Binding of Trichophyton rubrum mannan to human monocytes in vitro. We recently reported that the mannan component of Trichophyton rubrum cell wall (TRM) has an inhibitory influence on cell-mediated immune function in vitro. We now describe experiments designed to identify the target cell for this effect of TRM. T. rubrum mannan labeled with fluorescein (FITC-TRM) was incubated with peripheral blood mononuclear leukocytes, monocytes, or lymphocytes. Binding and uptake of the FITC-TRM were monitored by fluorescence microscopy and flow cytometry. Approximately 10% of mononuclear leukocytes were stained with this reagent and the fluorescent cells appeared to be monocytes by morphology. Virtually all purified monocytes and no purified lymphocytes stained with FITC-TRM. Flow cytometry to analyze FITC-TRM monocyte-specific binding of FITC-TRM involved the use of a phycoerythrin-labeled anti-CD14 antibody to identify monocytes. The only cells stained with FITC-TRM were those stained with the monocyte-specific antibody. The ability of monocytes to endocytose mannan was assessed by fluorescence microscopy. Cells were exposed to FITC-TRM and washed, and the staining pattern recorded periodically over a 48-h incubation period. After 15 min, staining was homogeneous and involved the entire cell surface; by 30 min, "patching" was observed; by 90 min, bright granules had formed along the cell border and a large number of small granules were present in the cytoplasm; by 8-12 h, the fluorescent granules were enlarged in size and reduced in number; by 24-36 h, the intensity of cytoplasmic fluorescence began to diminish; and, after 48 h, all fluorescent staining had disappeared. An additional feature of staining during the 8-12-h period was the appearance of a large round bright spot in the nuclear region of each cell, which may represent nucleolar staining. A role for "mannan receptors" is suggested by observations that FITC-TRM binding was prevented by unlabeled TRM or pretreatment of the monocytes with trypsin. Our finding that monocytes selectively and specifically bind TRM appears to identify the monocyte rather than the lymphocyte as the target cell for the inhibitory effect of mannan on cell-mediated immune function.


MR imaging of extracranial nerve sheath tumors. We retrospectively reviewed MR images of 32 histologically proven extracranial nerve sheath tumors (NSTs). There were 23 benign (10 neurofibromas, 13 schwannomas) and 9 malignant NSTs. On T1-weighted images (T1WIs) tumors were isointense or slightly hyperintense to muscle. On T2-weighted images (T2WIs) (11 lesions) and enhanced T1WIs (1 intraspinal lesion), a target pattern with peripheral hyperintense rim and central low intensity was seen in 12 of 23 (52%) benign NSTs (5 of 10 neurofibromas and 7 of 13 schwannomas). This pattern corresponded histologically to peripheral myxomatous tissue and central fibrocollagenous tissue. The pattern was absent in lesions with cystic, hemorrhagic, or necrotic degeneration. These tumors were hyperintense and variably inhomogenous on T2WIs. Malignant NSTs were hyperintense and variably inhomogenous on T2WIs and mimicked benign variably inhomogeneous lesions unless involvement of contiguous structures was visualized. A target pattern was not visible in malignant lesions. Magnetic resonance imaging cannot distinguish schwannomas from neurofibromas, and benign tumors may mimic malignant NSTs when cystic, hemorrhagic, and necrotic degeneration is present. A target pattern may be visualized in some benign NSTs, and evaluation of this sign with assessment of location and growth along nerves may help to avoid confusion with other lesions.


Human papillomavirus (HPV) types specific of epidermodysplasia verruciformis detected in warts induced by HPV3 or HPV3-related types in immunosuppressed patients. Epidermodysplasia verruciformis (EV) is characterized by an abnormal genetic predisposition to infection with specific types of human papillomavirus (HPV). Specific defects of the cell-mediated immunity and/or of the control of HPV infection in keratinocytes are assumed to be involved in the development of the disease. As a model to test this hypothesis, we have studied the prevalence of EV-specific HPV in skin warts of 56 immunosuppressed patients. All main types of cutaneous HPV (HPV1, 2, 3, 4, 10, and 28) responsible for skin warts in the general population were detected by blot hybridization. EV-specific HPV (HPV5, 20, and 23) were detected in three patients. Four additional patients were found infected with HPV49, first characterized in the course of this study, and found to be related to EV HPV. A most important finding was that HPV5, 20, 23, and 49 were always codetected with HPV3 or the related types HPV10 and 28. None of the specimens showed the typical clinical morphology of EV lesions. In none of these specimens was the specific cytopathic effect of EV recognized; instead that of HPV3 and related types was seen. No evidence for productive EV HPV DNA replication was obtained for the three specimens that could be further analyzed by in situ hybridization. Our data suggest that HPV3 infection favors infection with EV HPV in immunosuppressed patients but that the full expression of EV HPV is usually restricted as in the general population.


[Investigation of two cases of adenoid cystic carcinoma of the esophagus]. A 59-year-old man and a 55-year-old man with adenoid cystic carcinoma of the esophagus are presented. They were also treated with esophagectomy but whose prognosis were significantly different. The former has been still alive for 3.5 years and the later died at 7 months after the operation. In the patient with good prognosis, the tumor was 1.6 x 1.2 cm in size and pathologically invaded muscularis propria (mp) with no metastases to lymph nodes (pathological stage I). It has been reported that the prognosis of adenoid cystic carcinoma of the esophagus is poorer than that of other histological type of carcinomas. However, it is suggested that if the tumor can been resected when it is small in size with no metastases, it will be able to expect for good prognosis as in this presentation.


The molecular mechanisms of cardiovascular and renal regulation by endothelin peptides. In summary, we have seen that endothelins are potent cardiovascular and renal regulatory peptides. Cardiovascular regulation by endothelin requires complex spatial and temporal regulation of endothelin gene expression and the coordinated interplay of numerous signaling pathways. Although the precise physiologic role for endothelin peptides in the cardiovascular and renal systems remains uncertain, two general models can be proposed, as follows. (1) Endothelin appears to act as an autocrine or paracrine hormone involved in long-term (hours to days) regulation of cardiovascular and renal function in normal physiology. Similar regulation occurs in response to other cardiovascular hormones such as angiotensin II, atrial natriuretic peptides, and catecholamines. In this respect it is noteworthy that endothelin also interacts with other hormonal systems that affect cardiovascular status, such as renin-aldosterone and atrial natriuretic peptide (see references 9, 161, 162, and 213-217). (2) Endothelin might also function as a proinflammatory peptide, being locally produced at sites of vascular damage and injury. The fact that endothelin secretion is stimulated by cytokines, growth factors, and transforming growth factor beta is consistent with this role. In addition, the mitogenic actions of endothelin could contribute to vascular remodeling in the inflammatory response. A similar, defensive role has been proposed for other regulatory peptides. Endothelin has also been implicated in the pathogenesis of numerous disorders such as hypertension (see references 28, 56, 168, 184, and 224 through 226), cerebral and myocardial vasospasm (see references 180, 223, and 227 through 233), acute renal failure, and cyclosporine nephrotoxicity. It is clear that the development of specific ECE inhibitors and endothelin receptor antagonists will enable definitive experiments addressing the role of endothelin in normal physiology and the putative role of endothelin in the development of hypertension and other cardiovascular disorders. Further identification and biochemical analysis of signaling networks evoked by endothelin, in conjunction with physiologic studies, should provide a detailed understanding of the complex biologic events regulated by endothelin peptides.


Biologic activities of parathyroid hormone (1-34) and parathyroid hormone-related peptide (1-34) in isolated perfused rat femur. Parathyroid hormone-related peptide (PTHrP) has substantial homology with both human and rat parathyroid hormone (66% and 73%, respectively) in the first 15 amino acids. PTHrP (1-34) stimulates cyclic adenosine monophosphate (cAMP) release in bone and kidney, and these effects are felt to occur through interaction with the parathyroid hormone (PTH) receptor. Differences in the biologic potency between rat PTH(1-34) and human PTH(1-34)--and between PTHrP and PTH--have been described in a variety of experimental systems. In this study, we compared the bioactivity of these three amino-terminal synthetic fragments on the stimulation of cAMP formation in an isolated perfused rat femur preparation. Dose-response experiments demonstrated that PTHrP(1-34) was more potent in stimulating cAMP release than human PTH(1-34), whereas PTHrP(1-34) and rat PTH(1-34) were equipotent. Despite the fact that the extraction of immunoreactive rat PTH(1-34) and human PTH(1-34) was the same, rat PTH(1-34) was more potent in stimulating adenylate cyclase activity than human PTH(1-34). These data show that the isolated perfused rat femur preparation is an effective method for evaluation of the effects of PTH and PTHrP. Despite significant structural differences in the binding domain between rat PTH(1-34) and PTHrP(1-34), the effects of rat PTH(1-34) and PTHrP(1-34) are similar. Because the structure of rat PTHrP(1-34) and human PTHrP(1-34) are identical, and because it is desirable to utilize homologous systems to study the potency and effects of test peptides, it would appear that rat PTH(1-34) is the most appropriate peptide for comparison with PTHrP in rat-based experimental systems.


Cytomegalovirus colitis in an older woman, successfully treated with ganciclovir. Cytomegalovirus (CMV) infections are usually found in immunocompromised persons and rarely present in those who are immunocompetent. We report a case of CMV colitis in an elderly woman presenting with delirium, prolonged fever, and diarrhea. Treatment with ganciclovir, an antiviral agent, resulted in resolution of the colitis within 3 weeks without noticeable side effects. Older adults may be more susceptible to CMV infection by virtue of age-related changes in immune function. The possibility of CMV colitis should be considered in an elderly person with persistent fever, diarrhea, and negative stool cultures.


Identification of nitric oxide reductase activity in Rhodobacter capsulatus: the electron transport pathway can either use or bypass both cytochrome c2 and the cytochrome bc1 complex. Several strains of Rhodobacter capsulatus have been shown to possess a nitric oxide reductase activity (reaction product nitrous oxide) after anaerobic phototrophic growth, but not after aerobic growth. The reductase is associated with the cytoplasmic membrane and electrons can reach the enzyme via the cytochrome bc1 complex. However, use of appropriate strains has shown that neither the latter, cytochrome c2 nor cytochrome c' is essential for the reduction of nitric oxide. Inhibition by myxothiazol of nitric oxide reduction in a strain that lacks a cytochrome c2 establishes that in phototrophically grown R. capsulatus the cytochrome bc1 complex is able to transfer electrons to an acceptor that is alternative to cytochrome c2. Electron transport to nitric oxide from NADH or succinate generated a membrane potential. When isoascorbate plus 2,3,5,6-tetramethyl-p-phenylenediamine (DAD) was the electron donor a membrane potential was not generated. This observation implies that nitric oxide is reduced at the periplasmic surface of the membrane and that the reductase is not proton translocating.


