Different effect of testosterone on polypotential stem hematopoietic stem cells and immunocompetent B-lymphocytes]

A research was made to study the dynamics of the proliferative, colony-forming and migration capacity of stem hemopoietic cells in (CBA X C57Bl) F1 hybrid mice under the influence of testosterone propionate, 10 mg/100 g, as well as the migration of immunocompetent B lymphocytes from the bone marrow to the spleen and the accumlation of their progeny, antibody-producing cells, in the spleen. The immunodepressive effect of testosterone was manifested by a decrease in the migration of B cells and the number of antibody-producing cells in the spleen. On the contrary, testosterone had a stimulating effect on the functional activity of stem hemopoietic cells, increasing their proliferation and migration. Under conditions of the suppressed erythropoietic differentiation of multipotent stem hemopoietic cells the injection of testosterone resulted in an increase in the number of antibody-producing cells in the spleen. This suggests that the stimulation of erythropoiesis and immunosuppression, induced by testosterone, are interconnected and determined by the direct action of the hormone on the cellular cycle of the stem cells, as well as by their prevailing differentiation towards the erythroid series, resulting in the decrease of their differentiation into B cells.     

Nifedipine inhibits the hypoxia-induced decrease in plasma renin activity in conscious dogs.

Acute hypoxia induces a decrease in plasma renin activity (PRA), mediated, e.g., by an increase in adenosine concentration, calcium channel activity, or inhibition of ATP-sensitive potassium channels. The decrease in PRA results in a decrease in angiotensin II (AngII) and plasma aldosterone concentration (PAC). This study investigates whether these hypoxia-induced mechanisms can be inhibited by the L-type voltage-dependent calcium channel antagonist nifedipine. Eight conscious, chronically tracheotomized dogs received a low sodium diet (0.5 mmol Na x kg body wt(-1) x day(-1)). The dogs were studied twice in randomized order, either with nifedipine infusion (1.5 microg x kg body wt(-1) x min(-1), Nifedipine) or without (Control). The dogs were breathing spontaneously: first hour, normoxia [inspiratory oxygen fraction (FiO2)=0.21]; second and third hour hypoxia (FiO2=0.1). In Controls, PRA (6.8+/-0.8 vs. 3.0+/-0.5 ngAngI x ml(-1) x min(-1)), AngII (13.3+/-1.9 vs. 7.3+/-1.9 pg/ml), and PAC (316+/-50 vs. 69+/-12 pg/ml) decreased during hypoxia (P<0.05). In Nifedipine experiments, PRA (6.5+/-0.9 vs. 10.5+/-2.4 ngAngI x ml(-1) x min(-1)) and AngII (14+/-1.1 vs. 18+/-3.9 pg/ml) increased during hypoxia, whereas the decrease in PAC (292+/-81 vs. 153+/-41 pg/ml) was blunted (P<0.05). These results foster the idea that the hypoxia-induced decrease in PRA involves L-type calcium channel activity.     

Promoting effect of bombesin on the cell proliferation in the rat endocrine pancreas during the early postnatal period

The present work investigated whether orally administered bombesin influences cell proliferation in the endocrine pancreatic islets of rats during the suckling period and after weaning. Four series of pups were given bombesin diluted in milk (20 micrograms/kg, 3 times daily) or milk alone for 5 days during either the first, second, third or fourth postnatal week of life. Oral administration was used because bombesin-like peptides have been identified in the breast milk of mammals. 45 min before death, animals were given a single [3H]thymidine pulse injection. Tissue sections were processed for radioautography; DNA labeling and mitotic indices were estimated after counting at least 1000 endocrine cells per rat pancreas. In control rats, the labeling and mitotic indices in pancreatic islets dropped regularly from the first week to the fourth week of life (3.6% +/- 0.2% versus 1.9% +/- 0.1% and 0.46% +/- 0.09% versus 0.08% +/- 0.02%, respectively). Orogastric bombesin administration significantly increased the DNA labeling and mitotic indices at the end of the first week (+20% and +62%, respectively) and second week of life (+37.5% and +49%, respectively) (P less than 0.05 to P less than 0.005), but did not modify these parameters for the third and subsequent weeks of life. Therefore, this study provides evidence that bombesin stimulates DNA synthesis and cell division in pancreatic endocrine cells during the developmental period.     

