A contribution to the world selenium map.

Atomic absorption spectrophotometric method was used to determine the serum selenium levels of 86 healthy individuals. Variations in age, sex, and geographically different urban regions of Yugoslavia were investigated. A group of 63 healthy children, ages 8-15 yr, were examined. Mean +/- standard deviation of the serum selenium concentration was 57 +/- 9 micrograms/L; age and geographic area had no effect on the Se status of children, but the difference between boys and girls was significant (P less than 0.05). A group of 23 men from Zagreb, ages 22-37 yr, were examined. The group was divided into three age subgroups and no difference was found among these groups. The mean Se concentration was 69 +/- 18 micrograms/L, and a statistically significant difference was found only between the group of adults and the group of children (P less than 0.05).


Monitoring circulating B cells in patients with multiple myeloma at diagnosis or in plateau phase: how prevalent is light chain isotype suppression?

Peripheral blood lymphocytes from 25 patients with multiple myeloma at diagnosis or in plateau phase, and from 18 healthy subjects, were analysed for the surface expression of CD19 and immunoglobulin (Ig) light chain isotypes by dual parameter flow cytometry. The aim was to test for the existence of light chain isotype suppression (LCIS). LCIS is a hypothetical suppressor mechanism which supposedly operates in myeloma patients with stable disease, causing a drop in the number of circulating B cells bearing the same Ig light chain isotype as the myeloma paraprotein. Our experiments emphasized that the use of a non-Ig, pan-B cell marker, combined with dual antigen analysis, is necessary to distinguish Ig on B cells form (cytophilic) Ig on some non-B cells. We found good correlation between results obtained using polyclonal versus monoclonal anti-light chain antibodies, and washed blood versus peripheral blood mononuclear cells. There was no evidence of an abnormal ratio of CD19+, K+ to CD19+, lambda + cells in any of the patients. Thus, these patients did not manifest LCIS, and these experiments raise doubts about the existence of this phenomenon. The results also illustrate the problem that may arise in the interpretation of one parameter flow cytometry.


Selenium. Mechanistic aspects of anticarcinogenic action.

Selenium is increasingly recognized as a versatile anticarcinogenic agent. Its protective functions cannot be solely attributed to the action of glutathione peroxidase. Instead, selenium appears to operate by several mechanisms, depending on dosage and chemical form of selenium and the nature of the carcinogenic stress. In a major protective function, selenium is proposed to prevent the malignant transformation of cells by acting as a "redox switch" in the activation-inactivation of cellular growth factors and other functional proteins through the catalysis of oxidation-reduction reactions of critical SH groups of SS bonds. The growth-modulatory effects of selenium are dependent on the levels of intracellular GSH and the oxygen supply. In general, growth inhibition is achieved by the Se-mediated stimulation of cellular respiration. Selenium appears to inhibit the replication of tumor viruses and the activation of oncogenes by similar mechanisms. However, it may also alter carcinogen metabolism and protect DNA against carcinogen-induced damage. In additional functions of relevance to its anticarcinogenic activity, selenium acts as an acceptor of biogenic methyl groups, and is involved in the detoxification of metals and of certain xenobiotics. In its interactions with transformed cells at higher concentrations, it may induce effects ranging from metabolic and phenotypical changes, and partial renormalization to selective cytotoxicity owing to reversible or irreversible inhibition of protein and DNA synthesis. Selenium also has immunopotentiating properties. It is required for optimal macrophage and NK cell function. Its protective effects are influenced by synergistic and antagonistic dietary and environmental factors. The latter include a variety of toxic heavy metals and xenobiotic compounds, but they are also influenced by essential elements, such as zinc. The exposure to antagonistic factors must be minimized for the full expression of its anticarcinogenic potential.


Accidental selenium poisoning of growing pigs.

Chronic selenium (Se) toxicosis was diagnosed in two groups of growing pigs. Emaciation, loss of hair, necrotic areas in the skin, lesions of the coronary band and hooves, postnecrotic atrophic cirrhosis of liver, and lumbal poliomyelomalacia were the principal findings. High Se concentrations were detected in blood plasma. Addition of the calculated amounts of sodium selenite directly to the feedstuff instead to mineral premix was the cause of this intoxication.


Effects of anti-TNF monoclonal antibody infusion in patients with hairy cell leukaemia.

Tumour necrosis factor (TNF) can act as an autocrine growth factor for hairy cell leukaemia (HCL) cells. The TNF produced by the malignant clone may also inhibit normal haematopoiesis thereby contributing to the cytopenias observed in patients with the disease. We have studied the effects of infusing a murine monoclonal anti-TNF antibody in three patients with HCL. In two patients receiving 0.5 mg of antibody/kg on alternate days for 12 d, the drug was well tolerated. The third patient received 2 mg/kg on alternate days and developed symptoms of serum sickness by day 9. In two patients with severe B-lymphocytopenia, circulating CD19 and CD20 positive, B-cells were restored to normal, the majority of which were negative for the HCL-associated marker CD11c. B-lymphocyte recovery was associated with a rise in serum immunoreactive IL-6 and with an early rise in immunoreactive TNF. These short courses of anti-TNF MAb treatment had modest effect on the tumour burden, producing a reduction in splenomegaly in one patient. Exploration of the effects of more prolonged administration of higher dose anti-TNF antibody will only be feasible when less immunogenic MAbs are available.


Selenium status of patients with Balkan endemic nephropathy.

The selenium (Se) contents in common cereals in endemic and nonendemic areas in Serbia are very low. Plasma Se levels of both patients and healthy subjects, were also low, reflecting low Se intakes. Patients with Balkan endemic nephropathy (BEN) had significantly lower (p less than 0.05) plasma Se levels than healthy individuals, both from regions close to endemic areas, and from Belgrade. Mean plasma Se of BEN patients was slightly but insignificantly higher in samples taken immediately after dialysis than in those taken before, suggesting that very little of the Se present in plasma is dialyzable. Plasma SeGSH-Px activities before and after hemodialysis in both BEN and Nonendemic chronic renal failure (NCRF) patients were not significantly different, but BEN patients had lower enzyme activities than those with NCRF and healthy controls. In BEN patients, a significant correlation between plasma Se and SeGSH-Px activity was found.


Macrophage nitric oxide synthase: relationship between enzyme-bound tetrahydrobiopterin and synthase activity.

Nitric oxide synthase (NOS) (EC 1.14.23) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. The complex reaction carried out by NOS, which involves NADPH, O2, and enzyme-bound FAD, FMN, and tetrahydrobiopterin (BH4), has only recently begun to be elucidated. Herein we report the characterization of the pterin requirement of murine macrophage NOS. Although purified NOS activity was not dependent on BH4, activity was significantly enhanced by BH4 in a concentration-dependent fashion. NOS purified in the absence of added BH4 was found to contain substoichiometric concentrations of enzyme-bound pterin, where increased concentrations of bound pterin correlated with an increase in activity when assayed in the absence of exogenous BH4. However, NOS purified in the presence of BH4 followed by gel filtration exhibited a 1 mol of pterin:1 mol of NOS 130-kDa subunit stoichiometry and activity that was essentially independent of exogenous BH4. Experiments to probe a redox role for the pterin were carried out using pterin analogues. 6(R,S)-Methyltetrahydropterin was found to increase NOS activity in enzyme purified in the absence of BH4. However, the deaza analogue, 6(R,S)-methyl-5-deazatetrahydropterin, was not only incapable of supporting enzymatic turnover but also inhibited citrulline formation in a concentration-dependent manner. Overall, these results support a role for BH4 in the NOS reaction that involves stabilization of the enzyme and redox chemistry wherein a 1:1 stoichiometry between bound pterin and NOS subunit results in maximum activity.


A calcium and ATP sensitive nonselective cation channel in the antiluminal membrane of rat cerebral capillary endothelial cells.

The patch-clamp technique was applied to the antiluminal membrane of freshly isolated capillaries of rat brain (blood-brain barrier). With 1.3 mM Ca2+ in the bath, excision of membrane patches evoked ion channels, which could not be observed in cell-attached mode. The channel was about equally permeable to Na+ and K+ ions, but not measurable permeable to Cl- and the divalent ions Ca2+ and Ba2+. The current-voltage curve was linear in the investigated voltage range (-80 mV to +80 mV), and the single-channel conductance was 31 +/- 2 pS (n = 22). The channel open probability was not dependent on the applied potential. Lowering of Ca2+ to 1 microM or below on the cytosolic side inactivated the channels, whereas addition of cytosolic ATP (1 mM) inhibited channel activity completely and reversibly. The channel was blocked by the inhibitor of nonselective cation channels in rat exocrine pancreas 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10 microM) and by the antiinflammatory drugs flufenamic acid (greater than 10 microM) and tenidap (100 microM), as well as by gadolinium (10 microM). Thus, these nonselective cation channels have many properties in common with similar channels observed in fluid secreting epithelia. The channel could be involved in the transport of K+ ions from brain to blood side.


Modulation of the platelet thromboxane A2 and aortic prostacyclin synthesis by dietary selenium and vitamin E.

Vitamin E and selenium (Se) interact synergistically as an important antioxidant defense mechanisms. Se, an essential component of glutathione peroxidase (GSH-Px) and vitamin E decompose fatty acid hydroperoxides and hydrogen peroxides generated by free radical reactions. Vitamin E and GSH-Px may modulate arachidonic acid metabolism and the activity of cyclooxygenase enzymes by affecting peroxide concentration. The balance between arterial wall prostacyclin (PGI2) production and platelet thromboxane (TX)A2 directly influences platelet activity. In order to elucidate the differential role of dietary vitamin E and Se in aortic PGI2 and platelet TXA2 synthesis, 1-mo-old F344 rats were fed semipurified diets containing different levels of vitamin E (0, 30, 200 ppm) and Se (0, 0.1, 0.2 ppm) for 2 mo. Thromboxane B2 (TXB2) and 6-keto-PGF1 alpha, were measured by radioimmunoassay (RIA) after incubation of whole blood and aortic rings at 37 degrees C for 10 and 30 min, respectively. Vitamin E deficiency reduced plasma vitamin E to 5-17% of control-fed rats, and supplementation in vitamin E-supplemented animals increased plasma GSH-Px by 17%, compared to vitamin E-deficient rats. Se and vitamin E supplementation did not have a similar effect on TXB2 and PGI2 synthesis. Se deficiency did not alter platelet TXB2 synthesis, but significantly decreased aortic PGI2 synthesis. It was necessary to supplement with both antioxidants in order to increase PGI2 synthesis. Se and vitamin E deficient groups had a higher TXB2/PGI2 ratio (0.17 +/- 0.08) compared to Se- and vitamin E-supplemented groups (0.03 +/- 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


High-affinity, equilibrative nucleoside transporter of pig kidney cell line (PK-15).