Tn916 insertion mutagenesis in Escherichia coli and Haemophilus influenzae type b following conjugative transfer. Transposon Tn916 was shown to be capable of direct conjugative transfer in broth and membrane matings between strains of Escherichia coli K12 and between E. coli K12 and Haemophilus influenzae type b. Only Tn916 was transferred, but Tn916 donor ability was not itself inheritable by the recipients and seemed to be associated with the presence of Tn916 on a non-conjugative pBR322-derived vector in the original donor strain. Transfer of Tn916 by conjugation was found to be an efficient method for producing insertion mutations in the chromosome of recipient cells. Although such insertions were unstable when the cells were grown under non-selective conditions, it was possible to show that over 40% of the isolated Tn916 insertions in the chromosome of E. coli K12 were in gene(s) concerned with histidine biosynthesis, implying that there is a partial hot-spot for Tn916 insertion on the E. coli K12 chromosome. When a strain of H. influenzae type b was used as a recipient, out of approximately 1500 transconjugants tested, two mutants were isolated with insertions in genes controlling the expression of iron-regulated transferrin-binding proteins. These mutants constitutively produced major 76 kDa and minor 90 kDa proteins which bound transferrin, even when grown under iron-sufficient conditions. Tn916 insertion mutagenesis, following transfer by conjugation, is a convenient method for isolating mutations in genes concerned with iron acquisition by this important human pathogen.


Effects of growth temperature on alginate synthesis and enzymes in Pseudomonas aeruginosa variants. Spontaneous variation of the level of alginate synthesis in Pseudomonas aeruginosa was associated with changes in the activity of all four enzymes leading to synthesis of GDP-mannuronic acid, the activated precursor for polymerization. For the high-alginate-producing variant 8821M, alginate yield and properties, as well as the levels of alginate enzymes, were dependent on growth temperature. In contrast, levels of alginate and enzymes in the mucoid parent strain 8821 were very low and near temperature-independent. The difference in the specific activity of GDP-mannose dehydrogenase (GMD), encoded by the algD gene, between the two strains was associated with the alginate biosynthetic ability and with the degree of activation of the algD promoter, measured using the algD-xylE transcription fusion on plasmid pVD2X. Maximal activity of the four enzymes was observed in strain 8821M grown at 30 degrees C, a temperature below the optimum for growth (35 degrees C). The effect of temperature on GMD activity could not be explained by the regulation of the algD promoter by temperature, since expression of pVDZX appeared to be more active at 35 degrees C, when the decrease of pVD2X copy number with increasing temperature was taken into account. The involvement of enzymes that catalyse steps downstream from the formation of the activated precursor should also be considered, as suggested by differences in the molecular mass of alginates synthesized by the two strains at various temperatures. Acetyl content of alginates increased as temperature decreased and strain 8821M produced the highest levels of acetylated polymers. The degree of acetylation appeared to be related to growth rate and could reflect acetyl-CoA availability.


Akuammine and dihydroakuammine, two indolomonoterpene alkaloids displaying affinity for opioid receptors. Akuammine [1], an indolomonoterpene alkaloid, which is the major component of the seeds of Picralima nitida, was reduced to dihydroakuammine [4]. This compound has structural analogy with eseroline [7], for which affinity for opiate receptors was reported. The present investigation showed that 1 and 4 also bind (with lower affinity however) to mu and kappa opiate receptors. 1H- and 13C-nmr spectra of 1 and 4 have been fully assigned by 2D nmr experiments.


The liberating effects of RN-to-BSN education. This study investigated whether postlicensure baccalaureate education was associated with decreased acceptance of oppressed status for nursing and the self, with behavioral changes in job performance, and in joining a professional organization. Selected writings of Freire (1970a, 1970b, 1973) concerning education of the oppressed were used to form the conceptual framework. Baccalaureate education was conceptualized as a means of overcoming oppression-related behaviors and attitudes in nurses. The criterion measures were self-reports of job performance, joining the American Nurses Association (ANA), self-concept measurements, and perception of nursing measured by a semantic differential. Except for self-concept, which remained unchanged, all the findings were in the predicted direction supporting the conceptual framework.


Competencies of novice nurse educators.

Competencies of novice nurse educators. Appropriate preparation for the nurse faculty role has been debated for many years. Currently, the master of science in nursing (MSN) is considered to be the minimal acceptable preparation. The focus of most MSN programs, however, is advanced knowledge and skill in clinical nursing. The purpose of this study is to identify the perceptions of novice nurse faculty concerning nurse faculty competencies, to ascertain the extent to which novice nurse faculty demonstrate the competencies, and to determine mechanisms through which competencies are obtained. The findings indicate that many novice nurse faculty are not educationally prepared for the faculty role.


The affective outcomes of course work on computer technology in nursing. At the University of Iowa, nursing students are introduced to computer technology as one unit in a course designed to explore nursing as a profession. A single group, pretest/posttest design was used to evaluate changes in attitudes associated with the course work. Relationships of attitudes scores and eight background variables were studied. Posttest attitudes scores were significantly higher than pretest scores. The attitudes scores were positively related to skills scores at a statistically significant level of .05, with no significant relationship between attitudes scores and knowledge base scores. Four of the eight background factors were related to the attitudes scores at a statistically significant level of .05.


The NCLEX-RN and nurse educators.

The NCLEX-RN and nurse educators. The involvement of nurse educators with recent NCLEX-RN changes was studied. One hundred and twenty administrators from associate (ADN) and baccalaureate (BSN) degree programs across the country responded to a questionnaire regarding educator knowledge about, involvement in, and opinion of the change process. Findings revealed that administrators from both program types felt ill-informed regarding NCLEX-RN changes prior to their implementation, and they expressed a desire to have had input in the change process. While administrators had varied opinions regarding the passing standard change and the content change, they were strongly opposed to the manner in which change occurred.


Ways of knowing among beginning students in nursing. Twenty-one students in their first quarter of nursing school were interviewed to determine the utility of the typology for ways of knowing developed by Belenky, Clinchy, Goldberger, and Tarule (1986) for understanding the cognitive development of nursing students; the differences in epistemological perspectives by gender, age, and ethnicity; and the forces in nursing school that encourage developmental transitions. The typology was found to be applicable for Caucasian women and provided important insights on differences among groups of students. Some of the implications for providing supportive educational environments are presented.


Cognitive coaching for nurse educators.

Cognitive coaching for nurse educators. Major changes within the work setting may be required in order to meet the requisite conditions for a successful peer coaching program. A cadre of teachers and administrators who are committed to faculty professional development is fundamental to the implementation and success of this type of program. The peer coaching approach to faculty development allows for personal and professional growth through trust, openness, and curiosity. Learning is promoted as a never-ending, lifelong process. Additionally, discussion and critique of others' views are promoted. These aid in the refinement of individual thinking and enhance individual competencies. Peer cognitive coaching is a strategy that involves the use of human resources in an effort to enhance the education of both teachers and students. Cognitive coaching acknowledges nurse educators as professionals and encourages them to function as self-regulating learners.


Metabolic and histologic investigation of the nature of nephrocalcinosis in children with hypophosphatemic rickets and in the Hyp mouse. To investigate the biochemical nature of nephrocalcinosis in children with hypophosphatemic rickets treated with orally administered phosphate and vitamin D, we studied five such patients, aged 3.7 to 12.3 years, during treatment and again 3 days after it had been discontinued. Treatment was associated with significant increases in mean serum phosphate concentration and urine phosphate/creatinine ratio, from 0.71 to 1.03 mmol/L and from 3.61 to 9.42 mmol/mmol, respectively. Significant correlation was found between urine phosphate/creatinine and oxalate/creatinine ratios (r = 0.670; p less than 0.01); however, the mean urine oxalate/creatinine ratio of 65.0 mumol/mmol while patients were taking phosphate orally was not significantly different from the ratio of 59.0 mumol/mmol when treatment was discontinued. Kidney biopsy specimens from three of the patients showed that the renal calcifications were located mainly intratubularly and were composed exclusively of calcium phosphate. In a further investigation of the nature of phosphate-induced nephrocalcinosis, six 6-week-old male Hyp mice, the murine analog of the human disease, received oral phosphate therapy with drinking water for 48 days; six others served as control animals. Mice in the experimental group excreted more phosphate (p less than 0.001) and less calcium (p less than 0.01) than control mice did, and medullary nephrocalcinosis, with a high kidney calcium content, developed (p less than 0.001). Histologic sections showed that the renal calcifications were located intratubularly and were composed of calcium phosphate. We conclude that, both in children with hypophosphatemic rickets and in the Hyp mouse, the development of nephrocalcinosis is associated with high oral phosphate intake and subsequent deposition of calcium phosphate precipitates in the kidney.


Extraintestinal rotavirus infections in children with immunodeficiency. Some rotavirus strains, including vaccine candidates, have been demonstrated to cause hepatitis in immunodeficient and malnourished mice and to grow in human liver cells. To determine whether rotavirus spreads outside the intestine in naturally infected children, we examined tissues from four immunodeficient children affected with severe combined immunodeficiency disease, acquired immunodeficiency disease syndrome, or DiGeorge syndrome. Chronic rotavirus-related diarrhea, which persisted until death, had also developed in each child. Using indirect immunoperoxidase techniques, we identified rotavirus antigen in the liver and kidney with a hyperimmune guinea pig antiserum prepared to double-shelled rotavirus particles. Similar immunostaining with an antiserum to a rotavirus nonstructural protein (NS26) provided evidence of active virus replication. The observed reactivity was eliminated specifically when serial sections were immunostained with the same antiserum that had been absorbed with either double-shelled rotavirus particles or NS26. Immunostaining was not observed in the liver of children with other diseases (alpha 1-antitrypsin deficiency, inspissated bile syndrome, and acute rejection of a transplanted liver). These findings demonstrate that rotavirus infections in children can extend beyond the intestinal tract. Further studies are warranted to determine whether extraintestinal rotavirus replication occurs in children without severe immunodeficiency, such as malnourished children.


Fatal disseminated infection with human herpesvirus-6. A 13-month-old immunocompetent girl had fever, rash, and multisystem disease, and she eventually died of cardiac failure. Autopsy revealed intracellular viral inclusions of the herpesvirus group, with results of in situ hybridization positive for human herpesvirus-6. This is apparently the first case of fatal disseminated herpesvirus-6 infection.


