Elevated 4-hydroxylation of estradiol by hamster kidney microsomes: a potential pathway of metabolic activation of estrogens. Characterization of enzymes mediating the formation of catecholestrogens (CE) by hamster kidney is of importance because of the proposed role of CE in renal cancer induced in this species by estrogens. We have reexamined the potential of hamster kidney to convert estradiol (E2) to 2- and 4-hydroxylated CE because of recent evidence of the limitations of assays used in previous studies, in particular in measuring 4-hydroxylation of estrogens. Under conditions optimized for NADPH-dependent activity, hamster kidney microsomes exhibited high levels of both E2-2- and E2-4-hydroxylase activities. Evidence that the two activities depend on different forms of cytochrome P-450 was obtained by the demonstration that 2- and 4-hydroxylation of E2 were affected differentially 1) by chronic treatment of hamsters with E2 and 2) by fadrozole hydrochloride, a selective cytochrome P-450 inhibitor. NADPH-dependent 2-hydroxylation of E2 from control and E2-treated hamsters, measured by a direct product isolation assay, was 1 order of magnitude higher (apparent maximum velocity, 24-32 and 6-12.5 pmol/mg protein.min in control and E2-treated hamsters, respectively) than that reported previously using radioenzymatic assays. NADPH-dependent 4-hydroxylation of E2 in controls approached and in E2-treated hamsters exceeded 2-hydroxylation of E2 (apparent maximum velocity, 17-21 and 7.5-19 pmol/mg protein.min in control and E2-treated hamsters, respectively). Thus, estrogen treatment reversed the ratios of NADPH-dependent E2-2-/4-hydroxylase activities by causing a much greater decline in 2- than 4-hydroxylation of E2 (P less than 0.007, by analysis of variance). Fadrozole hydrochloride caused a marked dose-dependent decrease in 2-hydroxylation of E2, in contrast to a small nondose-dependent inhibition of 4-hydroxylation. Under conditions optimized for peroxidatic organic hydroperoxide-dependent activity, hamster kidney microsomes generated 2- and 4-hydroxylated CE in similar amounts. The amounts of the two CE and, consequently, the ratios remained unaffected by estrogen treatment (1:0.9 and 1:1.0 in control and E2-treated hamsters, respectively). Thus, this study establishes that CE can be generated in the same tissue by three different pathways, i.e. NADPH-dependent E2-2-hydroxylase, NADPH-dependent E2-4-hydroxylase, and organic hydroperoxide-dependent E2-2/4-hydroxylase activities. We also show that these three activities can be regulated differentially and are, thus, probably mediated by different forms of cytochrome P-450. In hamster kidney, the potential to generate 4-hydroxylated CE metabolites with distinct properties could be a factor in this tissue's vulnerability to estrogen-induced carcinogenesis.


Evidence for atrial natriuretic peptide-(5-28) production by rat placental cytotrophoblasts. Atrial natriuretic peptide (ANP), a 28-amino acid peptide, is produced and secreted by cardiac atriocytes to modulate cardiovascular and renal functions. We report here the production of ANP-(5-28) and its 15K mol wt (M(r)) presumptive precursors by cytotrophoblasts of rat placentae. Placental tissues were collected from Sprague-Dawley fetal rats on days 12, 16, 18, and 20 of gestation and acid extracted for immunoreactive (ir) ANP assay. The contents of placental irANP increased over the course of fetal growth, with the highest amount (mean +/- SE, 1083 +/- 125 pg/tissue; n = 7) found near term. Sephadex G-50 gel chromatographic profiles of the placental extract revealed a major peak of irANP coeluted with the 3K M(r) of synthetic rat (r) ANP-(1-28) and a minor peak in the position consistent with that of 15K M(r). HPLC analysis of the 3K M(r) species showed a single peak of immunoreactivity, which eluted with a retention time similar to that of rANP-(5-28). In placental sections, irANP and pro-ANP mRNA were localized by immunoperoxidase staining and colorimetric in situ hybridization in a subpopulation of placental cytotrophoblasts, but not in syncytiotrophoblasts or chorionic cells. Northern blot analysis showed a single band of pro-ANP mRNA in rat placental tissues similar in size to that found in the heart (approximately 0.85 kilobases), with the highest level of pro-ANP mRNA signal detected in the placentae of 16-day gestation fetuses. Our findings suggest that ANP is expressed and produced by a small population of rat placental cytotrophoblasts and that the 15K M(r) precursor peptide is extensively processed into the N-terminal-truncated form of ANP-(5-28) in the tissue.


A desiccation-related Elip-like gene from the resurrection plant Craterostigma plantagineum is regulated by light and ABA. The resurrection plant Craterostigma plantagineum tolerates an extreme loss of cellular water. Therefore this plant is being studied as model system to analyse desiccation tolerance at the molecular level. Upon dehydration, new transcripts are abundantly expressed in different tissues of the plant. One such desiccation-related nuclear gene (dsp-22 for desiccation stress protein) encodes a mature 21 kDa protein which accumulates in the chloroplasts. Sequence analysis indicates that dsp-22 is closely related to early light inducible genes (Elip) of higher plants and to a carotene biosynthesis related gene (cbr) isolated from the green alga Dunaliella bardawil. In contrast to other desiccation-related genes, light is an essential positive factor regulating the expression of dsp-22: ABA-mediated gene activation leads to the accumulation of the transcript only in the presence of light. During the desiccation process, light acts at the transcriptional and post-transcriptional levels. The implications of these different controls and the possible role of the dsp-22 protein in the desiccation/rehydration process are discussed.


Sensory mother cell division is specifically affected in a Cyclin-A mutant of Drosophila melanogaster. Cyclin proteins are one of the important components of the mechanism regulating mitosis in eukaryotic cells. We isolated a Drosophila Cyclin-A mutant in which the progenitor cells of the peripheral nervous system (the sensory mother cells) do not divide properly, causing the loss and other abnormalities of mechanosensory organs in the adult fly. Sequence analysis of the mutant genome reveals that a P element is inserted into the first intron of the Cyclin-A gene. A 13 kb wild-type genomic DNA containing the Cyclin-A transcription units rescued the mutant phenotype when introduced into the mutant fly. The regulation of cell type specific expression of the Cyclin-A gene is discussed.


Lac repressor with the helix-turn-helix motif of lambda cro binds to lac operator. Lac repressor, lambda cro protein and their operator complexes are structurally, biochemically and genetically well analysed. Both proteins contain a helix-turn-helix (HTH) motif which they use to bind specifically to their operators. The DNA sequences 5'-GTGA-3' and 5'-TCAC-3' recognized in palindromic lac operator are the same as in lambda operator but their order is inverted form head to head to tail to tail. Different modes of aggregation of the monomers of the two proteins determine the different arrangements of the HTH motifs. Here we show that the HTH motif of lambda cro protein can replace the HTH motif of Lac repressor without changing its specificity. Such hybrid Lac repressor is unstable. It binds in vitro more weakly than Lac repressor but with the same specificity to ideal lac operator. It does not bind to consensus lambda operator.


Solubility and posttranslational regulation of GP130/F11--a neuronal GPI-linked cell adhesion molecule enriched in the neuronal membrane skeleton. GP130 (renamed contactin) has previously been identified by its detergent insolubility and retention with the actin-containing "membrane skeleton" isolated from chicken neurons and brain. The contactin sequence predicted a transmembrane and cytoplasmic domain for the molecule. Recently, F11 was shown to have an identical sequence except for the C terminus, and it was predicted to be linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) group. Here we describe that GP130 can be released both from brain membranes and the detergent-insoluble membrane skeleton by a phosphoinositol-specific phospholipase C (PI-PLC) indicating that F11 and GP130/contactin are probably identical and that surprisingly the lipid anchor is partly or totally responsible for its non-ionic detergent insolubility. The "membrane skeleton" is a rich source of GPI-linked glycoproteins as judged by 1) most glycoproteins can be released by a PI-PLC and 2) most [3H]ethanolamine-labeled glycoproteins are present in, or enriched in the membrane skeleton. Thus, detergent insolubility appears to be a characteristic of GPI-anchored glycoproteins. No evidence has been obtained that GP130/F11 is released or secreted in vivo or in culture. In addition, GP130/F11 has an unusually long half-life in culture of greater than 3 days. The structure of the neuronal membrane skeleton and the potential function of GPI-anchored glycoproteins is discussed.


Lymphokine profile and activation pattern of two unrelated antigen- or idiotype-specific T suppressor cell clones. Two T suppressor (Ts) clones of different specificity have been analyzed for their lymphokine spectrum. BVI/5 is an I-Ek-restricted bovine serum albumin (BSA)-specific Ts cell clone from a CBA/J mouse tolerized by low doses of BSA. It affects directly or indirectly the function of BSA-specific T helper (Th) cells. The Ts cell clone 178-4 from a BALB/c mouse is I-Ed restricted and recognizes the public J558 Id on B cells. It prevents alpha(1----3)dextran B 1355S (Dex)-specific IgG antibody production and drives Dex-specific J558 idiotype-bearing B cells into an anergic B IgG memory cell state. Both Ts cell clones thus cause specific suppression, yet in different experimental systems using different effector mechanisms. Upon stimulation with concanavalin A or fixed CD3-specific monoclonal antibody, both clones produce high levels of interferon (IFN)-gamma and tumor necrosis factor (TNF) but in contrast to Th1 cells no interleukin (IL)-2. Both clones produce low levels of IL-3 and IL-6 but no IL-4, IL-5 and IL-9. Furthermore, unlike Th2 cells, both clones do not respond to IL-1. The mechanism of the idiotype-specific induction of anergy in Dex-specific B IgG memory cells by 178-4 Ts cells is not yet understood. BVI/5 Ts cells suppress in vitro the BSA-specific proliferation of the BSA-specific Th cell clone 83/1, as well as the response of BSA-primed CBA/LN cells. Whereas the suppressive effect on 83/1 cells is due to IFN-gamma alone the suppression of BSA-specific lymph node cells can be simulated neither by IFN-gamma nor the combination of IFN-gamma and TNF. Thus these mediators cannot account for the antigen-specific suppression by BVI/5 Ts cells in polyclonal in vitro responses from lymph node cells and probably not for the induction of in vivo unresponsiveness.


A protective NOD islet-infiltrating CD8+ T cell clone, I.S. 2.15, has in vitro immunosuppressive properties. Type 1, insulin-dependent diabetes mellitus (IDDM) appears to result from a T cell-dependent destruction of insulin-producing pancreatic beta cells. In non-obese diabetic (NOD) mice and in other rodent models of human IDDM, final expression of disease may be controlled by protective, as well as, destructive T cell influences. Previously, a CD8+ T cell clone, I.S. 2.15, was isolated directly from islets of disease-resistant male NOD mice. Upon transfer to young NOD recipients, the non-cytolytic I.S. 21.5 T cell clone, confers in vivo protection from two forms of accelerated IDDM. The present study demonstrates that I.S. 2.15 T cells induce in vitro immunosuppression. The suppressive effects of I.S. 2.15 T cells are mediated through soluble factor(s) and are independent of T cell activation, cell contact, antigen specificity or the major histocompatibility complex (MHC). By polymerase chain reaction (PCR), I.S. 2.15 T cells contain mRNA species encoding for the potentially immunosuppressive cytokines, interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta). The T cell suppressive effects engendered by I.S. 2.15 T cells closely mimic those observed with TGF-beta. Moreover, I.S. 2.15-induced immunosuppression correlates with intracellular levels of TGF-beta mRNA. These results establish that immunoregulatory T cells are present within islets in IDDM-resistant NOD mice and may impact on final disease expression through the production of soluble mediator(s).


