoassay) and by immunoradiometric assay with monoclonal luteinizing hormone antibodies (luteinizing hormone-immunoradiometric assay) during gonadotropin-releasing hormone agonist treatment. In the in vitro study bead-attached pituitary cells that were desensitized with a continuous infusion of 10(-7)mol/L gonadotropin-releasing hormone responded to 50 mmol/L K+. In the in vivo study gonadotropin-releasing hormone binding sites and rat luteinizing hormone beta-messenger ribonucleic acid in the pituitary decreased during gonadotropin-releasing hormone agonist treatment, but serum levels of rat luteinizing hormone did not decrease. In addition, a disparity between luteinizing hormone-radioimmunoassay and luteinizing hormone-immunoradiometric assay was demonstrated during gonadotropin-releasing hormone agonist treatment. Pituitary desensitization in response to gonadotropin-releasing hormone agonist may not be wholly receptor mediated and a nonreceptor process may be involved.


Studies of connexin 43 and cell-to-cell coupling in cultured human uterine smooth muscle. The aim of this study was to assess the presence and the permeability of gap junctions between human uterine smooth-muscle cells in culture. The uterine smooth muscles obtained from term-pregnant women were cultured. The presence of gap junction was evaluated by immunocytochemistry with gap junction protein antibodies and by measuring input resistance and intercellular spread of lucifer yellow. These measures also evaluated the permeability of gap junctions. Octanol, isoproterenol, dibutyryl cyclic adenosine monophosphate and forskolin were applied to the cultures to assess their effects on the permeability of gap junctions. During culture, immunocytochemical staining of gap junction protein (connexin 43) was increased and input resistance was decreased on day 2 of culture versus day 21 (18.4 +/- 7.87 M omega day 2; 3.8 +/- 1.76 M omega, day 21; p < 0.001). However, the decrease in input resistance was related to cell density rather than time in culture (16.4 +/- 5.01 M omega, single cells on days 1 and 2; 5.3 +/- 2.35 M omega, high-density cultures on days 1 and 2; p < 0.001). Octanol increased input resistance and intercellular spread of lucifer yellow in confluent cultures; isoproterenol, dibutyryl cyclic adenosine monophosphate, and forskolin did not. The increased staining of connexin 43 and the decreased input resistance during culture are evidence of elevated number of gap junctions between cells. The rapid and reversible increase in input resistance and decrease in spread of lucifer yellow by octanol are the result of decreased permeability of gap junctions. These two methods of modulation of gap junctions in human uterine smooth muscles are thought to be major mechanisms for the control of uterine contractility.


Recurrent maternal virilization during pregnancy caused by benign androgen-producing ovarian lesions. Benign causes of maternal virilization in pregnancy, such as luteoma of pregnancy and hyperreactio luteinalis, are generally believed to resolve completely post partum and not to recur. We present the fifth case in the literature of recurrent maternal virilization in pregnancy. These lesions should be viewed as potentially recurrent and cases should be managed accordingly.


Failure of ritodrine to prevent preterm labor in the sheep. The purpose of this study was to determine whether continuous infusion of ritodrine could prevent preterm delivery in sheep. Sheep in preterm labor induced by RU 486 (mifepristone) received infusions of either ritodrine (n = 5) or saline solution (n = 5), and the progress of labor was monitored. beta 2-Adrenergic receptor density and function (agonist-induced cyclic adenosine monophosphate production) was measured in myometrial samples from both groups. Ritodrine initially inhibited labor contractions. This inhibition was only maintained for 16 hours, after which both the amplitude and frequency of electromyographic bursts and contractions returned. The failure of the myometrium to respond to ritodrine (desensitization) was associated with significant reductions in agonist-induced cyclic adenosine monophosphate production and beta 2-adrenergic receptor concentration in myometrial tissue collected from these animals compared with the saline solution-treated controls. Continuous infusion of ritodrine to sheep in preterm labor produces only a transient inhibition of contractions. This desensitization is caused by a down-regulation of myometrial beta 2-adrenergic receptors.


Long-term vascular volume expansion maintains elevated thoracic duct lymph flow. To investigate the mechanisms responsible for edema seen during pregnancy, we tested whether lymph vessels are able to pump high volumes over long periods of time. In six chronically catheterized nonpregnant ewes, we examined left thoracic duct lymph flow rate and fluid balance responses to the administration of a balanced isotonic solution at a rate of 1 L/hr for 20 hours. Because estrogen administration decreases lymphatic contractility against outflow pressure, we also administered conjugated estrogens (Premarin) during the last 3 hours of the fluid infusion (experimental time 17 to 20 hours). After volume loading for 16 hours, the mean +/- SEM lymph flow rate, blood volume, and arterial pressure rose 100% +/- 26%, 20% +/- 2.3%, and 16% +/- 8.1%, respectively. Vascular compliance decreased significantly and, as evidenced by a lack of body weight changes, interstitial fluid volume failed to change (p < 0.05, analysis of variance). The transcapillary oncotic pressure difference increased by 2 mm Hg; venous pressure increased by 5.2 mm Hg. These data suggest that transcapillary forces favored fluid movement into the interstitium. Lymph flow rate remains elevated after blood volume expansion to a level similar to that described during pregnancy in sheep. A transient decrease in urinary output (approximately 20%) occurred with no changes in lymph flow rate, arterial pressure, or blood volume. Lymph flow rate is able to compensate for the increased capillary filtration observed during prolonged blood volume expansion.