Cytomegalovirus and Pneumocystis carinii pneumonia in children with acquired immunodeficiency syndrome. To ascertain the effect of cytomegalovirus (CMV) infection on the course of Pneumocystis carinii pneumonia (PCP) in children with acquired immunodeficiency syndrome (AIDS), we reviewed the charts of all children with AIDS who also had a lung biopsy specimen or a bronchoalveolar lavage specimen cultured for CMV at the time PCP was diagnosed. The data indicate that children with AIDS and PCP whose cultures are positive for CMV do not have a poorer prognosis during a first episode of PCP compared with children whose cultures are negative for CMV.


Effects of dexamethasone on the hypothalamic-pituitary-adrenal axis in preterm infants. The effects of dexamethasone therapy on the hypothalamic-pituitary-adrenal axis were tested in 10 premature infants with bronchopulmonary dysplasia by means of the corticotropin-releasing hormone stimulation test before therapy and on the seventh day of therapy. Adrenocorticotropic hormone and cortisol levels were determined by radioimmunoassay. There was significant suppression of the hypothalamic-pituitary-adrenal axis after 7 days of a currently used dexamethasone treatment regimen. A site of suppression was located at the level of the pituitary gland.


Eicosanoids content in small intestinal mucosa of children with celiac disease. Celiac disease (CD) is characterized by diarrhea, growth retardation, and weight loss in genetically susceptible subjects on a gluten-containing diet. The exact pathogenesis of CD is still obscure, but it is considered to be immunologically mediated. We have previously shown elevated prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) content in small intestinal mucosa obtained from active celiac children. In the present study, we found significantly elevated PGE2, leukotriene B4 (LTB4), and leukotrienes C4, D4, and E4 (LTC4D4E4) content in small bowel mucosa from children suffering from CD on a gluten-containing diet in comparison to control subjects. PGE2 was 25,278 +/- 7,761 vs. 4,478 +/- 426 pg/mg of protein (mean +/- SEM), respectively. LTB4 was 8,807 +/- 3,706 vs. 403 +/- 63 pg/mg of protein (mean +/- SEM), respectively. LTC4D4E4 was 15,369 +/- 4,085 vs. 2,998 +/- 279 pg/mg of protein (mean +/- SEM), respectively. We conclude that the elevated content of arachidonic acid metabolic products via cyclooxygenase and lipoxygenase pathways may contribute to the diarrhea and may be involved in the pathogenesis of mucosal injury.


Extensive hepatic necrosis in a premature infant. A fatal case of fulminant hepatic failure that occurred in the neonatal period is reported in a premature infant born after 27 4/7-weeks' gestation. Immediately after birth the infant had severe hypoxia and hypotension resulting from birth asphyxia, hypovolemic shock, and septicemia. At autopsy, histological appearance of the liver showed virtually total hepatocellular necrosis without features of fibrosis. Although the exact cause of hepatocellular injury cannot be fully ascertained, it is assumed that hypoxia and hypotension must have been the predominant factors leading to massive hepatic necrosis.


Freeze-dried, cross-linked bovine type I collagen: analysis of properties. This study was undertaken to assess the physical and biological properties of freeze-dried cross-linked bovine type I collagen and to assess its potential for use in the guided tissue regeneration method of treatment of periodontal disease in human adult subjects. The modulus of elasticity, swelling ratio, and biodegradation rate were investigated. The collagen sponge was implanted subdermally into Sprague-Dawley rats and a histological study carried out at 2, 7, 21, 35, and 49 days post implantation. Growth of human gingival and periodontal ligament derived fibroblasts on collagen sponge was assessed, as well as the effect of bovine collagen supernatants upon gingival and periodontal fibroblast cultures. The physical properties of the collagen sponge were consistent with good handling qualities and, therefore, it was appropriate for use at a surgical site. The histological study demonstrated a reduction in thickness of the collagen at 21 days; at 35 days there was a hazy appearance of the collagen remnants; and at 49 days the graft material had been completely replaced with fibrous tissues. The in vitro response of human gingival and periodontal fibroblasts to bovine collagen showed that, after 21 days, confluent fibroblast growth was observed around and underneath the sponge. The effect of bovine collagen supernatants upon fibroblasts demonstrated an apparent proliferative effect of the supernatant with both gingival and periodontal ligament fibroblasts. However, the non-parametric Friedman test revealed no significant differences between dilutions or time points. The overall findings provide encouraging evidence of the safety of freeze-dried cross-linked bovine collagen sponge in the surgical treatment of periodontal disease.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunity to self-antigens in periodontal disease. Auto-antibody to collagen, previously detected in periodontal disease, may represent either a response to local tissue damage or be the manifestation of a disturbance of the host immune response induced by the periodontal flora and its products. In an effort to distinguish between these two hypotheses, this study was undertaken to determine circulating IgG auto-antibody levels in 41 periodontal-disease patients against 12 self-antigens (salmon DS-DNA, calf SS-DNA, human and bovine thyroglobulin, rabbit proteoglycan, horse myoglobin, bovine myosin, actin, fetuin, human transferrin, cytochrome C, and human Type I collagen) and compare them to those in 21 periodontal disease-free subjects. None of the detected IgG auto-antibody levels were significantly different between periodontal disease and control sera (Mann-Whitney U-test, P greater than 0.05) except for human Type I collagen (P less than 0.05). Fifty-six percent of patients and 38% of controls were "broad responders;" i.e., 50% or more of the auto-antibody levels were higher than the median values of the control group; however, these values were not significantly different using the chi-square test. It was concluded that the destruction of connective tissue components is the primary driving force in the induction of the enhanced auto-antibody response found in periodontal disease. This response is apparently secondary to the primary bacterial infection which remains the major etiologic event.


The occurrence of periodontitis-related microorganisms in relation to titanium implants. The peri-implant space was examined for the presence of periodontitis-related microorganisms in 19 patients. All patients had been fitted with osseointegrated titanium implants which were clinically sound at the time of examination and all had been prescribed daily 0.2% chlorhexidine mouthrinse. In 17 patients, two contralateral fixtures in patients were selected, the remaining 2 patients had only one fixture, and associated mucosal clinical variables assessed for plaque accumulation, gingival inflammation, bleeding tendency, and probing depth. The bacteriological composition of submucosal plaque samples was investigated using non-selective media and selective media for Actinobacillus actimomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Prevotella intermedia (Pi). The peri-implant tissues were healthy. Twenty-two of 37 sites had a greater proportion of anaerobes than aerobes. Aa was detected at one site. Pg was not cultivated, and Pi was present in 7 of 37 sites, comprising between 0.4% and 60.9% of the total anaerobic viable counts. Since the presence of any of these organisms may play a contributory role to the loss of the fixture, it is recommended that the submucosal plaque of implants be monitored regularly for the presence of these periodontitis-associated species.


On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. 1. The mechanism of action of picrotoxin on GABA receptor channels in rat sympathetic neurones has been investigated with whole-cell clamp. In addition, the action of picrotoxin on single GABA channels has been examined in outside-out membrane patches from these cells. 2. Picrotoxin, at concentrations which dramatically reduced the amplitude of whole-cell GABA currents, did not alter the spectral time constants or single-channel conductance estimated by analysis of GABA-activated current noise. This was observed at potentials both negative and positive to the GABA reversal potential (i.e. for both inward and outward GABA currents). In control conditions, the slow and fast time constants from GABA noise were 40 +/- 14 ms and 2 +/- 0.4 ms, while the estimated single-channel conductance was 14 +/- 2 pS. In the presence of picrotoxin, the time constants and estimated single-channel conductance were 41 +/- 5 ms, 2.7 +/- 0.6 ms and 15 +/- 2.3 pS. 3. Picrotoxin did not alter the shape of the whole-cell GABA current-voltage relationship, indicating that the steady-state block was not voltage dependent. The lack of effect of picrotoxin on the GABA noise spectra and the lack of outward rectification makes it unlikely that picrotoxin acts by a simple voltage-dependent (or voltage-independent) channel blocking mechanism. In the presence of picrotoxin the reversal potential for GABA remained at approximately 0 mV in symmetrical chloride. 4. Distributions of total burst durations, obtained from single-channel records with low concentrations of GABA, were fitted with three or four exponential components. Picrotoxin had no consistent effect on the time constants of the total burst length distributions. It also did not alter the amplitude of the main conductance state. However, picrotoxin did reduce the frequency of channel openings. 5. The application of brief ionophoretic pulses of GABA, to cells under whole-cell voltage clamp, revealed that the rate of onset of block by picrotoxin was accelerated in the presence of GABA. In the absence of agonist, picrotoxin produced a more slowly equilibrating block. 6. Our data are consistent with a mechanism whereby picrotoxin binds preferentially to an agonist bound form of the receptor and stabilizes an agonist-bound shut state. This could, for example, mean that picrotoxin enhances the occurrence of a desensitized state or an allosterically blocked state.


Calcium influx and calcium current in single synaptic terminals of goldfish retinal bipolar neurons. 1. The calcium influx pathway in large synaptic terminals of acutely isolated bipolar neurons from goldfish retina was characterized using Fura-2 measurements of intracellular calcium and patch-clamp recordings of whole-cell calcium current. 2. Depolarization of bipolar cells with high [K+]o resulted in a sustained, reversible increase in [Ca2+]i in both synaptic terminals and somata. Removal of external calcium abolished the response, as did the addition of 200 microM-cadmium to the bathing solution, indicating that the rise in [Ca2+]i was due to entry of external calcium. Dihydropyridine blockers of voltage-gated Ca2+ channels also blocked the influx, and the Ca2+ channel agonist Bay K 8644 potentiated influx, implicating voltage-activated, dihydropyridine-sensitive channels in the influx pathway. 3. Under voltage clamp, depolarization from a holding potential of -60 mV evoked a slowly inactivating inward current that began to activate at -50 to -40 mV and reached a maximal amplitude between -20 and -15 mV. This current was identified as a calcium current because it decreased when the extracellular calcium concentration was lowered, increased when barium was the charge carrier, and was blocked by 200 microM-external cadmium. The current was substantially blocked by 1 microM-nitrendipine and potentiated by 0.1 microM-Bay K 8644, as expected for L-type Ca2+ channels; it was unaffected by omega-conotoxin. No evidence for transient or rapidly inactivating Ca2+ current was found. 4. At a given level of potassium depolarization, both the amplitude and the speed of increase in [Ca2+]i were greater in synaptic terminals than in somata. For instance, depolarization by 32.6 mM-potassium caused an increase in intracellular calcium of 400 +/- 23 nM in terminals and 180 +/- 20 nM in somata (mean +/- S.E.M., n = 73 terminals, n = 30 somata), with maximal rates of change of 40 +/- 3 and 12 +/- 2 nM/s, respectively. 5. The contribution of terminal and somatic currents to the total whole-cell Ca2+ current was determined under voltage clamp by local application of calcium or of blocking agents. While there was no qualitative difference between currents in terminals and somata, synaptic terminals accounted for 64 +/- 3% (mean +/- S.E.M., n = 12) of the total whole-cell calcium current, and somata accounted for 39 +/- 2%. Thus, the density of Ca2+ current was higher in the terminal, accounting for the greater magnitude and speed of Ca2+ influx observed in terminals in Fura-2 experiments.(ABSTRACT TRUNCATED AT 400 WORDS)