Nucleoside transport was determined in the cloned porcine kidney cell line PK-15 which exhibits properties of tubular cells. The cells did not express any Na(+)-dependent, concentrative nucleoside transport. They exhibited only nitrobenzylthioinosine-sensitive equilibrative nucleoside transport. Their transport activity, however, was only about 10% of that observed in many other mammalian cell lines. This low transport activity correlated with a relatively low number of high-affinity nitrobenzylthioinosine binding sites (5 x 10(3)/cell). Furthermore, although the equilibrative transporter of PK-15 cells exhibited a similar broad substrate specificity as the equilibrative nucleoside transporters of other mammalian cells, it exhibited a much higher affinity for certain nucleosides, especially cytidine and uridine, than the latter. The Michaelis-Menten constants for zero-trans transport and equilibrium exchange of uridine in ATP-depleted cells were about the same (about 40 microM), indicating equal mobility of the nucleoside-loaded and empty carrier. Concentrative nucleoside transport was detected in one set of PK-15 cultures, but was found to be due to mycoplasma contamination.


Monoclonal antibodies against defined epitopes of the human transferrin receptor cytoplasmic tail.

Three murine monoclonal antibodies (mAbs) against the 61-residue amino-terminal cytoplasmic tail of the human transferrin receptor (TR) have been produced by immunization of mice with recombinant human TR produced in a baculovirus expression system. Mutant human TRs expressed in chick embryo fibroblasts (CEFs) with point mutations or deletions in their cytoplasmic tails have been used to map the epitopes defined by each of the mAbs. One mAb, H68.4, previously shown to block receptor internalization, binds proximal to the carboxy-terminal side of the YTRF internalization signal of TR. The second mAb, H73.2, binds near to the carboxy-terminal side of the H68.4 epitope, whereas the third mAb, 160.1, binds closer to the transmembrane region. H68.4 and H73.2 are auto-antibodies consistent with their epitopes mapping to a region of the human TR that has an identical amino acid sequence to the mouse TR. All three mAbs crossreact with the cytoplasmic tail of Chinese hamster TR. Double labelling of recombinant human TRs on chick embryo fibroblast (CEF) cell membrane preparations with B3/35 and H68.4 antibody-gold conjugates established that receptors in clathrin-coated pits were not labeled with H68.4, implying that associated coated pit proteins may block binding of this mAb.


Inhibitors of protein and RNA synthesis block the cytotoxic effects of oxygenated sterols.

Oxygenated derivatives of cholesterol are known to exhibit potent cytotoxic effects against many different cell types. The cellular basis of this cytotoxicity is not understood. Using two murine cancer cell lines (the EL4 lymphoma and the K36 leukemia cell line) and two oxygenated sterols (7-ketocholestanol and 25-hydroxycholesterol), our laboratory attempted to determine whether the cytotoxic action of oxysterols was mediated by a mechanism requiring protein or RNA synthesis. The addition of 5 microM 7-ketocholestanol or 25-hydroxycholesterol to the culture medium regularly caused the viable cell count to fall below 10-20% of control within 48-72 h. In the presence of inhibitors of protein or RNA synthesis, however, cell viability was consistently and significantly increased in a dose-dependent manner. For cultures of EL4 cells grown in the presence of 5 microM 7-ketocholestanol, for example, the addition of appropriate concentrations of cycloheximide, puromycin, emetine, and actinomycin increased the percentage of viable cells from a control value of less than 6% to 66%, 28%, 76% and 42%, respectively. Qualitatively similar results were obtained with the K36 cell line. Additional studies revealed that macromolecular synthesis inhibitors, while effective in inhibiting protein or RNA synthesis to varying degrees, did not affect the cellular uptake of 7-keto[3H]cholestanol, suggesting that their ability to protect cells against oxysterol-induced cytotoxicity was not due to an inhibition of the cellular oxysterol uptake. These observations suggest that the cytotoxicity of oxygenated sterols may be mediated by mechanisms requiring de novo protein or RNA synthesis and that oxysterol-induced cytotoxicity may provide a useful system for the identification of proteins involved in cell death.


Correction of the apical membrane chloride permeability defect in polarized cystic fibrosis airway epithelia following retroviral-mediated gene transfer.

We are studying the introduction and expression of the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA into cultured human airway epithelial cells as a model for gene therapy of cystic fibrosis. In this paper, we show that the chloride transport defect at the apical membrane is corrected in vitro in differentiated ion-transporting CF airway epithelial cells that exhibit polarized properties similar to those found in vivo. Using a retroviral vector containing a copy of the normal CFTR cDNA, we infected cultures of proliferating, cystic fibrosis CFT1 cells and found that correction was maintained following differentiation into a polarized epithelial sheet. At least partial correction of the Cl- transport defect was preserved in CFT1 cells for periods of up to 6 months without selection for maintenance of the retroviral provirus. These results suggest that it may be feasible to target proliferating cells in the lung using retroviral vectors for treatment of CF lung disease.


Haplotypes in SS patients from Nigeria; characterization of one atypical beta S haplotype no. 19 (Benin) associated with elevated HB F and high G gamma levels.

We have determined the haplotypes of 669 beta S and 109 beta A chromosomes from numerous members of 297 Nigerian families of various ethnic backgrounds. Among the beta S chromosomes, haplotype 19 was detected in 93.2%, haplotype 17 in 3.4%, and haplotype 20 in 0.1%, while 2.4% represented atypical haplotypes. As many as 60.6% of the beta A chromosomes exhibited haplotype 19 mutations, 8.2% had haplotype 3, and 1.8% had haplotype 20. Two siblings with elevated Hb F and G gamma levels were heterozygous for a beta S chromosome with haplotype 19 and a second chromosome with a hybrid haplotype (termed 19 B). In this hybrid chromosome, haplotype 3-like locus control region (LCR) [hypersensitive site-2 (HS-2)] sequences are in juxtaposition to those of the 5' flanking region of the G gamma promoter of a beta S chromosome with haplotype 19. The presence of this hybrid chromosome is associated with high G gamma values in individuals with both sickle cell anemia (SS) and sickle cell trait (AS); it closely resembles another hybrid beta S chromosome, termed 19 A, observed in a previously reported Turkish SS patient who was homozygous for this chromosome and had high Hb F and high G gamma values. In both instances, it is hypothesized that the haplotype 3-like sequences of the LCR HS-2 contain genetic determinants that can combine with factors produced during hematopoietic stress, resulting in increased gamma-globin gene expression.


Serum selenium level in patients with colorectal cancer.

Serum selenium levels were determined by fluorometric procedure in 37 patients of both sexes suffering from colorectal cancer. The diagnosis was verified with histopathological examination during surgical treatment. The values found were 46.8 +/- 11.2 micrograms/L. The control group consisted of 230 healthy persons from the same environment as the group of patients. The values found were 64.2 +/- 11.5 micrograms/L. The results of this study are compared with the results of the other research groups analyzing the level of selenium in colorectal cancer.


Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on circulating platelets.

The effect on platelets of rhG-CSF was studied in 20 healthy volunteers with the thrombometer, a specially developed device which is described in detail. Additionally, conventional aggregation tests were performed. Low doses of rhG-CSF enhance functional platelet activity, as shown by significant acceleration of the occlusion of the thrombometer channel. Similar results were found in conventional aggregation tests utilizing collagen for induction. At G-CSF concentrations of 0.1 and 1.0 ng/ml the time to response was significantly accelerated and the maximum response was observed in a higher proportion of platelets. However, the second phase of aggregation induced by epinephrine was significantly inhibited by 1.0 ng/ml G-CSF. The activation of platelets may be beneficial in thrombocytopenia, but it may also increase platelet turnover and platelet loss. Further investigation is needed to clarify the mechanism by which G-CSF exerts its effects on platelets.


Serum selenium and glutathione-peroxidase activities and their interaction with toxic metals in dialysis and renal transplantation patients.

Selenium, aluminum, cadmium, and magnesium concentrations and glutathione-peroxidase activities in sera of 35 healthy individuals, 30 renal transplants, and 30 hemodialysis patients were measured. Serum selenium, aluminum, and cadmium concentrations in both groups of patients were higher than the controls (p less than 0.001), whereas the serum glutathione-peroxidase levels were lower (p less than 0.001). According to our results, it can be concluded that the patients receiving hemodialysis are subjected to more toxic elements than the transplantation patients. These findings imply that dietary selenium supplement may be suggested in renal failure for the detoxification of elements, such as cadmium and mercury. The essential trace element selenium takes part not only in the direct protection of endothelial cells against the accumulation of aggressive oxygen species, but also in the prevention of the toxic effects of cadmium or in the modulation of the active calcium transport.


Renewal and differentiation of keratinocytes cultured on dead de-epidermalized dermis.

Human keratinocytes grown at an air-liquid interface on dead de-epidermalized dermis exhibit a pattern of organization similar to that seen in vivo. Cell renewal is limited to the basal layer. The cell cycle time determined after 7 days of culture, using a percentage labelled mitoses (PLM) technique, was about 15 h. This result is comparable with published data for cultivated keratinocytes but is shorter than the parameter proposed for epidermis in vivo. Appearance of labelled cells in the granular layer was observed 4 days after pulse labelling. Despite this high cell renewal, a normal cell differentiation with expression of various keratinization markers was maintained.


Prostacyclin-mediated protection by angiotensin-converting enzyme inhibitors against injury of aortic endothelium by free radicals.

We report here the protective effect of the angiotensin converting enzyme inhibitors captopril and ramiprilat against damage due to oxygen free radicals on aortic endothelium in a superfusion cascade system. Oxygen free radicals were generated by electrolysis of Krebs solution. Acetylcholine was infused through the donor aortic segment and relaxation of the detector aortic ring was used to indicate the release of endothelium-derived relaxing factor (EDRF). The percentage of relaxation before and after electrolysis was compared to calculate the relaxation index. Electrolyzed Krebs solution impaired the release of EDRF, as shown by a reduction in the relaxation index with a concomitant decrease in the tissue levels of superoxide dismutase and 6-keto PGF1 alpha and increase in malondialdehyde in the donor vessel. Captopril (100 microM), 15 microM ramiprilat and 30 nM iloprost had a protective effect as shown by a significantly smaller reduction in the relaxation index and the level of 6-keto PGF1 alpha and by an attenuation of the production of malondialdehyde. In addition, 1 microM indomethacin almost eliminated the protection by captopril. We conclude that both the SH-containing angiotensin-converting enzyme inhibitor captopril and the non-SH-containing ramiprilat, as well as iloprost, a stable analog of prostacyclin, can protect rabbit aortic endothelium against damage due to oxygen free radicals. The mechanism of such protection may be partly associated with the facilitation of the release of prostacyclin and consequent reduction in lipid peroxidation.