Novel immunologic strategies in ovarian carcinoma. The purpose of our study was to develop new biologic systems for the treatment or diagnosis of patients with ovarian carcinoma through expansion of T-cell lines from the tumor-infiltrating lymphocytes of patients with ovarian carcinoma in low-dose recombinant interleukin-2 in sufficient numbers for treatment and human monoclonal antibodies that recognize cell-surface tumor-associated antigen determinants on ovarian carcinoma cells. Technologic advances in tumor immunology and new data presented in relation to ovarian carcinoma were used to develop T-cell lines for the treatment of advanced ovarian carcinoma patients. Logarithmic expansion of T-cell lines was performed in a hollow-fiber bioreactor, and a pilot clinical trial was initiated to treat ovarian carcinoma patients with intraperitoneal tumor-infiltrating lymphocytes plus low-dose recombinant interleukin-2. Human hybridomas were produced by fusion of regional lymph node B cells with a heteromyeloma cell line SPATZ 4. Two ovarian carcinoma patients have been treated with tumor-infiltrating lymphocytes expanded to 1 x 10(10) to 1 x 10(11) with manageable side effects and evidence of biologic activity. Human monoclonal antibodies have been developed that recognize tumor-associated antigen determinants. Recombinant interleukin-2-expanded tumor-infiltrating lymphocytes and human monoclonal antibodies recognize different molecular entities on tumor cells and act by different mechanisms. These approaches may be complementary to one another in future treatment strategies for ovarian carcinoma.


Polymerase chain reaction for the detection of the varicella-zoster genome in ocular samples from patients with acute retinal necrosis. We used the polymerase chain reaction to detect the virus genome in ocular samples from patients with clinically diagnosed acute retinal necrosis. Four samples from four patients with acute retinal necrosis, and five samples from three patients with other ocular diseases (sarcoidosis, rhegmatogenous retinal detachment, and epiretinal membrane of unknown origin) were evaluated. The samples consisted of aqueous humor, vitreous, or subretinal fluid. Primers were specific for varicella-zoster virus, herpes simplex virus, or cytomegalovirus. The varicella-zoster virus genome was detected in three of the four samples from patients with acute retinal necrosis. Among these three positive samples, two had PstI-site-less point mutation, strains that have been described only in Japan and of low prevalence. Samples from patients with diagnoses other than acute retinal necrosis yielded negative results when varicella-zoster virus primer was used. No sample was positive for herpes simplex virus or cytomegalovirus primers.


Cathepsin D in invasive ductal NOS breast carcinoma as defined by immunohistochemistry. No correlation with survival at 5 years. Cathepsin D expression was assessed by immunohistochemistry in 59 node-negative and 77 node-positive infiltrative ductal not otherwise specified (NOS) breast carcinomas and compared with overall survival at 90 months. Cancer cells in 60% (81/136) of the tumors expressed cathepsin D. In the stroma of 33% (18 of 55) cathepsin D negative tumors, numerous strongly cathepsin D positive, benign macrophage-like cells were found. Multivariate analysis showed no significant correlation of cathepsin D expression and overall survival for all patients for node-negative and node-positive patients and for patients with vimentin-positive and -negative tumors. However, in node-negative but not in node-positive patients, a trend for better survival for patients with cathepsin-positive vimentin-negative tumors and worse survival for those with cathepsin-positive vimentin-positive tumors was noted. Due to the low number of patients in these subgroups, neither trend reached significance. Cathepsin D expression was independent of patient age, size, and histologic grade of tumor, and vimentin expression. However, in the node-positive group, negative correlation of cathepsin D and vimentin expression was found. We suggest that prognostic significance of cathepsin D in infiltrative ductal NOS breast carcinomas may be associated with the pathway of its synthesis rather than with its mere presence in tumor cells.


Characterization of hu-PBL-SCID mice with high human immunoglobulin serum levels and graft-versus-host disease. A chimeric model consisting of severe combined immune deficiency (SCID) mice populated with human peripheral blood leukocytes (PBL) has recently been described (bu-PBL-SCID mice). These reports indicated a limited reconstruction of the transferred human immune system and functionality of the human graft. Herein we described modifications of the PBL transfer method that minimize transfer time and cellular manipulations, leading to a more effective population of SCID mouse recipients. Severe combined immune deficiency mice given 15 x 10(6) PBL had human IgG serum levels reaching 2 to 5 g/l, and all mice had detectable human anti-tetanus toxoid antibody levels when they received cells from donors with such levels. These transfers were associated also with clinical and histologic evidence of graft-versus-host disease, suggesting responsiveness of the human graft in the recipients. When Epstein-Barr virus seropositive (EBV+) donors were used, the chimeric mice also showed a high incidence of fatal lymphoproliferative disease 1 to 3 months after transfer of 15 x 10(6) PBL. The high level of immunoglobulin synthesis and immunoresponsiveness of the human cells with this transfer procedure may expand the use of these chimeric mice for the manipulations of human immune cells in vivo.


Human papillomavirus genomes in male urethral cells. Sixty-four samples of urethral cells from male sexual partners of women with genital human papillomavirus (HPV) infection were analyzed for the presence of HPV types 6, 11, 16, and 18 by polymerase chain reaction (PCR) followed by slot blot hybridization. Additional samples from 37 of these subjects were analyzed for the presence of viral cytopathic effects by conventional cytology. By PCR, HPV DNA was detected in 21% (14/64) of samples. By cytology, 16% (6/37) of the samples showed cellular changes consistent with HPV infection. Polymerase chain reaction and cytology results were concordant for presence and absence of HPV in 5 and 28 cases, respectively. Three additional HPV-positive cases were obtained with PCR in the cytologically negative samples. The cytologic abnormalities were found to be associated with the presence of both low-risk HPV types and meatal acetoreactivity. On the contrary, HPV DNA positivity by PCR was unrelated to viral type and peniscopic findings. Urethral HPV infection was detected by PCR in 30% of males with visible penile lesions and in 18% of those without. These results indicate that PCR analysis of urethral samples is a helpful adjunct to cytology for the detection of HPV DNA in absence of cytologic evidence of infection.


