Prickly pear (Opuntia sp.) pectin reverses low density lipoprotein receptor suppression induced by a hypercholesterolemic diet in guinea pigs. The effects of prickly pear pectin on plasma LDL metabolism were investigated by feeding guinea pigs either a diet containing 15 g/100 g lard and 0.25 g/100 g cholesterol (LC diet) or the LC diet in which cellulose was partially replaced (2.5 g/100 g) by prickly pear pectin (LC-P diet). The LC-P diet lowered plasma LDL cholesterol concentrations by 33% (P < 0.001). Low density lipoprotein composition was modified by intake of prickly pear pectin; the relative percentages of free and esterified cholesterol were lower and triglycerides were higher in LDL from animals fed the LC-P diet (P < 0.05). Intake of prickly pear pectin did not affect hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity; however, hepatic free and esterified cholesterol concentrations were lowered by 46 and 64%, respectively. Hepatic apolipoprotein B/E receptor expression (Bmax) was 60% higher in animals fed the LC-P diet (P < 0.01). Similar to the in vitro data, receptor-mediated LDL fractional catabolic rates were 190% higher in animals fed the LC-P diet (P < 0.05), whereas apolipoprotein LDL flux rates were not affected. Apolipoprotein LDL pool size and fractional catabolic rates exhibited a significant correlation (r = -0.52, P < 0.01). These data indicate that an increase in apolipoprotein B/E receptor expression is a major metabolic response by which intake of prickly pear pectin decreases plasma LDL concentrations.


HL-60 cells can be made copper deficient by incubating with tetraethylenepentamine. A system for studying copper deficiency was developed in a cell culture model. HL-60 cells were incubated with three chelators known to bind copper. One chelator, tetraethylenepentamine (TEPA), reduced cellular copper levels and the activities of two copper-requiring enzymes, Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and cytochrome c oxidase. The specificity of the chelator was assessed by incubating cells with both copper and TEPA and, in other experiments, with zinc and TEPA. Copper levels, Cu/Zn-SOD activity and cytochrome c oxidase activity were restored to control values when copper and TEPA were added to cultures simultaneously, indicating the TEPA was responsible for reducing these aspects of copper metabolism. Incubating with both zinc and TEPA reduced copper levels relative to the control, but did not reduce Cu/Zn-SOD activity to the same extent as TEPA alone. The chelation of copper was a time-dependent process that was stable for at least 4 d. Cell growth and viability were not affected by TEPA. Respiratory burst activity, an indicator of differentiation, was not affected by TEPA, demonstrating that the reduction of Cu/Zn-SOD activity was due to copper chelation and not due to changes in Cu/Zn-SOD protein levels that occur during differentiation. Loss of copper, as well as a reduction of the activity of two copper-requiring enzymes, provides evidence that TEPA is a useful compound for creating a functional copper deficiency in cell culture.


Dietary corn oil and guar gum stimulate intestinal crypt cell proliferation in rats by independent but potentially synergistic mechanisms. The effects of corn oil, guar gum and cellulose on mucosal proliferation were investigated in rats. Animals were allocated to three groups and fed a fiber-free diet or diets containing 100 g/kg of cellulose or guar gum. Each group was subdivided to receive corn oil at 40 or 80 g/kg. The crypt cell production rate (CCPR) was determined after 28 d. Consumption of guar gum or corn oil led to greater CCPR in the ileum and cecum. In a second experiment, animals were allocated to two groups and fed diets containing either cellulose or guar gum (100 g/kg). Each group was again subdivided to receive either corn oil (80 g/kg) or minimal lipid (linolenic acid, 10 g/kg). The trophic effect of guar gum occurred even in the low lipid-fed group, indicating that guar gum exerts a positive effect on cell turnover independently of any interaction with luminal lipid. However, the highest CCPR occurred in animals fed guar gum and corn oil. Postprandial enteroglucagon and gastrin concentrations were highest in animals fed both guar gum and corn oil. Thus, corn oil and guar gum exert independent trophic effects on the intestinal mucosa. The combination of effects led to a three- to four-fold increase in colon mucosal CCPR.


Japanese soybean paste miso scavenges free radicals and inhibits lipid peroxidation. Japanese soybean paste miso, which has been reported to prevent gastric and mammary cancer and chronic nephritis, was demonstrated by electron spin resonance spectrometry using 5,5'-dimethyl-1-pyrroline-N-oxide as a scavenger of free radicals. Fifty mg/ml of miso scavenged 100% of 1,1-diphenyl-2-picrylhydrazyl radicals (3.9 x 10(15) spins/ml); 45 mg/ml quenched 92% of hydroxyl radicals (7.9 x 10(16) spins/ml); and 50 mg/ml quenched 50% of superoxide anion (6.7 x 10(16) spins/ml). In the system of rat cerebral cortex homogenate supplemented with 2 mM each of Fe2+ and ascorbic acid, 90% and 82% of the hydrogen and carbon-centered radicals having 1.7 x 10(13) spins/ml and 3.9 x 10(13) spins/ml, respectively, were quenched by 180 mg/ml of miso. The thiobarbituric acid-reactive substances, an index of lipid peroxidation in the brain, was inhibited by 10 mg/ml of miso. These results showed that miso acts as an antioxidant by scavenging free radicals.


p53 immunohistochemistry in malignant fibrous histiocytomas and other mesenchymal tumours. In this study we analysed by immunohistochemistry the expression of p53 protein in 14 malignant fibrous histocytomas (MFHs), 22 other types of sarcoma (eight leiomyosarcomas, four rhabdomyosarcomas, four liposarcomas, two fibrosarcomas, two chondrosarcomas, one malignant schwannoma, and one dermatofibrosarcoma protuberans), and 25 non-malignant mesenchymal lesions (eight dermatofibromas, four cases of nodular fasciitis, three leiomyomas, three fibromatoses, two epithelioid.leiomyomas, two neurofibromas, one schwannoma, one myositis ossificans, and one giant cell tumour of tendon sheath). Four MFHs and nine other types of sarcoma (four leiomyosarcomas, two chondrosarcomas, one liposarcoma, one fibrosarcoma, and one dermatofibrosarcoma protuberans) showed nuclear positivity for p53. Of the benign soft tissue lesions, p53 positivity was observed in two fibromatoses, one nodular fasciitis, and one dermatofibroma. The number of p53-positive cells in these benign lesions was considerably smaller than that in most of the p53-positive sarcomas. The p53 positivity in MFHs and other types of sarcoma indicates that p53 gene alterations may play a part in the neoplastic transformation of these tumours. The occurrence of p53 positivity in benign mesenchymal lesions suggests that sometimes p53 protein may accumulate in cells without an associated malignancy. Because of this, p53 immunoreactivity cannot, by itself, be used as a criterion of malignancy. According to our results, p53 positivity in over 1 per cent of tumour cells in mesenchymal lesions favours malignancy.


The inhibiting effect of catecholamine-melanins on UV-induced lecithin peroxidation. It was found that the yield of thiobarbituric acid reactive substances in UV-irradiated liposome membranes was significantly suppressed in the presence of catecholamine-melanins, indicating their ability to inhibit lipid peroxidation. The extent of inhibition depended on the type and concentration of melanin polymers. Melanin-copper complexes inhibited lecithin photooxidation less effectively than copper-free melanins derived from the same precursor. The antioxidant efficiency of melanins appears to be related to the levels of intrinsic and photo-induced free radical centers in the melanin polymer, as well as to accessibility of these centers for active species formed during irradiation of liposomes.


Refractory periodontitis associated with abnormal polymorphonuclear leukocyte phagocytosis and cigarette smoking. To learn if refractory periodontitis may be associated with defects in peripheral blood polymorphonuclear leukocyte (PMN) function, phagocytosis and chemotaxis were analyzed in 31 otherwise healthy patients and 12 unaffected controls. When compared to controls, no chemotactic defects to 10 nM f-Met-Leu-Phe (fMLP) were detected. In contrast, phagocytosis was significantly impaired (P < 0.001). The mean rates of adhesion and ingestion of opsonized Staphylococcus aureus by PMNs were 7.1 +/- 1.7 (+/- SD) and 1.4 +/- 0.5 bacteria/100 PMNs/minute respectively for patients, and 11.0 +/- 2.4 and 3.1 +/- 0.6 for unaffected, healthy controls. While the quality of oral hygiene and access to dental care were high, a retrospective search for associated environmental variables showed that 90% (28 of 31) of the refractory patients were smokers. The frequency of smokers is particularly striking, since only 21% of adults in Minnesota use tobacco regularly. These data suggest that there is a strong association between a peripheral blood PMN defect and refractory periodontitis. Furthermore, these studies suggest that tobacco use may contribute to this association.


Binding sites for [3H]-melatonin in human platelets. A number of in vitro effects of melatonin on human platelets were revealed in previous studies. In order to examine whether high affinity binding sites for [3H]-melatonin were present in membrane preparations of human platelets, a rapid filtration procedure through Whatman GFB paper was employed. Maximal melatonin binding was attained within 3 hr at 0 degree C. Scatchard analysis indicated a single population of binding sites with a dissociation constant (Kd) = 4.1 +/- 0.5 nM and maximal number of binding sites (Bmax) = 24.2 +/- 1.9 fmol/mg protein (mean +/- SEM of five experiments). When various indole analogs were tested for their ability to inhibit [3H]-melatonin binding, the following Ki (nM) were obtained: 6-chloromelatonin (11.4), 2-iodomelatonin (22.0), melatonin (24.7), 5-methoxytryptophol (49.9), N-acetylserotonin (68.9), 6-hydroxymelatonin (78.2), 5-methoxytryptamine (184). Serotonin was a potent inhibitor of [3H]-melatonin binding with a Ki = 20.6 nM. Except for 2-methylserotonin and alpha-methylserotonin, a number of serotonin agonists and antagonists tested did not affect melatonin binding to platelet membranes. Binding experiments carried out at either 0800 or 2000 did not reveal time-dependent differences in Kd or Bmax. The results suggest that high affinity melatonin acceptors are present in human platelets.


Infection and dysfunction of monocytes induced by experimental inoculation of calves with bovine immunodeficiency-like virus. Three calves were experimentally inoculated with bovine immunodeficiency-like virus (BIV) to examine BIV pathogenesis. Inoculated calves produced specific antibody that could be detected from 3 to 5 weeks up to 1 year postinoculation (pi). Virus was isolated from peripheral blood mononuclear cells (PBMC) 3-4 weeks pi by syncytia assay. Thereafter, the virus could be continually isolated. BIV could be isolated from monocytes but not from T cells. Likewise, monocytes could be infected with BIV in vitro. Various monocyte functions of these BIV-infected calves and age-matched uninfected calves were tested; superoxide anion release, phagocytic activity, and chemotactic responsiveness of monocytes were depressed in BIV-infected calves compared with control calves. A slight delay in the humoral immune response against mouse serum protein was also evident. During the observation period of approximately 1 year, no significant clinical symptoms could be observed. One calf, however, was killed at 15 months pi. At the time of necropsy, BIV could be isolated from PBMC as well as from cells of the spleen, liver, and lymph nodes.