Ionic currents in ventricular myocytes isolated from the heart of a patient with idiopathic cardiomyopathy.

Whole-cell configuration of the patch-clamp technique was applied to record ionic currents from human ventricular myocytes isolated from a cardiac transplant patient with idiopathic cardiomyopathy. Inward calcium current, transient outward current, and inward rectifier potassium current were recorded, while no discernible delayed rectifier potassium current was observed. Thus the currents that underlie electrical activity in human ventricular myocytes appear to resemble those reported earlier in canine and rabbit ventricular myocytes.


Ion channels for communication between and within cells.

The development of patch-clamp procedures for measuring single-channel current fluctuations are described. The application of these techniques for studying secretion is discussed.


A predictive model for substrates of cytochrome P450-debrisoquine (2D6).

Molecular modeling techniques were used to derive a predictive model for substrates of cytochrome P450 2D6, an isozyme known to metabolize only compounds with one or more basic nitrogen atoms. Sixteen substrates, accounting for 23 metabolic reactions, with a distance of either 5 A ("5-A substrates", e.g., debrisoquine) or 7 A ("7-A substrates", e.g., dextromethorphan) between oxidation site and basic nitrogen atom were fitted into one model by postulating an interaction of the basic nitrogen atom with a negatively charged carboxylate group on the protein. This acidic residue anchors and neutralizes the positively charged basic nitrogen atom of the substrates. In case of "5-A substrates" this interaction probably occurs with the carboxylic oxygen atom nearest to the oxidation site, whereas in the case of "7-A substrates" this interaction takes place at the other oxygen atom. Furthermore, all substrates exhibit a coplanar conformation near the oxidation site and have negative molecular electrostatic potentials (MEPs) in a part of this planar domain approximately 3 A away from the oxidation site. No common features were found in the neighbourhood of the basic nitrogen atom of the substrates studied so that this region of the active site can accommodate a variety of N-substituents. Therefore, the substrate specificity of P450 2D6 most likely is determined by the distance between oxidation site and basic nitrogen atom, by steric constraints near the oxidation site, and by the degree of complementarity between the MEPs of substrate and protein in the planar region adjacent to the oxidation site.(ABSTRACT TRUNCATED AT 250 WORDS)


The respiratory sensation at high altitude.

The respiratory sensation was studied in Nepal at four different altitudes, 1377 m before and after the ascension, 2800 m, 3900 m and 530 m. Dyspnea was noted at each altitude for the nine subjects. They had to rate 4 external resistive loads between 2.5 and 13 cm H2O.l-1.s, presented in 2 pairs, a low and a high one. The discrimination between the loads i.e. the subject's sensitivity was obtained from Sensory Decision Analysis. These subjects were compared to six normal ones observed at sea level while breathing air, an hypoxic mixture (FIP2:11%) and air in a cold environment (-6 degrees C). In these protocols, the load perception was not modified. The 2 populations exhibited a similar sensitivity when observed in normal conditions. At exertion and with altitude, the nine subjects demonstrated a progressive increase in dyspnea, rated with visual analog scales. At rest, the perception of the loads was not altered but slightly improved with altitude for 6 subjects. The other 3 subjects (2 subjects with clinical impairment, important dyspnea and pulmonary oedema) showed an impairment of the perception. The sensitivity to the loads was similar before and after the ascension for the well adapted subjects to altitude. In conclusion, the respiratory sensation is not impaired with altitude in well adapted subjects and transient hypoxia does not result in change in load perception. An impairment in load perception observed in some subjects is probably related to the secondary effects of chronic hypoxia, i.e. cerebral and/or pulmonary suboedema.


Changes in microsomal phospholipid fatty acids composition in cholestatic rats.

In the rat, the effect of the bile duct ligation on liver microsomal phospholipid fatty acid composition and on phosphatidylcholine, phosphatidylserine and phosphatidylinositol pattern were studied. After two days of cholestasis, microsomal phospholipid fatty acids showed a decrease in linoleic, stearic and arachidonic acids and an increase in oleic and docosahexaenoic ones, as compared to controls. Phosphatidylcholine showed an increment in oleic and palmitic acid content and a concomitant decrease in arachidonic acid. Phosphatidylserine showed a progressive increase while phosphatidylinositol showed a progressive decrease in all fatty acids. Eight-days post-cholestatic rats showed a marked increase in oleic acid, whereas linoleic, arachidonic, stearic and palmitic acids concentration decreased. Phosphatidylcholine showed a global decrease in its fatty acid content, except for oleic which is increased. Phosphatidylserine showed an increase over the two-days cholestasis fatty acids values. Phosphatidylinositol decreased in most fatty acids except in docosahexaenoic acid that recovered normal values. It was concluded that cholestasis produced significative changes in the fatty acid composition of the major phospholipids constituents of the microsomal membranes.


The neonatal testosterone surge: a comparative study.

In the rat an abrupt discharge of testicular testosterone in the newborn male figures prominently in the development of mechanisms controlling gonadotropin secretion, sexual behavior, and also promotes the functional differentiation of the accessory sex glands. In this study we detail the temporal characteristics of this surge in the rat, and we provide comparative data documenting a similar surge-like appearance of testosterone in neonatal male mice, recently foaled male horses, and newborn human infants. Although the physiological and behavioral significance of this phenomenon for species other than the rat remains to be determined, the apparently ubiquitous appearance of the neonatal testosterone surge suggests that it may be of special significance in the sexual differentiation of many mammalian species.


Cardiovascular and hormonal responses to ANP infusion in the guinea-pig: effects of angiotensin-converting enzyme inhibition with perindopril.

In the guinea-pig, perindopril inhibited plasma angiotensin converting enzyme (ACE) by 90% when given orally at 2 mg/kg/day during 10 days. Mean blood pressure and plasma aldosterone, cortisol and vasopressin concentrations were not modified by this treatment, while plasma renin activity (PRA) and plasma angiotensin I concentrations increased significantly. The same parameters were studied using a constant intravenous 30 min-infusion of atrial natriuretic peptide (ANP) (0.1 micrograms.kg-1min-1). This dose of ANP infused to anesthetized guinea-pigs induced a significant decrease in mean blood pressure (about -20%) in control and in perindopril treated animals. In ANP infused animals, plasma aldosterone and cortisol concentrations decreased similarly in both groups by about -50%, whereas plasma vasopressin concentrations increased in controls (+169%) but not in perindopril treated guinea-pigs. An increase in PRA and plasma angiotensin I concentrations was observed in both groups after the infusion of ANP. Thus, when ANP demonstrated an potent hypotensive effect a concomitant increase in PRA occurred. The rise observed in vasopressin concentration in control animals was probably mediated by angiotensin II. The fall in plasma aldosterone and cortisol concentrations observed after ANP infusion demonstrated a direct potent action of ANP at the adrenal levels.


Dietary sucrose supplementation fails to modify fat deposition in lean or obese rats.

The effects of sucrose supplementation on body composition and heat production were studied in lean, dietary (cafeteria diet) and genetically (Zucker fa/fa) obese adult (60 days) rats. Sucrose supplement (29 kJ) for 10 days did not result in significant changes in the pattern of energy (fat) deposition or carcass composition. There were no alterations, either, in heat production measured by direct calorimetry. Under the conditions studied, sucrose intake did not affect lipid deposition or thermogenesis.


Induction in Gallus domesticus of experimental hypercholesterolemia by saturated fat. Effects on cholesterogenic enzyme activity.

The effect of coconut oil supplementation to the diet (10 or 20%) on lipid levels in plasma and liver as well as on the cholesterogenic enzyme activity were studied in 14-day-old chicks. Treatments for 1 or 2 weeks did not interfere in the growth rate of animals nor in the liver weight. The 10% coconut oil group showed a significant increase of plasma cholesterol after 2 weeks of treatment, while after 1 week the increase was not statistically significant. The 20% coconut oil group increased plasma cholesterol from the first week. Triacylglycerol content increased after each coconut oil supplementation to the diet during the first week. Hepatic cholesterol did not change significantly after any treatment assayed. No significant difference was observed in the cholesterogenic activity, measured as hepatic 3-hydroxy-3-methylglutaryl-CoA reductase, so that this study provides a perfect model of hypercholesterolemic animals without changes in their cholesterogenic ability.


Tissue composition in persistent dietary obesity after early and adulthood overfeeding in the rat.

The objective of this study was to assess the effects of prolonged cafeteria-diet feeding on tissue composition in adult rats comparing those that had been overfed in early life and then in adulthood with a group that was only overfed in adulthood, and to determine whether any alterations were related to the high energy diet per se or to obesity. In addition to following the body weight changes in detail, tissue masses and composition were determined at selected points of this long term dietary experiment. The marked changes in body weight and tissue composition of cafeteria-fed obese rats were sustained for at least 84 days after returning to the standard diet, and the obesity was exaggerated if these animals were pre-exposed to the palatable diet in early life. Three patterns of tissue composition in response to cafeteria feeding could be discerned: Liver and brown adipose tissue developed cell hypertrophy without apparent hyperplasia. In contrast, the retroperitoneal white adipose depot developed hyperplasia whereas the intestine and kidneys were not associated with marked changes in lipid composition. These adaptations were not recovered to control levels by prolonged standard diet feeding. Previous obesity influenced the adaptations in adipose tissue during the subsequent return to standard diet feeding, and differences between cafeteria-induced obese animals became more apparent when the cafeteria diet was removed. These results indicated the important effects of early dietary experience in subsequent responses to overfeeding.


Effect of starvation on the in vivo intestinal absorption of sugars and amino acids in young chickens (Gallus domesticus).

The effect of different patterns of starvation (acute and intermittent) on the in vivo intestinal absorption of glucose and tryptophan during the first days of Gallus domesticus chicks life was measured. Both acute and intermittent starvation increase duodenal absorption of glucose and jejunal absorption of tryptophan. Intermittent starvation tends to reduce the effect of age which normally decreases intestinal uptake of nutrients. This effect is clearer for glucose absorption than for tryptophan absorption.


[Changes in lymph microcirculation in appendicular peritonitis].

When studied in experiment (63 dogs) and in clinical settings (116 patients), microlymphatic channel in appendicular peritonitis (AP) was found impaired, while lymphatic drainage of large-molecular substances and metabolites coming from the abdominal cavity appeared activated. Methods of correction of AP-induced abnormalities in microlymphocirculation have been developed which imply three levels of the impact: microcirculation, affected organ, lymphatic system. The clinical trial of the methods proved their efficacy even in the most severe AP forms.


Hormonal induction of c-fos and HSP68 mRNAs on an isolated coronary perfused adult rat heart.