Purine 8-bromination modulates the ribonuclease L binding and activation abilities of 2',5'-oligoadenylates. Possible influence of glycosyl torsion angle

Analogs of the 2',5'-linked adenylate trimer diphosphate (pp5'A2'p5'A2'p5'A or 2-5A) containing 8-bromoadenosine in the first, second, third, first and third, or second and third nucleotide positions (from the 5' terminus) were synthesized and found to vary dramatically in their ability to bind to and activate the RNase L of mouse L cells. Whenever the 8-bromoadenosine residue was substituted for adenosine in the first or 5'-terminal residue, there resulted a marked decrease in ability to bind to the 2-5A-dependent endonuclease. A similar result was obtained when the second adenosine nucleotide was replaced by 8-bromoadenosine. To the contrary, all analogs that bore an 8-bromoadenosine (br8A) in the third or 2'-terminal position were bound about as well as parent 2-5A to RNase L. Additionally, the 5'-diphosphate pp5'A2'p5'A2'p5' (br8A) was 10 times more effective than 2-5A as an inhibitor of translation. An increase in stability could not explain this significantly enhanced ability since the 2'-terminally brominated analog showed a similar half-life to 2-5A itself. Finally of particular interest was the analog monophosphate p5'A2'p5'(br8A)2'p5'(br8A) which possessed nearly 10% of the translational inhibitory activity of 2-5A triphosphate itself. These results suggest that changes in the base-sugar torsion angles of 2-5A may modulate both binding to and activation of mouse L cell RNase L.     

A CD1a+/CD11c+ subset of human blood dendritic cells is a direct precursor of Langerhans cells.

Based on the relative expression of CD11c and CD1a, we have identified three fractions of dendritic cells (DCs) in human peripheral blood, including a direct precursor of Langerhans cells (LCs). The first two fractions were CD11c+ DCs, comprised of a major CD1a+/CD11c+ population (fraction 1), and a minor CD1a-/CD11c+ component (fraction 2). Both CD11c+ fractions displayed a monocyte-like morphology, endocytosed FITC-dextran, expressed CD45RO and myeloid markers such as CD13 and CD33, and possessed the receptor for GM-CSF. The third fraction was comprised of CD1a-/CD11c- DCs (fraction 3) and resembled plasmacytoid T cells. These did not uptake FITC-dextran, were negative for myeloid markers (CD13/CD33), and expressed CD45RA and a high level of IL-3Ralpha, but not GM-CSF receptors. After culture with IL-3, fraction 3 acquired the characteristics of mature DCs; however, the expression of CD62L (lymph node-homing molecules) remained unchanged, indicating that fraction 3 can be a precursor pool for previously described plasmacytoid T cells in lymphoid organs. Strikingly, the CD1a+/CD11c+ DCs (fraction 1) quickly acquired LC characteristics when cultured in the presence of GM-CSF + IL-4 + TGF-beta1. Thus, E-cadherin, Langerin, and Lag Ag were expressed within 1 day of culture, and typical Birbeck granules were observed. In contrast, neither CD1a-/CD11c+ (fraction 2) nor CD1a-/CD11c- (fraction 3) cells had the capacity to differentiate into LCs. Furthermore, CD14+ monocytes only expressed E-cadherin, but lacked the other LC markers after culture in these cytokines. Therefore, CD1a+/CD11c+ DCs are the direct precursors of LCs in peripheral blood.     

