A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Characterization of the glycosylation sites in yeast external invertase. I. N-linked oligosaccharide content of the individual sequons

External invertase is the product of the SUC2 gene of Saccharomyces cerevisiae. The deduced sequence of this enzyme (Taussig, R., and Carlson, M. (1983) Nucleic Acid Res. 11, 1943-1954) reveals it to contain 14 potential N-linked glycosylation sites, or sequons, although only 9-10 appear to be glycosylated (Trimble, R. B., and Maley, F. (1977) J. Biol. Chem. 252, 4409-4412). To determine the location of the glycosylated sequons, external invertase was deglycosylated with endo-beta-acetylglucosaminidase H and its component peptides analyzed by both fast atom bombardment mass spectrometry (FABMS) and classical peptide isolation procedures. By use of the former technique most of the glucosamine-containing sequons could be located and by the latter sufficient amounts of small glucosamine-containing peptides were isolated to enable their quantitation. From the combined FABMS and glucosamine analyses, it was established that eight of the sequons in a subunit of invertase are either completely or almost completely glycosylated, while five others are glycosylated to the extent of about 50% or less. In the case of two overlapping sequons (4 and 5), which include Asn92-Asn93-Thr-Ser, only the first Asn was glycosylated. Thus, all but one of the sequons of external invertase are glycosylated to some extent, giving an appearance of only 9-10 N-linked oligosaccharides/subunit. The sequence identity of both external and internal invertase was verified by FABMS and by peptide sequence analysis. In only one site was an amino acid found to differ from that deduced from the DNA sequence of the SUC2 gene. This occurred at position 390 where a proline was found in place of alanine, which could result from a single base change in the triplet specifying the latter amino acid.     

Technique for hand-rearing and reintroducing rejected cotton-top tamarin infants

During a one year period, seven rejected cotton-top tamarin infants were hand-reared. A hand-rearing program was developed which included the use of heated surrogates, a non-human primate infant formula, special nipples, and a systematic reintroduction into a family group. This regimen was quite successful. The hand-rearing process yielded a success rate of 75.0%, while the reintroduction process had a success rate of 80.0% for reintroduction into a family group and 100.0% survival rate.     

Release of Monoamines from Striatum of Rat and Mouse Evoked by Local Application of Potassium: Evaluation of a New In Vivo Electrochemical Technique

Local application of K+ via micropressure-ejection, coupled with in vivo electrochemical detection, was used to study stimulated release from monoaminergic nerve terminals in the striatum of anesthetized rats and mice. K+-evoked releases were reversible, reproducible, and dose-dependent. In contrast, releases of electroactive species could not be evoked by local ejection of Na+. The magnitudes and time courses of K+-evoked releases recorded from the caudate nucleus of mice were greater than those seen in rats. Local application of nomifensine, a putative catecholamine reuptake blocker, augmented the magnitudes and time courses of K+-evoked releases. Releases were also recorded from brain regions adjacent the striatum; these signals were always smaller than those seen in the caudate nucleus and had amplitudes that showed good correspondence to the relative degree of dopaminergic input to these areas. These data, taken together with other information in the literature, suggest that this new technique is well suited for in situ studies of monoamine release and reuptake in intact animals.     

Amino acid sequence of Manduca sexta adipokinetic hormone elucidated by combined fast atom bombardment (FAB)/tandem mass spectrometry

Combination of Fast Atom Bombardment Tandem Mass Spectrometry with Amino Acid Analysis assigns the amino acid sequence of the Manduca sexta adipokinetic hormone as pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-GlyNH2. Similarities and differences with other invertebrate hormones and with mammalian glucagon are discussed.     

Influence of the H-2u haplotype on immune function in F1 hybrid mice. I. Antigen presentation

Previously, we reported that myelin basic protein (MBP) peptide 1-37 contains an encephalitogenic epitope for PL/J mice, and MBP peptide 89-169 is encephalitogenic for SJL/J mice. (SJPL)F1 hybrid mice do not respond to immunization with these peptides in a co-dominant manner because the encephalitogenic response to peptide 1-37 dominates. To examine this phenomenon more closely, we tested the ability of MBP-primed parental or F1 T cells to respond to MBP or MBP peptides in the context of PL, SJL, or F1 antigen-presenting cells (APC). It was found that the F1 T cells responded to either the protein or the peptides when these were presented in the context of F1 or PL APC. However, F1 T cells would not respond to MBP in the context of SJL APC, although the latter cells were functionally intact. This effect was not antigen-specific because SJL APC would not present ovalbumin or PPD to primed F1 T cells. F1 T cells from mice immune to the strongly antigenic bacterium Listeria monocytogenes responded to bacterial antigens presented by SJL APC, although at a significantly lower level compared to the results obtained when these antigens were presented by F1 or PL APC. This finding implied that unbalanced antigen presentation was a quantitative rather than a qualitative phenomenon. When F1 hybrid mice from other strain combinations were tested, a similar effect was observed whenever one of the parental strains was PL/J. This effect was mapped to the MHC in MHC-congenic B10 mice.     