Early and progressive helper T-cell dysfunction in feline leukemia virus-induced immunodeficiency. Cats infected with the highly pathogenic feline leukemia virus isolate FeLV-FAIDS develop an immunodeficiency syndrome characterized by progressive loss of CD4+ T cells and eventual pan-lymphocyte depletion. Prior to the decline in circulating CD4+ cells, infected cats are unable to mount primary antibody responses to T-dependent antigens. We investigated whether the altered ability of helper T cells to produce cytokines necessary for B cell growth and differentiation might be a primary event in the pathogenesis of FeLV-FAIDS infection. We found that as early as 9 weeks after infection, lymphocytes from cats with normal CD4+ cell numbers produced significantly lower levels of B-cell stimulatory factors. This deficit became progressively more severe with time. By contrast, resting B cells isolated from infected cats did not differ from controls in the ability to undergo activation and differentiation to antibody-secreting cells. The results suggest that a progressive defect in Th function occurs prior to CD4+ T-cell depletion early in the course of FeLV-FAIDS induced immunodeficiency.


Antibody to HIV-1, HTLV-I, and HCV in three populations of rural Haitians. The seroprevalence of antibodies to HIV-1, HTLV-I, and HCV was evaluated in three populations from northern rural Haiti: 1,727 patients attending the hospital for symptoms suggestive of HIV disease, 228 consecutive surgical patients, and 500 pregnant women were tested. HIV-1 seroprevalence was 6.1 and 4.0% in the last two groups, respectively, and 39.3% in the symptomatic population. Associated symptoms of wasting, cough, and diarrhea and a clinical diagnosis of AIDS were significantly predictive of HIV-1 seropositivity. Antibody to HTLV-I seroprevalence ranged from 2.2-5.3% in pregnant women, surgical patients, and HIV-seronegative symptomatic patients and was similar among the three groups when stratified by age. In contrast, HIV-1 seropositivity and HTLV-I seropositivity were significantly associated. The prevalence of confirmed antibody to HCV was low and not associated with either HIV-1 or HTLV-I seropositivity.


Natural history of anal cytologic abnormalities and papillomavirus infection among homosexual men with group IV HIV disease. Previous studies have demonstrated a high prevalence of anal cytologic abnormalities as well as anal human papillomavirus (HPV) infection among homosexual men with group IV HIV disease. However, the natural history of these changes in this population has not yet been studied. To this end, 37 homosexual men with group IV HIV disease attending an outpatient HIV clinic were followed at approximately 9-month intervals for an average of 17 months, using anal cytology, anoscopy, anal biopsy, and anal HPV DNA hybridization. During the study, the proportion of the 37 subjects with anal cytologic abnormalities increased from 27 to 65%. The proportion of subjects with any grade of anal intraepithelial neoplasia rose from 8 to 32%, with high-grade anal intraepithelial neoplasia increasing from 0 to 16%. The proportion of subjects with anal HPV infection increased from 60 to 89%, and infection with multiple HPV types was noted in at least 48%. We conclude that a large proportion of homosexual men with group IV HIV disease develop anal cytologic abnormalities, including anal intraepithelial neoplasia, over a short period of time. Together with a rapidly increasing incidence of anal cancer among single, never-married men in the San Francisco Bay area, these results suggest that these men may be at significant risk of development of anal cancer.


Reperfusion injury in bone: effects of CV-3611, a free radical scavenger, on ischemic revascularized bone grafts in rats. Oxygen-derived free radicals have been shown to play an important role in reperfusion injury. The protective effect of CV-3611, a new free radical scavenger, on reperfusion injury in an ischemic revascularized hind limb model in rats was examined. Warm ischemia (25 degrees C) was produced by vascular pedicle clamping and sustained for 0, 3, and 6 hr. Histologic and fluorochrome bone-labeling analyses demonstrated improved overall viability of osteocytes, osteoblasts, and marrow cells in the CV-3611-treated group compared to controls. The CV-3611-treated group had statistically significant improvement in the ratio of lacunae, maintained osteogenetic ability, and preserved normal growth plate architecture after 6 hr of ischemia. The control group showed local central areas of disorganization by 3 hr and complete destruction of the growth plate with early growth arrest after 6 hr of ischemia. These results indicate that administration of CV-3611 prior to reperfusion can prevent reperfusion damage in bone tissue and maintain osteogenetic ability. This technique may have clinical application for reducing the complications of prolonged ischemia to bone tissue.


Roles of structural domains in the morphology and surface anchoring of the tetragonal paracrystalline array of Aeromonas hydrophila. Biochemical characterization of the major structural domain. The tetragonally arranged S-layer of Aeromonas hydrophila contains two morphological domains. The mature S-layer protein of A. hydrophila has a subunit molecular weight of 52,000, and has been reported to contain two structural domains. Here a mutant has been isolated which produces an S-layer of subunit molecular weight 38,650 as determined by sedimentation analysis. This truncated S-protein was exported via the periplasm to the cell surface, but could not self-assemble into a tetragonal array or be anchored to the cell surface. Instead the truncated protein formed cup-like structures which were purified and characterized biochemically. Automated Edman degradation showed that the truncated protein comprised the amino-terminal structural domain of the S-protein. This domain had an increased hydrophobic amino acid content relative to the wild-type protein, and contained approximately 42% beta-sheet, 10% alpha-helix, and 19% beta-turn. Differences in alpha-helix and beta-turn contents between the wild-type and truncated proteins were observed when the effects of pH and SDS were examined, indicating that the carboxy terminus influences the effects of environmental change on the conformation of the S-protein. This lesser carboxy-terminal array also appears to be required for both correct array morphology, and array anchoring, while the greater amino-terminal domain appears to comprise the major morphological core of the surface array.


Preliminary crystallographic study of bluetongue virus capsid protein, VP7. Bluetongue virus serotype 10 (BTV-10) VP7, expressed by insect cells infected with the recombinant baculovirus, has been purified and crystallized. Two crystal forms suitable for X-ray analysis have been obtained. Type I crystals belong to space group P6(3)22 with a = b = 95.2 A, c = 181.0 A, alpha = beta = 90 degrees gamma = 120.0 degrees, and contain a single subunit in the crystallographic asymmetric unit. They diffract to dmin = 3.0 A. Type II crystals belong to space group P2(1) with a = 69.4 A, b = 97.1 A, c = 71.4 A, beta = 109.0 degrees, and contain a trimer in the crystallographic asymmetric unit. They diffract to dmin = 2.1 A. These results, together with solution studies, show that the molecule is a trimer.


Crystallization and preliminary X-ray diffraction studies of a peroxidase from barley grain. Crystals suitable for X-ray diffraction analysis of both glycosylated and non-glycosylated forms of a barley peroxidase have been grown. The crystals of the glycosylated peroxidase have been grown by the hanging drop vapour diffusion method using polyethylene glycol 4000 as the precipitant in the presence of n-propanol and potassium iodide at pH 8.5. The crystals are needles belonging to the orthorhombic spacegroup P2(1)2(1)2(1) with unit cell dimensions a = 62.95 A, b = 66.24 A and c = 70.78 A. There is one barley peroxidase molecule in the asymmetric unit. The crystals contain approximately 38% solvent and appear to be stable to lengthy X-ray exposure. They diffract to beyond 1.9 A.


Activity and distribution of phosphoinositidase C in rat sciatic nerve. The hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) by rat sciatic nerve cytosolic phosphoinositidase C [phosphoinositide-specific phospholipase C (PIC)] was studied at neutral pH and at ionic concentrations that approximate intracellular conditions. The principal water-soluble product formed was shown to be inositol trisphosphate by anion exchange chromatography. The maximum hydrolysis rate (2.5 nmol/min/mg protein) was achieved at less than 100 nM Ca2+. Hydrolysis was markedly increased to 15 nmol/min/mg protein by inclusion of K+ in the reaction mixture. In the presence of 200 mM K+, the optimum Ca2+ was increased to approximately 600 nM. Higher Ca2+ concentrations progressively inhibited PIP2 hydrolysis. Mg2+ also inhibited the reaction, but the presence of equimolar amounts of ATP and Mg2+ had no effect. Appreciable degradation of phosphatidylinositol-4-phosphate (PIP) also occurred in the nanomolar Ca2+ range, whereas breakdown of phosphatidylinositol (PI) required millimolar Ca2+. The presence of PIP but not PI inhibited PIP2 hydrolysis. Upon subcellular fractionation of nerve, more than 50% of recovered PIC activity was in the cytosol and about 20% was located in a myelin-enriched fraction. Using PIP2 as substrate, PIC activities in nerves from normal and streptozotocin-induced diabetic animals were not different. However, the myelin-associated enzyme from diabetic animals was more labile to freezing and thawing.


Mechanisms of nerve growth factor mRNA regulation by interleukin-1 beta in hippocampal cultures: role of second messengers. Cytokines such as interleukin-1, which are found in the brain after trauma, regulate expression of nerve growth factor (NGF) mRNA and protein in hippocampal cultures. We have investigated possible mechanisms by which Il-1 beta regulates NGF in hippocampal cells. The induction of NGF mRNA by Il-1 beta was blocked by a receptor antagonist indicating that this effect is receptor mediated. Il-1 beta elicited a dramatic induction of c-fos mRNA and a slight elevation of c-jun mRNA in a time dependent manner which may allow for a role in the induction of NGF mRNA expression. We examined whether specific second messenger pathways were involved in mediating the action of Il-1 beta in the hippocampus. Activation of cAMP with forskolin or treatment with 8-Br-cAMP had no effect on NGF mRNA levels. Moreover, exposure of hippocampal cultures to Il-1 beta evoked no change in cAMP levels, indicating that this second messenger system played little or no role in the regulation of NGF expression by Il-1 beta in these cells. Further, interleukin-1 elicited no change in membrane inositol phosphate turnover, nor did it affect intracellular calcium levels. Treatment of cell cultures with the phorbol ester PMA elicited an increase in NGF mRNA, suggesting that activation of protein kinase C (PKC) may mediate NGF mRNA expression. However, prolonged treatment of cultures with PMA to desensitize PKC did not eliminate the Il-1 beta induction of NGF mRNA. Il-1 beta, therefore, did not appear to activate NGF expression via cAMP, Ca2+, or a PKC isoform that is downregulated by prolonged PMA treatment. However, a phosphorylation event may be involved in the signal transduction mechanism, as treatment with okadaic acid to inhibit protein phosphatase 2a potentiated the induction of NGF mRNA by Il-1 beta. The results presented indicate that Il-1 beta acts via its receptor to induce a rise in NGF expression. Identification of the specific second messenger pathway has remained elusive; however, a phosphorylation event appears to be intermediary. Moreover, the induction of c-fos and c-jun may represent a final common path in activation of NGF gene expression by different signals such as Il-1 beta and PMA.