Novel suppression of an L-type calcium channel in neurones of murine dorsal root ganglia by 2,3-butanedione monoxime. 1. Voltage-activated currents through calcium channels in primary cultures of murine dorsal root ganglion cells (DRG) were studied with the whole-cell and cell-attached patch recording techniques. 2. The chemical phosphatase 2,3-butanedione monoxime (BDM) reversibly reduced the amplitude of L-type calcium current (ICa) in a dose-dependent manner; at a concentration of 20 mM, BDM caused a 47% suppression of ICa. 3. Application of 10 mM-8-bromo-cyclic AMP or 50 microM-isoprenaline onto DRG treated with BDM completely restored ICa to the pre-BDM level. 4. In striking contrast, bath application of Bay K 8644 (0.5-5 microM) had no effect on the BDM-suppressed ICa. As expected, Bay K 8644 alone caused a two- to threefold increase of the maximal ICa and shifted its I-V relationship to the left. Interestingly, if a cell was first exposed to Bay K 8644 further treatment with 20 mM-BDM resulted in 100% suppression of ICa. This suggests that Bay K 8644 changes the conformation of the calcium channel to one which is more sensitive or more accessible to the action of the phosphatase. 5. Pre-treatment of DRG with an activator of protein kinase C, 12-O-tetradecanoyl-phorbol-13-acetate, did not antagonize BDM's effect on ICa. 6. The depressant action of BDM on ICa was distinct from that of nifedipine in that it did not exhibit use dependence. 7. When single calcium channel currents were recorded in cell-attached patches (barium as the charge carrier), bath application of BDM reduced the percentage of time that the channel spent in the open state. 8. Superfusion with 8-bromo-cyclic AMP restored the ensemble macroscopic 'ICa' to the pre-BDM amplitude. This was due to a dramatic enhancement of the frequency of channel openings. 9. We suggest that BDM acts through the cytoplasm to alter cyclic AMP-dependent protein kinase modulation of neuronal L-type calcium channels. The brief, high-frequency openings which 8-bromo-cyclic AMP activates in the presence of BDM may reflect a rapid phosphorylation-dephosphorylation sequence which controls channel gating.


Alpha 1-adrenergic effects on intracellular pH and calcium and on myofilaments in single rat cardiac cells. 1. The cellular effects of alpha 1-adrenoceptor stimulation by phenylephrine were studied in the presence of propranolol in single cells isolated from the ventricles of rat hearts. 2. Phenylephrine (10-100 microM) induced a biphasic pattern of inotropism in these cells: a transient negative followed by a sustained positive inotropic effect as usually observed in cardiac tissues. 3. In Snarf-1-loaded cells, phenylephrine induced an alkalinization. This effect was reversible on wash-out and inhibited by prazosin, an alpha 1-adrenoceptor antagonist. 4. The alpha 1-adrenoceptor-mediated increase in intracellular pH (pHi) was 0.1 pH unit in HEPES buffer containing 4.4 mM-NaHCO3 and in Krebs buffer containing 25 mM-NaHCO3. 5. The alkalinization was blocked by the Na(+)-H+ antiport blocker, ethylisopropylamiloride (EIPA). 6. The recovery from an acidosis induced by a NH4Cl pre-pulse was accelerated by phenylephrine. The phenylephrine-induced alkalinization was attributed to activation of the Na(+)-H+ antiport. 7. Despite its ability to increase pHi, phenylephrine did not alter Ca2+ current amplitude and kinetics. 8. Ca2+ transients recorded in Indo-1-loaded cells were not augmented by phenylephrine. Diastolic calcium level was decreased. 9. In single skinned cells, the Ca2+ sensitivity of the contractile proteins was increased by a pre-treatment with phenylephrine even when the alpha 1-adrenoceptor-mediated alkalinizing effect had been prevented by EIPA. 10. These results lead us to propose that the alpha 1-adrenergic-induced positive inotropic response of heart muscle could result from an increased sensitivity of the myofilaments to Ca2+ ions. This alpha 1-adrenoceptor-mediated Ca2+ sensitization could result both from an intracellular alkalinization and from a direct effect on contractile proteins.


Proton-induced sodium current in freshly dissociated hypothalamic neurones of the rat. 1. The proton-gated current was investigated in freshly dissociated ventromedial hypothalamic (VMH) neurones from 4-week-old Wistar rats, under whole-cell configuration by the use of the 'concentration-clamp' technique which combines intracellular perfusion with the rapid exchange of external solution within 1-2 ms under a single-electrode voltage-clamp condition. 2. The proton-gated current increased in a sigmoidal fashion as extracellular pH (pHo) decreased. In external solution containing 2 mM-Ca2+, the threshold of current activation was at pHo 6.5, and the maximum response appeared at pHo 4.1-3.9. The dissociation constant (Kd) and Hill coefficient were 10(-4.9) M (pHo = 4.9) and 1.5 respectively. 3. Decreasing extracellular Na+ concentration reduced the proton-gated current. The current reversed direction at the Na+ equilibrium potential (ENa), indicating that it was carried by Na+. 4. The activation phase kinetics of proton-induced current was single exponential. The time constant of activation (tau a) did not have a potential dependence but decreased slightly by decreasing pHo. The inactivation phase kinetics was two-exponential. The time constant of inactivation (tau i) consisted of fast and slow components (tau if and tau is, respectively). Like tau a, both tau if and tau is did not have any potential dependence, but they slightly increased with decreasing pHo. 5. The steady-state inactivation curve, constructed by decreasing pHo from various conditioning pHos to 4.1, revealed that the proton-induced current had a half-maximum inactivation at pHo 6.2. 6. The proton-induced current was suppressed as the extracellular Ca2+ concentration ([Ca2+]o) increased from almost free (0.01 mM) to 80 mM. Increasing [Ca2+]o increased tau a, but slightly decreased both tau if and tau is. 7. Recovery of proton-induced current from complete inactivation of proton-induced current depended on the degree of pHo change. A bigger change in pHo induced faster recovery than a smaller change. 8. External divalent cations inhibited the proton-induced current, and the inhibitory potency was in the order of Mn2+ greater than Co2+ greater than Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. 9. Tetrodotoxin (TTX) at relatively low concentration (less than 10(-7) M) did not inhibit the peak amplitude of the proton-induced current, but at a higher concentration (10(-6) M) it slightly inhibited the peak amplitude of the current and accelerated the inactivation process. Scorpion toxin markedly increased the peak amplitude of the proton-induced current and prolonged the inactivation phase. The tau is was also increased by scorpion toxin in a concentration-dependent manner. Veratridine had no effect on the proton-induced current. 10. The membrane properties of the proton-operated channel were similar to those of the voltage-gated Na+ channel rather than the Ca2+ channel.


Cyclic AMP-mediated regulation of excitation-contraction coupling in canine gastric smooth muscle. 1. Agonists known to increase cyclic AMP levels in gastrointestinal smooth muscles were studied in isolated circular muscles of the canine antrum to investigate the mechanisms of the inhibitory effects of these agents. 2. Muscles were electrically active, generating typical slow wave activity. Cytosolic Ca2+ ([Ca2+]cyt; measured by Indo-1 fluorescence) and tension increased in response to slow waves. 3. Stimulation by isoprenaline (via beta 2-receptors) or forskolin, in the presence or absence of acetylcholine, inhibited the plateau phase and reduced phasic [Ca2+]cyt and contractile responses. 4. Vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP), had similar effects to isoprenaline and forskolin. 5. Increases in the plateau phase of slow waves and the associated increases in [Ca2+]cyt and tension caused by direct activation of voltage-dependent Ca2+ channels by Bay K 8644 (0.1 microM) were also reduced by forskolin. 6. Isoprenaline and forskolin induced negative chronotropic effects, but VIP increased frequency. 7. At a given level of [Ca2+]cyt, contractions were greater under control conditions than in the presence of isoprenaline, VIP and CGRP, suggesting that part of the inhibition produced by these agents may be due to decreased Ca2+ sensitivity of the contractile apparatus. 8. Experiments performed on alpha-toxin-permeabilized muscles confirmed that cyclic AMP-dependent effects involve reduced Ca2+ sensitivity of the contractile apparatus. Addition of cyclic AMP (3-300 microM) caused a reduction in Ca(2+)-induced contraction at a constant level of Ca2+ (pCa 5.5). 9. These results suggest that increased cyclic AMP and probably subsequent activation of protein kinase A: (i) decrease [Ca2+]cyt and contraction by an inhibition of Ca2+ influx during slow waves, and (ii) decrease the sensitivity of the contractile apparatus to [Ca2+]cyt. The membrane effects might occur directly by inhibition of Ca2+ channels or indirectly by increasing the open probability of K+ channels which would tend to cause premature repolarization of slow waves.


Kinetic analysis of the GABAB-mediated inhibition of the high-threshold Ca2+ current in cultured rat sensory neurones. 1. The action of baclofen on the voltage-gated Ca2+ current (ICa) was studied, using cultured neurones of the newborn rat dorsal root ganglia (DRG). Two major categories of ICa were identified: a small transient current activated positive to -60 mV (low voltage-activated ICa) and a large and slowly inactivating current activated positive to -30 mV (high voltage-activated ICa). 2. Baclofen reversibly blocked the high voltage-activated ICa and slowed the activation phase of the current in a concentration-dependent manner (0.5-50 microM). The half-maximal effective concentration was about 1.5 microM as measured by a peak of ICa. On the contrary, a high concentration of baclofen (100 microM) had no detectable effect on the low voltage-activated ICa. 3. The baclofen-sensitive component of the high voltage-activated ICa was largely inactivated by a depolarized holding potential (Vh) of -40 mV, whereas the baclofen-resistant component was not affected by a change in Vh ranging from -110 to -30 mV. 4. The high voltage-activated ICa had two components of current decay: an inactivating component and a quasi-sustained component, with time constants about 420 and 1220 ms, respectively. The time constant of decay for the inactivating component was not affected by replacement of external Ca2+ with Ba2+, whereas that for the quasi-sustained component was markedly prolonged, suggesting that the decay of this component may be due to Ca(2+)-induced block rather than voltage-dependent inactivation. A high concentration of baclofen (50 microM) selectively blocked the inactivating component. 5. The decay phase of the baclofen-sensitive component of the high voltage-activated ICa was best fitted by a sum of two exponentials, with 29.2 and 481 ms for the fast and slow components, respectively. The time constants of the two components were not affected by an increase in the concentration of baclofen, whereas the amplitudes changed concentration-dependently. 6. The slowed activation of the high voltage-activated ICa by baclofen was partially reversed by a large depolarizing pre-pulse. However, such an acceleration of the current was similarly observed in the control solution. Furthermore, the actual current size increased by the pre-pulse was similar in both the control and baclofen-containing solutions. 7. These results suggest that baclofen selectively blocks the inactivating component of the high voltage-activated ICa which forms a rapid rising phase of this current, thus slowing the activation phase of the total high voltage-activated ICa.