Immunobiological studies on experimental visceral leishmaniasis. II. Adherent cell-mediated down-regulation of delayed-type hypersensitivity response and up-regulation of B cell activation. Visceral leishmaniasis or kala-azar is characterized by a variety of immunopathological consequences in man. The most remarkable of these are the depression of cell-mediated immunity and polyclonal B cell activation. The consequences observed in man could be induced in a murine model by inoculating the causative agent, Leishmania donovani. The cell-mediated response was studied in this murine model in terms of the delayed-type hypersensitivity (DTH) response toward leishmania antigen in a progressive infection. BALB/b (H-2b) mice showed progressive enhancement in the DTH response, whereas BALB/c (H-2d) mice showed strong DTH at the onset which gradually disappeared (defined as DTH-negative phase) and reappeared again at the later stage of infection. Adoptive transfer of enriched populations of splenic T cells from infected BALB/c mice together with parasite antigen into the footpad of syngenic normal recipients produced a dramatic enhancement in the DTH response, except at the onset of the DTH-negative phase. These observations indicate that adherent cells have a role in suppression of the cell-mediated immune response and also that another mechanism operates at the onset of the DTH-negative phase. This DTH-negative phase was not caused by depletion of DTH-mediating cells from the repertoire, but rather by suppression mediated by a subset of T cell evolved in the course of infection. Characterization on the basis of lymphokine production of the T cells mediating the DTH response and of T cells mediating suppression of the DTH response showed them to be of Th1 and Th2 type, respectively. Studies also indicated that at the onset and the later stages of infection suppression was mediated by adherent cells, but at the onset of DTH-negative phase, in particular, suppression was mediated by Th2 cells. Furthermore, experiments also showed that adherent cells from infected mice gained another property, that of driving B cells, in a T cell-dependent manner.


Characterization of molecular components associated with surface immunoglobulin M in human B lymphocytes: presence of tyrosine and serine/threonine protein kinases. To characterize the signal transduction through the antigen receptor (AgR) on human B lymphocytes, we analyzed its association with other molecular components. The surface IgM (sIgM) complex isolated in digitonin contains two surface expressed polypeptides--the previously described Ig alpha and Ig beta proteins--covalently linked to each other in a 48/39-kDa heterodimer. We show herein that the human sIgM complex isolated from the Burkitt's lymphoma cell line, Ramos, or from dense tonsillar B cells contains additional molecules--160 kDa and 75 kDa in size--and enzymatic activities able to phosphorylate on tyrosine as well as serine/threonine residues the 39-, 48-, 75- and 160-kDa polypeptides. By specific immunoprecipitation with antibodies to src-family kinases, we consistently detected p56lyn in the sIgM complex. In the Ramos cell line, both p56lck and p59fyn activity were also observed, although to a much lesser extent than p56lyn. These kinases are associated with sIgM before cell stimulation. As shown by two-dimensional electrophoresis, they interact in a tight complex with multimeric forms of the Ig alpha and Ig beta components. The kinases are active in vitro but must be highly regulated in vivo: Western blotting with anti-phosphotyrosine antibodies revealed that stimulation of the AgR on viable B cells increased detectable phosphotyrosine residues on the components present in the sIgM complex. Based on these phosphorylation changes, the 39-, 48-, 75- and 160-kDa molecules are likely to be functionally active elements in an IgM complex crucial for the transduction of the antigenic signal.


Co-crosslinking Fc epsilon RII/CD23 and B cell surface immunoglobulin modulates B cell activation. Previous studies have shown that a highly multivalent from of anti-IgD or anti-IgM, prepared by conjugating the respective antibodies to dextran, causes extensive B cell proliferation with ng/ml concentrations of the anti-immunoglobulin (Ig). A modification of this system has been exploited to investigate the effect of co-crosslinking the Fc epsilon RII and surface Ig by binding DNP to the dextran backbone (DNP-dextran) and employing a DNP-specific monoclonal IgE of either rat or mouse origin. Addition of anti-IgD-(H delta a/1)[DNP-dextran] or anti-IgM-[DNP-dextran] to purified, resting murine B cells resulted in B cell proliferation over a broad dose (0.03-30 micrograms/ml). Addition of DNP-specific rat or mouse IgE dramatically modulated the proliferative response. Proliferation in response to doses greater than 0.3 microgram/ml H delta a/1-[DNP-dextran] was consistently reduced in a dose-dependent manner in the presence of increasing amounts of IgE while proliferation to lower concentrations of H delta a/1-[DNP-dextran] was slightly enhanced or not influenced at all by the IgE anti-DNP. Interleukin-4 (IL-4) significantly increased the IgE effect, in line with its known enhancing effects on Fc epsilon RII levels. Experiments measuring Ig production rather than proliferation demonstrated that in the presence of IgE anti-DNP, B cells produced lower amounts of immunoglobulin (IgG1 or IgM) in response to an anti-Ig signal. Control experiments demonstrated that the IgE effect on proliferation was blocked by monoclonal anti-Fc epsilon RII, but not anti-Fc gamma RII, thus demonstrating the necessity for IgE/Fc epsilon RII interaction. In addition, the necessity for co-crosslinking was shown by the inability of IgE anti-DNP to affect the proliferative response to H delta a/1-dextran even in the presence of various doses of DNP-dextran. These results demonstrate that co-crosslinking of sIg and the Fc epsilon RII results in an altered B cell response to anti-Ig mediated activation. IL-4 does not ablate this inhibition, in contrast to the effect of co-crosslinking Fc gamma RII and surface Ig, suggesting a model whereby IgE can modulate its own production.


Attenuation of autoimmune disease and lymphocyte accumulation in MRL/lpr mice by treatment with anti-V beta 8 antibodies. MRL-MP-lpr/lpr mice are afflicted by a severe systemic autoimmune disease that is aggravated by the lpr mutation resulting in the accumulation of phenotypically abnormal lpr cells (CD3+CD4-CD8-) in all lymphoid issues including hyperplastic lymph nodes. Given that products of the T cell receptor V beta 8 gene family are overrepresented among lpr cells, different schedules aimed at selectively decreasing the frequency of lpr cells were designed. First, continuous administration of the monoclonal antibody F23.1 (specific for V beta 8 products) resulted in a significant depletion of V beta 8+ cells and prevented the manifestation of lymph accumulation at the same time as it reduced the serological, clinical, and histopathological signs of autoimmune disease. Along the same line, administration of either F23.1 or two different anti-F23.1 anti-idiotypic antibodies to MRL/Mp-lpr/lpr mothers elicited, in the offspring, the production of antibodies sharing a recurrent idiotype with F23.1 and resulted in long-term amelioration of autoimmunity and lymphadenopathy. Thus, a strategy aimed at specifically reducing the frequency of a subset of lpr cells proved successful in mitigating the autoimmune process.


Presence of murine fetal liver cells capable of being induced to differentiate in vitro into T cell receptor-positive cells. Mouse fetal liver cells were analyzed for the surface expression of T cell markers. Fetal liver cells prepared from mouse embryos at 14.5 days of gestation contained a small number of CD4+ cells (1.4%), but virtually no cells positive for any other T cell markers such as CD8, CD3 and T cell receptor (TcR). When a fetal liver cell suspension prepared from BALB/c(male) x AKR(female) F1 embryos at 14.5 days of gestation was cultured in medium supplemented with culture supernatants of both WEHI-3 and concanavalin A-stimulated rat spleen cells, TcR alpha beta+ and CD4+ cells were generated, whereas CD8+ and TcR gamma delta+ cells were hardly detectable. Most of TcR alpha beta+ and CD4+ cells were H-2d+, thus clearly showing their fetal origin. Treatment with anti-CD4, anti-CD3 or anti-TcR alpha beta antibodies plus complement or electronic sorting to remove cells expressing these markers failed to inhibit the generation of T cell marker-positive cells following culture in vitro. On the other hand, depletion of Thy-1.2+ cells reduced their generation. These findings indicate the presence of some progenitor T cells in fetal liver with the Thy-1+, CD3-, CD4-, CD8-, TcR- phenotype, which can be induced to differentiate into TcR alpha beta+ cells in the presence of specific humoral supplements without the influence of the thymus.


Differential effect of transforming growth factor beta on the synthesis of Th1- and Th2-like lymphokines by human T lymphocytes. (TGF)-beta is a pluripotent cytokine exerting differential effects on distinct components of the immune response. The present report, based on lymphokine determination in culture supernatants and Northern blot analysis of lymphokine mRNA, demonstrates that TGF-beta 2 markedly inhibits interleukin (IL)-4 and IL-5 synthesis by polyclonally activated human T cells in the absence of any significant effect on (IFN)-gamma, lymphotoxin or IL-2, suggesting a modulatory effect of TGF-beta 2 on the interferon Th1/Th2) balance of immune responses. The inhibitory effect of TGF-beta on IFN-gamma production by unfractionated peripheral blood mononuclear cells is likely to reflect the blunting of natural killer cell activation by TGF-beta.


Commitment to the T cell receptor-alpha beta or -gamma delta lineages can occur just prior to the onset of CD4 and CD8 expression among immature thymocytes. Two types of T lymphocytes, distinguishable by their surface expression of either the gamma delta or the alpha beta T cell receptor (TcR) for antigen, populate the periphery in the adult. In addition, immature precursors of both T cell types can be found in the thymus. While it is generally accepted that these two cell types represent distinct lineages, it is not known at which developmental stage these lineages diverge. The most mature thymocyte precursor population not yet expressing T lineage-specific surface markers (i.e. CD3, CD4, and CD8) is known to be capable of generating TcR-alpha beta T cells, and has been thought to be preprogrammed into the TcR-alpha beta lineage at an earlier developmental stage. We now show that this late-stage precursor is capable of giving rise to cells of both the TcR-alpha beta and -gamma delta lineages, both in vitro after intrathymic transplantation, and in vitro in simple culture medium or medium with cytokines. Thus it appears that the divergence of TcR-alpha beta and -gamma delta cells can occur at a relatively late stage of intrathymic development, just prior to the onset of CD4 and CD8 expression in most cells.


pH dependency of the binding of [3H]BAY U 3405 and various non-labelled ligands to the thromboxane A2/prostaglandin H2 receptor of human platelet membranes. [3H]BAY U 3405 was used to characterize the effect of acidic and alkaline pH values on the binding of the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor of human platelet membranes. The specific binding of [3H]BAY U 3405 largely increased upon acidification up to pH 5.8. Saturation binding studies revealed an increase in binding affinity without change in the number of binding sites. At pH 7.4 the Kd was 8.7 +/- 3.7 nM (Bmax = 6.6 +/- 0.6 pmol/mg protein) compared to 1.2 +/- 0.2 nM (Bmax = 6.1 +/- 0.6 pmol/mg protein) at pH 5.8. A more than 10-fold higher rate of association was observed at pH 5.8 compared to pH 7.4, while the rate of dissociation showed only minor changes. The kinetically derived dissociation constant was 1 nM (pH 5.8) and 9.6 nM (pH 7.4). The pH dependency of the binding of structurally different non-labelled ligands to the TXA2/PGH2 receptor was evaluated by inhibition studies at pH 5.8 and pH 7.4. BAY U 3405, daltroban, CTA2, and U 46619 showed significantly higher affinities at pH 5.8. In contrast, I-PTA-OH and GR 32191 had a higher affinity at pH 7.4. No significant difference was seen with SQ 29548 at the observed pH values. A second protonable group within the molecules I-PTA-OH, GR 32191, and SQ 29548 might be responsible for the observed differences.