Amplified expression of streptomyces endo-beta-N-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product

The endo-beta-N-acetylglucosaminidase H (Endo H) gene from Streptomyces plicatus has been cloned into the Escherichia coli plasmid pKC30 (Shimatake, H., and Rosenberg, M. (1981) Nature 272, 128-132), thus placing expression of this gene under control of the strong lambda promoter pL. The construction, pKCE3, which includes a properly positioned E. coli ribosome binding site from the lac operon (Robbins, P.W., Trimble, R. B., Wirth, D.F., Hering, C., Maley, F. Maley, G. F., Das, R., Gibson, B.W., and Biemann, K. (1984) J. Biol. Chem. 259, 7577-7583), was used to transform an E. coli strain lysogenic for a lambda prophage containing a temperature-sensitive repressor. By shifting cultures of pKCE3 lysogens to 42 degrees C, the production of Endo H commenced and was linear for about 1 h. Enzyme yields were amplified 150-fold above those obtained from comparable cultures of S. plicatus and represented 3 to 4% of total cellular protein, which enabled purification of Endo H to homogeneity by a rapid fourstep procedure. Although most of the cloned Endo H was secreted into the periplasmic space by E. coli, its 4 kDa leader sequence peptide (Robbins et al. (1984] was only partially removed during processing. As a result the purified pKCE3 Endo H was a heterogeneous population of molecules with an average molecular mass of 31 kDa compared to the 28.9 kDa fully processed product normally secreted by S. plicatus. Despite the residual approximately 2 kDa of leader sequence on the cloned pKCE3 product, there were no detectable differences in either the substrate specificity or the stability characteristics of the enzyme purified from E. coli or from S. plicatus. Of particular value for studies on glycoproteins was the finding that the genetically engineered Endo H was completely free of proteolytic contaminants.     

Refinement of HLA gene mapping with induced B-cell line mutants

The lymphoma cell line BJAB.B95.8.6 was gamma-irradiated to induce mutations of major histocompatibility complex (MHC) encoded genes. Cloned wild-type cells were phenotyped HLA-A1, A2, B 13, 1335, Bw4, Bw6, Cw4, DR5, DRw52, DQwl, DQw3, DPw2, DPw4, GLO1^*1, PGM3^*2-1, and ME1^*0 and possessed two apparently normal chromosome 6s prior to mutagenesis. Loss mutants were selected 5 days after 3 Gy gamma-irradiation employing three complement-fixing monoclonal antibodies specific for HLA-A2 (TÜ101) and Bw4 (TÜ48, TÜ109). Fifteen independently arising mutants were isolated and cloned. Typing with monospecific alloantisera and cell-mediated lympholysis revealed the presence of HLA-A1, 835, Bw6, Cw4, DR5. DRw52, DQw3, and DPw4 specificities on all mutant clones. HLA-A2, B13, and Bw4 were absent. Mutants differed in their expression of class 11 antigens. One group retained DQw1 and DPw2, another was DQw1^–, DPw2⁺, and a third was DQw1^–, DPw2^–. Karyotyping of the wild-type line and selected mutant clones showed that the loss of HLA specificities correlated with deletions which map the HLA-A and -B loci directly to the distal part of the 6p2l.33 region and the class II genes to the region 6p21.33 (proximal) to 6p21.31 (distal) on the short arm of chromosome 6.Abbreviations used in this paper: CML cell-mediated lympholysis - CTX cytotoxicity - DBBA direct bacterial binding assay - EBV Epstein-Barr virus - GLO glyoxalase - IBBA indirect bacterial binding assay - LU lytic units - ME1 cytoplasmic malic enzyme - MHC major histocompatibility complex - MOAB monoclonal antibody - NADP nicotinamide-adenine dinucleotide phosphate - PGM3 phosphoglucomutase isozyme 3 In partial fulfillment of Ph.D. thesis requirements.     

Electro-fusion of mesophyll protoplasts ofAvena sativa

In order to assay the viability of electrically fused mesophyll protoplasts ofAvena sativa a technique was developed to determine adenylate levels in single protoplasts and fusion products. The results demonstrate that the intracellular ATP/ADP ratios are identical before and after fusion (values between 1.4 and 1.8) and that the time of the rounding up process is directly related to the ATP level of the hybrid. This was shown by the manipulation of the intracellular ATP/ADP ratio in the light using different effectors. Hybrids with an ATP/ADP ratio of 2.3 needed 54 s to round up completely; in the presence of antimycin (inhibition of both oxidative and light-dependent cyclic electron flow: ATP/ADP=1.1) or dibromothymoquinone (plastoquinone antagonist: ATP/ADP=1.0) the time for rounding up was slightly increased (64 s and 76 s respectively), whereas after preincubation with antimycin, dichlorophenyldimethylurea (inhibition of oxidative and light-dependent electron flow) or uncouplers (ATP/ADP=0.19–0.32) this process needed 128–153s for completion. These results are discussed in relation to the viability of electrically induced fusion products and to energy-dependent events involved in the process of fusion.