Transient growth signals which can be related to protein synthesis and cellular growth are of particular interest in the heart because of the incidence of cardiac hypertrophy in man. The isolated coronary perfused adult rat heart or the so-called Langendorff preparation, is an useful model in exploring not only protein synthesis but also c-fos/c-myc protooncogene and Heat Shock Protein (HSP) gene expression. Phenylephrine infusion in this preparation induces c-fos expression whether the heart is beating or reversibly or irreversibly arrested by solutions enriched in KCl. Norepinephrine has the same effect. Quantitative analysis with slot blots shows that in both cases the adrenergic effect has a dual origin since it is inhibited both by propranolol, a beta-adrenergic antagonist, and terazosine, a soluble alpha 1-adrenergic antagonist. We conclude that the isolated heart is a useful tool to explore the early changes in gene expression which occur in this tissue in response to various physiological stimuli.


GABAA receptor channels: from subunits to functional entities.

GABAA receptor channels mediate postsynaptic inhibition. The functional diversity of these receptors rests on differences in subunit composition and on a large repertoire of subunits. Subunit expression patterns in the brain have been found to predict in vivo compositions of GABAA receptors. In addition, molecular determinants underlying the differential binding properties of allosteric ligands to receptor subtypes have been identified.


Influence of thyroid hormone deficiency in DNA contents in different areas of adult rat brain.

We have studied DNA concentrations in various regions of the adult rat brain in prolonged hypothyroidism of different origin. In both normal and hypothyroid rats hypophysis, cerebellum and hypothalamus contained the highest concentrations of DNA. Hypothyroidism produced a slight decrease in DNA content but this effect varied according to the different regions. These variations were due to changes in their maturation period, a critical and specific target for thyroid hormone action after birth.


Human thymic epithelial cells in serum-free culture: nature and effects on thymocyte cell lines.

Thymic epithelial cells (TEC) have been cultured for several months and/or for 4 to 5 transfers in a growth factor-defined serum-free medium without concurrent growth of other cell types. The use of monoclonal antibodies and alpha MAM-6 indicated that the majority of TEC were of medullary origin. The vast majority of cells were positive for LFA-3 and class I, and class II expression was low or absent. Supernatants from the cultures were shown to contain IL-1 beta, IL-6, and M-CSF. Coculture of cloned subpopulations of thymocytes and TEC showed effects of TEC and of secreted ILs on thymocyte proliferation. High percentages of TEC were able to bind DN, DP, or SP thymocyte populations, partly via CD2-LFA-3 adhesion. Thus, it is possible to culture TEC without unknown serum factors and with maintenance of functional activities.


Effect of naloxone-reversible immobilization stress on the adrenal acetylcholinesterase activity in mice.

In male mice immobilized during 30 min, statistically significant increase in adrenal acetylcholinesterase (Ache) activity--up to 154% of control value--has been observed. Naloxone pretreatment (10 mg/kg b.w., ip.) suppressed that increase, Ache activity remaining at the level of non-immobilized, saline treated mice. This suggests the role of opioid peptides in this change. Ganglionic blockade by hexamethonium markedly inhibited Ache activity in adrenals of non-immobilized and of immobilized mice. Opioid peptides secreted from the splanchnic nerve terminals seem to be involved in the control of cholinergic mechanisms.


Biochemical characterization of particles from rat striatum containing dopamine, cholecystokinin or both.

Cholecystokinin (CCK) containing particles were isolated from rat striatum. After differential and isopycnic sucrose density gradient centrifugation, CCK containing particles equilibrated around 1.2 M sucrose. Dopamine (DA) containing particles equilibrated around the same buoyant density, but the colocalization of a cytosolic as well as a mitochondrial enzyme marker and the low enrichment at this density indicated an incomplete isolation of CCK containing particles from other subcellular organelles. Alternatively, differential centrifugation and gel filtration on Sephacryl S-1000 did yield advanced isolation of CCK containing vesicles. These vesicles also contain particle-bound DA. During hypo-osmotic lysis, these CCK and/or DA containing particles behaved like synaptic vesicles. Therefore, it was concluded that CCK containing vesicles from rat corpus striatum were indeed isolated and characterized and that the same or similar vesicles contain DA as well as CCK.


A striational muscle antigen and myasthenia gravis-associated thymomas share an acetylcholine-receptor epitope.

The coincidence of autoantibodies against the acetylcholine receptor (AChR) and muscle striational antigens (SA) is a characteristic finding in thymoma-associated myasthenia gravis (MG), but their origins are still unresolved. Some common muscle antigens that were shown to be targets of anti-SA autoantibodies in thymoma-associated MG have also been detected in normal or neoplastic thymic epithelial cells, suggesting that the release of (eventually altered) antigens from the thymic tumors could elicit SA autoimmunity. In contrast to this model, we report here that titin, which is a recently reported target of SA autoimmunity, is not expressed in thymomas. In addition, we show that skeletal muscle type-II fibers exhibit a striational immunoreactivity with monoclonal antibody mAb155, which was previously identified to label a very immunogenic cytoplasmic epitope of the AChR and neoplastic epithelial cells of MG-associated thymomas. We conclude from these findings that titin autoimmunity in thymoma-associated MG is either due to a molecular mimicry mechanism involving tumor antigens (other than titin) or is a secondary phenomenon following release of titin from muscle. Based on the common immunoreactivity of the AChR, a striational antigen and thymoma, we suggest as the pathogenetic mechanism of thymoma-associated MGa "circulus vitiosus" in which SA autoimmunity could help maintain the AChR autoimmunity that is primarily elicited by the thymomas.


Antigenic analyses of Sugi basic protein by monoclonal antibodies: I. Distribution and characterization of B-cell-tropic epitopes of Cry j I molecules.

Using 23 monoclonal antibodies raised against Sugi basic protein (SBP, major allergen of Japanese cedar pollen), composed of Cry j I and Cry j II, analyses of B-cell-tropic epitopes of Cry j I were performed. The following results were obtained. (1) As far as the mAbs were used, no major cross-reactive determinants were detected between Cry j I and Cry j II molecules. (2) 21 of the 23 mAbs were specific for Cry j I, and the anti-Cry j II mAbs were classified into four groups by their fine specificities, suggesting that Cry j I bears at least four antigenic determinant regions. (3) Cry j I molecules were found to take a monomeric form in solution and to display no repeating antigenic epitopes on their surfaces. (4) Some of the determinants seemed to be located in the interior of a Cry j I molecule, and when the Ag is coated on a plastic plate, the determinants become exposed on its surface. (5) Binding of human IgE antibodies to Cry j I and Cry j II was blocked by some of the obtained mAbs, suggesting that these epitopes recognized by the mAbs might have an important role in human allergic response against the cedar pollen.


[Simple and generalized first order differential equations. Their use in a few macrophysiology domains].

First order differential equations formulate the simplest physiological models and, therefore, are endowed with wide applicability. They owe this advantage to the principle of parcimony: according to Occam's razor, the most general explanations are a priori the best. First order equations express the essential properties of the system. However, they often are inappropriate for representing complementary facts characteristic of the real objects. Any refinement of the model requires the inclusion of higher order terms. Our goal is to arrive at a general method for addressing a variety of problems. Here, we chose the example of the Windkessel model to introduce the mathematical framework. The input was simplified without loss of generality. We also proposed a possible analytical approach and compared its results with those of the numerical methods. This led to the improvement of the latters. Finally, some prominent examples in the field of macrophysiology were discussed.


Heterogeneity of 5-HT responsiveness in different segments of rabbit arteries.

The variation in sensitivity to serotonin (5-HT) as assessed by pD2 was investigated in isolated 25 arterial segments of different vascular regions obtained from rabbits. The responses of the vascular rings which were opened by a transverse cut to form rectangular strips were recorded isometrically. pD2 values for 5-HT of thoracic and abdominal aortic segments were significantly greater than those of visceral arterial branches. Maximal contractions produced by 5-HT in visceral segments were higher than those of thoracic and abdominal aortic segments. There was a positive correlation between the pD2 values for 5-HT and the radii and the thickness of the tunica media layer (r = 0.62 and r = 0.54, respectively, P less than 0.05). The results indicate that the activity of 5-HT varies regionally in different segments of the rabbit arteries and the sensitivity to 5-HT of the segments decreases as the radii of the vessels diminish.


Antigenic analyses of Sugi basic protein by monoclonal antibodies: II. Detection of immunoreactive fragments in enzyme-cleaved Cry j I.

The 4 anti-Cry j I mAbs showing an epitope specificity different from each other, 046, 029, 026 and 027, were selected to analyze the structure of the antigenic determinant for each mAb on a Cry j I molecule. Immunoreactive fragments in enzyme-cleaved Cry j I were detected by means of the adsorption on the mAb column and of the binding to the mAbs on Elisa. The mAb 026 was found to be reactive to the fragments containing a Cry j I N-terminal region obtained by V8 protease or pepsin digestion, but not to those by lysylendopeptidase digestion. The mAb 027 was found to be capable of binding to the fragments containing a linear structure of Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser-Leu-Ser-Lys, which were generated by V8 protease, lysylendopeptidase or pepsin digestion. Furthermore, the synthetic peptide Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser- Leu-Ser-Lys-Arg could bind to 027, but not to 026, and could inhibit the binding of 027 to Cry j I or to its immunoreactive fragments. No fragments capable of reacting to the mAbs 046 and 029 could be found in this study, suggesting that 046 and 029 recognize a conformationally constituted epitope of Cry j I molecule which is destroyed by enzymatic cleavage. The epitope recognized by the mAbs 027 or 026 was found to be located in conformationally hidden parts of the molecule which was exposed to react to the mAbs only after the physicochemical or enzymatic treatment.


Antiallergic activity of 6-(2-cyclohexylethyl)-[1,3,4]thiadiazolo- [3,2-a]-1,2,3-triazolo-[4,5-d]pyrimidin-9(3H)-one (DS-4574) in rats.

The antiallergic activity of DS-4574 was evaluated in several commonly used rat models for allergic diseases. In passive cutaneous anaphylaxis, DS-4574 given intravenously and orally induced dose-dependent inhibition with ID50 values of 0.55 and 2.8 mg/kg, respectively. In contrast, this compound had no antagonistic activity against the histamine- and serotonin-induced cutaneous vascular permeability. In lung anaphylaxis, DS-4574 inhibited pulmonary function changes induced by the antigen in a dose-dependent manner when it was given intravenously and orally, the ID50 values being 0.04 and 0.89 mg/kg, respectively. DS-4574 also inhibited antigen-induced histamine and leukotriene release in passive peritoneal anaphylaxis following oral administration. In addition, this compound prevented antigen-induced histamine release in passively sensitized mast cells in vitro. These potent activities of DS-4574 in in vivo and in vitro models of immediate-type hypersensitivity reactions suggest that this compound could be useful in the treatment of allergic diseases including asthma.


Development of Hodgkin's disease in the course of primary Sjögren's syndrome.

