Nocturnal adrenocorticotropin and cortisol secretion depends on sleep duration and decreases in association with spontaneous awakening in the morning. It is still discussed controversially to what extent the nocturnal activity of the hypothalamus-pituitary-adrenocortical system depends on sleep and awakening in the morning. Therefore, we investigated the association of plasma ACTH and cortisol levels with undisturbed nocturnal sleep and spontaneous awakening in 14 healthy male subjects (between 2300 h and 1100 h). Between sleep onset and 476.9 min after sleep onset mean plasma cortisol level was significantly (P < 0.01) higher (210 +/- 15 vs. 155 +/- 9 nmol/L) in the group with a shorter (476.9 +/- 15.0 min; n = 7; mean +/- SEM) than in the group with a longer total sleep time (596.9 +/- 14.4 min; n = 7). Spontaneous awakening in the morning was not linked to the presence of any specific sleep stage or to rising plasma ACTH and cortisol levels. However, spontaneous awakening was followed by a brief rise in plasma ACTH and cortisol in both groups. Thereafter, during wakefulness plasma ACTH and cortisol abruptly declined in all subjects irrespective of the time of awakening. The slope of the plasma ACTH and cortisol curves differed significantly (ACTH: P < 0.001; cortisol: P < 0.002, for all subjects) comparing the time after awakening (until 1100 h) with a time interval of identical length before awakening. We conclude that the duration of sleep and nocturnal ACTH and cortisol secretion are interrelated. Furthermore, the data suggest that the endogenous early morning activation of the hypothalamus-pituitary-adrenocortical system is terminated by mechanisms closely associated with awakening.


Normative data for the steroidogenic response of mineralocorticoids and their precursors to adrenocorticotropin in a healthy pediatric population. The responses of mineralocorticoids and their precursors 1 h after a 0.25-mg bolus of ACTH has not previously been established in infancy or childhood. We report the steroidogenic responses of pregnenolone, progesterone (Prog), deoxycorticosterone (DOC), corticosterone (B), 18-hydroxycorticosterone (18OHB), and aldosterone (A) measured 1 h after a 0.25-mg bolus of ACTH in 102 healthy children who were divided into 5 age groups: group 1 (< 1 yr; n = 22), group 2 (1-5 yr; n = 22), group 3 (6-12 yr; n = 15), group 4 (early to midpuberty; n = 21), and group 5 (late puberty; n = 22). Baseline pregnenolone levels were constant throughout childhood; however, there was a significant fall in the stimulated level after the first year of life (group 1 vs. 2, P < 0.0125). Baseline Prog levels rose significantly with the onset of puberty (group 3 vs. 4, P < 0.0125), but levels did not increase after ACTH stimulation during puberty. Both baseline and stimulated levels of Prog, DOC, and 18OHB were significantly higher in group 3 males than in group 3 females (P < 0.05). Stimulated levels of DOC and corticosterone were constant during childhood, the only exception being the fall in the stimulated level of both steroids with the onset of puberty in males (group 3 vs. 4, P < 0.0125). The baseline level of 18OHB also fell with the onset of puberty in males (P < 0.0125), but a similar fall was not seen in females or in the stimulated level of 18OHB in either sex. The stimulated aldosterone level was higher in group 1 males than in group 2 males (P < 0.0125); a similar difference was not observed in females. The differences that we observed confirm the importance of specific age- and sex-related reference data when patients with possible abnormalities of mineralocorticoid synthesis are evaluated.


Adrenocorticotropin interferes with transforming growth factor-beta-induced growth inhibition of neocortical cells from the human fetal adrenal gland. The effects of transforming growth factor-beta (TGF-beta) and ACTH on growth, as indicated by [3H]thymidine incorporation into DNA, of primary cultures of neocortical cells from the human fetal adrenal gland were studied. TGF-beta inhibited, in a dose- and time-dependent manner, the growth of fetal neocortical cells, and ACTH significantly blunted the inhibitory effects of TGF-beta on growth of these cells. ACTH did not block the inhibitory effects of TGF-beta on growth of fetal adrenal fibroblasts or liver cells; neither ACTH nor TGF-beta had any effect on growth of fetal kidney cells. Thus, it appears that growth regulation of the neocortex may differ strikingly from that of the fetal zone of the human fetal adrenal, in which ACTH and TGF-beta have been reported recently to have additive inhibitory effects on cell proliferation.


Accuracy in the diagnosis of parotid tumours. Parotid lesions are often not easily classified. Important information is gathered by conventional and instrumental investigation. But these data are often insufficient to make a correct diagnosis. The authors have compared these techniques. In particular, they have evaluated their accuracy in identification of site and histology. 60 patients with parotid tumours underwent the same instrumental diagnostic techniques (echography, sialography, computed tomography and fine needle aspiration biopsy). In regard to the identification of the site, the highest percentage was achieved by computed tomography (98%). Echotomography has shown an accuracy of 83%, and sialography 87%. Fine needle aspiration biopsy has allowed the definition of the histiotype in 98% of cases. Computed tomography, echotomography and sialography have not allowed the definition of histiotype in malignant tumours, but in the case of benign tumours computed tomography has permitted a diagnosis in 29 cases (57% of benign tumours). Echotomography and sialography in 25 (49%) and 26 cases (51%), respectively.


Polyamine involvement in functional activation of human macrophages. Polyamines naturally occur in all living beings and play an important role in the regulation of cell proliferation, differentiation, and functional stimulation of terminally differentiated cells. Our studies, using specific inhibitors of polyamine biosynthesis such as alpha-difluoromethylornithine and methylglyoxal-bis[guanylhydrazone] to prevent polyamine accumulation, have indicated that polyamines are associated with functional activation of human macrophages. Both inhibitors diminished the respiratory burst activity of macrophages induced by lipopolysaccharide and interferon gamma. The methylglyoxal-bis]guanylhydrazone] inhibitory effect was concentration-dependent and could be reversed by spermine, which is the final product of polyamine biosynthesis.


A new, one-step assay on whole cell suspensions for peroxidase secretion by human neutrophils and eosinophils. The degranulation of neutrophils and eosinophils is frequently monitored by assaying myeloperoxidase (MPO) and eosinophil peroxidase (EPO) activity in the cell-free supernatant of degranulating cells, after removal of the cells by centrifugation. This procedure leads to underestimation of the extent of degranulation, since both peroxidases tend to stick to cell surfaces, to test tube walls, and to particulate stimuli used to elicit degranulation, because of their highly cationic nature. In this paper we describe a method for assaying MPO and EPO secretion in whole cell suspensions that avoids separation of the cells from the incubation medium. The least toxic and thus safest among the sensitive peroxidase substrates, 3,3',5,5'-tetramethylbenzidine (TMB), was employed for peroxidase assay. The method we describe here is applied to the detection of peroxidase release by neutrophil and eosinophil cell suspensions incubated in either polypropylene test tubes or flat-bottomed microtiter plate wells. Because of the omission of the centrifugation step, the TMB method offers two major advantages over the currently used techniques: (1) higher estimates of degranulation, which permits the use of a smaller number of cells (in the microassay version, 150,000 neutrophils and 50,000 eosinophils) and smaller amounts of the secretagogues, and (2) rapidity, since the degranulation assay can be performed immediately on completion of the cell incubation with the secretagogue.


Organic peroxides inhibit neutrophil leukotriene B4 biosynthesis. Leukotriene B4, an autacoid metabolite of arachidonic acid produced by polymorphonuclear neutrophils, induces chemokinesis, chemotaxis, and adhesion of these cells at sites of inflammation. Because neutrophil infiltration is a self-limited process, we hypothesized that oxidized lipid products of neutrophil-damaged tissue might inhibit leukotriene B4 biosynthesis, thereby preventing additional neutrophil infiltration and limiting peroxidative tissue damage. Erythrocyte ghosts exposed to a hydrogen peroxide-generating system served as a model of peroxidized tissue in inflammation and inhibited neutrophil leukotriene B4 production by 50% compared with unoxidized ghosts. Organic peroxides, including tert-butylhydroperoxide, peracetic acid, and linoleic hydroperoxide, resembling the product(s) of tissue membrane peroxidation in lipid solubility and catalase resistance, inhibited leukotriene B4 biosynthesis in a dose-dependent manner (50% inhibitory concentration of 3.9 microM compared to 530 microM for H2O2). Biosynthetic steps prior to the 5-lipoxygenase did not appear to be the site of inhibition. Likewise, the step after the 5-lipoxygenase, the leukotriene A4 hydrolase, was not primarily involved. Thus a possible mechanism for controlling the influx of neutrophils and their oxidative damage during inflammation may be inhibition of the 5-lipoxygenase by catalase-resistant lipid peroxides released by tissue membranes.


Nerve growth factor (NGF) prevents the shift in ocular dominance distribution of visual cortical neurons in monocularly deprived rats. The hypothesis that NGF could play a role in the plasticity of the developing mammalian visual cortex was tested in monocularly deprived (MD) rats. In particular, we have asked whether an exogenous supply of NGF could prevent the changes in ocular dominance distribution induced by monocular deprivation. Hooded rats were monocularly deprived for 1 month, starting at postnatal day 14 (P14), immediately before eye opening, by means of eyelid suture. In eight rats, only monocular deprivation was performed; in eight rats, monocular deprivation was combined with intraventricular injections of beta-NGF, and in three rats, with intraventricular injections of cytochrome C. Injections (2 microliters) were given every other day for a period of 1 month. Single neuron activity was recorded in the primary visual cortex of MD rats, MD rats treated with NGF, and MD rats treated with cytochrome C at the end of the deprivation period, and in normal rats of the same age. We found that monocular deprivation caused a striking change in the ocular dominance distribution of untreated MD rats, reducing binocular cells by a factor of two and increasing by a factor of eight the number of cells dominated by the nondeprived eye. In MD NGF-treated rats, the ocular dominance distribution was indistinguishable from the normal. Cytochrome C treatment was completely ineffective in preventing the ocular dominance shift induced by monocular deprivation. To test whether NGF affected cortical physiology or interfered with transmission of visual information, we evaluated in NGF-treated rats the spontaneous discharge and the orientation selectivity. We found these functional properties to be in the normal range. We conclude that NGF is effective in preventing the effects of monocular deprivation in the rat visual cortex and suggest that NGF is a crucial factor in the competitive processes leading to the stabilization of functional geniculocortical connections during the critical period.


