Stimulation of human B cells specific for Candida albicans for monoclonal antibody production. The stimulation and immortalisation of human peripheral blood B lymphocytes specific for Candida albicans antigen were investigated. An in vitro immunisation system was employed which involved pretreatment of mononuclear cells with L-leucyl L-leucine methyl ester which removes the suppressive effects of CD8+ T cells, NK cells and monocytes. The remaining cells, CD4+ T cells, B cells and dendritic cells, were cultured with antigen and a mixture of cytokines. A mixture of IL-2, -4 and -6 was found to be optimal for antibody production as determined by an Elispot assay. Transformation of the activated B cells by Epstein Barr virus was found to be optimal after 2 days and lines secreting anti-Candida antibodies were established. These lines could form the basis for specific monoclonal antibody production by generating hybridomas, or by a newly described technique whereby cDNA encoding antibody Fab regions is transferred into phage display libraries. The overall strategy might be generally applicable for the generation of human monoclonal antibodies to infectious agents.


Generation of furazolidone radical anion and its inhibition by glutathione. Furazolidone is a nitrofuran drug which causes dilated cardiomyopathy in turkeys and serves as an important model of human dilated cardiomyopathy. Although extensively investigated, the chemical mechanism by which furazolidone produces injury remains unknown. In this work we used electron paramagnetic resonance (EPR) spectroscopy to show that furazolidone was reduced to its corresponding nitro anion radical by ascorbate and hypoxanthine. Glutathione prevented the generation of this anion radical. These results document directly, with EPR spectroscopy, the presence of furazolidone anion radical during biochemical reduction and suggest a protective role of glutathione in furazolidone-induced injury. These data enhance our understanding of furazolidone metabolism and may be useful in defining its role in furazolidone-induced dilated cardiomyopathy.


Nicotinamide-induced activity of alkaline phosphodiesterase I toward tumor-derived cultured cells from neurofibromatosis patients. Membrane-bound alkaline phosphodiesterase I was investigated in control fibroblasts and tumor-derived fibroblasts from patients with neurofibromatosis. Alkaline phosphodiesterase I activity of tumor-derived cells increased in a dose response to nicotinamide (0-9 mM) in culture; 5'-thymidine p-nitrophenyl phosphate was used as substrate. The enzyme activity increased 1.7-fold after 30 h of incubation with 9 mM nicotinamide, and after 3 weeks increased 5-fold. The nicotinamide-dependent enhancement of alkaline phosphodiesterase I activity was inhibited by actinomycin D, which specifically blocks RNA synthesis, but not by cycloheximide. These results suggest that the increase in the enzyme activity caused by nicotinamide was due to induction of alkaline phosphodiesterase I at the transcriptional level in tumor-derived cells. The metabolic effect of nicotinamide on alkaline phosphodiesterase I may be related to tumorigenicity in neurofibromatosis.


The function of (Na(+)-K+)ATPase in the beta cell: characterization of the enzyme in a glucose-responsive insulinoma. (Na(+)-K+)ATPase is necessary for the maintenance of the membrane potential. The activity of this enzyme was studied in purified plasma membranes from a glucose-responsive rat insulinoma. Ouabain-sensitive (Na(+)-K+)ATPase activity showed expected ATP dependency with a Km of 0.4 mM. It was also dependent on Mg2+ (Km range 70-80 microM). In the presence of Mg and ATP, half-maximal activity was obtained at a Na concentration of 30 mM and the enzyme activity increased sigmoidally with a Hill coefficient of 1.5. No direct effect on enzyme activity was observed with the insulin secretagogues glucose, fructose, glyceraldehyde, and ketoisocaproate, or with dibuturyl-cAMP and the phosphodiesterase-inhibitor isobutyl methyl xanthine. It is concluded that (Na(+)-K+)ATPase is not directly influenced by known secretagogues associated with insulin release by the beta cell.


Sequential administration of camptothecin and etoposide circumvents the antagonistic cytotoxicity of simultaneous drug administration in slowly growing human colon carcinoma HT-29 cells. We compared the cytotoxicity of simultaneous and sequential combination chemotherapy with camptothecin and etoposide, in slowly growing human colon carcinoma, HT-29 cells. Simultaneous treatments of HT-29 cells with etoposide and camptothecin produced no marked enhancement of cytotoxicity over single agent administration. This finding demonstrates antagonism of one drug's cytotoxicity over the other. When these studies were repeated in sequential treatment protocols, we observed that antagonism could be circumvented if the period between individual drug administration was separated by 6-8 h. The cytotoxicity that was observed with this approach was never more than additive and the order of camptothecin or etoposide administration did not significantly affect the extent of combined cytotoxicity observed. The protective effect of simultaneous camptothecin and etoposide exposure was not due to reduced formation or alterations in the rate of cleavable complex reversal, and protection persisted for a considerably longer period of time than DNA strand breaks. Protection correlated with the kinetics of DNA and RNA synthesis inhibition produced by either drug. Remarkably, full cytotoxic protection could be afforded by one drug over the other, in the presence of only partial inhibition of DNA or RNA synthesis (50-60%). Our findings suggest that sequential rather than simultaneous administration of topoisomerase I and II inhibitors in future cancer chemotherapy schedules will enhance cytotoxicity over single-agent administration.


In vitro karyotypic and immunophenotypic characterisation of primitive neuroectodermal tumours: similarities to malignant gliomas. Monoclonal antibody (Mab) mediated immunotherapy of brain tumours requires the identification of tumour-restricted cell surface antigens. We have characterised four primitive neuroectodermal tumours, which included pineoblastoma, medulloblastoma and ependymoblastoma cultures, that demonstrated in vitro evidence of malignant behaviour (anchorage-independent growth and nu/nu xenograft tumour formation). The cytogenetic findings ranged from normal G-banded and Q-banded karyotypes through mixed near-diploid/hyperdiploid. These cultures resembled the cell surface immunophenotypic spectrum of malignant gliomas. They were distinguished from normal glia in vitro by the expression of restricted fetal mesenchymal, neuronal, myoblastic, melanocytic, epidermal, chondrocytic, lymphoid and epithelial antigens. Certain antigens appeared sufficiently represented among central nervous system (CNS) neoplasms to afford potential targets for Mab-mediated immunotherapy.


Cytochrome c oxidase and purine nucleotides in skeletal muscle in tumour-bearing exercising rats. We have previously shown that spontaneous physical exercise can delay the onset of experimental anorexia and cachexia and retard tumour growth and we now report the effects on the energy metabolism in skeletal muscle. Exercising tumour-bearing animals (TBE) had an increased maximal capacity for oxygen uptake expressed as Vmax of the cytochrome c oxidase compared with their tumour-bearing sedentary controls (TBS) [mean (S.E.) 289.9 (30.7) vs. 141.6 (11.0); P less than 0.05] but an unchanged Km value. The TBS animals had a depressed Vmax as compared with non-tumour-bearing sedentary controls (CS) [141.6 (11.0) vs. 210.1 (15.1); P less than 0.05]. Most of the purine nucleotides in the 'glycolytic' anterior tibial muscle were significantly altered in the TBE animals compared with the TBS animals, but in the mainly 'oxidative' soleus muscle only the level of inosine monophosphate (IMP) was changed. The results indicate that physical exercise can normalise the oxidative capacity and improve the energy state in skeletal muscle in the tumour-bearing host.


Comparative studies on the effects of doxorubicin and differentiation inducing agents on B16 melanoma cells. The differentiation-inducing activity of doxorubicin on B16 melanoma cells grown in vitro was compared with that of other known differentiation inducers, such as theophylline, retinoic acid, and melanocyte-stimulating hormone (MSH). At drug concentrations resulting in cytostatic effects, doxorubicin and theophylline induced morphological changes (dendritic-like structures with a terminal melanin granule) with an enhancement of total melanin content and tyrosinase activity. Retinoic acid did not alter melanin content and cell morphology, although it affected cell growth. MSH enhanced total melanin content and tyrosinase activity, with no significant morphological changes. Flow cytometric analysis showed that MSH led to an accumulation of cells in G1 phase whereas doxorubicin induced an accumulation of cells in G2 + M. Studies on DNA content in doxorubicin-treated cells, selected on the basis of a morphologically differentiated pattern, showed a clustering of these cells in G2 + M, probably due to a cytokinesis block. Thus doxorubicin can induce cell differentiation comparable with other differentiation inducers.


Chronic oral etoposide in non-small cell lung carcinoma. 25 consecutive inoperable or extended non-small cell lung cancer (NSCLC) patients (19 non-chemotherapy pretreated, 6 non-heavily pretreated) were given oral etoposide, 50 mg/m2/day for 21 successive days, every 4 weeks. 5 partial responses (PR), 9 disease stabilizations were achieved; the overall response rate of 20% (95% confidence interval, 4% to 36%) or 26% in non-pretreated patients. Median survival and PR duration probabilities were 6.7 months and 6.3 months, respectively. Alopecia excepted (96% of patients), non-haematological toxicity was mild. Haematological toxicity WHO grade II+III mainly consisted of leukopenia (28%).


Plasma itraconazole concentrations in neutropenic patients after repeated high-dose treatment. Two different treatments with repeated oral high doses of itraconazole were tested for 10 days in 20 neutropenic patients, 10 receiving 400 mg per day and 10 receiving 600 mg per day. In each group 5 patients were treated for acute leukaemia and 5 patients were recipients of autologous bone-marrow transplantation (ABMT). Itraconazole plasma concentrations were assayed by high-performance liquid chromatography. Statistical analysis disclosed a significant interaction between the dispensed dose and the patient types. The difference between the two doses of itraconazole was greater in the ABMT than in the leukaemia patients. After 10 days at 600 mg per day all the ABMT patients had an itraconazole plasma concentration higher than 250 micrograms/l. Therefore, 600 mg per day seems more efficient to obtain a therapeutic level of itraconazole in ABMT patients but this needs to be confirmed for all the neutropenic patients.


Intraductal breast cancer: review of 183 consecutive cases. The aim was to review a series of 183 intraductal breast cancer to define the diagnostic features and therapeutic outcome. Patients' average age was 54 years. Diagnostic procedures employed were clinical examination, mammography in 173 cases and fine-needle aspiration cytology in 98 cases. The sensitivity of clinical examination was 0.61, of mammography 0.74, of fine needle aspiration cytology 0.70. The sensitivity of clinical examination and mammography associated was 0.93. The surgical options adopted were: conservative surgery 80 cases, mastectomy 103 cases. Conservative surgery was followed by breast irradiation in 34 cases. Axillary dissection was performed in 122. 97 cases have been reviewed histologically. 60% of ductal carcinoma in situ (DCIS) were multifocal and 22% multicentric. Local recurrence, all infiltrating, occurred in the same breast after conservative surgery in 8 cases, 3 of which had received postoperative radiotherapy, and in 3 patients after mastectomy. Contralateral breast cancer was recorded in 13 cases, being synchronous in 4 (infiltrating in 3, DCIS in 1) and metachronous in 8 (all infiltrating). 3 patients died of breast cancer. The present series confirms the risk of ipsilateral cancer recurrence after conservative surgery but there are no significant differences relating to mammographic pattern, size, histological type, margin involvement and radiotherapy.