The role of corticotrophin releasing factor in relation to the neural control of adrenal function in conscious calves. 1. Adrenal responses to intra-aortic infusions of pure synthetic ovine corticotrophin releasing factor (CRF), and to electrical stimulation of the preganglionic sympathetic innervation, have been investigated in functionally hypophysectomized conscious calves, in the presence and absence of a specific CRF antagonist. 2. CRF exerted a substantial steroidogenic effect on the adrenal gland of functionally hypophysectomized calves when infused intra-aortically at a dose (1.3 ng min-1 kg-1) below that which caused any fall in the arterial blood pressure. This response was significantly reduced, but not abolished by a concomitant infusion of CRF-antagonist into the aorta. 3. The steroidogenic effect of CRF was significantly reduced in the presence of exogenous adrenocorticotrophic hormone (ACTH) (2 ng min-1 kg-1, I.V.) and the surviving response was completely abolished by CRF-antagonist. 4. Stimulation of the peripheral end of the splanchnic nerve at 4 Hz in functionally hypophysectomized calves given exogenous ACTH produced a rise in mean adrenal output of the same order of magnitude as did exogenous CRF under the same conditions. The response to splanchnic nerve stimulation was apparently unaffected by CRF-antagonist although release of endogenous CRF from the gland was significantly increased thereby. 5. These results indicate that release of CRF from the adrenal gland during splanchnic nerve stimulation in the calf does not contribute significantly to the steroidogenic response thereto.


Intracellular pH regulation in isolated cochlear outer hair cells of the guinea-pig. 1. The intracellular pH (pHi) regulation mechanisms of the outer hair cell (OHC) isolated from the guinea-pig were studied using fluorescence ratio imaging microscopy. 2. The OHC pHi in the resting condition was 7.26 +/- 0.08 (mean +/- S.D., n = 49) when the standard solution buffered with HEPES-Tris was superfused. 3. Exposure to 25 mM-NH4+ in the absence of HCO3- caused biphasic changes in pHi; a transient increase (7.89 +/- 0.14, n = 22) followed by a slow decrease (7.57 +/- 0.12; mean +/- S.D.). Removal of external NH4+ by introducing the N-methyl-D-glucamine (NMDG+) solution in the absence of HCO3- markedly acidified the pHi to 6.38 +/- 0.12 with little pHi recovery. Subsequent application of the standard Na+ solution restored the pHi to the initial value. The recovery was inhibited by 0.5 mM-amiloride but not by 0.3 mM-DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). 4. In the presence of HCO3-, removal of both external NH4+ and Na+ promptly caused an intracellular acidification followed by a pHi recovery. The pHi recovery from an acid load was inhibited by 0.3 mM-DIDS or 10 microM-NPPB (5-nitro-2-(3-phenylpropyl-amino)-benzoate). However, the pHi in the steady state in the presence or absence of HCO3- was not altered by addition of 0.5 mM-amiloride or NMDG+ solution. 5. The intracellular buffering power obtained from the NH4+ exposure and withdrawal was -15.1 +/- 8.7 mM (pH unit)-1 (n = 6) and -14.3 +/- 5.8 mM (pH unit)-1, respectively. 6. Replacement of external Cl- with gluconate in the HCO3- solution increased the pHi from 7.22 +/- 0.12 to 7.51 +/- 0.20 (n = 6), which was inhibited by 0.3 mM-DIDS. Moreover, addition of DIDS to the HCO3- solution increased the pHi by 0.13 +/- 0.08 (n = 8). 7. When the external standard solution buffered with HEPES-Tris was replaced with the HCO3- solution, the basal pHi (7.27 +/- 0.10) was promptly acidified to 6.87 +/- 0.10 then relaxed slowly to 7.00 +/- 0.15 (n = 16). 8. The pHi showed an initial alkalinization and a subsequent slow acidification after the HCO3(-)-free standard solution replaced the HCO3- solution. The slow acidification was inhibited by low external Cl- concentration or by addition of 0.3 mM-DIDS.(ABSTRACT TRUNCATED AT 400 WORDS)


The nature of fuel provision for the Na+,K(+)-ATPase in porcine vascular smooth muscle. 1. The specific contributions of aerobic glycolysis and oxidative metabolism to Na+ pump activity were quantitated in porcine carotid arteries under aerobic conditions. 2. Active reaccumulation of potassium by potassium-depleted tissues could be supported by oxidative metabolism alone, anaerobic metabolism in the presence of glucose, or a combination of oxidative metabolism and aerobic glycolysis, but not under anaerobic conditions in the absence of glucose. 3. Increasing levels of potassium added to potassium-depleted arteries under aerobic conditions resulted first in stimulation of aerobic lactate release which saturated at 0.028-0.036 mumol min-1 g-1, which was then followed by a stimulation of oxidative metabolism. This behaviour is opposite to the classic Pasteur effect. 4. The dependence of potassium uptake and lactate release on the concentration of potassium added to potassium-depleted arteries ('potassium re-entry concentration') under aerobic conditions were qualitatively similar. The K0.5 (concentration at which the velocity is half-maximally activated) and Vmax (the maximum velocity) for lactate release were 1.2 +/- 0.3 mM and 0.037 mumol min-1 g-1, respectively; those for K+ uptake were 4.3 +/- 0.4 mM and 0.399 mumol min-1 g-1. 5. The stoichiometric ratio between potassium uptake and ATP as calculated from lactate release approximated theoretical values of 2:1 (assuming 1 ATP per lactate) when potassium re-entry concentrations were less than 2 mM; higher concentrations of potassium produced ratios up to 9:1. 6. Physiological pump rates, as determined by potassium efflux studies, corresponded to potassium re-entry concentrations of less than or equal to 2 mM, the same potassium re-entry concentrations where the stoichiometry between potassium transport and aerobic glycolysis approximated the theoretical ratio of 2:1. Increases in oxidative metabolism were not detected in this range, but were detected at potassium re-entry concentrations of greater than or equal to 4 mM. 7. It was concluded that at physiological Na+ pump rates, aerobic glycolytic metabolism supported the N+,K(+)-ATPase; at higher pump rates, oxidative metabolism was required for pump support as well.


The role of GABAA and GABAB receptors in presynaptic inhibition of Ia EPSPs in cat spinal motoneurones. 1. The role of GABAA and GABAB receptors in presynaptic inhibition was studied by examining the effect of local application of antagonists by ionophoresis during intracellular recording of presynaptic inhibition of compound and unitary group Ia afferent excitatory postsynaptic potentials (EPSPs) in gastrocnemius motoneurones. 2. Ionophoresis of the GABAA antagonist bicuculline methochloride (BMC) was found to block presynaptic inhibition of both compound and unitary EPSPs by up to 85%. BMC also substantially reduced, and occasionally abolished, the late part of the inhibitory postsynaptic potential (IPSP) evoked in motoneurones by the conditioning stimulation. The early part of this IPSP was found to be sensitive to ionophoresis of strychnine hydrochloride. 3. Ionophoresis of 2-OH-saclofen caused a reduction in presynaptic inhibition of compound EPSPs by 5-25% but had no effect on the IPSP evoked in motoneurones by the conditioning stimulation. 4. Ionophoresis of the GABAB antagonist (-)-baclofen reduced the amplitude of unconditioned EPSPs; however it had little effect on presynaptic inhibition. 5. It was concluded that at the Ia afferent-motoneurone synapse presynaptic inhibition is mediated primarily through the activation of GABAA receptors. The activation of GABAB receptors appears to play only a minor role in presynaptic inhibition at this synapse. This contrasts with the relative ease with which (-)-baclofen can reduce transmitter release from Ia afferent terminals and suggests that the receptors activated by (-)-baclofen are predominantly extrasynaptic.


Mechanism of pHi regulation by locust neurones in isolated ganglia: a microelectrode study. 1. We have measured membrane potential (Em) and intracellular pH (pHi), and sodium and chloride activities (aNai and aCli) in exposed dorsal unpaired median neurones in isolated metathoracic ganglia from the desert locust, Schistocerca gregaria using eccentric double-barrelled ion-sensitive microelectrodes. 2. In the absence of added HCO3- the steady-state pHi was 7.21 +/- 0.13 (mean +/- S.D.) at a mean membrane potential of -37 +/- 7.0 mV (S.D.) (n = 44 cells). The pHi was always more alkaline than predicted for passive H+ distribution. 3. The pHi recovery from acid loads, induced by weak acid application or weak base removal, was pHi dependent and associated, in both the presence and absence of added CO2-HCO3-, with a transient increase in aNai. 4. In the absence of added HCO3-, application of the Na(+)-H+ exchange blocker amiloride or external Na+ removal caused intracellular acidification. Also in the absence of added HCO3- the inhibitor SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) caused an acidification of about 0.2 pH units which was not additive to the effects of the removal of external Na+. 5. We found that the application of a CO2-HCO3(-)-containing solution increased the rate of pHi recovery from acidification. 6. Intracellular chloride was decreased by intracellular acidification in the presence of added CO2-HCO3-. In the presence of amiloride, intracellular Cl- depletion inhibited pHi regulation. 7. Simultaneous application of SITS (160 microM) and removal of CO2-HCO3- revealed a continuous underlying acid load of 0.03-0.05 pH unit min-1. 8. We conclude that locust neurones possess at least two pHi-regulating mechanisms which operate against a continuous acid load. One is a Na(+)-H+ exchanger which can be blocked by amiloride, while the second is a Na(+)-dependent Cl(-)-HCO3- exchanger. The latter mechanism appears to be able to operate in the absence of added HCO3- and can recover pHi to around pH 7.4; it is probably the main pHi regulating mechanism. The Na(+)-H+ exchanger appears to activate at more acid pHi and being less energy efficient may serve a protective role.


