Anti-myeloperoxidase autoantibodies in patients with necrotizing glomerular and alveolar capillaritis. We conducted a prospective study of 651 Mediterranean patients from Catalonia (Spain) with well-defined forms of systemic vasculitis, connective tissue diseases, and renal and pulmonary disorders to determine the prevalence and clinical value of antineutrophil cytoplasmic autoantibodies (ANCA) with myeloperoxidase (MPO) specificity (MPO-ANCA). ANCA were first tested by indirect immunofluorescence on ethanol-fixed neutrophils. When a positive result was obtained, then MPO-ANCA were identified by performing the immunofluorescence assay again on neutrophils from a voluntary donor known to have a complete and selective deficiency of MPO. This disorder was detected by automated flow cytochemistry with the Technicon system and was further verified by cytochemical and biochemical studies. We detected MPO-ANCA in 61 of 70 (87%) patients with a perinuclear pattern (p-ANCA), but in none of 25 with a cytoplasmic pattern (c-ANCA). These results were corroborated by enzyme-linked immunosorbent assay (ELISA) using human purified MPO as a substrate. On immunofluorescence microscopy, all patients with MPO-ANCA were found to have a typical and restrictive immunostaining pattern. In our study, while c-ANCA were mainly found in patients with biopsy-proven Wegener's granulomatosis, MPO-ANCA identified those with idiopathic and polyarteritis nodosa-associated necrotizing and crescentic glomerulonephritis. In addition, pulmonary hemorrhage with necrotizing alveolar capillaritis as the main morphologic substrate occurred frequently among patients with MPO-ANCA, including three affected by polyarteritis nodosa and three who had pulmonary hemorrhage as the only clinical finding. On the other hand, these antibodies could be also detected in 30% of patients with a proven diagnosis of anti-glomerular basement membrane (GBM) disease.(ABSTRACT TRUNCATED AT 250 WORDS)


Specific genetic analysis of microscopic tissue after selective ultraviolet radiation fractionation and the polymerase chain reaction. A method using selective ultraviolet radiation fractionation followed by polymerase chain reaction (PCR) can analyze specific cell subsets present on a microscope section. Direct ultraviolet radiation of fixed and stained tissue sections prevents subsequent amplification by PCR. An "umbrella" or dot placed physically over small numbers of pure cell populations selected by microscopic examination protects these cells from the ultraviolet inactivation. The DNA in these protected cells can be specifically amplified while no signal is derived from the unprotected surrounding cells. Specific amplification was demonstrated by detecting human papillomavirus sequences only if infected cells were protected. Similarly, loss of heterozygosity at the p53 locus was documented by selective dotting of normal or tumor cells. The method allows the specific and sensitive molecular genetic analysis of small numbers of cells histologically identified and selected under the microscope.


Characterization of glomerular epithelial cell matrix receptors. Integrin matrix receptors on glomerular epithelial cells (GEC) may play an important role in adhesion of GEC to the glomerular basement membrane (GBM) and in the maintenance of normal glomerular permeability. Therefore, the author determined the types of matrix receptors present on cultured rat GEC and examined their interactions with several components of the extracellular matrix. Beta 1 integrin matrix receptors were detected on all three glomerular cell types in rat kidney in vivo and at areas of cell-cell contact on cultured GEC. Glomerular epithelial cell adhesion to types I and IV collagen was slightly greater than to laminin and fibronectin. Adhesion to fibronectin was significantly inhibited by a synthetic peptide containing the RGD adhesion sequence. Immunoprecipitation of lysates of surface-iodinated GEC showed the presence of alpha 3 beta 1 integrin. Chromatography of lysates on immobilized collagen showed alpha 3 beta 1 integrin and a 70- to 75-kd protein band as the collagen receptors on GEC. Chromatography on the 120-kd cell-binding fragment of fibronectin disclosed only alpha 3 beta 1 as a specific fibronectin receptor. Antibody to the beta 1 integrin chain inhibited adhesion to laminin and collagen. These studies demonstrate that in vitro, as in vivo, GEC appear to express only alpha 3 beta 1 integrin. Furthermore, this matrix receptor is capable of mediating GEC adhesion to collagen, fibronectin, and laminin, components of the GBM, and presumably plays a similar role in promoting GEC adhesion to GBM in vivo.


Transforming growth factor-beta and transforming growth factor beta-receptor expression in human meningioma cells. The transforming growth factor-beta (TGF beta) family in mammals includes three closely related peptides that influence proliferation and numerous physiologic processes in most mesenchymal cells. In this study, Northern blots, immunohistochemistry, TGF beta radioreceptor assays, TGF beta receptor affinity labeling and [3H] thymidine incorporation were used to evaluate whether primary cell cultures of human meningiomas synthesize the three TGF beta isoforms, bear TGF beta receptors, and respond to TGF beta. Transcripts for TGF beta 1 and 2 were detected in the three cases analyzed. Transforming growth factor-beta 1 immunoreactivity was detected in three of six cases, and TGF beta 2 and 3 immunoreactivity were detected in each case analyzed. Media conditioned by cells cultured from six meningiomas also contained latent TGF beta-like activity. Transforming growth factor-beta receptor cross-linking studies identified TGF beta binding sites corresponding to the type 1, type 2, and type 3 receptors on meningioma cells. Treatment with active TGF beta 1 produced a statistically significant reduction in [3H] thymidine incorporation after stimulation with 10% fetal calf serum and epidermal growth factor in all six cases studied.


Basic fibroblast growth factor in Dupuytren's contracture. Lesions excised from nine patients undergoing surgery for Dupuytren's contracture (DC) and three normal fascia were examined for the presence of the angiogenic protein basic fibroblast growth factor (basic FGF). Endothelial cell proliferation assays established basic FGF-like activity in extracts of DC. Western blotting confirmed the presence of an 18,000-dalton protein which was localized in the lesions by immunohistochemical staining. All of the cells implicated in the progression of the disease (endothelial cells, fibroblasts, and myofibroblasts) contain the growth factor. Endothelial cells within the narrowed or occluded vessels, as well as fibroblasts surrounding these vessels, stained intensely positive. In situ hybridization using an antisense probe for human basic FGF and its receptor's (FGFR-1) mRNA established the major difference between normal and DC tissues: their levels are significantly higher than in the normal tissues. Thus the cells in DC also express both basic FGF and FGFR-1, suggesting a potential autocrine/paracrine role for basic FGF in the pathogenesis of DC. This finding is thus the first description of a nontumoral proliferative disease that can be directly associated with increased basic FGF mRNA. The possibility that therapies can be developed on the basis that basic FGF and its receptor are expressed in DC is discussed.


Direct inhibitory mechanisms of halothane on canine tracheal smooth muscle contraction. Halothane directly relaxes airway smooth muscle. To determine the direct inhibitory mechanisms of halothane on canine tracheal smooth muscle contraction, the effects of this anesthetic on the levels of several intracellular second messengers were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i), Ca2+/phospholipid-dependent protein kinase (PKC) translocation, and intracellular cyclic adenosine monophosphate concentration ([cAMP]i). When carbachol (1 microM) was used to increase [Ca2+]i to the same concentration as that induced by high-K+ (72.7 mM), the carbachol-induced contraction was more than twice as great, indicating that carbachol enhances the sensitivity of contractile elements to Ca2+ or activates a Ca(2+)-independent mechanism. Similarly, 12-deoxyphorbol 13-isobutylate, a potent PKC activator, markedly potentiated high-K(+)-induced muscle contraction without an increase of [Ca2+]i. The addition of halothane (0.33, 0.75, 1.15, and 1.47 mM) decreased [Ca2+]i and the muscle tension induced by carbachol. However, the decrease of muscle tension was more marked than that of [Ca2+]i at the higher concentrations. Although [Ca2+]i in the presence of verapamil and carbachol was not affected by halothane, the anesthetic markedly decreased muscle force by decreasing the "Ca2+ sensitization" or the Ca(2+)-independent enhancement of tension observed with carbachol. Halothane (0.75 and 1.47 mM) significantly released the membrane-associated PKC to cytosol, which decreased PKC activity. [cAMP]i of the smooth muscle stimulated by carbachol was moderately but significantly increased by halothane. However, when equivalent relaxation was induced with forskolin, which acts via adenylate cyclase activation, a much higher [cAMP]i was observed, which suggests that halothane acts via an additional pathway.(ABSTRACT TRUNCATED AT 250 WORDS)


Pentoxifylline-mediated reduction of superoxide anion production by human spermatozoa. The release of superoxide anions from sperm suspensions prepared for in vitro fertilization was measured by superoxide dismutase-inhibitable reduction of ferricytochrome C in response to phorbol myristate acetate. The superoxide anion production was found to be within the range of 0.21-3.76 nmoles O2- per 10(6) spermatozoa. Spermatozoa pre-incubated in the presence of 3.7 or 10 mM of pentoxifylline for 30 min at 37 degrees C released less (P less than 0.001) superoxide anions than did the untreated-control spermatozoa. It has been concluded that the inhibitory effect of pentoxifylline on superoxide anion production may be a factor contributing to the improvement of sperm preparation before insemination, and may, thereby, increase the fertility potential.


Relationship between nonspecific bronchial responsiveness to methacholine and peripheral mononuclear leukocyte beta-adrenergic receptor function in young drug-naive subjects. Asthma is associated with dysfunction of the beta-adrenergic receptor adenylyl cyclase signal transduction pathway. It has been argued that this results from receptor down-regulation by beta-agonist therapy. This study examined the relationship between nonspecific bronchial responsiveness (NSBR) to methacholine (Newcastle dosimeter method) and beta-adrenergic receptor density (Bmax) and affinity (%KH) in membranes from peripheral blood mononuclear leukocytes (MNL) in 12 male (27.3 +/- 1.7 yr old) and 14 female (31.4 +/- 1.7 yr old) drug-naive subjects with and without symptoms of mild intermittent wheezing. None had ever smoked or received any antiasthma medication. "Hyperresponsive" subjects were defined as those (n = 11) whose simplified slope of FEF50 (calculated as the percent fall in FEF50 divided by the dose of methacholine) was more than one SD above the mean for asymptomatic subjects. The log of the slope was reproducible (repeatability coefficient = 0.43) on two nonconsecutive days. Multiple regression analysis (overall R2 = 0.57) revealed negative relationships between the log of the slope and both Bmax (p = 0.016) and %KH (p = 0.011). Analysis of variance confirmed a lower mean (+/- SEM) value of %KH in "hyperresponsives" (45.7 +/- 5.5%) than in "normoresponsives" (60.4 +/- 4.1%, p = 0.04) with a similar trend for Bmax (hyperresponsives = 33.5 +/- 4.1 fmol/mg, normoresponsives = 45.9 +/- 7.1 fmol/mg, p = 0.18). These relationships between bronchial responsiveness, Bmax, and %KH cannot be explained by drug therapy, and they provide further evidence that there is an intrinsic impairment in the function of beta-adrenergic receptors on peripheral MNLs from subjects with high levels of nonspecific bronchial responsiveness.