Expression of the L2 and E7 genes of the human papillomavirus type 16 in female genital dysplasias. The expression of the E7 and L2 genes of HPV 16 was studied in benign and precancerous female genital lesions to evaluate their role in the development of dysplasias. Ninety biopsy specimens from 70 patients, selected on basis of dot blot DNA hybridization, were included in immunohistochemical and in situ hybridization analyses. In the HPV 16 DNA positive cases, L2 mRNA and E7 mRNA were detected in biopsies from 24 and 21 patients, respectively. L2 mRNA was found in eight of 16 cases of condyloma and mild dysplasia, and in 13 of 14 cases of moderate to severe dysplasia. The figures for E7 mRNA were 6/16 and 13/14, respectively. We found L2 mRNA in four of 12 normal or condylomatous specimens and E7 mRNA in only one of these. The detection rates for L2 and E7 mRNAs increased along with the severity of the lesions (P = 0.0064 and P = 0.0001, respectively). The L2 protein was found in one condyloma and in 12 dysplasias, eight of which were moderate or severe. The L2-antibody-reactive cells were localized in superficial layers of the epithelium. The detection rate for L2 mRNA and especially for E7 mRNA increased along with the histopathologic grade of the lesion.


Localization of the human cytomegalovirus 2.7-kb major early beta-gene transcripts by RNA in situ hybridization in permissive and nonpermissive infections. During the early phase of a human cytomegalovirus (HCMV) infection, the 2.7-kb early gene is by far the most abundantly transcribed RNA. Using strand-specific 32P or digoxigenin-labeled riboprobes derived from a subgenomic fragment of the HCMV Towne 2.7-kb early gene, we have performed Northern blot analysis and RNA in situ hybridization on human and mouse fibroblasts infected with HCMV and on 23 formalin-fixed paraffin-embedded sections of tissue obtained at autopsy. By Northern blot analysis, expression of the 2.7 kb early gene was found only in permissive infections. In contrast, specific hybridization was detected in both permissive and nonpermissive cells by RNA in situ hybridization. In nonpermissive cells, hybridization was weak and predominantly nuclear. In permissive cells, strong nuclear and cytoplasmic hybridization was noted. Specific hybridization to cells with and without cytopathic changes was detected with the anti-sense probe in CMV infected tissue obtained at autopsy. When the sense riboprobe was employed, no specific hybridization was found under nondenaturing conditions. These results suggest that in situ hybridization with a probe directed at the 2.7-kb early gene is an effective method of detecting both permissive and nonpermissive HCMV infections in different stages of infection and in localizing the expression of the major early gene in various cell types.


Rat kidney Na-K pumps incorporated into low-K+ sheep red blood cell membranes are stimulated by anti-Lp antibody. A genetic dimorphism of sheep red blood cells characterized by differences in the intracellular K+ concentration of mature red blood cells (low-K+ or high-K+ cells) reflects differences in their Na-K pumps and is known to be linked to the ML blood group system. We investigated the relationship of Na-K pumps in red blood cells from sheep of the low-K+ phenotype with an antigen, Lp, that is restricted to low-K+ cells. Anti-Lp antibody stimulates the Na-K pumps in these cells presumably by relieving inhibition of the pumps by Lp. The questions addressed were as follows: is Lp a molecular entity distinct from pumps and, if so, can it interact with pumps of exogenous origin? Rat kidney Na-K pumps were incorporated by fusion of microsomes into either low-K+ or high-K+ sheep red blood cells. The activity of the exogenous kidney pumps was distinguished from that of the endogenous red blood cell pumps by the low sensitivity of rodent pumps to ouabain. Anti-Lp stimulated by > 50% rat kidney pumps incorporated into immature low-K+ sheep cells. This indicates that Lp is a distinct molecular entity free to dissociate from endogenous pumps and inhibit exogenous pumps. Anti-Lp did not stimulate kidney pumps incorporated into mature low-K+ cells but did stimulate kidney pumps following in vitro maturation of microsome fused reticulocytes, probably reflecting restriction of lateral movement of pumps and antigens by the cytoskeleton in mature cells.


Beta-adrenergic inhibition of Na-K-Cl cotransport in lymphocytes. Lymphocytes contain a homogeneous population of beta 2-adrenoceptors. However, their physiological role in the regulation of lymphocyte function, especially early in the activation/proliferation process, remains unclear. To study the role of beta-adrenergic activation on the regulation of membrane transport, we examined 86Rb uptake in the Jurkat human lymphoma cell line. 86Rb uptake was predominantly accounted for by bumetanide-sensitive uptake (74 +/- 2% total 86Rb uptake) and ouabain-sensitive uptake (20 +/- 2%). Bumetanide potently inhibited 86Rb uptake (50% inhibitory concentration = 210 +/- 40 nM), and bumetanide-sensitive uptake was dependent on extracellular sodium and chloride, consistent with uptake via Na-K-Cl cotransport. The beta-adrenergic agonist isoproterenol (10 microM) mediated a 50 +/- 11% reduction in bumetanide-sensitive uptake without a significant alteration in ouabain-sensitive uptake. The effect of isoproterenol was potent (50% effective concentration = 4.42 +/- 1.70 nM), stereoselective, and was inhibited by the beta-adrenergic antagonist nadolol. The effect of isoproterenol was mimicked by permeant adenosine 3',5'-cyclic monophosphate analogues but not by pindolol. These data indicate that beta-adrenoceptor activation decreases 86Rb uptake in lymphocytes via inhibition of Na-K-Cl cotransport. Modulation of this transporter could be one mechanism by which beta-adrenergic agonists regulate lymphocyte function.