Gene structure and expression of the mouse 5-HT2 receptor. Serotonin (5-hydroxytryptamine, 5-HT) is an important neurotransmitter which mediates numerous physiological functions. Using the SacI-EcoRI restriction fragment of the rat brain 5-HT2 receptor cDNA as a probe, we have screened a mouse brain cDNA library, created by random priming and constructed in SWAJ vectors, and have isolated a cDNA encoding a 1.4 kb open reading frame which codes for a functional mouse 5-HT2 receptor identified from pharmacological binding profiles and coupling of phosphoinositide formation in a stably transfected fibroblast cell line. The deduced amino acid sequence is 97.4% identical to the rat 5-HT2 receptor. Using the same 5-HT2 receptor cDNA probe, ten positive genomic clones were isolated from two mouse genomic libraries constructed in the pWE15 cosmid vector and the EMBL-3 phage vector. Extensive mapping and sequencing of these genomic clones indicate the mouse 5-HT2 receptor coding region spans over 20 kb and is composed of three exons split by two introns. Northern blot analysis shows one band of 5-6 kb in the mouse brain, but not in the heart, lung, liver, or kidney total RNA. Southern analysis of mouse liver genomic DNA shows a simple pattern of digestion by several restriction enzymes, which suggests that one copy of the 5-HT2 receptor gene may exist in the mouse genome.


Growth control of cultured microglia.

Growth control of cultured microglia. Microglia, the resident macrophages of the brain, typically react to injuries or chronic diseases with proliferation and expression of differentiated features, such as production of cytokines associated with inflammatory events. Regulation and control of microglial cytokine expression, therefore, is a major focus of scientific interest. It has been shown that GMCSF and Il-3 are potent mitogens for microglia. Moreover, Il-3 and other cytokines are products of microglia. It is shown here that interleukin-1 (Il-1) as well as tumor necrosis factor (TNF alpha) increased microglial proliferation in mixed astrocyte-microglial cultures but had no mitogenic effects on isolated microglia. Lipopolysaccharide (LPS), the bacterial endotoxin, irreversibly inhibited microglial cell division in both mixed astrocyte-microglial cultures and in isolated microglial cultures. By contrast, the corticosteroids hydrocortisone and aldosterone and the synthetic glucocorticoid dexamethasone reversibly inhibited microglial proliferation. They also antagonized the stimulatory effects of Il-3 and granulocyte macrophage colony-stimulating factor (GMCSF). Estradiol and progesterone had no significant effects on mixed cultures but inhibited microglial proliferation in isolated cultures. Conditioned media from mixed cultures, isolated cultures, from the WEHI-2B cell line, or from fresh (serum-supplemented) media stimulated microglial proliferation to various extents. In summary, cytokine-mediated microglial proliferation can be down-regulated by a variety of steroid hormones. Along with their unimpaired access to brain cells in general, corticosteroids likely maintain an inhibitory tonus on microglial proliferation. It is hypothesized that this inhibition is overcome locally and temporally in brain injury and repair.


Altered genetic response to beta-adrenergic receptor activation in late passage C6 glioma cells. Previous studies have demonstrated variability in the phenotype of rat C6 glioma cells. In the present study, we compared morphology, growth rate, and beta-adrenergic regulation of gene expression in early (P39-47) and late (P55-90) passage C6 cells. Morphological changes were observed in five independently derived, late passage populations. In four of the five, the untreated cells were more polygonal than the fibroblast-like parental cells, and only a small fraction exhibited process outgrowth after dbcAMP treatment. Untreated cells from the fifth late passage population had longer cytoplasmic processes than parental cells and responded to dbcAMP with further process outgrowth. All late passage populations had shorter generation times than the parental cells. In early passage cells, treatment with the beta-adrenergic agonist, isoproterenol (IPR), resulted in an increase in c-fos mRNA and a decrease in c-jun mRNA (Gu-bits RM, Yu H: J Neurosci Res, 30:625-630, 1991). Both of these immediate early gene responses were irreversibly lost between P50 and P55. Additional differences in basal or IPR-induced mRNA levels were observed for beta-APP, GFAP, NGF, and PPE, but not for a number of other mRNAs. These results are discussed in relationship to previously described differences in the ability of early and late passage C6 cells to accumulate cAMP (Mallorga P, et al.: Biochim Biophys Acta 678:221-229, 1981).


[Effects of sodium bicarbonate on the end-tidal CO2,PaCO2, HCO3-, PH and cerebral blood flow]. To estimate the quantitative reactivity of cerebral blood flow (CBF), the effects of sodium bicarbonate on the end-tidal CO2, arterial PaCO2, HCO3-, PH and CBF were examined. The CBF was measured by 133Xe inhalation method with ring type SPECT (HEADTOME). Activation study with sodium bicarbonate administration was performed after 30 minute of resting study, and the reactivity of each parameters was investigated. The arterial HCO3- and PH increased with similar reactivity, but PaCO2, end-tidal CO2 and CBF in the non-injured hemisphere changed with irregular reactivity. The excellent correlation between PaCO2 and end-tidal CO2 was vanished by the administration of sodium bicarbonate. The reactivity of CBF did not correlate with reactivity of PaCO2 and end-tidal CO2, but correlated with arterial HCO3- and PH. Thus the measurement of arterial HCO3- and PH may be indispensable to estimate the CBF reactivity with the administration of sodium bicarbonate.


Plasma catecholamines in pulmonary tuberculosis. Fifty pulmonary tuberculous patients (minimal; moderate and far-advanced), 18 TB-healed persons and 15 healthy control subjects were examined for plasma levels of adrenaline (AD), noradrenaline (NA), dopamine (DA), ACTH and cortisol. The estimated hormones were found to be increased significantly with the severity of the disease suggesting that the stress of infection plays a role in induction of enzymes responsible for catecholamines synthesis with subsequent stimulation of ACTH and cortisol secretion. Noradrenaline appeared the most effective in this respect.


Angiotensin II stimulates endothelin-1 secretion in cultured rat mesangial cells. The present study was designed to test two hypotheses: (1) that angiotensin II (Ang II) stimulates endothelin-1 secretion in cultured rat mesangial cells and (2) that atrial and brain natriuretic peptides (ANP and BNP) inhibit the above-mentioned secretion in these cells. Ang II stimulated immunoreactive (ir) endothelin-1 secretion in a concentration-dependent manner between 10(-8) M and 10(-7) M. The protein kinase C (PKC) inhibitors from two chemical classes, H7 and staurosporine, inhibited secretion following such stimulation. The stimulatory effect of Ang II was also abolished in the PKC-depleted cells. Rat ANP(1-28) and rat BNP-45, which are the respective major circulating forms of ANP and BNP in rats, potently inhibited Ang II-stimulated endothelin-1 secretion in a concentration-dependent manner. Inhibition by ANP and BNP of Ang II-stimulated endothelin-1 secretion was paralleled by an increase in the cellular level of cyclic guanosine 5'-monophosphate (GMP). The addition of a cyclic GMP analogue, 8-bromo cyclic GMP, reduced the stimulated endothelin-1 secretion. Rat ANP(5-25) was less effective that rat ANP(1-28) with respect to inhibiting ir-endothelin-1 secretion and increasing cellular cyclic GMP. These findings indicate that Ang II stimulates endothelin-1 secretion in cultured rat mesangial cells by a mechanism probably involving activation of PKC, and that rat ANP and BNP inhibit this stimulated secretion through a cyclic GMP-dependent process.


Localization of phosphoinositide-specific phospholipase C-alpha in porcine kidney. The presence of multiple forms of phosphoinositide-specific phospholipase C (PLC), an important enzyme in the cell signal transduction, suggests that specialized functions of tissues and cells may require different modes of PLC regulation. In the present study, we have purified a 54-kDa heparin-binding protein from a 4 M guanidine hydrochloride extract of porcine kidney, and identified it as one of isoenzymes of PLC on the basis of its partial amino acid sequence. Among 194 determined sequences of the porcine protein, 186 residues were identical with those deduced from nucleotide sequence of the cDNA encoding rat PLC-alpha. The subcellular distribution of porcine renal PLC-alpha was examined by Western blotting by using a specific antibody against the purified protein. Quantitation of the Western blots revealed that 70% of PLC-alpha was membrane-associated. Immunohistochemical studies showed a specific localization of PLC-alpha in epithelial cells of distal tubules and collecting ducts of normal porcine kidney, but not in other cells composing the nephron. Moreover, the highest expression of PLC-alpha was observed in apical membranes in these epithelial cells. Thus, this form of PLC is considered to have a specific role in the signal transduction process related to regional renal tubular functions.


Glomerular mesangial cells and inflammatory macrophages ingest neutrophils undergoing apoptosis. Apoptosis or programmed cell death of senescent neutrophils leading to their uptake by phagocytes is a general mechanism by which neutrophils may be removed from inflamed sites in vivo, promoting resolution rather than persistence of inflammation. We now report morphological evidence of neutrophil apoptosis leading to uptake by glomerular cells in rats with experimental glomerulonephritis. In addition to confirming that inflammatory macrophages take up apoptotic neutrophils, these studies indicated that glomerular mesangial cells can also participate in this mode of neutrophil clearance. Furthermore, human neutrophils which had been "aged" in vitro so as to undergo apoptosis were ingested by 31.5 +/- 1.3% (mean +/- SE) of cultured human mesangial cells, but there was minimal recognition of freshly isolated neutrophils (2.2 +/- 0.1%). Centrifugal elutriation of aged neutrophil populations yielded fractions with varying degrees of apoptosis (from 11.1 to 79.4%). Uptake of these fractions (by 8.2% to 59.8% of mesangial cells) was closely correlated with apoptosis (r = 0.96, P less than 0.0001). This demonstrated that recognition was dependent upon apoptosis, as in previous reports of macrophage recognition of aged neutrophils. However, by contrast, a partial requirement for serum was observed. These data indicate a hitherto unexpected function for the mesangial cell in clearance of senescent neutrophils from the glomerulus which may supplement inflammatory macrophage uptake of leucocytes undergoing apoptosis.


Evidence for the presence of a K-dependent acidifying adenosine triphosphatase in the rabbit renal medulla. To date direct evidence for the presence of a H-K-ATPase in the medulla comes from proton and potassium transport studies performed on K-restricted animals and K dependent ATP hydrolysis and Rb uptake in both normal and K-depleted animals. The present work examines K-dependent acidification in the medulla of rabbits on normal K diets. A membrane vesicle preparation was developed that was enriched for apical membranes derived from the renal medulla. Adenosine triphosphate (ATP)-dependent vesicular acidification was present and the extent of vesicular acidification was dependent on ambient K concentration. Moreover, ATP hydrolysis was dependent on ambient K concentration. K-dependent acidification was inhibited by the specific inhibitor of the gastric H-K-ATPase, SCH28080. However, significant acidification was observed in the absence of K that was not inhibited by SCH28080. The data suggest that an H-K-ATPase similar to the gastric H-K-ATPase is present in the renal medulla of rabbits on a normal K diet. The component of acidification and ATP hydrolysis that is independent of K concentration likely represents the previously characterized vacuolar H-ATPase.