Effect of Sp-cAMP on sperm motility in patients with unexplained infertility. Sperm motility is one of the most important semen parameters affecting the fertility of an individual. Sp-cAMP, a thiophosphate analogue of c-AMP, is a phosphodiesterase inhibitor controlling the intracellular c-AMP levels which in turn influences sperm motility. The results in our laboratory on a group of suspected subfertile men and fertile donors showed significant enhancement of sperm motility after incubation of washed sperm with various doses of Sp-cAMP.


Prognostic significance of tumor proliferative fraction and DNA content in stage I non-small cell lung cancer. Analyses of tumor DNA content and proliferative fraction by flow cytometry have been useful as prognostic determinants in a variety of solid tumors. The significance of this analysis in Stage I (T1N0M0 and T2N0M0) non-small cell lung carcinoma (NSCC) is unestablished. We determined DNA content (ploidy) and proliferative fraction (percentage S phase) on 44 surgically resected Stage I NSCC specimens obtained between 1977 and 1982. All cases had a minimum follow-up of 5 yr. Of the 44 cases, 27 were adenocarcinomas, 15 squamous cell carcinomas, and 2 large cell carcinomas. Of these, 32 (73%) had T1N0M0 lesions and 12 (27%) had T2N0M0 lesions. Overall 5-yr survival was 70%. All patients surviving 5 yr were free of detectable tumor. Patients with T1N0M0 lesions had an 81% 5-yr survival, but those with T2N0M0 lesions had a 42% 5-yr survival (p = 0.009). Analysis of tumor DNA content revealed 35 diploid tumors (79%) and 9 aneuploid tumors (21%). The 5-yr survival for diploid tumors was 77% compared with a 44% 5-yr survival in aneuploid lesions (p = 0.048). The median proliferative fraction was 6%. All patients with a percentage S phase less than 6% survived 5 yr, and 41% (9 of 22) of those greater than 6% survived 5 yr (p less than 0.001). When 8% S phase was used as a cutoff, 93% (28 of 30) below the cutoff survived 5 yr but only 21% (3 of 14) above the cutoff survived 5 yr (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of smoked marijuana of varying potency on ventilatory drive and metabolic rate. Ventilatory responses to hypercapnia in experienced marijuana smokers have previously been shown to decrease, increase, or not change acutely after marijuana. In one study, minute ventilation (VE) and O2 consumption (VO2) increased but hypoxic ventilatory response did not change after smoking marijuana. We further investigated the effects of marijuana of increasing potency (0, 13, and 20 mg THC) on ventilatory and mouth occlusion pressure (P0.1) responses to hypercapnia and hypoxia in 11 young, healthy men who smoked marijuana regularly but refrained from any smoked substance, alcohol, caffeine, or other drugs for greater than or equal to 12 h before study. Ventilatory and P0.1 responses to hypoxia and hypercapnia were measured on 3 separate days before and 5 and 35 min (hypoxia) and 15 and 45 min (hypercapnia) after smoking. In a companion 3-day study, 12 young male habitual marijuana smokers underwent measurements of VE, VO2, and CO2 production (VCO2) before and 5 to 135 min after smoking marijuana containing 0, 15, or 27 mg THC. None of the active marijuana preparations caused significant changes in ventilatory or P0.1 responses to either hypercapnia or hypoxia or in resting VE, VO2 or VCO2. We conclude that smoking marijuana (13 to 27 mg THC) has no acute effect on central or peripheral ventilatory drive or metabolic rate in habitual marijuana smokers. These conclusions cannot be applied to infrequent users of marijuana without further study.


The Singapore Polyposis Registry.

The Singapore Polyposis Registry. The Singapore Polyposis Registry was established in 1989 in Singapore General Hospital. The aim is to provide a central registry service to all doctors in Singapore to facilitate in identification, surveillance and management of families and individuals at high risk of getting colorectal cancer from FAP (Familial Adenomatous Polyposis) and HNPCC (Hereditary Non-polyposis Colorectal Cancer). Both have an autosomal dominant inheritance that gives rise to colorectal cancer at any early age if untreated. They account for 5-6% of all colorectal cancers. Sixteen FAP families with 139 members have been evaluated. Fifty-eight members are affected and 81 are at risk or unaffected. Those who have been screened positive have a much lower risk of cancer (13%) compared with those who presented with the disease (89%) and death from colorectal cancer in the corresponding groups were nil and 58%. Eight HNPCC families with 36 affected and 170 at-risk members have been registered. Colonoscopic surveillance have just started: one case of Dukes' A cancer in a 26 year-old patient, and two cases with polyps have been diagnosed. Advances in molecular genetics and the identification of APC (Adenomatous Polyposis Coli) gene in the FAP locus of Chromosome 5 have made it possible to diagnose FAP genetically. This has important impact on management in terms of prenatal diagnosis and dietary and chemoprevention programmes in addition to surgical intervention.


Systematics of the Lactobacillus population on rat intestinal mucosa with special reference to Lactobacillus reuteri. The systematics of the Lactobacillus population of the intestines of 88 different rats was studied; 80 rats had been fed on fermented oat-meal soup (Molin et al. 1992). One-hundred-twenty-two Lactobacillus strains from the intestinal mucosa were phenotypically classified together with twenty-eight reference strains of Lactobacillus and Leuconostoc, using 49 unit characters. Data were examined using Jaccard coefficient, and unweighted pair group algorithm with arithmetic averages. Two major and eleven minor clusters were defined at the 76% SJ-similarity level: Cluster 1 included thirty isolates which could not be identified further, but had resemblance to the type strains of L. jensenii, L. gasseri, L. crispatus, and to some extent to L. acidophilus. Cluster 12 including fifty-four intestinal isolates was identified as L. reuteri; and so was cluster 13 (five isolates). Isolates of the major clusters were found in all parts of the intestines. The genomic homogeneity of the L. reuteri isolates was scrutinized by endonuclease restriction analysis of the chromosomal DNA, and the isolates could be divided into six genomic strains.


Isolation and characterization of respiration-deficient mutants from the pathogenic yeast Candida albicans. The isolation of several respiration deficient mutants of the pathogenic yeast Candida albicans is described. These show greatly reduced respiration rates, loss of cytochromes aa3 and b, and reduced growth rates. All of the mutants had lost the ability to assimilate a wide range of carbon sources. Ultrastructural studies showed reduced development of mitochondrial cristae in the mutants. The mutants can be divided into three classes depending on their respiration responses to the addition of cyanide.


[A case of hyperosmolar nonketotic coma occurring during chemotherapy in lung cancer]. A 61-year-old woman was admitted to our hospital because of hoarseness and abnormal shadow on chest X-ray. We diagnosed this patient as large cell carcinoma of the right upper lobe of the lung; T3N3M1 Stage IV. She was treated with OK-432, CDDP and CQ. On the 6th day after 2nd cycle chemotherapy, she was confused, and we diagnosed her as a case of hyperosmolar nonketotic coma (HNC) on the 7th day. Unfortunately, she died on the 8th day, after 20 hours of treatment for HNC. She suffered from chronic dehydration due to trouble with left recurrent nerve palsy. Although continuous intravenous hyperalimentation was used, she had severe HNC. HNC might be one complication in chemotherapy for patients with malignancy.


Microscopic polyarteritis. Report of a case with cutaneous involvement and antimyeloperoxidase antibodies. Microscopic polyarteritis is a systemic small-vessel vasculitis that primarily involves the kidneys but may also affect the skin and other organ systems. This unique vasculitis represents one of the vasculitides with antineutrophil cytoplasmic antibodies, usually of the perinuclear immunostaining pattern (P-ANCA, antimyeloperoxidase antibodies). The objective of this case report is to describe microscopic polyarteritis, the use of antineutrophil cytoplasmic antibodies, and the unique cutaneous histopathologic features of our patient. We describe a patient with clinical findings consistent with microscopic polyarteritis, the presence of antimyeloperoxidase antibodies, and a specific cutaneous leukocytoclastic vasculitis. This report further characterizes the histopathologic features by demonstrating that the anatomic location of the cutaneous vasculitic lesions is at the level of the dermal arteriolar vessels and postcapillary venules. Cutaneous arteriolar leukocytoclastic vasculitis may prove to be a histologic hallmark of microscopic polyarteritis when it presents in the skin. However, further case comparisons and histopathologic investigations are needed. The consequences of this systemic vasculitis, if not adequately treated, may be life threatening. Recognition of clinical features, use of antineutrophil cytoplasmic antibodies, and demonstration of a cutaneous arteriolar leukocytoclastic vasculitis may aid in the diagnosis and subsequent treatment of patients followed up by dermatologists for vasculitic ulcers.


JC virus associated meningoencephalitis in an immunocompetent girl. JC virus is most commonly acquired during childhood, and no clinical illness has been associated with primary infection, which is assumed to be asymptomatic. The only disease associated with JC virus to date is progressive multifocal leucoencephalopathy (PML), which is usually caused by viral reactivation in immunocompromised adults. A chronic meningoencephalitis associated with an active JC virus infection in an immunocompetent 13 year old girl is described.


Cytomegalovirus prevalence in pregnant women: the influence of parity. Over 20,000 women attending for antenatal care at three London hospitals were prospectively studied to determine the prevalence of cytomegalovirus (CMV) antibodies; 54.4% of these women were CMV seropositive. Ethnic group was strongly associated with CMV status: 45.9% of white women were seropositive, 88.2% of Asian, and 77.2% of black women (African/Caribbean ethnic origin). Among 12,159 white women born in the British Isles, seropositivity was independently associated with increasing parity, older age, lower social class, and being single at antenatal booking. The findings are consistent with the hypothesis that, in the UK, child to mother transmission of infection plays a significant part in the acquisition of CMV infection in adult life.


New aerosol delivery system for neonatal ventilator circuits. There is mounting evidence that a variety of drugs delivered as aerosols are likely to be of benefit in neonatal units. To avoid many of the problems associated with the use of jet nebulisers in ventilator circuits, a chamber was designed to be used in conjunction with a metered dose inhaler (MDI). The dimensions (4 cm x 11 cm) were chosen in an attempt to maximise drug delivery. In vitro studies were subsequently performed in order to determine the optimum operating conditions. Sodium cromoglycate delivered via this system was collected on a filter placed between the tip of an endotracheal tube and a model lung. The dose delivered was determined by means of an ultraviolet spectrophotometric assay. Using a Draeger Babylog 8000 ventilator it was found that drug delivery as maximised by actuating the device just before the inspiratory cycle when the chamber was placed adjacent to the endotracheal tube and by using a long (one second) inspiratory time. Under these conditions 1.5-2% of the original dose was deposited upon the filter at tidal volumes of 11-22 ml. When considered in terms of body weight this is many times the equivalent dose delivered to adults from an MDI. Effective drug delivery to the filter was confirmed using a radiolabelled aerosol. Radiolabelled studies delivering aerosol to the lungs of intubated rabbits demonstrated that deposition aerosol was distributed uniformly between lobes when corrected for the weight of each lobe. In conclusion, the device appears likely to deliver significant, reproducible quantities of drug to the lower respiratory tract while being simple to use.