Calcium signaling in endothelia: cellular heterogeneity and receptor internalization. The vasoactive factors thrombin, bradykinin (BK), and ATP are released in response to tissue damage and inflammation and act on endothelium to modulate vascular perfusion. We have investigated the second messenger response of endothelium activated by these agonists and, in particular, the mechanism of desensitization to BK. Fura-2 fluorescence ratio imaging of calf pulmonary artery endothelial cells (CPAE) revealed 5- to 10-fold increases on intracellular Ca (Cai) in response to these agents. Maximal doses caused Cai to increase from 52 to 248 nM (thrombin), 556 nM (BK), and 643 nM (ATP). Agonists elicited a rapid (within 30 s) increase of Cai due to release of Ca from intracellular stores followed by a secondary elevation of Cai dependent on entry of external Ca. The temporal characteristics of the Cai responses to all agonists were heterogeneous from cell to cell, and, interestingly, repeated stimulation gave identical signature responses from individual cells, although the amplitude of the Cai response decreased to thrombin and especially bradykinin but not for ATP. This decrease was agonist specific because ATP elicited large increases of Cai after thrombin or BK desensitization. Maximal desensitization was obtained with BK applied for 5-10 min followed by a rest of < 10 min before restimulation. Although desensitization primarily reduced the elevation of Cai due to the release of the internal store, entry of extracellular Ca was also reduced. Cells responded heterogeneously to desensitization in that those with prominent extracellular Ca entry responded most strongly upon a second stimulation with BK. Because desensitized cells still responded to ATP with an increase of Cai, the desensitization was controlled at a step prior to the activation of phospholipase C. Desensitization occurred by a reduction of BK receptor number; a 10-min BK pretreatment reduced [3H]BK binding to receptors by 70% (from 14,600 receptors/cell, Km = 5 nM, to 5,300). As surface receptor numbers decreased, internalized receptors increased as assayed by an acetic acid wash. The time course of the receptor internalization was similar to the decrease in Cai response to BK. We conclude that the vasoactive agonists thrombin, BK, and ATP increase the second messenger Cai in endothelial cells and that a desensitized Cai response occurs with BK, but not with ATP, due to downregulation and endocytosis of the BK receptor.


Extracellular nucleotides elevate [Ca2+]i in rat osteoblastic cells by interaction with two receptor subtypes. Extracellular nucleotides, through interaction with specific cell-surface receptors, mediate a variety of biological responses, including elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in a number of cell types. The effects of nucleotides on [Ca2+]i in the rat osteoblastic cell line UMR-106 were studied by fluorescence spectrophotometry of indo-1-loaded cells. In response to ATP (100 microM), [Ca2+]i rose to peaks 228 +/- 16 nM (n = 59) above baseline (85 +/- 3 nM) before returning to near basal levels. Half-maximal elevation of [Ca2+]i was observed at an ATP concentration of 3 +/- 1 microM, consistent with a high-affinity interaction. The response arose primarily by release of Ca2+ from internal stores. UTP, ADP, and 2-methylthioadenosine 5'-triphosphate also induced Ca2+ transients, whereas adenosine, AMP, CTP, and TTP did not, demonstrating specificity. Responsiveness to adenosine 5'-O-(3-thiotriphosphate) and inhibition by Mg2+ of the response to ATP indicated that signaling was not dependent on nucleotide hydrolysis. Ca2+ responses to ADP, ATP, and UTP, added sequentially or simultaneously, were consistent with the presence of two distinct P2-purinoceptor subtypes, both linked to Ca2+ mobilization. ADP appeared to interact selectively with one receptor, whereas ATP and UTP interacted selectively with the other. After maximal stimulation with ATP, subsequent responses to ATP were abolished. However, removal of ATP from the extracellular medium rapidly restored responsiveness, suggesting that, with continued receptor occupation, there is time-dependent inactivation of the Ca2+ signaling pathway. Our findings indicate that extracellular nucleotides elevate [Ca2+]i in osteoblastic cells through interaction with two receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)


Single-channel behavior of a purified epithelial Na+ channel subunit that binds amiloride. The apical membrane of high electrical resistance epithelia, which is selectively permeable to Na+, plays an essential role in the maintenance of salt balance. Na+ entry from the apical fluid into the cells is mediated by amiloride-blockable Na(+)-specific channels. The channel protein, purified from both amphibian and mammalian sources, is composed of several subunits, only one of which the 150-kDa polypeptide, specifically binds the Na+ transport inhibitor amiloride. The goal of the present study was to investigate whether the isolated amiloride-binding subunit of the channel could conduct Na+. The patch-clamp technique was used to study the 150-kDa polypeptide incorporated into a lipid bilayer formed on the tip of a glass pipette. Unitary conductance jumps averaged 4.8 pS at 100 mM Na2HPO4. Open times ranged from 24 ms to several seconds. The channel spent most of the time in the closed state. Channel conductance and gating were independent of voltage between -60 and +100 mV. Amiloride (0.1 microM) decreased the mean open time of the channel by 98%. We conclude that the 150-kDa subunit of the amiloride-blockable Na+ channel conducts current and may be sufficient for the Na+ transport function of the whole channel.