We report two patients with a definite diagnosis of primary Sjögren's syndrome who developed Hodgkin's disease. Clinical and laboratory features of this transformation comprised prolonged fever, the appearance of lymphadenopathy together with loss of serum autoantibodies and a reduction in serum gammaglobulin levels. We know of only one well documented case of such an association. From these observations, it seems reasonable to include Hodgkin's disease in the clinical spectrum of the lymphoproliferative disorders that may occur in the course of primary Sjögren's syndrome.


Elective versus emergency surgery for patients with colorectal cancer.

A prospective study of 570 patients presenting with colorectal cancer over a 6-year period was undertaken. Of these, 363 were admitted electively and 207 presented as emergencies. The outcome following elective admission was more favourable than after emergency admission. In the elective group the proportion of resected tumours was greater (77 versus 64 per cent, P less than 0.001), the operative mortality rate lower (9 versus 19 per cent, P less than 0.001) and the 5-year disease-related survival rate higher (37 versus 19 per cent, P less than 0.001). These differences may relate to the greater resection rates in the elective situation. Results of surgical intervention might be improved if emergency colorectal operations were undertaken by surgeons with more experience of this type of surgery.


Post-hatching developmental changes in the ultrastructure of the duodenal absorptive epithelial cells in 1, 10 and 60-d-old chickens, with special reference to mitochondria.

1. Post-hatching developmental changes in the ultrastructure, cell area and histological RNA content of duodenal absorptive epithelial cells were observed in 1, 10 and 60-d-old White Leghorn (WL) and broiler (BR) chickens, with special reference to the mitochondria (Mt). 2. Epithelial cells in 1-d-old WL and BR had a well-developed Golgi area and a dense cluster of rod type Mt near the cell surface and at the infranuclear region. In BR, cells included well developed profiles of endoplasmic reticulum but fewer supranuclear vacuoles than those of WL, in which numerous free ribosomes were also found. Some Mt in BR showed a bud-like protrusion from the main body (tadpole type Mt). These suggest that the fine structural maturation of the epithelial cells in WL is involved in the process of cell maturation but epithelial cells of BR have almost matured at hatching. 3. In 10-d-old WL and BR, supranuclear vacuoles had disappeared and Mt increased in number. In WL, in addition to a decrease in free ribosomes, Mt developed to the tadpole type and further to dumbbell type ones. In BR, Mt also aggregated at the perinuclear region and some of them had developed from dumbbell shapes to a thick doughnut type. These findings indicate that epithelial cells in both breeds are more developed ultrastructurally than those in 1-d-old and that epithelial cells in BR are more activated for digestive and absorptive functions than those in WL. 4. In 60-d-old WL and BR, Mt showed various types with thinner matrices than 10-d-old, suggesting that the cell structure and function had reached a stable state. 5. BR revealed higher values for cell area and RNA content than WL at every age and RNA content of both breeds were maximal at 10 d, followed by 60 d. 6. Mt changed their shapes from rod type to tadpole type. The latter then developed to doughnut type via dumbbell type, related to an increase of epithelial cell functions. 7. Epithelial cells of BR are thus more highly activated than WL cells at each age and reach morphological and physiological maturation around 10 d.


Mechanism for the recruitment of macrophages to cancer site. In vivo concentration gradient of monocyte chemotactic activity.

Tumor stroma is characterized by the development of new blood vessels, an inflammatory cell infiltration, and a fibrotic reaction. The inflammatory component of tumor stroma plays an important role in the modulation of tumor expansion. In this respect, macrophages constitute a major part of the inflammatory cell infiltration and can exert cytotoxic activity against tumor cells. The accumulation of macrophages in the vicinity of the tumor suggests their recruitment from circulating blood monocytes through the local release of chemotactic factors for monocytes. To detect the existence of a concentration gradient of monocyte chemotactic activity (MCA) between the tumor vicinity and blood vessels, malignant pleural effusions defined by the local presence of cancer cells were evaluated for quantification of MCA. Unlike nonmalignant pleural effusions, malignant pleural effusions were characterized by the presence of increased levels of MCA, and in lung adenocarcinoma (a cancer with high inflammatory cell infiltration), pleural levels of MCA were significantly greater than in small cell lung carcinoma (a cancer with low inflammatory cell reaction). An MCA concentration gradient between pleural fluid and plasma was present in malignant effusions because pleural MCA levels in all cancer types were significantly greater than MCA levels in the plasma of the same patients. Thus, an increased local level of MCA is a feature of cancers with high inflammatory cell infiltration, and the presence of an in vivo concentration gradient of MCA suggests the direct role of this biologic activity in recruiting blood monocytes to the cancer site.


Disposition characteristics of macromolecules in the perfused tissue-isolated tumor preparation.

Disposition characteristics of model macromolecules with different physicochemical characteristics and macromolecular prodrugs of mitomycin C, namely mitomycin C-dextran conjugates, were studied in tissue-isolated tumor preparations of Walker 256 carcinoma with the use of a single-pass vascular perfusion technique. In constant infusion experiments, all radiolabeled macromolecules accumulated in the tumor tissue, but the degree and pattern of distribution greatly varied, depending on their electric charges. Positively charged macromolecules were markedly accumulated compared with those that were neutral or negatively charged. In addition, their concentrations were significantly higher in viable than in necrotic regions, while neutral and negative compounds were distributed in necrotic rather than in viable regions. Pharmacokinetic analysis of tissue concentration-time courses of positively charged diethylaminoethyl and neutral dextrans showed that their movement occurred by convective fluid flow, and that high tissue accumulation of positively charged macromolecules could be explained by strong binding due to electrostatic interaction. For neutral and anionic macromolecules with negligible affinity to the tissue, it was suggested that the final concentration gradient between the viable and necrotic regions was decided by their tissue fluid content. Thus, the present study revealed the basic disposition characteristics of macromolecules in tumor tissue relative to their physicochemical properties.


Cyclin D1 messenger RNA is inducible by platelet-derived growth factor in cultured fibroblasts.

In fibroblasts in culture, the levels of cyclin D1 mRNA are growth regulated. In mouse and in human fibroblasts, both serum and platelet-derived growth factor increase cyclin D1 mRNA levels with similar kinetics of induction. Insulin-like growth factor 1 by itself does not induce cyclin D1 expression, and an antisense oligodeoxynucleotide to the insulin-like growth factor 1 receptor RNA does not affect the growth-regulated levels of cyclin D1 mRNA.


Broad spectrum neuropeptide antagonists inhibit the growth of small cell lung cancer in vivo.

The proliferation of small cell lung cancer (SCLC) cells appears sustained by multiple autocrine and paracrine circuits involving Ca2+ mobilizing neuropeptides. Consequently, broad spectrum neuropeptide antagonists which inhibit SCLC growth in vitro have been suggested as potential anticancer agents. Here we evaluated this hypothesis using xenografts of WX322 cells, a SCLC cell line that responds to multiple Ca2+ mobilizing neuropeptides. The broad spectrum neuropeptide antagonists [Arg6,D-Trp7,9,MePhe8]substance P(6-11) and [D-Arg1,D-Phe5,Trp7,9Leu11[substance P were shown to inhibit the growth of WX322 xenografts in nude mice. Similar results were obtained with xenografts of the SCLC cell line H69. The results indicate that broad spectrum neuropeptide antagonists can inhibit the growth of SCLC in vivo and suggest that these antagonists could be useful in the treatment of SCLC.


Synthesis of lysine-containing fragments of the Proteus mirabilis O27 O-specific polysaccharide and neoglycoconjugates therefrom.

Amide-linked lysine mono- and di-uronic acid fragments of the O-specific polysaccharide from P. mirabilis O27 have been synthesised. N epsilon-Boc-L-lysine tert-butyl ester was condensed with 2-azidoethyl glycosides of glucuronic acid and beta-D-GlcpNAc-(1----3)-beta-D-GlcpA. Transformation of the products into 2-acrylamidoethyl glycosides, followed by deprotection using trifluoroacetic acid, gave the target monomers that were converted into high-molecular-weight copolymer-type neoglycoconjugates.


The polarity of editing within a multiple gRNA-mediated domain is due to formation of anchors for upstream gRNAs by downstream editing.

Seventeen kinetoplast minicircle-encoded and nine maxicircle-encoded gRNA genes have been identified. Six overlapping minicircle-encoded gRNAs mediate editing for the 5'-pan-edited MURF4 gene and two for the 5'-edited COIII gene. The pan-edited RPS12 mRNA is edited by seven minicircle-encoded gRNAs and one maxicircle-encoded gRNA. The 3'-most gRNA in each domain forms an anchor with unedited mRNA, whereas upstream gRNAs form anchors only with edited mRNA, thereby explaining the observed 3' to 5' polarity of editing within an editing domain. We suggest that a role of G-U base pairs is to allow breathing of the edited mRNA-gRNA hybrid and formation of the upstream anchor hybrid.


Generation of unexpected editing patterns in Leishmania tarentolae mitochondrial mRNAs: misediting produced by misguiding.

We have analyzed the generation of unexpected patterns of RNA editing, i.e., those not following a strict 3' to 5' progression, which occur in junction regions between fully edited and preedited sequences. Evidence is presented that these patterns are generated by misediting due to specific events of misguiding. Misediting can occur through the interaction of inappropriate gRNAs with mRNAs or appropriate gRNAs in an incorrect fashion. Four possible mechanisms for the generation of misedited sequences are presented. Chimeric molecules have been detected in steady-state mitochondrial RNAs that are composed of misguiding gRNAs covalently linked to mRNAs at misediting sites by the 3' oligo(U) tail. We propose that misediting within junction regions can be corrected by appropriately acting gRNAs.


Chemical modification of recombinant human granulocyte colony-stimulating factor by polyethylene glycol increases its biological activity in vivo.

Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) produced in Escherichia coli was chemically modified by polyethylene glycol (PEG) of molecular weights 4,500 or 10,000. The neutrophils observed at 32 hours after intravenous injection of the rHuG-CSF modified with PEG (4,500) or PEG (10,000) to mice were, respectively, 2.5 times and 5 times more than that observed after the injection of the unmodified rHuG-CSF. These results show that the covalent attachment of PEG to rHuG-CSF enhanced its pharmacological activity in vivo and that the modification with the larger PEG molecule is more effective to enhance the in vivo activity of rHuG-CSF.


Neoadjuvant chemotherapy with cisplatin, vincristine, and bleomycin and radical surgery in early-stage bulky cervical carcinoma.