Reversible mitochondrial myopathy with cytochrome c oxidase deficiency. Two siblings, a boy and a girl born in a nonconsanguineous marriage, presented with a similar clinical course. Sucking and breathing difficulties appeared within a few weeks of birth. Clinical examination revealed profound muscular hypotonia, hepatomegaly, increased serum creatine kinase activities, and lactic acidosis. Both infants were treated with gavage feeding, the boy also needing ventilatory support. Clinically they improved gradually. Now, the boy aged 4 years and the girl aged 28 months are free of clinical signs. Muscle biopsy specimens taken at 3 months showed, in both, ragged red fibres, abnormal mitochondria, and reduced cytochrome c oxidase (COX) staining. Biochemical analysis showed COX activity to be reduced to about 25% of the normal mean. The second biopsy specimen from the boy at 16 months was normal on morphological examination, but the girl's second specimen at 13 months still showed abnormal features. These cases are examples of the rare benign reversible COX deficiency. Early diagnosis is crucial to provide intensive treatment until spontaneous clinical improvement appears.


Effect of sequential and daily continuous hormone replacement therapy on indexes of mineral metabolism. Continuous regimens of estrogen-progesterone have recently been favored over sequential regimens because of a lower incidence of withdrawal bleeding. To determine whether the beneficial effects of sequential hormonal therapy on bone metabolism are preserved with the newer continuous regimens, we studied indexes of skeletal metabolism and changes in bone mineral density during a 1-year prospective trial. Our subjects were randomized to one of three treatment groups: those in group C-2.5 were treated with 0.625 mg of conjugated estrogen with 2.5 mg of micronized medroxyprogesterone acetate daily continuously; group C-5 received 0.625 mg of conjugated estrogen and 5.0 mg of micronized medroxyprogesterone acetate daily continuously; and group S-5 received 0.625 mg of conjugated estrogen on days 1 through 25 and 5 mg of micronized medroxyprogesterone acetate on days 14 through 25. At 1 year, all groups demonstrated a significant decrease in indexes of bone formation turnover, including decrements in alkaline phosphatase levels of 11% to 30% and in osteocalcin levels of 45% to 60%. Intact parathyroid hormone levels rose 10% to 20%, with a concomitant near-significant decrement in ionized calcium levels at 12 months. In addition, there were significant decrements in the 24-hour urinary calcium-creatinine ratios and hydroxyproline-creatinine ratios of 13% to 28%, measures of bone resorption. Linear regression analyses showed that the subjects with the high bone resorption achieved the greatest increment in bone mineral density in response to hormone therapy. The daily continuous estrogen-progesterone regimens are as efficacious as sequential hormonal therapy in decreasing indexes of bone turnover and stabilizing bone mineral density of the spine and proximal femur.


[Effects of a specific thromboxane A2 synthetase inhibitor on lymphocyte and neutrophil functions in adult intractable asthmatics]. To clarify whether thromboxane A2 (TXA2) is involved in type III and IV allergy, or so called "cell-mediated allergy", we studied the effect of a specific TXA2 synthetase inhibitor, sodium ozagrel (OKY-046) on peripheral blood mononuclear cells and neutrophils in adult intractable asthmatics. The results revealed, firstly, that lymphocyte blastogenesis and interleukin-2 (IL-2) production from peripheral blood mononuclear cells stimulated by PHA and Candida antigen in intractable asthmatics was significantly suppressed dose-dependently by OKY-046. Secondly, there was a tendency that neutrophil chemotactic factor (NCF) and eosinophil chemotactic factor (ECF) from peripheral blood mononuclear cells stimulated by Candida antigen in intractable asthmatics were suppressed by OKY-046. Thirdly, leukotriene (LTC4) and superoxide (O2-) production from peripheral blood neutrophils in intractable asthmatics was significantly suppressed dose-dependently by OKY-046. That is, OKY-046 has a suppressive effect on type IV allergy caused by lymphocyte activation and on mediator release from neutrophils. These results suggest that TXA2 plays an important role in the development of bronchial asthma and OKY-046 might be a useful drug in the treatment of intractable asthmatics.


[Urinary leukotrienes levels in asthmatic children]. In order to investigate the possible contribution of leukotrienes to the airway hypersensitiveness and inducibility of asthmatic spells, the probable temporal correlation of the urinary leukotriene levels (U-LTB4 and U-LTC4) were estimated along with the peripheral neutrophil counts in two groups of asthmatic children with or without an inflammatory sign of the elevated CRP. The first group consisted of 6 asthmatic children possessing inflammatory signs and symptoms of fever and peripheral leukocytosis. They all were hospitalized. The second group of 6 patients of light asthmatic attack without any signs and symptoms of acute inflammation were studied in the Outpatient Department. In the first group, U-LTB4 was as high as 258.6 +/- 88.9 ng/mmol Cr. during the attacks while U-LTB4 levels of the second group was as low as 62.2 +/- 32.20 ng/mmol Cr. The difference was statistically significant at p less than 0.01. Further mathematical analysis revealed a positive correlation of U-LTB4 to the peripheral neutrophils counts at r = 0.71. Thus, it was concluded that the elevation of U-LTB4 levels in the first group was strongly related to infections, and their asthmatic spells were thought to represent an infection-induced type. On the contrary, U-LTC4 levels in the samples during the asthmatic attacks were increased in 2 of the 6 of the first group. They both represented grave asthmatic spells. In the remainder, U-LTC4 levels did not rise enough to induce spasmodic contractions of the bronchial smooth muscles. Thus, it was also discussed that the most appropriate timing for urine collection for the study of U-LTs is some time following an asthmatic attack.


Identification and localization of atrial natriuretic factor receptors in human spermatozoa. Specific binding sites for atrial natriuretic factor (ANF) have been detected and localized in viable human spermatozoa through radioreceptor analysis and autoradiography, respectively. Radiotracer uptake was time and concentration dependent. Scatchard analysis of saturation data showed a single class of ANF receptors with a kd of 2.5 nM and a Bmax of 1.03 fmol/10(6) sperm 2.5 min-1, corresponding to about 620 molecules per sperm. Nonreducing SDS-PAGE analysis after covalent cross-linking of sperm bound 125I-ANF evidenced a single displaceable (i.e., specific) band with an apparent molecular weight of 135-140kD. In 125I-ANF bound spermatozoa, optical autoradiography showed an exclusive distribution of silver grains covering the midpiece region. The effects of ANF binding on ionic homeostasis and cyclic nucleotide metabolism, which modulate a number of sperm cellular processes, could make this factor play outstanding roles in gamete physiology.


Sciatic neuropathy associated with persistent sciatic artery. Persistent sciatic artery is a congenital vascular anomaly of the arterial supply to the lower extremity. Thrombosis, distal embolization, aneurysmal dilatation, and rupture of this vessel with compression of the sciatic nerve have been recorded. Although rare in occurrence, complications of persistent sciatic artery should be included in the differential diagnosis of sciatic neuropathy. We present a case of an acute sciatic neuropathy secondary to pseudoaneurysm formation of a persistent sciatic artery. We demonstrate the diagnostic usefulness of magnetic resonance imaging.


Early Guillain-Barré syndrome without inflammation. A patient with typical acute Guillain-Barré syndrome died 72 hours after his first symptoms occurred, and an autopsy was performed 8 hours after his death. Extensive sampling of cranial and peripheral nerves, sensory ganglia, and autonomic nerves showed only minimal inflammatory lymphocytic and macrophage infiltrates. This case, one of the earliest studied extensively, represents an extreme example of a noninflammatory mechanism that has been proposed in some cases of Gullain-Barré syndrome.


Retinal periphlebitis in patients with acquired immunodeficiency syndrome with cytomegalovirus retinitis mimics acute frosted retinal periphlebitis. Acute frosted retinal periphlebitis is an inflammatory condition of unknown origin characterized by marked perivenular infiltration in otherwise healthy patients. We encountered seven patients with acquired immunodeficiency syndrome who exhibited visual loss associated with an unusual diffuse retinal periphlebitis very similar in appearance to acute frosted retinal periphlebitis. Each patient developed a thick inflammatory infiltrate surrounding the retinal venules, creating a frosted appearance. Two cases were bilateral. All patients had areas of more typical cytomegalovirus retinitis in their involved eye(s). Five of six patients treated with ganciclovir sodium showed improvement not only of the cytomegalovirus retinitis but also of the periphlebitis. Although we do not have histopathologic evidence that cytomegalovirus was the cause of these cases of periphlebitis, we believe that periphlebitis may be a previously unrecognized finding of cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome. So far, there is no evidence implicating cytomegalovirus as the cause of acute frosted retinal periphlebitis in healthy patients.


Immune responsiveness and risk of illness in U.S. Air Force Academy cadets during basic cadet training. It has been proposed, but not confirmed, that environmental stressors alter immune function and increase the risk of viral infection among healthy individuals. We evaluated this hypothesis, examining the relationship among stress, immune function, and illness in 96 first-year U.S. Air Force Academy cadets during orientation and 4 weeks later during the stressful environment of Basic Cadet Training (BCT). Perceived stress and well-being levels of cadets were assessed via questionnaire. Immune responsiveness was analyzed by PHA-stimulated thymidine uptake in mononuclear leucocytes and by serologic evidence of reactivation of the Epstein-Barr virus (EBV). We documented significant declines in in vitro PHA-induced lymphocyte transformation (-35%; p less than 0.05) and subjective well-being (-19%; p less than 0.05) from orientation to BCT with corresponding, significant increases in perceived stress (+32%; p less than 0.05). Despite significantly altered in vitro immune responsiveness, there was no serologic evidence of EBV reactivation nor was there an association between these measures and risk of illness as determined by medical chart review and self-reported symptoms. These results suggest that reduced in vitro immune responsiveness during a moderate stressor may not necessarily lead to an increased risk of infection and/or reactivation of EBV in normal individuals.


Allozyme and mitochondrial DNA analysis of a hybrid zone between white-tailed deer and mule deer (Odocoileus) in west Texas. Thirty allozyme loci and 35 mitochondrial DNA (mtDNA) restriction sites were examined in 24 white-tailed deer and 46 mule deer from a hybrid zone in West Texas. A common mtDNA genotype is shared by all of the mule deer with 67% of the white-tailed deer. At the albumin locus, 13% of the white-tailed deer and 24% of the mule deer are heterozygous, sharing alleles that are otherwise species-specific in allopatric populations; 7% of the mule deer are homozygous for the allele that is characteristic of allopatric white-tailed deer. Gene flow appears to have been bidirectional, with greater genetic introgression into mule deer. The mtDNA data suggest that matings between white-tailed and mule deer have occurred in the past. Despite evidence of genetic introgression, analysis of multilocus genotypes indicates that none of the deer examined is an F1 hybrid. Production of such hybrids appears to be generally uncommon in North American deer; management plans that assume otherwise should be reconsidered.


