Renin release responses to in vitro prostaglandin challenge in the summer-active ground squirrel, Spermophilus lateralis

1. Using a renal cortical slice preparation from the summer-active ground squirrel, Spermophilus lateralis, resting renin release (RR) and tissue cyclic AMP content (TcAMPC) levels were found to be significantly higher and lower, respectively, than those previously reported for its hibernating counterpart. 2. At a 10(-5) M dose, PGE2 but not PGE1, PGF2-alpha or PGA1, significantly stimulated RR in the summer-active ground squirrel (SAGS). 3. Addition of agents which normally increase TcAMPC significantly potentiated the effect of PGE1, while preventing that of PGE2, on RR and TcAMPC. 4. Opposite TcAMPC changes may mediate the in vitro RR responses to PGE1 and PGE2 administration in the SAGS.     

Humoral factors released during trauma ofAplysia body wall

1. Preliminary, general chemical characteristics of substances in artificial sea water (ASW) washed through stimulated body wall (SBW) and in hemolymph taken from noxiously stimulated animals (SHL) were consistent with those of classical neurotransmitters, amino acids, and small- to medium-sized peptides. 2. 5-Hydroxytryptamine (5HT) and acetylcholine (ACh), unlike SBW and SHL, caused relaxation when perfused into isolated body wall. FMRFamide produced a biphasic response--brief contraction followed by prolonged relaxation. 3. Small cardioactive peptide (SCPB) caused body wall contractions similar to those produced by SBW and SHL, except that SCPB contractions displayed more desensitization and were completely blocked by 30 mM CoCl2. SCPB and SBW contractions were synergistic. 4. Dopamine caused persistent body wall contractions similar to those of SBW and SHL. Dopamine contractions were reduced but not blocked by 30 mM CoCl2. Unlike SBW activity, dopamine activity was reduced by alkalinization. 5. Glutamate and taurine produced strong but usually short-lasting body wall contractions. Adenosine, octopamine, arginine vasotocin, and cholecystokinin (CCK-8) caused weak or variable contractions. Met-enkephalin and somatostatin caused no obvious body wall responses. 6. When superfused over the fully sheathed abdominal ganglion, FMRFamide, met-enkephalin, glutamate, aspartate, and taurine reduced the magnitude of the gill-withdrawal reflex elicited by siphon nerve stimulation. 7. Taken together with earlier results, these data suggest a preliminary framework for trauma signal pathways. It is proposed that stress hormones (perhaps including FMRFamide, SCPs, 5HT, and dopamine) are released into hemolymph from neuroendocrine cells. Effective amounts of active intracellular solutes such as amino acids may also be released by extensive cellular rupture. Various humoral signals produce slow effects that contribute to hemostasis, balling up, increased cardiac output, and reflex suppression.     

Immunophenotyping of acute myeloid leukaemia: relevance of analysing different lineage-associated markers

The immunophenotype of 135 previously untreated patients with FAB defined acute myeloid leukaemia (AML) was studied at diagnosis. The panel of reagents included monoclonal antibodies (MoAb) recognising myeloid-associated determinants (CD11, CD13, CD14, CD33 and others) as well as MoAb directed towards lymphoid antigens (CD7, CD10, CD19) and TdT. The results indicate that CD13 and/or CD33 are consistently expressed in AML and only rarely in ALL blasts (131/135 + ve cases, versus 4/130 in ALL). Lymphoid antigen expression was rarely detected when CD10 and CD19 were investigated in AML (0.9% and 2% + ve cases, respectively), whereas significant positivities were found for TdT and CD7 (20% and 10% respectively). Concerning FAB subtypes, two new MoAb (LAM3 and LAM7) proved very useful in the specific recognition of AML with monocytic features. The phenotype CD13+ and/or CD33+, CD9+, HLA-DR- was found to be almost exclusive for M3 AML. The response to induction chemotherapy was analysed in CD7+ and in TdT+ patients. In the latter group a statistically significant lower response rate was found with respect to TdT-ve-AML patients.     