Evidence for pulsatile secretion of human atrial natriuretic peptide in healthy subjects. The secretion pattern of human atrial natriuretic peptide (hANP) was investigated in 8 healthy male volunteers. Blood was drawn in 2 minute intervals for 90 minutes and in a second study in 4 minute intervals for 180 minutes. Base-line hANP plasma levels varied in a pulsatile secretion pattern of regular pulses every 9 +/- 1 minutes. Additionally long-term oscillations of 34 +/- 4 minutes were found. These data suggest that under basal conditions hANP varies in a pulsatile fashion.


Dehydroepiandrosterone (DHEA) levels in patients with prostatic cancer, heart diseases and under surgery stress. DHEA levels in patients with prostatic cancer were significantly lower, but total and free testosterone (T) significantly higher as those in an age-matched control group. Therefore, the calculated quotients DHEA/free T and DHEA/T were especially different between both groups. DHEA and DHEAS levels in patients with heart diseases were also significantly lower but cortisol (F) levels were significantly higher as those in a control group. The quotients DHEA/F and DHEAS/F were also of greater significance between both groups than the hormone values alone. The response of DHEA and F levels in patients undergoing surgery showed an increase of both steroids under surgery. On the second postoperative day, however, F levels were still significantly higher but DHEA levels were significantly lower as the initial values. The differences between the initial values and those on the second postoperative day of F and DHEA showed a significant correlation, i.e. the higher the elevation of F levels above the initial values the greater was the diminution of DHEA levels below the initial values.


Differential increase in activity of acid phosphatase induced by phosphate starvation in Tetrahymena. We have studied the effects of phosphate starvation on the levels and distributions of activities of acid phosphatase and beta-hexosaminidase in cultures of Tetrahymena thermophila. The cells were grown in synthetic nutrient medium and refed every day with fresh medium. After 4 days of growth in the complete medium, the cultures were divided into two portions. One received complete medium and the other phosphate-free, but otherwise complete, medium. Population densities and activities of acid phosphatase and beta-hexosaminidase in cells plus medium and in cell-free samples were determined in aliquots removed every day before medium replacement. In cultures having complete medium the enzyme levels remained fairly constant; in the phosphate-starved cultures both total and extracellular activities of acid phosphatase increased sixfold. beta-Hexosaminidase levels remained essentially unaltered in both cases. These results indicate that phosphate starvation can induce differential increase in acid phosphatase activity in cultures of Tetrahymena. Somewhat less than 50% of the total activities of both enzymes are found in the cell-free extracellular fluid at any time.


Inhibition of mouse lymphocyte proliferative response by glycosphingolipids from Leishmania (L.) amazonensis. The effect of a purified preparation of glycosphingolipids (GSLs) extracted from Leishmania (L.) amazonensis amastigotes on murine lymphocytes was investigated. GSLs inhibited both concanavalin A (Con A)- or lipopolysaccharide-induced [3H]thymidine uptake by normal and immunized BALB/c mouse lymph node cells. The effect of total GSLs was dose dependent. Total GSLs also suppressed the two-way mixed lymphocyte reaction of BALB/c x C57BL/6 cells and the antigen-specific response of immunized mouse cells. Six pure bands as well as a pool containing a mixture of GSLs with five to seven sugar residues separated by a combination of HPLC and preparative HPTLC were tested and shown to be inhibitors of Con A stimulation. These results suggest that parasite glycosphingolipids may play an immunologically relevant role in leishmaniasis.


The cellular immune response to heat shock proteins. T lymphocytes, which are central to almost every immune response, frequently recognize microbial hsp60. Such cells could provide an early defense mechanism against pathogenic microbes. However, T cells also recognize epitopes of hsp60 shared by microbe and host. Not only conventional alpha/beta T cells respond to hsp60; gamma/delta T cells do so, as well. In fact, certain gamma/delta T cells seem to have a particular preference for this molecule. Recognition of stressed host cells expressing hsp60 could facilitate the scavenger function of the T cell system. On the other hand, such recognition could be involved in autoimmune disease.


Effects of hypertension and hyperlipidemia on the myocardium and coronary vasculature of the WHHL rabbit. This study was undertaken to study the effects of hyperlipidemia and hypertension on the coronary circulation and on the myocardium of Watanabe heritable hyperlipidemic (WHHL) rabbits. Surgery to induce hypertension by the one-kidney, one-clip technique was performed on the WHHL rabbits at 3 months of age. At 3 and 6 months after surgery, the right and left coronary arteries and the left ventricle and posterior papillary muscle from normotensive and hypertensive animals were assessed. Atherosclerotic involvement was found at the coronary origin in 94% of the arteries evaluated. Lesions were usually confined to the proximal 1-2 mm of the coronary artery. The prevalence of coronary atherosclerosis in the WHHL rabbit was found to be higher than previously reported in rabbits of the same age. Hypertension-induced muscular and vascular changes such as left ventricular hypertrophy, medial thickening of the arteries, and hyaline arteriolosclerosis were found in most of the hypertensive animals. These changes were rarely seen in the normotensive rabbits. Characteristics of ischemia and cell injury such as eosinophilic fibers, fiber vacuolization, and contraction band necrosis were found more often in hypertensive than in normotensive WHHL rabbits. Confluent areas of severe necrosis indicative of myocardial infarction were not found; myocardial damage was diffuse and involved individual cells and small microscopic areas. This model may be valuable in further studies of coronary artery disease and myocardial injury that result from the combination of hypercholesterolemia and hypertension.


Meta-analysis of efficacy of zinc acexamate in peptic ulcer. Zinc acexamate (ZAC) is a new drug for the treatment of peptic ulcer. The present study was performed in order to evaluate the clinical efficacy of ZAC in peptic ulcer, using a meta-analysis of all randomized clinical trials performed with this drug. Eighteen studies were reviewed, but only 13 were considered in the final analysis. The total number of patients was 757. Control groups included placebo or H2 receptor antagonist drugs. Healing rate, assessed by endoscopy, was selected as the criterion for evaluating drug efficacy. The meta-analysis was performed using a modified version of the Mantel-Haenszel method. ZAC proved to be better than placebo in the treatment of peptic ulcer (pooled odds ratio: POR = 5.55; 95% confidence interval: 95% CI = 2.20-14.04) and not different from H2 receptor antagonist drugs when compared in patients with gastric (POR = 1.14; 95% CI = 0.47-2.72), duodenal (POR = 0.97; 95% CI = 0.13-7.33) or both ulcer types (POR = 1.10; 95% CI = 0.74-1.64). The present results show that ZAC is an effective drug for the treatment of peptic ulcer.


Presence and properties of acyl coenzyme A synthetase for medium-chain fatty acids in rat intestinal mucosa. A system was developed for assay of acyl coenzyme A (acyl CoA) activity on medium-chain fatty acids in rat intestinal mucosa. Using this system, we compared the characteristics of octanoyl (C8:0) CoA synthetase activity and palmitoyl (C16:0) CoA synthetase activity. Palmitoyl CoA synthetase activity as a function of palmitate concentration followed Michaelis-Menten kinetics, but octanoyl CoA synthetase activity as a function of octanoate concentration showed a biphasic reaction curve. The distributions of octanoyl CoA synthetase activity and palmitoyl CoA synthetase activity along the gastrointestinal tract were similar, both activities being present mainly in the middle portion of the small intestine. Incubation of octanoate with a homogenate of intestinal mucosa revealed that octnoate, like palmitate, is incorporated into phospholipids and triglycerides after its CoA activation. Oral administration of medium chain triglycerides induced a nearly 2-fold increase in octanoyl CoA synthetase activity in rat intestinal mucosa, whereas oral administration of long chain triglycerides did not affect the palmitoyl CoA synthetase activity. These results indicate that acyl CoA synthetase for medium-chain fatty acids in rat intestinal mucosa plays a key role in the utilization of medium-chain fatty acids for lipid synthesis, and that it is regulated in a different manner from acyl CoA synthetase for long-chain fatty acids.


[Prevalence of hereditary pathologies in residents of the Kirov Province]. Medical-genetic study was carried out in the population of Kirov Province (population size about 120.000). 203 families with 334 affected with hereditary disorders were registered. The correctness of pathology classification for the inheritance type was confirmed by segregational analysis. The load of hereditary diseases in the population was: 1.25 +/- 0.06 for autosomal dominant, 1.37 +/- 0.07 for autosomal recessive and 0.22 +/- 0.06 for X-linked recessive disorders. It is suggested that variability in the values of the load of autosomal recessive disorders is determined to the large extent by genetic structure of the population.


[Cloning and complementation analysis of the Escherichia coli gpr locus, influencing DNA replication of certain lamdoid phages]. Escherichia coli DNA fragments which suppressed gpr27 and gpr2 mutations in the earlier proposed gprA and gprB genes, respectively, were cloned within phage vectors. Mutations gpr2 and gpr27 restrict DNA replication of some lambdoid phages and are located in the region of dnaK, J genes. DNA-DNA hybridization showed that the cloned fragments correspond to the region where gpr mutations were genetically mapped and, in some cases, do not include dnaK, J genes. These results provide the evidence that gprA and gprB genes may be physically separated from dnaK, J genes.


A broad bean cDNA clone encoding a DNA-binding protein resembling mammalian CREB in its sequence specificity and DNA methylation sensitivity. A plant cDNA has been cloned that encodes a DNA-binding protein displaying a nucleotide (nt) sequence specificity similar to that of the mammalian cyclic AMP response element-binding protein/activating transcription factor (CREB/ATF) family of mammalian proteins. This cDNA was cloned in Escherichia coli from a broad bean (Vicia faba) cDNA expression library using a recognition site probe. The deduced amino acid (aa) sequence of the recombinant cDNA-encoded protein, called VBP1, has a basic region adjacent to a leucine zipper motif, of the type seen in the DNA-binding domains of many eukaryotic DNA-binding proteins, including mammalian CREB/ATF. Although this aa sequence has homology to regions of deduced aa sequences of other cloned plant cDNAs, it is distinct in both the derived primary structure and in its nt sequence specificity. VBP1, as well as proteins in nuclear extracts of V. faba with similar nt sequence specificity, have their binding to DNA suppressed more than tenfold by cytosine methylation at the CREB/ATF consensus sequence.


Genomic structure of the putative BTF3 transcription factor. On the basis of the amino acid (aa) sequence of the putative BTF3 transcription factor, two complementary DNAs coding for the two proteins, BTF3a and BTF3b, have been cloned from a HeLa cell cDNA library. The two proteins are identical, except that BTF3b lacks the first 44 N-terminal aa present in BTF3a. These proteins originate from the same gene, which contains seven exons, and are the products of alternative splicing. The promoter region, containing a putative TATA box(es) and a CAAT box sequence, was identified. In addition, three other BTF3-like genes were characterized.