Uptake and incorporation in pteridines of externally supplied GTP in normal and pigment-deficient eyes of Drosophila melanogaster. Some aspects of the synthesis of drosopterins in the eyes of Drosophila melanogaster have been studied in flies with different levels of white gene expression. The activity of GTP cyclohydrolase was found to differ between wild-type and yellow-eyed mutants in vivo but not in vitro. To elucidate the uptake of substrate, we measured the removal of labeled GTP from the incubation medium by excised pupal eyes and followed the subsequent fate of this label. It was found that GTP was dephosphorylated to guanosine extracellularly before label was taken up by the eye tissue. The uptake was much lower in yellow and white eyes than in wild-type eyes, and in the latter, a considerable part of the label was present in pteridine compounds. The strain differences in the uptake of label seem to be due to different rates of intracellular utilization of guanine derivatives in pteridine synthesis. We suggest that this is caused by a hampered transport of precursor (possibly GTP) in white and zeste eyes through the membrane of red pigment granules.


Atrial natriuretic peptide decreases hepatic and cardiac blood content, but increases intestinal blood content in supine humans. The authors evaluated in humans whether atrial natriuretic peptide (ANP) alters the regional distribution of blood in capacitance vessels. Eight healthy male volunteers (mean age: 30 years, range: 24-39) were studied twice. On different days and in a randomized, double blind fashion they received either alpha h-ANP (99-126), 25 micrograms intravenously followed by infusion of 0.1 microgram kg-1 min-1, or vehicle. Changes of regional blood content in heart, liver, and intestine were evaluated at 3-min intervals using autologous radioactively (99mTc) labeled red cells. Calf circumference (strain gauge), central venous pressure, and heart rate were recorded continuously while arterial pressure (oscillometry), hematocrit, ANP and cGMP plasma concentrations were determined intermittently. Exogenous ANP increased plasma concentrations of ANP (49 pg ml-1 +/- 8 SE to 614 +/- 190) and cGMP (1.7 pmol ml-1 +/- 0.2 to 30.8 +/- 4.4). This elicited significant and profound decreases in liver (-11%) and cardiac (-10%) radioactivity, contrasted by a smaller but significant increase (+4%) of intestinal radioactivity. These changes became gradually apparent about 15 min during ANP administration and reached their nadir at the end of the infusion period. Central venous pressure significantly decreased by 3.4 cm H2O and calf volume by 0.3 ml/100 ml while hematocrit increased by 2.6%. All changes were at least partly reversed when ANP administration ceased. Of note, two subjects developed a near syncope with abrupt bradycardia and arterial hypotension following an initial gradual decrease in cardiac counts and central venous pressure. We conclude that in humans ANP markedly alters the regional blood distribution in the capacitance vasculature as blood content decreased profoundly in both heart and liver, but increased in the intestine, albeit to a lesser extent. Accordingly, a redistribution of blood away from the heart represents another unique mechanism by which ANP can exert its cardiovascular actions.


Oscillations of nitric oxide concentration in the perturbed denitrification pathway of Paracoccus denitrificans. The metabolism of nitric oxide in Paracoccus denitrificans has been studied using a Clark-type electrode. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and the SH reagent N-ethylmaleimide, both of which released nitric oxide from cells respiring nitrite, were found to be efficient inhibitors of nitric oxide reductase activity. Control experiments with another uncoupler, pentachlorophenol, showed that the inhibitory effect of CCCP was not the result of a decrease in membrane potential. The denitrification pathway in cells with partly inhibited nitric oxide reductase, or in a reconstituted system containing purified nitric reductase and membrane vesicles, exhibited marked sustained oscillations of nitric oxide concentration. The occurrence of the oscillations was strictly dependent on the initial concentration of nitrite. The observed oscillatory kinetics is considered to reflect two regulatory signals destabilizing the denitrification pathway, namely the inhibition of nitric oxide reductase by nitric oxide and/or by nitrite.


Baroreflex and beta-adrenoceptor function are diminished in rat cardiac hypertrophy due to volume overload. We investigated whether cardiac hypertrophy induced by volume loading influences baroreflex sensitivity. Aortic insufficiency (AI) was induced in male Wistar rats by graded disruption of the aortic valve, which, after 2 weeks, resulted in a 30% increase in heart/body weight or left-ventricular/body weight ratio compared with control animals. Baroreflex sensitivity was assessed in conscious animals by measuring the heart rate (HR) responses to the changes in mean arterial pressure (MAP) induced by phenylephrine and nitroprusside sodium at 2 weeks. The slopes of the HR vs MAP plots obtained with phenylephrine and nitroprusside decreased significantly with increasing heart weight/body weight ratio (correlation coefficient r = 0.625 and 0.526, respectively). In isolated right atria from AI animals baseline rate was higher, and the isoproterenol effect on sinus rate was significantly smaller than in atria from control animals, indicating a dysfunction of the beta-adrenoceptor pathway. The data show that baroreflex dysfunction associated with a down-regulation of the beta-adrenoceptor pathway of the sinus node develops simultaneously with volume overload-induced hypertrophy in the absence of overt heart failure.


Inositol monophosphatase is a highly conserved enzyme having localized structural similarity to both glycerol 3-phosphate dehydrogenase and haemoglobin. The cDNA coding for an inositol monophosphatase in the oocytes of the African clawed frog, Xenopus laevis, has been isolated and sequenced. The predicted primary structure of this enzyme is markedly conserved when it is compared with its mammalian functional homologues; up to 84% of the amino acid residues are identical, and conservative substitutions increase the similarity to 95%, suggesting that this sequence represents the most parsimonious primary structure for the protein to maintain not only catalytic activity but also perhaps the facility to interact with other macromolecules. Two regions of the protein, each of about 11 residues and separated by about 90 residues, have been identified as a consensus found also in glycerol 3-phosphate dehydrogenase (EC 1.1.1.8). One of these regions is also found to be particularly conserved in the alpha-globin of birds and reptiles; birds and some turtles are known to modulate the oxygen affinity of their haemoglobin with inositol polyphosphate in the same way as with 2,3-bisphosphoglycerate in other species. This region is also conserved in the beta-globin of most species, beginning with lysine-82, which is known to participate in the binding of organic phosphates. These regions of the inositol monophosphatase may represent motifs for the binding of its substrate.


Intrinsic factor receptor during fetal development of the human intestine. Intrinsic factor receptor activity was observed in mucosal homogenates from whole small intestine and colon of 10-19-week fetuses, whereas it was only detected in the distal part of the small intestine of a 25-week fetus. The receptor-intrinsic factor-cobalamin complex was eluted into the void-volume position when ileum mucosal extract was assayed for receptor activity by gel filtration after incubation with either fetal gastric extract or human gastric juice. The intrinsic-factor-binding capacity of intestinal mucosal extracts ranged from 2.6 to 30.5 fmol/mg and was correlated with the gestational age of six fetuses. The dissociation constant of the receptor for the intrinsic factor-cobalamin complex was estimated at 0.24-0.36 nM at pH 7.4. In conclusion, intrinsic-factor-receptor activity was detected in the whole intestine in 10-19-week fetuses, whereas it was only present in the distal ileum at the end of fetal development.


Purification and characterization of calponin phosphatase from smooth muscle. Effect of dephosphorylation on calponin function. Calponin, a thin-filament protein of smooth muscle, has been implicated in the regulation of smooth-muscle contraction, since in vitro the isolated protein inhibits the actin-activated myosin MgATPase. This inhibitory effect, and the ability of calponin to bind to actin, is lost after its phosphorylation by protein kinase C or Ca2+/calmodulin-dependent protein kinase II [Winder & Walsh (1990) J. Biol. Chem. 265, 10148-10155]. If this phosphorylation reaction is of physiological significance, there must be a protein phosphatase in smooth muscle capable of dephosphorylating calponin and restoring its inhibitory effect on the actomyosin MgATPase. We demonstrate here the presence, in chicken gizzard smooth muscle, of a single major phosphatase activity directed towards calponin. This phosphatase was purified from the soluble fraction of chicken gizzard by (NH4)2SO4 fractionation and sequential chromatography on Sephacryl S-300, DEAE-Sephacel, omega-amino-octyl-agarose and thiophosphorylated myosin 20 kDa light-chain-Sepharose columns. The purified phosphatase contained three polypeptide chains of 60, 55 and 38 kDa which were shown to be identical with the subunits of SMP-I, a smooth-muscle phosphatase capable of dephosphorylating the isolated 20 kDa light chain of myosin but not intact myosin [Pato & Adelstein (1983) J. Biol. Chem. 258, 7047-7054]. Consistent with its identity with SMP-I, calponin phosphatase was classified as a type-2A protein phosphatase. Of several potential phosphoprotein substrates examined, calponin proved to be kinetically the best, suggesting that calponin may be a physiological substrate for this phosphatase. Finally, dephosphorylation of calponin which had been phosphorylated by protein kinase C restored completely its ability to inhibit the actin-activated MgATPase of smooth-muscle myosin. These observations support the hypothesis that calponin plays a role in regulating the contractile state of smooth muscle and that this function in turn is controlled by phosphorylation-dephosphorylation.


Expression in Escherichia coli and characterization of a recombinant 7Fe ferredoxin of Rhodobacter capsulatus. The 7Fe ferredoxin of Rhodobacter capsulatus (FdII) could be expressed in Escherichia coli by cloning the fdxA gene coding for FdII downstream from the lac promoter. The expressed recombinant ferredoxin appeared as a brown protein which was specifically recognized in E. coli cell-free extracts by anti-FdII serum. The purified recombinant ferredoxin was indistinguishable from R. capsulatus FdII on the basis of its molecular, redox and spectroscopic properties. These results indicate that the [3Fe-4S] and [4Fe-4S] clusters were correctly inserted into the recombinant ferredoxin.


Use of experimental isotope-exchange fluxes in reversible enzyme and membrane transport models, assessed by simultaneous computer simulation of unidirectional and net chemical rates. Steady-state rate equations for unidirectional (isotope-exchange) rates can become so complex, even for rather simple (reversible) enzyme or membrane transport models, that they are useless for detailed data analysis. In this paper a procedure is described for simultaneous simulation of net (chemical) and isotope-exchange rates. The method employs an expanded version of the basic model to monitor explicitly the fate of the label in an experiment. The procedure is quite general, and can be used for steady-state as well as transient kinetic situations, or it can be used in conjunction with existing interactive computer programs for steady-state model analysis. Three numerical examples are presented. First, it is shown, using the conventional (Post-Albers) model for Na+/K(+)-ATPase, that the change in concentration of a labelled intermediate after a change in experimental conditions does not in general reflect the change in the total concentration of that intermediate, and thus labelled intermediate concentrations may be misleading. Second, using a standard co-transport model and a prototype active-transport model (equivalent to a ligand-ATPase), it is shown that the ratio of tracer transport fluxes at steady state yields transport stoichiometries which depend on the experimental conditions, are different from the net apparent stoichiometries, and whose changes with conditions are also different from that of the net stoichiometries. It follows that conclusions drawn on the basis of experimentally determined tracer fluxes should be viewed with some caution. Specifically, a measured influx stoichiometry ligand/ATP (in the ATPase case) of higher than 1:1 does not necessarily imply the existence of more than one site for either ligand on the enzyme.