Serotonin agonists increase transferrin levels via activation of 5-HT1C receptors in choroid plexus epithelium. Choroid plexus epithelial cells are enriched in mRNA for proteins such as the iron carrier transferrin, which acts as a trophic factor in the brain. Choroid plexus epithelial cells also have a high density of 5-HT1C receptors linked to activation of the phosphoinositide (PI) hydrolysis second messenger system. The present studies show that the 5-HT1C/5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) potently increases PI hydrolysis and the levels of transferrin in primary cultures of rat choroid plexus epithelial cells. These effects are blocked by the 5-HT1C/5-HT2 receptor antagonists mesulergine and mianserin, but not by the 5-HT2 receptor-selective antagonist spiperone. Similarly, mesulergine and mianserin, but not spiperone, block the increases in transferrin levels and PI hydrolysis elicited by 5-carboxamidotryptamine (5-CT), a 5-HT1 receptor-selective agonist, and by serotonin. We conclude, therefore, that 5-HT1C receptor activation in the choroid plexus leads to an increase in the production of transferrin. By promoting transferrin synthesis in the choroid plexus, 5-HT may indirectly influence brain development and differentiation.


Trigeminal and dorsal root ganglion neurons express CCK receptor binding sites in the rat, rabbit, and monkey: possible site of opiate-CCK analgesic interactions. 125I-Bolton-Hunter sulfated cholecystokinin-8 was used to localize and characterize cholecystokinin (CCK) receptor binding sites in trigeminal and dorsal root ganglia, and in the spinal cord of the rat, rabbit, and monkey. In the rabbit and monkey, a substantial number, 90 +/- 21% and 24 +/- 8%, respectively, of trigeminal and dorsal root ganglion neurons express CCK binding sites. In the spinal cord, the highest concentration of CCK receptors is found in laminae I and II, which is the major termination site of dorsal root ganglia neurons expressing CCK receptor binding sites. Neonatal capsaicin treatment of the rat results in a 70% decline in CCK receptor binding sites in laminae I and II of the spinal cord, indicating that dorsal root ganglia neurons are a major source of CCK receptors in the spinal cord. Pharmacological experiments using selective CCK-A and CCK-B receptor antagonists demonstrate that CCK-B is the prominent CCK receptor subtype in trigeminal and dorsal root ganglia neurons in the rat, rabbit, and monkey. In the rat and rabbit spinal cord, CCK-B binding sites are the prominent subtype, whereas in the monkey cord, CCK-A is the prominent receptor subtype. These results demonstrate that CCK-B receptors are expressed by a substantial percentage of dorsal root ganglion neurons at all spinal levels, and that CCK may antagonize opiate analgesia at the level of the primary afferent neuron itself.


Vasoactive intestinal peptide and noradrenaline exert long-term control on glycogen levels in astrocytes: blockade by protein synthesis inhibition. Vasoactive intestinal peptide (VIP) and noradrenaline (NA) have been previously shown to promote glycogenolysis in mouse cerebral cortex (Magistretti, 1990). This action, which is fully expressed within a few minutes, is exerted on astrocytes (Sorg and Magistretti, 1991). In the present article, we report a second, temporally delayed, action of VIP or NA in primary cultures of mouse cerebral cortical astrocytes; thus, following glycogenolysis, an induction of glycogen resynthesis is observed, resulting, within 9 hr, in glycogen levels that are 6-10 times higher than those measured before the application of either neurotransmitter. This effect of VIP or NA is concentration dependent and, for NA, is mediated by adrenergic receptors of the beta subtype. The continued presence of the neurotransmitter is not necessary for this long-term effect, since pulses as short as 1 min result in the doubling of glycogen levels 9 hr later. The induction of glycogen resynthesis triggered by VIP or NA is dependent on protein synthesis, since both cycloheximide and actinomycin D abolish it entirely. The ability to elicit glycogenolysis is not sufficient per se to trigger the induction of glycogen resynthesis. Thus, two glycogenolytic agents such as methoxamine, an alpha 1-adrenergic agonist, and phorbol 12,13-dibutyrate, both acting via protein kinase C activation, are unable to induce glycogen resynthesis. This observation, taken together with the fact that dibutyryl-cAMP application also results in enhanced glycogen resynthesis, strongly suggests that the long-term effect of VIP or NA is mediated by the cAMP second-messenger pathway.(ABSTRACT TRUNCATED AT 250 WORDS)


An updated study of taconite miners and millers exposed to silica and non-asbestiform amphiboles. This is the second update of a study of 3,444 taconite miners and millers who were first exposed to taconite, with associated exposures to silica and nonasbestiform amphiboles, in the period 1947 through 1958. Previous analyses of deaths through 1977, and again through 1983, showed no significant excess deaths from any specific causes. The present study continues the follow-up through 1988, adding 14,748 person-years of observation and 261 death certificates for analysis. The population, reduced to 3,431 because of the detection of 13 earlier duplications, has now been observed for 101,055 person-years, with 1,058 deaths and 1,039 death certificates. Death certificates were obtained for 98.2% of those known to be dead. The total number of deaths was significantly fewer than expected. Based on US rates, the standardized mortality ratio (SMR) was 83 (ie, 83% of expected). Based on Minnesota death rates, it was 91. With both US and Minnesota death rates, the SMRs for malignant neoplasms, cancer of the respiratory tract, cancer of the digestive system, heart disease, nonmalignant respiratory disease, and cirrhosis of the liver were all below 100. Slightly elevated SMRs were found for cancer of the colon, cancer of the kidney, and lymphopoietic cancer. These elevations were not statistically significant. Separate analyses were made of total deaths, lung cancer deaths, and kidney cancer deaths in men who had worked with taconite for time periods of less than 1 year, 1-5 years, 5-10 years, and over 10 years, during observation periods less than 10 years, 10-20 years, and over 20 years.(ABSTRACT TRUNCATED AT 250 WORDS)


[Immunohistochemical study of basement membrane in pleomorphic adenomas of the parotid gland: comparison between primary treated tumor and recurrent tumor]. We immunohistochemically investigated the distributions of components of the basement membrane (BM) (type IV collagen and laminin) and alpha smooth muscle actin in twelve primary pleomorphic adenomas of the parotid gland, as compared with six recurrent benign pleomorphic tumors (recurrent type). There were no differences between the two types of the tumor in the distributions of the BM components and actin, whereas the recurrent type of tumor have numerous myxochondroid areas histologically. The localization of type IV collagen in the tumor tissue was almost the same as that of laminin. Actin was identified in the occasional myoepithelial cell. In the tumor tissues, the components of BM were most densely localized in the areas surrounding solid clusters of epithelial cells, outer cells of glandular structures and occasional cells in the myxoid area, but the stroma of the myxochondroid areas did not take up stain. BM was densely defined between the tumor capsule and epithelial cells, but the tumor capsule was not bordered by myxochondroid areas with BM. These results suggest that loss of BM between the capsule and myxochondroid areas may be one of the causes of postoperative recurrence of pleomorphic adenomas.


A persistent sodium current in rat ventricular myocytes. 1. The tight seal, whole-cell, voltage-clamp technique was used to record currents from single ventricular myocytes acutely dissociated from adult rat hearts. Subtraction of currents recorded in the presence and absence of tetrodotoxin (TTX, 50 microM) revealed a small, persistent, inward current following a much larger, transient, inward current. 2. Both currents were sodium currents because they reversed close to the sodium equilibrium potential and were depressed when choline was substituted for extracellular sodium. 3. The persistent sodium current could be recorded when the transient current had been inactivated with conditioning depolarization. Only slight inactivation of the persistent current occurred during depolarizing pulses lasting up to 900 ms. 4. A lower concentration of TTX (0.1 microM) blocked the persistent sodium current while having little effect on the transient sodium current. 5. The persistent sodium current was activated at more negative potentials than the transient sodium current. It cannot have been a window current because it was recorded at positive potentials when the transient current was completely inactivated. 6. Because the persistent and transient sodium currents had a different voltage dependence and sensitivity to TTX, it was concluded that different channels are responsible for the two currents.


A symptom management program for psychotically ill adolescents and their families: preliminary clinical outcomes. The author describes the preliminary results of a clinical pilot study of a symptom management program for adolescents who are recovering from a nondrug-induced psychotic episode. The program was designed as a follow-up treatment for adolescents who are living at home with their families. Preliminary findings showed that the program was effective in increasing the adolescents' ability to recognize symptoms of their illnesses and to develop coping strategies to manage their symptoms.


The inhibitory action of cyclic AMP on responses to carbachol dependent on calcium stores in rat gastric smooth muscle. 1. The effects of cyclic AMP on contraction and Ca(2+)-activated K+ currents induced by carbachol (CCh), caffeine and inositol 1,4,5-trisphosphate (InsP3) were examined in intact and skinned smooth muscle fibres and in whole-cell voltage-clamped smooth muscle cells of the rat stomach. Intracellular Ca2+ level, [Ca2+]i, was monitored in intact muscle fibres loaded with Fura-2. 2. In intact muscle fibres, dibutyryl cyclic AMP, 8-bromo-cyclic AMP and forskolin inhibited a phasic contraction induced by CCh (100 microM) much more extensively than that induced by caffeine (30 mM) in Ca(2+)-free solution containing 2 mM-EGTA. A rise in [Ca2+]i evoked by CCh was also reduced by dibutyryl cyclic AMP. 3. In skinned muscle fibres, InsP3 (40 microM) produced a contraction of amplitude similar to that evoked by caffeine (30 mM) in Ca(2+)-free solution containing 0.05 mM-EGTA. Cyclic AMP suppressed the InsP3-induced contraction to a much greater extent than that induced by caffeine. 4. In cells voltage-clamped at 0 mV, CCh (100 microM) evoked a transient Ca(2+)-activated outward K+ current in 61% of cells tested. After wash-out of CCh, caffeine (10 mM) evoked a similar K+ current in all cells. In cells loaded with cyclic AMP (100 microM), the percentage of cells responding to CCh was reduced to 26% and the magnitude of current response tended to decrease. Cyclic AMP caused a small increase in the caffeine-induced K+ current. 5. An outward current was elicited immediately after the patch membrane was ruptured at a holding potential of 0 mV, using a patch pipette containing InsP3 (40 microM), in 76% of cells tested. In cells treated with dibutyryl cyclic AMP, the percentage of cells responding to InsP3 was reduced to 50% and the magnitude of current response tended to decrease. 6. In intact muscle fibres loaded with Fura-2, the relationship between [Ca2+]i and tension development shifted to the right in the presence of dibutyryl cyclic AMP. In skinned muscle fibres, cyclic AMP shifted the pCa-tension relation to the right, suggesting that cyclic AMP inhibits the contractile machinery directly. 7. These results suggest that the inhibitory effect of cyclic AMP on muscarinic receptors mediating both the contraction and the Ca(2+)-activated K+ current, is partly due to the inhibition of InsP3-induced Ca2+ release from intracellular stores in rat gastric smooth muscle cells.


Agriculture and environment.