Isotope lung imaging.

Isotope lung imaging. The contribution of isotope imaging to the investigation of respiratory disease has been primarily limited to the detection of ventilation and perfusion abnormalities and the investigation of inflammation or cancer. In recent years, there has been a growing interest in the metabolic function of the lung and in measurements of alveolar capillary permeability in a variety of conditions. This review will deal with these newer applications of isotope techniques in the lung in addition to developments in ventilation-perfusion imaging.


In situ expression of helper-free avian leukosis virus (ALV)-based retrovirus vectors in early chick embryos. Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.


Cytochemical and stereological analysis of rat cortical astrocytes during development in primary culture. Effect of prenatal exposure to ethanol. This study has investigated the effect of prenatal alcohol exposure on the qualitative and quantitative ultrastructure of proliferating and differentiated astrocytes in primary cultures as well as on the cytochemical activity of several subcellular phosphatase markers, including acid phosphatase, uridine diphosphatase, thiamine pyrophosphatase, 5'-nucleotidase and glucose-6-phosphatase. The astrocytes were obtained from 21-day-fetuses of both control and alcohol-fed rats. Our results show that several cell components, such as mitochondria, rough endoplasmic reticulum and lysosomes, exhibit qualitative and/or quantitative ultrastructural changes during the process of astrocyte maturation. In some cases these morphological changes are accompanied by variations in the cytochemical activity of enzymes located in these and other cell components, suggesting that these enzymes, and therefore the functional state of these organelles, are modulated during astrocyte development. When prenatally exposed to ethanol, both proliferating and differentiated astrocytes showed striking ultrastructural alterations compared with controls, including an increment of lysosomes as well as a decrease in the values of stereological parameters relative to mitochondria, rough endoplasmic reticulum and Golgi apparatus. Cytochemical analysis of these cells indicates that prenatal exposure to ethanol decreased the activities of all the enzymes tested, except for acid phosphatase, which was increased in both groups of treated astrocytes. These results suggest that prenatal exposure to ethanol could affect astrocytes during development in two different but probably complementary ways: a) by causing a delay in astrocyte maturation and, b) by inducing a direct toxic effect on these cells.


Expression of FGFR-4 mRNA in developing mouse tissues. We have previously reported the isolation of human FGFR-4 cDNA encoding a receptor for aFGF and shown that the expression patterns of FGFR-1 through FGFR-4 mRNAs are overlapping, but distinct in human fetal tissues (Partanen et al., 1991). In order to define cells and tissues expressing FGFR-4 more accurately, we have cloned mouse FGFR-4 cDNA and used it in Northern and in situ hybridization of mouse embryonic tissues. Our results show that the 3.5 kb FGFR-4 mRNA is expressed in day 10 to full term embryos and newborn mice, with maximal expression levels during days 14-16 p.c. Adult mice express the FGFR-4 mRNA predominantly in the liver, kidney and lung. In in situ hybridization of 10-13 day p.c. mouse embryos, FGFR-4 mRNA is present primarily in tissues of mesodermal origin and in the developing lung and gut. Particularly the mesenchymal tissue in association with the first pharyngeal arch and along the prevertebrae (in developing myotomes) which will differentiate into muscle tissue express high amounts of FGFR-4 mRNA. In addition, the developing metanephros contains FGFR-4 transcripts. These results suggest that FGFR-4 may be particularly important for the differentiation of skeletal muscle and endodermally derived organs.


[Survival and development of neoplasms in 56 patients with idiopathic hemochromatosis]. Fifty-six patients with idiopathic hemochromatosis (50 men and 6 women, mean age 49.7 +/- SD 11.1 and 60.8 +/- SD 10.3 years respectively) were studied and followed up for a median period of 44 months (range 3-168). At basal liver biopsy 44 patients had cirrhosis and 12 fibrosis. Iron depletion was achieved in 39 of the 46 cases who underwent iron removal by erythrocytapheresis. Eighteen patients died, 11 from malignancy and 7 from other causes. A total of 14 malignant neoplasms were observed (6 hepatocellular and 8 extrahepatic), of which 6 (5 hepatocellular and 1 pancreatic) were already evident at enrollment. Cumulative survival rates at 3, 5 and 8 years were 75.4%, 64.2% and 54.4% respectively, significantly lower than those in the general population. Probabilities of developing cancer in the 50 patients without cancer at diagnosis were 5.5%, 14.9% and 44.4% at 3, 5 and 8 years respectively.


Stable transfection of androgen receptor and MMTV-CAT into mammalian cells: inhibition of cat expression by anti-androgens. A hormone-inducible transcriptional system has been established, based on the stable transfection of the rat androgen receptor (rAR) and a reporter plasmid containing the mouse mammary tumour virus promoter linked to the chloramphenicol acetyltransferase gene (pMMTV-CAT) into steroid receptor-negative CV-1 cells. First, the rAR was stably introduced into CV-1 cells. Single clones were tested for stable expression of functionally active AR by analysing the effect of dihydrotestosterone on induction of transiently transfected pMMTV-CAT. Stable transfection and the expression of AR was confirmed by steroid-binding assays. In a second step, a clone expressing physiological amounts of AR protein (30 fmol/mg protein) was stably transfected with pMMTV-CAT to yield a permanent cell line that stably expresses functional AR and MMTV-CAT sequences. This cell line provides a powerful tool for the efficient and accurate determination and quantification of the effects of androgens and anti-androgens on reporter gene transcription. This was demonstrated by investigating the action of the three anti-androgens hydroxyflutamide, casodex and cyproterone acetate. The three compounds were shown to reverse the effects of the androgen R1881 on gene expression but were themselves devoid of agonistic activity.


Effects of progesterone on gonadotropin-releasing hormone receptor concentration in cultured estrogen-primed female rat pituitary cells. Acute (0.5-4 h) treatment of estradiol (E)-primed female rat pituitary cells with progesterone (P) augments gonadotropin-releasing hormone (GnRH)-induced LH release, whereas chronic (48 h) P-treatment reduces pituitary responsiveness to the hypothalamic decapeptide. Dispersed E-primed (48 h, 1 nM) rat pituitary cells were cultured for 4 or 48 h in the presence of 100 nM P to assess the effects of the progestagen on GnRH receptors and on gonadotrope responsiveness to the decapeptide. P-treatment (4 h) significantly augmented GnRH-receptor concentrations (4.44 +/- 0.6 fmol/10(6) cells) as compared to cells treated only with E (2.6 +/- 0.5 fmol/10(6) cells). Parallel significant changes in GnRH-induced LH secretion were observed. The acute increase in GnRH-receptor number was nearly maximal (180% of receptor number in cells treated with E alone) within 30 min of P addition. Chronic P-treatment (48 h) significantly reduced pituitary responsiveness to GnRH as compared to E-treatment. The GnRH-receptor concentrations (3.9 +/- 0.6 fmol/10(6) cells), however, remained elevated above those in E-primed cells. GnRH-receptor affinity was not influenced by any of the different treatments. These results indicate that the acute facilitatory P-effect on GnRH-induced LH release is at least chronologically closely related to an increase in GnRH-receptor concentration. The chronic negative P-effect on pituitary responsiveness to GnRH, however, shows no relation to changes in available GnRH receptors.


ACTH/MSH like peptides in the treatment of cisplatin neuropathy. The neurological toxicity seen in patients treated with cisplatin in most cases concerns ototoxicity and peripheral neuropathy. Thus far, the pathogenesis of cisplatin neuropathy remains obscure. Yet the fact that cisplatin affects mainly the sensory peripheral nerve fibers points towards an involvement of the dorsal root ganglia. In a rat model of cisplatin neuropathy, following a cumulative dose of approx. 12 mg/kg cisplatin the sensory nerve conduction velocity began to slow as compared to age-matched controls. Peptides derived from ACTH and MSH are known to exert neurotrophic effects. In vivo they facilitate postlesion repair mechanisms in the peripheral nervous system by enhancing the early sprouting response of the damaged nerve. Surprisingly, chronic treatment with a synthetic ACTH4-9 analog not only prevented cisplatin neurotoxicity following a low or high dose regimen, but also counteracted already existing cisplatin-induced neurotoxicity. Stimulated by these findings a randomized, double blind, placebo-controlled study was performed to assess the efficacy of the peptide in the prevention of cisplatin neuropathy in women suffering from ovarian cancer. The threshold of vibration perception (VPT) was used as the principal measure of neurotoxicity. Following 6 cycles of chemotherapy the VPT had increased more than 8-fold in women receiving placebo as co-medication. Whereas the VPT in women receiving 1 mg/m2 body surface ACTH4-9 analog before and after each cisplatin cycle only increased less than 2-fold. No side effects of the peptide treatment were observed and the clinical response to the chemotherapy was similar in all treatment groups. Collectively these preclinical and clinical data suggest that treatment based on non-endocrine fragments of ACTH/MSH may be a therapeutic option in the treatment of cisplatin neuropathy.


Leiomyosarcomas have a poorer prognosis than mixed mesodermal tumours when adjusting for known prognostic factors: the result of a retrospective study of 423 cases of uterine sarcoma. To determine the survival data for the various tumour types of uterine sarcoma and determine the influence of various prognostic factors on survival. Retrospective analysis of all uterine sarcoma cases registered in the 15 year period 1967-1981. West Midlands Regional Cancer Registry, serving a catchment area of 2.6 million women. 423 women registered as having a uterine sarcoma; 367 of these were associated with the two main histological types, leiomyosarcomas (LMS) and mixed mesodermal tumours (MMT). Duration of survival was taken as the primary endpoint. The overall 5-year survival for uterine sarcomas in this series was 31%, with the major prognostic indicator being tumour stage. Survival for mixed mesodermal tumours is similar to other sarcomas despite a tendency towards less differentiation, wider dissemination and a greater age of the patient at diagnosis. Multivariate analysis shows that for cases with similar stage, age and grade, mixed mesodermal tumours have a better prognosis than leiomyosarcomas. These results demonstrate the danger of considering each variable in isolation when the relation between variables can lead to spurious significance or lack of significance because of the imbalances in the numbers between groups of prognostic importance. This study underlines the need for an adequate inspection of the intra-abdominal contents at the time of hysterectomy for uterine fibroids.