Importance of various antioxidant enzymes for cell stability. Confrontation between theoretical and experimental data. A theoretical model was developed taking into account the production and destruction of oxygen-derived free radicals. The steady state of the system was derived by using the rate equations of these reactions, and the stability of the system was tested. In the simplified model, only one stable steady state was found. However, we know that glutathione peroxidase can be inhibited by hydroperoxides, and, when incorporated into the model, this effect led to a complex situation with the presence of some stable and some unstable domains according to the concentration of either the enzyme or the hydroperoxide. This qualitative description of the system was compared with experimental data on the protection given by three antioxidant enzymes, and concordance of data was found which allows some quantification of the system. A general view of the efficiency of the three antioxidant enzymes and of the stability of the system according to their concentrations could be produced.


Rat surfactant protein D enhances the production of oxygen radicals by rat alveolar macrophages. Rat surfactant protein D (SP-D) was shown to enhance the production of oxygen radicals by rat alveolar macrophages. This enhancement, which was determined by a lucigenin-dependent chemiluminescence assay, was maximal after 18 min at an SP-D concentration of 0.2 micrograms/ml. Surfactant lipids did not influence the stimulation of alveolar macrophages by SP-D, whereas the oxygen-radical production of these cells induced by surfactant protein A was inhibited by the lipids in a concentration-dependent manner.


Trypanosoma cruzi: inhibition of metacyclogenesis by mannose. Metacyclogenesis of Trypanosoma cruzi epimastigotes was evaluated in a medium supplemented with Triatoma infestans intestinal homogenate in the presence of sugars and derivates as are mannose, galactose, fucose, N-acetylglucosamine, mannose 6-P, and fructose 1,6-P at a concentration of 25 mM. Only mannose significantly inhibited metacyclogenesis. Sodium metaperiodate and trypsin treatment of the intestinal homogenate also inhibited differentiation. In our opinion there exists a proteinic factor in the intestine of the vector that promotes metacyclogenesis and is incorporated by the parasite. Treatment of the intestinal homogenate with alkaline phosphatase had no effect. Instead, high ionic strength in the medium (0.4 M NaCl) strongly inhibited metacyclogenesis indicating that, in these conditions, the possible binding of the differentiation factor to the parasite surface was inhibited.


Discrete subcellular localization of phosphoinositidase C beta, gamma and delta in PC12 rat pheochromocytoma cells. Phosphoinositidase C activity was revealed in nuclei isolated from PC12 rat pheochromocytoma cells incubated with tritiated phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Phosphoinositide breakdown was found to be optimal at neutral pH and Ca++ concentrations ranging from endogenous levels to millimolar values. To characterize the enzymes involved, three monoclonal antibodies directed against the beta, gamma and delta phosphoinositidase C isoforms were employed. A combination of Western blot immunochemical analysis on cytoplasmic and nuclear fractions and of in situ immunocytochemistry on intact cells and isolated nuclei indicated that phosphoinositidase C gamma, though predominantly cytoplasmic, was present in both cell compartments. On the contrary, phosphoinositidase C beta was exclusively localized in the nucleus, whereas phosphoinositidase C delta was restricted to the cytoplasm. These data suggest that inositol lipid breakdown is controlled by different phosphoinositidase C isozymes in the various cell compartments, and support the notion that a separate phosphoinositide signalling system is located in the nucleus.


Heterogeneity among human nasopharyngeal carcinoma cell lines for inflammatory cytokines mRNA expression levels. Using polymerase chain reaction (PCR), we confirmed the expression of interleukin-1 alpha (IL-1 alpha) by the human nasopharyngeal carcinoma (NPC) cell line C15 without contribution of either human IL-1 beta or mouse IL-1 alpha in the biological activity previously found in C15. However we showed that IL-1 alpha was not expressed in all NPCs. IL-1 beta and/or tumor necrosis factor (TNF)-alpha genes could also be activated, independently from the number of Epstein Barr Virus (EBV) copies harbored by the cells. Interestingly, the primary tumor C15 showed a profile of TNF-sensitive tumor while C17, C18 and C19 which were derived from metastasis have a typical profile of TNF-resistant cells. Furthermore, the inflammatory cytokines whose genes are classically induced by IL-1 and TNF were found expressed only in C17 and C19 suggesting another level of heterogeneity among NPCs.


Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2. To clarify the function of the receptor binding protein for inositol hexakisphosphate (IP6), we obtained a partial amino acid sequence from the purified protein and a partial nucleotide sequence from a cDNA clone of the gene. The sequences are essentially identical to those of the alpha-subunit of the clathrin assembly protein AP-2. The IP6 receptor protein analyzed by SDS-PAGE contains a series of subunits which are the same as those of AP-2. Antibodies to AP-2 react with the IP6 receptor protein in immunoblot analysis.


Amphotropic retroviruses with a hybrid long terminal repeat as a tool for gene therapy of cystic fibrosis. We have made two retroviral vectors encoding the bacterial beta-galactosidase (lacZ) as a marker gene and a long terminal repeat (LTR) containing an enhancer of the polyoma F101 virus [symbol: see text]. One vector, [symbol: see text], can be used as a test vector in grafting, lineage analysis and gene therapy studies. The other, [symbol: see text] carries an additional unique cloning site in which a gene of interest can be cloned. Titration experiments showed that in human epithelial cell lines, [symbol: see text] produces a transcriptionally active integration more often than the commonly used BAG vector with the wild type LTR. Human epithelial cells in primary culture could be successfully infected. Our data suggest that gene therapy protocols requiring infection in situ, such as in the case of cystic fibrosis, will be hampered by the relatively low local titres that can be achieved at present.


Nucleotide-activated chloride channels in lysosomal membranes. Lysosomal membrane vesicles purified from rat liver contain a basal chloride conductance that was enhanced in the presence of ATP, non-hydrolysable ATP-analogs and, to a lesser extent, GTP. Other nucleotides, including AMP, ADP and cAMP, as well as CTP and UTP were not effective. Following fusion of the vesicles with an artificial phosphatidylethanolamine/phosphatidylserine bilayer, we found that ATP gamma S dramatically increased the incidence of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS)-sensitive chloride channels with a unitary slope conductance of approx. 40 pS in 300 mM/50 mM KCl buffers and 120 pS in symmetrical 300 mM KCl buffers. Since similar results were obtained with AMP-PNP, the results indicate that lysosomes contain a chloride permeable ion channel that is activated by ATP through allosteric interaction.


Calcitonin gene-related peptide: an autocrine growth factor with regulatory activity in vitro. We show that an autocrine system for calcitonin gene-related peptide (CGRP) exists in F9 teratocarcinoma cells. Synthesis of CGRP by F9 cells was demonstrated by measuring the peptide concentration in cells and medium and by determining specific mRNA in cells. During six days of culture, CGRP secretion did not vary significantly in the medium, while intracellular CGRP and CGRP mRNA levels increased. F9 cells contained a CGRP-sensitive adenylate cyclase system and CGRP increases the accumulation of cAMP in the culture medium. Interestingly affinity purified antibodies against CGRP specifically inhibited growth of F9 cells by 50%. CGRP therefore stimulates F9 cell growth by an autocrine process, suggesting that CGRP may be a growth factor during early embryogenesis.


IL-3 and GM-CSF induce the expression of the inositol trisphosphate receptor in K562 myeloblast cells. When treated with IL-3 plus GM-CSF, K562 myeloblast cells acquired the ability to mobilize nonmitochondrial stores of intracellular Ca2+ in response to added Ins (1, 4, 5) P3. Untreated K562 cells are capable of sequestering intracellular Ca2+ but released none of this Ca2+ in response to Ins (1, 4, 5) P3. Untreated K562 cells were shown to have no detectable specific [3H] Ins (1, 4, 5) P3 binding sites and no InsP3 receptor mRNA as assayed by Northern blot and PCR. However, following IL-3 and GM-CSF treatment, both a single class of low nM KD Ins (1, 4, 5) P3 binding site and a 10 kb InsP3 receptor mRNA were detectable. The results suggest that IL-3 and GM-CSF regulate the expression of the Ins (1, 4, 5) P3 receptor gene.


Comparative effects of protein phosphatase inhibitors (okadaic acid and calyculin A) on human leukemia HL60, HL60/ADR and K562 cells. Inhibitors of protein phosphatases 1/2A (okadaic acid and calyculin A) exhibited differential cytotoxicity toward three human leukemia cell lines, in an increasing order of resistance, HL60 less than HL60/ADR less than K562 cells. Cytotoxicity of the toxins was associated with marked mitotic arrest of the cells, characterized by chromatid scattering/overcondensation and abnormal mitotic spindles. In all cases, calyculin A was more potent than okadaic acid. Protein phosphorylation experiments in intact cells revealed that HL60/ADR, the adriamycin-resistant variant, showed a higher overall phosphorylation of nuclear proteins than the drug-sensitive parental HL60, and that phorbol ester (protein kinase C activator) and calyculin A appeared to more specifically stimulate phosphorylation of p66 and p60, respectively. It was suggested that the toxins might be useful in delineating mechanisms underlying certain properties of cancer cells (such as multidrug resistance, mitosis and differentiation) related to protein phosphorylation/dephosphorylation reactions.


Cell cycle dependent gene expressions and activities of protein phosphatases PP1 and PP2A in mouse NIH3T3 fibroblasts. We determined the mRNA levels and the activities in nuclear and non-nuclear fractions of protein phosphatase type 1 (PP1) and type 2A (PP2A) through the cell cycle in synchronized mouse NIH3T3 fibroblasts. The mRNA level for PP1 alpha was gradually elevated in late G1 phase, began to decrease in M phase, and reached the control level with entering into the next G1 phase. The mRNA level for PP2A was rapidly increased in early G1 phase, kept at the high level, and decreased after S phase. In nuclear fractions of cells, spontaneous activities of both PP1 and PP2A were gradually increased until M phase and rapidly decreased with entering the next G1 phase, while in non-nuclear fraction such dramatic alterations were not observed. Potential activities of PP1 in both fractions revealed by Co(2+)-trypsin treatment showed an oscillaion patterns similar to those of the spontaneous activities. These results strongly suggest that cell cycle dependent gene expressions and activities of PP1 and PP2A play roles in DNA synthesis and mitosis during the cell cycle.