Agriculture and environment. Agriculture came into existence about 15,000 years ago and passed through different stages of gathering, hunting, hoe culture, and sedentary agriculture, followed by modern agriculture. It began simultaneously in five world centers concentrating on different crops, most suited to the respective areas, and it was affected by changes in climate which determined the distribution of populations and their occupations. With the increase in population, the need for food progressively increased, necessitating introduction of modern agriculture to enhance production. The indiscriminate and faulty use of advanced technology to meet the food demand of fast-increasing populations added to its share to the destruction of the environment. The two approaches that contributed to this were horizontal expansion and vertical expansion. The former resulted in deforestation, desertification, soil erosion, land salting, and water logging, as well as the frequent occurrence of droughts and famines; the latter destroyed the soil structure and fertility through decreased microbial populations, fish culture, wildlife and bird sanctuaries, in addition to producing water and soil pollution and serious health hazards in human beings and fauna. The real culprit of this geobiologic devastation is the population growth rate, which needs to be contained at safer levels.


Treatment of resistant rheumatoid arthritis with minocycline: an open study. In a 48-week open trial, 18 patients with active rheumatoid arthritis (RA), resistant to second line agents, received 200 mg minocycline daily. Twelve patients completed 48 weeks of therapy. Statistically significant improvement was noted in almost all variables of disease activity. Three patients discontinued therapy because of lack of improvement, 2 patients because of side effects and one patient was lost to followup. Cytofluorographic analysis revealed a significant decrease in expression of a T cell activation antigen (gp 26). Our data suggest that minocycline could be a useful therapeutic agent in RA.


Zileuton, a 5-lipoxygenase inhibitor in rheumatoid arthritis. The effects of zileuton, a new 5-lipoxygenase inhibitor, on leukotriene generation and clinical response in rheumatoid arthritis (RA) was studied in a 4-week randomized double blind placebo controlled study at 2 academic rheumatology centers. Zileuton decreased the mean (+/- SEM) ionophore induced synthesis of leukotriene B4 at Week 1 by 70% from 191.2 +/- 28.5 to 57.5 +/- 17.0 ng/ml. A parallel suppression of all major 5-lipoxygenase pathway products was observed. An improvement in clinical variables was observed in the zileuton and placebo treated population. No unique toxicity was identified in this study. Zileuton inhibited 5-lipoxygenase in RA with a suggestion of clinical response with limited toxicity.


Acute calcific periarthritis in scleroderma. Three patients with limited cutaneous scleroderma (CREST syndrome) and acute calcific periarthritis are presented. "Chalky" bursal effusion in one patient demonstrated masses of calcium hydroxyapatite crystals. In contrast to idiopathic acute calcific periarthritis, resorption of periarticular calcifications after resolution of acute attacks occurs both less frequently and incompletely in scleroderma associated acute calcific periarthritis.


Disparate effects of representative dithiocarbamates on selected immunological parameters in vivo and cell survival in vitro in female B6C3F1 mice. Recent studies indicate that sodium methyldithiocarbamate is immunotoxic. Major effects of this compound in female B6C3F1 mice include decreased thymus weight, increased spleen weight, and decreased natural killer (NK) cell activity. The effects of other dithiocarbamates on these parameters are not known, and the immunotoxic potential of this important class of compounds is uncertain. In the present study, the effects of sodium methyldithiocarbamate (SMD), sodium diethyldithiocarbamate (DEDTC), and disodium ethylene-bis(dithiocarbamate)(EBD) on thymus weight, spleen weight, and NK cell activity were compared in female B6C3F1 mice. SMD caused significant loss of thymic weight following oral administration at 200, 225, or 300 mg/kg/d for 7 d and caused significant suppression of splenic NK cell activity at doses of 150, 225, or 300 mg/kg/d for 7 d. In contrast, a dose of 1000 mg/kg/d of DEDTC was required to decrease significantly thymus weight or increase spleen weight, and the only significant change produced by EBD was a slight increase in spleen weight at a dose of 675 mg/kg/d. EBD and DEDTC did not affect NK cell activity at any dose tested. Dithiocarbamates are known to be cytotoxic for a variety of cell types, and it seemed possible that SMD might be more potent in vivo than EBD or DEDTC because it was more cytotoxic than these compounds. However, direct measurement of the cytotoxicity of all three compounds toward splenocytes and thymocytes in vitro demonstrated that SMD and EBD are approximately equally potent (EC50 from 6.1 to 10.5 microM), whereas DEDTC is much more potent (EC50 from 0.13 to 0.15 microM). Of the three compounds examined in this study, only SMD affected thymus weight, spleen weight, and splenic NK cell activity in vivo. Thus, this pattern of immunological effects is not produced by all dithiocarbamates. In addition, the data demonstrate that differences in the potency of SMD, DEDTC, and EBD in vivo do not correlate with their relative cytotoxic potencies in vitro.


Strategies for blood screening for the hepatitis C virus and for the human immunodeficiency virus in high risk groups. For many infectious agents, seroprevalence rate is low but has serious consequences and must therefore be kept out of donated blood supplies. However, screening to ensure the safety of blood supplies has an associated very high cost. For example, in blood banks, detection of all the harmful items in a large number of samples is an expensive and tedious process. The laboratory and statistical approaches to obtain significant savings by the pooling method were discussed from 1943, recently, there have been further discussions of pooling sera as a means to determining the HIV seroprevalence rate in the general population or the weed out all HIV-positive individuals in blood screening. Here we describe a simple mathematical method to weed out all HIV, and HCV seropositive units. The method is designed to maximize possible savings. Two examples illustrate the application of this method in determining the number to be pooled in each stage, and the resulting savings. When the prevalence rate is lower than 2 percent. our method offers savings of over 80 percent.


Nonfunctioning pituitary adenoma.

Nonfunctioning pituitary adenoma. A total of 14 patients (8 males and 6 females) with nonfunctional pituitary adenomas were diagnosed and underwent surgical intervention at the Kaohsiung Medical College Hospital between 1986 and 1991. Their ages ranged from 20 to 68 years with an average of 49.7 years. Eight adenomas were composed of small polygonal cells with chromophobic cytoplasm and other six were of large cells with pale eosinophilic granular cytoplasm. Immunohistochemical staining had negative staining of prolactin (PRL), growth hormone (GH), adrenal corticotropic hormone (ACTH) and thyroid stimulating hormone (TSH) in all cases, but scattered or wide spread cells containing alpha-subunit of glycoprotein hormones were found in 6 cases. Hormonal hypersecretion was absent in all of the clinical and biological pictures, as was reactivity to immunohistochemical examination. There were either very few signs of secretory activity, or none at all. CT and MRI scanning revealed 13 macroadenomas (92.9%) and one microadenoma. In seven cases, local invasion or suprasellar extension resulted in decreased visual acuity, but the most common clinical symptoms were progressive headache (78.6%), and visual disturbance (50.0%). After surgical intervention, symptoms disappeared in 74.2% of the patients.


[Quantitative evaluation of deglutition in the upper esophagus by scintigraphy]. To evaluate the function of deglutition quantitatively, radionuclide transit in the and upper esophagus was examined. Ten ml of water containing 185 MBq 99mTc pertechnetate was put into the mouth and isotopic counts were measured every 0.2 second in the oral cavity, upper and lower cervical esophagus, and upper mediastinum. The subjects were studied twice, once while sitting and once supine. Esophageal transit was evaluated with time-activity curves obtained in each region of interest. In twelve healthy volunteers transit of the radionuclide was significantly delayed in supine position compared with sitting position. Even while the subject was supine, the radionuclide was propelled into the lower cervical esophagus in 0.8 second and upper mediastinum in 1.4 second in the healthy volunteers. In the patient, who had reconstruction of the esophagus through posterior mediastinum, radionuclide transit was not different from that of healthy volunteers. Whereas in the patient, who had the reconstruction through retrosternal space, the transit was remarkably delayed. The esophageal scintigraphy was safe and sensitive enough to evaluated the function of deglutition quantitatively.


The glucose-6-phosphatase system in the retina. The retinal glucose-6-phosphatase system was studied in rabbits. Following subcellular fractionation of the retina by differential centrifugation, the microsomal fraction was used for spectrophotometrical assay of the phosphate release. Assays were performed with intact and Triton X-100-treated preparations. Intactness of the microsomal preparations was assessed by using 2 mM mannose-6-phosphate as a substrate. Latencies towards glucose-6-phosphate and mannose-6-phosphate were 13.1 +/- 4.1% (n = 5) and 92.5 +/- 2.8% (n = 5) respectively; the difference between them was significant (P < 0.001). Pyridoxal-5'-phosphate specifically inhibited the retinal glucose-6-phosphate translocase activity at concentrations of 5-10 mM. Postnatal development was studied at postnatal days 1, 10, 17, 25, 36, 46, 54 and 70. At the postnatal 17th day, the retinal glucose-6-phosphatase specific activity was 60.1 +/- 3.8 (n = 3) nmol/min/mg protein which was one-third of the adult level [173.6 +/- 26.2 (n = 6) nmol/min/mg protein] (P < 0.001). This finding suggested that, in the rabbit, development of this enzyme system might coincide with development of retinal glucose catabolism.


Superoxide dismutase activity in cataractous lenses. The relationship between free oxygen radicals and cataract formation has been discussed. The behavior in lenses of the enzyme superoxide dismutase (SOD), which eliminates active oxygen, and glutathione (GSH), which has a defensive action against cataract, were investigated. SOD activity in human cataractous lenses and in porcine lenses was measured by electron spin resonance spectrometry. GSH was measured by high performance liquid chromatography. SOD and GSH significantly decreased in human lenses with senile cataracts as the cataracts advanced. The SOD and GSH values showed a positive correlation. The SOD activity in human lenses was localized dominantly in the lens epithelium and was also found in the shallow layer of the cortex. SOD activity appears to act as a barrier against photooxidation.


[Pulmonary distribution of 99mTc-technegas--a comparative study of radioactive inert gases]. 99mTc-technegas (99mTc-gas), which consists of fine particles, is produced in carbon crucibles burned at 2500 degrees C. On this study, the particle size of 99mTc-gas was measured and the pulmonary distribution of 99mTc-gas was assessed in 28 patients with various pulmonary diseases. Most particles were 5-30 nm in diameter as determined by electron microscopy. In a clinical study, about 37 MBq of 99mTc-gas was inhaled three times during deep breathing in a sitting position. In a comparative study with radioactive inert gases (133Xe, 81mKr), 99mTc-gas showed a similar distribution to the inert gas in most patients, although some with obstructive disease showed hot spots in the lung fields. In patients with severe obstructive change, marked deposits of 99mTc-gas was noted in the central airways, but 99mTc-gas penetrated to the peripheral lung field. This result suggests that 99mTc-gas can be used to evaluate ventilatory function even in patients with chronic obstructive pulmonary diseases.


