Sequential sodium-proton exchange in thrombin-induced human platelets

Thrombin stimulation of human platelets initiates a membrane depolarization attributable to a Na+ influx into, and an alkalinization of, the cytoplasm, both of which follow a similar rapid time scale and thrombin-dose dependence. These responses precede secretion of the contents of the dense granules (serotonin) and, after 1 minute, of lysosomes (beta-glucuronidase). We have evaluated these parameters in the presence of 2H2O in order to determine if the Na+ influx and H+ efflux are sequential or simultaneous. NMR evidence indicates that 2H2O equilibration in rapid, and virtually complete within the 3 min prestimulation platelet equilibration period. In response to an 0.05 U/ml addition of thrombin, the rate of depolarization is 70-80% slower in 2H2O than in H2O. The time to reach maximal depolarization is 5 to 10 seconds longer in 2H2O, the extent of depolarization 60% inhibited, and the pH change 85% inhibited. The serotonin secretion is unaltered, while the beta-glucuronidase secretion is 130-180% enhanced. Dimethylamiloride inhibits the Na+ influx and the pH change completely. These results suggest that the Na+ and H+ fluxes across the plasma membrane are interdependent but neither simultaneous nor electroneutral. Furthermore, granule secretion, previously shown by us to be independent of the existent Na+ gradient, depends on the cytoplasmic K+ and H+ concentrations.     

IFN-gamma regulates the isotypes of Ig secreted during in vivo humoral immune responses

The lymphokine IFN-gamma has been shown in vitro to stimulate IgG2a secretion and inhibit IgG1 and IgE secretion by LPS-activated B lymphocytes. To determine whether IFN-gamma has a similar isotype regulatory role in vivo, we studied the abilities of rIFN-gamma and a mAb to IFN-gamma to modify the isotypes of Ig secreted in mice injected with a goat antibody to mouse IgD, which by itself induces large increases in levels of serum IgG1 and IgE and a relatively small increase in serum IgG2a. Multiple injections of IFN-gamma substantially inhibited production of IgG1 and IgE, and stimulated production of IgG2a in affinity purified goat antibody specific for mouse IgD-treated mice; anti-IFN-gamma antibody blocked the effects of IFN-gamma and in fact enhanced IgG1 and IgE secretion and inhibited the IgG2a response in these mice. The role of IFN-gamma in the selection of isotypes of Ig produced in response to injection of mice with the bacterium Brucella abortus (BA) was also studied, because killed, fixed BA are known to stimulate IFN secretion and a predominantly IgG2a antibody response. Anti-IFN-gamma antibody strongly suppressed IgG2a secretion and stimulated IgG1, but not IgE, secretion in BA-immunized mice. BA suppressed IgG1 and IgE secretion and enhanced IgG2a secretion in affinity purified goat antibody specific for mouse IgD-injected mice; treatment of these mice with anti-IFN-gamma antibody reversed the effects of BA on IgG1 and IgG2a secretion, but not the suppressive effect of BA on IgE secretion. These observations demonstrate that IFN-gamma has an important and perhaps unique physiologic role in the stimulation of IgG2a secretion and in the suppression of secretion of IgG1, whereas bacterial antigens can suppress IgE secretion by other mechanisms in addition to IFN-gamma secretion.     

Anti-Ig-mediated proliferation of human B cells in the absence of protein kinase C

Cross-linking of surface Ig has been shown to stimulate phosphatidylinositol hydrolysis in murine B cells, leading to increases in [Ca2+]i and activation of protein kinase C (PKC). Preliminary evidence suggests that a similar activation mechanism occurs in human B cells. We wished to examine whether anti-Ig antibody-stimulated human B cell proliferation is as dependent upon the presence of PKC as is anti-Ig-mediated murine B cell proliferation. Using highly purified, small, dense peripheral-blood B lymphocytes from healthy adult donors, we confirmed that PMA, a direct activator of PKC, is a potent mitogen for human B cells that synergizes with anti-mu antibody. Furthermore, we demonstrated that PMA treatment abolishes detectable cellular stores of immunoreactive PKC. However, after such depletion of cellular PKC, anti-mu antibody is still capable of delivering a proliferative signal to human B cells. It is unlikely that this signal occurs solely on the basis of increases in [Ca2+]i, because the calcium ionophore A23187 does not induce a proliferative response in PMA-treated B cells similar in magnitude to that seen with anti-mu. Additionally, the finding that pretreatment of B cells with PMA ablates the ability of anti-Ig antibody to mobilize intracellular and extracellular calcium also suggests that the ability of PMA to enhance anti-Ig mediated stimulation does not depend on elevations of [Ca2+]i induced by anti-Ig. Together, these observations suggest that anti-Ig signaling of human B cells may occur via other pathways in addition to the phosphatidylinositol system of calcium influx and PKC activation.     