Structure and expression of two isoforms of the murine calmodulin-dependent protein phosphatase regulatory subunit (calcineurin B). Murine cDNAs representing distinct genes for the regulatory subunits of calmodulin-dependent protein phosphatase (CaM-PrP) were cloned from a testis library, using probes prepared by PCR amplification of brain and testis mRNA. The cDNA sequence of the brain-specific isoform (beta 1) encodes a 170 amino acid protein (M(r) approximately 19.3 kDa), whereas that for the testis isoform (beta 2) contains 179 residues (M(r) approximately 20.7 kDa); these two sequences show approximately 80% amino acid identity. An oligonucleotide probe for the brain isoform hybridized to a single mRNA of 3.6 kilobases (kb) in many tissues, whereas using the beta 2 probe, two mRNAs of 1.8 and 0.8 kb were detected only in testis. The mRNA for the testis-specific isoform increases markedly during development, its pattern being virtually identical to that of mRNA for a testicular form of the catalytic subunit (alpha 3). These data are consistent with the biological co-regulation of catalytic and regulatory subunits of a testis-specific isoenzyme during germ cell maturation.


Nucleoside diphosphate kinase does not directly interact with tubulin nor microtubules. Nucleoside diphosphate kinase has been shown to play a role in proliferation and development. Microtubules have been evoked as a possible target of NDP kinase action; in particular it was proposed that NDP kinase could regulate the cellular pool of polymerizable GTP-tubulin by direct phosphorylation of tubulin bound GDP. We show that this reaction does not occur in vitro and also that NDP kinase does not bind to microtubules both in the presence and absence of MAPs. Thus, any possible physiological effect of NDP kinase on microtubule dynamics is exerted only by modulating the concentrations of free guanine nucleotides in the vicinity of microtubules.


Induction of differentiation of the human promyelocytic cell line HL-60 by activin/EDF. A human promyelocytic cell line, HL-60, treated with activin/EDF was found to differentiate into monocyte/macrophage-like cells. This was shown not only by morphology but by the loss of myeloperoxidase granules and the appearance of nonspecific esterase. Dose-dependent inhibition of the differentiation by follistatin, an activin-binding protein, confirmed that it was indeed caused by activin. Thus, activin/EDF exerts its effect on hematopoietic cells not only on erythroid differentiation but also on at least a part of myeloid cell differentiation.


N-terminal truncation of salmon calcitonin leads to calcitonin antagonists. Structure activity relationship of N-terminally truncated salmon calcitonin fragments in vitro and in vivo. Structural requirements for binding to the bone calcitonin (CT) receptor and for CT bioactivity both in vitro and in vivo were assessed for a series of N-terminally truncated, N alpha-acetylated, fragments of salmon calcitonin (sCT). Sequential deletion of amino acid residues from the amino-terminus of [Ala7]sCT-(2-32) peptide amide first led to partial agonists and, upon deletion of residues 1 to 7, to a high affinity antagonist, N alpha-acetyl-sCT-(8-32)-NH2. The presence of two separate domains within the sCT sequence is proposed: (I) a binding domain comprising residues 9-32 and (II) an activation domain requiring residues 3 to 6. N alpha-acetyl-sCT-(8-32)-NH2, in several bioassays including plasminogen activator release from LLC-PK1 cells (pA2 = 7.31), cAMP production in UMR-106-06 cells (pA2 = 7.81) and in the fetal rat long bone resorption assay showed potent antagonistic properties.


Molecular cloning and expression of an adenosine A2b receptor from human brain. A novel receptor cDNA was isolated from a human hippocampal cDNA library. The encoded polypeptide contains structural features consistent with its classification as a G protein-coupled receptor and shares 45% homology with the human A1 and A2a adenosine receptors. Chinese hamster ovary K1 cells expressing this receptor showed marked stimulation of adenylate cyclase when treated with 1mM adenosine. There was no response to ligands selective for A1 and A2a receptors but the general adenosine agonist N-ethylcarboxyamidoadenosine (NECA) caused a 10 fold increase in cyclic AMP accumulation with an EC50 of approximately 0.9 microM. This effect was inhibited by the adenosine receptor antagonist theophylline. Specific binding of A1 and A2a selective agonists and NECA was not detected. It is proposed that the novel receptor is a human brain adenosine A2b receptor subtype.


The oxygenated flavohaemoglobin from Escherichia coli: evidence from photodissociation and rapid-scan studies for two kinetic and spectral forms. The kinetics of dissociation and reassociation of the oxygenated species of Escherichia coli flavohaemoglobin (Hmp) were studied using stopped-flow rapid-scan and flash photolysis spectrophotometry at 25 degrees C. The oxygenated compound(s) form rapidly on mixing oxygen with the NADH-reduced flavohaemoglobin. On exhaustion of NADH, with residual oxygen, decay occurs in two phases to give a form in which haem b and flavin are oxidized. Spectral changes during this process suggest a direct release of O2- from the oxy form. Photodissociation of the oxygenated species generates the unliganded protein, which recombines with oxygen to give two spectrally and kinetically distinct forms. The reversibility of the oxygen reaction and the rapid reassociation kinetics after photodissociation confirm the haemoglobin-like features of this protein.


Neuroendocrine differentiation in male breast carcinomas. The presence of neuroendocrine differentiation, as expressed by cellular chromogranin immunoreactivity, was investigated in paraffin-embedded tissue material from 51 consecutive cases of male breast carcinoma. From six of these cases electron microscopic studies were included. Chromogranin-immunoreactive cells were present in solid cords and delineated tubular structures. Ultrastructurally, dense core secretory granules could be detected. The expression of neuroendocrine differentiation was 45%, which is between two and eight times higher than reported for female breast carcinomas by other investigators. The present findings suggest that male breast carcinoma is an exclusive tumour disease showing both similarities and discrepancies when compared to its female counterpart.


Some bacterial parameters influencing the neutrophil oxidative burst response to Pseudomonas aeruginosa biofilms. Persistence of bacteria in spite of a normal host immune system and relevant antibiotic treatment is a key problem in many chronic infections, such as the bronchopulmonary P. aeruginosa infection in cystic fibrosis patients. The capability of bacteria to establish themselves in microcolonies or biofilms is an important protective mechanism of the microorganisms. We examined the human PMN oxidative burst response to P. aeruginosa in biofilm and in planktonic form. The PMN chemiluminescence response to P. aeruginosa in biofilms was reduced to 30.5-47.5% (p less than 0.04) and the superoxide response to 85.9% (p less than 0.02) of the response to equivalent numbers of planktonic bacteria. Mechanical disruption of the biofilms before the assays elicited a significantly increased response in the chemiluminescence experiments and to nonopsonized biofilms in the superoxide anion experiments. We conclude that biofilm bacteria, although able to stimulate the PMN, result in a reduced, suboptimal response leading to lack of efficient eradication of the bacteria in the chronic infection.


Ibotenic acid mediates neurotoxicity and phosphoinositide hydrolysis by independent receptor mechanisms. Ibotenic acid (Ibo) has been shown to have agonist activity at both the N-methyl-D-aspartate (NMDA) and trans-ACPD or metabolotropic quisqualate (Qm) receptor sites in several systems. Both of these receptor sites have been implicated in excitotoxicity. Like NMDA neurotoxicity, Ibo neurotoxicity can be enhanced by glycine and blocked by MK-801. Ibo induced stimulation of phosphoinositide (PI) hydrolysis, on the other hand, is unaffected by either of these treatments. We therefore conclude that Ibo is capable of acting at both NMDA and trans-ACPD receptors in the CNS, although only activation of NMDA receptors is involved in Ibo neurotoxicity. This conclusion leads us to postulate that stimulation of phosphoinositide hydrolysis is neither necessary nor sufficient for neurotoxicity.


Variable natural killer function of tumour-infiltrating lymphocytes from breast carcinomas. Samples were obtained from 36 breast cancer patients: 24 tissue samples from carcinomas, six samples from non-cancerous breast tissue and 22 samples of peripheral blood (PB). Natural killer (NK) activity was tested against the classical target K-562 and the breast carcinoma cell line T-47D, using a microcytotoxicity assay measuring inhibition of uptake of 3H-thymidine by target cells. The active effector cells against both target cell types were identified as CD-56-positive large granular lymphocytes. PB lymphocytes from breast cancer patients showed normal NK activity against both K-562 (mean inhibition: 75.4%, controls: 78.1%) and T-47D (mean inhibition: 77.6%, controls: 78.0%). Tumour-infiltrating lymphocytes (TIL) from 24 breast carcinomas showed considerable variation in NK activity against K-562: only two cases (8%) had no activity (less than or equal to 5% inhibition), 15 cases (63%) had moderate activity (15-48% inhibition), and seven cases (29%) showed full NK activity (defined as greater than or equal to 50% inhibition). TIL from three carcinomas were also tested against T-47D and gave 21-46% inhibition. The NK activity of TIL declined with time in culture with one notable exception where there was full NK activity after seven days in culture. NK activity of TIL showed significant positive correlation with increasing degree of lymphocytic infiltration of the tumour, and correlated also with PB NK activity. No correlation was apparent with stage of disease. The NK activity was usually lower in the TIL than the PB sample when tested in parallel. This difference was probably caused by experimental conditions as well as different relative numbers of natural killer cells. In six cases tested, infiltrating lymphocytes from carcinoma and normal tissue from the same breast were found to have similar NK activities. We conclude that the non-specific cytotoxic activity of TIL from breast carcinomas covers the whole range from complete lack of function to full capacity comparable with NK activity of PB lymphocytes. The level of activity was moderately correlated with the degree of lymphoid infiltration but was not related to stage of disease. Differences in functional capacity of TIL may be of importance in predicting possible benefits from immunomodulating therapy.


High-affinity uptake of GABA and glutamate decarboxylase activity in rat primary somatosensory cortex after sciatic nerve injury. We have studied the changes in the GABAergic system in the rat somatosensory cortex 1-14 d after sensory deprivation of the hind-limb representation area. Glutamate decarboxylase (GAD) activity was measured in the individual cortical layers using serial sections cut on a freezing microtome parallel to the cortical surface. Gamma-aminobutyric acid (GABA) high-affinity uptake was studied in cortical homogenates of the hind-limb representation area. There was a less than or equal to 13% decrease in GAD activity in layer II-IV in both cortical hemispheres 3 d after sciatic nerve injury. In contrast, we found that high-affinity uptake of GABA is not affected. The data mirror only small changes in GABAergic transmission, probably as a result of the methods employed. These changes correspond to electrophysiological studies suggesting that peripheral manipulation of the somatosensory system, e.g., nerve transection, is accompanied by changes in GABAergic transmission.


Comparison of HPV detection in parallel biopsies and cervical scrapes by PCR. PCR was used to detect HPV DNA in cervical scrapes of 23 women with cervical cancer. Compared with PCR-assisted HPV DNA detection in parallel biopsies, the sensitivity was 81% and the specificity 100%. We conclude that cervical scrapes can be used for detection of HPV DNA in women with cervical cancer.