Beta 1 and beta 2 adrenergic agonists in exocrine pancreatic secretion in the rabbit

The effect of Dobutamine (a beta 1-adrenergic agonist) and Terbutaline (a beta 2-adrenergic agonist) on exocrine pancreatic secretion was studied in anaesthetized rabbits, simultaneously controlling pancreatic blood flow and blood pressure. The secretion of fluid and ions (bicarbonate, sodium and potassium) was unaffected by the infusion of Dobutamine (8 micrograms.kg-1.min-1) or Terbutaline (10 micrograms.kg-1.min-1). Neither were pancreatic blood flow or mean blood pressure altered. Dobutamine or Terbutaline depress the function of the acinar cells, amylase secretion being more affected by the action of Terbutaline. The results show that beta 1 and beta 2-adrenergic stimulation has no effect on the ductular cells but does decrease the secretion by the acinar cells.     

AViscum album oligosaccharide activating human natural cytotoxicity is an interferon γ inducer

Summary CommercialViscum album extract Helixor-M contains a dialysable oligosaccharide (HM-BP) that activates natural killer (NK) cytotoxicity against K562 tumour cells when preincubated with human peripheral blood mononuclear cells (PBMC) for 72 h. The activated effector cells were exclusively found in the monocyte/macrophage subpopulation. However, when peripheral non-adherent cells (PNAC) were preincubated with HM-BP for 72 h the NK cytotoxicity of CD56⁺CD3^– NK cells was activated. This discrepancy was found to be due to the release of prostaglandin E₂ from activated monocytes/macrophages, which blocked activation of the cytotoxicity of NK cells. Analysis of the supernatant culture medium after 72 h preincubation demonstrated that HM-BP induced release of interferon (IFN) from T cells (preferentially from CD3⁺CD4⁺ cells) and of tumour necrosis factor (TNF) from monocytes/macrophages. Release of IFN was the crucial step for activation of NK cytotoxicity since enhancement of NK cytotoxicity during pretreatment of PBMC or PNAC with HM-BP was completely blocked in the presence of anti-IFN antibodies. Anti-interleukin-2, anti-TNF or anti-IFN antibodies had no effect on the HM-BP-induced enhancement of NK cytotoxicity. The activation of the NK cytotoxicity of nonadherent cells by interleukin-2 treatment was found to be synergistic to the enhancement of NK cytotoxicity by treatment with HM-BP.     

Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA

We report the cloning, nucleotide (nt) sequence and expression of the cDNA (pah) encoding phenylalanine hydroxylase (PAH) of Drosophila melanogaster. The strong hybridization signals observed in genomic blots when D. melanogaster DNA was probed with 32P-labeled human pah cDNA, indicated the existence of a high degree of sequence similarity between the pah genes of both species. The length of the pah genomic fragment is about 30 to 40 kb. The cDNA contains 84 bp of the 5'-untranslated region, 1359 bp of the protein-coding region and 87 bp of the 3' region, with only one polyadenylation signal. The isolated cDNA is probably full-length, since the size of the D. melanogaster PAH mRNA is 1.5 kb. At the nt level, the similarity of the D. melanogaster cDNA with human and rat pah cDNAs is 57.9% and 58.1%, respectively. The highest similarities are restricted to the nt sequence coding for the presumed hydroxylation domain. There is no nt sequence similarity between the first three exons of the human pah gene and an equivalent fraction of the D. melanogaster pah gene. At the amino acid (aa) level, the similarity in the presumed hydroxylation domain is 88.5%, in which two motifs of the structure AGLLSSXXXL are found, where X represents any aa. It was interesting to notice the conservation of aa 408, 311 and 280, where mutations are associated with phenylketonuria in humans. We observed, moreover, that, as it occurs in humans and rats, the expression of the D. melanogaster pah gene is tissue-specific and temporally regulated.     

Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution.

The development of a dedicated two-dimensional gel electrophoresis system is described that provides superior performance in terms of high resolving power and enhanced gel-to-gel reproducibility. Isoelectric focusing is performed in a 1-mm capillary tube with a 0.08-mm thread, optimized for this application, incorporated along its length prior to polymerization of the gel matrix. The isoelectric focusing gel is 4% T, 2.6% C to minimize sieving of proteins and promote adhesion of the gel to the thread. The thread incorporated in the isoelectric focusing matrix prevents gel stretching and breakage during its application to the second dimension. An optimum ampholyte pH range has been defined based on 1600 polypeptides present in a transformed fibroblast cell lysate and verified using a variety of other cell types. The length of time required to complete an electrophoretic separation in the second dimension was found to depend on buffer conductivity establishing the importance of high quality electrophoresis grade reagents devoid of contaminating salts. To ensure reproducibility of electrophoretic separations, it is critical to maintain a strict control of temperature during the second dimension separation. This prevents altered migration of some polypeptides relative to neighboring polypeptides that have constant Rfs over a broad temperature range. It was also determined that to obtain the maximum information from a complex protein mixture it is critical to use a large format 22- x 22-cm two-dimensional electrophoretic system. Using the optimized two-dimensional electrophoretic system and computerized gel analysis, it was determined that molecular weight estimates of polypeptides differed by approximately 350 daltons between gels, while isoelectric point estimates differed by approximately 0.03 pH units between gels. Using the two-dimensional electrophoresis system described, approximately 1000 polypeptides can be routinely detected from silver-stained 10% polyacrylamide gels or 1600 polypeptides from autoradiographs of 35S-methionine-labeled polypeptides.     