Neoadjuvant chemotherapy consisting of 2-3 courses of cisplatin, vincristine, and bleomycin was used in the primary treatment of 36 consecutive patients with locally advanced early-stage cervical carcinoma [International Federation of Gynecology and Obstetrics (FIGO) stages Ib or IIa; tumor size, greater than or equal to 4 cm]. The effectiveness of the preoperative chemotherapy was evaluated in the surgical specimens. Among the 33 evaluable patients, the overall clinical response rate was 84.8%, which included a complete response in 8 patients (24.2%) and a partial response in 20 subjects (60.6%). No residual tumor was found in the surgical specimens obtained from 2 complete responders. This therapy induced varying degrees of tumor shrinkage and rendered radical surgery feasible in all evaluable cases despite the initial bulky size of the lesions. No significant difference was observed in the response rate according to age and disease stage. Lymph-node metastases were found after chemotherapy in 18.2% (6/33) of the patients. Grade II and III hematological toxicities occurred in 23.3% of the 90 chemotherapy cycles completed. Nausea and vomiting occurred to a mild to moderate degree in 75 (83.3%) cycles. These preliminary results suggest that the administration of induction chemotherapy involving two to three courses of cisplatin, vincristine, and bleomycin prior to surgery is effective in reducing the tumor volume and in providing better circumstances for surgical removal of the early yet bulky cervical tumors and results in tolerable toxicity. This protocol is now undergoing prospective randomized trials to test its impact on long-term survival.


Phase I trial of a 72-h continuous-infusion schedule of fazarabine.

Fazarabine (Ara-AC), a structural analog derived from the antitumor nucleoside cytosine arabanoside (Ara-C) and 5-azacytidine (5-AC), was studied in a phase I clinical trial. Doses ranging from 0.2 to 2.0 mg m-2 h-1 were given intravenously over 72 h every 28 days. The maximum tolerated dose (MDT) was 2.00 mg m-2 h-1. The dose-limiting toxicity was myelosuppression, with granulocytopenia being quantitatively more important than thrombocytopenia or anemia. Nonhematologic toxicity was minimal. Associated with the solvent dimethylsulfoxide (DMSO) was a bitter taste and a garlic-like odor.


Factors influencing the reaction of alpha 1-fetoprotein with concanavalin A and Lens culinaris agglutinin in crossed affinoimmunoelectrophoresis.

Concanavalin A (Con A) and lentil lectin (LCA) analysis of alpha-fetoprotein (AFP) glycosylation heterogeneity is used in a variety of clinical situations. We studied the influence of analytical conditions on the separation of AFP glycoforms by using lectin-crossed affinoimmunoelectrophoresis, regardless of the AFP concentration, which can vary over a wide range in biological fluids. We defined the optimal concentration of Con A (2 g/L) and LCA (0.35 g/L) in the first-dimension gel, together with the optimum antigen (AFP)/antibody ratio in the second-dimension gel. The presence of protein in the diluent used for AFP samples was found to change the shape of crossed affinoimmunoelectrophoresis patterns without changing the percentage composition of AFP fractions. The within-run CV was less than 4% for both lectins, and the between-run CV was less than 6.3%. The minimal quantity of AFP that provided a visible pattern with both lectins was 4 ng, corresponding to 50 microL of an 80 micrograms/L AFP sample. These technical conditions allow the cellular origin of AFP to be determined, regardless of the concentration in the sample. Typical AFP lectin patterns of secreting tumors are compared with fetal and cord serum AFP.


Macroamylases: differences in activity against various-size substrates.

Hyperamylasemia caused by macroamylases can lead to the overdiagnosis of acute pancreatitis. We examined whether interference from macroamylase is less in assays that use high-molecular-weight rather than oligosaccharide substrates. We hypothesized that high-molecular-weight substrates would be sterically excluded from macroamylasemic complexes and thus would be hydrolyzed less efficiently. Eighteen macroamylasemic samples were assayed by using red-dyed amylopectin or blue-dyed starch as polysaccharide substrates or by using maltoheptaose or maltotetraose as oligosaccharide substrates. The oligosaccharide substrates gave comparable results (y = 0.81x + 83); we observed consistently lower activities for amylopectin than for maltotetraose (y = 0.32x + 38). We observed no bias among methods when nonmacroamylasemic specimens were analyzed. The mechanism of this difference was examined by adding anti-human pancreatic amylase antibodies to hyperamylasemic serum samples from patients without macroamylasemia and purified human pancreatic or salivary isoamylases. In each case, polyclonal and monoclonal antibodies lowered amylase activity more in assays with complex polysaccharides than in those with oligosaccharides. The use of high-molecular-weight substrates diminishes interference, and detection of suspected macroamylasemia may be possible through comparison of activities determined from automated methods that use different substrates.


Hemoglobin A1c by HPLC with the Pharmacia Mono S HR 5/N cation-exchange column: influence of sample protein load on optimal chromatographic conditions.

The Pharmacia Mono S HR 5/5 column has been optimized for hemoglobin A1c analysis by HPLC by using a much smaller column load, decreased buffer flow rate, and a steeper gradient than was used in previously described methods. Superior chromatographic separation, shorter analysis time, and a greatly extended column life have resulted.


Antigenic determinants of the 70-kDa subunit of the Ku autoantigen.

Autoantibodies against Ku antigen were found in subsets of sera from patients with rheumatic diseases. The Ku autoantigen was characterized as a DNA-binding protein complex composed of two subunits, 70 and 86 kDa. In this study, we report the amino acid sequences of the 70-kDa subunit that are important for interactions with a monoclonal and autoimmune antibodies. Full-length cDNA and numerous 5' and 3' deletion mutants were expressed in bacteria and the immunoreactivity of the fusion proteins was analyzed by Western blotting. The reactivity of the monoclonal antibody depended on the region between Ile321 and Phe350. Ten autoimmune sera were tested for reactivity with deletion mutants in immunoblots. The reactivity of six sera strongly depended on the C-terminal amino acids and four sera did not show such dependence; however, these C-terminal sequences did not react with the sera when expressed alone. These results strongly suggest the conformational nature of the Ku autoepitopes. Interestingly, the DNA-binding activity of this Ku protein subunit analyzed by Southwestern blot depended on the same C-terminal amino acids that were involved in interactions with autoantibodies, indicating that anti-Ku autoantibodies are directed to conformationally intact Ku antigen. Reactivities of the autoimmune sera with Met1-Arg115, Met116-Val149, and Val149-Arg586 were also observed. These results demonstrate that different amino acid regions can be involved in interactions with autoimmune antibodies.


Decreased monocyte-mediated angiogenesis in scleroderma.

Scleroderma is a disease characterized by proliferative vascular lesions in which monocytes/macrophages may play a key role. Monocytes were isolated from 14 scleroderma patients and 11 normal controls and cultured with or without lipopolysaccharide (LPS) (5 micrograms/ml). Monocyte-conditioned medium was assayed in the rat corneal bioassay for angiogenesis. Conditioned medium from normal monocytes was nonangiogenic, as was conditioned medium from scleroderma monocytes. While conditioned medium from LPS-activated normal monocytes was potently angiogenic in 11/13 corneas, conditioned medium from LPS-activated scleroderma monocytes was angiogenic in only 3/14 corneas. Levels of the angiogenic cytokine tumor necrosis factor-alpha (TNF-alpha) were measured in conditioned medium from scleroderma and normal monocytes. TNF-alpha levels were not significantly different in patient and control groups and thus do not account for the decreased angiogenic activity exhibited by scleroderma monocytes. As monocytes require activation to produce angiogenic activity, we determined the cell surface binding of monoclonal antibodies to activation-related (HLA-DR, 3D8, and 8D7) and other (Leu-M5) markers on monocytes by radioimmunoassay. Monocytes were cultured alone, with LPS (5 micrograms/ml), or with interferon-gamma (IFN) (200 units/ml). The usual increase in binding of anti-HLA-DR on stimulation of scleroderma monocytes with IFN was slightly less than that of controls. IFN-stimulated monocytes bound less anti-8D7 than controls. Anti-3D8 and anti-Leu-M5 binding was comparable in both groups. These results suggest that scleroderma monocytes do not produce normal levels of angiogenic activity with LPS stimulation, have some altered markers of activation on their cell surfaces, and may thus contribute to the aberrant vascular proliferation found in this disease.


Effects of granulocyte colony-stimulating factor in modifying mortality from Pseudomonas aeruginosa pneumonia after hemorrhage.

Alterations in immune function occurring after hemorrhage and trauma may contribute to the high occurrence rates of nosocomial pneumonia, multiorgan system failure, morbidity, and mortality in this setting. Therapy with granulocyte colony-stimulating factor (G-CSF) can increase neutrophil numbers and function, and enhance resistance to infection in experimental and clinical settings associated with abnormal immune function. To investigate whether treatment with G-CSF could increase resistance to pneumonia after hemorrhage, we bled mice 30% of the blood volume and treated them with various doses of G-CSF, starting either immediately or 2 days after hemorrhage. Pseudomonas aeruginosa pneumonia was induced by the intratracheal instillation of 2 x 10(7) colony-forming units of P. aeruginosa 4 days after blood loss, and mortality was assessed over the next 7 days. Treatment of mice with 100 or 500 micrograms/kg/day G-CSF, but not with 50 micrograms/kg/day, resulted in significant increases in the numbers of circulating polymorphonuclear cells. Platelet counts significantly decreased in mice given 500 micrograms/kg/day G-CSF. Mice given 100 micrograms/kg/day G-CSF starting 2 days after blood loss had improved outcome compared with vehicle-treated controls (38% survival rate in the G-CSF treated group vs. 8% in controls, p less than .05). There also was a trend toward an improved survival rate in mice treated with 50 micrograms/kg/day G-CSF for 4 days after hemorrhage (46% survival rate in G-CSF treated vs. 17% in controls). G-CSF prophylactically administered after hemorrhage can improve survival from pneumonia due to P. aeruginosa. However, the protection afforded by G-CSF was highly dependent on the dosing schedule used.


Hemodynamic, renal, and hormonal actions of aprotinin in an ovine model of septic shock.

Previous studies documented activation of protease enzymes such as the plasma kallikrein-kinin system in endotoxemia and sepsis, both in experimental animals and in patients. We investigated the actions of aprotinin (a protease inhibitor that binds to plasma kallikrein) on systemic hemodynamics and renal function, in an ovine model of septic shock. Aprotinin was infused intravenously in high dosage (1 x 10(6) kallikrein inhibitor units [KIU] loading, 200,000 KIU/hr), commencing 30 mins after surgical induction of sepsis (cecal ligation and puncture). In the control group (n = 6), there were significant decreases with time in BP and systemic vascular resistance, an increase in pulmonary artery pressure, reductions in creatinine clearance and sodium excretion, and an increase in plasma renin activity. In aprotinin-treated animals (n = 6), none of these changes occurred. There were significant between-group differences (controls vs. treatment) for mean arterial pressure, pulmonary artery pressure, and plasma renin activity. In this large animal model of septic shock, which reproduces the important features of clinical sepsis, treatment with aprotinin after the initiation of sepsis appears beneficial in relation to systemic hemodynamics, renal function, and hormonal stimulation, with no evidence of adverse effects.