Native and oxidized LDL enhances production of PDGF AA and the surface expression of PDGF receptors in cultured human smooth muscle cells. Animal studies have demonstrated that hypercholesterolemia leads to the development of fibromuscular atherosclerotic lesions that are characterized by the intimal accumulation of cholesterol esters in macrophage foam cells and focal proliferation of smooth muscle cells (SMCs). There is now convincing evidence that formation of foam cells occurs as a result of macrophage uptake of oxidized low density lipoprotein (LDL), but the processes linking hypercholesterolemia to activation of SMC growth are less clear. In the present study, we demonstrated that native as well as oxidized LDL stimulates DNA synthesis in cultured human SMCs. Both native and oxidized LDL enhances the expression of platelet-derived growth factor (PDGF) A-chain transcripts in the cells, suggesting that the mitogenic effect of the lipoprotein preparations may be due to activation of autocrine or paracrine PDGF loops. Preincubation of SMCs with native and oxidized LDL also increased the expression of PDGF alpha- and beta-receptors on SMCs and enhanced the responsiveness of the cells to exogenous PDGF. The maximal stimulatory effect of oxidized LDL occurred at a concentration of 3 micrograms/ml, whereas that of native LDL occurred at 10 micrograms/ml, but otherwise no difference was observed between the native and oxidized LDL preparations. The mitogenic effects of LDL disappeared if the cells were exposed to the lipoprotein preparations for more than 4 hours and was also effectively inhibited by superoxide dismutase. The present results suggest that LDL may influence the growth of SMCs by modulating the expression of growth-regulatory genes in the cells.


An unusual giant cell lesion in a child with vitamin D-resistant rickets. This paper reports the presence of a focus of giant cells in a sinus tract associated with an abscessed primary tooth in a patient with vitamin D-resistant rickets. The relevance of this giant cell lesion to the systemic disorder is discussed.


p65 fragments, homologous to the C2 region of protein kinase C, bind to the intracellular receptors for protein kinase C. Receptors for activated protein kinase C (RACKs) have been isolated from the particulate cell fraction of heart and brain. We previously demonstrated that binding of protein kinase C (PKC) to RACKs requires PKC activators and is via a site on PKC that is distinct from the substrate binding site. Here, we examine the possibility that the C2 region in the regulatory domain of PKC is involved in binding of PKC to RACKs. The synaptic vesicle-specific p65 protein contains two regions homologous to the C2 region of PKC. We found that three p65 fragments, containing either one or two of these PKC C2 homologous regions, bound to highly purified RACKs. Binding of the p65 fragments and PKC to RACKs was mutually exclusive; preincubation of RACKs with the p65 fragments inhibited PKC binding, and preincubation of RACKs with PKC inhibited binding of the p65 fragments. Preincubation of the p65 fragments with a peptide resembling the PKC binding site on RACKs also inhibited p65 binding to RACKs, suggesting that PKC and p65 bind to the same or nearby regions on RACKs. Since the only homologous region between PKC and the p65 fragments is the C2 region, these results suggest that the C2 region on PKC contains at least part of the RACK binding site.


Sequential NMR resonance assignment and secondary structure of ferrocytochrome c553 from Desulfovibrio vulgaris Hildenborough. Two-dimensional nuclear magnetic resonance spectroscopy was used to assign the proton resonances of ferrocytochrome c553 from Desulfovibrio vulgaris Hildenbourough at 37 degrees C and pH = 5.9. Only a few side-chain protons were not identified because of degeneracy or overlap. The spin systems of the 79 amino acids were identified by DQF-COSY and HOHAHA spectra in H2O and D2O. Sequential assignments were obtained from NOESY connectivities between adjacent amide, C alpha H, and C beta H protons. From sequential NH(i)----NH(i + 1) and long-range C alpha H(i)----NH(i + 3) connectivities, four stretches of helices were identified (2----8, 34----46, 53----59, 67----77). Long-range NOE between residues in three different helices provide qualitative information on the tertiary structure, in agreement with the general folding pattern of cytochrome c. The heme protons, including the propionate groups, were assigned, and the identification of Met 57 as sixth heme ligand was established. The dynamical behavior of the ring protons of the six tyrosines was analyzed in detail in terms of steric hindrance. The NMR data for ferrocytochrome c553 are consistent with the X-ray structure for the homologous cytochrome from D. vulgaris Miyazaki. On the basis of the secondary structure element and of observed chemical shift due to the heme ring current, a structural alignment of eukaryotic and prokaryotic cytochromes c is proposed.


Action of derivatives of mu-conotoxin GIIIA on sodium channels. Single amino acid substitutions in the toxin separately affect association and dissociation rates. We have studied binding and block of sodium channels by 12 derivatives of the 22-residue peptide mu-conotoxin GIIIA (mu-CTX) in which single amino acids were substituted as follows: Arg or Lys by Gln, Gln-18 by Lys, Asp by Asn, and HO-Pro by Pro. Derivatives were synthesized as described by Becker et al. [(1989) Eur. J. Biochem. 185, 79]. Binding was measured by displacement of labeled saxitoxin from eel electroplax membranes (100 mM choline chloride, 10 mM HEPES-NaOH, pH 7.4). Blocking kinetics were evaluated from steady-state, single-channel recordings from rat skeletal muscle sodium channels incorporated into planar, neutral phospholipid/decane bilayers (200 mM NaCl, 10 mM HEPES-NaOH, pH 7.0). Blocking events generally appeared as periods of seconds to minutes in which current through the single channel was completely eliminated. A notable exception was seen for the substitution Arg-13-Gln for which the "blocked" events showed measurable conductances of about 20-40% of the open state. The substitution of Arg-13 reduced binding to electroplax membranes to undetectable levels and increased the apparent dissociation constant determined for skeletal muscle channels by greater than 80-fold compared with the native peptide. Other substitutions caused smaller decreases in affinity. The decreased potency of the toxin derivatives resulted both from increases in the rates of dissociation from the channel, and from decreases in association rates. Our data support the suggestion by Sato et al. [(1991) J. Biol. Chem. 265, 16989] that Arg-13 associates intimately with the binding site on the channel. In addition, our results suggest that certain residues affect almost exclusively the approach and docking of the toxin with its binding site, others appear to be important only to the strength of the association once binding has taken place, and yet others affect both.


Photoaffinity labeling study of the interaction of calmodulin with the plasma membrane Ca2+ pump. Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca(2+)-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (peptide C28WC*), respectively. The localization of the contact regions between calmodulin and the N- and C-terminal portions of the peptides was the aim of this study. The three peptides were N-terminally blocked with a 3H-labeled acetyl group to facilitate the identification of labeled fragments after isolation and digestion. The binding site for phenylalanine 25 was identified in the N-terminal domain of calmodulin while the phenylalanine derivative in position 9 labeled the C-terminal domain. Fluorescence studies using the dansylated N- and C-terminal halves of calmodulin and peptide C20W corresponding to the first 20 amino acids of the calmodulin-binding domain showed that only the C-terminal lobe of calmodulin had high affinity for the peptide (KD in the nanomolar range).


Oxidation of vitamin E during iron-catalyzed lipid peroxidation: evidence for electron-transfer reactions of the tocopheroxyl radical. Incubation of phosphatidylcholine liposomes containing the biological antioxidant alpha-tocopherol (alpha-TH) with xanthine, xanthine oxidase, and FeCl2 caused alpha-TH oxidation to alpha-tocopherol quinone (alpha-TQ) and 8a-hydroperoxytocopherone (2). In addition, 4a,5-epoxy-8a-hydroperoxytocopherone (3), 7,8-epoxy-8a-hydroperoxytocopherone (4), and their respective hydrolysis products 2,3-epoxy-alpha-tocopherol quinone (6) and 5,6-epoxy-alpha-tocopherol quinone (7) also were formed. alpha-TQ was the major product at less than 20% alpha-TH oxidation, whereas epoxides were the predominant products when alpha-TH was more extensively oxidized. 8a-(Alkyldioxy)tocopherones 1, which are formed when peroxyl radicals oxidize alpha-TH in other systems and which are precursors to alpha-TQ, were not found. 8a-Hydroxytocopherone (5), rather than 8a-(alkyldioxy)tocopherones 1, appeared to be the precursor to alpha-TQ. Approximately 30% of the alpha-TH consumed was regenerated by treatment of samples with ascorbic acid or nordehydroguaiaretic acid (NDGA) at pH 3, but not at pH 7. The stability of the ascorbic acid- and NDGA-reducible species and pH dependence for regeneration matched those of 8a-hydroxytocopherone (5) and contrasted with the properties of the tocopheroxyl radical (alpha-T.). Incubation of liposomes containing alpha-TH with the diphenylpicrylhydrazyl (DPPH) radical, which oxidizes alpha-TH to alpha-T. in high yield, formed an ascorbic acid-reducible species with properties identical to those of compound 5. The results indicate that phospholipid peroxyl radicals oxidize alpha-T. to epoxides, 8a-hydroperoxytocopherone (2), and the tocopherone cation (alpha-T+), which hydrolyzes to 5, the immediate precursor to alpha-TQ.(ABSTRACT TRUNCATED AT 250 WORDS)


Relationship between local and global stabilities of proteins: site-directed mutants and chemically-modified derivatives of cytochrome c. The methionine 80 sulfur-heme iron bond of rat cytochrome c, whose stability is decreased by mutating the phylogenetically invariant residue proline 30 to alanine and increased when tyrosine 67 is changed to phenylalanine, recovers its wild-type characteristics when both substitutions are performed on the same molecule. Titrations with urea, analyzed according to the heteropolymer theory [Alonso, D. O. V., & Dill, K. A. (1991) Biochemistry 30, 5974-5985], indicate that both single mutations increase the solvent exposure of hydrophobic groups in the unfolded state, while in the double mutant this conformational perturbation disappears. Similar increases in solvent exposure of hydrophobic groups are observed when the sulfur-iron bond of the wild-type protein is broken by alkylation of the methionine sulfur, by high pH, or by binding the heme iron with cyanide. The compensatory effects of the two single mutations do not extend to the overall stability of the protein. The added loss of conformational stability due to the single mutations amounts to 7.3 kcal/mol out of the 9 kcal/mol representing the overall free energy of stabilization of the native conformation of the wild-type protein. The folded conformation of the doubly mutated protein is only 2 kcal/mol less stable than that of the wild type. These results indicate that the double mutant protein is able to retain the essential folding pattern of cytochrome c and the thermodynamic stability of the methionine sulfur-heme iron bond, in spite of structural differences that weaken the overall stability of the molecule.