Endometrial response in sequential cyclic therapy assessed with associated hysteroscopy and histology. A morphologic study was performed on the endometrium in 37 asymptomatic postmenopausal women under effects of cyclically administered oestrogens. Eighty-seven postmenopausal women were taken as control group. Medroxyprogesterone acetate (MPA), 10 mg daily, was administered in association with two types of oestrogen replacement therapy: conjugated equine oestrogens 0.625 mg (CEE) or transdermal 17 beta-oestradiol 0.05 mg (E2-TTS). Endometrial biopsies were taken under hysteroscopic control before treatment and on days 8-12 of combined therapy at the 6th month. Follow-up at 12 and 18 months was only performed in 8 and 5 patients, respectively, with transdermal 17 beta-oestradiol treatment. Various types of endometrial response were identified from atrophy to hyperplasia and secretory patterns. No atypical hyperplasia was found. All cases of simple or complex hyperplasia showed a regression after increased MPA dosage treatment (20 mg). This work is aimed at investigating the endometrial response during sequential cyclic therapy by using morphologic criteria based on hysteroscopy and histology. A large number of patients with hyperplasia can be detected with target biopsy under hysteroscopy, thus playing an important role in the management of patients during replacement therapy in research protocols.


Effect of elevated extracellular glucose concentrations on calcium ion uptake by cultured rat vascular smooth muscle cells. Blood flow autoregulation is impaired in early diabetes mellitus, predisposing the microvasculature to injury. Blood flow autoregulation is in part a myogenic response that is critically dependent on Ca2+ uptake via voltage-sensitive calcium channels in vascular smooth muscle cells (VSMC). Recent evidence suggests that impairment of blood flow autoregulation in diabetes may be responsive to variations in glycemic control. The present study thus examined the independent effect of an elevated extracellular glucose concentration on Ca2+ uptake by cultured rat VSMC in vitro. A threshold glucose concentration of 15-20 mmol/l markedly and maximally depressed basal and voltage sensitive Ca2+ channel activated (BAY K 8644, 10(-7) M) Ca2+ uptake. This effect was apparent within 3 h of incubating VSMC with the high glucose medium and was maximal after 48 h incubation. Osmotic control media containing either mannitol or L-glucose did not inhibit Ca2+ uptake by VSMC, thus confirming the effect was specific for elevated extracellular glucose concentrations and unrelated to changes in extracellular osmolality. Glucose-induced inhibition of basal and voltage-sensitive transmembrane fluxes of Ca2+ in VSMC may provide a metabolic mechanism for impaired calcium-dependent blood flow autoregulatory responses in early diabetes mellitus.


Renal cortical endosomes participate in the degradation of insulin. It has long been thought that metabolism of insulin by kidney proximal tubule cells takes place only in lysosomes. But previous studies with the perfused rat kidney and a proximal tubule-like cultured kidney cell line suggested that insulin degradation occurred in a nonlysosomal compartment. In this study we show that endosomes isolated from rat kidney cortex degrade insulin and that this process is ATP and pH dependent.


Interest in and limits to the utilization of reporter genes for the analysis of transcriptional regulation of nitrate reductase. Reporter gene techniques and mutant analysis were used to identify the molecular basis of the regulation of the expression of nitrate reductase (NR) by nitrate and nitrate-, or ammonium-derived metabolites (N-metabolites), in the true diploïd species Nicotiana plumbaginifolia and in the amphidiploïd species Nicotiana tabacum. The N. plumbaginifolia mutant E23 results from the insertion of a Tnt1-like retrotransposon (Tnp2) in the first exon of the single-copy nia gene, which encodes nitrate reductase. One of the resulting transcripts ends in the 5' LTR (long terminal repeat) sequence of this retrotransposon, and another one in the 3' LTR. Nitrate and N-metabolites modulate the expression of these truncated transcripts, indicating that intron splicing and termination processes are not essential to these regulatory events. A GUS reporter sequence was transcriptionally linked to the promoter of the nia-1 gene of N. tabacum. This fusion was functional in transient expression assays done with protoplasts derived from mesophyll cells of N. tabacum. However none of the regulatory mechanisms known to affect steady-state levels of the nia-1 transcript were operative under these experimental conditions. Transgenic plants carrying either this fusion or translational fusions of GUS linked to the promoter of either the nia-1 or nia-2 gene of N. tabacum were obtained by Agrobacterium-mediated transfer. A low proportion of the transgenic plants (22 out of 105 independent transformants) expressed GUS activity although at a low level. Only 4 plants exhibited a detectable level of GUS mRNA. The concentration of this mRNA increased significantly in an NR-deficient background, indicating regulation by N-metabolites. Only 2 plants, however, showed regulation (induction) by nitrate. Attempts to use aux2 or nptII reporter sequences linked to either the nia-1 or nia-2 promoter as marker genes for the selection of regulatory mutants of the nitrate assimilation pathway were unsuccessful because of our inability to isolate transgenic plants in which these reporter genes were properly regulated by nitrate. The implications of these results are discussed.


Influence of DNA repair defects (rad1, rad52) on nitrogen mustard mutagenesis in yeast. Nitrogen mustard (HN2) mutagenesis of a plasmid-borne copy of the Saccharomyces cerevisiae SUP4-o gene was examined in a repair-proficient yeast strain and isogenic derivatives defective for excision (rad1) or DNA double-strand break (rad52) repair. The excision repair deficiency sensitized the cells to killing by HN2 and abolished mutation induction. Inactivation of RAD52 had no influence on the lethality of HN2 treatment but diminished the induced mutation frequency by 50% at all doses tested. DNA sequence analysis of HN2-induced SUP4-o mutations suggested that RAD52 contributed to the production of basepair substitutions at G.C sites. The rad52 defect appeared to alter the distribution of G.C-->A.T transitions in SUP4-o relative to the distribution for the wild-type strain. This difference did not seem to be due to an effect of RAD52 on the relative fractions of HN2-induced transitions at localized (flanked by A.T pairs) or contiguous (flanked by at least one G.C pair) G.C sites but instead to an influence on the strand specificity of HN2 mutagenesis. In the repair-proficient strain, the transitions showed a small bias for sites having the guanine on the transcribed strand and this preference was eliminated by inactivation of RAD52.


Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1. Previous genetic analyses indicated that translational frameshifting in the--1 direction occurs within the run of six adenines in the sequence 5'-TTAAAAAACTC-3' at nucleotide positions 305-315 in IS 1, where the two out-of-phase reading frames insA and B'-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84-87. IS 1 mutants with a 1 bp insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84-87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-Lys-Lys-Leu. An IS 1 mutant with the DNA segment 5'-CTTAAAAACTC-3' at positions 305-315 carrying the termination codon TAA in the B'-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The beta-galactosidase activity specified by several IS 1-lacZ fusion plasmids, in which B'-insB is in-frame with lacZ, showed that the region 292-377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu-Lys-Lys-Leu encoded by the DNA segment 5'-TTAAAAAACTC-3', indicating that--1 frameshifting does occur within the run of adenines.


DNA sequences required for translational frameshifting in production of the transposase encoded by IS1. The transposase encoded by insertion sequence IS1 is produced from two out-of-phase reading frames (insA and B'-insB) by translational frameshifting, which occurs within a run of six adenines in the -1 direction. To determine the sequence essential for frameshifting, substitution mutations were introduced within the region containing the run of adenines and were examined for their effects on frameshifting. Substitutions at each of three (2nd, 3rd and 4th) adenine residues in the run, which are recognized by tRNA(Lys) reading insA, caused serious defects in frameshifting, showing that the three adenine residues are essential for frameshifting. The effects of substitution mutations introduced in the region flanking the run of adenines and in the secondary structures located downstream were, however, small, indicating that such a region and structures are not essential for frameshifting. Deletion of a region containing the termination codon of insA caused a decrease in beta-galactosidase activity specified by the lacZ fusion plasmid in frame with B'-insB. Exchange of the wild-type termination codon of insA for a different one or introduction of an additional termination codon in the region upstream of the native termination codon caused an increase in beta-galactosidase activity, indicating that the termination codon in insA affects the efficiency of frameshifting.


The cauliflower mosaic virus 35S promoter is regulated by cAMP in Saccharomyces cerevisiae. The cauliflower mosaic virus 35S promoter confers strong gene expression in plants, animals and fission yeast, but not in budding yeast. On investigating this paradox, we found that in budding yeast the promoter acts through two domains. Whereas the upstream domain acts as a silencer, the downstream domain couples expression to the nutritional state of the cells via the RAS/cAMP pathway. Point mutations indicate that two boxes with similarity to the cAMP regulated element (CRE) of mammalian cells mediate this response. Gel retardation assays show that, in both yeast and plant protein extracts, factors bind to this promoter element. Therefore, transcriptional activation appears to be highly conserved at the level of transcription factors and specific DNA target elements in eukaryotes. This offers new ways to investigate gene regulation mechanisms of higher eukaryotes, which are not as amenable to genetic analysis as yeast.


Exogenous supply of nerve growth factor prevents the effects of strabismus in the rat. It has recently been reported that exogenous supply of nerve growth factor prevents the effects of monocular deprivation both in rats and in cats. Here we have extended these experiments to the case of strabismus. Repeated intraventricular injections of nerve growth factor were performed in rats made surgically strabismic early in the critical period. At the end of the critical period the ocular dominance distribution of visual cortical neurons was assessed in strabismic untreated, strabismic nerve growth factor-treated and strabismic Cytochrome C-treated (control) rats by means of extracellular recordings. We found that in rats surgical strabismus causes a consistent loss of binocular neurons. By contrast the treatment with nerve growth factor maintains the normal ocular dominance distribution of neurons in the primary visual cortex. We conclude that nerve growth factor exogenously supplied prevents the effects induced by surgical strabismus in rats and suggest that nerve growth factor has a role in visual cortical plasticity.


K(+)-evoked dopamine release depends on a cytosolic Ca2+ pool regulated by N-type Ca2+ channels. Membrane depolarization evoked by 25-40 mM K+ elicited an immediate increase of somatic and neuritic [Ca2+]i in cultured dopaminergic neurons as measured by digital fluorescence microscope imaging. The rise of neuritic [Ca2+]i was inhibited by N-type but not L-type Ca2+ channel blockers, while the rise of somatic [Ca2+]i was prevented by both L- and N-type Ca2+ channel blockers. Similarly, depolarization-induced [3H]dopamine release was selectively attenuated by N-type Ca2+ channel blockers. The present results suggest that [3H]dopamine release from mesencephalic neuronal cell cultures relates to a Ca(2+)-dependent mechanism regulated by N-type channels located in the vicinity of the exocytotic sites within neuritic processes.


