Effects of membrane physical parameters on hematoporphyrin-derivative binding to liposomes: A spectroscopic study

Summary Physical parameters of membrane bilayers were studied for their effect on the binding of hematoporphyrin derivative (Hpd), which is used as a sensitizer in photodynamic therapy of cancerous tissues. The purpose of this study was to clarify which parameters were relevant, under physiological conditions, to the selectivity of Hpd binding to cancer cells. Fluorescence spectroscopy was used to measure the relative partitioning of the dye between the lipid and aqueous media. Increasing the microviscosity of the liposomes' membranes by various bilayer additives results in a strong reduction of Hpd binding, to an extent independent of the specific additive. The effect of temperature near the physiological value as well as the effect of cross membrane potential are small. Surface potential does not affect the binding constant, indicating that the binding species does not carry a net electric charge.     

NIH 3T3 cells transfected with human tumor DNA lose the transformed phenotype when treated with swainsonine

Phenotypic expression of transformation was inhibited by swainsonine at concentrations which affect the late stages of glycoprotein processing but not growth of cells. In the presence of swainsonine, NIH 3T3 fibroblasts transfected with human tumor DNA (al-l) no longer grew in soft agar or expressed complex type oligosaccharides characteristic of transformed cells. Thus, it appears that glycoproteins with fully processed oligosaccharides are necessary for the maintenance of the transformed phenotype in these cells.     

The relationship of calcium and parathyroid hormone in their effect on heart cells

Parathyroid hormone (PTH), the plasma concentration of which is raised in uremia, has been suggested as one of the agents responsible for the myocardial changes commonly seen in uremia. The effect of intact [1–84] PTH on rat heart cells grown in tissue culture has been studied. Addition of the hormone to the media significantly stimulated beating rate. The stimulation was directly proportional to the amount of PTH in the medium. Excessively high concentration of PTH caused immediate cessation of the beating, which was reversed by the addition of calcium to the medium. The extent of stimulation by PTH was inversely proportional to the calcium concentrations in the medium. Isoproterenol and phenylephrine at excessively high concentrations in the medium did not mimic the PTH effect either alone or together with PTH. When beating ceased due to verapamil the effect was not reversed by the addition of calcium to the medium.Calcium added to the myocytes seen after beating ceased reversed the effect and the cells started to beat again. Cells kept for a longer period in the arrested state were not revived by the addition of calcium.     

Induction of T4 DNA ligase in a recombinant strain of Escherichia coli

The kinetics of cell growth and foreign protein production, as well as factors affecting protein stability, were studied and optimized in batch and fed-batch fermentations of a recombinant strain of Escherichia coli. The pL promoter from bacteriophage lambda under the control of a temperature-sensitive cl represser, with the entire construct integrated into the E. coli chromosome through the use of a defective bacteriophage lambda lysogen, was used to direct the synthesis of T4 DNA ligase. The biphasic fermentations consisted of a primary growth phase at 30 degrees C followed by an induction phase which was initiated by shifting the temperature to 42 degrees C. In the fed-batch fermentations, additional nutrients were added at the time of initiating induction. Maintenance of sufficiently high concentrations of the organic substrates (glucose and casamino acids) during the induction phase was required for continued cell growth at 42 degrees C. Such growth was essential for T4 DNA ligase formation and in vivo stability. Hence, fed-batch fermentations produced the highest yield of the foreign protein Commensurate with providing lower total amounts of substrates. In such cases, high cell densities (6 g dry wt/L) with substantial intracellular levels of T4 DNA ligase (4.6% total cellular protein, or 2.7% of the dry biomass) were achieved.     

N-linked oligosaccharide changes with oncogenic transformation require sialylation of multiantennae

Glycopeptides derived from NIH 3T3 fibroblasts and these cells transformed by transfection with human DNA containing oncogene H-ras were analyzed by 500-MHz 1H-NMR spectroscopy and binding to immobilized lectins. The cells were metabolically labeled with D-[3H]glucosamine or L-[3H]fucose and the glycopeptides included in Bio-Gel P-10 (Mr 5000-3500) were separated into neutral and charged fractions on DEAE-cellulose. The major portion (80%) of these [3H]fucose glycopeptides from the non-transformed NIH 3T3 fibroblasts were neutral or contained one or two charged residues, whereas 90% of the glycopeptides from the transformed cells contained two or more charged residues. The structure of the predominant neutral glycopeptide from the non-transformed NIH 3T3 cells was determined by 1H-NMR spectroscopy to be tetraantennary containing terminal Gal alpha 1----3. (formula; see text) This structure was verified by binding to the immobilized alpha-Gal-specific lectin, Griffonia simplicifolia I and leukoagglutinating phytohemagglutinin from Phaseolus vulgaris (L-PHA), which binds certain tri- or tetraantennary glycopeptides. In contrast, the structure derived by NMR spectroscopy of one of the predominant charged glycopeptides from the transformed cells was triantennary containing terminal NeuNAc alpha 2----3 in addition to Gal alpha 1----3. (formula; see text) In attempting to verify this structure by lectin-binding properties it was found that removal of NeuNAc alpha 2----3 reduced the affinity to L-PHA - agarose. The other major glycopeptides of the transformed cells which were more charged also cotained NeuNAc alpha 2----3 but no NeuNAc alpha 2----6 or Gal alpha 1----3. A tentative structure was proposed for the major glycopeptide of the first charged class from NIH 3T3 cells on the basis of lectin-binding properties and the NMR spectrum which showed, in addition to NeuNAc alpha 2----3, the presence of NeuNAc alpha 2----6 and Gal alpha 1----3. On the basis of the NMR spectrum and other results, it is concluded that the presence of tetraantennary oligosaccharides are not sufficient for the transformed oligosaccharide phenotype. Rather, the tri- or tetraantennae must be sialylated in alpha 2----3 linkage, on more than one antennae, when properties of transformation are expressed in NIH 3T3 cells. Prior to transformation the tetraantennary oligosaccharides of these cells are terminated in alpha-Gal residues, whereas after transformation alpha-Gal residues appear to be replaced by NeuNAc alpha 2----3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of Chronic Lithium Treatment on 5-Hydroxytryptamine Autoreceptors and Release of 5-[3H]Hydroxytryptamine from Rat Brain Cortical, Hippocampal, and Hypothalamic Slices