Aspartic acid-66 is the only essential negatively charged residue in the putative hydrophilic loop region of the metal-tetracycline/H+ antiporter encoded by transposon Tn10 of Escherichia coli. Of the 16 acidic amino acid residues located in the hydrophilic region of the metal-tetracycline/H+ antiporter of transposon Tn10, five glutamic acids and three aspartic acids are conserved among the tetracycline/H+ antiporters of Gram-negative bacteria. When these conserved acidic residues were each replaced by a neutral polar residue, glutamine or asparagine, only the Asp66 substitution mutants completely lost their transport activity. The substitution of Glu274, Asp120, Glu181, or Asp38 caused significant reduction of the transport activity, whereas the substitution of the other three residues had no detectable effect on the activity. These findings led to the conclusion that only Asp66 is essential for the transport function.


Structural characterization of the 5' regions of the human phenylalanine hydroxylase gene. Human phenylalanine hydroxylase (PAH) is expressed in a liver-specific manner and catalyzes the enzymatic conversion of phenylalanine to tyrosine. Genetic deficiency of PAH results in the autosomal-recessive disorder phenylketonuria (PKU). Through the application of genomic and cDNA cloning, primer extension studies, SI mapping experiments, and PCR methodologies, the transcription initiation (CAP) site has been identified and the 5'-flanking region determined. The most upstream CAP site for the human hepatic PAH gene transcript is located 154 nucleotides upstream of the first translation codon. The genomic and cDNA sequences analyzed demonstrated that the previously reported cDNA sequence, phPAH247 [Kwok et al. (1985) Biochemistry 24, 556-561], contained a 164-nucleotide cloning artifact at its 5'-end. The 319 base pair region immediately upstream of the CAP site is characterized by the lack of a proximal TATA box and the presence of sequences similar to GC boxes, CACCC boxes, CCAAT boxes, activator protein 2 (Ap-2) sites, partial glucocorticoid response elements (GREs), and partial cyclic AMP response elements (CREs). This suggests that the human PAH gene has a TATA-less promoter regulated by multiple transcription factors.


Parameters affecting the elongation block by RNA polymerase II at the SV40 attenuator 1 in vitro. We have previously suggested that folding of the SV40 attenuator RNA 1 into two hairpin elements leads to a block of transcription elongation. Using site-directed mutagenesis, we created three templates that either strengthened or weakened the proposed hairpin structures. The mutated and wild-type templates were cloned downstream from the adenovirus 2 MLP, and transcription patterns were compared among the templates, in cell-free extracts. In this report, we show that at the standard temperature (30 degrees C) the elongation block occurs at elevated KCl concentrations (0.5-1.2 M KCl) while at high temperature (65 degrees C), although the transcription elongation rate increases, the block to elongation occurs at lower KCl concentrations (less than 0.5 M KCl) as well. Since under the conditions tested the extent of the elongation block is directly dependent on the stability of the hairpin structure of the RNA, as assessed by a comparison of transcription of the wild-type and mutated templates, we suggest that elevated KCl concentrations and high temperatures are favored conditions for optimizing the processes whereby the hairpin structures are recognized by the polymerase as an attenuation signal. Moreover, the present experiments indicate that cellular elongation factors are not directly involved in modulating the extent of the elongation block at the SV40 attenuator 1 in vitro. The SV40 attenuator 1 is the only eukaryotic system known to date which has been shown to have structural characteristics so similar to rho-independent terminator in prokaryotes. We discuss the similarities between the mechanisms which lead to elongation blocks at these eukaryotic and prokaryotic sites.


Variant forms of the pig lutropin/choriogonadotropin receptor. The cloning and sequencing of porcine lutropin/choriogonadotropin (LH/hCG) receptor messenger RNAs have shown the presence of a full-length receptor (pLHR-A) and of shorter variants lacking either the transmembrane and the intracellular domains (pLHR-B and pLHR-C) or only the transmembrane domain (pLHR-D). Moreover, immunoblotting of testicular membrane extracts has detected 85-, 68-, and 45-48-kDa proteins reacting with antireceptor antibodies. Transfection experiments were performed to assign the protein species to the various messenger RNAs and to study the function of the various receptor species. COS-7 and L-cells transfected with an expression vector encoding full-length receptor pLHR-A yielded a protein of apparent molecular mass of 105 kDa. This corresponded to the complete receptor which had undergone a different glycosylation pattern to that found in testis, since after digestion with peptide N-glycosidase F both the 105-kDa COS-7 protein and the 85-kDa testicular glycoprotein yielded a holoprotein of approximately 63 kDa. Transfection with pLHR-A also yielded a high proportion of the 68-kDa glycoprotein which was shown by digestion with endoglycosidase H to be a high-mannose precursor of the full-length receptor. The existence of a large pool of precursor species in both transfected cells and Leydig cells evokes possible physiological regulations at the level of receptor maturation.(ABSTRACT TRUNCATED AT 250 WORDS)


Chemiluminescence in the crude extracts of soybean seedlings. Postulated mechanism on the formation of hydroperoxide intermediates. It has been reported that weak chemiluminescence (CL) from crude extracts of soybean seedlings is remarkably enhanced with the addition of various aldehydes (Biochim. Biophys. Acta 1058, 209-216). The reactivity of certain emitter(s) with oxygen species was examined in the autoclaved extracts of seedlings. When samples were reduced by the addition of hydrosulfite, two different types of reactivities in CL were defined. One type showed an initial rapid increase and a subsequent fast decay in CL upon mixing with oxygen. This rapid increase in CL intensity was independent of the presence of aldehydes, and was significantly suppressed by SOD. However, the subsequent slow decay phase in CL was dependent on the presence of aldehydes. In the sample reduced more moderately by borohydride, the same slow decay of CL appeared upon mixing with acetaldehyde and oxygen. This second type of CL was not inhibited by active oxygen scavengers. Hydrogen peroxide added to unreduced (oxidized) samples also elicited CL. Three types of primary emitters may be oxidized to form transient hydroperoxide, and excited for light emission by slightly different ways: two of them are excited by abstraction of one atomic oxygen from the hydroperoxy intermediate with aldehyde or hydrogen peroxide, leading to formation of an excited hydroxide intermediate. The third is excited directly on the binding of superoxide anion to the reduced primary emitter.


Chemoreception in hydra: specific binding of glutathione to a membrane fraction. Specific binding of reduced [35S]glutathione (GSH) was measured using a crude membrane fraction of Hydra vulgaris (attenuata). The specific binding shows both rapid displaceable and nondisplaceable components. Rapid displaceable binding accounted for 20% of the total specific binding. Data from saturation binding experiments indicates half maximal total specific binding occurs at 2 microM GSH which is similar to reported EC50 values from behavioral experiments. Calcium is required for displaceable binding of GSH to the putative receptor. Oxidized glutathione (GSSG), an antagonist of the GSH-activated feeding response, and S-methylglutathione (GSM), an agonist of the feeding response, inhibit the binding of radiolabeled GSH to the putative receptor. Glutamate, a putative competitive antagonist of the GSH-activated feeding response in hydra, does not inhibit the specific binding of radiolabeled GSH to the receptor and must therefore block the feeding response by a mechanism other than competitive inhibition.


Inhibition of different steps of the ubiquitin system by cisplatin and aclarubicin. Ubiquitin is involved in such fundamental cellular processes as cell cycle control, DNA repair, protein degradation and stress responses. We previously reported that cisplatin could inhibit the ubiquitin-ATP-dependent proteolysis and ubiquitination. We further investigated the effect of various antitumor agents on the ubiquitin system and found that aclarubicin (ACR) inhibits the ubiquitin-ATP-dependent proteolysis but not the ubiquitination process. We found that ACR as well as cisplatin inhibited the ubiquitin-ATP-dependent proteolytic activity of rabbit reticulocytes. The IC50 values of these agents were 52 and 90 microM, respectively. Although cisplatin inhibits the conjugation of ubiquitin to proteins through the inhibition of a ubiquitin-activating enzyme, ACR, at 120 microM, does not. Thus, the antitumor agents affecting the ubiquitin system could be classified into two groups; one is represented by cisplatin, which inhibits the ubiquitination of the proteins, and the other is ACR, which does not inhibit the ubiquitination but does inhibit the ubiquitin-ATP-dependent proteolysis. Mitomycin C belongs to the latter group.


Glutathione-binding proteins identified by monoclonal antibodies which depress the behavioral response evoked by glutathione in Hydra. Hydra shows at least 5 components of the behavioral response (R1-R5) which are evoked at different concentrations of S-methylglutathione (GSM). We have prepared several monoclonal antibodies (mAbs), each of which depressed specific responses and visualized specific structures. In this paper, we analyzed molecules participating in the signaling pathway of R5 response with use of three mAbs (J245, J5 and J5/1), all of which depressed the response. A behavioral analysis of the response in the presence of mixtures of these mAbs suggested that J245 and J5 both acted on a component of the receptor-effector system which was not affected by J5/1. Correspondingly, an immunoblotting analysis showed two 220 kDa proteins which reacted with both J245 and J5 but not with J5/1. ELISA analyses also showed that the J245 antigens formed only a portion of the J5 antigens. A labeled major peak was found in the 220 kDa protein fraction by gel permeation HPLC after immunoprecipitation of the J245 antigen photolabeled with [35S]-S-(p-azidophenacyl)-glutathione. Competition of the photolabeling by the ligands GSM and L-glutamate (a competitive inhibitor of the R5 response) indicated dissociation constants for their binding to the protein of 55 and 90 microM, respectively. These values were consistent with those expected from behavioral experiments. The 220 kDa proteins therefore appear to be candidates for the receptor molecules mediating R5.