Conditions for antitumor cytotoxic T-lymphocyte formation and inhibition of their activity by T-suppressors in mixed culture of human lymphocytes and tumor cells

Conditions for antitumour autolytic T lymphocytes (CTL) induction during human mixed lymphocyte tumour cell culturing (MLTC), as well as the patterns of CTL activation abolition in the experimental system by suppressor cells were investigated. The responders used in MLTC were peripheral blood lymphocytes of colorectal cancer patients fractionated by means of multilayer Percoll gradients centrifugation (the density of layers being 1.077, 1.067, 1.056 g/ml). The cells of the second fraction collected in the density interphase of 1.077-1.067 g/ml (more than 90% of population belonging to T lymphocytes), when used as responders in MLTC, developed an autolytic activity against autologous and allogeneic tumour target cells. The cell of the first fraction (collected in the interphase of 1.067-1.056 g/ml) added to the second fraction cells at the beginning of MLTC, prevented the following CTL induction. The first fraction contained T-suppressor cells capable of strongly interfering with antitumour CTL activity.     

Glycated albumin and glycated hemoglobin levels in normal pregnancy

Glycated albumin levels showed a progressive increase during normal pregnancy. The mean values (mole hexose/mole protein) were 1.68 +/- 0.27 (n = 15) in nonpregnant women, 1.83 +/- 0.21 (n = 11) in first trimester, 2.00 +/- 0.41 (n = 13) in second trimester, and 2.42 +/- 0.49 (n = 15) in third trimester. Glycated hemoglobin levels indicated a biphase pattern with low values at midpregnancy (controls 0.29 +/- 0.05, first trimester 0.30 +/- 0.04, second trimester 0.27 +/- 0.05, and third trimester 0.33 +/- 0.04). The data suggest that glycated albumin reflects the decreased glucose tolerance in pregnancy better that glycated hemoglobin levels. The reasons for the differing pattern of the two glycated proteins are discussed.     

The adaptor disabled-2 binds to the third psi xNPxY sequence on the cytoplasmic tail of megalin

The cytoplasmic tail (CT) of megalin possesses several functional motifs likely to participate in protein-protein interactions within the proximal tubular epithelial cell (PTEC) of the kidney. One such interaction is with the phosphotyrosine interaction domain (PID) of the adaptor protein disabled-2 (Dab2), a mitogen-responsive phosphoprotein, which interacts via its PID with Psi xNPxY (where Psi represents a hydrophobic residue) motifs on its binding partners. Megalin CT has three such motifs; it has been established that there is no interaction of Dab2 with the first (from N to C) (Biochem. J. 3 (2000) 613). Here, we analyse in real-time the binding of recombinant megalin CT, and of synthetic peptide sequences encompassing the second and third Psi xNPxY motifs, to Dab2PID in real-time using surface plasmon resonance (SPR). We report a binding affinity of DabPID for megalin CT of K(D) = 2.6 x 10(-7) +/- 5.3 x 10(-8). Direct binding and competition studies indicate that this interaction is with the third Psi xNPxY motif. The dissociation of Dab2 from the third Psi xNPxY peptide was significantly slower than that from the second (k(off) (mean +/- S.E.M.) (per s) = 0.002 +/- 0.002 vs. 0.007 +/- 0.002, P < 0.05). Synthetic peptide sequences encompassing the third Psi xNPxY but not the second inhibited Dab2PID binding both to intact megalin CT and to the third Psi xNPxY motif. Tyrosine phosphorylation of either motif did not exert a major effect upon competition efficacy. We further demonstrate for the first time the presence of Dab2 expression in primary human PTEC.     

Separation and properties of the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus

We have developed methods for separating the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus. The total membrane fraction from ethylenediaminetetraacetic acid-lysozyme-treated cells was resolved into three major fractions by isopycnic density centrifugation. Between 85 and 90% of the succinate dehydrogenase and cyanide-sensitive reduced nicotinamide adenine dinucleotide oxidase activity was found in the first (I) fraction (rho = 1.221 g/ml) and 80% of the membrane-associated 2-keto-3-deoxyoctonate was found in the third (III) fraction (rho = 1.166 g/ml). The middle (II) fraction (rho = 1.185 g/ml) appeared to be a hybrid membrane fraction and contained roughly 10 to 20% of the activity of the enzyme markers and 2-keto-3-deoxyoctonate. No significant amounts of deoxyribonucleic acid or ribonucleic acid were present in the three isolated fractions, although 26% of the total cellular deoxyribonucleic acid and 3% of the total ribonucleic acid were recovered with the total membrane fraction. Phosphatidylethanolamine made up the bulk (60 to 70%) of the phospholipids in the membrane fractions. However, virtually all of the phosphatidylserine and cardiolipin were found in fraction I. Fraction III appeared to contain elevated amounts of lysophospholipids and contained almost three times the amount of total phospholipid as compared with fraction I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved approximately 40 polypeptides in the total membrane fraction. Two-thirds of these polypeptides were enriched in fraction I, and the remainder was enriched in fraction III. Fraction II contained a banding pattern similar to the total membrane fraction. Electron microscopy revealed that vegetative cells of M. xanthus possessed an envelope similar to that of other gram-negative bacteria; however, the vesicular appearance of the isolated membranes was somewhat different from those reported for Escherichia coli and Salmonella typhimurium. The atypically low bouyant density of the outer membrane of M. xanthus is discussed with regard to the high phospholipid content of the outer membrane.     

Development of acetabulum after closed reduction in developmental hip dysplasia

The radiographic study of acetabulum development after closed reduction of 87 congenitally dislocated hips in 64 patients who were treated between 1980 and 1990 was conducted retrospectively. An average patient's age at the moment of closed hip reduction was 16 months (range between 6 to 24 months). On the average, patients were 16 months old (range between 6 to 24 months) at the moment of closed hip reduction. Development of bony acetabulum was followed for 8 years by measuring the AC angle. According to the age at the moment of closed reduction patients were sorted into three groups, the first group from 6 months to 12 months old (39 hips), the second group from 12 to 24 months old (28 hips) and the third group from 24 to 36 months old (20 hips). At the beginning of the treatment mean values of the AC angle in the first group were 37.2 degrees +/- 3.31 degrees; in the second group 39.7 degrees +/- 3.2 degrees; and in the third group 43.8 degrees +/- 2.7 degrees. The final values of the AC angle in the first group were 14.7 degrees +/- 3.21 degrees, in the second group 21 degrees +/- 3.02 degrees; and in the third group 24 degrees +/- 3.76 degrees. Normally and mildly dysplastic hips were achieved in 80.5% treated hips, while in 19.5% treated hips medium and serious dysplasia retarded. The acetabulum development in dysplastic hip after retained stable concentric reduction was the same as in the normal hip. The operative reconstruction of acetabulum should not be done before the third year of life.     

Immunological aspects of retinoids in humans. II. Retinoic acid enhances induction of hemolytic plaque-forming cells

The effects of retinoic acid (RA) on the induction of antibody-producing cells from human tonsillar lymphocytes sensitized to sheep erythrocytes (SRBC) have been evaluated. Our results indicated that 10(-5) to 10(-7) M RA caused up to a three-fold increase in the number of plaque-forming cells (PFC) and a qualitative increase in the size of the plaques during the induction of PFC in 5- to 7-day cultures. Enhancement also occurred when tonsil cells were preincubated with RA for 24 hr and then washed, or when RA was added any time in the first 4 days after initiation of the culture. When T- and B-cell fractions were pretreated with RA for 24 hr, washed, and recombined with SRBC, RA-induced augmentation of PFC occurred only in conjunction with RA treatment of the B-cell fraction. Pretreatment of the T-cell fraction had no effect on PFC induction or on the RA-enhanced response when the B-cell fraction was simultaneously treated with RA. Other experiments suggested that RA did not modulate PFC induction by influencing regulatory functions of adherent accessory cells. Our study demonstrates that RA can enhance human antibody responses and shows that this effect is not caused by increased activity of T cells or adherent accessory cells, but is instead the result of a direct effect of RA on B-cell populations.     

Segregation of rapidly acetylated histones into a chromatin fraction released from intact nuclei by the action of micrococcal nuclease.