The hemodynamic status of preascitic cirrhosis: an evaluation under steady-state conditions and after postural change. To assess the hemodynamic status of patients with compensated cirrhosis, mean arterial pressure, cardiac index and peripheral vascular resistance and markers of central (plasma concentrations of atrial natriuretic factor) and arterial volemia (plasma norepinephrine concentration, plasma renin activity) were studied in 10 patients and 10 healthy control subjects under steady-state conditions (after 2 hr of standing) and after assumption of the supine position (30, 60, and 120 min). After standing, neither hemodynamics nor markers of effective volemia differed significantly between controls and patients. By evaluating the areas under the curve during the 2 hr of supine posture, the increase in cardiac output and plasma natriuretic factor and the decrease in peripheral vascular resistance were greater in patients (2.59 +/- 0.43 [S.E.M.] L/min/hr; 32.8 +/- 7.2 pg/ml/hr -1,103 +/- 248.4 dyn.sec/cm5/hr, respectively) than in controls (0.53 +/- 0.24 L/min/hr, p = 0.005; 17.4 +/- 4.7 pg/ml/hr, p = 0.005; -265.5 +/- 206.2 dyn.sec/cm5/hr, p = 0.02). The declines in heart rate, plasma norepinephrine concentration and plasma renin activity did not differ significantly. Mean arterial pressure did not significantly change. Our results suggest that during periods of upright posture, cirrhotic patients in the preascitic stage, who are known to have expanded blood volume, compensate for dilatation of the splanchnic vascular bed through total hypervolemia. The latter becomes excessive during recumbency, leading to supernormal increases in venous return, central volemia and cardiac index. The decline in peripheral vascular resistance appears to be a compensatory mechanism to maintain steady arterial blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)


Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor. Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free modified Dulbecco's modified Eagle's medium containing 10 mmol/L nicotinamide and 10 ng/ml epidermal growth factor. Each colony consisted of cells that had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically these cells were stained with albumin and transferrin. Ultrastructurally these cells had mitochondria, peroxisomes and desmosomes, indicating that they were derived from hepatocytes. When 6 x 10(5) cells were plated on 35-mm dishes, about 5.5 colonies/mm2 were observed. This result suggested that about 1.5% of adult rat hepatocytes has the potential for multiple replications and of forming a focal colony. These cell populations had higher proliferative activities than surrounding hepatocytes. DNA synthetic activity could not be inhibited by 2% dimethyl sulfoxide. Flow cytometric analysis showed that both 2N and 4N nuclei synthesized their DNA until day 4 but that the number of 2N nuclei rapidly increased at day 5. This result correlated with the observation of the appearance of small cell populations indicating that the cells of these focal colonies were predominantly diploid.


Genomic organization, chromosomal localization, and independent expression of human cyclin D genes. Murine cDNA clones for three cyclin D genes that are normally expressed during the G1 phase of the cell cycle were used to clone the cognate human genes. Bacteriophage and cosmid clones encompassing five independent genomic loci were partially sequenced and chromosomally assigned by an analysis of somatic cell hybrids containing different human chromosomes and by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes. The human cyclin D1 gene (approved gene symbol, CCND1) was assigned to chromosome band 11q13, cyclin D2 (CCND2) to chromosome band 12p13, and cyclin D3 (CCND3) to chromosome band 6p21. Pseudogenes containing sequences related to cyclin D2 and cyclin D3 mapped to chromosome bands 11q13 and 6p21, respectively. Partial nucleotide sequence analysis of exons within each gene revealed that the authentic human cyclin D genes are more related to their mouse counterparts than to each other. These genes are ubiquitously transcribed in human tumor cell lines derived from different cell lineages, but are independently and, in many cases, redundantly expressed. The complex patterns of expression of individual cyclin D genes and their evolutionary conservation across species suggest that each family member may play a distinct role in cell cycle progression.


Molecular cloning and chromosomal mapping of CCND genes encoding human D-type cyclins. A human D-type cyclin gene (CCND1/cyclin D1/PRAD1) was previously isolated by virtue of its ability to complement a triple G1 cyclin (Cln) deficiency of Saccharomyces cerevisiae and was also identified as a candidate BCL1 oncogene. We now report the molecular cloning of two additional human D-type cyclin genes, CCND2 (cyclin D2) and CCND3 (cyclin D3). All three human D-type cyclin genes encode small (33-34 kDa) proteins that share an average of 57% identity over the entire coding region and 78% in the cyclin box. The D-type cyclins are most closely related to cyclin A (39% identity) and cyclin E (36%), followed by cyclin B (29%) and cyclin C (21%). Isolation and characterization of genomic clones revealed two pseudogenes corresponding to CCND2 and CCND3, respectively. All three cyclin D genes are interrupted by an intron at the same position. CCND2 has been mapped to chromosome 12p13, and CCND3 has been mapped to chromosome 6p21.


The human IL-1 receptor antagonist gene (IL1RN) maps to chromosome 2q14-q21, in the region of the IL-1 alpha and IL-1 beta loci. The IL-1 receptor antagonist (IL-1RN) is a protein that binds to IL-1 receptors and inhibits the binding of IL-1 alpha and IL-1 beta. As a consequence, the biological activity of these two cytokines is neutralized in physiological and pathophysiological immune and inflammatory responses. In this study, using a panel of somatic rodent-human cell hybrids, we show that the gene for the human interleukin-1 receptor antagonist (IL1RN) maps to the long arm of chromosome 2. Previously, we described a length variation polymorphism within the second intron of the IL-1RN gene (Steinkasserer et al., 1991, Nucleic Acids Res. 19: 5095). Segregation of this, together with an IL-1 alpha polymorphism, was followed in a panel of five CEPH families. Linkage analysis permitted the mapping of the IL-1RN gene to band q14-q21 in the region for the IL-1 alpha and IL-1 beta loci. This study supports the view that an early gene duplication event resulted in the creation of an interleukin-1 gene family.


Genomic sequence and chromosomal location of human interleukin-11 gene (IL11). The genomic sequence of human interleukin-11 (IL11) has been isolated based on its sequence homology with a cDNA clone encoding primate IL11. The human IL11 genomic sequence is 7 kb in length and consists of five exons and four introns. The IL11 gene has been localized to the long arm of human chromosome 19 at band 19q13.3-q13.4 by in situ hybridization. Several potential transcriptional control sequences that may play an important role in the control of IL11 gene expression have been identified within the 5'-flanking region of the human IL11 gene. The 5'-flanking region of the human IL11 gene contains sequences similar to those present in the 5'-regulatory regions of other cytokine genes. Four repeats of a 30-bp DNA segment were found in the fourth intron, and several copies of ATTTA and Alu repetitive sequences were identified in the 3'-noncoding region of the human IL11 gene. A DNA sequence (ACATGGCAAAACCC) that has 71% similarity with the IL1-responsive element of the IL6 gene was found in the 3'-flanking region of the human IL11 gene. The two polyadenylation sites located at nucleotide positions 6762 and 5591 correspond to the 2.5- and 1.5-kb IL11 transcripts expressed in IL1-induced PU-34 cells. The availability of the human IL11 genomic sequence should aid in studies of the regulatory mechanisms of IL11 gene expression in different cell types and the role of IL11 expression in the hematopoietic microenvironment.


Identification of a CA repeat at the TCRA locus using yeast artificial chromosomes: a general method for generating highly polymorphic markers at chosen loci. The creation of a comprehensive genetic map in human has been limited by the lack of highly polymorphic markers spaced evenly throughout the human genome. We have utilized yeast artificial chromosomes (YAC) containing large human DNA inserts to help identify highly polymorphic (CA)n repeats at a chosen locus. The DNA of a YAC containing the locus was subcloned in M13 vectors, and the recombinants were screened at high stringency to detect preferentially long (CA)n repeats (n greater than 20). These repeats, which are the most likely to be highly polymorphic, were then studied to confirm both the level of polymorphism and their precise genetic location. This strategy has permitted the identification of a new, highly polymorphic CA repeat (77% heterozygosity) at the T cell receptor alpha chain (TCRA) locus on chromosome 14q. It provides a powerful marker for assessing the role of this locus in the susceptibility to autoimmune and infectious diseases. This approach should permit the development of highly polymorphic markers at any targeted locus and rapidly improve the current human genetic map.


Localization of the gene coding for ventricular myosin regulatory light chain (MYL2) to human chromosome 12q23-q24.3. Human myosin light chain-2 (MYL2) is an important protein involved in the regulation of myosin ATPase activity in smooth muscle. In cardiac muscle, the precise role of MYL2 is not well understood; however, an increase in ventricular MYL2 is observed during myocardial hypertrophy in cardiac patients with valve stenosis. The chromosomal location of the gene coding for MYL2 was identified using a cloned cDNA for human MYL2. Southern blot analysis of DNA from a human/rodent somatic cell hybrid mapping panel showed that the BamHI fragment that hybridized with this cDNA probe was concordant with chromosome 12. The 768-bp cDNA was hybridized to human metaphase chromosomes. The results revealed a significant clustering of silver grains over chromosome 12 bands q23-q24.3, indicating that the gene coding for MYL2 is located in this region.


cDNA sequence of human p11 calpactin I light chain. The cDNA encoding full-length human p11 calpactin I light chain has been cloned and subjected to DNA sequencing. The open reading frame specifies a 97-amino-acid residue protein that surprisingly is identical to the p11 sequences of two mammalian ungulate species, cow and pig. However, the previously reported p11 polypeptide sequences of mouse and rat exhibited 8-9% nonidentity to human p11. These mammalian sequence comparison results are unexpected in view of current molecular cladistic theories that suggest a closer relationship between primates and rodents, rather than primates and ungulates. The mouse p11 gene has been previously mapped to chromosome 3 at a position syntenic with a centromeric-proximal region on human chromosome 1, and the human p11 cDNA clone is likely to be useful in physical mapping on chromosome 1.


Down-regulation of E1a expression by E3 gene products in group C adenoviruses. Mutant group C adenoviruses defective in expression of the E3 transcription unit were found to overexpress E1a proteins relative to wild-type adenoviruses. This result suggests that one or more proteins encoded in the E3 region (present in wild-type viruses) down-regulate E1a expression. This interpretation was confirmed by transfection experiments in which a plasmid expressing the E3 region reduced expression of E1a in 293 cells. Experiments to examine the molecular basis of this down-regulation of E1a suggest that E3 protein products interfere with the translation of viral mRNA molecules.


Somatic cell mapping of T-cell receptor CD3 complex and CD8 genes in cattle. Bovine genes encoding T-cell receptor, CD3, and CD8 molecules have been mapped to syntenic groups using bovine x rodent hybrid somatic cells. T-cell receptor alpha and delta chains were assigned to bovine syntenic group U5, and the beta and gamma genes were syntenic with each other and with markers on U13. CD3E and CD3D genes were syntenic with each other and located to bovine syntenic group U19. CD8 was most concordant with markers of syntenic group U16, although the concordancy was only 85% and the assignment must be regarded as tentative. The comparative gene maps of human chromosome 7, bovine syntenic group U13, and mouse chromosomes 6 and 13 suggest extensive evolutionary conservation.