Intracellular pH and Na+/H+ antiport activity of cultured skin fibroblasts from diabetics. Increased leucocyte Na+/H+ antiport activity has previously been demonstrated in both hypertensive subjects and Type 1 diabetic patients with nephropathy and may indicate a predisposition to hypertension in such diabetic patients. We have studied intracellular pH and Na+/H+ antiport activity in cultured skin fibroblasts from diabetic patients with and without nephropathy, together with non-diabetic controls to assess if such differences persisted in cultured cells. Fibroblasts from diabetic patients with nephropathy were significantly more alkaline [median (range): 6.90 (6.82 to 7.07)] compared to both normoalbuminuric diabetic patients [6.81 (6.75 to 6.89)] or normal controls [6.82 (6.77 to 6.93)] (P < 0.001 for both). This was associated with a raised Na+/H+ antiport activity in cells from patients with nephropathy when intracellular pH (pHi) was clamped to pH 6.5, without any differences in the maximal transport capacity of the antiport at pHi 6.2. Using both intracellular pH and Na+/H+ antiport activity at pHi 6.5, patients with nephropathy were separated from uncomplicated subjects with a sensitivity of 92% and a specificity of 100%. In conclusion, the raised Na+/H+ antiport activity in cells from patients with diabetic nephropathy persists despite passaging in vitro, thus indicating a heritable component, and results mainly from an increased apparent affinity of the antiport for intracellular H+.


Studies on re-entrant arrhythmias and ectopic beats in excitable tissues by bifurcation analyses. A phase-plane bifurcation analysis is a useful way to theoretically understand how various types of arrhythmias may arise from excitable tissues. In this paper, we have performed phase-plane bifurcation analysis to characterize arrhythmogenic states in excitable tissues. To achieve this, we have first formulated a model which is simple enough to be mathematically tractable, yet captures the non-linear features of cardiac excitation and conduction. In this model, single cells are connected in a circular fashion by gap conductances. Each cell carries the following two types of currents: a passive outward current and an inward "excitable" current which contains an activation and an inactivation gate. The activation gate is responsible for the upstroke of action potential and inactivation gate is responsible for the termination of the plateau potential. With this model, we have constructed bifurcation diagrams as a function of a bifurcation parameter. The parameter chosen as the bifurcation parameter has the property of raising maximum diastolic potential while shorting the refractory period. Our analysis revealed the existence of three distinct multi-stable phases in certain ranges of the bifurcation parameter: (1) bistability between a rotor and a quiescent state, (2) bistability between rotor and ectopic beats, and (3) three stable states co-existing among quiescent state, rotor, and ectopic beats. In these three regions, external impulses exert very distinct effects: In region 1, a brief current pulse can annihilate a re-entrant arrhythmia to quiescence. To initiate re-entry from a quiescent tissue, however, it takes two pulses (a primary pulse followed by a premature pulse at a site different from the "primary" site). In region 2, a brief pulse can convert a re-entrant arrhythmia to ectopic beats. To convert the ectopic beats back to circus movement, these beats have to be suppressed by a few brief current pulses to initiate one-way propagation. Depending on the frequency and strength of impulses in region 3, the tissue can switch back and forth among quiescence, circus movement, and ectopic beats. For comparison, we have also included a more complete Beeler-Reuter cardiac cell model in our analysis and obtained essentially the same results. From the behavioral similarities of these models, we conclude that re-entrant and ectopic arrhythmias must be intrinsic properties of excitable tissues and external stimuli can convert one mode of arrhythmia to another in the multistability regions.(ABSTRACT TRUNCATED AT 400 WORDS)


Changes in myocardial beta-adrenergic receptors during acute rejection of heterotopically transplanted rat hearts. To evaluate changes of the myocardial beta-adrenergic receptors in acute cardiac graft rejection, the density and binding affinity value of the myocardial beta-adrenergic receptors in heterotopically transplanted rat isografts and allografts were analyzed. Hearts from Fisher rat donors were transplanted either to the Fisher rats (isografts) or to Lewis rats (allografts). Histologic examination of the allografts showed mild to moderate rejection on the seventh and fourteenth days and showed severe rejection on the twenty-first day after transplantation. The density values in the allografts and isografts similarly increased significantly (p < 0.05) above the normal level on the seventh and fourteenth days after transplantation. The density in allografts on the twenty-first day decreased significantly (p < 0.05) below the normal level, while that in isografts remained at the normal level. In contrast, the binding affinity value of myocardial beta-adrenergic receptors in both isografts and allografts did not change after transplantation. These results demonstrated that myocardial beta-adrenergic receptors presented upregulation in mild to moderate rejection, whereas these receptors presented downregulation in severe rejection. The data suggested that downregulation of myocardial beta-adrenergic receptors plays a major role in decreased cardiac contractility during severe rejection, but not during mild and moderate rejection.


Role of the carotid sinus in response of integrated venous system to pulsatile and nonpulsatile perfusion. Effects of pulsatile and nonpulsatile perfusion on capacity system were investigated in 33 open-chest mongrel dogs with extracorporeal circulation. In 28 dogs with intact carotid sinus nerves, after changing from nonpulsatile to pulsatile systemic perfusion the mean arterial pressure decreased by 14 +/- 1.5 mm Hg, peripheral resistance by 423 +/- 15 dyne.cm-5.sec, and venous blood volume by 3.3 +/- 0.4 ml/kg. After changing from pulsatile perfusion to nonpulsatile systemic perfusion the mean arterial pressure increased by 15 +/- 2.2 mm Hg, the peripheral resistance by 431 +/- 15 dyne.cm-5.sec, and venous volume by 3.9 +/- 0.5 ml/kg. The same effects were observed in five dogs with bilateral isolated carotid pulsatile perfusion or nonpulsatile perfusion and systemic nonpulsatile perfusion. These effects were abolished after denervation of pressor receptors and pharmacologic blockade of alpha and beta receptors. It is proposed that with nonpulsatile perfusion carotid sinus-mediated vasoconstriction occurs in the downstream part of the capacitive system, where more pronounced wall musculature with increased number of adrenergic receptors is present. The throttle effect caused by this "resistance" part of venous circulation leads to increase of pressure in the upstream section of the capacitive system, compensates for the constrictive effect of the sympathetic tone, and leads finally to an increased volume of the capacity system. We conclude that venous response to different perfusion modalities is critically dependent on intact carotid sinus nerves and adrenergic receptors in the veins.


Rearrangements of T-cell receptor delta, gamma and beta genes in acute myeloid leukemia coexpressing T-lymphoid features. In order to investigate the role of T-cell receptor (TcR)-delta and TcR-gamma gene rearrangements and/or deletions in acute myeloid leukemia (AML) coexpressing T-cell-associated antigens (i.e. CD2 and/or CD4 and/or CD7), we examined blasts from a selected group of 56 AML cases (25 children, 31 adults) coexpressing either of these antigens without cytoplasmic CD3 expression. Forty-four typical AML cases (7 children, 37 adults) without T-cell associated antigens were further studied as controls. Germline configuration of the TcR-delta gene was observed in 91 out of the total of 100 AML cases investigated. Eight of nine cases with rearranged or deleted TcR-delta genes coexpressed T-cell-associated antigens. Blast cells of 7/9 cases were classified as FAB M1, two as FAB M2. In six of these cases TcR-gamma gene rearrangements were also detected. TcR-delta alterations were predominantly found in children whose blasts coexpressed T-lymphoid associated antigens (6/25, 24%), but were rarely detected in adult AML with or without coexpression of T-cell antigens (2/31 and 0/37, respectively).


The monocytic cell line SKM-1 strongly expresses the myeloperoxidase gene. A newly established human monocytic cell line, SKM-1, showed strong expression of myeloperoxidase mRNA, to the same extent as in HL-60 cells. We studied the cell morphology and myeloperoxidase expression of this cell line, which was established from a patient with myelodysplastic syndrome who had an abnormal chromosome on the upstream region of 17p13. Electron micrographs showed the cells to have a fragile and irregular cell surface. SKM-1 cells were peroxidase-positive. About 60% of myeloperoxidase (MPO) was released to the culture fluid from SKM-1 cells but only a few percent of MPO was released from HL-60 cells into the culture fluid. The predominant mRNA size of SKM-1 myeloperoxidase was 3.3 kb although there was a smaller size as well. Fluorescent in situ hybridization of MPO mRNA showed strong staining in 5% to 10% of SKM-1 cells and of bone marrow cells from patients with myelogenous leukemia, while all cells from HL-60 were positive.


Lineage alteration in precursor B cell acute lymphoblastic leukemia following T cell lymphoblastic lymphoma. A 10-year-old girl was diagnosed with lymphoblastic lymphoma; staging evaluation revealed a large mediastinal mass and normal peripheral blood and bone marrow morphology. Tumor cell immunologic marker analysis and Southern blot gene rearrangement studies demonstrated a T-cell lineage. She achieved a complete remission following multi-agent chemotherapy; however, 19 months following initial diagnosis while on maintenance therapy, she presented with typical acute lymphoblastic leukemia (ALL). The bone marrow was replaced by lymphoblasts, though the mediastinum was normal and there was no peripheral lymphadenopathy. Repeat immunophenotypic and genotypic studies demonstrated a precursor B-cell ALL lineage without expression of the T-cell surface antigens present on the original neoplasm. Repeat genotypic analysis showed immunoglobulin heavy and light chain gene rearrangements without the T-cell receptor gamma and beta gene rearrangements noted in the original lymphoblastic lymphoma. The complete alteration of lineage in these lymphoblastic processes suggests the de novo occurrence of a second neoplasm or, alternatively, an ALL relapse from a lineage-uncommitted neoplastic lymphoid progenitor cell.


[Chemotherapy with cisplatin, adriamycin, etoposide and ifosfamide in microcytic lung carcinoma]. The aim of this study was to analyze the results obtained in the treatment of small cell lung cancer (SCLC) with the PAVI chemotherapy protocol (cisplatin, adriamycin, etoposide and ifosfamide). Over a period of 3 years, 41 patients with a mean age of 57 years were treated. Twenty-two patients were considered as having limited disease (LD) and 19 disseminated disease (DD). Survival was studied by the Kaplan and Meier method. The percentage of complete response (CR) achieved was 42%, LD 52% and 27% for DD, with partial responses (PR) being achieved in 50%, 43% in LD and 60% in DD. With a mean follow up of 32 months, the mean 2 length of response was 13 months in the patients with CR and 9 months in those with PR. The median of survival in LD was 22 months and 10 months for patients with DD. Prolonged survival of over 2 years, was only achieved in LD (16%). Five patients died in relation with the treatment. Hematologic toxicity was doses-limited with the greatest toxicity being found in patients with DD under the Karnofsky index (KI). The PAVI protocol is effective in the treatment of small cell lung cancer and a good median of survival may be achieved in patients with limited disease. Toxicity is elevated and is fundamentally found in patients with disseminated disease and under the Karnofsky index, with its use not being recommended in these cases.