Cyclic nucleotide-dependent regulation of agonist-induced calcium increases in mouse megakaryocytes. 1. The regulatory effects of cyclic AMP and cyclic GMP on ADP- and thrombin-induced increases in [Ca2+]i were studied in mouse bone marrow megakaryocytes. Changes in [Ca2+]i were continuously monitored in single Fura-2-loaded cells using microspectrofluorometry, and cyclic nucleotides were directly introduced into the single cells using the whole-cell patch-clamp technique. 2. ADP increased [Ca2+]i in a concentration-dependent fashion, and its threshold concentration was in the order of 0.01 microM. A low dose of ADP (below 0.1 microM) induced a transient response of [Ca2+]i which recovered to original levels during the stimulation. A high dose of ADP (0.3-10 microM) induced a biphasic response of [Ca2+]i with an initial peak and a plateau lasting until the end of the stimulation. Repeated stimulation with the same dose of ADP induced a reduced response, probably as a result of desensitization. 3. Thrombin increased [Ca2+]i in a concentration-dependent manner. The time courses of the responses were different from those caused by ADP. Thrombin-induced responses lacked the initial sharp peak observed in ADP-induced responses, and caused a sustained response. 4. The ADP-induced increase in [Ca2+]i was antagonized by the presence of prostaglandin E1 (PGE1, 100-1000 nM), in the medium, and by direct injection of cyclic AMP (100-500 microM) or cyclic GMP (500 microM) into the megakaryocyte. When 500 microM-cyclic AMP was injected into the cells, the rise of [Ca2+]i induced by ADP was reduced by 85%. Effects of these antagonists were inhibited by treatment with a protein kinase inhibitor, H-8. Thrombin-induced increases in [Ca2+]i were reduced by direct injection of cyclic AMP or cyclic GMP. 5. ADP could induce an increase in [Ca2+]i in the absence of external Ca2+. The time course of the response was essentially similar to that observed in the normal condition (1 mM-CaCl2), but the size of the response was reduced by 33%. Thus, 67% of the rise in [Ca2+]i induced by ADP could be accounted for by calcium mobilization from internal storage pools. The presence of NiCl2 (5 mM) duplicated the effects of external Ca2+ removal, suggesting the involvement of a Ca2+ influx pathway, which could be inhibited by Ni2+ in ADP stimulation. 6. Injection of cyclic AMP or cyclic GMP reduced ADP-induced increases in [Ca2+]i under conditions of inhibited Ca2+ influx by NiCl2 (5 mM).(ABSTRACT TRUNCATED AT 400 WORDS)


Histamine H3-receptor activation augments voltage-dependent Ca2+ current via GTP hydrolysis in rabbit saphenous artery. 1. Actions of histamine on the voltage-dependent Ba2+(Ca2+) currents (IBa, ICa) were investigated using the whole-cell patch-clamp technique on dispersed smooth muscle cells from the rabbit saphenous artery. 2. Histamine (half-maximal dose, EC50 = 530 nM) augmented the IBa evoked by a brief depolarizing pulse (100 ms duration; to +10 mV from a holding potential of -80 mV) in a concentration-dependent manner. The maximum augmentation was obtained with 30 microM-histamine (1.29 times control). This augmentation of IBa was inhibited by the H3-antagonist, thioperamide (Ki = 30 nM, slope of the Schild plot = 1.0), but not by H1- or H2-antagonists (mepyramine or diphenhydramine, or cimetidine, respectively). 3. An H3-agonist, R alpha-methylhistamine (EC50 = 93 nM), also augmented IBa in a concentration-dependent manner at a holding potential of -80 mV and the maximum augmentation (1.25 times control) was obtained with 10 microM. This augmentation was also inhibited by thioperamide, but not by the above H1- and H2- antagonists. 4. Intracellularly applied 500 microM-guanosine 5'-triphosphate (GTP) enhanced, but 1 mM-guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) abolished, the histamine-induced augmentation of IBa. When one of the non-hydrolysable GTP analogues, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; greater than 5 microM), guanylyl-imidodiphosphate (GMP-PNP; 200 microM) or guanylyl (beta, gamma-methylene)-diphosphonate (GMP-PCP; 1 mM) was intracellularly applied, the IBa amplitude evoked without the application of histamine was not affected, but the excitatory effect of histamine on IBa was reversed to an inhibition. Pre-treatment with pertussis toxin (PTX: 300 ng/ml and 3 micrograms/ml) did not modify the histamine-induced responses in the absence or presence of GTP gamma S. 5. 4 beta-Phorbol 12,13-dibutylate (PDBu) increased the amplitude of IBa. However, this action of PDBu was not enhanced by the application of GTP (500 microM) in the pipette, but additional application of histamine further increased the amplitude of IBa. Pre-treatment with a potent non-selective protein kinase inhibitor, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H-7; 100 microM), did not modify the histamine-induced current augmentation or inhibition observed in the presence or absence of intracellular GTP gamma S.(ABSTRACT TRUNCATED AT 400 WORDS)


Effect of myoplasmic pH on excitation-contraction coupling in skeletal muscle fibres of the toad. 1. The effect of pH on excitation-contraction coupling in skeletal muscle of the toad was examined using a skinned fibre preparation which gives ready access to the intracellular environment while still allowing stimulation of Ca2+ release by the normal voltage-sensor mechanism. 2. In each fibre, depolarization-induced responses (produced by changing the ions in the bathing solution) were examined first at pH 7.1, and then at another pH between 6.1 and 8.0. At all pH levels examined, the first depolarization elicited a large response which was slightly greater (pH 7.6 and 8.0) or smaller (pH 6.6 and 6.1) than that at pH 7.1. The size of the first depolarization-induced response varied with pH in almost exactly the same manner as did the maximum Ca(2+)-activated response. The duration of the depolarization-induced response at all other pH levels was longer than at pH 7.1. 3. Repeated depolarizations (30 s or more apart) produced similar responses at pH 7.1, but at all other pH levels examined the second and subsequent responses became progressively smaller. The reasons for this decrease were different at low and high pH. 4. Examination of the size of the depolarization-induced response after reloading the depleted sarcoplasmic reticulum (SR) with Ca2+ in solutions at various pH levels, indicated that the SR Ca2+ pump operated more poorly at pH 6.6 and 6.1 than at pH 7.1. This can account for the wide and successively smaller depolarization-induced responses at acidic pH. 5. Examination of the size of the depolarization-induced response after 5 min bathing without stimulation in a very low [Ca2+] solution (-log10[Ca2+] (pCa) greater than 8.0, 1 mM-BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), indicated that the sarcoplasmic reticulum (SR) lost far more Ca2+ at pH 7.6 and 8.0 than at pH 7.1 or 6.1. This indicates that SR is 'leakier' at alkaline pH. 6. Exposure of fibres heavily loaded with Ca2+ to solutions at pH 8.0 invariably induced a large response within 1-6 s. This response was unaffected by inactivation of the voltage sensors and was blocked completely by 2 microM-Ruthenium Red. This response and the 'leak' at alkaline pH are consistent with previous studies of the pH dependence of Ca(2+)-activated opening of single Ca2+ release channels in lipid bilayers. 7. These findings indicate that depolarization can induce massive Ca2+ release at both acidic and alkaline pH, provided that the SR is normally loaded with Ca2+, and give further insight into the intracellular events underlying fatigue in skeletal muscle.


Barium- or quinine-induced depolarization activates K+, Na+ and cationic conductances in frog proximal tubular cells. 1. Frog proximal tubular cells were fused into giant cells. We measured membrane potential (Vm), its changes (delta Vm), and current-induced voltage changes (delta psi) in single cells, during control and experimental states. Each cell served as its own control. 2. In the presence of a physiological Ringer solution, the transference number for potassium (tK) was 0.50. Barium (3 mM) reduced membrane conductance (Gm) by 50%; low-Cl- solutions and low-Na+ solutions also diminished Gm, by 52 and 30%, respectively. The association of barium and low-NaCl solutions decreased Gm to approximately 38% of control, indicating that the impermeant substitute of a physiological ion may interact with other pathways; alternatively, blockade of steady-state conductances may activate physiologically silent processes. 3. In an attempt to enhance the contribution of the partial K+ conductance (GK) to Gm, fused cells were exposed to low-Cl- solutions, containing in addition 0.1 mM-methazolamide, to inhibit the rheogenic Na(+)-HCO3-symport, and 1 microM-amiloride, to block Na+ conductance (GNa). tK went up to 0.83. 4. The high tK preparation was challenged with barium (3 mM) or quinine (Quin, 1 mM). These blockers produced large depolarizations (approximately 60 mV), however, although Gm decreased along early- and mid-depolarization, Gm plateaued and eventually it increased with larger and larger depolarization. 5. Depolarization-associated increase in Gm reflects activation of other conductances. These are Na+, cationic, and K+ conductance(s) poorly sensitive to quinine or barium. In the presence of Ba(2+)- or Quin-induced depolarization, injection of depolarizing current produces delayed increase in conductance. 6. Depolarization-induced activation of cationic conductance (Gcat) and GNa results in enlargement of the K+ electrochemical potential difference, to about 70 mV; this difference allows recycling of K+ ions outwards, since a GK is still detected and may contribute up to 38% of the total conductance.


Background current in sino-atrial node cells of the rabbit heart. 1. The Ca2+ current, K+ current, hyperpolarization-activated current, Na(+)-K+ pump current and the Na(+)-Ca2+ exchange current were all blocked by appropriate blockers and the remaining time-independent currents were investigated in single pacemaker cells of the rabbit sino-atrial node using the whole-cell patch clamp technique. 2. Exchanging the bathing solution from Tris-hydroxymethyl-aminomethane hydrochloride (Tris) Na+ free to 150 mM-Na+ induced an inward current and the slope conductance of the current-voltage relationship increased from 0.45 +/- 0.18 to 0.87 +/- 0.33 nS (n = 71) at -50 mV. The remaining conductance in Tris Na(+)-free solution was essentially the same when Tris was substituted with tetraethylammonium (TEA) or N-methyl-D-glucamine (NMG). The current density of the Na(+)-dependent inward current obtained by subtracting the current in Tris Na(+)-free from that in 150 mM-Na+ solution was 0.73 +/- 0.21 pA/pF (n = 71) at -50 mV. We called this current the Na(+)-dependent background current. 3. The membrane conductance was reduced by lowering the temperature of the external solution from 36 to 23 degrees C. In Tris Na(+)-free solution, the temperature-sensitive component was outward at all potentials, whereas it showed a reversal potential at around -20 mV in 150 mM-Na+ solution. This reversal potential was interpreted as a sum of the Cs+ efflux and Na+ influx, by comparing the Na(+)-dependent inward currents obtained at 36 degrees C and those at 23 degrees C. 4. Divalent cations (2 mM-Ni2+, 1 mM-Ba2+ or 2 mM-Ca2+) reduced only the outward current in the Tris Na(+)-free solution, while in the 150 mM-Na+ solution, they reduced both the inward and outward components of the current which had a reversal potential of around -10 mV. 5. Amiloride depressed the membrane conductance in 150 mM-Na+, Cs+ or Rb+ external solution, though only at negative membrane potentials, which suggests amiloride has a voltage-dependent effect on the background current. 6. Removal of Cl- from the external solution or the addition of a Cl- channel blocker (4,4'-dinitrostilbene-2,2'-disulphonic acid disodium salt, DNDS) failed to affect the membrane conductance. 7. When the monovalent cation-dependent inward current was measured by subtracting the current in the Tris solution from those recorded in the various monovalent cation solutions, the current amplitude decreased in the order: Rb+ greater than K+ greater than Cs+ greater than Na+ greater than Li+, which suggests a poor cation selectivity of this current system.(ABSTRACT TRUNCATED AT 400 WORDS)