Salt supplementation does not alter the pressor effect of blocking atrial natriuretic peptide in nucleus tractus solitarii. We have previously shown that microinjection of monoclonal antibody to atrial natriuretic peptide (ANP) into the caudal nucleus tractus solitarii causes a pressor response in salt-sensitive spontaneously hypertensive rats (SHR) fed a basal (1%) salt diet, suggesting that endogenous ANP in this region may be involved in the centrally mediated regulation of blood pressure in this model. The present study tested the hypothesis that the pressor effect of blocking endogenous ANP in caudal nucleus tractus solitarii is enhanced by dietary salt supplementation in salt-sensitive SHR. Monoclonal antibody to ANP (0.55 micrograms) in 50 nl artificial cerebrospinal fluid or control immunoglobulin G was microinjected into the caudal nucleus tractus solitarii of conscious salt-sensitive SHR, salt-resistant SHR, and Wistar-Kyoto rats fed 1% or 8% salt diets for 3 weeks. Microinjection of the monoclonal antibody into the caudal nucleus tractus solitarii evoked similar increases in mean arterial pressure in salt-sensitive SHR on both 1% and 8% salt diets and in salt-resistant SHR on a 1% salt diet but had no effect in Wistar-Kyoto rats. In contrast, microinjection of control immunoglobulin G into this brain area did not alter mean arterial pressure or heart rate in any experimental group. Thus, endogenous ANP in caudal nucleus tractus solitarii mediates tonic control of blood pressure in both salt-sensitive and salt-resistant SHR but not in Wistar-Kyoto rats, and this effect is independent of the salt sensitivity of hypertension and of dietary salt intake.


Cell extracts of Candida albicans block adherence of the organisms to endothelial cells. Cell extracts of Candida albicans were fractionated by concanavalin A affinity chromatography. Eluted mannosylated proteins (fraction II) and nonbinding, nonmannosylated proteins (fraction I) were collected and assayed directly for inhibition of adherence of C. albicans to endothelium. Fraction II blocked blastospore adherence to endothelial cells. Fraction I blocked both blastospore and germ tube adherence to endothelial cells. Monoclonal antibody OKM-1 (anti-CR3) and an anti-C. albicans monoclonal antibody, CA-A (anti-CR2), reacted in Western blots with proteins from fraction I, suggesting the presence of the CR2- and CR3-like proteins that have been previously identified on C. albicans germ tubes.


MHC class-I-restricted auto-tumor-specific CD4+CD8- T-cell clones established from autologous mixed lymphocyte-tumor-cell culture (MLTC). Autologous mixed lymphocyte-tumor cell cultures (MLTC) were initiated with cytokine (IFN gamma and TNF alpha)-treated ex-vivo tumor cells of lung, ovarian, breast and stomach carcinomas. The cytokine-treated tumors expressed class-I but not class-II molecules. Although the proportion of CD8+ lymphocytes increased in the bulk culture of MLTCs, in 5/7 experiments the majority of the established T-cell clones were CD4+. Among the CD8+ clones a high proportion (77%) was cytotoxic, while the proliferative response was more frequent among the CD4+ clones (70%). In 4/26 cytotoxic T-lymphocyte (CTL) clones (3/17 CD4+ and 1/9 CD8+), derived from a patient with class I+ class II- stomach carcinoma, lysis was restricted to the autologous tumor cells. These auto-tumor-specific clones did not lyse the autologous ConA blasts, the 5 allogeneic ex-vivo tumors, the NK-sensitive K562 or the relatively sensitive Daudi cells. The cytotoxicity of these clones was inhibited by pre-incubation of the tumor cells with W6/32 (alpha-class I) MAb, or by preincubation of the lymphocytes with OKT3 (alpha-CD3) MAb. The alpha-CD4 (OKT4) MAb had only a marginal effect on the CD4+ clones, while the lytic function of the CD8+ clone was inhibited by the alpha-CD8 (OKT8) MAb. The 3 CD4+ CTL clones also responded with proliferation to the autologous tumor cells. This proliferative response was inhibited by the presence of W6/32 MAb. Our results indicate that the auto-tumor lysis exerted by CD4+ CTL clones was restricted by the class-I antigens, and that the CD4 molecules of the clones were not essential for the lytic interaction.


Optimization of culture conditions for activation and large-scale expansion of human T lymphocytes for bispecific antibody-directed cellular immunotherapy. We investigated the optimal culture conditions (i.e., activation procedure, medium composition and type of culture vessel) for rapid in vitro expansion of large numbers (greater than 5 x 10(9) of blood T lymphocytes. These expanded lymphocytes can be targeted to be cytotoxic to ovarian carcinoma cells with a bispecific monoclonal antibody (BsAb) specific for CD3 and for the ovarian carcinoma-associated antigen MOv18. Both phytohemagglutinin (PHA) and monoclonal antibody (MAb) CD3 induced rapid T-cell proliferation, although the growth kinetics after PHA activation were slightly faster. A 50-fold increase in cell number was obtained after 14 and 16 days for PHA and CD3 MAb, respectively. The induction of BsAb-directed cytolysis was faster after CD3 MAb than after PHA activation of lymphocytes, but became similar around day 20. A mixture of media consisting of 78% RPMI 1640, 20% AIM-V and 2% human plasma (Mix-med) yielded better results than 100% AIM-V medium. Culture of lymphocytes in polyolefin bags, compared with tissue culture flasks, or cryopreservation did not affect lymphocyte yield and function. In most cultures the proportion of CD8+ lymphocytes increased, suggesting a growth advantage of CD8+ over CD4+ lymphocytes in this culture system. A protocol employing PHA activation, Mix-med and polyolefin bags has been used successfully to activate and expand blood lymphocytes for the first 5 patients entered into a phase-I/II clinical trial for the intraperitoneal treatment of ovarian carcinoma using CD3 x anti-MOv18 BsAb-directed T lymphocytes.


Plasma docosahexaenoic acid levels in various genetic forms of retinitis pigmentosa. In 188 patients from separate families with various forms of retinitis pigmentosa (RP) and 91 normal subjects, plasma fatty acids were measured as a percentage of total plasma fatty acids, and their concentrations were determined using capillary-column gas-liquid chromatography. After controlling for the effects of age and gender, those with RP had significantly lower (P less than 0.01) mean plasma percentages and concentrations of the omega-3 fatty acids: 18:3 omega 3 (alpha-linolenic acid), 22:3 omega 3 (13,16,19 docosatriaenoic acid), and 22:6 omega 3 (docosahexaenoic acid, DHA) compared with the group of normal subjects. The mean percentages were reduced 15%, 14%, and 10%, respectively, below the mean percentages in normal subjects. Analysis by genetic type revealed that the X-linked and isolate forms of RP had significantly lower (P less than 0.01) mean percentage values for DHA (18% and 17%, respectively). Dominant and recessive forms of RP had DHA levels close to normal. Mean absolute plasma DHA concentrations in X-linked RP were not significantly different from the concentrations in the control subjects, although these levels were significantly lower in patients with isolate RP. These data identify the possibility that some forms of RP may have alterations in plasma omega-3 fatty acid metabolism resulting in decreased plasma DHA content. These observations await additional confirmation using an analysis of the fatty acid content of specific erythrocyte phospholipid classes.


Effect of steroids and nonsteroidal antiinflammatory agents on human ocular fibroblast. 5-fluorouracil and steroids have been used to suppress excessive scar formation after glaucoma filtering surgery (GFS). Steroidal and nonsteroidal antiinflammatory drugs (NSAIDs), which are inhibitors of arachidonic acid (AA) pathway, both limit fibroblast activity and reduce inflammation. In this experiment, the ability of corticosteroid (dexamethasone sodium phosphate), cyclooxygenase inhibitor (piroxicam), lipoxygenase inhibitor (ferulic acid), and dual cyclo/lipoxygenase inhibitor (phenidone) to inhibit human Tenon's fibroblast proliferation was evaluated in culture. After human Tenon's fibroblast cell lines were established, a complete dose-response curve was done for the representative compounds for 8 days. Fibroblast attachment and proliferation were quantified by Coulter counter, hexosaminidase, and tritiated thymidine uptake assays. All four drugs inhibited attachment and proliferation at high concentrations. Phenidone was the most effective, with inhibition occurring within the 0.001-0.1 mmol/l range. It also was the only drug that showed inhibition at the antiinflammatory range in vivo. Dexamethasone, piroxicam, and ferulic acid did not inhibit fibroblast attachment and proliferation until doses well above those required to inhibit AA biosynthesis were attained. Only dexamethasone showed increased potency with incubation time. Overall, the NSAIDs showed antiproliferative activity comparable to or better than that of the steroids. Because the potency of steroids increases over time, these drugs may be more beneficial if given prior to initiation of inflammation. These results suggest that NSAIDs may be useful as both antiinflammatory and antiproliferative agents in preventing bleb failure after GFS.


Frequency of HLA-DPB1 alleles, including a novel DPB1 sequence, in the Northern Ireland population. HLA-DPB1 allele frequencies in 150 unrelated normal individuals from Northern Ireland were determined using oligonucleotide typing methods. HLA-DPB1*0401 was the most common allele in the population possessed by 75.3% of subjects, followed by DPB1*0201 (20.7%). In addition to these alleles, only HLA-DPB1*0402, -DPB1*0301, and -DPB1*0501 were present in subjects at frequencies greater than 10%. The results in this study are in broad agreement with other Caucasoid studies, but there is regional and ethnic variation in HLA-DP allele frequencies. Three DPB1 alleles were found to be in linkage disequilibrium with HLA-DR antigens determined by RFLP, namely, DPB1*0101 with DRw17 (Dw24 associated) RFLP, DPB1*0501 with DRw13-Dw19 RFLP, and DPB1*1901 with DRw13-Dw18 (Dw25 associated) RFLP. One individual revealed a novel DPB1 pattern of probe reactivity, which following DNA sequencing was found to be HLA-DPB1*2001. To assess the system used and to compare consistency of results between laboratories, 62 cell lines were oligotyped for HLA-DP. The results revealed the system described here to be extremely accurate and showed excellent agreement of HLA-DP typing results for cell lines between laboratories.


Cross-species reactivity of the anti-idiotype anti-OKT3 cascade between mice and humans. The administration of murine mAb specific for the CD3 epsilon subunit of the TCR complex (OKT3) has been demonstrated to engender in humans an anti-OKT3 idiotypic cascade. This study used murine-derived anti-OKT3 (Ab2) as a bioreagent to determine whether this Ab2 and polyclonal anti-(anti-OKT3) (Ab3) generated in some human kidney transplant patients are idiotypically connected. Two anti-OKT3 mAbs G-880 (IgG1) and M-12 (IgM) were derived by immunizing BALB/c mice with the OKT3-secreting hybridoma. The two mAbs exhibited specificity for OKT3 F(ab)'2 idiotypic determinants. Both mAbs were tested for their ability to inhibit OKT3 induced mitogenesis and to block FITC-OKT3 binding to cell surface CD3 epsilon chain. The M-12 mAb inhibited OKT3-induced mitogenesis and blocked (approximately 60%) the binding of OKT3 to peripheral blood (PBL) T-cell CD3 epsilon chain in flow cytometry. In contrast, the G-880 mAb did not inhibit mitogenesis and only weakly blocked OKT3 binding to CD3 epsilon chain (approximately 12%). Sera of kidney transplant recipients who received OKT3 antirejection therapy and who developed antiidiotypic anti-OKT3 antibodies could be divided into two subgroups exhibiting anti-OKT3 activity: (a) those who had similar specificity as M-12 and failed to enhance the M-12 inhibition of OKT3 binding to PBL T-cell CD3 epsilon chain when added as a third component (n = 3), and (b) those with anti-OKT3 antibodies with idiotype specificity dissimilar to M-12 and who were able to increase the (maximum 60%) inhibition obtained with M-12 in the OKT3 to T-cell CD3-binding assay (n = 4). From these observations, we conclude that M-12 had the characteristics of an Ab2 beta and G-880 that of an Ab2 alpha. Additionally, there was an idiotypic connectivity of mouse-derived M-12 anti-OKT3 (Ab2) and OKT3-engendered human polyclonal anti-(anti-OKT3) (Ab3), in that three of seven patients examined had human serum IgG antibodies that specifically recognized M-12 idiotypic determinants as demonstrated in ELISA.