Clinical application of the Nd:YAG laser bipolar dissector in intra-abdominal surgery. A laser bipolar dissector (LBD) using a 1064 nm Nd:YAG laser energy source that provides hemostatic dissection using low-powered laser energy (15-25 W) has been developed. Clinical applications have included a partial hepatectomy (n = 5), a cholecystectomy (n = 4), an appendectomy, lymphnode dissection, and division of adhesions. The LBD was effective in resection of noncirrhotic liver and reduced the number of ligations required. There were no direct complications related to using the LBD. Operative time and blood loss were difficult to compare to conventional methods. The LBD may have applications for partial hepatectomies and other intra-abdominal operations.


Stimulation of the Na+,K(+)-ATPase activity of K562 human erythroleukemia cells by triiodothyronine. The effect of triiodothyronine (T3) on Na+,K(+)-ATPase activity of K562 human erythroleukemic cell was studied to understand why the erythrocyte sodium pump activity is decreased in hyperthyroidism. Na+,K(+)-ATPase activity of K562 cell lysates was assayed by measuring the release of inorganic phosphate (Pi) from ATP. Na+,K(+)-ATPase activity of K562 cell grown in the presence of T3 for 48 hours was significantly higher than that of control (0.98 +/- 0.05 mumol Pi h-1 mg protein-1 vs 0.82 +/- 0.10 mumol Pi h-1 mg protein-1, p < 0.05). The Na+,K(+)-ATPase activity could be stimulated in a time- and concentration-dependent manner; maximum stimulatory effect of T3 was seen at a concentration of 10(-7) mol/L. When an inducer [cytosine-beta-D-arabino-furanoside (ARA-C)] was added to the culture medium, the K562 cells showed signs of differentiation and synthesised haemoglobin. At the same time, the Na+,K(+)-ATPase activity remained high. We conclude that T3 stimulates Na+,K(+)-ATPase activity of K562 cells and in the presence of T3 during differentiation, the enzyme activity remains high.


N-terminal truncated analogs of men 10376 as tachykinin NK-2 receptor antagonists. Three N-terminal fragments of the selective tachykinin NK-2 receptor antagonist MEN 10376 (H-Asp-Tyr-DTrp-Val-DTrp-DTrp-Lys-NH2) have been synthesized and tested in several mammalian tissues in order to establish the minimum length of the peptide chain for maintenance of the antagonist activity. Biological activity has been determined on the rabbit pulmonary artery (RPA) and hamster trachea (HT) preparations, chosen as representative of the NK-2A and NK-2B receptor subtypes, respectively, and on the rabbit bronchus (RB), guinea-pig bronchus (GPB), human urinary bladder (HuUB), human ileum (HuI) and human colon (HuC) preparations to verify the previously described NK-2A character of these tissues. The N-terminal tetrapeptide was inactive in the RPA and HT, while the N-terminal hexa- and penta- peptides maintained antagonist activity in all preparation investigated. The selectivity of the latter two peptides confirms that the receptor expressed in RB, GPB, HuUB, HuC and HuI tissues is of the NK-2A type.


Methionine-enkephalin in a porcine endometrial cell line and its responsiveness to potassium depolarization. Immunoreactive methionine-enkephalin (ir-MENK) has been identified in the porcine uterine fluid and endometrium. Previously, we have established a porcine endometrial cell line of epithelial origin (PE-1) by transfecting primary endometrial cells with temperature sensitive SV40 DNA. The current study was conducted to identify and characterize ir-MENK present in PE-1 cells, and to investigate the effect of KCl depolarization on the kinetics of ir-MENK secretion. PE-1 cells were cultured at 33C until confluency was reached (33C cells), after which they were incubated at 40C for 2 days (40C cells). Ir-MENK in PE-1 cells was analyzed by Sephadex G-15 gel filtration and reverse phase (RP)-HPLC. Analysis of 40C cell extract by Sephadex G-15 and RP-HPLC indicated that the major portion of ir-MENK present in PE-1 cells was eluted at a position similar to that of synthetic MENK. The effect of temperature on ir-MENK synthesis in PE-1 cells was examined by measuring ir-MENK content in 33C and 40C cells over a 14-day culture period. Compared to 33C cells, 40C cells maintained higher and steadier levels of ir-MENK, suggesting that synthesis of ir-MENK is temperature sensitive. KCl stimulated ir-MENK secretion at all concentrations tested (5-60 mM for 60 min), with 30 mM being the optimal concentration. Temporal analysis of ir-MENK secretion showed that incubation for 60 min with 30 mM KCl allowed maximal secretion. Secretion of ir-MENK from PE-1 cells resulted in depletion of ir-MENK in cell content. These results demonstrate that PE-1 cells contain ir-MENK which is biochemically similar to synthetic MENK, PE-1 cells synthesize ir-MENK in a temperature sensitive manner, and these cells secrete ir-MENK upon KCl stimulation.


Isolation of enteroviruses and adenoviruses in continuous simian cell lines. The ability of five simian cell lines--BGM, LLC-MK2, MA104, Vero and BSC-1--to isolate enteroviruses and adenoviruses from faeces was assessed and compared with that of primary or secondary rhesus monkey kidney (RhMK) and HEp2 cultures. For enteroviruses, LLC-MK2 and BGM were best of the five lines, detecting 77% and 73% respectively of 88 strains isolated, but neither was as good as RhMK, which detected 88%. Both Vero and BSC-1 detected less than 50%. All cell types were good at isolating poliovirus and coxsackie B strains, although LLC-MK2 and BGM were slightly better than MA104, Vero, BSC-1 and HEp2 for the latter. Greatest variation in sensitivity occurred with coxsackie A9 and echovirus isolates, with all lines being less sensitive than RhMK. However, the combined isolation rate for BGM, LLC-MK2 and HEp2 was the same as that for RhMK and HEp2, although the strains not detected by each cell combination were different. Both Vero and BSC-1 were reasonable for detecting adenoviruses, but neither was as good as HEp2. The reasons for variations in results, and the future use for such cell lines for isolating viruses from clinical and environmental samples, are discussed.


Low-molecular-weight heparin exerts an early antithrombotic effect in small arteries and veins following severe trauma. Administration of low-molecular-weight heparin (LMWH) and standard heparin was studied by evaluating vessel bleeding, patency, and thrombotic material following severe vascular trauma. Arteriotomy and intimectomy or venotomy and intimectomy were performed on small rabbit arteries or veins in two separate blinded studies. All vessels were closed using a continuous microvascular suture. Patency and weight of thrombotic materials were evaluated 2 hr after reperfusion. In the arterial study, two groups of 23-24 arteries were treated with saline or LMWH systemically. Bleeding times were 89 +/- 15 sec in the control group and 103 +/- 27 sec in the LMWH group; there was no significant difference between the groups. Patency was significantly increased in the LMWH group (79%) compared to the control group (52%). The weight of thrombotic material in the LMWH group (1.39 +/- 0.20 mg per artery) was significantly different compared to the control group (2.19 +/- 0.22 mg per artery). In the venous study, 65 veins were divided into three groups (21-23 vessels/group) and treated with systemic saline, heparin, or LMWH. Bleeding times in the conventional heparin group (37 +/- 7 sec), the control group (23 +/- 3 sec), and LMWH group (22 +/- 4 sec) were not significantly different. The patency rates were significantly increased in the heparin (42%) and LMWH (39%) groups compared to the control group (0%). The weight of thrombotic material in each vein was significantly less in the LMWH (1.07 +/- 0.24 mg) and heparin (1.78 +/- 0.52 mg) groups than in the control group (3.78 +/- 0.29 mg).(ABSTRACT TRUNCATED AT 250 WORDS)


[The role of oxygen free radicals in asbestos cytotoxicity]. Despite numerous experiments, the cytotoxicity of asbestos is still far from being well understood. There is an increasing evidence which supports the contribution of free oxy-radicals resulting from the phagocytosis of asbestos fibres concerning macrophages neutrophils, pulmonary epithelial and endothelial cells. Both in vitro and in vivo studies revealed an asbestos-induced generation of hydrogen peroxide, hydroxyl radical and superoxide anion. The intensity of the process varies with the physico-chemical properties of the asbestos fibres. Oxygen free radical activity accounts for the lipid peroxidation of cell membranes as well as for the damage of structural proteins and DNA. However, the cells contain antioxidant enzymes such as catalase, superoxide dismutase, etc., which prevent the toxic effect of oxidants. Their activity increases when the cells are exposed to asbestos.


A putative anaerobic coproporphyrinogen III oxidase in Rhodobacter sphaeroides. I. Molecular cloning, transposon mutagenesis and sequence analysis of the gene. A mutant of Rhodobacter sphaeroides, N1, has been isolated which is incapable of photosynthetic growth and, instead of synthesizing bacteriochlorophyll, N1 excretes coproporphyrin III into the growth medium. Using conjugative gene transfer, several clones were isolated from a R. sphaeroides gene library which restored normal pigment synthesis and photosynthetic growth to N1. Using transposon Tn5 mutagenesis, the gene was located to a 1.05 kb EcoRI fragment. Sequence and transcription analysis defined the position and expression of an open reading frame of approximately 920 bp, which is proposed as the anaerobic coproporphyrinogen III oxidase dedicated to bacteriochlorophyll biosynthesis.


Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes. Three genes from Pseudomonas aeruginosa involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrc genes lie at the thr locus of the P. aeruginosa chromosome map (31 min) and are likely to be organized in a bicistronic operon. The encoded proteins are quite similar to the Hom and TS proteins from other bacterial species. The thrB gene was located by pulsed-field gel electrophoresis experiments at 10 min on the chromosome map. The product of this gene does not share any similarity with other known ThrB proteins. No phenotype could be detected when the chromosomal thrB gene was inactivated by an insertion. Therefore the existence of isozymes for this activity is postulated. HDH activity was feedback inhibited by threonine; the expression of all three genes was constitutive. The overall organization of these three genes appears to differ from that in other bacterial species.


Plasma-cell dyscrasia with polyneuropathy. The spectrum of POEMS syndrome. The POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes) syndrome and osteosclerotic myeloma (polyneuropathy and sclerotic bone lesions) may both be manifestations of plasma-cell dyscrasia, but the interrelation of these diseases is not clear. We therefore set out to define the clinical spectrum of disease in patients with plasma-cell dyscrasia and polyneuropathy who have the complete or incomplete form of the POEMS syndrome or osteosclerotic myeloma. Among 2714 patients with plasma-cell dyscrasia who were identified between 1973 and 1989, we reviewed the cases of those with polyneuropathy and plasma-cell dyscrasia who fulfilled the criteria for the POEMS syndrome or osteosclerotic myeloma. Thirty-eight patients (1.4 percent) with a median age of 51 years were identified, 22 of whom were male. By definition, all had polyneuropathy (37 combined sensorimotor, and 1 primarily motor). Other findings included osteosclerotic bone lesions (82 percent), skin abnormalities (58 percent), lymphadenopathy (42 percent), papilledema (37 percent), peripheral edema (29 percent), hepatomegaly (24 percent), splenomegaly (21 percent), and ascites (11 percent). Thirty-three patients (87 percent) had an abnormal M protein in serum or urine (17 had IgA lambda, and 12 IgG lambda). Five patients fulfilled all the criteria for the POEMS syndrome. The estimated five-year survival in the 38 patients was 60 percent, which was significantly better than the 20 percent survival in 869 patients with multiple myeloma (P < 0.05). The clinical course was similar among the patients with the complete form of the POEMS syndrome and those with the incomplete form. Plasma-cell dyscrasia with polyneuropathy is a rare multisystem disease that often presents with osteosclerotic bone lesions. The differentiation of the POEMS syndrome from so-called osteosclerotic myeloma with peripheral neuropathy appears to have no clinical value.