[The role of a GTP-binding protein in coupling of a muscarinic cholinergic receptor and Na,K-ATPase in myocardial sarcolemma]. The effects of the cholinergic agonist carbachol (Cch) and guanine nucleotides on the Na,K-ATPase and K-dependent p-nitrophenylphosphatase (K-p-NPPase) activities in rabbit and dog myocardial sarcolemma vesicles in the presence of the pore-forming antibiotic alamethicin (20 micrograms/ml), was studied. Cch (0.01-100 microM) inhibited the both enzymatic activities by 40-45% (IC50 = 0.3-0.5 microM) only after addition of GTP (50 microM) or its analogs: GTP gamma S (0.1-1.0 microM) and Gpp(NH)p (10 microM). The muscarinic acetylcholine receptor (mAchR) antagonist atropine (10 microM) blocked the effect of Cch. GTP gamma S alone produced a concentration-dependent decrease in the both Na,K-ATPase and K-p-NPPase activities by 40-45% (IC50 = 1-2 microM) with a lag period of about 3 minutes; this lag disappeared in the presence of the agonist. The GDP analog GDP beta S (0.01-100 microM) neither affected these activities nor promoted the inhibiting effect of Cch. Pretreatment of sarcolemmal vesicles with 20 micrograms/ml of pertussis toxin in the presence of 100 microM NAD abolished the inhibiting effect of Cch on the Na,K-ATPase and phosphatase activities. Under these conditions pertussis toxin catalyzed the ADP-ribosylation of alpha-subunits of the inhibitory GTP-binding protein (G1) which were identified immunochemically as alpha i2, alpha i3 and, possibly, alpha i1. The data obtained testify to the involvement of G1 in the mAchR-mediated inhibition of myocardial sarcolemmal Na,K-ATPase as well as in the signal transduction from the receptor to the enzyme.


[The role of calcium ions in the molecular mechanism of the protective mechanism of exogenous phosphocreatine]. The role of Ca2+ in the manifestation of the cardioprotective effect of phosphocreatine (PCr) on the ischemic myocardium was studied in isolated rat hearts perfused by the Langendorf method. Under ischemic cardiac arrest induced by a Ca(2+)-free perfusing solution PCr had no protective effect on the ischemic myocardium. PCr accelerated the postischemic restoration of contractility of hearts perfused with a solution containing 0.5 and 1.2 mM Ca2+. The structural analog of PCr, phosphoarginine, possessing a Ca(2+)-binding capacity similar to that of PCr, had no protective effect. The effects of PCr and Ca2+ on the package of sarcolemmal vesiculate lipids were studied by ESR spectroscopy. PCr induced a more dense package of membrane phospholipids at weakly acidic and neutral values of pH (but not at pH 8.5). Although at pH 5.5 Ca2+ did not affect the membrane structure, it potentiated the effect of PCr on sarcolemmal phospholipids. Thus, the protective effect of PCr on the ischemic myocardium is not linked with its ability to bind Ca2+; however, Ca2+ is an indispensable component of the mechanism underlying the protective effect of PCr on the ischemic myocardium.


The use of adrenocorticotrophic hormone (4-9) analog ORG 2766 in autistic children: effects on the organization of behavior. In a double-blind placebo-controlled crossover trial, 14 autistic children were treated with the neuropeptide ORG 2766, a synthetic analog of adrenocorticotrophic hormone (ACTH) (4-9). ORG 2766 treatment (20 mg per day during 4 weeks) was associated with an increased amount and an improved quality of the social interaction of the autistic children with a familiar experimenter. These changes in interaction were clinically relevant. Following treatment with ORG 2766 gaze and smile behaviors of child and experimenter showed stronger temporal contingencies. Further, after ORG 2766, stereotypies were temporally disconnected from verbal initiatives. The data supported the notion of a stimulating effect of ORG 2766 on social interaction. The implications of these findings for the endogenous opioid theory of autism are discussed.


Synthesis, antiviral and cytotoxic activity of 2'-deoxyuridines, 2'-fluoro-2'-deoxyuridines and 2'-arabinouridines containing 5-(1-hydroxy-2-halo-2-ethoxycarbonylethyl)-, 5-(1-hydroxy-2-iodo-2- carboxyethyl)- and 5-[1-hydroxy (or methoxy)-2-iodoethyl] substituents. The 5-[1-hydroxy-2-chloro-2-(ethoxycarbonyl)ethyl]-2'-deoxyuridine (7) and 5-[1-hydroxy-2-bromo-2-(ethoxycarbonyl)ethyl]-2'- fluoro-2'-deoxyuridine/uridine nucleosides (8, 9) were synthesized by the regiospecific addition of HOX (X = Br or Cl) to the vinyl substituent of the respective (E)-5-[2-(ethoxycarbonyl)-vinyl]-2'-deoxyuridines (6a-b) and uridine (6c). A related reaction of (E)-5-(2-carboxyvinyl)-2'-deoxyuridines (10a-b) and uridine (10c) with iodine and potassium iodate afforded the 5-(1-hydroxy-2-iodo-2-carboxyethyl) derivatives (11-13). 5-(1-Hydroxy-2-iodoethyl)-arabinouridine (18) was obtained by the reaction of (17) with iodine in the presence of the oxidizing agent iodic acid. Treatment of (18) with methanolic sulfuric acid afforded 5-(1-methoxy-2-iodoethyl)-arabinouridine (19) in 65% yield. Of the newly synthesized compounds, 7, 11 and 12 showed activity in vitro against HSV-1. The most active compound (12, ID50 = 0.1 microgram/ml) was 10 times less active than acyclovir (ID50 = 0.01 microgram/ml) against HSV-1. Compounds 7 and 11 were cytotoxic to L1210 cells in culture, exhibiting an ED50 of 7.2 and 4.7 micrograms/ml respectively, relative to melphalan (ED50 = 0.15 microgram/ml), but were inactive against the KB cell line.


PA-III rat prostate adenocarcinoma cells (review). PA-III is one of the four transplantable cell lines propagated in vitro in cell monolayer cultures from four spontaneously developed prostate cancer tumors in aged germfree and conventional Lobund-Wistar (L-W) rats (Pollard Model). Our data documented the presence of IGF-I and glucocorticoid but the absence of IGF-II, insulin and androgen receptors in PA-III cells. Activation of IGF-I receptors by IGF-I, IGF-II and insulin stimulated the proliferation of PA-III cells. Activation of glucocorticoid receptors by dexamethasone inhibited the proliferation of PA-III cells. PA-III cells contained urokinase-like activity and expressed the mRNA for urokinase gene which was negatively regulated by glucocorticoids. The above results suggest that PA-III cells could be a useful model for the study of glucocorticoid effects on prostate cancer cells. In addition, these data are discussed in relationship to the capacity of PA-III cells to produce the osteoblastic reaction when transplanted on L-W rat skeleton. A model outlining the putative paracrine interactions between PA-III cells and rat osteoblasts is proposed.


PAF receptors and G-proteins in human blood eosinophils and neutrophils. Identification of G-proteins and coupling of PAF receptors to G-proteins have been examined in the membranes of human blood eosinophils and neutrophils. Heterotrimeric G-proteins, Gi and GS, were present in both cell types, as demonstrated by immunoblotting and ADP-ribosylation with pertussis toxin. In addition, a group of low molecular mass (18-28 kDa) monomeric G-proteins was also identified. Pertussis toxin and GTP gamma S attenuated the specific binding of [3H]PAF, suggesting the occurrence of coupling between pertussis toxin-sensitive Gi protein and PAF receptors in eosinophils and neutrophils.


Occupational lung diseases in the industrializing and industrialized world due to modern industries and modern pollutants. Although most new 'high tech' industrial processes are developed in industrialized countries, many of these technologies are eventually transferred to the industrializing countries. Many of these new technologies are associated with the use of respiratory toxins. However, there has been little study of acute or chronic health effects of work in these industries. The semiconductor industry illustrates many of these issues. The past decade has been increasing globalization of semiconductor manufacturing. Semiconductor manufacturing uses many chemicals with extremely high respiratory toxicity, including gases such as arsine and phosphine, strong acids and bases, dopants and photoactive chemicals. In semiconductor manufacturing, gases and chemicals are strictly controlled, but little is known about the occurrence of respiratory symptoms or disease in this industry. Potential acute respiratory effects of these exposures include mucous membrane irritation, tracheobronchitis, pulmonary edema and death. Chronic effects may include airway sensitization and possibly respiratory cancer. Movement of 'high tech' industries to less industrialized countries may not be accompanied by the same degree of attention to the control of workplace exposures. The shortage of adequately trained health and safety personnel, greater attention to safety than to health issues, and the unorganized and unskilled workforce in industrializing countries may exacerbate this situation. More research is needed on the health effects of exposures in rapidly changing industries such as semiconductor manufacturing, and the results of this research must be communicated and safe practices implemented worldwide.


Properties of the guinea-pig lung platelet-activating factor receptor encoded by the cloned cDNA. Platelet-activating factor (PAF) is a naturally occurring phospholipid possessing proinflammatory and shock-inducing activities. Although PAF exerts these functions via specific receptors, the molecular structures of the receptor has not been clarified. We have isolated a cDNA coding for PAF receptor through expression cloning. The PAF receptor expressed in Xenopus laevis oocytes and COS-7 cells showed pharmacological properties consistent with those in previous reports. Sequence analysis revealed that PAF receptor belongs to the superfamily of G-protein-coupled receptors.


Synthesis and action of PAF in human eosinophils. We compared the level of PAF precursor as well as the synthesis and the catabolism of PAF in eosinophils with those in neutrophils. The modes of action of PAf on these two types of cells were also compared.


The early bactericidal activity of rifabutin measured by sputum viable counts in Hong Kong patients with pulmonary tuberculosis. Previously untreated patients with smear-positive pulmonary tuberculosis were randomly allocated to treatment with 600, 300, 150 or 75 mg doses of rifabutin (LM427, ansamycin), 600, 300 or 150 mg of rifampicin, 300 mg isoniazid or to no drug daily for 2 days. The fall in viable counts of Mycobacterium tuberculosis in sputum collections during the 2 days, termed the early bactericidal activity (EBA), was estimated from counts of colony-forming units (cfu) on selective 7H-11 agar medium. The EBA for rifabutin ranged from -0.039 (an increase in counts) to 0.049 log10 cfu/ml/day whereas the EBA increased from 0.071 for 150 mg rifampicin to 0.293 log10 cfu/ml/day for 600 mg rifampicin and was 0.43 log10 cfu/ml/day for 300 mg isoniazid. The difference between the EBAs for rifabutin and rifampicin just attained significance (P = 0.05) suggesting that rifabutin was inactive or less active than rifampicin against the extracellular bacilli in pulmonary cavities. Peak plasma concentrations of rifabutin after the initial doses were found to be proportional to dose size and were approximately 7 times lower than those after the same dose size of rifampicin. The lower EBA of rifabutin as compared to rifampicin is probably due to the low plasma concentrations which are not fully compensated for by slightly greater antituberculosis activity of rifabutin in vitro.