Identification of anti-CD3 antibodies that do not modulate antigen but induce mitogenesis and block the mixed lymphocyte reaction. Using a panel of anti-CD3-TCR monoclonal antibodies (OKT3 A-E), it appears possible to separate the ability to cause surface antigen modulation from inhibition of MLR or induction of mitosis. OKT3D, an antibody that recognizes the CD3 antigen at a site that can be differentiated from the epitopes recognized by other members of this panel by competition binding, does not cause antigen modulation when incubated with human T cells for up to 3 days. Despite this, OKT3D is mitogenic and is capable of blocking MLR. Two different isotypes were produced from the OKT3D clone, IgG1 and IgG2b. The IgG2b isotype of OKT3D blocked MLR even in individuals unable to respond mitogenically to this antibody. Use of members of this panel may now permit dissection of the types of signals delivered by the CD3-TCR complex inducing mitosis, receptor modulation, and other T-cell responses.


Adriamycin-induced recall of radiation pneumonitis and epilation in lung and hair follicles of mouse. The influence of Adriamycin and Actinomycin D on the expression of residual damage following irradiation of mouse thorax was evaluated. Drugs were given i.v. at various times starting on the same day, up to 3 months after irradiation, and the mortality from lung damage up to 160 days or epilation up to 98 days after irradiation were noted. Adriamycin (1.2 mg/kg in two equal doses), which on its own did not cause pneumonitic deaths, did so when combined with doses of local thoracic radiation as small as 6 Gy. The dose effect factor was greater if Adriamycin was given at 1 or 2 months (1.68) rather than on the same day (1.49). A reduction in the latent period to death was also observed with a minimum period of 50-60 days after irradiation rather than the normal 80-160 days. Adriamycin enhanced the epilation response to radiation, but only at or above threshold radiation doses. There was no reduction in the latency. Actinomycin D had no dose-modifying effects on the radiation response of both lung and hair follicles. The interaction between irradiation and Adriamycin seen when the interval between the two modalities is long, as it was in this study, may be mechanistically similar to the "radiation recall" phenomenon described in the clinic.


Factor I-dependent inactivation of human complement C4b of the classical pathway by C3b/C4b receptor (CR1, CD35) and membrane cofactor protein (MCP, CD46). Proteolytic inactivation of C4b is a crucial step for regulation of the classical complement pathway. A plasma protease factor I and membrane cofactors, C3b/C4b receptor (CR1) and membrane cofactor protein (MCP), participate in the regulation of cell-bound C4b although the physiological potency of these cofactors remains unknown. We have examined the optimal conditions of the factor I-mediated C4b regulatory system using purified cofactors. CR1 being a cofactor at a cofactor/C4b ratio less than 0.1 (w/w), fluid phase C4b, and methylamine-treated C4 (C4ma) were degraded by factor I into C4bi: minimal Cd4 was generated in the fluid phase. Liposome-bound C4b (LAC4b), on the other hand, was degraded into C4c and C4d. CR1 showed two optimal pHs (6.0 and 7.5) for fluid phase C4b, but one (6.0) for LAC4b, and in both cases low conductivity conditions enhanced the C4bi generation. CR1 cofactor activity was barely influenced by the NP-40 concentration. On the other hand, MCP degraded C4b and C4ma, as a factor I-cofactor, more efficiently into C4c and C4d. Though MCP cofactor activity, like that of CR1, was enhanced under low conductivity conditions, it has only one optimal pH, 6.0, in both fluid and solid phases. Furthermore, as in the case of C3b cleavage, a sufficient NP-40 concentration to solubilize membrane was needed for MCP to express full cofactor activity for C4b, in contrast to CR1. MCP was less potent for C4b inactivation than for C3b inactivation, while CR1 acted as a slightly more effective cofactor for C4b cleavage than for C3b cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)


Maturation of murine erythroleukemia cells committed to differentiation requires protein kinase C. Treatment of murine erythroleukemia cells (MELC) attached to fibronectin-coated dishes with dimethyl sulfoxide causes the cells to become committed to the erythroid differentiation pathway. These cells mature extensively and acquire the characteristics of erythroid cells. The cells lose their cell-surface fibronectin receptors and accumulate red cell-specific membrane proteins, such as band 3, in amounts comparable to those in erythrocytes. Previous studies of MELC have shown that the presence of protein kinase C (PKC) is required for commitment to differentiation, but that the level of PKC activity declines progressively during maturation. In this study, we have established a role for PKC in the maturation of MELC committed to differentiation. Our results show that down-regulation of PKC by addition of phorbol 12-myristate 13-acetate (PMA) to committed MELC blocks subsequent maturation of the cells. Treatment of MELC with the PKC inhibitors H7 and sphingosine had similar effects. Down-regulation of PKC was assayed by measuring cytosolic PKC activity as well as by Western blotting using PKC antibodies. MELC maturation was monitored by loss of the cell-surface fibronectin receptor, release of cells from fibronectin plates, and accumulation of the band 3 anion transport protein. Immunoprecipitation of surface-labeled proteins by an anti-fibronectin receptor (integrin) antibody showed that PMA-treated cultures had more fibronectin receptor protein than untreated cultures 6 days post-induction. As a result, cultures of committed MELC treated with PMA remained attached to fibronectin-coated plates, whereas non-PMA-treated cells were released into the culture medium. Furthermore, PKC-depleted cells accumulated much smaller amounts of band 3 protein and band 3 mRNA than did non-PKC-depleted controls. Our results show that although PKC activity declines progressively during post-commitment maturation of MELC, its continued presence is critical for the process of cellular maturation.


The effects of sphingosine on sarcoplasmic reticulum membrane calcium release. In this study, we report that sphingosine is a potent inhibitor of sarcoplasmic reticulum (SR) calcium release. Evidence is presented demonstrating a direct effect of sphingosine on the SR ryanodine receptor. Calcium release from "skinned" rabbit skeletal muscle fibers and isolated junctional SR derived from the terminal cisternae (TC) was measured in response to caffeine, doxorubicin, 5'-adenylyl-beta,gamma-imidodiphosphate or calcium. Sphingosine inhibited caffeine-induced release in a dose-dependent manner with an IC50 of 0.1 microM for the single muscle fibers and 0.5 microM for the isolated TC vesicles. Near complete blockage of TC calcium release rate was observed with 3 microM sphingosine. Neither sphingomyelin nor sphingosylphosphorylcholine had any effect at the 3 microM level, suggesting that the sphingosine effect was specific. Doxorubicin-induced calcium release and spontaneous calcium release were also blocked by sphingosine. Sphingosine was also capable of stimulating calcium transport in the isolated TC vesicles without an effect on Ca-ATPase activity. Ruthenium red was not capable of substantial additional stimulation of calcium transport nor inhibition of calcium release beyond the action of sphingosine. Sphingosine's blockage of calcium release was not reversed by the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2- methylpiperazine dihydrochloride, suggesting that the action of sphingosine on calcium release was not dependent on ryanodine receptor phosphorylation. Sphingosine significantly increased (8-fold) the Kd for specific [3H]ryanodine binding to TC membranes and decreased the Bmax with a dose dependence similar to the inhibition of calcium release, but sphingosine did not affect the pCa tension relationship of skinned skeletal muscle fibers. These data are consistent with a direct effect of submicromolar sphingosine on the ryanodine receptor. Substantially higher concentrations of sphingosine (30-50 microM) or sphingosylphosphorylcholine (10-20 microM) were capable of inducing calcium release by themselves. Preliminary data indicate that the transverse tubule and not the SR contain substantial sphingomyelinase activity consistent with a transverse tubule source of sphingosine production. Considering that sphingosine is found in micromolar concentrations in some cells, our data indicate that sphingosine generated by the transverse tubule membranes may be a physiologically relevant mechanism for modulating SR calcium release.


Arginine transport in mitochondria of Neurospora crassa. Transport of arginine into mitochondria of Neurospora crassa has been studied. Arginine transport was found to be saturable (Km = 6.5 mM) and to have a pH optimum of pH 7.5. Mitochondrial arginine transport appeared to be facilitated transport rather than active transport because: (i) the arginine concentration within the mitochondrial matrix after transport was similar to that of the reaction medium, and (ii) uncouplers and substrates of oxidative phosphorylation did not affect the transport rate. The basic amino acids ornithine, lysine, and D-arginine inhibited arginine transport. The arginine transport system could be irreversibly blocked by treating mitochondria with the reactive arginine derivative, N-nitrobenzyloxycarbonyl-arginyl diazomethane.


Cloning, sequencing, and expression of the Escherichia coli peptide methionine sulfoxide reductase gene. The gene encoding peptide methionine sulfoxide reductase was cloned from an Escherichia coli genomic library using an oligonucleotide probe based on the amino-terminal sequence of the protein. The nucleotide sequence revealed that the gene codes for a polypeptide of 212 amino acid residues with a calculated molecular weight of 23,314. The protein has been overexpressed in E. coli and is present as a soluble active species.


The role of arrestin and retinoids in the regeneration pathway of rhodopsin. Phototransduction results from a cascade of reactions that culminate in a neuronal signal. Photoisomerization of rhodopsin's chromophore, 11-cis-retinal to all-trans-retinal, leads to the formation of the activated photoproduct metarhodopsin II (Meta II). Subsequently, Meta II initiates the excitation events by activating many copies of the rod cell-specific G-proteins (Gt or transducin). To terminate the signal, the long-lived Meta II must be quenched. Deactivation of Meta II involves phosphorylation by rhodopsin kinase followed by the binding of arrestin. In order to recycle rhodopsin for phototransduction, arrestin must dissociate, and the chromophore must be replaced. In this study, we show that the reduction of the photolyzed chromophore all-trans-retinal to all-trans-retinol is essential for recycling photoactivated rhodopsin. Once this reduction has occurred, the arrestin blockade of the receptor is removed, the chromophore site becomes accessible for regeneration, and the phosphates can be hydrolyzed. If the reduction does not occur, we demonstrate that free all-trans-retinal can react with the apoprotein to form pseudo-photoproducts that are spectrally identical to the photoinduced metarhodopsin species (Meta I/II/III). The Meta II-like product, M380, interacts tightly with arrestin and kinase, however, it does not measurably interact with Gt. The persistent blockade by arrestin and the low affinity for Gt together prevent activation of the visual cascade. Therefore, any insufficiency in the reduction of all-trans-retinal to all-trans-retinol may lead to the accumulation of M380-arrestin in situ, which may effect adaptational processes.