Ca2+ mobilization by extracellular ATP in rat cardiac myocytes: regulation by protein kinase C and A. Activation of protein kinase C (PKC) modulates the mobilization of intracellular Ca2+ induced by extracellular ATP in rat ventricular myocytes. Pretreatment of myocytes with PKC activators attenuated both the ATP-induced Ca2+ transient and the noradrenergic potentiation of the Ca2+ response. Various PKC activators decreased both the basal cAMP level and the cAMP levels that had been elevated by norepinephrine, forskolin, or 3-isobutyl-1-methylxanthine. The inhibitory effects of PKC activators were reversed by the PKC inhibitor staurosporine. The ATP-induced Ca2+ response is an integrated response resulting from ATP eliciting an inward cation current (IATP), cellular depolarization, Ca2+ influx through Ca2+ channels, and Ca2+ release from the sarcoplasmic reticulum. We used the whole cell voltage-clamp technique to investigate which steps of this integrated response are affected by PKC. PKC activators did not significantly affect the IATP. In contrast, PKC activators decreased the basal Ca2+ current (ICa) or Ba2+ current and the beta-adrenergic-stimulated ICa. These results suggest that PKC-induced suppression of the ATP-induced Ca2+ response and the beta-adrenergic-potentiated Ca2+ response is achieved at least partially by decreasing the intracellular cAMP level and ICa.


Acute variations in extracellular pH modulate transduction pathways of PTH in rat proximal tubule. An increase in circulating parathyroid hormone (PTH) has been shown to enhance the capacity for the kidney to excrete an acid as well an alkaline load, which suggests that changes in systemic acid-base status may modulate the effect of the hormone on bicarbonate absorption in proximal tubule. In the present study, we tested the possibility that acute variations in extracellular pH (pHe), obtained by modifying bicarbonate concentration at constant PCO2 (40 mmHg), may modulate the responses of intracellular messengers coupled to PTH receptors in a preparation of freshly isolated proximal tubule fragments. Variations in pHe, which induced parallel variations in intracellular pH (pHi), did not affect unstimulated values for adenosine 3',5'-cyclic monophosphate (cAMP) production, inositol trisphosphate accumulation, or cytosolic free Ca2+ concentration. In contrast, reducing pHe from 7.4 to 7.2 elicited a decrease of the PTH-induced cAMP production, whereas increasing pHe from 7.4 to 7.6 enhanced it. The ability for cholera toxin and forskolin (which both bypass PTH receptors) to stimulate cAMP formation was diminished at pHe 7.2 and enhanced at pHe 7.6 (the increase did not achieve statistical significance in the presence of forskolin), suggesting that variations in pHe and/or pHi may affect per se adenylyl cyclase activity. Conversely, reducing pHe from 7.4 to 7.2 enhanced the PTH-induced inositol trisphosphate accumulation and rise in cytosolic free Ca2+ whereas increasing pHe from 7.4 to 7.6 had opposite effects.(ABSTRACT TRUNCATED AT 250 WORDS)


Single calcium channel currents of arterial smooth muscle at physiological calcium concentrations. Entry of Ca through voltage-dependent Ca channels is an important regulator of the function of smooth muscle, cardiac muscle, and neurons. Although Ca channels have been extensively studied since the first descriptions of Ca action potentials (P. Fatt and B. Katz. J. Physiol. Lond. 120: 171-204, 1953), the permeation rate of Ca through single Ca channels has not been measured directly under physiological conditions. Instead, single Ca channels have typically been examined using high concentrations (80-110 mM) of another divalent charge carrier, Ba, so as to maximize the amplitude of the single-channel currents. Calculations of unitary currents at 2 mM Ca indicated that the single-channel currents would be immeasurably small (i.e., < 0.1 pA). We provide here the first direct measurements of single Ca channel currents at a physiological Ca concentration. Contrary to earlier estimates, we have found that currents through single Ca channels in arterial smooth muscle are 0.1-0.3 pA at 2 mM Ca and physiological membrane potentials. These relatively large unitary currents permit direct measurement of Ca channel properties under conditions that do not distort their function. Our data also indicate that Ca permeates these channels at relatively high rates in physiological Ca concentrations and membrane potentials.


Role of angiotensin II and alpha-adrenergic receptors during estrogen-induced vasodilation in ewes. Estradiol-17 beta (E2 beta) produces uterine and systemic vasodilation in nonpregnant ewes without altering mean arterial pressure (MAP). Mechanisms responsible for maintaining MAP and thus uterine blood flow (UBF) may include activation of the renin-angiotensin and/or adrenergic systems. We therefore investigated the effects of systemic blockade of angiotensin II (ANG II) and/or alpha-adrenergic receptors in nonpregnant, castrated ewes, using saralasin (Sar) and/or phentolamine (Phen) in the presence or absence of intravenous E2 beta (1.0 microgram/kg). In nonestrogenized ewes neither antagonist alone had substantial cardiovascular effects; however, Sar + Phen decreased systemic vascular resistance (SVR) 20 +/- 7.4% (SE) and increased heart rate (HR) 50 +/- 19% (P < 0.01); MAP and UBF were unaffected. Following E2 beta treatment SVR fell 17 +/- 2.4% (P < 0.01), UBF increased more than fourfold, and MAP was unchanged. Compared with E2 beta alone, Phen + E2 beta decreased SVR 42 +/- 4.7%, and MAP fell 11 +/- 1.8% (P < 0.05) despite 40-50% increases in HR and cardiac output (P < 0.05). Responses to Sar + E2 beta were similar to E2 beta alone, except for a fall in MAP, whereas responses to Sar + Phen + E2 beta resembled those of Phen + E2 beta. E2 beta-induced uterine vasodilation was unaltered by Sar and/or Phen. During E2 beta-induced vasodilation, MAP is maintained by enhanced activation of the alpha-adrenergic and renin-angiotensin systems; however, uterine vascular responses to E2 beta are independent of both systems and perfusion pressure.