Actions of perchlorate ions on rat soleus muscle fibres. 1. The effects of perchlorate (ClO4-) on contraction have been studied in rat soleus muscle fibres using (i) potassium (K+) contracture and (ii) two-microelectrode-point voltage clamp techniques. 2. Membrane potentials (Vm) at all external [K+] were 3-5 mV more negative in ClO4-. The hyperpolarization could not be attributed to a change in Na+, K+, or Cl- permeability, or to an effect on the Na(+)-K+ pump. 3. ClO4- shifts the voltage dependence of tension activation, and contraction threshold, to more negative membrane potentials without altering maximum tension. Consequently, twitches and submaximal K+ contractures were potentiated, whereas tetanic contractions and 200 mM-K+ contractures were unaltered. 4. The decay of K+ contractures during steady depolarization with ClO4- developed a slow exponential phase with an average time constant of 6.05 +/- 0.76 min at -38 mV, and 1.68 +/- 0.15 min at -19 mV. This slow component was (a) under the rapid control of the surface Vm and (b) did not depend on external Ca2+. 5. Inactivation of E-C coupling was measured with a test 200 mM-K+ depolarization following 3-10 min depolarizations in conditioning solutions containing 20-120 mM-K+. ClO4- induced a negative shift in the curve-relating test K+ contracture amplitude to conditioning Vm but did not alter the rate of repriming of tension upon repolarization. 6. The results suggest that ClO4- increases the amount of activator produced during depolarization and thus allows the slow inactivation step in excitation-contraction (E-C) coupling to be reflected in the decay of K+ contracture tension. 7. A 'perchlorate contracture', which did not depend on the activation of E-C coupling, was observed. The contracture depended on external Ca2+, but not on voltage-dependent Ca2+ channels or Na(+)-Ca2+ exchange.


A discharge group for chronically mentally ill. Easing the way. 1. Chronically mentally ill clients have unique needs for discharge planning due to their lack of resources and poor social skills. 2. A weekly group focused on discharge needs can assist patients in planning and solving problems. 3. A psychoeducational approach is used, including video tapes, role playing, group discussion, and guest speakers. 4. Topics for discussion include medication compliance, employment, housing, loneliness, aftercare, and fear of failure.


Role of progesterone and oestradiol in the regulation of uterine oxytocin receptors in ewes. The interaction between oestrogen and progesterone in the regulation of the uterine oxytocin receptor in sheep was evaluated by measuring the binding of oxytocin to membrane preparations of caruncular and intercaruncular endometrium and myometrium. Ovariectomized ewes were assigned in groups of five to each cell of a 4 x 2 factorial design. The four treatments were (a) vehicle (maize oil) for 12 days, (b) progesterone (10 mg day-1) for 9 days, (c) progesterone for 9 days followed by maize oil until day 12 and (d) progesterone for 12 days. The two oestradiol treatments consisted of the administration of implants in the presence or absence of oestradiol. The ewes were killed on day 10 (group b) or day 13 (groups a, c and d) for collection of uterine tissues. The response of the caruncular and intercaruncular endometrium to the treatments was similar. In the absence of oestradiol, treatment with progesterone continuously for either 9 or 12 days reduced the concentration of the oxytocin receptor in comparison with both the control and the progesterone withdrawal group (in which values were similar). The presence of oestradiol reduced the receptor concentrations in control and both 9- and 12-day continuous progesterone treatment groups, but enhanced the concentration in the progesterone withdrawal group. The myometrial oxytocin receptors responded in a similar way to those in the endometrium to progesterone treatment alone, but the addition of oestradiol produced no further effect. In conclusion, progesterone and oestradiol caused downregulation of the endometrial oxytocin receptor. On the other hand, progesterone withdrawal, similar to that which occurs during luteolysis, increased receptor density in the presence of oestradiol. Progesterone may influence the response of the myometrium to oxytocin by causing a reduction in receptor density.


Features of female reproductive senescence in tamarins (Saguinus spp.), a New World primate. Cyclical changes in concentration of plasma progesterone, urinary oestrone-conjugates and urinary luteinizing hormone (LH) were compared in young and older cotton-top tamarins (Saguinus oedipus) and saddle-backed tamarins (S. fuscicollis). A group of six young adult tamarin females (4-5 years of age) was sampled over eight periods of 6-8 weeks and six older (14-20 years of age) females were sampled over thirteen periods. There was hormonal evidence of ovulation in all of the sampling periods for young females; in five of thirteen periods, older females displayed no evidence of ovulation. Of the six older females, two were anovulatory in one sampling period, while one female displayed no evidence of ovulation in any of three sampling periods. Generally, females over 17 years of age either did not ovulate or displayed abnormally long periods of moderate concentrations of progesterone and oestrone conjugates. Basal concentrations of LH differed in individuals, but were not always higher in older females. In contrast to patterns of reproductive senescence in other primates, older, anovulatory tamarins displayed moderate concentrations of urinary oestrone conjugates (5-50 micrograms/mg creatinine) and plasma progesterone (8-19 ng/ml), both of which are hormones of probable luteal origin in these species. This result suggests continued production of steroids by the luteal cells of the prominent interstitial gland in reproductively senescent tamarins. This suggestion was reinforced by histological examination of the ovaries of four older, anovulatory females; few primary follicles were found. Three females had no normal antral follicles, but all females had large luteal masses. The presence of functional luteal cells in the older ovaries, which do not experience regular follicular development, may distinguish ovarian ageing in New World primates from that of Old World primates.


Long-term effects of porcine zonae pellucidae immunocontraception on ovarian function in feral horses (Equus caballus). Ten feral mares free-roaming in Maryland, USA, were inoculated with porcine zonae pellucidae (PZP) protein before the breeding season for three consecutive years (1988-90). Ovarian function was monitored for 51 days during the peak of the breeding season after the third annual PZP inoculation, in seven of these mares and in four untreated control mares, by means of urinary oestrone conjugates and nonspecific progesterone metabolites. None of the ten inoculated mares became pregnant in 1990, compared with 55% of 20 control mares, which included two of the four monitored for ovarian function. Three of the untreated mares demonstrated apparent normal ovarian activity, characterized by preovulatory oestrogen peaks, concurrent progesterone nadirs at ovulation, breeding activity, and luteal-phase progesterone increases after ovulation. Two of the seven monitored PZP-treated mares demonstrated ovulatory cycles that did not result in conception. One was pregnant as a result of conception in 1989 and demonstrated a normal, late-gestation, endocrine profile. The remaining four PZP-treated mares revealed no evidence of ovulation, and urinary oestrogen concentrations were significantly depressed. The experiments indicated that (i) a third consecutive annual PZP booster inoculation is greater than 90% effective in preventing pregnancies in mares and (ii) three consecutive years of PZP treatment may interfere with normal ovarian function as shown by markedly depressed oestrogen secretion.


Concentrations of endothelial-cell-stimulating angiogenesis factor, a major component of human uterine angiogenesis factor, in human and bovine embryonic tissues and decidua. Embryonic development involves the establishment of new patterns of vascular growth in the fetus and within the lining of the womb. A factor, human uterine angiogenesis factor, has been purified from the decidua and stimulates the growth of blood vessels in collagen sponge implants and in the chick chorioallantoic membrane. Evidence is presented that suggests that a major active component of human uterine angiogenesis factor is an activator of latent matrix metalloproteinases, of low M(r), called endothelial-cell-stimulating angiogenesis factor and that this factor is present in substantial quantities in a number of embryonic tissues.


Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors. Cells isolated from the ejaculates of a high proportion of patients exhibiting oligozoospermia are characterized by generation rates of reactive oxygen species that considerably exceed those obtained for the normal fertile population. The purpose of this study was to resolve the cellular source of this enhanced activity. Semen samples from a cohort of oligozoospermic patients and a group of fertile controls were fractionated on discontinuous Percoll gradients to generate three cell populations (0, 50 and 100%) of differing density. For each fraction, both the steady-state and the phorbol-ester-induced chemiluminescent signals were significantly (P less than 0.001) greater for the oligozoospermic samples than for the fertile controls. In the fertile donors, leucocytes comprised the major source of reactive oxygen species, particularly in the low-density Percoll fractions; in oligozoospermic patients, however, spermatozoa were identified as a second major source of reactive oxygen species. Particularly striking was an intense phorbol-ester-induced chemiluminescent signal generated by oligozoospermic spermatozoa, purified by passage through isotonic Percoll and free of leucocyte contamination, which was 167 times greater than the median signal generated by the corresponding fraction from the fertile controls (P less than 0.001). These results emphasize the importance of spermatozoa as a major source of reactive oxygen species in oligozoospermia and have implications for the diagnosis and treatment of this condition, as well as for the design of appropriate diagnostic strategies.