Regulation of sarco-endoplasmic reticulum Ca(2+)-ATPases during platelet-derived growth factor-induced smooth muscle cell proliferation. The role of the sarco-endoplasmic reticulum Ca(2+)-ATPases (SERCA) in the regulation of cell proliferation by Ca2+ was investigated by testing the effect of platelet-derived growth factor (PDGF) on cultured pig aorta smooth muscle cells. For this purpose, the PDGF-mediated rise in the Ca2+ concentration was first examined for its ability to induce the formation of prostaglandins from the specific membrane enzyme, cyclooxygenase. In parallel experiments, similar conditions (10 ng/ml PDGF for 24 h) were used to investigate the smooth muscle cell membrane SERCA2 isoforms. Total SERCA2 activity rose by 472% as reflected by their specific formation of phosphorylated intermediate (E approximately P). This rise correlated with an increase in the amount of SERCA2 proteins (100 kDa) as shown by Western blotting. With isoform-specific anti-SERCA2-a and anti-SERCA2-b antibodies, we demonstrated that the increase in total SERCA2 proteins concerned the minor isoform SERCA2-a, which rose 10-fold, whereas SERCA2-b proteins were not affected. Lastly, Northern blotting using riboprobes showed that PDGF treatment increased the SERCA2-a mRNA species by 82%, and concomitantly decreased the SERCA2-b mRNA by 28%, as a result of isoform switching. We conclude that up-regulation of the SERCA2-type Ca(2+)-ATPases occurs in PDGF-treated smooth muscle cells, which suggests that this enzymatic system plays an essential part in cell proliferation.


Interleukin 1 induces NF-kappa B through its type I but not its type II receptor in lymphocytes. It is not known whether one or both of the interleukin 1 (IL1) receptors mediates the induction of the DNA-binding protein NF-kappa B. Nuclear extracts of the murine lines EL4.NOB.1 and 70Z/3, which bear the type I (80 kDa) and type II (67 kDa) IL1 receptor, respectively, were analyzed by an electrophoretic mobility shift assay. A 265-base pair sequence of the human serum amyloid A gene or a synthetic oligonucleotide each containing the NF-kappa B site were used as the DNA probes. IL1 induction of NF-kappa B was rapid (optimal at 15-30 min) and transient in both cell types. The IL1 receptor antagonist (IL1ra), which binds strongly to the type I receptor, inhibited the NF-kappa B response in both cell lines. IL1ra did not bind to the type II receptor on 70Z/3 cells as judged by competition for binding with 125I-IL1 alpha. When 125I-IL1ra binding to 70Z/3 cells was measured, a small number (10/cell) of high affinity sites (Kd = 5 x 10(-12) M) were detected. These were likely to have been type I receptor because an antibody to this inhibited the NF-kappa B induction in 70Z/3 cells (as well as EL4). Potential signal transduction mechanisms involving protein kinase C or oxygen radicals were studied. Phorbol 12-myristate 13-acetate induced NF-kappa B with a similar time course to IL1 in 70Z/3 but only after 4 h in EL4.IL1 was unaffected by a protein kinase C inhibitor (staurosporine). H2O2 did not mimic IL1, and IL1 was not inhibited by an antioxidant. The type I receptor mediates the induction of NF-kappa B in response to IL1 via a signaling mechanism that still remains to be identified.


Chromosomal localization and organization of the murine genes encoding the beta subunits (AIC2A and AIC2B) of the interleukin 3, granulocyte/macrophage colony-stimulating factor, and interleukin 5 receptors. Chromosomal genes for two mouse homologous beta subunits (AIC2A and AIC2B) of the interleukin-3, granulocyte/macrophage colony-stimulating factor, and interleukin-5 receptors were characterized. Both AIC2A and AIC2B genes were present on a 250-kilobase MluI restriction fragment and were mapped on murine chromosome 15 (these loci were provisionally designated as Il3rb-1 (AIC2A) and Il3rb-2 (AIC2B)), closely linked to the c-sis locus. Both genes consist of 14 exons and span about 28 kb each. The major transcription initiation sites of both genes were mapped at 194 bp from the initiation codon. These genes are 95% identical up to 700 bp from the transcription initiation sites. Potential recognition sequences for hemopoietic transcription factors including GATA-1 and PU.1 in addition to a TATA-like sequence are present in the 5'-flanking region. A stretch of 20 bp including the initiation site is homologous to the corresponding region of the erythropoietin receptor and the interleukin-7 receptor genes and to the initiator sequence of the adeno-associated virus P5 promoter, suggesting a possible role in transcription initiation. Comparison of the exon/intron boundaries of AIC2A and AIC2B genes with those of other members of the cytokine receptor superfamily reveals a conserved evolutionary structure. Isolation of various forms of AIC2 cDNAs reveals differential splicing of the transcripts.


Characterization of a P-type Ca(2+)-ATPase from Flavobacterium odoratum. In most bacterial cell types studied, low intracellular free calcium is maintained by a variety of secondary exchangers which utilize transmembrane ion gradients. Prokaryotic calcium ATPases appear to be extremely uncommon, and none have been reported in Gram-negative organisms. We demonstrate ATP-dependent calcium uptake in everted membrane vesicles of Flavobacterium odoratum, a common Gram-negative soil and water bacterium. Calcium is transported with an apparent initial rate of 10 nmol/min mg of protein. It is inhibited by 20 microM orthovanadate, a specific P-type ATPase inhibitor, but significantly, it is unaffected by the addition of N-ethylmaleimide, N,N-dicyclohexylcarbodiimide, valinomycin, or nigericin. Because the Ca(2+)-ATPase makes up a high proportion of the total ATPase activity it is easily detected by a soluble ATP hydrolysis assay, with an initial rate for calcium-dependent ATPase activity in vesicles of 25-40 nmol/min.mg at pH 7.8 and 25 degrees C. The calcium-dependent activity is preferentially solubilized by the detergent C12E8 and can be precipitated at 55-80% ammonium sulfate in a fraction free of other contaminating ATPase activities. This partially purified fraction is enriched 15-fold and demonstrates an apparent Km for calcium of 2 microM, and for ATP of 130 microM. The IC50 for vanadate is 1.6 microM. These values are similar to those obtained for the eukaryotic sarcoplasmic reticulum calcium ATPase. The enzyme is rapidly phosphorylated by [gamma-32P]ATP in a calcium-dependent, vanadate-inhibitable manner. The phosphorylated species migrates with an apparent molecular mass of 60 kDa by NaDodSO4-polyacrylamide gel electrophoresis, and the phosphoryl group is sensitive to alkaline conditions, a characteristic of the acylphosphate linkage found in ATPases. These data demonstrate that the majority of calcium transport in F. odoratum is facilitated by a P-type ATPase.


Synergistic actions of epidermal growth factor-urogastrone and vasopressin in cultured aortic A-10 smooth muscle cells. In cultured rat aorta-derived A-10 cells, epidermal growth factor-urogastrone (EGF-URO) acts synergistically with arginine vasopressin (AVP) to augment the AVP-mediated release of 3H-arachidonate (3H-AA) from 3H-AA prelabeled cells. On its own, EGF-URO had no effect on AA release and had no effect on calcium influx or efflux either in the absence or presence of AVP. The synergistic action of EGF-URO was not affected by actinomycin D, cycloheximide, indomethacin, by the diacylglycerol lipase inhibitor U-57,908, or by the tyrosine kinase inhibitors genistein (GS) and tyrphostin (TP). TP did, nonetheless, completely abrogate 3H-thymidine incorporation triggered in the presence of EGF-URO. Although EGF-URO stimulated an increase in calpactin-II (lipocortin-I) phosphorylation in permeabilized cells, no such increase was detected in intact cells exposed to EGF-URO either alone or in combination with AVP, under conditions where EGF-URO augmented the action of AVP. The phospholipase A2 inhibitor, mepacrine, had no effect on AVP-mediated AA release, but abolished the synergistic action of EGF-URO. We conclude that in contrast with our previous results with gastric smooth muscle strips, wherein EGF-URO acts via the diacylglycerol lipase-mediated metabolism of diacylglycerol, and in keeping with observations with cultured mesangial cells, EGF-URO acts synergistically with AVP in A-10 cells via the activation of phospholipase A2. This synergistic action of EGF-URO does not appear to be due to increased levels of cyclooxygenase and would appear not to require increased tyrosine kinase activity.


Phage typing of coagulase-negative staphylococci. Seventy-nine staphylococcal strains isolated from blood cultures (57 coagulase-negative staphylococci (CNS) and 22 S. aureus) and 308 CNS isolated from the skin of healthy donors were phage typed. S. epidermidis and S. capitis were readily typed with 91 strains out of 124 and 24 strains out of 43 strains being successful. Species such as S. haemolyticus, S. hominis and S. simulans could be moderately phage typed. Others gave only a few strains capable of being typed, such as S. saprophyticus and S. sciuri. Under our experimental conditions the S. warneri, S. xylosus and S. cohnii could not be typed with our set of phages.


[Vascular endoprosthesis. A new indication in the surgery of the iliac artery]. We have used endoprosthesis (Palmaz Schatz) after balloon angioplasty of iliac arterial stenoses or thromboses, in order to increase the immediate patency and to prevent the recurrence of stenosis. Our series gathers 24 patients operated with endovascular procedures over a period of 2 years: 22 men, 2 women--extreme ages 42 to 78 years, average age 63.5 years--Clinical stage: 22 at stage I, 1 at stage III, 1 at stage IV. Arteriographic findings: 8 primary iliac lesions (6 stenoses and 2 thromboses), 11 external iliac lesions (stenoses). All these lesions were atheromatous. One of them had recurred after angioplasty. Usual technique: balloon angioplasty of the stenosis, assessment on a fluoroscopic screen and angioscopy of the result, decision to insert the Palmaz Stent if defects are seen on the image. Repatency of impassable lesions with a YAG laser was carried out in 2 cases. The indication of an endoprosthesis was established on the basis of the radiological image in 17 cases, of the angioscopic image in 4 and systematically in 10 cases of recurrence of stenosis, iliac thrombosis or associated surgery. Associated surgery: 2 femoropopiteal bypass grafts, 3 femorofemoral bypass grafts, 1 deep plasty, 1 superficial femoral recanalization with laser, 1 lymbar sympathectomy. Postoperative results: 1 death due to MI (78-year-old diabetic woman), 1 thrombosis treated with femorofemoral bypass. Middle-term results: after 6 to 24 months, average time lapse 13 months. The comparison of the ankle pressure indices and of the pre- and postoperative sonographic findings shows an indisputable hemodynamic improvement.