It has been previously shown that micrococcal nuclease digestion and subsequent fractionation of hen oviduct nuclei generates fractions enriched (first supernatant fraction - 1SF) and depleted (second supernatant fraction - 2SF) in ovalbumin genes, while a third fraction, the pellet fraction, contains about the same level of this gene as whole chromatin (Bloom and Anderson (1978) Cell 15, 141-150). We have utilized this fractionation method in an attempt to assess the extent and kinetics of histone acetylation associated with chromatin from the 1SF, 2SF, and pellet fraction. Hepatoma Tissue Culture (HTC) cells were labelled for 30 minutes in vivo with 3H-acetate, nuclei isolated and the chromatin fractionated. The specific activity of the histones in the 1SF was slightly greater than that of the 2SF (1.2 to 1.6 fold difference) independent of the length of nuclease digestion. If the labelling period is followed by short (10 to 60 minute) treatment of the cells with sodium butyrate, the more rapidly as well as more extensively acetylated histones are also preferentially found in the 1SF. This is in part the result of segregation of chromatin particles into the 1SF as the histones associated with these particles become hyperacetylated. That is, the extent of histone acetylation regulates the distribution of chromatin in the 1SF, 2SF and pellet fraction.     

Glycolate oxidase isoforms are distributed between the bundle sheath and mesophyll tissues of maize leaves

Glycolate oxidase (EC 1.1.3.15) activity was detected both in the bundle sheath (79%) and mesophyll (21%) tissues of maize leaves. Three peaks of glycolate oxidase activity were separated from maize leaves by the linear KCl gradient elution from the DEAE-Toyopearl column. The first peak corresponded to the glycolate oxidase isoenzyme located in the bundle sheath cells, the second peak had a dual location and the third peak was related to the mesophyll fraction. The mesophyll isoenzyme showed higher affinity for glycolate (Km 23 micromol x L(-1)) and a higher pH optimum (7.5-7.6) as compared to the bundle sheath isoenzyme (Km 65 micromol x L(-1), pH optimum 7.3). The bundle sheath isoenzyme was strongly activated by isocitrate and by succinate while the mesophyll isoenzyme was activated by isocitrate only slightly and was inhibited by succinate. It is concluded that although the glycolate oxidase activity is mainly attributed to the bundle sheath, conversion of glycolate to glyoxylate occurs also in the mesophyll tissue of C4 plant leaves.     

Replacement of the helper function of T cells by an RNA-containing antigen-specific lysis factor]

The lymph node viable cells suspension of immunized mice was centrifugated. The supernatant was chromatographed in Sephadex G-200, and fractions were deproteinized. The deproteinized third fraction (Mol wt 30000) stimulated specifically the plaque-forming cells of intact mice immunized by SRBC. It restored the capacity to antibody production in the lethally irradiated intact mice protected by the syngeneic bone marrow. The activity of this fraction disappeared following treatment with RNA-ase, but not with DNA-ase or trypsin. The first and the second deproteinized fractions of the supernatant inhibited non-specifically the viable lymph node cells of the immunized animals in the intact mice immunized with SRBC.     

Antiviral and antiproliferative activity of human fibroblast interferon fractions

The affinity chromatography of Human crude beta-interferon preparations on Blue Dextran Sepharose columns resulted in isolation of several fractions with different ratio of antiviral to antiproliferative activities. The results of investigation of two of these fractions are described in this report. The first of them was eluted by 1N NaCl in 0.01 M tris buffer at pH 7.8, the second was eluted by 1 M NaCl, 50% methylethylenglycol in 0.01 M tris-HCl buffer at pH 7.8. The first of the fractions possessed presumably antiproliferative and the second presumably antiviral activity. Both fractions induced the increase of 2'5'-oligoadenylatesynthetase activity in cells although the inducing activity of the first fraction was about 6-fold higher than that of the second one as compared with their antiviral activities. The obtained results indicate that purification of interferon preparation for interferons main antiviral activity may lead to the loss of the great part of antiproliferative material.     