Fermentation parameters of Peptostreptococcus productus on gaseous substrates (CO,H2/CO2)

Intrinsic growth and substrate uptake parameters were obtained for Peptostreptococcus productus, strain U-1, using carbon monoxide as the limiting substrate. A modified Monod model with substrate inhibition was used for modeling. In addition, a product yield of 0.25 mol acetate/mol CO and a cell yield of 0.034 g cells/g CO were obtained. While CO was found to be the primary substrate, P. productus is able to produce acetate from CO₂ and H₂, although this substrate could not sustain growth. Yeast extract was found to also be a growth substrate. A yield of 0.017 g cell/g yeast extract and a product yield of 0.14 g acetate/g yeast extract were obtained. In the presence of acetate, the maximum specific CO uptake rate was increased by 40% compared to the maximum without acetate present. Cell replication was inhibited at acetate concentrations of 30 g/l. Methionine was found to be an essential nutrient for growth and CO uptake by P. productus. A minimum amount of a complex medium such as yeast extract (0.01%) is, however, required.     

Annulate lamellae: comparison of antigenic epitopes of annulate lamellae membranes with the nuclear envelope

Laminin derived from the Engelbreth-Holm-Swarm (EHS) tumor and a lamininlike molecule synthesized by RN22 Schwannoma cells both stimulate rapid neurite outgrowth, consistent with a common neurite-promoting site. However, antilaminin antisera can only inhibit the activity of the EHS laminin. The blocking antibodies in such sera are directed against the terminal heparin-binding domain of the laminin long arm (Edgar, D., R. Timpl, and H. Thoenen. 1984. EMBO [Eur. Mol. Biol. Organ.] J. 3: 1463-1468). These epitopes are demonstrated by immunoblotting to be part of the A chain and to be absent in RN22 laminin, showing (through metabolic labeling) that the cells synthesized little if any 440-kD A chain. This indicates that the antibody inhibition was probably due to steric hindrance, a common neurite-promoting site, apparently not being antigenic in native molecules. Antibodies raised against a 25-kD proteolytic fragment derived from the long arm of laminin were then used as probes to identify other potential neurite-promoting structures. Although these antibodies do not cross-react with native laminin, they recognized the B chains of denatured EHS and RN22 molecules on immunoblots. The antibodies also bound to the large proteolytic fragment, derived from the long arm of laminin that contains the neurite-promoting site, thus inhibiting its activity. Taken together, these results point to the localization of normally nonantigenic, defined, B chain sequences within or close to the neurite-promoting site of laminin.     

Intercalation of anthracyclines into living cell DNA analyzed by flow cytometry.

Anthracyclines (ANT) are used in the treatment of leukemia and other cancers. These drugs have been shown to intercalate between the strands of DNA. In the present study, we show that the amount of ANT intercalated into DNA can be determined by measuring the fluorescence resonance energy transfer (FRET) between Hoechst 33342 (H33342) and ANT bound to DNA. The transfer efficiency was found to depend on the amount of disposable ANT but was independent of the amount of H33342 bound to DNA over a wide range of H33342 concentrations. The method was adapted for flow cytometric measurement of FRET in whole living cells and was used to evaluate the degree of intercalation of daunorubicin (DAU) and idarubicine (IDA) into DAU-sensitive and DAU-resistant leukemic cell lines. ANT intercalation into DNA was affected by factors which modify the intracytoplasmic concentration of ANT, and it was shown that the action of ANT and the resistance to ANT could not be attributed solely to the intercalative effect of the drugs. The method has advantages over previously described methods and represents a useful complementary tool in studies on the mode of action of ANT and the mechanisms of chemoresistance.