Phosphorylation of myosin-II regulatory light chain by cyclin-p34cdc2: a mechanism for the timing of cytokinesis. To understand how cytokinesis is regulated during mitosis, we tested cyclin-p34cdc2 for myosin-II kinase activity, and investigated the mitotic-specific phosphorylation of myosin-II in lysates of Xenopus eggs. Purified cyclin-p34cdc2 phosphorylated the regulatory light chain of cytoplasmic and smooth muscle myosin-II in vitro on serine-1 or serine-2 and threonine-9, sites known to inhibit the actin-activated myosin ATPase activity of smooth muscle and nonmuscle myosin (Nishikawa, M., J. R. Sellers, R. S. Adelstein, and H. Hidaka. 1984. J. Biol. Chem. 259:8808-8814; Bengur, A. R., A. E. Robinson, E. Appella, and J. R. Sellers. 1987. J. Biol. Chem. 262:7613-7617; Ikebe, M., and S. Reardon. 1990. Biochemistry. 29:2713-2720). Serine-1 or -2 of the regulatory light chain of Xenopus cytoplasmic myosin-II was also phosphorylated in Xenopus egg lysates stabilized in metaphase, but not in interphase. Inhibition of myosin-II by cyclin-p34cdc2 during prophase and metaphase could delay cytokinesis until chromosome segregation is initiated and thus determine the timing of cytokinesis relative to earlier events in mitosis.


Use of direct injection precolumn techniques for the high-performance liquid chromatographic determination of the retinoids acitretin and 13-cis-acitretin in plasma. A previously developed highly sensitive high-performance liquid chromatographic method for the determination of retinoids, using direct injection of large plasma volumes, on-line solid-phase extraction and ultraviolet detection, was improved and fully validated for the determination of acitretin and 13-cis-acitretin in plasma samples. The addition of acetonitrile to improve the recovery was performed on-line by a T-piece, avoiding any cis-trans isomerization which could occur when acetonitrile was added prior to storage in the autosampler. About 30 injections could be made onto one precolumn despite the large injection volume (1 ml of plasma containing the internal standard). Full automation was attained by the use of automated precolumn replacement. In addition, forward- and back-flush purging of the precolumn enhanced the longevity of the analytical column. This consisted of three coupled C18 columns of 125 mm length each. The quantification limit was 0.3 ng/ml, using ultraviolet detection at 360 nm, and the mean inter-assay precision was 3.8% for the two compounds.


Effect of atrial natriuretic peptide on potassium-stimulated aldosterone secretion: potential relevance to hypoaldosteronism in man. Atrial natriuretic peptide (ANP) has been shown to suppress aldosterone secretion under certain circumstances, although the physiological significance of this is uncertain. We wondered if ANP would suppress potassium-stimulated aldosterone secretion in man and, if so, whether we might find high circulating levels of ANP in patients with the syndrome of acquired hypoaldosteronism. We studied seven healthy young subjects under two conditions: 1) infusion of KCl (0.5 mmol/kg) over 45 min, and 2) KCl infused with ANP (0.01 microgram/kg.min) for 60 min. We also evaluated ANP levels in eight elderly subjects with the syndrome of acquired hypoaldosteronism, as defined by hyperkalemia (mean serum K+, 5.3 +/- 0.1 mmol/L) associated with inappropriately low aldosterone levels (216 +/- 50 pmol/L). In the normal subjects, ANP almost completely suppressed the aldosterone response to KCl infusion (P less than 0.001, by analysis of variance) despite a similar rise in the serum potassium level with KCl alone (0.70 +/- 0.07 mmol/L) and KCl plus ANP (0.75 +/- 0.09 mmol/L). PRA fell slightly during KCl plus ANP treatment, but did not change during the infusion of KCl alone. ANP levels were approximately 800 pmol/L during the ANP infusion studies. Endogenous ANP levels in the hyperkalemic patients with hypoaldosteronism were markedly elevated at 1186 +/- 340 pmol/L (compared to 93 +/- 10 pmol/L in healthy elderly controls), a level that would be capable of suppressing the potassium-mediated aldosterone response. Exogenous infusion of ANP suppressed the aldosterone response to hyperkalemia, and ANP levels were found to be markedly elevated in a group of patients with hyperkalemia and hypoaldosteronism. We suggest that ANP may contribute to clinically significant hypoaldosteronism and hyperkalemia in the syndrome of acquired hypoaldosteronism.


Assessment of growth hormone (GH) axis in Turner's syndrome using 24-hour integrated concentrations of GH, insulin-like growth factor-I, plasma GH-binding activity, GH binding to IM9 cells, and GH response to pharmacological stimulation. The GH axis was studied in Turner's syndrome (TS) patients. Thirty-seven prepubertal TS patients and 42 normally growing girls (NGG; 5.5-16.3 yr old), of whom 13 were prepubertal, were studied by 24-h continuous blood withdrawal and provocative tests. The 24-h integrated concentrations of GH (IC-GH), FSH (IC-FSH), and insulin-like growth factor-I (IC-IGF-I) as well as the IC-IGF-I/IC-GH ratio were determined. An increase in IC-GH with age and progression of puberty was found in NGG, but not in TS. IC-GH in the NGG was significantly higher than that in age-matched TS patients. Estrogen replacement therapy normalized IC-GH levels in 6 TS patients in whom these levels were subnormal for age. A positive correlation between IC-GH and IC-FSH or IC-estradiol was found in NGG (r = 0.462; P less than 0.01), but not in TS patients. The IC-IGF-I/IC-GH ratio was significantly higher in the TS than in the NGG group. Serum GH-binding activity and serum GH binding to IM9 cells in the TS group did not differ from those in the normal group. We hypothesize that the growth retardation of TS results from a combination of insufficient GH secretion, mainly due to sex steroid deficiency, and an end-organ resistance to IGF-I. IGF-I receptor studies are needed to test this speculation about IGF-I resistance.


Reduction of vasomotor symptoms and bone mineral density loss with combined norethindrone and long-acting gonadotropin-releasing hormone agonist therapy of symptomatic endometriosis: a prospective randomized trial. The hypoestrogenic state induced by gonadotropin-releasing hormone agonists (GnRHa) has been shown to suppress symptomatic endometriosis effectively but to elicit vasomotor symptoms and loss of bone mineral density. The role of norethindrone as a supplement to GnRHa in eliminating such side effects was assessed by enrolling 20 patients with symptomatic endometriosis diagnosed laparoscopically in a randomized, prospective, double-blinded trial. All patients received the long-acting GnRHa leuprolide acetate 3.75 mg im every 4 weeks for 24 weeks. Ten patients self-administered norethindrone 5 then 10 mg by mouth daily, whereas the remainder self-administered placebo tablets. Results of this study showed that combination therapy was as effective as GnRHa alone in significantly reducing circulating gonadotropin and estrogen levels (P less than 0.01), extent of visible endometriotic implants (P less than 0.01), and painful symptoms (P less than 0.01). Marked vasomotor and vaginal symptoms experienced by patients given GnRHa alone were minimized in those receiving GnRHa with norethindrone. Lumbar spine bone mineral density loss, measured by dual energy x-ray absorptiometry, was significantly reduced and more completely reversed in patients receiving combination therapy (P less than 0.05). A reversible decrease in high density lipoprotein-cholesterol and increase in low density lipoprotein:high density lipoprotein ratio was noted only in the patients receiving combination therapy, but not in those receiving GnRHa only. The addition of norethindrone to GnRHa is an effective means of treating symptomatic endometriosis while ameliorating side effects induced by GnRHa alone.


Effects of a long-term treatment with alacepril on left ventricular hypertrophy and function in patients with essential hypertension. The authors examined the effects of a long-term treatment with the angiotensin-converting enzyme inhibitor, alacepril, with respect to the reversal of left ventricular hypertrophy and the improvement of left ventricular function. Ten uncomplicated essential hypertensive patients with left ventricular hypertrophy, aged 53 +/- 8 years, were treated with alacepril alone for 12 months. All patients underwent echocardiography to to assess left ventricular dimensions and function before and after the treatment. After the treatment, blood pressure was decreased significantly from 163 +/- 14.1/98 +/- 4.2 to 142 +/- 20.3/86 +/- 11.0 mm Hg (each, P less than .01), whereas heart rate did not change (66 +/- 6 versus 69 +/- 8 beats/min). The left ventricular mass index was decreased significantly from 146 +/- 27 to 119 +/- 29 g/m2 (P less than .01). Ejection fraction, fractional shortening, peak shortening rate, and peak lengthening rate all improved significantly after the treatment. There was a significant inverse relationship between fractional shortening and end-systolic wall stress before the treatment (r = .63, P less than .05), and this relationship did not change after the treatment. It is concluded that alacepril improved both left ventricular systolic and diastolic function without causing any consistent augmentation of left ventricular contractility.


Long-standing dermatological manifestations in a patient with chronic heavy metal intoxication. A patient with chronic metal intoxication is described, presenting during four years after the cessation of her exposure to industrial substances, maculo-papular eruptions with several ulcerated lesions and excoriations on her abdomen and buttocks. She also had pallor of her face, greyish-dark discoloration of the hair, while the fingernails were brittle and sensitive. Scrupulous physical examination excluded further cutaneous involvement. The immunological workup revealed both phenotypic and functional defects in cellular immunity.


A case of lymphadenosis benigna cutis of the earlobe: an immunohistochemical study. We report a 38-year-old woman who developed an erythematous nodule on her earlobe after wearing pierced-type 18-carat gold earrings. Biopsy specimens revealed dense cellular infiltration predominantly composed of lymphocytes and plasma cells in the dermis and subcutaneous tissue with formation of lymph follicles. Morphological and immunohistochemical analyses were compatible with lymphadenosis benigna cutis.


Orthognathic surgery for the treatment of chronic self-mutilation of the lips. Self-mutilation by lip chewing is an uncommon problem, but may result in severe mutilation, infection and loss of tissue, with associated scarring. Many modalities of treatment have been proposed in the literature with variable success and morbidity to the patient. Alternative techniques involving orthognathic surgery to create an anterior open bite are described which may result in cessation of self-mutilation. A case is presented with a 13-year follow-up, the anterior open bite is still present, and no recurrence of self-mutilation.


Establishment of stable CD8+ suppressor T cell clones and the analysis of their suppressive function. Stable CD8+ suppressor T cell (Ts) clones were established by a relatively simple method. Keyhole limpet hemocyanin (KLH)-primed spleen cells from C3H mice were depleted of B cells and CD4+ T cells by panning and cytotoxic treatment, and the resulting CD8+ T cells were periodically stimulated with antigen and irradiated syngeneic spleen cells followed by manifestation in interleukin-2 (IL-2) containing medium. T cell clones with a definite suppressor function were established by limiting dilution. They were defined as classical effector type Ts of CD8+ phenotype as they had constant and definite suppressor functions in antigen-induced T cell proliferation and specific antibody response against T cell-dependent antigens without detectable cytotoxic activity against both antigen presenting cells (APC) and helper T cells (Th). They showed no helper activity for B cells and produced no detectable helper type lymphokines such as IL-2 and IL-4. CD8+ Ts clones were able to inhibit the antigen-induced IL-2 production of normal and cloned T cells. Their suppressive activity was antigen-nonspecific and major histocompatibility complex-unrestricted. CD8+ Ts clones were also able to suppress the proliferative response of Th clones induced by immobilized anti-T cell receptor (TcR) and anti-CD3 mAbs but not the response induced by concanavalin A (ConA) and IL-2. All the CD8+ T cell clones established independently utilized the TcR V beta 8 gene. Syngeneic antigen presenting cells could induce proliferation of these CD8+ clones, which was blocked by anti-CD8 and anti-I-Ak monoclonal antibody (mAb) but not by anti-class I mAbs. The stimulation of CD8+ Ts clones with immobilized anti-CD3 resulted in the release of a suppressor factor(s) that potently inhibited the antigen-induced proliferation of CD4+ Th clones and the in vitro secondary antibody formation.