Effect of an omental wrap on the healing and vascularity of compromised intestinal anastomoses.

Adult Wistar rats were used to investigate the ability of an omental wrap to limit leakage from compromised intestinal anastomoses. Under ketamine anesthesia, a section of small bowel was divided and then reanastomosed using a "control" anastomosis, a "deficient" anastomosis, or an "ischemic" anastomosis, plus or minus the addition of a wrap of omentum. Initially 10 rats were randomly assigned to each group. Nineteen of the 20 rats with unwrapped compromised anastomoses died within six weeks, compared with five deaths in the rats protected by an omental wrap (Fisher's exact test; P less than 0.01). The experiment was then repeated with a sample of rats from each anastomotic group being sacrificed for histologic examination on days 2 to 7, 10, 14, and 42. At the time of sacrifice a dye was injected into the omental vasculature to determine its contribution to the healing anastomosis. An anastomosis could be demonstrated between omental and bowel wall vessels by the third postoperative day. At one week the infarcted bowel edges were being resorbed and the omentum formed a fibrotic cylinder aligning the separated ends of bowel wall. At six weeks the scar became more contracted and the bowel mucosa had started to grow onto its luminal surface. It is concluded from this study that the omental wrap is protective to a compromised anastomosis by providing a biologically viable plug to prevent early leakage and a source of granulation tissue and neovasculature for later wound repair.


New antiepileptic drugs: from serendipity to rational discovery.

Antiepileptic drug discovery has made enormous progress from the serendipity and screening processes of earlier days to the rational drug development of today. The modern era of research began with the recognition that enhancement of inhibitory processes in the brain might favorably influence the propensity for seizures, gamma-aminobutyric acid (GABA) being the main inhibitory transmitter. Work in this field led to the development of vigabatrin, which inhibits the enzyme responsible for the degradation of GABA. More recently, research has focused on the therapeutic potential of blocking excitatory amino acids--in particular glutamate. Of the three receptors for glutamate, the N-methyl-D-aspartate (NMDA) receptor is considered the one of most interest in epilepsy, and research on a series of competitive NMDA receptor antagonists--especially those that are orally active--is in the forefront of antiepileptic drug development today. A further alternative for diminishing neuronal excitability is to modulate sodium, potassium, or calcium channels. The latter are especially implicated in absence seizures.


Role of nonacidic endosomes in recycling of ADH-sensitive water channel structures.

Toad urinary bladder epithelial cells respond to the hormone ADH by increasing the water permeability of their luminal membrane. This action is mediated by insertion into the apical membrane of specific water channels. In the absence of ADH these channels appear to be present in tubular cytoplasmic vesicles as morphologically distinctive intramembrane structures called particle aggregates. ADH induces these vesicles to fuse with the apical membrane, transferring their aggregate-water channels into the apical membrane. When ADH stimulation is removed (ADH reversal), aggregates and fluid-phase markers from the mucosal bath appear in water-permeable vesicles in the cytoplasm. We have examined the fate of fluid-phase markers and aggregates with time after ADH reversal. Although the fluid-phase markers horseradish peroxidase and colloidal gold are initially found predominantly in tubular vesicles near the apical surface, by 30 min the markers were found in perinuclear multivesicular bodies (MVBs) of heterogeneous size and shape. These MVBs appear to be nonacidic since they fail to accumulate DAMP. Acid phosphatase (AcPase) was undetectable in these structures. After 60 min, labeled MVBs tended to be smaller, and some of these structures displayed DAMP accumulation and AcPase activity. By evaluation of uncleaned replicas it was possible to localize recycled aggregate-water channels with respect to internalized fluid-phase markers. Thirty minutes after retrieval from the apical surface in tubular vesicles, aggregates could be localized to both the central body and tubular projections of labeled MVBs. At 60 min following reversal, most MVBs had a reduced number of aggregates compared with 30 min, and compact structures could be identified that contained markers but no detectable aggregates. These observations show that aggregates and fluid-phase markers enter a nonacidic endosomal compartment with an MVB morphology following ADH reversal. At extended times following reversal, labeled MVBS having lysosomal characteristics and labeled MVBs having no detectable aggregates can be found, suggesting that aggregates are sorted or degraded prior to this stage.


Morphology of sympathetic preganglionic neurons innervating the superior cervical ganglion in the chicken: an immunohistochemical study using retrograde labeling of cholera toxin subunit B.

Preganglionic sympathetic neurons (SPNs) in the chicken were demonstrated immunohistochemically using cholera toxin subunit B (CTb) as a retrograde tracer. After injection of CTb-solution into the superior cervical ganglion, labeled SPNs were mainly found in the ipsilateral sympathetic preganglionic column of Terni (the column of Terni), with only a few in the intermediate zone. They were observed from the caudal half of the 15th cervical segment to the rostral tip of the 3rd thoracic segment. Cell somata of SPNs were loosely packed within the column of Terni, where they had an elliptic shape with the long axis oriented rostrocaudally. In the horizontal plane three kinds of dendrites could be discriminated on the basis of their orientation. Longitudinally oriented dendrites emanated from the rostral and the caudal poles of the SPNs. Medially oriented dendrites were observed to cross the midline and enter the contralateral column of Terni, where they further branched to form a loose dendritic plexus; some extended beyond the lateral limit of the contralateral column of Terni to reach the intermediate zone. Laterally oriented dendrites formed periodically arranged dendritic bundles projecting into the intermediate zone. The present findings provide a detailed account of the dendritic organization of SPNs in the chicken, and suggest that avian SPNs share certain structural features in common with mammalian SPNs.


Axonal trajectories of single Forel's field H neurones in the mesencephalon, pons and medulla oblongata in the cat.

We studied axonal trajectories of single Forel's field H (FFH) neurones (n = 19) in the mesencephalon, pons and medulla by systematic antidromic threshold mapping in cats and differentiated them into two major types. Type I neurones were characterized by projections to the oculomotor nucleus (IIIn) and type II neurones by lack of projections to the IIIn. 2. Type I neurones (11/19) were further classified into three subtypes by the lowest level of projections; type Ic (n = 3) which projected to the cervical cord and type Ib (n = 7) which terminated at the ponto-medullary level and type Ia (n = 1) at more rostral level. In the mesencephalon, stem axons passed just lateral to the IIIn and projected collaterals to the IIIn and the ventral part of the periaqueductal gray matter. In the lower brain stem, stem axons of type Ib and Ic neurones passed in the dorsal part of the reticular formation or in the medial longitudinal fasciculus and projected collaterals to the dorsal part of the nucleus reticularis pontis caudalis (NRPC) and the nucleus reticularis gigantocellularis (NRG) and the reticular formation underlying the nucleus prepositus hypoglossi (PH) and the raphe region. Projections to the superior colliculus were observed in two cases. 3. Type II neurones (8/19) were classified into 2 type IIb projecting to the ponto-medullary reticular formation and 6 type IIc projecting to the cervical spinal cord. In the mesencephalon, stem axons passed through a more lateral region than those of type I and projected collaterals to the mesencephalic reticular formation and the red nucleus. In the lower brain stem, the stem axons passed in the ventral part of the reticular formation corresponding to the central tegmental tract and projected collaterals to the ventral part of the NRPC and NRG. Projections to the interstitial nucleus of Cajal, the inferior olive and the reticular formation underlying the PH were also observed. 4. The dorsal and ventral location of, respectively, stem axons of type I and type II neurones in the lower brain stem was confirmed in a larger number of neurones in experiments with restricted mapping. 5. There was not much difference in location of cell bodies of type I (totally n = 50) and type II (n = 46) neurones. The proportion of spinal-projecting neurones were larger in type II (21/46, 46%) than in type I (7/50, 14%) neurones.


Prevalence of anti-HCV in subjects of various age groups.

812 serum samples from 382 males and 430 females from various age groups were examined. All the samples were tested for anti-HCV hepatitis C virus, anti-HBc and HBsAg with an enzymeimmunoassay. The total serum prevalence was 2.9% for anti-HVC, 22.2% for anti-HBc and 4.6% for HBsAg. The seropositivity rates of anti-HCV and anti-HBc tended to increase with age, while for HBsAg a more regular pattern was observed for the different subject groups. The fact that anti-HCV are more frequently found together with HBV markers confirms the existence of similar modes of transmission of the two viruses.


Mechanisms accounting for the impaired natural-killer cell activity in refractory anaemia with excess of blasts.

Natural killer (NK) cells were analyzed in 38 untreated patients with refractory anaemia with excess of blasts (RAEB), using cytotoxicity assays and immunofluorescence with monoclonal antibodies. We found that patients with RAEB have normal numbers of peripheral blood and bone marrow NK cells. NK cells from RAEB patients express very low natural-killer cell activity (NKa) which may be increased significantly with recombinant alpha-interferon and recombinant interleukin-2, although it remains below the lower limit of the control range. The cells exhibit normal tumour cell binding capacity, but fail to release sufficient amounts of natural-killer cytotoxic factors (NKCFs) upon their interaction with NK-sensitive K562 cell targets or their stimulation with phytohaemagglutinin. Our results suggest that defective NKa in RAEB patients may be due, at least in part, to impaired release of functionally active NKCFs. This disturbance is probably the result of some intrinsic defect of RAEB NK cells in NKCF production, storage, and/or release. The possibility of an impairment in the activation signal provided by the stimulatory K562 cells cannot be excluded, although it seems unlikely. We postulate that this abnormality might represent a manifestation of dysplastic haemopoiesis. Further studies are certainly needed to investigate whether other defective mechanisms are also implicated in the determination of the low NKa in patients with RAEB.


Effect of tandemly repeated AGG triplets on the translation of CAT-mRNA in E. coli.

It has been shown that tandems of rare arginine codons AGG have a strong inhibitory effect on translation of mRNA in E. coli [5]. This has been explained by the rate-limiting interaction of these codons with the less abundant tRNA(AGG) [6]. In this study tandemly repeated AGG triplets were introduced into the chloramphenicol acetyltransferase (CAT) gene either upstream of the initiation ATG codon or downstream of it (both in frame and out of frame) and the expression of the modified genes was investigated. We report that the addition of AGG clusters resulted in a substantial inhibitory effect on CAT gene expression independently of their localization in mRNA. This inhibitory effect is explained by a competition of the tandem AGGAGG with the natural Shine-Dalgarno (SD) sequence (consensus AAGGAGGU) for the 3'-end of the 16S small ribosomal RNA (rRNA).


Parathyroid hormone activation of stretch-activated cation channels in osteosarcoma cells (UMR-106.01).

Cell-attached patches of membrane of osteoblast-like cells UMR-106.01 respond to bath application of parathyroid hormone (PTH) with an increase in the average activity, as well as the single channel conductance, of a stretch-activated non-selective cation channel. Correlations with whole cell membrane potential and conductance changes are considered.