Complex effects of arachidonic acid and its lipoxygenase products on cytosolic calcium in GH3 cells. The mechanisms by which arachidonic acid (AA) and its metabolites stimulate prolactin release from pituitary cells are not understood. Because Ca2+ ions are pivotal to exocytosis, we investigated the effects of AA metabolites on intracellular calcium concentration ([Ca2+]i) and membrane ionic currents using dual-excitation microspectrofluorimetry and whole cell patch-clamp techniques in GH3/B6 pituitary line cells. AA (1 microM) had a biphasic effect on [Ca2+]i, mobilization of intracellular Ca2+, followed by stimulation of Ca2+ entry. Only the latter appeared to result from the degradation of AA through the lipoxygenase pathway. Indomethacin (Indo, 10 microM) and lipoxygenase products 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE, 1 microM), increased action potential duration (12-HETE) or frequency (5- and 15-HETE, Indo). These effects depended on inhibition of d-tubocurarine- or tetraethylammonium-sensitive K+ conductances and stimulation of voltage-dependent Ca2+ channels. Refilling of intracellular Ca2+ stores, and Ca2+ efflux, may also be stimulated. Our results demonstrate a control of [Ca2+]i by a second messenger (AA) and its metabolites (HETEs).


Oxoanions stimulate in vitro ovulation and signal transduction pathways in goldfish (Carassius auratus) follicles. The present study investigated the effects of a number of oxoanion compounds on in vitro ovulation of goldfish follicles and ovarian second messenger activities. Significant levels of ovulation were induced by 0.1 mM sodium chromate, 0.1 mM sodium metavanadate, 10 mM sodium molybdate, 0.1 mM sodium orthovanadate, 5 mM sodium selenate, 0.5 mM sodium tungstate, and 0.1 mM vanadyl sulfate. At levels that significantly stimulated ovulation, metavanadate, molybdate, orthovanadate, tungstate, and vanadyl sulfate also stimulated follicular phosphatidylinositol cycling and inhibited ovarian alkaline phosphatase activity. Moreover, the ovulation induced by these oxoanions was not inhibited by indomethacin (10 micrograms/ml), while ovulation induced by selenate and chromate was. In contrast, only vanadium-containing compounds significantly stimulated prostaglandin (PG) synthesis, and, in fact, selenate significantly inhibited PG production. Finally, only sodium molybdate- and vanadium-containing compounds appeared to increase follicular adenosine 3',5'-cyclic monophosphate content. While all oxoanions stimulated in vitro ovulation, they had differential effects on certain signal transduction pathways when tested at concentrations that stimulated in vitro ovulation. From the results, two basic groups could be delineated, one containing tungstate-, molybdate-, and vanadium-containing compounds and the other selenate and chromate. Thus the mechanism by which ovulation is induced by chromate and selenate may be different from that of vanadium-containing compounds, molybdate, and tungstate.(ABSTRACT TRUNCATED AT 250 WORDS)


Connexins and glucagon receptors during development of rat hepatic acinus. Levels (by immunoblotting) and cell distribution (by immunofluorescence) of connexins (Cxs) and glucagon receptors (GRs) were studied during the ontogeny of the rat liver. Cx32, the main rat liver gap junction protein, was present in fetal hepatocytes, and its abundance increased between the 19th and 21st gestational days. The major increase in Cx32 levels occurred between the 7th and 21st postnatal days, after which adult levels were reached. The adult pattern of distribution of Cx26 and Cx32 was established by the 21st postnatal day. Cx43 was present in the liver capsule and perisinusoidal cells, and its levels did not show significant variations throughout development. Levels of GRs were much lower in fetal than in adult livers. After birth, GRs increased progressively, reaching adult levels at about the 28th postnatal day. In adults, GR immunoreactivity on the plasma membrane of hepatocytes was localized to the region facing the sinusoid and showed a density gradient of distribution along the hemiacinus: high in pericentral regions and decreasing toward more glucogenically active cells located in periportal regions. Because cells of the hepatic acinus communicate via gap junctions, which are permeable to second messengers, we propose that signals transduced by GRs in cells expressing high levels of receptor might spread to cells expressing low levels of receptor, thus maximizing metabolic activation.


Role of surface complexed iron in oxidant generation and lung inflammation induced by silicates. Inhalation of silicates induces a variety of lung diseases in humans. The molecular mechanism(s) by which these dusts cause disease is not known. Because several naturally occurring mineral oxides have large amounts of transition metal ions on their surfaces, we tested the hypothesis that surface complexation of iron may be an important determinant of their ability to induce disease. Silica, crocidolite, kaolinite, and talc complexed considerable concentrations of Fe3+ onto their surfaces from both in vitro and in vivo sources. The potential biological importance of iron complexation was assessed by examining the relationship between surface [Fe3+] and the ability of silicates to mediate oxidative degradation of deoxyribose in vitro, induce a respiratory burst and elicit leukotriene B4 (LTB4) release by alveolar macrophages (AM) in vitro, and cause acute alveolitis after intratracheal insufflation. For these studies, three varieties of silicate dusts were used: iron-loaded, wetted (unmodified), and deferoxamine-treated to remove Fe3+. The ability of silicates to catalyze oxidant generation in an ascorbate/H2O2 system in vitro, to trigger respiratory burst activity and LTB4 release by AM, and to induce acute lung inflammation in the rat all increased with surface complexed Fe3+. The results of these studies suggest that surface complexation of iron may be an important determinant in the pathogenesis of disease after silicate exposure.