Effects of traumatic brain injury in rats on binding to forebrain opiate receptor subtypes. Sprague-Dawley rats were subjected to a moderate level (2.2 atm) of traumatic brain injury (TBI) using fluid percussion. Injured animals were allowed to survive posttrauma for periods of 5 min, 3 h, and 24 h. The effect of TBI on binding to forebrain opiate receptors was assessed using quantitative receptor autoradiography, and compared to a sham control group. Binding of [3H]DAGO to mu receptors in neocortex and the CA1 pyramidal layer of the hippocampus was significantly decreased in the 24-h group (p less than 0.05). [3H]Bremazocine binding to kappa receptors was unchanged at 5 min and 24 h, but showed large decreases 3 h after TBI in the CA1 pyramidal layer (65%, p less than 0.05) and dentate gyrus (43%, p less than 0.05). Levels of delta binding (measured with [3H]DSLET) and lambda binding (measured with [3H]naloxone) were unaffected by TBI. These data support previous suggestions of a role for endogenous opioids in TBI, and provide further evidence that mu and kappa opioid receptor subtypes in neocortex and hippocampus may have different functions in TBI.


Prevalence of hepatitis B and C and HIV antibodies in children in a Romanian orphanage. Two hundred and fifteen children in an orphanage in Romania were examined for serum markers of present or past hepatitis B and C virus and HIV infection. In total, 183 children (85.1%) had at least one marker (HBsAg, anti-HBs or anti-HBc) of hepatitis B virus infection. An HBsAg carrier state was diagnosed in 38 (20.8%) of the infected children. Among the carriers 24.3% were HBeAg carriers, 51.4% had anti-HBe and 24.3% had neither HBe antigen nor antibody. Nine children (4.2%) had antibodies to hepatitis C virus. All sera were negative in tests for HIV antibodies. False-positive reactions represented a considerable problem with these sera. Six percent of the sera gave false-positive reactions in indirect ELISA tests for hepatitis C and HIV. Sera giving false-positive reactions had rather high serum IgG levels. The results of this study indicate that these children have been heavily exposed to hepatitis B virus and to a certain degree to hepatitis C virus, while there were no cases of HIV infection in this orphanage.


The immediate early genes of human cytomegalovirus upregulate expression of the cellular genes myc and fos. Human cytomegalovirus (HCMV) is an important pathogen of the lung. We determined whether the HCMV immediate early genes (IE1 and IE2) can alter the regulation of the cellular immediate early genes (c-fos and c-myc). Plasmid constructs containing the promoter-regulatory regions c-myc or c-fos upstream of the reporter gene, chloramphemicol acetyl transferase, were co-transfected into T cells (Jurkat cells), monocytes/macrophages (THP-1 cells), or human fibroblast cells with plasmid constructs containing the promoter-regulatory region of the HCMV IE genes upstream of the bona fide IE1, IE2 or IE+2 genes; a plasmid that contained no IE coding region was used as a control. These studies show that both products of the HCMV IE genes markedly upregulated expression of the cellular c-fos and c-myc genes. The viral effects of individual proteins (IE1 or IE2) were dependent both on the promoter-regulatory region of the cellular gene and the cell type. In all cells, the combination of IE1 and IE2 further upregulated both cellular genes, suggesting a synergistic effect of IE1 with IE2. Both of the c-myc promoters (P1 and P2) were up-regulated by the HCMV IE gene products. IE1 and IE2 also upregulated the cells' endogenous c-myc and c-fos genes, as determined by amounts of the respective mRNAs. These studies show that HCMV can markedly alter cellular IE gene expression and that the effects of HCMV IE1 and IE2 proteins are dependent both on the promoter-regulatory region of the cellular gene and the type of cell in which the interaction occurs.


The effect of human eosinophils on cultured human nasal epithelial cell activity and the influence of nedocromil sodium in vitro. Although there is increasing evidence of a pathogenic role for eosinophils in the airway epithelium, there is little direct evidence which demonstrates that eosinophils influence epithelial cell activity in humans. We have cultured human nasal epithelial cells in vitro and studied the effect of isolated human eosinophils on the ciliary beat frequency (CBF) and cell membrane integrity of these cells after incubation in the absence or presence of 0.1 microM phorbol 12-myristate 13-acetate (PMA) or 0.1 mg/ml opsonized latex beads and the absence or presence of 10(-5) M nedocromil sodium. CBF was monitored by an analogue contrast-enhancement technique, and cell damage was assessed by release of 51Cr from the cells. Cell cultures were also assessed for the percentage of eosinophil cationic protein (ECP) released into the medium at the end of incubation. Neither 0.1 microM PMA, 0.1 mg/ml opsonized latex beads, 10(-5) M nedocromil sodium, nor eosinophils alone altered the CBF of the epithelial cells. PMA-stimulated eosinophils, however, attenuated the CBF significantly, from 10.2 +/- 0.3 to 8.8 +/- 0.4 Hz (P less than 0.05) after 15 h of incubation. Similarly, opsonized latex bead-stimulated eosinophils led to a significant attenuation of CBF from 9.2 +/- 0.3 to 8.4 +/- 0.3 Hz (P less than 0.05), 6.9 +/- 0.5 Hz (P less than 0.001), and 7.5 +/- 0.3 Hz (P less than 0.001) after 2, 15, and 24 h of incubation, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Differential regulation of the genes encoding platelet-derived growth factor receptor and its ligand in rat lung during microvascular and alveolar wall remodeling in hyperoxia. The growth factors that operate while the lung remodels in hyperoxia are not known. At the lung periphery, high oxygen levels cause cell hypertrophy and hyperplasia, and this results in a thickening of the alveolar-capillary membrane and the walls of its associated microvessels. The present study examines gene expression of platelet-derived growth factor (PDGF) receptor and its ligand in this region of the lung of rats breathing 87% oxygen and compares this with the levels of expression in normal lung. In similar peripheral lung tissue, the proliferative response of specific cell populations has been assessed by [3H]thymidine incorporation and autoradiography. Normal lung expresses PDGF alpha-receptor subunit transcripts of 6.5 and 4.7 kb and PDGF beta-receptor transcripts of 5.5 and 4.5 kb. PDGF A-chain transcripts of 2.9, 2.3, and 1.7 kb are also expressed, each being at 10-fold higher levels than the single 3.5-kb transcript detected for PDGF B-chain. Within hours of breathing high concentrations of oxygen, mRNA levels change rapidly for the PDGF receptor subunits. These levels return to normal after 1 day and then decline over the next 28 days of exposure. PDGF A-chain mRNA increases 12 to 18 h after exposure, but then returns to normal levels. It is the PDGF B-chain mRNA that responds most to hyperoxia by increasing 10-fold on day 3. This increase immediately precedes the proliferative response on day 4 of microvascular adventitial fibroblasts, precursor smooth muscle cells, and epithelial cells but not smooth muscle cells, which do not proliferate until day 28.


Neutral endopeptidase of a human airway epithelial cell line recovers after hypochlorous acid exposure: dexamethasone accelerates this by stimulating neutral endopeptidase mRNA synthesis. Hypocholorous acid (HOCl) exposure of Calu-1 cells in situ leads to a relatively rapid and substantial decrease in whole cell neutral endopeptidase (NEP) activity that may result from the internalization of NEP from plasma membrane surfaces. To confirm this, and to assess the time course of changes in cell NEP after oxidant exposure and the potential influence of corticosteroid treatment on these, we evaluated Calu-1 NEP activity by high performance liquid chromatography and NEP-specific mRNA over the ensuing 48 h after HOCl in the presence or absence of 1 microM dexamethasone. Cells, grown to confluency in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, were exposed for 5 min to 100 microM HOCl and then maintained in culture for 48 h thereafter. Before exposure, some cell plates were cooled to 4 degrees C and/or incubated for 5 min in 1 mM sodium azide. In some experiments, post-HOCl changes in NEP-specific mRNA in the presence or absence of dexamethasone were also evaluated using Northern blot analysis. We found that sodium azide at 4 degrees C totally blocked the effect of HOCl on Calu-1 NEP (n = 6). In the absence of sodium azide, NEP activity spontaneously recovered to preexposure levels within 24 h. This recovery occurred 6 h earlier in the presence of 1 microM dexamethasone. Furthermore, dexamethasone increased NEP activity at 24 and 48 h after HOCl. Northern blot analysis indicated that NEP-specific mRNA did not change during spontaneous recovery, but was increased by dexamethasone 24 h after HOCl.(ABSTRACT TRUNCATED AT 250 WORDS)


Ontogeny of beta-adrenergic receptors in pulmonary arterial smooth muscle, bronchial smooth muscle, and alveolar lining cells in the rat. beta-Adrenergic receptors play an integral role in the modulation of cell function in the developing lung. In the rat, there are marked increases in beta receptor density in whole lung during postnatal maturation, but it is now known whether there are differential developmental changes in receptor density in specific cell types. Quantitative light microscopic autoradiography with [125I]iodocyanopindolol ([125I]ICYP) was used to determine maturational changes in beta-adrenergic receptor density in pulmonary arterial smooth muscle (ASM), bronchial smooth muscle (BSM), and alveolar lining cells (ALC) in rat lung during postnatal development (1 day to 6 mo). [125I]ICYP binding to whole lung sections revealed a single class of high-affinity receptors; agonist competitive binding studies suggested that the receptors are primarily of the beta 2 subtype. beta-Adrenergic receptor density in newborn (1 day) lung was lowest in ASM cells and was comparable in BSM cells and ALC. In contrast, in lungs from adult rats (3 mo), receptor density was similar in ASM versus BSM cells and was 2-fold greater in ALC. In addition, the maturational pattern of increasing receptor density differed in ASM compared with BSM and ALC. Receptor density in ASM increased 93% from 1 to 13 days, another 92% from 13 to 20 days, and was unchanged thereafter. In contrast, receptor density in BSM cells did not change from 1 to 13 days, but it increased 65% from 13 to 20 days, rose another 47% from 20 days to 3 mo, and increased an additional 24% from 3 to 6 mo.(ABSTRACT TRUNCATED AT 250 WORDS)


Gene expression from adeno-associated virus vectors in airway epithelial cells. Lung diseases such as cystic fibrosis (CF) might be treated by gene therapy using viral vectors delivered to the airway. One potential vector is the defective human parvovirus, adeno-associated virus (AAV). We examined the AAV p5 transcription promoter for gene expression in immortalized cell lines derived from the airway (IB3-1) or pancreas (CFPAC-1) of CF patients. AAV vectors expressing the prokaryotic genes cat (pAAVp5cat) or neo (pAAVp5neo) from the p5 promoter were evaluated after introduction into IB3-1 or CFPAC-1 cells by lipofection. In transient assays in both cell lines, the cat gene was expressed 5- to 10-fold more efficiently from the p5 promoter than from a simian virus 40 early gene promoter (pSVcat). IB3-1 cells were transformed stably to geneticin resistance by pAAVp5neo at a 5-fold higher efficiency than by an SVneo vector. The AAV inverted terminal repeat (ITR) region immediately upstream of the p5 promoter appears to have an enhancer effect and the promoter also contains a CREB site which confers a response to forskolin. In IB3-1 cells, expression of the cat gene from a p5 promoter was decreased about 5-fold by deletion of both the upstream ITR and the CREB site. The AAVp5neo vector was also packaged into AAV particles and used to infect IB3-1 cells as a transducing virus. Under these conditions, 60 to 70% of the cells could be stably transformed to geneticin resistance. Thus, AAV transducing vectors appear to be a highly efficient delivery system for stable integration and expression of genes in cultured airway epithelial cells.