A phase III comparison of etoposide/cisplatin with or without added ifosfamide in small-cell lung cancer. A total of 92 patients with small-cell lung cancer (SCLC) were randomized to receive cisplatin (80 mg/m2, day 1)/etoposide (100 mg/m2, days 1, 3, 5) (PE) or cisplatin (80 mg/m2, day 1)/etoposide (100 mg/m2, days 1, 3, 5)/ifosfamide (2 g/m2, days 1, 2, 3) (PEI) combination chemotherapy. After 2 courses of chemotherapy, patients with limited disease (LD) received chest irradiation of 40-50 Gy. Of the 89 patients who could be wholly evaluated, the overall response rate was 78% for PE and 74% for PEI therapy (NS). For all patients the complete response (CR) rates were 14 versus 21%, respectively, and 22 versus 30% for LD. However, the median survival times for all patients were 55 weeks for PE therapy versus 56 weeks for PEI therapy (NS). The 2-year survival rates were 15 and 17%, respectively, for all patients (NS). There was no difference in the duration of response between PE and PEI therapy in cases with CR or partial response. However, severe leukopenia (< 2,000/mm3) occurred more often after PEI (73%) than after PE (44%) therapy (p < 0.05). These results suggest that PEI is not superior to PE chemotherapy in SCLC. The use of ifosfamide in multimodality treatment regimens needs to be studied further.


[Efficacy of biliary endoprostheses using expandable metallic stents in obstructive jaundice--long-term results of three different types of stent]. Three types of expandable metallic stents were used to relieve obstructive jaundice in 59 patients. They consisted of 3 cases with benign stricture, and 56 with malignant obstruction including 28 of cholangiocarcinoma, 17 of pancreatic carcinoma, 9 of lymph node metastases, and 2 of gall bladder carcinoma. The median age of patients was 68.9 years. Of 56 cases with malignant obstruction, 51 cases were able to remove external drainage catheter. In these 51 cases, 35 patients died, and 16 are still alive. No significant difference was noted in the incidence of stent destruction or migration in three types of stents. The average survival period was 189.9 days in 35 patients who died after withdrawal of external drainage. Twenty-three of 35 patients had no recurrence of obstructive jaundice. Sixteen patients with malignant obstruction are still alive and have been observed for 22 to 764 days. The 30 day mortality rate was 6.8%. Three cases of acute cholecystitis were noted after procedure. It is warranted to say that endoprostheses using expandable metallic stents will be the major treatment of choice for biliary obstruction because of long term patency and low complication rate.


Pit-1-dependent expression of the receptor for growth hormone releasing factor mediates pituitary cell growth. In Snell (dw) and Jackson (dwJ) dwarf mice, mutations in the gene encoding Pit-1, a tissue-specific POU-domain transcription factor, lead to the absence of somatotroph, lactotroph and thyrotroph cells. Pre-somatotroph proliferation is stimulated by increased intracellular levels of cyclic AMP, normally induced by growth hormone releasing factor (GRF; refs 7-17). Here we report the cloning of mouse and rat complementary DNAs encoding a new member of the seven-transmembrane-helix, G-protein-coupled receptor family restricted to the pituitary gland, which mediates increases in intracellular cAMP and cAMP-dependent gene transcription in response to GRF. The receptor is expressed in a spatial and temporal pattern corresponding precisely to growth hormone gene expression, and neither is expressed in dw/dw mice. The pituitary hypoplasia in these mice thus appears to be due, at least in part, to the absence of GRF receptor, which is in turn due to the absence of functional Pit-1.


[Gianturco expandable metallic stents in the treatment of tumor thrombus in portal vein--preliminary clinical experience]. Two cases of hepatocellular carcinoma with protal vein invasion (Vp3) were successfully treated by Gianturco expandable metallic stents (GEMS). Each case was treated through the different approach, i.e. the percutaneous transhepatic or ileocolic venous route. GEMS was easily expanded within the protal vein and carcinoma thrombi were pushed against the walls, resulting in increase of portal blood flow. The GEMS might improve the impaired portal blood flow with hepatic failure and esophagogastric varices, in spite of the possibilities of dissemination and ingrowth of carcinoma thrombi.


Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume. Regulation of cell volume is essential for every cell and is accomplished by the regulated loss or gain of intracellular ions or other osmolytes. Regulatory volume decrease often involves the parallel activation of potassium and chloride channels. Overexpression of P-glycoprotein leads to volume-activated Cl- currents but its physiological importance for volume regulation is unclear. CIC-2 is a ubiquitously expressed Cl- channel activatable by non-physiologically strong hyperpolarization. We now show that CIC-2 can be activated by extracellular hypotonicity, which suggests that it has a widespread role in volume regulation. Domains necessary for activation by both voltage and volume are localized to the amino terminus. Mutations in an 'essential' region lead to constitutively open channels unresponsive to medium tonicity, whereas deletions in a 'modulating' region produce partially opened channels responsive to both hypo- and hypertonicity. These domains can be transplanted to different regions of the protein without loss of function.


Phosphorylation of the S. cerevisiae Cdc25 in response to glucose results in its dissociation from Ras. In the yeast Sacchromyces cerevisiae, addition of glucose to starved cells triggers a transient rise in the intracellular level of cyclic AMP that induces a protein phosphorylation cascade. The glucose signal is processed by the Cdc25/Ras/adenylyl cyclase pathway, where the role of Cdc25 is to catalyse the GDP-GTP exchange on Ras. The molecular mechanisms involved in the regulation of the activity of Cdc25 are unknown. We report here the use of highly selective anti-Cdc25 antibodies to demonstrate that Cdc25 is a phospho protein and that in response to glucose it is hyperphosphorylated, within seconds, by the cyclic AMP-dependent protein kinase. It is also demonstrated that, concomitantly with hyperphosphorylation, Cdc25 partially relocalizes to the cytoplasm, reducing its accessibility to membrane-bound Ras. These results are of general significance because of the highly conserved sequence of Ras-guanyl nucleotide exchange factors from yeasts to mammals.


Potassium transport into plant vacuoles energized directly by a proton-pumping inorganic pyrophosphatase. Potassium is accumulated in plant vacuoles against an inside-positive membrane potential. The mechanism facilitating energized K+ transport has remained obscure. However, electrogenic activity of the inorganic pyrophosphatase (H(+)-PPase) at the vacuolar membrane is dependent on cytoplasmic K+, raising the possibility that the enzyme translocates K+ into the vacuole. Membrane currents generated by the H(+)-PPase were measured (using a patch clamp technique) in intact vacuoles isolated from Beta vulgaris storage tissue. A significant orthophosphate-dependent outward current mediated by the enzyme in reverse mode is evoked only when potassium is present at the vacuolar face of the tonoplast, suggesting that potassium is a translocated ion. Furthermore, current-voltage analysis of the effects of extravacuolar potassium and pH on the reversal potential of the H(+)-PPase-generated current points to direct translocation of K+ and H+ by the enzyme. Thus the H(+)-PPase represents a distinct class of eukaryote translocase and could facilitate vacuolar K+ accumulation in vivo.


Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. In recent reports attention has been drawn to the extensive amino acid homology between pig heart, yeast, and Escherichia coli aconitases (EC 4.2.1.3) and the iron-responsive element binding protein (IRE-BP) of mammalian cells [Rouault, T. A., Stout, C. D., Kaptain, S., Harford, J. B. & Klausner, R. D. (1991) Cell 64, 881-883.; Hentze, M. W. & Argos, P. (1991) Nucleic Acids Res. 19, 1739-1740.; Prodromou, C., Artymiuk, P. J. & Guest, J. R. (1992) Eur. J. Biochem. 204, 599-609]. Iron-responsive elements (IREs) are stem-loop structures located in the untranslated regions of mRNAs. IRE-BP is required in the posttranscriptional regulation of ferritin mRNA translation and stabilization of transferrin receptor mRNA. In spite of substantial homology between the amino acid sequences of mammalian mitochondrial aconitase and IRE-BP, the mitochondrial protein does not bind IREs. However, there is a second aconitase, found only in the cytosol of mammalian tissues, that might serve as an IRE-BP. To test this possibility, we have prepared sufficient quantities of the heretofore poorly characterized beef liver cytosolic aconitase. This enzyme is isolated largely in its active [4Fe-4S] form and has a turnover number similar to that of mitochondrial aconitase. The EPR spectra of the two enzymes are markedly different. The amino acid composition, molecular weight, isoelectric point, and the sequences of six random peptides clearly show that these physicochemical and structural characteristics are identical to those of IRE-BP, and that c-aconitase is distinctly different from m-aconitase. In addition, both cytosolic aconitase and IRE-BP can have aconitase activity or function as IRE-BPs, as shown in the following paper and elsewhere [Zheng, L. Kennedy, M. C., Blondin, G. A., Beinert, H. & Zalkin, H. (1992) Arch. Biochem. Biophys., in press]. This leads us to the conclusion that cytosolic aconitase is IRE-BP.


Osteogenin and recombinant bone morphogenetic protein 2B are chemotactic for human monocytes and stimulate transforming growth factor beta 1 mRNA expression. Subcutaneous implantation of demineralized bone matrix initiates a sequence of developmental events, which culminate in endochondral bone formation. During early stages of development of matrix-induced implants, ED1, Ia-positive monocytes-macrophages were observed, suggesting that in the initial phases of the endochondral bone formation cascade, the bone-inductive protein osteogenin and related bone morphogenetic proteins (BMPs) might serve as potent chemoattractants to recruit circulating monocytes. In this investigation, we demonstrate that at concentrations of 10-100 fg/ml (0.3-3 fM), native bovine osteogenin and recombinant human BMP-2B (rhBMP-2B) induce the directed migration of human blood monocytes in vitro. This chemotactic response was associated with expression of BMP binding sites (receptors) on monocytes. About 750 receptors per cell were detected with an apparent dissociation constant of 200 pM. Both osteogenin and rhBMP-2B at higher concentrations (0.1-30 ng/ml) stimulated mRNA expression for an additional regulatory molecule, type beta 1 transforming growth factor (TGF-beta 1) in human monocytes. TGF-beta 1, in turn, is known to induce a cascade of events leading to matrix generation. Monocytes stimulated by TGF-beta are known to secrete a number of chemotactic and mitogenic cytokines that recruit endothelial and mesenchymal cells and promote their synthesis of collagen and associated matrix constituents. TGF-beta 1 in concert with these other cytokines and matrix components regulates chemotaxis, mesenchymal proliferation, differentiation, angiogenesis, and controlled synthesis of extracellular matrix. Our results demonstrate that osteogenin and related BMPs through their profound effects on monocyte recruitment and cytokine synthesis may promote additional successive steps in the endochondral bone formation cascade.