Insulin inhibits beta-adrenergic responses in fetal rabbit lung in explant culture. Infants of diabetic mothers are at increased risk of a number of problems at birth. Among these problems are increased risks of respiratory distress syndrome and transient tachypnea of the newborn. Because surfactant synthesis, surfactant secretion, and lung fluid resorption are all mediated in part by beta-adrenergic responses, we asked if excess insulin interferes with the beta-adrenergic response cascade in fetal lung. Lungs from fetal rabbits (26 day) were grown in explant culture in hormone-supplemented culture medium. The explants were harvested after 48 h exposure to hormones and processed for determination of beta-adrenergic receptor concentration, guanine nucleotide regulatory proteins (Gs, Gi), beta-agonist stimulated adenosine 3',5'-cyclic monophosphate (cAMP) generation, cAMP-dependent phosphodiesterase activity, and choline incorporation into phosphatidylcholine. Although insulin did not change the concentration of beta-adrenergic receptors, it decreased the ability of isoproterenol to stimulate cAMP generation. Increase in stimulation over basal was similar in explants treated with dexamethasone and dexamethasone plus insulin, but absolute levels of isoproterenol-stimulated cAMP were less in the explants treated with dexamethasone plus insulin. We speculate that insulin inhibition of cAMP generation may be important in the pathogenesis of the respiratory problems of infants of diabetic mothers.


Thrombin receptor 14-amino acid peptide binds to endothelial cells and stimulates calcium transients. We examined the binding characteristics of the recently described thrombin receptor amino-terminal peptide, SFLLRNPNDKYEPF (T. K. H. Vu, D. T. Hung, V. I. Wheaton, and S. R. Coughlin. Cell 64: 1057-1068, 1991), termed TRP-14, and its effect in activating intracellular calcium transients in pulmonary vascular endothelial cells. Binding of 125I-labeled TRP-14 was found to be saturable with a affinity constant of 2 microM and maximum binding of 41 pmol/mg of cell protein. The 125I-labeled TRP-14 also interacted with bovine pulmonary microvessel endothelial cells, human umbilical vein endothelial cells, and porcine pulmonary artery smooth muscle cells. Binding of 125I-labeled diisopropylphosphoryl (DIP)-alpha-thrombin, which is catalytically inactive but binds to thrombin receptors, was not inhibited by TRP-14 or vice versa, indicating that TRP-14 did not compete for the alpha-thrombin binding site(s) on the endothelial cell surface. TRP-14 (> 1 microM) increased the concentration of intracellular calcium ([Ca2+]i) in endothelial cells with kinetics similar to the increase in [Ca2+]i triggered by alpha-thrombin. In contrast, DIP-alpha-thrombin did not increase [Ca2+]i and also did not prevent the rise in [Ca2+]i induced by the subsequent challenge with either TRP-14 or alpha-thrombin. Because the generation of TRP-14 by the proteolytically active forms of thrombin stimulated a rise in endothelial [Ca2+]i, TRP-14 may be the agonist responsible for the activation of the alpha-thrombin receptor in pulmonary vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Mechanism of PGE2-induced cell swelling in distal nephron segments. The effects of prostaglandin (PG) E2 on cell swelling were studied in isolated perfused tubules of rabbit kidney. PGE2 (1 microM) added to the bath induced cell swelling by 13.4, 7.2, and 9.6% in the connecting tubule, distal convoluted tubule, and cortical collecting duct, respectively, but it had no effect on the proximal convoluted tubule and cortical thick ascending limb. The response was dose dependent in the range of 1 nM to 1 microM. PGI2 exerted a similar effect, but PGF2 alpha had no effect. The swelling was completely blocked by basolateral Na+ removal and was attenuated by bilateral Cl- removal, suggesting that the swelling was mediated by basolateral Na+ entry in association with Cl- entry. In all segments except proximal tubule, PGE2 caused an initial transient peak followed by a sustained increase in intracellular Ca2+. Intracellular Ca2+ chelation or inhibition of Ca2+ release from intracellular stores abolished the PGE2-induced cell swelling, but extracellular Ca2+ removal did not. An inhibitor of the Na(+)-Ca2+ exchanger (3',4'-dichlorobenzamil, 100 microM) in the bath completely inhibited PGE2-induced cell swelling. Neither furosemide (1 mM) nor amiloride (1 mM) added to bath abolished the response, indicating that neither Na(+)-K(+)-2Cl- cotransport nor Na(+)-H+ exchange is involved in the action of PGE2. The swelling response to PGE2 was observed even in the presence of ouabain, indicating that the effect of PGE2 is independent of Na(+)-K(+)-adenosinetriphosphatase inhibition. Nicardipine added to bath partially inhibited the swelling response.(ABSTRACT TRUNCATED AT 250 WORDS)


Rubidium absorption and proton secretion by rabbit outer medullary collecting duct via H-K-ATPase. Multiple lines of evidence support the hypothesis that the outer medullary collecting duct from the inner stripe (OMCDi) possesses a functional proton-potassium-activated adenosinetriphosphatase (H-K-ATPase). To examine the effect of inhibition of H-K-ATPase on Rb efflux, we measured the Rb tracer rate efflux coefficient (KRb) across the OMCDi of animals adapted to a K-restricted diet using the selective K-competitive H-K-ATPase inhibitor, Sch 28080. Sch 28080 (10 microM) did not significantly alter transepithelial voltage (VT) but significantly decreased KRb by 41%. We further examined the effect of 10% peritubular CO2 on KRb and the subsequent effect of Sch 28080 (10 microM) on KRb. After exposure to 10% CO2 for 120 min, vehicle-treated tubules exhibited a small but significant increase in KRb without a significant change in VT. In contrast, 10 microM Sch 28080 significantly decreased KRb by 44% without affecting VT. The lack of an effect of H-K-ATPase inhibition on VT in the presence of either 5% or 10% CO2 was in marked contrast to the effect of carbonic anhydrase inhibition (CAI). CAI consistently and significantly decreased VT either in the presence of 5% or 10% CO2. To address whether H-K-ATPase also participates in proton secretion we examined the effect of Sch 28080 (10 microM) on net bicarbonate absorption by the OMCDi of rabbits fed a normal rabbit ration and rabbits adapted to a K-restricted diet.(ABSTRACT TRUNCATED AT 250 WORDS)