Administration of recombinant bovine interferon-alpha I at the time of maternal recognition of pregnancy inhibits prostaglandin F2 alpha secretion and causes luteal maintenance in cyclic ewes. The antiluteolytic protein, ovine trophoblast protein-1, which is secreted by sheep embryos at about the time of the maternal recognition of pregnancy, exhibits significant structural homology with alpha interferons. Experiments were conducted to examine the effects of intra-uterine and systemic administration of a recombinant bovine interferon-alpha I (rboIFN-alpha I) upon the interoestrus interval, endometrial oxytocin receptor concentrations and secretion of prostaglandin (PG) F2 alpha in cyclic ewes. In Expt 1, each ewe had a cannula placed in the tip of a uterine horn ipsilateral to a corpus luteum, 7 days after an induced oestrus. From day 9 after oestrus until day 19, ewes received either 200 (n = 4), 667 (n = 5) or 2000 (n = 9) micrograms/24 h of rboIFN-alpha I, meclofenamic acid (n = 4) or vehicle (n = 11). Other ewes received 2000 micrograms rboIFN-alpha I/24 h (n = 5) between days 12 and 15 only. All ewes were killed on day 19. Mean luteal phase, as determined by daily plasma progesterone measurements, was significantly longer (P less than 0.01) and mean concentrations of 13,14-dihydro-15-keto PGF 2 alpha (PGFM) in plasma were lower (P less than 0.05) in ewes receiving 667 or 2000 micrograms rboIFN-alpha I between days 9 and 19, or 2000 micrograms between days 12 and 15, than in animals from other treatment or control groups. A similar protocol was used in Expt 2, in which further ewes received either 2000 micrograms rboIFN-alpha I/24 h (n = 5) or vehicle (n = 5) by bolus infusions twice a day into one uterine horn. Mean luteal phase was significantly (P less than 0.05) longer in treated than in control animals, but differences in PGFM concentrations were not significant. In Expt 3, after a synchronized oestrus, ewes received either 2.5 mg rboIFN-alpha I by i.m. injection twice a day between days 12 and 15 (n = 10), 2.5 mg rboIFN-alpha I by i.m. injection twice a day between days 9 and 15 (n = 11), i.m. injection of vehicle alone twice a day (n = 20), or continual intra-uterine infusion of 2 mg rboIFN-alpha I/day between days 12 and 15 (n = 7). The mean luteal phase of ewes receiving rboIFN-alpha I by intrauterine infusion or i.m. injection between days 9 and 15 was significantly longer than for animals from the other two groups (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)


Fetal-maternal bleed in the second trimester of pregnancy. A report of two cases. Two cases of nontraumatic fetal-maternal bleeding occurred in the second trimester. Both presented with mild, lower quadrant tenderness similar to round ligament pain, illustrating the potential for a misdiagnosis.


Characterization of type V collagenase (gelatinase) in synovial fluid of patients with inflammatory arthritis. Gelatin degrading matrix metalloproteinases in synovial fluid from 21 patients with inflammatory arthritis were shown to consist of two distinct gene products, 92 and 70 kDa gelatinases. The gelatinolytic activity of 92 kDa enzyme, which is released from stimulated neutrophils, was positively correlated to neutrophil count in the fluid. By contrast, 70 kDa molecule did not correlate with neutrophil cell count. Purification of these enzymes revealed they could degrade type XI collagen, a cartilage component resistant to interstitial collagenase. The elevated levels of 92 kDa gelatinase in rheumatoid arthritis samples compared to osteoarthritis suggest a role of this enzyme in cartilage destruction.


H+ extrusion by an apical vacuolar-type H(+)-ATPase in rat renal proximal tubules. The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)


Ion channels activated by swelling of Madin Darby canine kidney (MDCK) cells. According to previous studies hyposmotic swelling of Madin Darby Canine Kidney (MDCK) cells leads to a marked decrease of cell membrane resistance. The present study has been performed to identify the underlying ion channels using the patch-clamp technique: reduction of extracellular osmolarity to 230 mmol/liter leads to a transient activation of K+ channels and a sustained activation of anion channels. The K+ channels are inwardly rectifying with a single-channel slope conductance of 56 +/- 3 pS at -50 mV (cell negative) and of 29 +/- 2 pS at 0 mV PD across the patch (150 mmol/liter K+ in pipette). The same channels are activated by an increase of intracellular calcium activity, as shown previously. The anion channels display a single-channel slope conductance of 41 +/- 4 pS at -50 mV (cell negative) and of 25 +/- 3 pS at 0 mV PD across the patch (150 mmol/liter Cl- in pipette). The channel is anion selective and conducts both bicarbonate and chloride with a preference for bicarbonate. Its open probability is not affected by changing intracellular calcium from 0.1-10 mumol/liter. The channels observed explain the effects of cell swelling on PD, ion selectivity and resistance of the cell membrane in MDCK cells.


Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles. Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (kH+) was determined by addition of vesicles (pHi = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 x 10(-3) (gastric parietal cell stimulation-associated vesicles; SAV) and 5 x 10(-3) (duodenal brush-border membrane vesicles; dBBMV) to 11 x 10(-3) sec-1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mM, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in kH+ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na+/H+ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pHi less than pHo) 22Na+ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K(+)-diffusion potential ([K+]i much much greater than [K+]o). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K(+)-ionophore valinomycin, indicating that the native K+ permeability is rate limiting. In the presence of valinomycin, the K(+)-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K+ permeability. Valinomycin was without effect, but the K+/H+ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes.(ABSTRACT TRUNCATED AT 250 WORDS)


Synthesis and degradation of the mRNA of the Tn21 mer operon. The mercury resistance locus encoded by Tn21 on the monocopy IncFII plasmid R100 (merTn21) consists of a metal-responsive activator/repressor, merR, which controls initiation of a polycistronic message that includes genes for the uptake (merTPC) and reduction (merA) of Hg2+ and merD, which may also play a minor regulatory role. Comparison of the relative abundance of the 5' and 3' ends of the merTPCAD transcript revealed a strong transcriptional gradient in the operon, consistent with previous observations of lower relative abundance of the more promoter-distal gene products. In vivo mRNA degradation rates varied only slightly for the different genes: however, the rates of mRNA synthesis varied considerably from the beginning to the end of the operon. Specifically, mRNA corresponding to the promoter-proximal genes, merTPC, achieved a maximum in vivo synthesis rate between 60 and 120 seconds after induction; this rate was maintained for approximately ten minutes. In contrast, the synthesis rates of mRNA corresponding to the promoter-distal genes merA and merD, were initially fivefold lower than the rates of the promoter-proximal genes for the first five minutes after induction, and then rose gradually to approximately 50% of the merTPC synthesis rates. These data suggested that early after induction only 20% of the transcripts initiating at merT proceed beyond merC. At later times after induction approximately 50% of the transcripts proceed beyond merC. Nuclease end mapping did not reveal any discrete termination events in the merPCA region, thus, premature termination may occur at many sites.


McrBC: a multisubunit GTP-dependent restriction endonuclease. McrBC-mediated restriction of modified DNA has been studied extensively by genetic methods, but little is known of its molecular action. We have used overproducing plasmid constructs to facilitate purification of the McrBL and McrC proteins, and report preliminary characterization of the activity of the complex. Both proteins are required for cleavage of appropriately modified DNA in vitro, in a reaction absolutely dependent on GTP. ATP inhibits the reaction. The sequence and modification requirements for cleavage of the substrate reflect those seen in vivo. The position of cleavage was examined at the nucleotide level, revealing that cleavage occurs at multiple positions in a small region. Based upon these observations, and upon cleavage of model oligonucleotide substrates, it is proposed that the recognition site for this enzyme consists of the motif RmC(N40-80)RmC, with cleavage occurring at multiple positions on both strands, between the modified C residues. In subunit composition, cofactor requirement, and relation between cleavage and recognition site, McrBC does not fit into any of the classes (types I to IV) of restriction enzyme so far described.


Accumulation of p53 tumor suppressor gene protein: an independent marker of prognosis in breast cancers. Mutations of the tumor suppressor gene p53 have been identified in breast cancer cell lines, and some breast carcinomas are detectable by immunohistochemical assay because of p53 protein accumulation. This study was designed to determine whether p53 protein accumulation in breast cancers correlates with p53 gene mutation, with survival, and with five pathobiologic factors associated with prognosis. IgG1 monoclonal antibody to human p53 protein (PAb 1801) and immunohistochemical methods were used to detect p53 protein accumulation in archival formalin-fixed, paraffin-embedded, randomly selected carcinomas. We studied 295 invasive ductal carcinomas from the Massachusetts General Hospital; 151 were determined to be sporadic (not hereditary). We also studied 97 invasive ductal carcinomas--21 sporadic and 76 familial (hereditary)--from Creighton University. In addition, we examined 31 archival in situ carcinomas, 15 snap-frozen invasive ductal carcinomas, primary cell cultures from three benign breast tissue samples, and breast carcinoma cell lines MDA-MB-231 and MDA-MB-468. Nuclear p53 protein was observed in 16% of the 31 in situ carcinomas, 22% of the 172 sporadic carcinomas, 34% of the 50 tumors from patients with familial breast cancer, 52% of the 23 tumors from patients with the familial breast and ovarian cancer syndrome, and all three tumors from two patients with the Li-Fraumeni syndrome. There was complete concordance between p53 gene mutation and p53 protein accumulation in the 15 snap-frozen carcinomas and in both breast carcinoma cell lines. Statistically significant associations of p53 protein accumulation with estrogen receptor negativity and with high nuclear grade were found. There were statistically significant associations, independent of other prognostic factors, between p53 protein accumulation and metastasis-free and overall survival, for randomly accrued and for both sporadic and familial tumors. Immunohistochemically detected p53 protein accumulation was an independent marker of shortened survival and was seen more often in familial than in sporadic carcinomas. Our findings also suggest a correlation between p53 protein accumulation and p53 gene mutation.


Int-2, an autocrine and/or ultra-short-range effector in transgenic mammary tissue transplants. Previous studies have shown associations between overexpression of int-2 messenger RNA (mRNA) and murine mammary tumors and between amplification of the int-2 genomic locus and human breast cancers. The Int-2 protein (fibroblast growth factor 3) is a member of the heparin-binding growth factor family of proteins. The export of these growth factors from cells may depend on the presence of amino terminal sequences containing hydrophobic signal peptides. Although Int-2 has a putative signal sequence, it is not known whether or how this protein is secreted from cells. Assuming that the Int-2 protein is secreted from mammary epithelial cells in a basolateral direction so that it is available to affect adjacent cells, we investigated whether it acts in a paracrine manner, exerting its effect externally on adjacent cells, or in an autocrine manner, exerting its effect internally within the same cell. Using in situ hybridization with 35S-labeled RNA antisense probes that specifically detect mRNA coding for the Int-2 protein, we determined the cell-specific localization of int-2 mRNA expression in the mammary gland of transgenic FVB/N mice overexpressing int-2 mRNA. Then we transplanted pieces of mammary epithelial tissue expressing int-2 mRNA into the mammary fat-pad of wild-type, syngeneic animals. The mammary glands of host animals were examined as whole-mounts and as histologic sections 2-6 months after transplantation. In situ hybridization was used to confirm which cells continued to express int-2 mRNA following transplantation. Int-2 mRNA expression in transgenic mice was localized to the mammary epithelial cells. Transplants expressing int-2 mRNA were similar to wild-type transplants in that they had no observable effect on either the growth or the morphology of host mammary epithelium. Abnormal growth occurred only in transplanted tissue expressing int-2 mRNA but not in adjacent host mammary epithelium. Given the limitations of our experimental system and the limited information available to date on the secretion of Int-2 protein, these results suggest that, although the Int-2 protein contains a putative signal peptide, it may act primarily as an autocrine or as an ultra-short-range paracrine growth factor in mammary epithelium.