Immunodepressive effect of phentyrin]

A study was made of the immunodepressive activity of phentirin (dichlohydrate o/p-di (2-cholorethyl) aminophenyl/D,L-tyrosine). The preparation was administered to mice in a dose of 50 mg/kg, per os. Experimental results showed phentirin to produce a marked immunodepressive action on the transplantation immunity and the production of plaque- and rosette-forming cells. Immunodepressive action of phentirin was more pronounced in comparison with azaprine under the same experimental conditions.     

Distribution of Ca2+-ATPase, ATP-dependent Ca2+-transport, calmodulin and vitamin D-dependent Ca2+-binding protein along the villus-crypt axis in rat duodenum

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca2+-ATPase activity and ATP-dependent Ca2+-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na+ + K+)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca2+-transport in basolateral membranes was highest in fraction II (8.2 +/- 0.3 nmol Ca2+/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca2+-transport activity (0.9 +/- 0.1 nmol Ca2+/min per mg protein). The distribution of high-affinity Ca2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca2+-transport. The activity of Na+/Ca2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca2+-transport system, the Na+/Ca2+ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.     

Estimation of chlorophyll a distribution in the photosynthetic pigment systems I and II of the blue-green alga Anabaena variabilis.

Chlorophyll a distribution in pigment systems I and II was estimated with the blue-green alga Anabaena variabilis by two methods: first, with intact cells using delayed light emission as an index reaction; second, by measuring the 2,6-dichlorophenolindophenol-Hill reaction and the cytochrome c photooxidation in membrane fragments. The first estimation indicated that 0.053+/-0.014 of total chlorophyll a functions as a component of pigment system II, and the second method 0.086+/-0.012. Though the values were somewhat different in the two methods, both estimations indicated that pigment system II chlorophyll a occupies a very small fraction of total chlorophyll a.     

Regulation of the growth of Epstein-Barr virus-infected B cells: temporal profile of the in vitro development of three distinct cytotoxic cells

The time course of the appearance of cytotoxic cells was examined in cocultures of E-rosetting (E+) cells and EBV-infected non-T cells (4:1 ratio) from the blood of VCA-positive healthy adults. Classical HNK-1+ NK cells were present at the initiation of the cultures and they produced 76 +/- 2% specific 51Cr-release from K-562 cells, but they did not effectively lyse the NK-resistant Daudi cells, nor did they kill autologous EBV-induced lymphoblasts (LCLEBV). The NK activity decreased during the first week in culture to 40 +/- 7% cytotoxicity. At the same time, nonspecific cytotoxic cells capable of killing Daudi as well as K562 developed and persisted into the third week in culture when it declined. This later nonspecific cytotoxicity was mediated by 4F2+, T8-, HNK-1- activated E+ cells. After 10 days in culture, killing of autologous LCLEBV increased continuously, from 4 +/- 3% at Day 10 to 38 +/- 4% by Day 22. The cytotoxicity to LCLEBV was mediated by classical T8+ CTL, and it was antigen specific and at least partially HLA Class I restricted. The regression of BEBV growth that occurs in E+/BEBV cocultures coincides with the development of this CTL-mediated cytotoxicity.     

Studies on the relationship of hepatic anion-binding proteins and sulfobromophthalein-glutathione conjugation in normal and phenobarbital-treated rats

The enzyme activity which conjugates sulfobromophthalein with glutathione was separated from rat liver supernate by Sephadex G-75 gel filtration, and assayed by two different methods; paper electrophoresis and spectrophotometry. The enzyme activity was found mainly in the second protein fraction, and less than 5% of the activity in the first or third protein fractions. In vitro mixtures of sulfobromophthalein, [³H]glutathione and rat liver supernate showed that the major part of [³H]glutathione was detected in the first and second protein fractions, and the remainder in the third protein fraction. Phenobarbital treatment caused an increase of the enzyme activity, sulfobromophthalein and [³H]glutathione, in the second protein fraction.