Enalaprilat handling by the kidney: barrier-limited cell entry. The angiotensin-converting enzyme inhibitor enalaprilat is formed in vivo in liver and kidney by esterolysis of the antihypertensive drug enalapril. To gain insight into the renal elimination of enalaprilat, we carried out multiple-indicator dilution experiments in the isolated perfused rat kidney. Kidneys were perfused single pass with an amino acid-supplemented Krebs-Henseleit buffer containing 20% bovine red blood cells and 4% bovine serum albumin, at a flow rate of 0.11 +/- 0.02 (SD) ml.s-1 x g-1. A bolus of 51Cr-labeled red blood cells (vascular red blood cell indicator), 125I-labeled albumin (vascular plasma indicator), L-[14C]glucose (interstitial space indicator), and [3H]-enalaprilat was injected into the renal artery, and timed samples of venous blood (up to 1 min) and urine (up to 10 min) were collected. The data were analyzed using a variable-transit-time, space-distributed model with modifications accounting for glomerular filtration and the observed 14% protein binding of enalaprilat; the glomerular filtration rate (GFR) estimated from L-glucose clearance was 9.0 +/- 2.9% of total plasma flow. The ratio of renal clearance of unbound enalaprilat to GFR was 1.56 +/- 0.29, indicating both glomerular filtration and net tubular secretion of enalaprilat. Unidirectional influx from plasma to tubular cells exceeded tubular secretion by a factor of 2.2 +/- 0.5. Thus only about one-half of the enalaprilat taken up by the tubular cells was excreted into urine, with the remainder refluxing into the capillary blood stream, indicating bidirectional permeation of enalaprilat across the basolateral tubular membrane.


EDRF-angiotensin II interactions in rat juxtamedullary afferent and efferent arterioles. The in vitro blood-perfused juxtamedullary nephron technique was utilized to determine the contribution of endothelium-derived relaxing factor (EDRF) to resting renal arteriolar caliber and to evaluate the interaction between EDRF and angiotensin II (ANG II) in renal microvascular control. Video microscopy was employed to visualize rat afferent and efferent arterioles and to measure their responses to blockade of nitric oxide (NO), which has been shown to account for much of the biological action of EDRF. The NO synthesis inhibitor, N omega-nitro-L-arginine (L-NNA), elicited vasoconstriction in a concentration-dependent manner, with 1,000 microM L-NNA significantly reducing both afferent (16 +/- 3%) and efferent (13 +/- 1%) diameters. This concentration of L-NNA also blocked the vasodilator response to 10 microM acetylcholine, while responsiveness to sodium nitroprusside was maintained. Vasoconstrictor responses to 1,000 microM L-NNA were attenuated in kidneys from rats pretreated with enalaprilat or losartan, reducing afferent diameter by 7 +/- 1 (n = 8) and 3 +/- 1% (n = 10) of control, respectively. Efferent arteriolar responses to L-NNA were similarly attenuated by losartan. The constrictor response to 10 nM ANG II was not exaggerated by L-NNA, suggesting that ANG II does not stimulate EDRF synthesis. These observations indicate that EDRF is continuously released in a quantity sufficient to affect both afferent and efferent arterioles of juxtamedullary nephrons in vitro. Furthermore, ANG II blockade attenuates the vasoconstriction elicited by L-NNA, suggesting that EDRF interacts with the renin-angiotensin system to control juxtamedullary afferent and efferent arteriolar resistance.


Effects of angiotensin II on proximal tubular cells stably transfected with the c-mas oncogene. Angiotensin II (ANG II) normally induces cellular hypertrophy in proximal tubular epithelium by engaging receptor systems that use a G-protein-signaling mechanism. The c-mas oncogene also encodes part of a superfamily of vasoactive peptide receptor-like moieties that couple to G proteins. To determine whether the stable expression of the c-mas gene might alter or modify the induction of cellular hypertrophy by ANG II in tubular epithelium, a rat c-mas cDNA was cloned into the pSV2 expression vector for use in cell transfection. Scatchard analysis of ANG II binding revealed no significant differences in ANG II receptor number or in the dissociation constant between pSV2mas-transfected or wild-type MCT cells, but rather an increase in the number of receptors not replaceable by known inhibitors. ANG II also induced proliferation in pSV2mas-transfected MCT cells that was not blocked by conventional inhibitors and increased intracellular levels of inositol trisphosphate. ANG II, furthermore, did not increase de novo protein synthesis in pSV2-transfected MCT cells and failed to lower their intracellular concentration of adenosine 3',5'-cyclic monophosphate, both expected parameters of cellular hypertrophy. Our findings demonstrate that expression of c-mas in tubular epithelium can modulate tubular cell phenotype toward proliferation rather than hypertrophy. This effect is likely mediated by a reshuffling of the heterogeneity of ANG II receptors on the cell surface, or perhaps by the emergence of a new ANG II receptor, followed by alterations in the process of signal transduction.