A controlled study of rifabutin and an uncontrolled study of ofloxacin in the retreatment of patients with pulmonary tuberculosis resistant to isoniazid, streptomycin and rifampicin. Hong Kong Chest Service/British Medical Research Council. A study of rifabutin in the retreatment of patients with chronic pulmonary tuberculosis whose strains of tubercle bacilli were resistant to all three of the drugs isoniazid, streptomycin, and rifampicin, and usually to others as well, was undertaken in 22 Chinese patients in Hong Kong. They were arranged in 11 pairs such that the resistance pattern of the strains for both patients was the same or closely similar. One of each pair was allocated at random to receive rifabutin (B) daily and the other rifampicin (R) daily. The two members of each pair were also prescribed the same or similar companion drugs, selected on the basis of the results of susceptibility tests and of the history of previous antituberculosis chemotherapy. The bacteriological results showed no evidence of a sustained benefit in any patient. A temporary response was seen on sputum smear examination in 14 patients (7B, 7R), of whom 10 (5B, 5R) had a period of smear-negativity, and on sputum culture examination in 10 patients (5B, 5R), of whom 3 (2B, 1R) had a period of culture-negativity. The duration of response was longer for the B patient in 5 pairs and for the R patient in 5. Two patients (both B) had rifabutin-susceptible strains on admission to the study; their temporary responses were among the best and were associated with the emergence of rifabutin resistance, suggesting that rifabutin may have contributed to their response. A total of 17 patients, including 7 not in the paired comparison, were subsequently retreated with ofloxacin; 10 showed a response, disease becoming and remaining quiescent in 3.(ABSTRACT TRUNCATED AT 250 WORDS)


Hetrazepine PAF antagonists.

Hetrazepine PAF antagonists. Hetrazepines such as WEB-2086 or WEB-2170 are potent, specific, competitive PAF antagonists binding at the same site as the PAF molecule. Despite their structural similarity to known hypnotic drugs they lack hypnotic actions. They almost certainly bind not only cell-surface but also intracellular PAF receptors, which may explain their activity in disease models in which adherence of neutrophils or other cell types to the vascular endothelium plays a role.


Intramembrane helix-helix association in oligomerization and transmembrane signaling. In spite of our greatly expanded knowledge of the primary structures of transbilayer receptor proteins, our knowledge of the tertiary and quaternary structures that define the biological activity of these receptors is scant. If we assume that the transmembrane regions of receptor proteins form stable alpha-helices regardless of the mechanism of insertion, a two-stage model of protein folding can be applied. For a multiple-helix protein, the two-stage model would predict that stable helical formation would be followed by an association of the helices to form the appropriate tertiary/quaternary structure. The two-stage model of protein folding is supported by various experiments with bacteriorhodopsin demonstrating that separate proteolytic fragments of bacteriorhodopsin can be refolded separately and can specifically recognize each other in order to associate and form a biologically active molecule. At the level of the bilayer, we propose that the energetics required for the association and packing of the helical transmembrane regions of a multiple-helix protein should not be significantly different from the association of separate single-helix proteins into an oligomer. Given the homogeneity in primary and secondary structure of the transmembrane regions of single-helix proteins, the association of multiple monomers may physically define high vs low affinity states and be a plausible mechanism of signal transduction. Increasing data suggest that oligomerization of receptor proteins may be involved in signal transduction. The transmembrane domains of receptor proteins appear to contain information critical to signaling and may be involved in a close contact site between receptors. This observation allows the two-stage model of protein folding for multiple-helix proteins to be directly applied to the oligomerization of single-helix receptor proteins. In addition, significant data suggest that the ectodomains and cytoplasmic domains are also involved in signaling and oligomerization of receptor molecules. In effect, the present data suggest that the most plausible model, both mechanistically and energetically, is one that includes both oligomerization and a global allosteric conformational change involving all of the defined domains of the receptor molecule. An oligomerization/conformational change model would predict that new sites of close contact would occur between the domains of the receptor molecule, some of which may be between the transmembrane helices. Therefore, experimenters should be able to generate peptides or small molecules that can specifically interfere with either the oligomerization or generation of new close-contact sites involved in the conformational change of the receptor that leads to signaling.(ABSTRACT TRUNCATED AT 400 WORDS)


Long-term use of gamma-linolenic acid-enriched fat emulsion (PFE 4501): case report. A new gamma-linolenic acid-enriched fat emulsion (PFE 4501) was given for 13 mo as part of a home parenteral nutrition regimen to a 28-yr-old patient with cystic fibrosis. Blood biochemistry, hematological data, and fatty acid profile were followed. No side effects were reported, and pulmonary function tests remained stable. gamma-Linolenic acid appears to be safe for clinical use and may benefit patients with derangements in essential fatty acid metabolism. The potential role of a gamma-linolenic acid-enriched intravenous fat emulsion is discussed.


Extracellular matrix and neurite outgrowth. The complex relationship between neuronal cells and the extracellular matrix molecules with which they interact both positively and negatively is currently being investigated on many fronts. Major areas of experimental emphasis include the characterization of an increasing number of extracellular matrix and cell surface associated molecules, the identification of receptors for these molecules, and the analysis of the function of extracellular matrix molecules with respect to neuronal process outgrowth.


Establishment of a liaison service for pregnant opiate-dependent women. A pregnancy liaison and outreach service was developed as part of a community drug team to attract pregnant drug-dependent women into treatment. Women were provided with information on harm reduction, safer drug use and offered treatment for drug dependence. In total, 43 women (45 pregnancies) presented to the service over a 30-month period. Thirty-four women began out-patient treatment of opiate dependence. There was a significant reduction in maternal methadone dose by delivery. Of the 45 pregnancies, four women had therapeutic abortions, two first trimester and two second trimester spontaneous abortions and 34 live births (three women remained untraceable). All the pregnancies that continued to the third trimester proceeded uneventfully with a consequent live birth. Mean gestational age was 37.9 +/- 3.7 weeks, mean birth weight was 2863 +/- 526 g, 10 of the babies were small for gestational age. Fifteen babies required medication for neonatal withdrawal symptoms; mothers whose babies exhibited withdrawal symptoms were using significantly higher doses of methadone at delivery than mothers whose babies did not withdraw. The importance of providing this type of service for women drug users in the 1990s is discussed.


A comparison of predictors of treatment drop-out of women seeking drug and alcohol treatment in a specialist women's and two traditional mixed-sex treatment services. The problem of high levels of client drop-out in drug and alcohol treatment is frequently reported in the literature. In the course of conducting an evaluation of a specialist women's treatment service, the inadequacy of the data on women-specific predictors of treatment drop-out was highlighted. Using a retrospective design, the characteristics of 160 women who left treatment less than 5 days after admission were compared to the 160 women who stayed longer than 5 days and were enrolled in the evaluation study. The findings of this study suggested that women who were employed, had a history of sexual assault (especially in adulthood), nominated alcohol as their drug of choice, were not married, older than 25 years of age and had demonstrated a sympathy with the agency's treatment philosophy were less likely to drop-out of treatment. In addition, for lesbian women, women with a history of sexual assault in childhood, and those with dependent children, attendance at a specialist women's service reduced the incidence of treatment drop-out.


Do smokers of lower tar cigarettes consume lower amounts of smoke components? Results from the Scottish Heart Health Study. Data on 1133 men and 1621 women who smoke solely cigarettes with a known tar yield are extracted from the baseline population survey of the Scottish Heart Health Study. The expired-air carbon monoxide (CO-E), serum thiocyanate and serum cotinine values are compared between smokers in three tar groups: low (below 13 mg/cig.), middle (14-15 mg/cig.) and high tar (above 15 mg/cig.). An index of tar consumption is calculated assuming that the intake of different smoke components relative to one another is in proportion to their concentration in the smoke. CO-E and cotinine are found to peak in the middle tar group. Thiocyanate only tends to increase from low to middle tar group for women and from middle to high tar group for men. Tar consumption increases with tar yield of the cigarette smoked, but the increase is much lower than would be expected. We conclude that the tar yield of a cigarette is not an accurate guide to the amount of smoke components consumed by its smoker. Health professionals should be aware of these limitations of tar yield as a measure of cigarette strength.


Characterization of cytoplasmic T3 binding sites by adsorption to hydroxyapatite: effects of drug inhibitors of T3 and relationship to glutathione-S-transferases. To facilitate studies of thyroid hormone (T3) binding to cytoplasmic proteins, we prepared monkey (M. fascicularis) liver cytosol (100,000g supernatant) and examined T3 binding using hydroxyapatite (HAP) separation. HAP adsorbs cytoplasmic and nuclear binding sites but not serum T4 binding proteins. Cytosol was incubated with [125I]T3 for 30 min at 4 degrees C and separated by adding an equal volume of HAP (15 g/100 mL). After a further incubation of 10 min, the HAP pellet was washed three times in buffer containing Triton X-100, 0.5%. With this method, a single class of T3 binding site was observed with Kd 15.8 +/- 1.2 nM, concentration 0.62 +/- 0.17 pmol/mg protein (n = 3, mean +/- SD). We used this assay to assess potential drug inhibitors of cytoplasmic binding and to evaluate the proposal that glutathione-S-transferases (GST) and cytoplasmic T3 binding proteins are identical. Displacement of [125I]T3 by unlabeled iodothyronines relative to T3 (100) was T4 58, Triac 7, rT3 7, Tetrac less than or equal to 1. This hierarchy indicates that this binding site is distinct from nuclear or serum binding sites. T3 binding was displaceable by nonsteroidal anti-inflammatory drugs (NSAID) and nonbile acid cholephils (NBAC). Half-inhibitory concentrations (microM, mean +/- SD, n greater than or equal to 3) were diclofenac 4.9 +/- 1.3, mefenamic acid 13.6 +/- 0.6, bromosulphthalein 45 +/- 3, iopanoic acid approximately 200. Amiodarone and furosemide were inactive up to 100 microM. No displacement was observed with cortisol or the bile acid taurocholate, up to 100 microM. Dithiothreitol, 5 mM, did not change binding affinity or capacity.(ABSTRACT TRUNCATED AT 250 WORDS)


Stimulation of bone collagen and non-collagenous protein synthesis by products of 5- and 12-lipoxygenase: determination by use of a simple quantitative assay. The influence of 5- and 12-lipoxygenase products on the rate of collagen and non-collagenous protein (NCP) synthesis by murine calvarial explants has been investigated using a new assay based on the resistance of native collagen to degradation by pepsin. The reproducibility and simplicity of this assay allows the quantitative estimation of the rate of bone formation in large numbers of cultures. Hydroxyeicosatetraenoic acids (HETEs) stimulated both the rate of collagen and NCP synthesis with maximal stimulation occurring at 10-100 pM. All leukotrienes stimulated collagen synthesis. LTB4, C4 and D4 showed similar dose-responses with maximal activity occurring at 100 pM. LTE4 was less potent only showing activity at 1-10 nM. Only LTD4 demonstrated the capacity to stimulate NCP synthesis with significant stimulation being seen at 10 nM. The extreme sensitivity of bone collagen and NCP synthesis to lipoxygenase products suggests that these mediators may play a physiological role in bone remodelling.