RYK, a receptor tyrosine kinase-related molecule with unusual kinase domain motifs. By using the polymerase chain reaction with degenerate oligonucleotides based on highly conserved motifs held in common between all members of the protein tyrosine kinase (PTK) family, a PTK-related sequence was isolated from murine peritoneal macrophage cDNA. Full-length clones have been isolated that encompass the entire coding region of the mRNA, and the predicted amino acid sequence indicates that the protein encoded has the structure of a growth factor receptor PTK (RTK). We have dubbed this molecule RYK (for related to tyrosine kinase). The RYK-encoded protein bears a transmembrane domain, with a relatively small (183 amino acid) extracellular domain, containing five potential N-linked glycosylation sites. The intracellular domain of RYK is unique among the broader family of RTKs and has several unusual sequence idiosyncrasies in some of the most highly conserved elements of the PTK domain. These sequence differences call into question the potential catalytic activity of the RYK protein.


Tumor necrosis factor alpha stimulates expression of adenovirus early region 3 proteins: implications for viral persistence. Human adenovirus (Ad) can cause persistent infections in humans. Early region 3 (E3) of the virus appears to be implicated in this phenomenon. This transcription unit encodes proteins that interfere in various ways with host cell functions, including (i) cell-surface expression of histocompatibility class I antigens (HLA), (ii) cell-surface expression of the epidermal growth factor receptor (EGF-R), and (iii) the biological activity of tumor necrosis factor alpha (TNF-alpha). We transfected the human cell line 293 with the entire E3 region of Ad2 and investigated the influence of the cytokines TNF-alpha and interferon gamma (IFN-gamma) on cell-surface expression of HLA class I and the EGF-R. Whereas IFN-gamma treatment induced expression of HLA to some extent but not that of the EGF-R, TNF-alpha treatment augmented the reduction of these cell-surface molecules. Subsequent studies on the mechanism of this effect showed a TNF-alpha-dependent upregulation of E3 protein (E3/19K) and mRNA. The significance of this phenomenon was confirmed in infection experiments. A dramatic increase in the amount of E3/19K, even after short induction with low doses of TNF-alpha could be demonstrated. The study provides evidence for an interaction between the immune system and Ad in which the virus takes advantage of an immune mediator to escape immunosurveillance of the host.


Long-term transformation of an inhibitory into an excitatory GABAergic synaptic response. For a constant membrane potential, a predominantly inhibitory GABAergic synaptic response is shown to undergo long-term transformation into an excitatory response after pairing of exogenous gamma-aminobutyric acid (GABA) with postsynaptic depolarization or pairing of pre- and postsynaptic stimulation. Current- and voltage-clamp experiments suggest that this synaptic transformation is due to a shift from a net increase of conductance to a net decrease of conductance in response to GABA. GABA-induced elevation of intracellular calcium is prolonged after the same stimulus pairing and may, therefore, contribute to this synaptic transformation via Ca(2+)-activated phosphorylation pathways. This synaptic transformation, which does not follow unpaired stimulus presentations, occurs in a neuronal compartment spatially separated from the soma, which also changes during stimulus pairing.


Cantharidin-binding protein: identification as protein phosphatase 2A. The toxic effects of cantharidin from blister beetles and its analogs, including the herbicide endothall, are attributable to their high affinity and specificity for a cantharidin-binding protein (CBP). An ammonium sulfate precipitate of mouse liver cytosol was purified by five chromatographic steps to isolate CBP in 14% yield and > 99% purity as monitored by [3H]cantharidin-binding activity. The purification factor of 2230-fold corresponds to a CBP content of 0.045% of the liver cytosolic protein. CBP is a heterodimer consisting of a 61-kDa alpha subunit and a 39-kDa beta subunit. Amino acid sequences of four peptides from CBP-alpha and three peptides from CBP-beta are identical with deduced amino acid sequences for the A alpha regulatory and C beta catalytic subunits, respectively, of protein phosphatase 2A (PP2A). This assignment of CBP as PP2A-AC from structural evidence is supported by biochemical studies with selective substrates and inhibitors. CBP dephosphorylation of phosphorylase alpha is sensitive not only to okadaic acid, as with PP2A, but also to cantharidin and its analogs, consistent with their potency in blocking the radioligand binding site of CBP. Okadaic acid is a potent inhibitor of [3H]cantharidin binding to CBP. PP2A is present in many mammalian tissues and in plants and is involved in regulatory phosphorylation-dephosphorylation events which modulate multiple cellular functions. Inhibition of PP2A activity may account for the diverse effects and toxicity of cantharidin and its analogs, including the herbicide endothall, in mammals and possibly plants.


Differential stereochemical requirements of mu vs. delta opioid receptors for ligand binding and signal transduction: development of a class of potent and highly delta-selective peptide antagonists. Opioid peptide analogs consisting entirely of aromatic amino acid residues and containing conformationally restricted phenylalanine derivatives in position 2 of the peptide sequence were synthesized and pharmacologically characterized in vitro. Both diastereoisomers of H-Tyr-(D or L)-NMePhe-Phe-Phe-NH2 (NMePhe is N alpha-methylphenylalanine) were mu-receptor-selective, were full agonists in the mu-receptor-representative guinea pig ileum assay, and were partial agonists in the mouse vas deferens assay, with the L-NMePhe2 analog displaying somewhat higher intrinsic activity than the D-NMePhe2 analog. Further conformational restriction at position 2 in the sequence, as achieved through substitution of D- or L-tetrahydro-3-isoquinoline carboxylic acid (Tic), produced a configuration-dependent differential effect on receptor selectivity and intrinsic activity, leading to a potent mu-selective mu agonist (the D-Tic2 analog) with increased intrinsic activity in the mouse vas deferens assay and to a potent delta-selective delta antagonist (the L-Tic2 analog). These results demonstrate that imposition of conformational constraints in a peptide not only may alter receptor selectivity but also may decrease, totally abolish, or even enhance intrinsic activity. The tetrapeptide H-Tyr-Tic-Phe-Phe-NH2 was a moderately potent full agonist in the guinea pig ileum assay and, thus, represents a compound with mixed mu-agonist/delta-antagonist properties. The corresponding peptide with a free C-terminal carboxyl group H-Tyr-Tic-Phe-Phe-OH showed high delta-receptor affinity (Ki delta = 1.2 nM), unprecedented delta selectivity (Ki mu/Ki delta = 1410), high potency as delta antagonist (Ke = 3-8 nM against various delta agonists in the mouse vas deferens assay) and, unlike other delta antagonists, had no mu-antagonist properties. The tripeptides H-Tyr-Tic-Phe-OH and H-Tyr-Tic-Phe-NH2 were also delta antagonists.


Interferon gamma rapidly induces in human monocytes a DNA-binding factor that recognizes the gamma response region within the promoter of the gene for the high-affinity Fc gamma receptor. Interferon gamma (IFN-gamma) transcriptionally activates several early-response genes in monocytes that are important for the ultimate phenotype of the activated macrophage. One of these genes is the high-affinity Fc receptor for IgG (Fc gamma RI). Recently, Pearse et al. [Pearse, R.N., Feinman, R. & Ravetch, J. V. (1991) Proc. Natl. Acad. Sci. USA 88, 11305-11309] defined within the promoter region of the Fc gamma RI gene an element, the gamma response region, which was necessary for IFN-gamma-induced enhancement of Fc gamma RI. In this report we describe the induction by IFN-gamma of a DNA-binding factor, FcRF gamma (Fc gamma RI DNA-binding factor, IFN-gamma induced), that specifically recognizes the gamma response region element. Electrophoretic mobility shift assays (EMSAs) demonstrated the presence of FcRF gamma in human monocytes within 1 min after exposure to IFN-gamma. On EMSA, FcRF gamma consisted of two complexes termed FcRF gamma 1 and FcRF gamma 2. The nuclear concentration of FcRF gamma rapidly increased, peaked at 15 min, and then fell after 1-2 hr. Dose-response studies revealed (i) as little as 0.05 ng of IFN-gamma per ml induced FcRF gamma, (ii) maximum activation occurred at 1 ng/ml, and (iii) steady-state levels of Fc gamma RI mRNA closely paralleled that of FcRF gamma. Since FcRF gamma was activated in cells normally not expressing Fc gamma RI RNA, other regulatory mechanisms must control Fc gamma RI-restricted tissue expression. Activation of FcRF gamma by IFN-gamma was inhibited by pretreatment with 500 nM staurosporin and 25 microM phenyl arsine oxide. These data suggest that a kinase and possibly a phosphatase activity are required for IFN-gamma-induced signaling of FcRF gamma in monocytes.


Odor-induced phosphorylation of olfactory cilia proteins. Stimulation of isolated rat olfactory cilia in the presence of [gamma-32P]ATP leads to a significantly enhanced incorporation of [32P]phosphate. Depending on the type of odorants applied, the induced phosphorylation is completely blocked by specific inhibitors of either protein kinase A or protein kinase C. Time-course experiments indicate that the odor-induced modification of ciliary proteins is transient; the intensity of labeling decayed over time (1-10 sec). Separation of ciliary proteins by SDS/polyacrylamide gel electrophoresis followed by autoradiography demonstrated that upon stimulation with lilial, a single polypeptide (50,000 Da) was phosphorylated; the size of the modified protein is in line with the hypothesis that odorant receptors are phosphorylated subsequent to activation by specific odors.


The delta-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization. A random primed expression cDNA library was constructed from the RNA of NG 108-15 cells. Pools of plasmid DNA were transfected into COS cells, which were screened for their ability to bind 3H-labeled Tyr-D-Thr-Gly-Phe-Leu-Thr, a tritiated agonist for the delta-opioid receptor. A cDNA was isolated that encodes a 371-amino acid-residue protein presenting all the structural characteristics of receptors that interact with guanine nucleotide-binding proteins. Noticeable features are (i) the high hydrophobicity of the encoded protein, (ii) its low sequence similarity to both catecholamine receptors and peptide-binding receptors, although it presents the typical aspartate residue involved in catecholamine binding of the first group and the characteristic short third cytoplasmic loop of the second group. When expressed in COS cells, the receptor exhibits pharmacological properties similar to those of the native receptor: high-affinity binding sites for 3H-labeled Tyr-D-Thr-Gly-Phe-Leu-Thr (Kd = 1.4 nM), stereospecific binding sites for the - enantiomers of levorphanol and naloxone, and the selectivity profile of a delta receptor, as determined by competition experiments with a set of mu-, delta-, and kappa-opioid ligands.