Growth characteristics of the copepodsEurytemora affinis andE. herdmani in laboratory cultures

Die calanoiden CopepodenEurytemora affinis (Poppe) undE. herdmani Thompson &Scott wurden über zahlreiche Generationen im Laboratorium gezüchtet. Mit einem Überschuß an Algennahrung vermehrt sichE. affinis in Salzgehalten zwischen 5 und 33 ‰ und bei Temperaturen zwischen 2° und 23,5° C.E. herdmani pflanzt sich in Salzgehalten unter 15 ‰ und bei 21,5° C und höheren Temperaturen nicht fort. Diese Ergebnisse stimmen überein mit Untersuchungen im natürlichen Lebensraum und zeigen, daßE. herdmani an kälteres und salzhaltigeres Wasser gebunden ist als die weiter verbreiteteE. affinis. An den Jungtieren, die von einzelnen Weibchen erhalten worden waren, wurden die Entwicklungszeitspannen vom Ei bis zum ersten Nauplius, ersten Copepoditen, Adultus und eiertragenden Weibchen ermittelt. In 20 ‰ Salzgehalt variiert die Generationszeit beiE. affinis von etwa 105 Tagen bei 2° C bis 9 Tagen bei 23,5° C. Die entsprechenden Generationszeiten fürE. herdmani betragen 73 Tage bei 2° C und 19 Tage bei 15° C. Die Körperlänge beider Arten nimmt bei niedrigeren Temperaturen zu. Die Errechnung des Zahlenverhältnisses der Geschlechter der Jungtiere einzelner Weibchen legt die Vermutung nahe, daß die Temperatur das Geschlechtsverhältnis beeinflußt. Überlebenszeiten wurden ermittelt für Weibchen, welche unter verschiedenen Temperatur- und Salzgehaltsbedingungen gehalten worden waren. Die Lebensspannen von Weibchen beider Arten übertreffen 100 Tage bei 2° C und nehmen bei höheren Temperaturen ab. Weibchen beider Arten vermögen noch lange nach der Kopulation fertile Eier zu produzieren. Diese Beobachtungen sprechen dafür, daß kleine Lokalpopulationen möglicherweise im Herbst kopulieren, dann überwintern und daß die Weibchen im Frühjahr ohne erneute Kopulation Junge produzieren.     

Depression and other psychiatric morbidity in carers of elderly people living at home.

OBJECTIVE--To describe the mental health of a community sample of carers of elderly people with dementia, depression, or physical disability and to compare that with the mental health of other adults living in the household and of those living alone. DESIGN--Assessment of psychiatric morbidity and physical disability with standardised questionnaire in randomly selected enumeration districts; subjects were interviewed at home. SETTING--London Borough of Islington. SUBJECTS--700 people aged > or = 65 and other coresidents. MAIN OUTCOME MEASURE--Depression measured with standardised interview. RESULTS--The prevalence of depression was not significantly higher in carers overall (15%) than in coresidents (11%). Being a woman carer was a significant predictor of psychiatric illness. Depression was more common in the carers of people with a psychiatric disorder than in coresidents (24% v 11%, P < 0.05) and in those living alone (19%). Depression was most common (47%) in women carers of people with dementia. CONCLUSION--The increase in psychiatric morbidity reported in carers of people with psychiatric disorders may reflect the lack of a confiding relationship.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Heligmosomoides polygyrus: CD4+ but not CD8+ T cells regulate the IgE response and protective immunity in mice.

Oral inoculation of BALB/c mice with infective larvae of Heligmosomoides polygyrus resulted in chronic infection characterized by the release of parasite eggs in the feces for several months. The actual number of eggs per gram of feces was dependent on the dose of the inoculum. Serum IgE in infected mice peaked at a level of greater than 70 micrograms/ml during Weeks 3 through 6 following inoculation, and high levels of IgE (greater than 40 micrograms/ml) persisted for over 14 weeks. Protective immune responses resulted in reduced egg production and the development of markedly fewer adult worms in the small intestines following a challenge inoculation. The role of CD4+ and CD8+ T cells in these responses was examined by depletion in vivo of either T cell subpopulation with rat mAb specific for the appropriate determinants. Mice treated with anti-CD4 during a primary infection had increased EPG which was due primarily to an increase in worm fecundity (eggs produced per adult female). A challenge inoculation of mice that had been cleared of the primary infection with an anthelmintic drug induced a protective response that reduced development of new adult worms by 70-80% and their fecundity by greater than 90%. This protective response was abrogated by injection of mice with anti-CD4. Serum IgE diminished when adult worms were removed after anthelmintic treatment. A more precipitous drop in serum IgE followed successive treatments of mice with an anthelmintic and anti-CD4. In addition, the anamnestic serum IgE response to a challenge inoculation was reduced by over 80% in anti-CD4-treated mice. Anti-CD8 treatment had no appreciable effect on the immunological or parasitological parameters measured following a challenge inoculation with H. polygyrus. Thus, CD4+ T cells regulate host protective immunity, worm fecundity, and IgE levels in an H. polygyrus infection. This experimental system may be particularly suitable for analysis of chronic nematode infections of humans and livestock because of the responsiveness of the parasite in vivo to changes in host immune function.     

Comparative investigation on the extractibility of acetylcholinesterase from normal and dystrophic muscles

Following combined administration of nonionic detergent and collagenase considerable differences were observed in extractibility of acetylcholinesterase activity from normal and from dystrophic rabbit muscles. The results obtained in the experiments carried out on vitamin-E deficient rats corroborated the authors' previous experiences obtained in vitamin-E deficient rabbits. However, as to the effect of the detergent, considerable differences were observed between the two species. If one compares the activities referred to 1 g muscle or to the total tissue mass it is clear that primarily the concentration of acetylcholinesterase was increased. The activity measured in the presence of butyrylthiocholine increased only to small extent in vitamin-E deficient dystrophy.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.