Interleukin-2 in bone marrow transplantation: preclinical studies. Interleukin-2 (IL-2) promotes the generation and proliferation of killer cells in the peripheral blood and bone marrow (BM) both in vitro and in vivo. When employed in a syngeneic bone marrow transplantation (BMT) setting and followed by IL-2 therapy, murine BM cells activated with IL-2 in vitro (ABM) demonstrate potent graft-versus-leukemia (GVL) and anticytomegalovirus effects. ABM cells retain the capacity to reconstitute the hemopoietic system both in normal and leukemic mice. This therapy does not cause graft-versus-host disease (GVHD). Human ABM cells carry out purging of leukemia without loss of progenitor cell activity in vitro. The purging ability of ABM can be augmented by interleukin-1, interferon, and tumor necrosis factor. IL-2 therapy stimulates the veto suppressor cell activity of T cell-depleted BM, and has reduced GVHD and permitted engraftment of mismatched allogeneic BM in murine models. Future studies should determine the optimum treatment schedules with IL-2 for improving the GVL effect in autologous BMT, and for abolishing GVHD in allogeneic BMT settings.


IAIMS development at Baylor College of Medicine. At Baylor College of Medicine, we are developing the technical and intellectual resources needed to realize the Integrated Academic Information Management System (IAIMS) concept fully. The substantial technical, organizational, and financial commitments involved demand that we align our efforts with the strategic purposes of the college. The support of science, therefore, has become the principal, but not exclusive, focus of Baylor's IAIMS effort. Even so, the information technology architecture we have created for biomedical research is proving valuable in other settings as well. And the infrastructure we are creating--the communications architecture and the linkages to information resources--serves many purposes in addition to those of research. The architecture accommodates a diversity of workstations, networks, and informational and computational servers. This will be the greatest possible chance of transferring the fruits of our Phase III development to other academic medical centers.


IAIMS at Columbia-Presbyterian Medical Center: accomplishments and challenges. The concept of "one-stop information shopping" is becoming a reality at Columbia-Presbyterian Medical Center. Our goal is to provide access from a single workstation to clinical, research, and library resources; university and hospital administrative systems; and utility functions such as word processing and mail. We have created new organizational units and installed a network of workstations that can access a variety of resources and systems on any of seventy-two different host computers/servers. In November 1991, 2,600 different individuals used the clinical information system, 700 different individuals used the library resources, and 900 different individuals used hospital administrative systems via the network. Over the past four years, our efforts have cost the equivalent of $23 million or approximately 0.5% of the total medical center budget. Even small improvements in productivity and in the quality of work of individuals who use the system could justify these expenditures. The challenges we still face include the provision of additional easy-to-use applications and development of equitable methods for financial support.


The impact of IAIMS at Georgetown: strategies and outcomes. Integration of multiple information systems of a medical center will change the way physicians work and practice medicine in the future. Several major steps must be taken by an institution to make this a reality. Since 1983, Georgetown has been engaged in an Integrated Academic Information Management System (IAIMS) project to bring together multiple sources of information that reside on different computers and database systems. Georgetown is developing a Biotechnology and Biomedical Knowledge Network that includes informational and clinical databases, scholar workstations, instruction on computer use, a campuswide network with local area network nodes, and a modular approach to systems integration. The IAIMS project, spearheaded by the medical library, has enabled a broad spectrum of health professionals to benefit directly from new, dynamic information services. The network is heavily used; in 1991, more than 2,100 individual users conducted more than 148,500 computer functions and more than 104,000 searches. There is economy of scale in high-volume use. Overall, the average search cost is $1.57; for high use databases the cost is $0.38, and for low use, it is $9.41. As described in this paper, IAIMS offers a cost-effective means of enhancing patient care by improving information services to physicians. At Georgetown, IAIMS has advanced the concept of integration, accelerated use of computers in education, increased user acceptance of advanced technologies, and established cost factors for providing information resources. While progress made in improving the transfer of medical information is impressive, it is clear that IAIMS requires several more years of support to achieve full implementation.


ACOGQUEST: the model phase of the IAIMS project of the American College of Obstetricians and Gynecologists. In 1990, the American College of Obstetricians and Gynecologists (ACOG) became the first national organization to receive a model phase Integrated Academic Information Management System (IAIMS) grant from the National Library of Medicine. The goal of the ACOG model phase project is to develop and test a prototype for an integrated system that will meet the needs of ACOG and NAACOG members in patient care, research, education, and administrative information. The model phase goal will be accomplished primarily through ACOGQUEST, an integrated approach to providing accurate, current, quality-filtered information to ACOG and NAACOG members in a variety of formats. Another method of information dissemination now being tested is a heuristic-based patient management database, which will include a concise, interactive display of ACOG-reviewed information that can be incorporated into patient records.


Prototyping an institutional IAIMS/UMLS information environment for an academic medical center. The paper describes a prototype information environment designed to link network-based information resources in an integrated fashion and thus enhance the information capabilities of an academic medical center. The prototype was implemented on a single Macintosh computer to permit exploration of the overall "information architecture" and to demonstrate the various desired capabilities prior to full-scale network-based implementation. At the heart of the prototype are two components: a diverse set of information resources available over an institutional computer network and an information sources map designed to assist users in finding and accessing information resources relevant to their needs. The paper describes these and other components of the prototype and presents a scenario illustrating its use. The prototype illustrates the link between the goals of two National Library of Medicine initiatives, the Integrated Academic Information Management System (IAIMS) and the Unified Medical Language System (UMLS).


Creating the future: IAIMS planning premises at the University of Washington. In September 1990, the University of Washington (UW) received a Phase I IAIMS Planning Grant from the National Library of Medicine and embarked upon a planning process involving the entire health sciences center. As a result of our relatively late entry into IAIMS planning, we have been able to learn from the experiences of other health sciences centers and to leverage our existing institutional efforts. Consequently, our progress has been rapid, and in a little over a year, we drafted a long-range plan and embarked on several related research and development projects. The hallmarks of our planning process include careful study of both the UW institutional environment and the experiences of other IAIMS institutions throughout the United States; broad, interdisciplinary participation of faculty, librarians, and administrators; an intensive educational process; a focus on people rather than technology; and, above all, leveraging of existing institutional and research projects that support our vision for the future.


VIP receptor subtypes in mouse cerebral cortex: evidence for a differential localization in astrocytes, microvessels and synaptosomal membranes. The binding characteristics of a monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) to the membranes of astrocytes, intraparenchymal microvessels and synaptosomes were analyzed in mouse cerebral cortex. Binding to astrocytes, studied in primary cultures, indicates the presence of a single class of high affinity binding sites with a Kd of 3.3 nM and a Bmax of 565 fmol/mg protein. The structurally related peptide secretin does not compete for sites labeled by M-[125I]VIP. In cultured astrocytes, VIP has been previously shown to promote glycogenolysis. Secretin, despite its lack of interaction with sites labeled by M-[125I]VIP, stimulates glycogenolysis with an EC50 of 0.5 nM, thus demonstrating the presence in astrocytes of functional secretin receptors independent from those for VIP. Trypsinization of the primary astrocyte cultures followed by replating as secondary cultures, reveals a second class of low affinity binding sites, with a Kd of 41.3 nM and a Bmax of 881 fmol/mg protein. Secretin does not compete for this class of low affinity binding sites either. Binding of M-[125I]VIP to intraparenchymal microvessels reveals the presence of two classes of binding sites with Kd of 1.4 and 30.3 nM, and Bmax of 7.1 and 73.8 pmol/mg protein, respectively. Similar to what is observed in primary or secondary astrocyte cultures, secretin does not interact with these sites. In this cell type VIP stimulates cAMP formation with an EC50 of 18 nM, while secretin is ineffective. Finally, in agreement with previous reports in rat and guinea pig cerebral cortex, two classes of binding sites are observed in synaptosomal membranes: a high affinity class with a Kd of 4.9 nM and a Bmax of 316 fmol/mg protein, and a low affinity class with a Kd of 42.8 nM and a Bmax of 1578 fmol/mg protein. In contrast to what is observed in non-neuronal membranes, in synaptosomal membranes, secretin effectively competes for sites labeled by M-[125I]VIP with an EC50 of approximately 150 nM. These results indicate that secretin may represent a useful tool to discriminate between neuronal and non-neuronal VIP binding sites, since it competes with M-[125I]VIP exclusively for the neuronal class of binding sites.


Differential effects of central and peripheral injection of interleukin-1 beta on brain c-fos expression and neuroendocrine functions. Cytokines such as interleukin-1 beta (IL-1 beta) alter the activity of the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes in the rat. However, the brain sites at which IL-1 beta exerts these effects have not been well identified. The present study sought to identify some of these sites, using c-fos protein expression as an index of cellular activation. We also attempted to determine possible differences between the effects of peripheral and central injection of IL-1 beta on the activation of specific brain areas. Castrated male rats received intravenous (i.v.) or intracerebroventricular (i.c.v.) injections of IL-1 beta through a jugular catheter or a permanent cannula implanted in the right lateral ventricle, respectively. Blood samples were taken before, as well as 30 and 120 min after i.v. or i.c.v. IL-1 beta infusion in order to measure plasma ACTH and LH levels. Immediately thereafter, the rats were anesthetized with pentobarbital, then perfused. Their brains were removed and postfixed for one hour. Thirty-microns frozen sections were cut and approximately every fourth tissue section was processed for c-fos expression by an avidin-biotin-peroxidase method. Both i.v. (1 microgram) and i.c.v. (100 ng) injection of IL-1 beta significantly increased plasma ACTH levels, but only i.c.v. treatment measurably inhibited LH secretion. I.c.v. infusion of the cytokine markedly augmented c-fos expression in the paraventricular nucleus (PVN) and the arcuate nucleus (ARC) of the hypothalamus. A large amount of CRF cells in the PVN contained labelled c-fos protein (as measured by a double labelling technique), which indicates that CRF perikarya in this hypothalamic region are activated by the central administration of IL-1 beta. In contrast, i.v. injection of IL-1 beta did not significantly alter c-fos expression in the PVN or the ARC of the hypothalamus. These results suggest that the increased HPA axis activity which follows the peripheral IL-1 beta administration, a phenomenon previously shown to depend on endogenous CRF, does not require immediate activation of hypothalamic CRF perikarya. Thus our results indicate that the stimulatory effect of blood-born cytokine may be exerted at the level of nerve terminals in the median eminence. In contrast, i.c.v.-injected IL-1 beta appears to activate the HPA axis through a stimulation of CRF neurons within the parvocellular part of PVN.(ABSTRACT TRUNCATED AT 400 WORDS)