A new approach to the molecular basis of neoplastic transformation in the brain. Gene transfer into living organisms has evolved as a powerful approach to study in vivo effects of specific genes and to devise animal models of hereditary disorders. We have been particularly interested in an approach to introducing transforming genes into the nervous system. Since specific promoter sequences for targeting the expression of a transgene to many cell types of the brain are not yet isolated, a suitable transgenic mouse model was not available for these experiments. This has prompted us to develop an alternative strategy for gene transfer into the brain. The rationale is to introduce foreign genes into fetal brain transplants using embryonic CNS as donor tissue and replication-defective retroviral vectors as genetic vehicles. This technique relies on the extraordinary organotypic differentiation capacity of neural grafts and the expression of retrovirally transmitted genes in different cell types of CNS transplants. In contrast to transgenic animals but analogous to sporadic tumour formation, target cells for the retroviral vector will develop in an environment of unmodified neural tissue. We have introduced a number of neurotropic oncogenes into fetal brain transplants to study potential effects of such genes on the brain. This review will summarize some of the findings which have emerged from this experimental study including the tropism of several genes for endothelial cells, attempts to identify cooperating combinations of transforming genes and an experimental model for primitive neuroectodermal tumours in neural grafts.


[The effect of a Coxsackie viral infection on the chromosomal apparatus of the cells of fetuses and newborn mice]. The Coxsackie virus B1 has been studied for its effect on the chromosome apparatus in cells of mouse feti and newborn mice in case of intraperitoneal infection of females on the 3d week of pregnancy. It is stated that the virus readily moves across the placental barrier and induces chromosome aberrations in the cells of feti, their number being 2.5 times higher than the control level. The maximal number of structural chromosome aberrations was registered during the first three days from the moment of infecting. Then a gradual decrease in the number of aberrant metaphases in the cells of newborn mice was observed. It had reached the value of the control level by the 17th day of observation.


Varicella-zoster virus encephalitis in acquired immunodeficiency syndrome: report of four cases. Four patients with acquired immunodeficiency syndrome, a 27-year-old female intravenous drug abuser and three males (two drug addicts aged 27 and 33 years and a 40-year-old homosexual) presented with a rapidly progressive encephalopathy. Two had generalized varicella-zoster virus skin infection, one had had a regressive thoracic zoster rash 7 months previously and one had no history of cutaneous eruption. Neuropathological examination revealed, in each case, multifocal necrotic changes with numerous, intranuclear Cowdry type A inclusion bodies in glial cells, endothelial cells, macrophages and neurons, within and around the lesions. These inclusion bodies were stained positively for varicella-zoster virus by immunocytochemistry and contained herpes virus nucleocapsids by electron microscopy. Molecular biology using the polymerase-chain-reaction method demonstrated viral genome. In one case, zoster-induced non-inflammatory vasculopathy involved medium sized leptomeningeal vessels and was associated with circumscribed areas of cortico-subcortical infarction. In another case, varicella-zoster virus encephalitis was associated with human immunodeficiency virus encephalitis and a secondary cerebral lymphoma. Multinucleated giant cells expressing human immunodeficiency virus proteins in their cytoplasm, were found in the lymphomatous deposits and in the varicella-zoster virus necrotic lesions. In these latter lesions, Cowdry type A inclusion bodies could be seen in the nuclei of some multinucleated giant cells confirming previous observations of MGCs co-infected by HIV and CMV, and supporting the hypothesis that DNA viruses interact with HIV, thus increasing its effect.


A further study on the stimulatory effect of peptide histidine methionine on growth hormone secretion in acromegaly: a dose-related study and a comparison with vasoactive intestinal peptide. We examined whether the GH-releasing effect of peptide histidine methionine (PHM) in acromegaly may be mediated by activation of pituitary receptors for vasoactive intestinal peptide (VIP), which is structurally similar to but more powerful than PHM in stimulating GH secretion in acromegaly. VIP (50 or 100 micrograms) or PHM (50, 100, or 200 micrograms) was given as an i.v. bolus to 11 patients with active acromegaly, and plasma GH levels were measured before and at intervals up to 120 min after the injection. A paradoxical GH response (> 50% and > 6 micrograms/l above the basal) to 50 or 100 micrograms of VIP was observed in 4 (36%) or 5 (45%) patients, respectively. 2 (18%) patients showed paradoxical GH responses to both 50 and 100 micrograms of PHM, and, interestingly, as many as 5 (45%) patients showed positive GH responses to 200 micrograms of PHM. 3 of these 5 responders to 200 micrograms of PHM were also responders to both doses of VIP. To add to, one of the responders to 100 micrograms of VIP did not show a positive GH response to even 200 micrograms of PHM. These results may suggest that in at least some acromegalics the PHM stimulation of GH secretion is mediated by activation of pituitary VIP receptors by PHM and/or by PHM binding to its specific receptors which may have appeared concomitantly with VIP receptors. However, the occasional heterogeneity of the VIP- and PHM-induced GH responses may suggest that on some somatotroph adenomas either VIP or PHM receptors may appear independently.


Pharmacologic characterization of the novel ligand [4,5-3H-Leu9]neurokinin-A binding to NK-2 receptors on hamster urinary bladder membranes. We synthesized a novel ligand [4,5-3H-Leu9]-Neurokinin A (3H-NKA, S.A 117-144 Ci/mmol), and evaluated its binding to hamster urinary bladder membranes (HUBM). The ligand bound to HUBM in a highly-specific (94 +/- 4%) and protein-dependent manner. Binding was rapid (k1 = 0.037 nM-1*min-1) and saturable (Bmax = 1210 +/- 177 fmol/mg protein), to a single population of high-affinity sites (KD = 2.41 +/- 0.15 nM, nH = 0.99 +/- 0.02). Binding was inhibited by non-hydrolyzable GTP analogs. Competition experiments with HUBM demonstrated the following rank order of potency: NKA > Kassinin > [beta-Ala8]-NKA(4-10) > [Nle10]-NKA(4-10) = Eledoisin = NKB > Physaelamin > Substance P. The selective NK-1 and NK-3 ligands, [Sar9-Met (O2)11]-SP, (+/-) CP96,345 and Senktide respectively, did not inhibit binding at 10 microM, whereas, the selective NK-2 antagonists: (+/-) SR-48,968 >> L-659,877 > R396 >> MEN-10,207 > MEN-10,376, inhibited binding in a competitive manner. In contrast, the low specific binding (< 30%) detected in guinea pig lung membranes, was not inhibited by selective NK-2 ligands. Over 30 ligands (0.1-10 microM) from other receptor classes, were not inhibitory. The data suggest that this new ligand binds with high-affinity and selectivity to homogeneous population of NK-2 receptors on HUBM but not on lung membranes, and is a suitable ligand to study NK-2 receptors.


Influence of CRF and alpha-MSH on the migration of human monocytes in vitro. The effects of stress in the modulation of immune responses are increasingly reported by a rapidly growing body of experimental and clinical data. Here we show that corticotropin releasing factor (CRF) stimulates 'in vitro' the migration of human monocytes, the maximum effect being obtained at 10(-14) M. On the other hand, another important neuropeptide of the stress response, alpha-melanocyte stimulating hormone (alpha-MSH), has no significant effect on the migration of monocytes. These findings show that one of the oldest immune responses is directly modulated by a key mediator of the stress response.


[Neurologic complications in high-grade non-Hodgkin's lymphomas. Clinico-neuropathological correlations]. The neurological and electroneurographic assessment was carried out in 56 patients with high-grade non-Hodgkin lymphomas. In 37 patients the neuropathological examination was also done. Polyneuropathy was the most frequent sign of nervous system involvement, observed in 40% of patients before chemotherapy and markedly enhanced by cytostatics. The signs of the central nervous system involvement were usually caused by lymphomatous infiltrations within leptomeninges, especially in lymphoblastic and immunoblastic lymphomas. The authors stress that cerebro-spinal fluid should be investigated several times for avoiding of false negative results. Signs of lymphomatous infiltrations disappeared after treatment, while signs of polyneuropathy due to chemotherapy increased during cytostatic treatment.


Al Eskan disease: Desert Storm pneumonitis. The authors observed an acute desert-related disease when the mixture of the fine Saudi sand dust and pigeon droppings triggered a hyperergic lung condition. It was further aggravated by various kinds of organic pathogenic components contributing to an opportunistic infection of the lung. These all lead to the recognition of a new clinicopathological entity, Desert Storm pneumonitis or Al Eskan disease. For the first time, the Saudi sand dust's elemental composition was studied by ultrastructural and microanalytical means. The authors concluded that, contrary to previous beliefs, sand particles less than 1 microns (0.1 microns to 0.25 microns) in diameter are present in substantial quantities in the Saudi sand and are pathogenic, causing hyperergia. Pathogenesis of the sand dust, induced hyperergia, and its immunopathologic background are highlighted.


CRH gene expression in the fetal rat is not increased after pharmacological adrenalectomy. A regimen of twice daily metyrapone injections (100 mg/kg), resulted in pharmacological adrenalectomy of pregnant rats and fetuses in utero, i.e. depression of plasma corticosterone and elevation of plasma adrenocorticotropic hormone (ACTH). Toxicity was minimal on days 14-17 of pregnancy, and increased with higher maternal weight and pregnancy progression. Corticotropin releasing hormone (CRH) messenger RNA abundance in the pregnant adults increased significantly within 48 h of metyrapone initiation. No change in CRH gene expression in the paraventricular nucleus of fetuses (days 17-18) was seen, even after 72 h of the regimen. This is compatible with the independence of CRH gene expression of glucocorticoid feedback in the fetal rat.


[The effect of cyclic nucleotides on the sensitivity of early mouse embryos to biogenic monoamine antagonists]. The effect of lipophilic cAMP analogs on the sensitivity of preimplantation mouse embryos of two strains to cytotoxic serotonin and adrenalin antagonists was studied. Dioctanoyl-cAMP significantly decreased the sensitivity of embryos to inmecarb and cyproheptadine: experimental embryos developed to the stage of morula or blastocyst, in contrast to control embryos incubated without this protector. A somewhat weaker effect was observed in experiments with propranolol: embryos incubated in the propranolol-containing medium after the addition of dioctanoyl-cAMP were capable of one to two cleavage divisions. 8-bromomonobutyryl-cAMP partially suppressed the inhibitory effect of cyproheptadine and did not affect the sensitivity of embryos to propranolol. These data suggest cAMP involvement in the regulatory activity of neurotransmitters in the early mouse embryos.