[Angiotensin-converting enzyme inhibitors as neutralizers of hydroxyl radical]. Angiotensin converting enzyme inhibitors are utilized in the treatment of essential hypertension and of chronic cardiac failure. They are also employed in the treatment of the myocardial lesion of ischemia-reperfusion, which involves oxygen free radicals. In the present study we investigated the possibility of three angiotensin converting enzyme inhibitors (captopril, enalapril, lisinopril) to act as hydroxyl radical scavengers. The rate constants for reactions of those compounds with .OH were determined using the deoxyribose method. All there compounds proved to be good scavengers of .OH with rate constants of about 10(10)M-1s-1 and are iron chelators specially enalapril. The fact that captopril possesses a thiol group does not confer an higher antioxidative capacity. These results suggest that scavenging of oxygen free radicals may be a possible mechanism contributing to the therapeutic effect of angiotensin converting enzyme inhibitors.


[A case of Klippel-Trenaunay-Weber syndrome accompanied by congenital hydrocephalus and micropolygyria]. We report a patient with the Klippel-Trenaunay-Weber syndrome accompanied by congenital hydrocephalus, which was slowly progressive and an Ommaya's reservoir was set up. However, the hydrocephalus remained stable even when the shunt was removed due to infection. Generalized tonic clonic convulsions had appeared from six months after birth and were treated with valproic acid. The electroencephalogram showed hypsarrhythmia. He died at eight months of age. Autopsy revealed extensive micropolygyria of the bilateral cerebral hemispheres and hydrocephalus. To our knowledge, it is rare for the Klippel-Trenaunay-Weber syndrome to be accompanied by congenital hydrocephalus, and there has been no previous report of its occurrence with micropolygyria.


Carbohydrates in herpesvirus infections. Carbohydrate structures constitute important factors in the pathogenesis of herpesvirus infections for three reasons: (i) Human herpesviruses specify several biologically active glycoproteins, where co-translational addition of carbohydrate units is essential for generation of functioning viral glycoproteins. (ii) Cellular carbohydrate structures are parts of the cellular receptor complex at least for some herpes-viruses. (iii) Peripheral carbohydrate structures are engaged in modulation of the immunological properties of herpesvirus glycoproteins. The present review will consider all these three aspects mainly for HSV-1 glycobiology, which has been most fully investigated so far. Due to the many structural similarities between glycoproteins from different human herpesviruses, many of the conclusions should also be applicable for other human herpesviruses.


Inhibition of HIV infection of resting peripheral blood lymphocytes by nucleosides. T cells in the peripheral blood are largely in the resting state and represent a significant pool of potential targets for HIV infection. The protection of these cells from infection is an important goal of nucleoside therapy. Resting PBL may not be protected effectively by such nucleosides as azidothymidine (AZT) since anabolic phosphorylation of thymidine nucleosides is reported to be limited in these cells. In this study we used DNA amplification procedures to follow HIV proviral DNA formation in resting T cells and to determine the ability of azidodeoxythymidine (AZT), dideoxyinosine (ddI), and dideoxycytosine (ddC) to inhibit this process. Experiments confirm that resting PBL synthesize HIV proviral DNA sequences. Drug titrations showed that this synthesis could be inhibited by nucleosides. ddC was the most potent drug, inhibiting transcription at the U5 region. ddI and AZT at similar concentrations (10 microM) did not inhibit. ddI in the concentration range of 10-100 microM was able to inhibit production of transcripts containing U3 and env sequences in resting cells. Similar inhibition levels were accomplished by AZT at 10-100-fold lower drug concentrations. These results demonstrate that resting T cells can be protected from HIV infection by nucleosides and that thymidine nucleosides are effective inhibitors despite the limited potential for anabolic phosphorylation.


Case report: diffuse gastrointestinal bleeding. Diffuse gastrointestinal bleeding in an immunodeficient patient is presented. Two hours after in vivo erythrocyte labeling, abnormal activity was observed in the wall of a distended colon. If this abnormal concentration had been luminal, a focal, surgically treatable lesion could not have been excluded. This pattern of hemorrhagic colonic lesions has been described pathologically, but not scintigraphically, in immune deficient patients.


[Inflammatory-type malignant fibrohistiocytoma of the mesocolon associated with colonic parasitosis due to Trichocephalus]. A case of inflammatory malignant fibrous histiocytoma of the mesocolon is reported. The lesion presented as a right lower quadrant abdominal mass, located at the mesocolon, with obstructive symptoms and accompanied by a severe infestation by Trichuris trichiura. It had a benign histologic appearance and was considered initially as an inflammatory and necrotic process with severe tissue eosinophilia secondary to the severe parasitosis. The tumor relapsed 13 months later at the anastomotic site and the patient died. Diagnostic, prognostic and therapeutic aspects of this rare tumor are discussed.


[Myeloperoxidase activity as an indicator of leukocyte infiltration into the ischemic area in the middle cerebral artery occlusion model of the rats]. Myeloperoxidase was solubilized with hexadecyltrimethylammonium bromide and myeloperoxidase activity was measured with a dianisidine-H2O2 assay in the ischemic area after middle cerebral artery occlusion in rats. This enzyme could be measured 6 hours after the occlusion of the middle cerebral artery, then showed two peaks, one after 8 hours and another after 18 to 24 hours in 2 phase fashion as could be measured as far as 7 days, which was the termination of this experiments. These findings suggest that leukocyte infiltration into the ischemic area play an important role for the extension of the ischemic area. Further study should undertake whether leukocyte depletion decrease the ischemic area or not.


[Alteration of atrial natriuretic peptide and cyclic GMP in cerebrospinal fluid in canine kaolin-induced hydrocephalus]. CSF and Plasma concentrations of atrial natriuretic peptide (ANP) and cyclic GMP (cGMP), which is regarded as a second messenger of ANP, were measured intermittently during the progress of canine kaolin-induced hydrocephalus. Data were analyzed being divided into three groups, normal, acute (within 2 weeks after intracisternal injection of kaolin suspension) and chronic (from 3 to 4 weeks after injection of kaolin suspension) stages of hydrocephalus. The presence of ventricular dilatation was evaluated by MRI or postmortal dissection. ANP, cGMP in CSF and CSF pressure significantly increased in the acute stage of hydrocephalus. In the chronic stage, ANP in CSF and CSF pressure had no statistical difference with data of the normal stage. Cyclic GMP in CSF kept significantly high value in the chronic stage of hydrocephalus. CSF concentrations of Na, CSF osmolarity, plasma ANP, plasma cGMP, plasma ADH, serum Na and serum osmolarity did not change significantly in the course of hydrocephalus. There was a significant positive correlation between ANP in CSF and CSF pressure. ANP in CSF did not correlate with degree of ventricular dilatation. Cyclic GMP in CSF did not correlate with ANP in CSF, nor with CSF pressure. These data suggest that concentration of ANP in CSF may alter directly or indirectly depending on CSF pressure in kaolin-induced hydrocephalus. And cGMP in CSF was suggested to depend not on ANP in CSF, but on other unknown factors in kaolin-induced hydrocephalus.


[A case of small epidermoid cyst in cauda equina with manifest regional hypesthesia]. This report concerns the 22-year-old female who has been suffering the paresthesia in the left buttock and thigh for three months. Neurological examination revealed nothing other than hypesthesia and hypalgesia which distributed in the third, fourth and fifth segments of left sacral region with the loss of anal reflex. Pain had not been noticed until when she came to our clinic. Myelography and MRI showed small irregular round mass occupying a third of the spinal canal behind the body of fourth lumbar spine. In the axial view of MRI, the mass was enhanced by the gadolinium (Gd) except for the small portion of its center. She underwent the surgical treatment which revealed the tumor entangling four nerves of cauda equina in its center as imaged in MRI. The tumor was epidermoid cyst which presumably caused the chemical meningitis and involved those nerves. That resulted in the sensory disturbance in the left sacral region, although the tumor was not large enough to compress the nerves.


Preparation and biodistribution in mice of [11C]carfentanil: a radiopharmaceutical for studying brain mu-opioid receptors by positron emission tomography. A potent mu-opioid agonist, [11C]carfentanil, was prepared by the methylation of carfentanil carboxylic acid with [11C]methyl iodide in order to study brain mu-opioid receptors by positron emission tomography. Synthesis (including purification) was completed within 25 min and the radiochemical yield was approximately 40%. The radiochemical purity of the product was more than 99% and its specific activity was 3.7-7.4 GBq/mumol. Biodistribution studies performed in mice after intravenous injection showed a high brain uptake and rapid blood clearance, so a high brain/blood ratio of 1.5-1.8 was found from 5 to 30 min. Regional cerebral distribution studies in the mouse showed a significantly higher uptake of [11C]carfentanil by the thalamus and striatum than by the cerebellum, with the radioactivity in the striatum disappearing more rapidly than that in the thalamus. Treatment with naloxone significantly reduced the uptake of [11C]carfentanil by the thalamus and striatum. These results indicate that [11C]carfentanil binds specifically to brain mu-opioid receptors.


Potent cytotoxic action of the immunotoxin SWA11-ricin A chain against human small cell lung cancer cell lines. The cytotoxic activity profile of an immunotoxin, SWA11-ricin A chain, recognising a cell-surface antigen associated with human small cell lung cancer (SCLC), was examined in detail using a panel of SCLC, non-SCLC and non lung tumour cell lines in tissue culture. SWA11-ricin A chain was potently and selectively active against three SCLC cell lines of both classic and variant morphologies, inhibiting the incorporation of 3H-leucine with an IC50 of 5 x 10(-11) M. At a concentration of 1 x 10(-8) M, the SWA11 immunotoxin could selectively eliminate in excess of 99.9% of clonogenic tumour cells. Intoxication proceeded rapidly following a 4 h lag phase; the initial rate of protein synthesis inhibition occurred with a t50 of 2 h and a t10 of 7 h. The cytotoxic activity of SWA11-ricin A chain was potentiated by 100-fold in the presence of the carboxylic ionophore monensin at 1 x 10(-7) M. Kinetic studies revealed that monensin enhanced the rate of protein synthesis inhibition by two-fold and eliminated the lag phase suggesting a rapid effect on either the rate or route of internalisation. Studies with SWA11 could detect no influence of monensin on the rate of antibody internalisation and a transient delay in the delivery of internalised antibody to lysosomes was observed by immunoelectron microscopy.