Dopamine enhances both electrotonic coupling and chemical excitatory postsynaptic potentials at mixed synapses. The transmitter dopamine reduces electrotonic coupling between retinal horizontal cells and increases their sensitivity to glutamate. Since in other systems single afferents establish mixed electrotonic and chemical excitatory synapses with their targets, dopamine might be expected there to depress one component of excitation while enhancing the other. This hypothesis was tested by applying dopamine locally in the vicinity of the lateral dendrite of the goldfish Mauthner cell (M cell) and monitoring the composite electrotonic and chemical excitatory postsynaptic potentials and currents evoked by ipsilateral eighth nerve stimulation. Dopamine produces persistent enhancements of both components of the postsynaptic response while it also increases input conductance. All these dopamine actions are prevented by superfusing the brain with saline containing the dopamine D1 receptor antagonist SCH-23390. Postsynaptic injections of the cAMP-dependent protein kinase inhibitor (Walsh inhibitor, or PKI5-24) block the dopamine-induced changes in synaptic transmission, implicating a cAMP-dependent mechanism. Furthermore, there is a dopaminergic innervation of the M cell, as demonstrated immunohistochemically with antibodies against dopamine and the rate-limiting enzyme in its synthetic pathway, tyrosine hydroxylase. Varicose immunoreactive fibers lie in the vicinity of the distal part of the lateral dendrite between the large myelinated club endings that establish the mixed synapses. As determined with electron microscopy, the dopaminergic fibers contain small vesicles, and they do not have synaptic contacts with either the afferents or the M cell, remaining instead in the synaptic bed. Taken together, these results suggest that dopamine released at a distance from these terminals increases the gain of this primary sensory input to the M cell, most likely through a phosphorylation mechanism.


Photoreceptors of mouse retinas possess D4 receptors coupled to adenylate cyclase. In the mouse, the light-sensitive pool of cAMP can be eliminated in the dark by application of the dopamine D2-like receptor agonists LY 171555 (quinpirole), (+)-N0437 (2-[N-(n-propyl)-N-2-(thienylethylamino)-5-hydroxytetralin]) , or (+)-3-PPP [3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride]. The rank-order affinity of the ability of the D2-like antagonists to block the action of LY 171555 matched that of the rat D4 receptor. Reverse transcription of retina mRNA followed by DNA amplification using D4-specific nucleotides demonstrates the presence of D4 mRNA in retina. In situ hybridization studies using D4-specific digoxygenin-labeled oligonucleotides or 35S-labeled UTP RNA probes demonstrate the presence of D4 mRNA in the photoreceptor cell layer and in the inner nuclear and ganglion cell layers. The modulation by D4 ligands of the dark level of light-sensitive cAMP in photoreceptors demonstrates the physiological coupling of the D4 receptor subtype.


Regulation of intrasteric inhibition of the multifunctional calcium/calmodulin-dependent protein kinase. A regulatory region involved in both autoinhibition and calmodulin (CaM) binding has previously been identified in the multifunctional Ca2+/CaM-dependent protein kinase (CaM kinase II). We have tested the role of various segments of the regulatory region in autoinhibition by the analysis of a series of truncation, substitution, and deletion mutants of the CaM kinase II alpha subunit (CaM kinase II alpha). Unexpectedly, the sequence Lys-Lys-Phe-Asn at positions 291-294, adjacent to the CaM binding domain, was found to be sufficient to maintain an inhibited state in a truncated form of the kinase. However, these residues are not essential in the context of the full-length protein, indicating the importance of additional residues from the overlapping CaM binding domain. We propose here a molecular model for CaM kinase II alpha based on the three-dimensional structure of the cAPK-PKI-(5-24) (protein kinase inhibitor fragment) complex. It is predicted from this model that autoinhibition is of the pseudosubstrate variety and that autophosphorylation of Thr-286 could occur by an intersubunit reaction in the holoenzyme complex.


A yeast protein phosphatase related to the vaccinia virus VH1 phosphatase is induced by nitrogen starvation. A phosphatase related to the vaccinia virus VH1 phosphatase has been cloned from Saccharomyces cerevisiae. The yeast phosphatase is related to the Schizosaccharomyces pombe cdc25 gene product and to a protein encoded by a mammalian open reading frame known as 3CH134, which is an immediate early gene responding to serum stimulation. The phosphatase activity of the yeast gene product appears to be restricted to the hydrolysis of phosphotyrosine-containing substrates, whereas the vaccinia phosphatase hydrolyzes both phosphoserine- and phosphotyrosine-containing substrates. The mRNA encoding the yeast phosphatase is dramatically induced by nitrogen starvation. Inactivation of the yeast phosphatase gene results in a decrease in growth rate.


Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic. Infection by certain human papillomavirus types is regarded as the major risk factor in the development of cervical cancer, one of the most common cancers of women worldwide. Analysis of the immunogenic and structural features of papillomavirus virions has been hampered by the inability to efficiently propagate the viruses in cultured cells. For instance, it has not been established whether the major capsid protein L1 alone is sufficient for virus particle assembly. In addition, it is not known whether L1, L2 (the minor capsid protein), or both present the immunodominant epitopes required for induction of high-titer neutralizing antibodies. We have expressed the L1 major capsid proteins of bovine papillomavirus type 1 and human papillomavirus type 16 in insect cells via a baculovirus vector and analyzed their conformation and immunogenicity. The L1 proteins were expressed at high levels and assembled into structures that closely resembled papillomavirus virions. The self-assembled bovine papillomavirus L1, in contrast to L1 extracted from recombinant bacteria or denatured virions, also mimicked intact bovine papillomavirus virions in being able to induce high-titer neutralizing rabbit antisera. These results indicate that L1 protein has the intrinsic capacity to assemble into empty capsid-like structures whose immunogenicity is similar to infectious virions. This type of L1 preparation might be considered as a candidate for a serological test to measure antibodies to conformational virion epitopes and for a vaccine to prevent papillomavirus infection.


Scorpion toxins targeted against the sarcoplasmic reticulum Ca(2+)-release channel of skeletal and cardiac muscle. We report the purification of two peptides, called "imperatoxin inhibitor" and "imperatoxin activator," from the venom of the scorpion Pandinus imperator targeted against ryanodine receptor Ca(2+)-release channels. Imperatoxin inhibitor has a M(r) of approximately 10,500, inhibits [3H]ryanodine binding to skeletal and cardiac sarcoplasmic reticulum with an ED50 of approximately 10 nM, and blocks openings of skeletal and cardiac Ca(2+)-release channels incorporated into planar bilayers. In whole-cell recordings of cardiac myocytes, imperatoxin inhibitor decreased twitch amplitude and intracellular Ca2+ transients, suggesting a selective blockade of Ca2+ release from the sarcoplasmic reticulum. Imperatoxin activator has a M(r) of approximately 8700, stimulates [3H]ryanodine binding in skeletal but not cardiac sarcoplasmic reticulum with an ED50 of approximately 6 nM, and activates skeletal but not cardiac Ca(2+)-release channels. These ligands may serve to selectively "turn on" or "turn off" ryanodine receptors in fragmented systems and whole cells.


Comparative inhibitory effects of dihydropyridines on platelet aggregation, calcium uptake and cyclic AMP concentration. We studied the in vitro effects of several calcium channel blockers from the dihydropyridine (DHP) family on platelet aggregation and endogenous serotonin secretion, calcium uptake and cyclic AMP (cAMP) concentration using washed rat platelets. We found that, after 1 min incubation, nifedipine (Nif), nitrendipine (Nit) and nisoldipine (Nis) inhibited the thrombin-induced platelet aggregation and serotonin secretion with IC50 of about 140, 5 and 2 mumol/l, respectively. Nis and Nit are thus much more active than Nif. We also found that the thrombin-induced Ca2+ uptake amounted to 2,600 +/- 326 pmol Ca2+/10(9) platelets in control conditions. In the presence of 10 mumol/l of the DHP, this uptake was decreased by 19, 49 or 77%, with Nif, Nit or Nis, respectively. Compound BAY K 8644 (BK) with known agonistic properties on the calcium channel had inhibitory effects on the studied parameters. These compounds were in the order of Nif < BK < Nit < Nis. When added to previously aggregated platelets, Nit caused them to deaggregate. These results seem to be similar to those obtained with cAMP analogues or adenylate cyclase activators. The platelet resting cAMP concentration was therefore measured in the presence of the DHP. A nonsignificant increase was found with 20 mumol/l Nif whereas significant increases of 20 and 68% as compared with controls were obtained with 20 mumol/l Nit and Nis, respectively. Partition studies between platelets and plasma lipoproteins indicated that the effects might be related to the lipophilicity of the compounds. These data suggest that these agents work on platelet activity by multiple effects located intracellularly or at the membrane level.(ABSTRACT TRUNCATED AT 250 WORDS)


Viral thymidine kinases and their relatives. Thymidine kinases were described for cellular life long before it was shown that they could also be encoded by viruses, but the viral thymidine kinase genes were the first to be sequenced. These enzymes have been extraordinarily useful to the researcher, serving first to help label DNA, then to get thymidine analogs incorporated into DNA for therapeutic and other purposes and more recently to move genes from one genome to another. Knowledge of the nucleotide and amino acid sequences of these enzymes has allowed some deductions about their possible three-dimensional structure, as well as the location on the polypeptide of various functions; it has also allowed their classification into two main groups: the herpesviral thymidine/eukaryotic deoxycytidine kinases and the poxviral and cellular thymidine kinases; the relationships of the mitochondrial enzyme are still not clear.


Diagnosis and treatment of the most common solid tumors in childhood. Childhood cancer is the leading cause of death from disease in children between 1 and 15 years of age. Since the introduction of modern multimodality therapy, the prognosis for solid tumors has improved dramatically, with 50% to 60% cure rates. Advances in cytogenetic and molecular genetic techniques are now being used for risk-based treatment in various tumors, including neuroblastoma and rhabdomyosarcoma. Further use of these research tools may lead to a better understanding of oncogenic molecular events, permitting treatment targeted to specific genetic lesions. This article focuses on the clinical features, treatment, and potential future research directions for this diverse group of diseases.


Pathogenicity of concurrent infection of pigs with porcine respiratory coronavirus and swine influenza virus. Combinations of porcine respiratory coronavirus (PRCV) and either of two swine influenza viruses (H1N1 or H3N2) were administered intranasally and by aerosol to six- to eight-week-old specific pathogen-free pigs. The clinical responses, gross respiratory lesions and growth performances of these pigs were studied and compared with those of single (PRCV, H1N1 or H3N2) and mock-infected animals. PRCV infection caused fever, growth retardation and lung lesions, but no respiratory symptoms. Infection with swine influenza viruses caused rather similar, mild symptoms of disease, with H1N1 infection being the least severe. Combined infections with influenza viruses and PRCV did not appear to enhance the pathogenicity of these viruses. Furthermore, viruses were isolated more frequently from tissues and nasal swabs taken from 'single' than 'dual' infected animals, suggesting a possible in vivo interference between replication of PRCV and swine influenza virus.