Lack of association among typical congenital hypertrophy of the retinal pigment epithelium, adenomatous polyposis, and Gardner syndrome. It has been recently documented that multiple bilateral pigmented lesions at the level of the retinal pigment epithelium may be an indicator of patients with familial adenomatous polyposis who are prone to develop intestinal cancer, particularly if there is a positive family history of these intestinal disorders. Although atypical, such lesions have been called congenital hypertrophy of the retinal pigment epithelium (CHRPE). This study was undertaken to determine whether the typical lesions of CHRPE, seen frequently by ophthalmologists, also were indicators of familial adenomatous polyposis. Review of charts and follow-up studies were performed on all patients diagnosed and coded as having solitary CHRPE or its multifocal variant (congenital grouped pigmentation; bear tracks). Patients and their physicians were contacted by telephone to complete a detailed questionnaire designed to detect signs or symptoms of familial adenomatous polyposis or Gardner syndrome among these patients with CHRPE and their relatives. Of the 132 patients with previously diagnosed CHRPE, there were none with familial adenomatous polyposis, Gardner syndrome, or intestinal cancer, and only one patient had a history of intestinal polyps. Among more than 2000 of their blood relatives, only 20 had intestinal polyposis or colonic cancer (1%). This is much lower than would be expected from a survey of patients with the typical fundus lesions seen with familial adenomatous polyposis. It appears that solitary CHRPE and congenital grouped pigmentation differ clinically from the multiple pigmented lesions seen with familial adenomatous polyposis and that patients with these conditions, as well as their relatives, are not at a greater risk of developing intestinal cancer.


An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. We attempted to develop an experimental animal model for peripheral neuropathic pain. Under sodium pentobarbital anesthesia, both the L5 and L6 spinal nerves (group 1) or the L5 spinal nerve alone (group 2) of one side of the rat were tightly ligated. For comparison, a parallel study was conducted with another group of rats (group 3) which received a partial tight sciatic nerve ligation, a paradigm developed previously as a neuropathy model. Withdrawal latencies to application of radiant heat to the foot were tested for the next 16 weeks in all 3 groups. Sensitivity of the hind paw to mechanical stimulation was tested with von Frey filaments. The general behavior of each rat was noted during the entire test period. Results suggested that the surgical procedure in all 3 groups produced a long-lasting hyperalgesia to noxious heat (at least 5 weeks) and mechanical allodynia (at least 10 weeks) of the affected foot. In addition, there were behavioral signs of the presence of spontaneous pain in the affected foot. Therefore, we believe we have developed an experimental animal model for peripheral neuropathy using tight ligations of spinal nerves. The model manifests the symptoms of human patients with causalgia and is compatible with a previously developed neuropathy model. The present model has two unique features. First, the surgical procedure is stereotyped. Second, the levels of injured and intact spinal segments are completely separated, allowing independent experimental manipulations of the injured and intact spinal segments in future experiments to answer questions regarding mechanisms underlying causalgia.


Rapid and sensitive detection of Leishmania kinetoplast DNA from spleen and blood samples of kala-azar patients. Following sequence analysis of a Leishmania donovani kinetoplast DNA (kDNA) minicircle, we have developed synthetic oligonucleotides for use in the polymerase chain reaction (PCR). With these primers, we have amplified L. donovani kDNA from splenic aspirates and blood samples taken from kala-azar patients. Treatment of the samples for PCR requires only limited DNA purification by lysis in SDS, digestion with proteinase K, phenol extraction and ethanol precipitation of the resulting nucleic acid. We have obtained amplified product routinely with DNA prepared from the equivalent of 2.5-25 microliters of splenic aspirate or of 50-500 microliters of blood from infected patients. In dilution experiments a visible product has been obtained on amplification of DNA from the equivalent of 2.5 x 10(-7) microliters of splenic material. We therefore propose the amplification of L. donovani kDNA by PCR as a rapid and highly sensitive method for the diagnosis of kala-azar.


Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24. Topoisomerases catalyse the interconversion of topological isomers of DNA and have key roles in nucleic acid metabolism. Human cells express two distinct type II topoisomerase isozymes, designated topoisomerase II alpha (170 kDa form) and topoisomerase II beta (180 kDa form). We have isolated cDNA clones encoding the beta isozyme from a human B-cell library. The proposed coding region for the topoisomerase II beta protein is 4,863 nucleotides long and would encode a polypeptide with a calculated M(r) of 182,705. The predicted topoisomerase II beta protein sequence shows striking similarity (72% identical residues) to that of the human alpha isozyme, and homology to topoisomerase II proteins from Drosophila, yeast and bacteria. Regions of greatest amino acid sequence divergence lie at the extreme N-terminus and over a C-terminal domain comprising approximately 25% of the total protein. We have quantified the level of topoisomerase II beta mRNA in a panel of human tumour cell lines of different origin using an RNase protection assay, and compared the level to that of topoisomerase II alpha mRNA. Topoisomerase II beta mRNA was expressed in haemopoietic, epithelial and fibroblast cell lines, although to different extents, with U937 cells (promonocytic leukaemia) showing a particularly high level. There was no obvious relationship in terms of level of expression between the topoisomerase II alpha and beta genes. We have localised the gene encoding topoisomerase II beta protein to chromosome 3p24 in the human genome.


SPO12 and SIT4 suppress mutations in DBF2, which encodes a cell cycle protein kinase that is periodically expressed. To help clarify the role of DBF2, a previously described cell cycle protein kinase, high copy number suppressors of the dbf2 mutation were isolated. Three open reading frames (ORF) have been identified. One ORF encodes a protein which has homology to a human small nuclear riboprotein, while the remaining two are genes which have been identified previously, SIT4 and SPO12. SIT4 is known to have a role in the cell cycle but the nature of the interaction between SIT4 and dbf2 is unclear. SPO12 has until now been implicated exclusively in meiosis. However, we show that SPO12 is expressed during vegetative growth, moreover it is expressed under cell cycle control coordinately with DBF2. SPO12 is a nonessential gene, but it becomes essential in a DBF2 delete genetic background. Furthermore, detailed analysis of the cell cycle of SPO12 delete cells revealed a small but significant delay in mitosis. Therefore, SPO12 does have a role during vegetative growth and it probably functions in mitosis in association with DBF2.


Nested chromosomal fragmentation in yeast using the meganuclease I-Sce I: a new method for physical mapping of eukaryotic genomes. We have developed a new method for the physical mapping of genomes and the rapid sorting of genomic libraries which is based on chromosome fragmentation by the meganuclease I-Sce I, the first available member of a new class of endonucleases with very long recognition sequences. I-Sce I allows complete cleavage at a single artificially inserted site in an entire genome. Sites can be inserted by homologous recombination using specific cassettes containing selectable markers or, at random, using transposons. This method has been applied to the physical mapping of chromosome XI (620 kb) of Saccharomyces cerevisi and to the sorting of a cosmid library. Our strategy has potential applications to various genome mapping projects. A set of transgenic yeast strains carrying the I-Sce I sites at various locations along a chromosome defines physical intervals against which new genes, DNA fragments or clones can be mapped directly by simple hybridizations.


Analysis of spliceosome assembly and the structure of a regulated intron in Drosophila in vitro splicing extracts. We characterize spliceosome assembly in Drosophila embryonic nuclear extracts. Further, we show that these extracts contain high levels of a 5' to 3' exoribonuclease activity allowing rapid, convenient protection mapping of 5' splice site and branchpoint sequences. We use this assay to show, for the first time, that a regulated arthropod intron uses a remote branchpoint strikingly similar in structure to those observed previously in regulated vertebrate introns. These results provide new evidence that both regulated and constitutive splicing are similar in detail in vertebrates and arthropods indicating that the powerful genetic systems for analysis of splicing regulation in Drosophila are likely to be directly informative for regulated splicing throughout metazoa. In addition, we report formation of a novel class of intron-dependent complexes. Behavior of these complexes indicates that they represent a mutually exclusive, kinetically competing pathway with spliceosome assembly. We propose that this competition represents the basis for a kinetic proofreading mechanism enhancing fidelity of intron recognition. We also discuss possible implications of this model for regulated splicing.


Selective cleavage of pyrophosphate linkages. Pyrophosphate linkages have a number of important roles in biology and are also formed chemically with great ease. They often are unwanted products, such as in the nonenzymatic oligomerization of mononucleotides. We have found that Zr(4+)- and Th(4+)-ions catalyze the symmetrical hydrolysis of pyrophosphate linkages. Oligonucleotide analogs linked by pyrophosphate bonds are substantially degraded in the presence of these metals, even at 0 degrees C. Conditions are described which permit the decapping of a pyrophosphate capped oligonucleotide. Oligodeoxynucleotides can be decapped by this procedure without cleavage of phosphodiester linkages. Oligoribonucleotides are susceptible to partial hydrolysis and require purification by HPLC after decapping.


The abolition of collagen gene expression in SV40-transformed fibroblasts is associated with trans-acting factor switching. The goal of this study was to determine whether alpha 2(1) procollagen gene expression is modulated by positive or negative trans-acting DNA-binding proteins. Previous studies have shown that a clone of SV40-transformed WI-38 fibroblasts (SVWI-38) does not produce any alpha 2(1) procollagen mRNA (Parker et al (1989), J. Biol Chem. 264, 7147-7152). In order to elucidate the mechanism(s) responsible for such inactivation, we have examined the activity of a transfected wild type COL1A2 promoter in SVWI-38 cells. A set of 5' promoter deletions was linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into SVWI-38 and other cell lines expression type I collagen. The resulting CAT assays confirmed the importance of several upstream regions for promoter activity and documented the decreased transcriptional activity from an exogenous COL1A2 promoter in the SVWI-38 cell line. Competition experiments with an excess of COL1A2 promoter DNA fragment and a constant amount of COL1A2/CAT construct displayed a linear relationship between excess COL1A2 fragment and CAT activity in SVWI-38 cells, suggesting the involvement of a titratable negative effector. Electrophoretic mobility shift assays revealed the presence of a specific DNA-protein complex which was present in SVWI-38 cells and almost absent in control fibroblasts. Methylation interference analysis mapped the region of binding of this factor between nucleotides -80 and -72, relative to the transcription start site. Thus the data presented provide strong evidence for the existence of a negative trans-acting factor that may play a role in the repression of COL1A2 expression in SVWI-38 fibroblasts.


[Cytochemical evaluation of blast cells in acute myeloblastic leukemia after induction of their maturation in liquid culture--diagnostic usefulness]. We have performed the cytochemical analysis (myeloperoxidase, ASD-chloroacetate and acid alpha-naphthylacetate esterases, Sudan black B) of blast cells from 25 acute leukemia patients, after 3, 5 and 7 days of liquid culture with conditioned medium from phytohemagglutinin stimulated leukocytes (PHA-LCM). In case of acute myeloid leukemia blast cells an increase of percentage of positive cells simultaneously with the enhancement of the cytochemical reactions was observed. This method may be useful for the precise diagnosis of poorly differentiated blasts with weak expression of cytochemical phenotype.