Molecular cloning of the full-length cDNA encoding the human calbindin-D9k.

The full-length cDNA encoding the human calbindin-D9k (CaBP-9k) has been cloned using reverse transcription/PCR methodology with rat- and bovine-derived primers and intestinal RNA. A core product, and both a 5' and 3' product encompassing the full-length cDNA were obtained. The clones include coding region for 79 amino acids, 57 nucleotides 5'-and 159 nucleotides 3'-non-coding region, and a poly(A) tail. The deduced protein sequence is homologous to other mammalian CaBPs. Northern analysis revealed the mRNA in human duodenum to be about 600 nucleotides in length. Expression levels in adult human tissue were substantially lower than in child, rat or porcine intestine.


Induction of an electrogenic transfer of monovalent cations (K+, NH4+) in thylakoid membranes by N,N'-dicyclohexylcarbodiimide.

The effect of N,N'-dicyclohexylcarbodiimide (DCCD) on photosynthetic electron transport and light-induced NH4+ and K+ uptake in the presence of ammonium or nigericin was studied. DCCD alone had no effect on either the electron transport or the uptake of protons. The simultaneous action of DCCD and low concentrations of ammonium or nigericin was shown to lead to a significant increase in the electron transport rate and a decrease in the steady-state uptake value of H+ and NH4+ or K+. The effect of DCCD on these processes was compared with the effect of the ionophore, valinomycin, which transports potassium and ammonium cations through membranes. The conclusion was made that 1.0-1.5 mol DCCD per mol chlorophyll activated the transfer system of monovalent cations (K+ and NH4+) in thylakoid membranes.


Molecular cloning and characterization of human endothelial nitric oxide synthase.

The constitutive calcium/calmodulin-dependent nitric oxide (NO) synthase expressed in vascular endothelium shares common biochemical and pharmacologic properties with neuronal NO synthase. However, recent cloning and molecular characterization of NO synthase from bovine endothelial cells indicated the existence of a family of constitutive NO synthases. Accordingly, we undertook molecular cloning and sequence analysis of human endothelial NO synthase. Complementary DNA clones predict a protein of 1,203 amino acids sharing 94% identity with the bovine endothelial protein, but only 60% identity with the rat brain NO synthase isoform. Northern blot analysis with an endothelial-derived cDNA identified a 4.6-4.8 kb mRNA transcript in HUVEC and in situ hybridization localized transcripts to vascular endothelium but not neuronal tissue.


Cell density-dependent expression of heparin-binding growth-associated molecule (HB-GAM, p18) and its down-regulation by fibroblast growth factors.

Heparin-binding growth-associated molecule (HB-GAM) is a developmentally regulated protein that is intensely expressed during the rapid postnatal growth phase of rat brain. The expression of HB-GAM studied in 12 cell lines was restricted to C6 rat glioma cell line and BALB/c 3T3 cells. In BALB/c 3T3 cells the expression of HB-GAM was enhanced in confluent and quiescent cell cultures. When the confluent cultures were treated with bFGF the expression of HB-GAM mRNA was strongly reduced and the protein disappeared rapidly from proliferating cells. The data presented suggest involvement of HB-GAM in cell differentiation phenomena rather than in cell proliferation.


Unusual effects of benzodiazepines and cyclodiene insecticides on an expressed invertebrate GABAA receptor.

We have previously reported [(1991) EMBO J. 10, 3239-3245] the sequence of an invertebrate gamma-aminobutyric acid (GABA) type A (GABAA) receptor polypeptide which forms homo-oligomeric GABA-gated, bicuculline-sensitive, chloride-ion channels upon heterologous expression. We now demonstrate that the benzodiazepines Ro5-4864 (4'-chlorodiazepam) and diazepam, that are active at mammalian peripheral benzodiazepine sites, and not those benzodiazepines specific for central sites, directly active the homo-oligomeric receptor and evoke larger maximal responses than those elicited by GABA. In addition, members of the cyclodiene class of insecticides block the channel of the receptor in a manner indistinguishable from that of picrotoxin.


Glycopeptide of P0 protein inhibits homophilic cell adhesion. Competition assay with transformants and peptides.

Expression of major myelin glycoprotein P0 by P0 cDNA transfection into C6 glioma cells promoted homophilic cell adhesion of the cells. After the dissociated cells were incubated for various times, the number of particles at each time point was measured. The total number of particles decreased to 24% in 60 min for transformant (C6P0) cells, in contrast to only 68% for control (C6P0') cells. To confirm the homophilic mechanism of adhesion, mixed-cell aggregation experiments were performed. Among the four synthetic peptides corresponding to a part of the P0 sequence used, only peptide 3 (residues 90-96), which contained a carbohydrate attaching site, caused considerable inhibition of cell aggregation (approximately 50%). In addition, the glycopeptide (residues 91-95) obtained from bovine P0 markedly inhibited cell aggregation (by approximately 85%).


Insulin-like growth factor II stimulates glucose transport in human skeletal muscle.

We investigated the effect of insulin-like growth factor II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) on 3-O-methylglucose transport in incubated human skeletal muscle strips. Increasing physiological concentrations of IGF-II stimulated glucose transport in a dose-dependent manner. Glucose transport was maximally stimulated in the presence of 100 ng/ml (13.4 nM) of IGF-II, which corresponded to the effect obtained by 100 microU/ml (0.6 nM) of insulin. Exposure of muscle strips to IGFBP-1 (500 ng/ml) inhibited the maximal effect of IGF-II on glucose transport by 40%. Thus, it is conceivable that IGF-II and IGFBP-1 are physiological regulators of the glucose transport process in human skeletal muscle.


Monitor peptide gene expression is increased by exogenous CCK in the rat pancreas and in a rat pancreatic acinar cell line (AR4-2J).

Monitor peptide (CCK-releasing peptide) mRNA increased on the administration of CCK in rat pancreas and the AR4-2J pancreatic cell line. Subcutaneous injection of CCK into rats at 8 h intervals increased the level of monitor peptide mRNA in the pancreas. Concomitant injection of CCK antagonist CR-1409 strongly decreased it. The monitor peptide mRNA was also increased by CCK in AR4-2J cells and was decreased by the antagonist. These findings suggest that the plasma CCK induced by prolonged intake of a high protein diet may be responsible for the adaptative increase in the monitor peptide as well as exocrine proteases in the pancreas.


Class II tubulin, the major brain beta tubulin isotype is polyglutamylated on glutamic acid residue 435.

Protein sequencing shows that porcine brain tubulin retains the N-terminal sequences of alpha and beta tubulin after a mild treatment with subtilisin. C-terminal peptides released by subtilisin were purified and characterized by automated Edman degradation and mass spectrometry. We confirm the polyglutamylation of alpha tubulin on glutamic acid residue 445 reported by others and show in addition that class II beta tubulin, the major beta tubulin isotype of adult brain, is also polyglutamylated. The substitution is restricted to glutamic acid residue 435. Thus all major tubulin isotypes of adult brain are subjected to polyglutamylation.


Induction of Ca2+/calmodulin-dependent NO synthase in various organs of rats by Propionibacterium acnes and lipopolysaccharide treatment.

Ca2+/calmodulin-dependent nitric oxide synthase was found to be induced during rat liver necrosis caused by administration of Propionibacterium acnes and E. coli lipopolysaccharide to rats. Examination of the specific induction of Ca2+/calmodulin-dependent NO synthase showed that the enzyme was induced in the lung, spleen and colon as well as the liver. Northern blot analysis revealed that the induction occurred at the transcriptional level.


Bleomycin-reactive iron in patients with acute non-lymphocytic leukemia.

Bleomycin-reactive iron was detected in the sera of six out of nine adults undergoing intensive chemotherapy for acute non-lymphocytic leukemia. In these individuals the corresponding transferrin saturation ranged from 96% to 113% and the serum ferritin from 775 to 9975 micrograms/l. Nontransferrin-bound iron has been postulated to be a factor in organ toxicity in iron overload conditions such as beta thalassemia and hereditary hemochromatosis by facilitating the production of tissue-damaging free radicals. We propose that bleomycin-reactive iron should be considered as a possible factor in organ dysfunction seen with intensive cancer chemotherapy.


Alpha 2-macroglobulin and other proteinase inhibitors do not interfere with the secretion of amyloid precursor protein in mouse neuroblastoma cells.

A series of proteinase inhibitors active against proteinases of all four major classes, including highly purified and well-characterized alpha 2-macroglobulin, added to the cell culture medium of murine Neuro 2a neuroblastoma cells did not interfere with APP secretase activity. We therefore advance the hypothesis that APP secretase activity is localized in an intracellular compartment.


Molecular cloning and characterization of a novel calcium channel from rabbit brain.

The complete amino acid sequence of a novel calcium channel (designated BII) from rabbit brain has been deduced by cloning and sequencing the cDNA. The BII calcium channel is structurally more closely related to the BI calcium channel than to the cardiac and skeletal muscle L-type calcium channels. Blot hybridization analysis of RNA from different tissues and from different regions of the brain shows that the BII calcium channel is distributed predominantly in the brain, being abundant in the cerebral cortex, hippocampus and corpus striatum.


Subunit interactions of the Go protein.

The monoclonal antibody, MONO, recognizes an epitope on the G protein alpha o-subunit [van der Voorn et al., submitted] and readily immunoprecipitates heterotrimeric Go proteins from solubilized, crude bovine brain membranes, as well as from a purified bovine brain G protein preparation. Upon incubation of the immunoprecipitates with GTP gamma S, all beta gamma-subunits are released from the alpha o-subunit. Thus, binding of MONO to the Go protein does not appear to interfere with release of bound GDP, binding of GTP gamma S or GTP gamma S-induced subunit dissociation. However, we have been unable to induce a similar dissociation of Go using its physiological activator, GTP. Surprisingly, we did not observe any dissociation of Go (bound to MONO) upon dilution in a range from 500 to 5 nM. Since an apparent Kd of alpha o-GDP for binding beta gamma of 340-390 nM has been reported [(1989) J. Biol. Chem. 264, 20688-20696] our results would suggest that binding of MONO to the alpha o-subunit induces an increased affinity of alpha o-GDP for beta gamma. Alternatively, these results could be explained if, under the conditions used, the Kd of alpha o-GDP for beta gamma were at least two orders of magnitude lower than estimated previously.


Single channel recording in the chloroplast envelope.

The patch-clamp technique was applied to study ion channels in the intact chloroplast envelope. Three channel types were characterized: two cation-selective, with a conductance (in 100 mM KCl) of 517 and 1016 pS, respectively, and one anion-selective with a conductance of 159 pS. All three channels showed voltage-dependent closures at both positive and negative membrane potentials.