The effect of acute and chronic lithium treatments on 5-hydroxytryptamine (5-HT, serotonin) release and on its regulation by presynaptic 5-HT autoreceptors was studied in [3H]5-HT preloaded superfused rat brain slices. The [3H]5-HT overflow evoked by a 30-s exposure to 65 mM K+ was increased after 3 weeks of ingestion of lithium-containing diet in the three brain areas examined. Acute injection of 4 mEq/kg lithium chloride did not affect 5-HT release. The K+-induced release observed in both control and chronically lithium-treated animals was Ca2+-dependent. Chronic lithium treatment was also found to be associated with a decrease in basal [3H]5-HT overflow in the cortex and hypothalamus but not in hippocampus [corrected]. The Ca2+-independent overflow induced by fenfluramine was also decreased in cortical slices from lithium-treated animals. The sensitivity of the inhibitory 5-HT autoreceptors was assessed by the response to the 5-HT agonist 5-methoxytryptamine. The results indicate a marked reduction in the maximal inhibition of [3H]5-HT release induced by 5-methoxytryptamine in slices obtained from animals which have been treated with lithium for 3 weeks. These data suggest that the functional down regulation of the prejunctional 5-HT sites may be responsible for the increase in K+-stimulated 5-HT overflow in brain slices of animals treated chronically with lithium.     

Prenatal care: a comparative evaluation of nurse-midwives and family physicians.

We evaluated the prenatal care provided to 44 low-risk women by nurse-midwives (NMs) at a special clinic of a large obstetric referral hospital and a sample of 88 low-risk women attended by family physicians (FPs) in their offices. The women were matched on the basis of date of delivery, age, parity, number of previous miscarriages, gravidity, socioeconomic status and delivery after 32 weeks'' gestation. The Burlington Randomized Controlled Trial criteria, which reflect community standards of care, were updated and used to assess the information, which was provided on standard provincial prenatal care forms. Scoring was carried out blindly, and interrater reliability was high. A highly significant difference was found in the proportions of NM and FP charts that were rated adequate, superior or inadequate: 77% v. 24%, 7% v. 16% and 16% v. 60% respectively. The rate at which procedures were omitted (leading to an inadequate score) in the categories of initial assessment, monitoring and management also varied between the two patient groups. These findings, even when considered in terms of several biases that may have resulted in the high proportion of NM charts rated at least adequate, suggest that NMs provide prenatal care to low-risk women that is comparable, if not superior, to the care provided by FPs.     

Plant-microbial interaction under gnotobiotic conditions: A scanning electron microscope study

Inoculation of canola seeds withPseudomonas putida GR12-2 stimulates root elongation under gnotobiotic conditions. Transformation ofP. putida GR12-2 with the broad-host-range plasmid pGSS15 abolishes the enhancement of root elongation. With scanning electron microscopy it was found that both transformed and nontransformedP. putida GR12-2 are capable of binding to canola seed coats. In addition, it was observed that 4 days after the initial inoculation the roots of bothP. putida GR12-2- and GR12-2/pGSS15-treated seedlings were free of adhering bacteria despite the fact that it was subsequently shown that both bacterial strains are capable of binding to roots. Thus, adhesion to roots is not necessary for the initial phase of enhanced root elongation that is induced byP. putida GR12-2 under gnotobiotic conditions.     

Influence of the structure of the lipid-water interface on the activity of hepatic lipase

Factors affecting the hydrolytic activity of purified rat hepatic lipase have been examined in mixed-monolayer systems. When nonsubstrate lipids [either egg sphingomyelin or beta-O-hexadecyl-gamma-O-(1-ocadec-9-enyl)-DL-phosphatidylcholine (OPPC-ether)] were used as inert matrices, hydrolytic activity for both triolein and dioleoylphosphatidylethanolamine was shown to decrease with increasing surface pressure (pi); negligible activity occurred at pi greater than or equal to 30 mN/m. Examination of the effect of introduction of cholesterol into either matrix containing 2 mol % triolein indicated that the mean molecular area decreased with increasing cholesterol and that, at pi = 24 mN/m, triolein was fully miscible in the sphingomyelin matrix at cholesterol concentrations less than or equal to 32.5 mol % and in the OPPC-ether matrix at cholesterol concentrations less than or equal to 49 mol %. Above these critical concentrations of cholesterol, the phase diagrams indicate transitions that suggest that triolein is forced out of the monolayer. Introduction of increasing amounts of cholesterol into either inert matrix increased the rate of hydrolysis of triolein by hepatic lipase, although by different degrees. There are at least two factors contributing to these effects: (1) condensation of the monolayer by cholesterol, thus increasing the total surface concentration of triolein at pi = 24 mN/m in the constant area surface balance, and (2) some change in triolein conformation and/or accessibility since at identical surface concentrations of triolein (8.7 +/- 0.1 pmol/cm2) and pi (24 mN/m) the rate of hydrolysis of triolein by hepatic lipase is 1.5-fold higher in the OPPC-ether matrix than in the egg sphingomyelin matrix.(ABSTRACT TRUNCATED AT 250 WORDS)