Dietary n-3 fatty acids reduce antibody-dependent cell cytotoxicity and alter eicosanoid release by chicken immune cells. The overall goal of the present study was to determine whether the incorporation of n-3 fatty acids into poultry rations would alter the immune response of broiler chickens. Female broiler chicks were fed a corn and soybean meal-based diet to which one of four dietary fats were added: lard (LA), corn oil (CO), flaxseed oil (SO), or menhaden fish oil (FO). The latter two fat sources are rich in n-3 polyunsaturated fatty acids (PUFA). Enriching the diet with n-3 PUFA did not alter the primary or secondary antibody response of broiler chickens to sheep red blood cells. Dietary fat source had no effect on antibody-dependent cell cytotoxicity (ADCC) by peripheral blood leukocytes, but ADCC by splenocytes was 50% lower in chickens fed SO and FO compared with LA and CO (P less than .005). As expected, the fatty acid profile of the isolated immune cells reflected the fatty acid composition of the dietary fats fed. Basal release and calcium ionophore (A23187)-stimulated (10 microM) release of thromboxane B were significantly lower (P less than .05) in the SO and FO groups compared with the LA and CO groups. Total leukotriene B release was not significantly altered by dietary fat source. In conclusion, feeding broiler chickens diets rich in n-3 PUFA reduced ADCC of splenocytes and altered eicosanoid production by isolated immune cells.


Research note: kinetic and inhibition studies with turkey acrosin. Acrosin was extracted from turkey spermatozoa and partially purified by chromatofocusing. Enzyme activity was tested over a pH range with three different substrates. In each case, the pH optimum was between pH 8 and 9. When N-alpha-benzoyl-DL-arginine-p-nitroanilide.HCl (BAPNA) was used as a substrate, the Km and Vmax were 1.17 +/- .05 x 10(-3) M and 1.50 +/- .07 x 10(4) mumol/min.milligram, respectively. Turkey acrosin amidase activity was inhibited by aprotinin, ovomucoid, soybean trypsin inhibitor, benzamidine, p-aminobenzamidine, and zinc.


Exogenous GTP increases cyclic GMP and inhibits thrombin-induced aggregation of washed human platelets: comparison with ATP, adenosine and guanosine. The effects of exogenous guanosine 5'-triphosphate (GTP), guanosine, adenosine 5'-triphosphate (ATP) and adenosine on platelet aggregation, serotonin secretion and cyclic nucleotide accumulation were studied using thrombin-stimulated washed human platelets. GTP (10 microM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion. The inhibition of aggregation was accompanied by an increase in platelet cyclic GMP. GTP did not affect cyclic AMP concentration. Adenosine (1 microM-1 mM) dose-dependently inhibited thrombin-induced aggregation and serotonin secretion, and increased cyclic AMP. ATP at high concentrations (100 microM-1 mM) inhibited aggregation and serotonin secretion, and 1 mM ATP increased cyclic AMP. Guanosine was relatively ineffective in preventing aggregation and serotonin secretion and did not affect cyclic GMP. The rank order of inhibition of thrombin-induced aggregation of washed human platelets was adenosine > GTP > ATP > guanosine. In conclusion, exogenous GTP inhibits thrombin-induced aggregation and serotonin secretion of washed human platelets by increasing cyclic GMP. The results raise the possibility of a cell membrane site of action for GTP in platelets which mediates the activation of soluble guanylate cyclase suggesting that GTP may have a local antithrombotic effect also in vivo.


Aluminium interferes with signal transduction in neuroblastoma cells. The effects of aluminium on inositol phosphate formation were examined in murine neuroblastoma cells labelled with [3H]-myo-inositol. In aluminium-pretreated cells, the bradykinin-triggered inositol triphosphate, IP3, release and the change in intracellular [Ca2+] were appreciably less compared with the control group. Stimulating digitonin-permeabilized cells with non-hydrolyzable guanosine 5'-[gamma-thio]-triphosphate, GTP[S], inositol phosphate formation decreased in the presence of aluminium. A primary target of aluminium toxicity may reside on the guanine nucleotide-binding protein(Gp)/phospholipase C system, at a site different from that of the GTP[S] binding site.


Effects of foetal treatment with methylazoxymethanol on noradrenergic synapses in rat cerebral cortex. Methylazoxymethanol (MAM)-induced cerebral hypoplasia resulted in a significant increase in densities of norepinephrine uptake sites in cerebral cortex, suggesting that norepinephrinergic axon terminals were compressed in the smaller brain volumes. The density of beta-adrenergic receptors in MAM-lesioned cerebral cortex was decreased probably due to down-regulation, while there were no changes in the proportions and affinities of agonist high-affinity sites and agonist low-affinity sites in the desensitized beta-receptors.


[HIV cofactors in the course of AIDS].

[HIV cofactors in the course of AIDS]. The responsibility of HIV in the occurrence of AIDS is definitely established, but "cofactors" are strongly suspected to intervene, which would explain the difference courses followed by the disease in one patient or the other. Some of these cofactors are related to the host or result from his behaviour. Thus, age at the time of HIV acquisition and the patient's HLA group are associated with differences in the speed of progression towards AIDS. Attitudes that lead to reexposure to the virus by the sexual or intravenous routes shorten the duration of the asymptomatic phase. Other cofactors are environmental in nature. Among the infectious agents, cytomegalovirus and some mycoplasmas have been the object of the most through studies. The responsibility of some physicochemical substances, such as cocaine, vitamin D and corticosteroids, acting as cofactors of increasing severity, mostly rests on data obtained in vitro and need clinical confirmation in man. However, the study of cofactors already seems to be a promising line of research aimed at understanding AIDS and hence its therapeutic approach.


High density of Ca(2+)-dependent K+ and Cl- channels on the luminal membrane of lacrimal acinar cells. Tight-seal whole-cell recording and Ca2+ imaging were simultaneously performed on cell clusters or individual acinar cells of rat lacrimal glands during application of the secretagogue acetylcholine. Activation of Ca(2+)-dependent K+ and Cl- currents was selectively followed as a function of time by placing the cell potential near the equilibrium potential for Cl- or for K+ ion, respectively. Upon acetylcholine application to cell clusters, K(+)- and Cl(-)-selective currents displayed a distinctive initial rise ("hump"). At this time, there was only a small elevation of Ca2+ concentration, [Ca2+]i, that was restricted to the luminal end of acinar cells. A quantitative analysis of Ca2+ and current signals during the hump suggested that the luminal membrane contained high densities of K(+)- and Cl(-)-selective channels, roughly 10 times higher than those found in the basolateral domain. Distinct luminal and basolateral membrane domains were preserved in isolated cells, but with less contrasted densities than in cell clusters. The results suggest that Ca(2+)-dependent K+ channels are implicated not only in the transfer of salt from the blood compartment to the interior of acinar cells, as commonly accepted, but also in the electrolyte secretion from the cell interior to the acinar lumen.


Regulation of an opioid-binding protein in NG108-15 cells parallels regulation of delta-opioid receptors. An opioid-binding protein has recently been purified from bovine brain and cloned, and its cDNA sequence has been obtained. Indirect evidence suggests that this protein has a role in opioid-receptor function. However, because direct testing of its function by expression of its cDNA has not yet been possible and because its structure bears no resemblance to G protein-coupled receptors, the role of this protein in opioid-receptor activity is still in question. An antibody raised to a portion of the predicted amino acid sequence of opioid-binding cell-adhesion molecule (OBCAM) specifically labeled the surface of NG108-15 cells, as visualized by immunofluorescence with confocal microscopy. Furthermore, chronic treatment of these cells with opioid agonist, which down-regulates opioid receptors, reduced OBCAM immunoreactivity (ir). Down-regulation of both opioid receptors and OBCAM-ir was greatest after chronic treatment of NG108-15 cells with delta-opioid agonists, as well as with nonselective agonists such as etorphine, whereas other agonists including [D-Ala2-N-MePhe4-Gly-ol]enkephalin, morphine, levorphanol, dynorphin A-(1-13), and U-50,488H were less effective or ineffective. Chronic treatment of NG108-15 cells with muscarinic agonists had no effect on OBCAM-ir. Furthermore, NG108-15 cells transfected with an antisense construct to OBCAM have a reduced density of opioid-binding sites as well as reduced OBCAM-ir. Taken together, these results strongly suggest that OBCAM has a role in opioid-receptor function in NG108-15 cells.


T-cell receptor beta-chain gene usage in the T-cell recognition of Mycobacterium leprae antigens in one tuberculoid leprosy patient. The beta chain of the T-cell antigen receptor present on 20 T-cell clones isolated from a tuberculoid leprosy patient was studied by gene rearrangement and PCR analysis. These T-cell clones all responded to Mycobacterium leprae-encoded protein antigens, and 8 of them specifically recognized peptides of the mycobacterial 65-kDa heat shock polypeptide (65hsp). All T-cell clones studied were HLA-DR-restricted (DR2 or -3). In the DR3-restricted group, 7 of 10 used a beta-chain variable region V beta 5 gene family member, whereas in the DR2-restricted group, 2 of 10 T-cell clones used a V beta 5 gene segment and 5 used the V beta 18 gene segment. The deduced amino acid sequences of the beta chain from 8 T-cell clones have revealed that 3 of 4 DR3-restricted T-cell clones expressed the V beta 5.1 gene segment whereas the fourth DR3-restricted T-cell clone employed a V beta 5 family member not previously described. The V beta 5.1-positive T-cell clones all recognized the same 65hsp peptide from residues 2 to 12. The N-D-N segment (where D is diversity) of the junctional region of these T-cell clones was very similar, despite different beta-chain joining gene segments. Of the 4 DR2-restricted T-cell clones investigated, 3 used the V beta 18 gene segment and recognized the 65hsp peptide from residues 418 to 427. In conclusion, within this panel of M. leprae-reactive T-cell clones, the DR3-restricted T-cell clones mainly used a V beta 5 gene segment, whereas the DR2-restricted clones employed preferentially the V beta 18 gene segment.


Evidence for a base-pairing interaction between U6 small nuclear RNA and 5' splice site during the splicing reaction in yeast. U6 small nuclear RNA (snRNA) is an essential factor in mRNA splicing. On the basis of the high conservation of its sequence, it has been proposed that U6 snRNA may function catalytically during the splicing reaction. If this is the case, it is likely that U6 snRNA interacts with the splice sites in the spliceosome to catalyze the reaction. We have used UV crosslinking to analyze the interactions of U6 snRNA with the splicing substrates during the yeast splicing reaction. Crosslinked products in which the central region of U6 snRNA was joined to the 5' splice site region of mRNA precursor and lariat intermediate were identified. The crosslinking sites were precisely located in one of these products. The results suggest a possible base-pairing interaction between U6 snRNA and the 5' splice site of the mRNA precursor.