Serological analysis of a small herd sample to predict presence or absence of animals persistently infected with bovine viral diarrhoea virus (BVDV) in dairy herds. In 10 herds containing animals persistently infected (PI) with bovine viral diarrhoea virus (BVDV) and nine herds without such animals the probabilities of obtaining at least two antibody-positive animals in a test sample of three or five animals selected among animals six to 18 months old were calculated. Among herds with PI animals these probabilities, with the exception of one herd, varied between 0.725 and 0.992 when samples of three animals were tested and between 0.977 and one when samples of five animals were tested. Among herds without PI animals the probabilities varied between 0 and 0.015 when samples of three animals were tested and between 0 and 0.048 when samples of five animals were tested. Thus, based upon a few blood samples, herds with PI animals and herds without PI animals could be distinguished with a high degree of accuracy.


Comparison of cortisol concentrations in saliva and plasma of dogs. It is increasingly important to have simple, non-invasive indicators of stress in animals. Studies in various species have shown that concentrations of cortisol in saliva relate closely to plasma levels of the free hormone; the aim of the present procedure was to show a similar correlation in the dog. Baseline blood and saliva samples were collected concurrently from six male beagles. Synthetic adrenocorticotrophic hormone was given and further samples were collected at 0.25, 0.5, one, two and 2.5 hours later. The results indicated a statistically significant correlation between the levels of cortisol in blood and saliva. Concentrations in saliva were between 5 and 10 per cent of those in plasma at each collection time. To demonstrate a response to a more natural stimulus, saliva samples were taken from a dog during exposure to a known stressor for that individual. The results showed a marked, delayed increase from baseline which was maintained for at least 0.5 hours after stressing.


[Herpes-simplex encephalitis: case example, diagnosis and therapy]. Herpes simplex encephalitis (HSE) is the most common nonepidemic cause of acute viral encephalitis. Since successful therapy depends on a high level of suspicion that HSE is present and on the early administration of antiviral treatment, knowledge of clinical and laboratory findings of HSE is of great importance. The clinical hallmark of HSE are signs of both focal and diffuse neurologic involvement. Our case report exemplifies the diagnostic problems that can occur in HSE-patients. The validity of the different ancillary examinations is discussed. Up to the present time brain biopsy has been the method of choice for a reliable early diagnosis of HSE. In the foreseeable future early diagnosis is likely to become available in a non-invasive way by the polymerase-chain reaction. Immediate antiviral therapy with acyclovir in HSE has proved to be useful in rigorously controlled trials. The clinical picture of the acyclovir-induced encephalopathy represents a disorder that can be probably avoided by means of a sufficient hydration.


Adsorption of whole saliva onto hydrophilic and hydrophobic solid surfaces: influence of concentration, ionic strength and pH. The influence of the concentration of salivary proteinaceous material from solutions of whole saliva on the kinetics of in vitro pellicle formation were studied together with the effects of ionic strength, pH and certain substrate characteristics. The pellicle formation was monitored by an automated Rudolph ellipsometer, equipped with a He-Ne laser (wavelength 632.8 nm). The substrates compared in the study were hydrophilic negatively charged silica surfaces and hydrophobic methylated silica surfaces. The results show that the adsorption of salivary proteins is a very rapid process on both types of surfaces. Part of the formed biofilm, however, desorbed upon rinsing, indicating that the proteinaceous material was adsorbed with varying binding strengths. Larger adsorbed amounts were recorded on hydrophobic than on hydrophilic surfaces. Increase of ionic strength caused larger amounts to be adsorbed on both types of surfaces but change of pH did not affect the adsorption on either of the studied surfaces. Ellipsometry was found to be a suitable technique to monitor the adsorption of salivary proteins at solid/liquid interfaces.


Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and cytochrome c. The crystal structure of a 1:1 complex between yeast cytochrome c peroxidase and yeast iso-1-cytochrome c was determined at 2.3 A resolution. This structure reveals a possible electron transfer pathway unlike any previously proposed for this extensively studied redox pair. The shortest straight line between the two hemes closely follows the peroxidase backbone chain of residues Ala194, Ala193, Gly192, and finally Trp191, the indole ring of which is perpendicular to, and in van der Waals contact with, the peroxidase heme. The crystal structure at 2.8 A of a complex between yeast cytochrome c peroxidase and horse heart cytochrome c was also determined. Although crystals of the two complexes (one with cytochrome c from yeast and the other with cytochrome c from horse) grew under very different conditions and belong to different space groups, the two complex structures are closely similar, suggesting that cytochrome c interacts with its redox partners in a highly specific manner.


Epidemiologic data linking diet to hyperlipidemia and arteriosclerosis. There is little debate that an elevated plasma cholesterol level, specifically an elevated plasma LDL cholesterol level, increases cardiovascular disease risk. Data from inter- and intrapopulation studies have clearly demonstrated that as total and LDL cholesterol levels increase, cardiovascular disease risk increases. Although this relationship is generally accepted, the specifies of the relationship generate debate. Relevant questions pertain to the actual level of plasma cholesterol at which cardiovascular disease risk is increased, whether the relationship holds true across all age groups and both sexes, and what contributions plasma HDL levels and the plasma LDL/HDL ratio make to cardiovascular disease risk independent of plasma LDL levels. Irrespective of these uncertainties, the evidence that elevated plasma LDL cholesterol levels constitute an independent risk factor for cardiovascular disease has been a major component in studying the genetic and environmental factors involved in hypercholesterolemia. Epidemiologic data reveal relationships between a number of dietary elements and elevated plasma cholesterol levels with the strongest relationships between dietary fatty acids, plasma cholesterol levels, and cardiovascular disease incidence. The data from a variety of epidemiologic investigations, both cross-cultural and cross-sectional, indicate that plasma total cholesterol levels are increased by saturated fat intake and obesity. HDL cholesterol levels are decreased by intakes of low-fat, high-carbohydrate diets, a high BMI, and lack of activity and increased by intake of dietary fat, alcohol, and physical activity. Controlled clinical trials have provided verification of these epidemiologic observations in practically every case.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatic clearance of plasma chylomicron remnants. Dietary lipid, following incorporation into chylomicrons, is rapidly removed from the blood by a two-stage process. Most of the triglyceride is taken up by extrahepatic tissue, particularly muscle and adipose tissue. The residual triglyceride and virtually all of the cholesterol ester is removed by the liver through the clearance of a particle called a chylomicron remnant. The remarkable rapidity and specificity of uptake of this particle seems to be due to its acquisition of apoE in the plasma. Uptake is mediated in part by the LDL receptor, the LRP (alpha a-macroglobulin receptor), and perhaps by a sieving mechanism that leads to trapping, but not endocytosis. Uptake is modulated by the type of apoE inherited, the amount of apoC present on the particle, and, perhaps, the phospholipid and fatty acyl chain composition of the particle. The process may be slowed in diabetes and hypothyroidism. The metabolic effects of the particle can be variable, depending on the composition of the diet, and this can affect whole body cholesterol metabolism significantly. Furthermore, even moderately prolonged residence of these particles in the circulation could contribute in a significant way to atherogenesis. Thus, the remnant particle and its uptake by the liver may be important links in determining the dietary contribution to the rate of atherosclerosis.


Restoring control: empowering older patients and their families during health crisis. Acute health crises and the care provided within the health care system often reduce control for older patients and their families. Drawing on the authors' research with hip fracture patients and work with home health teams, the discussion focuses specifically on older patients who require home health care following a hip fracture and who have family support. Successful recovery and rehabilitation of the older patient is contingent upon restoring the patients' perception of control over their recovery. Empowerment is achieved both through the content of interventions, and through the process by which health professionals work with patients and their families.


Aspects of bone healing and bone substitute incorporation. An experimental study in rabbit skull bone defects. After an initial assessment of the experimental approach this experimental project was undertaken to study the regenerative response and incorporation of various implanted materials in calvarial bone defects in rabbits. Four 5-mm full-thickness defects were trephined in the frontal and parietal bones of the rabbits. After removal of the circular bone plugs the defects were used as recipient sites for inlay bone tissue and bone substitutes. In five separate studies the impact of bone regeneration of autogeneic bone grafts or eight bone substitutes were evaluated mainly by contact radiography, light microscopy and morphometry. The observation periods were four and 15 weeks. The main findings were: Autogeneic bone chips offered only minor advantages over controls in the model used and, also, differences in bone regeneration between diversified amounts of bone chips were negligible. In contrast, after bone paste implantation a cellular and mature bone was rapidly produced. Natural bone mineral (Bio-Oss) and synthetic dense hydroxylapatite ceramic proved to be biocompatible and a definite long term bone regeneration around all implants regardless of granulae size or resorbability was observed. Initial bone regeneration in and around the Bio-Oss particles was more extensive. Demineralized bone matrix of membranous and enchondral origins displayed extensive osteoinductive capacity and early bone production significantly exceeded that of control groups. The embryonic origin implied minor effects on the initial regenerative response only. Lyophilized bone allografts of two embryonic origins showed a low osteoinductive potential and a similar fashion of bone regeneration. No marked difference between these groups were displayed. HTR-polymer alone or combined with membraneous mineralized autogeneic bone chips showed a more rapid early bone regeneration than the groups containing lactomer beads, resorbable gel and controls. Also, the HTR-material proved to be a well-tolerated implant material by the recipient tissue bed.


Glucose regulation of synaptic transmission in the dorsolateral septal nucleus of the rat. Intracellular recordings were made from neurons in the dorsolateral septal nucleus (DLSN) of rat brain slices. Lowering the concentration of extracellular glucose resulted in a concentration-dependent membrane hyperpolarization associated with a cessation of spontaneous firing. The amplitude of the excitatory postsynaptic potential (EPSP), inhibitory postsynaptic potential (IPSP), and late hyperpolarizing potential (LHP) evoked by a single stimulus applied to the fimbrial/fornix pathway was decreased when the concentration of glucose was reduced to 0-2 mM. Substitution of glucose with 2-deoxy-D-glucose (11 mM), an antimetabolite of glucose substrate, mimicked the effects of glucose depletion. Mannoheptulose (10-20 mM), a potent hexokinase blocker, and dinitrophenol (50 microM), a potent inhibitor of oxidative phosphorylation, produced both the hyperpolarization and inhibition of postsynaptic potentials, even in the presence of 11 mM glucose. The sulphonylureas, glibenclamide (10 microM) and tolbutamide (1 mM), did not antagonize the hyperpolarization and the inhibition of the postsynaptic potentials produced by glucose depletion. The amplitude of membrane depolarizations produced by pressure application of glutamate (10 mM) and the membrane hyperpolarizations produced by pressure application of either muscimol (1 mM) or baclofen (1 mM) were almost unchanged, even when glucose was reduced to 1-2 mM. These results indicate that intracellular glucose metabolism regulates the function of septal neurons, not only by changing the resting membrane potential, but also by presynaptically affecting neurotransmission between the hippocampal formation and the lateral septum.