Ionotropic excitatory amino acid receptors in discrete brain regions of kindled rats. A study was performed to examine the specific binding of excitatory amino acid (EAA) receptor subtypes in 5 brain regions of rats kindled from the amygdala or hippocampus, using extensively washed and Triton X-100-treated membranes. Seven days after the last amygdala kindled seizure, [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine maleate ([3H]MK-801) binding, which labels N-methyl-D-aspartate (NMDA)-sensitive receptor-linked cation channels, decreased significantly only in the amygdala of kindled rats compared to that of controls under equilibrium assay conditions. There was no significant change in [3H]MK-801 binding in the amygdala or hippocampus 7 days after the last hippocampal kindled seizure, or 28 days after the last amygdala kindled seizure. Nor was there a significant change in NMDA-sensitive [3H]glutamate, strychnine-insensitive [3H]glycine, [3H]spermidine, [3H]kainate or [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) binding in any brain region 7 days after the last amygdala kindled seizure, or in the hippocampus 28 days after the last amygdala kindled seizure. These results indicate that [3H]MK-801 binding sites labeling NMDA-sensitive receptor-linked cation channels in the amygdala undergo downregulation only transiently, but that none of the subcomponents of the NMDA receptor macromolecular complex exhibit enduring changes at steady state following the completion of amygdala kindling.


Antagonism of corticotropin-releasing factor receptors in the locus coeruleus attenuates shock-induced freezing in rats. Intracerebroventricularly administered alpha-helical CRF9-41, a corticotropin-releasing factor (CRF) receptor antagonist, is known to reduce a variety of stress-induced behavioral responses. This study examined in rats whether antagonism of CRF receptors in the region of locus coeruleus (LC) plays a role in reducing freezing induced by electric foot shock. Freezing is a well-characterized defensive response to stress and has been demonstrated to index an animal's degree of fear. A CRF-receptor antagonist, alpha-helical CRF9-41, bilaterally infused into the LC significantly reduced the duration of freezing at a dose as low as 0.20 micrograms. Additional experiments confirmed that 0.20 micrograms of alpha-helical CRF9-41 significantly reduced the duration of freezing only when cannulae were within the LC or in regions bordering the nucleus. Antagonist-treated rats with cannulae that did not impinge on the LC exhibited freezing at levels not different from vehicle-treated animals. These results strongly implicate CRF receptors located in the LC region in influencing the display of stress-induced behavior.


Effect of stress on hypothalamic insulin in rats. Four experiments examined the effects of stress on hypothalamic insulin and plasma hormones in rats. Two hours daily of immobilization (IM) stress for 2 and 4 days resulted in an increase in hypothalamic insulin. In contrast, 15 min of daily IM over 13 days or 48 h of continuous signalled shock avoidance did not alter hypothalamic insulin. These findings are interpreted to indicate that changes in hypothalamic insulin are part of the stress response. Possible reasons for the different effects of time and paradigm on the hypothalamic insulin responses to stress are discussed. Plasma insulin and glucose levels were not responsive to any of the stressors. Brief acute stress caused increases in the stress-responsive hormones ACTH, corticosterone, and prolactin, as expected, and these responses attenuated or disappeared with repeated or longer stress exposures.


[Combined therapy in patients with testicular germ cell tumors in the dissemination stage (pulmonary metastasis)]. The efficacy of combined cytostatic-surgical treatment was evaluated in a prospective study involving 70 patients with germinal tumors of the testis metastasizing into the lungs. Complete remission was achieved in 50 (71.4%) patients, 30 of these received only chemotherapy. Thoracotomy was performed in 14 patients with residual pulmonary CT finding, 6 of them underwent bilateral operations. Of the total 21 operations of the lungs residual malignant tumor was found 4 times. After a mean follow up period of 29.8 months since onset of treatment 48 patients (68.6%) survived. Within a mean of 11.4 months after onset of treatment 22 patients (31.4%) died. The authors consider thoracotomy with resection of the metastatic focus to be the only method for determining reliably the biological nature of the residual pulmonary lesion after chemotherapy. The result of the procedure determines the further measures, i.e. sequential chemotherapy or regular check ups. (Tab. 1, Fig. 6, Ref. 15.).


Detection of antibodies to human immunodeficiency virus (HIV-1) with the particle agglutination test. This study was designed to evaluate the sensitivity and specificity of the particle agglutination test (PA) for antibody detection and to determine whether it is possible to use this test as a screening routine for anti-human immunodeficiency virus type I antibodies instead of the enzyme linked immunosorbent assay (ELISA). Serum samples were collected from 5,142 subjects and tested with the above two tests. A total of 83 samples were observed to be ELISA-positive. However, only 36 were found to be seropositive by the PA test. These samples were then confirmed by the Western blot analysis and 28 were positive for HIV-1, 33 were negative and 22 gave indeterminate results. The most common indeterminate band was p15/17 and the p24 band came next. The sensitivity and specificity of the PA test were 100% and 99.9%. The results indicate that the PA test is not only a convenient technique but also has a high sensitivity and specificity. It can be used as a primary screening test for antibody against HIV-1 instead of the ELISA.


Serodiagnosis of tuberculosis by an enzyme-linked immunosorbent assay using mycobacterial antigen 60. This study was undertaken to assess the diagnostic utility of an enzyme-linked immunosorbent assay using mycobacterial antigen 60 in patients with pulmonary tuberculosis. This test was applied to 210 adults, including 111 patients with active pulmonary tuberculosis, 80 control subjects and 19 patients with inactive pulmonary tuberculosis. Although the serums from the patients with active tuberculosis has a higher mean value of IgG antibody concentration than the control group, there was a considerable overlap of serum IgG antibody level between the two groups. 200 ELISA unit (EU) was established as a reasonable cut-off value, which gave an 80.2% (89/111) sensitivity for the group with active pulmonary tuberculosis and a specificity of 76.3% (61/80) only for the control group. The patients with inactive pulmonary tuberculosis had a positive rate up to 47.4% (9/19) when 200 EU was chosen as a cut-off value.


Motor unit potential analysis under submaximal contraction: ADEMG study in normal and radiculopathy subjects. The purpose of this study was to investigate the motor unit potential (MUP) morphology in different degrees of muscle contraction. Fourteen normal volunteers and 8 patients with C7 radiculopathy were included. The extensor digitorum communis muscle was kept in minimal contraction and 5%, 10%, 30% and 50% of maximal contraction under the guidance of strain gauge. Automatic motor unit potential analysis was done by automatic decomposition electromyography for each epoch of muscle contraction in both normal and radiculopathic groups. The results revealed that as muscle contraction increased, the observed MUPs had higher amplitude, rising ratio and firing rate, while the duration became shorter, but no regular change was found in turns reading. These tendencies were found in both normal and radiculopathic groups. This study enhanced the understanding of morphology of MUPs with higher threshold, and proved the usefulness of ADEMG in differentiating normal from cervical chronic radiculopathy condition. It is suggested that the examiner should observe both lower and higher threshold MUPs during clinical needle examination to make diagnosis more accurately.


Shouldice inguinal hernioplasty.

Shouldice inguinal hernioplasty. Since 1978, 108 patients with 111 inguinal hernias have been treated with Shouldice hernioplasty in our hospital, including 97 males and 11 females with a mean age of 54 years. 85.6% of them were primary hernias and 14.4% of them were recurrent ones. Local anesthesia was performed in 67.6%. The average operation time was 84 min. Immediate postoperative complications were present in 4.5% of the patients. 93.5% of the patients were followed for an average period of 4.6 years. There was only one recurrence (1.0%). These results indicate that Shouldice hernioplasty is an effective repair for both primary and recurrent inguinal hernia in which most cases can be performed under local anesthesia with early recovery.


Mortality trends in the past 20 years in Pu-Li, Taiwan. This study was based on a survey of the death certificates of Pu-Li Town in Taiwan, issued over the past 20 years from 1966 to 1985. Age-adjusted mortality trends as well as cause-specific mortality trends were analyzed and compared with nationwide Taiwan data. As a whole, Pu-Li had a higher age-adjusted mortality than that of the overall Taiwan area. This finding may result from a higher mortality from tuberculosis in Pu-Li. Based on these data, the five leading causes of death in Pu-Li were cerebro-vascular disease, accident, heart disease, cancer and tuberculosis. Hypertension and diabetes were the 5th and 6th leading causes of death in 1985 and ranked 12th and 13th, respectively, in 1966. This data point out the increasing importance of hypertension and diabetes rates in Pu-Li. Tuberculosis and pneumonia had been controlled, ranking from the 1st and 3rd in 1966 to the 10th and 12th, respectively, in 1985. Suicide, cancer, and accident were usually coded as the single cause of death without other co-existent causes of death noted, so that there was in most instances not much difference between analyses based on the underlying cause of death and multiple causes of death. However, this was not true for hypertension and diabetes. If multiple causes of death were analyzed, only 34.5% of hypertension and 66% of diabetes were coded as the underlying cause of death. 37.2% of cerebro-vascular diseases co-existed with hypertension, and 20.3% of diabetes co-existed with hypertension.


[Circadian changes of atrial natriuretic peptide, cyclic AMP, cyclic GMP, sodium, blood pressure and heart rate in normal subjects]. To investigate the circadian change in the plasma atrial natriuretic peptide (ANP) concentrations and its relation to cyclic AMP (cAMP), cyclic GMP (cGMP) and serum sodium. This investigation was conducted on 22 normal subjects consisting of 16 men and 6 women with a mean age of 21.5 years. Blood samples, systolic blood pressure (SBP), and heart rate (HR), were collected and recorded every 8 hours during a 24 hour period. SBP and HR were highest at 1600 h (p less than 0.05). Plasma ANP concentrations at 0800 h (196.3 +/- 140.6 ng/L, p less than 0.01) and 1600 h (203.4 +/- 127.5 ng/L, p less than 0.01) were higher than at 2400 h (64.4 +/- 46.0 ng/L). Serum sodium, plasma cAMP, and cGMP were at the highest level at 1600 h (p less than 0.05). Plasma ANP correlated with plasma cAMP, cGMP, serum sodium, SBP, and HR (r = 0.378 - 0.419, p less than 0.05). This data supports the assumption that atrial wall distension seems to be a stimulus for ANP release.