Circular dichroism of platelet factor 4

The circular dichroism of platelet factor 4 was investigated and it was found to contain 15% alpha-helix, 25% beta-structure, and the rest of the molecule in unordered conformation. In the presence of heparin, no change in the circular dichroism was observed, suggesting no significant changes in the secondary structure of platelet factor 4 when heparin binds. The CD spectrum of platelet factor 4 was also investigated in the presence of increasing concentrations of guanidine hydrochloride. A two-state transition was observed with midpoints at 0.125 and 2.0 M guanidine hydrochloride. Based on gel filtration studies, the first unfolding transition was correlated with the dissociation of the tetrameric structure. This first unfolding domain was not observed in the presence of heparin, suggesting that heparin stabilizes the tetrameric structure. The second unfolding transition corresponds to the disruption of the overall secondary structure which is generally observed with most proteins. It is concluded that a relatively weak force of attraction holds the tetrameric structure of platelet factor 4 and the dissociation of the subunits is accompanied by loss of some helical secondary structure.     

Fluorometric detection of the bilayer-to-hexagonal phase transition in liposomes

We have used the fluorescent probe N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) to detect the bilayer-to-hexagonal phase transition. The fluorescence intensity of the probe was found to increase during the bilayer-to-hexagonal transition. The bilayer-to-hexagonal transitions of various types of phosphatidylethanolamine or cardiolipin measured by this method are consistent with results obtained by differential scanning calorimetry. To establish this method for wider use, agents known to alter the bilayer-to-hexagonal transition were examined, and the results are comparable with the published data. The added advantage of this fluorometric method over other currently available techniques is that it is applicable not only for aggregated lipid samples but also for dilute liposome suspensions. This is especially important when one of the components of the system under study can partition between lipid and aqueous phase. Since NBD is located near the headgroup region of the bilayer, it most likely detects the change of the environment surrounding that region. On the basis of our present study, it appears that NBD-PE is sufficiently sensitive to detect bilayer-to-hexagonal phase transition.     

Studies on the genus Fontinalis (Musci: Fontinalaceae)

Fontinalis welchiana, a new species in the concave-leaved group ofFontinalis restricted to Arkansas, Missouri, and Illinois, differs fromF. novaeangliae by its slender size, dimorphic leaves, erect and strongly concave branch leaves, and lightly papillose exostome with ventral lamellae that are strongly rounded at the corners.Fontinalis allenii is a synonym ofF. antipyretica var.oreganensis.     

Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. II. beta-Lipotropin (beta-LPH)-related peptides

We have studied the post-translational processing of POMC-derived peptides during fetal monkey pituitary development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Whole pituitary glands obtained from Day 50 and 55 fetal monkeys and separated lobes From Day 65 to 155 were extracted, fractionated, and analyzed for beta-melanotropin (beta-MSH), midportion beta-endorphin (beta-EP), and acetylated beta-EP immunoactivity. Separated adult pituitary lobes were analyzed for comparison. At Day 50, POMC-containing cells were located in both the anterior and intermediate pituitary lobes by immunofluorescence staining, the majority of these cells were localized in the anterior lobe. The Day 50 and 55 whole pituitaries contained predominantly beta-lipotropin (beta-LPH), gamma-lipotropin (gamma-LPH), beta-EP(1-31), and 2.2-kda beta-MSH. No acetylated products were found in Day 50 whole pituitary extracts. By Day 55, carboxy-shortened and acetylated beta-EPs were barely detectable in whole pituitary extracts. These forms were more apparent in the Day 65 separated neurointermediate lobe (NIL) extracts, and were similar to adult proportions by Day 80. The adult anterior lobe contained predominantly beta-LPH, beta-EP, and gamma-LPH. Adult NILs contained almost exclusively 2.2-kda beta-MSH, alpha-N-acetyl beta-EP(1-31) and alpha-N-acetyl beta-EP(1-27). The production of 2.2-kda beta-LPH in the monkey NIL indicates that monkey beta-LPH is different from rat beta-LPH in that it must contain the paired-basic cleavage site required for the formation of 2.2-kda beta-MSH that is known to be lacking in rat beta-LPH. Another finding was that monkey beta-EP contains a Tyr residue at position 27 as found in human beta-EP but appears to have the rat Gln substitution at position 31. The post-translational processing patterns characteristic of each lobe were well established by midterm fetal development (Day 80).     

Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-2 encoding the 57-kDa polypeptide and comparison to vma-1

In partially purified preparations of the vacuolar ATPase from Neurospora crassa, the two most prominent components are polypeptides of Mr = 70,000 and 60,000. We previously reported the isolation of the gene vma-1, which encodes the Mr = 70,000 polypeptide, and presented evidence that the polypeptide contains the site of ATP hydrolysis (Bowman, E. J., Tenney, K., and Bowman, B. J. (1988) J. Biol. Chem. 263, 13994-14001). We now report the isolation of a gene (designated vma-2), that encodes the Mr = 60,000 polypeptide. Analysis of the DNA sequence shows that the polypeptide has 513 amino acids and a molecular mass of 56,808 daltons (and will thus be referred to as the 57-kDa polypeptide). It is fairly rich in polar amino acids and has no apparent membrane-spanning domains. The vma-2 gene contains five short introns (55-71 bases), all clustered in the 5' end of the coding region. The gene maps to the right arm of linkage group II, near 5 S RNA gene 3. Thus, it is unlinked to vma-1 and to other known ATPase genes in N. crassa. The 57-kDa polypeptide shows 25% amino acid sequence identity with the vma-1 gene product. It shows essentially the same degree of similarity (25-28%) to both the alpha and beta subunits of F0F1 ATPases. Analysis of specific regions of the 57-kDa polypeptide, however, suggests it may have a function like that of the alpha subunit in F0F1 ATPases. The data indicate that all four types of ATPase polypeptides have evolved from a common ancestor and that the vacuolar-type ATPases have a structure surprisingly similar to that of the F0F1 ATPases.     

Effect of long-term Intralipid administration in mice

Intralipid was administered intravenously to mice at a level of 2 g kg-1 day-1 for 23 days. No alterations in phagocytic index, liver or spleen size were observed in the chronically injected mice as compared with control mice that received saline injections. Tissue distribution of 0.45 micron multilamellar liposomes of egg phosphatidylcholine:cholesterol (2:1) was similar in mice that had been chronically injected with Intralipid to that in control mice. Mice chronically given the same total amount of phospholipid in the form of 0.2 micron liposomes of phosphatidylcholine:cholesterol (2:1) rather than as a lipid-triglyceride emulsion showed altered tissue distribution of entrapped label with decreased liver uptake and increased splenic uptake, which is indicative of reticuloendothelial blockade. Tissue distribution of [14C]dipalmitoylphosphatidylcholine Intralipid was compared with that of [14C]dipalmitoylphosphatidylcholine 0.2 micron MLV of phosphatidylcholine:cholesterol (2:1). Intralipid was taken up 2- to 3-fold less by liver and 5- to 10-fold less by spleen than liposomes. Blood levels of Intralipid were higher than those of liposomes. [14C]dipalmitoylphosphatidylcholine Intralipid was eliminated from the body at a faster rate than [14C]dipalmitoylphosphatidylcholine liposomes. The lack of reticuloendothelial blockade caused by Intralipid as compared with liposomes appears to be related to its diminished uptake into reticuloendothelial tissues. This diminished uptake may be related to differences in apolipoprotein uptake of Intralipid, which is primarily in the form of a phospholipid monolayer, and liposomes, which have their phospholipid organized into a bilayer.     

Opsonic activity of antisera to ribosomal vaccine fractions with live and formalinized Pseudomonas aeruginosa

The opsonic capacity of antisera to Pseudomonas aeruginosa ribosomal vaccine fractions was determined by a chemiluminescent technique. Antiserum to a vaccine fraction ("peak A") containing lipopolysaccharide (antiserum A), and antiserum to a vaccine fraction ("peak B"), which did not contain detectable amounts of lipopolysaccharide (antiserum B), were used to opsonify live or formalin-treated bacteria. Polymorphonuclear leukocytes were then stimulated by the opsonified bacteria in the presence of the chemiluminigenic probe, luminol, resulting in the observed chemiluminescence. The data obtained indicated that the antisera had comparable opsonic activity with live (untreated) bacteria. However, antiserum B had far less opsonic activity than did antiserum A when formalinized bacteria were used. Owing to the effects of formaldehyde on protein, these results were interpreted as evidence to suggest that the opsonic activities of the two antisera are dependent on different antigens on the bacterial cell surface. Antiserum A activity is probably dependent on lipopolysaccharide to a great extent, whereas antiserum B activity is most likely dependent primarily on a protein(s).     

The localisation of an Mr 74,000 major chromatin antigen on native salivary chromosomes of Drosophila melanogaster

An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci.     

Metal ion requirements and other aspects of the reaction catalyzed by M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli

M1 RNA, the RNA subunit of ribonuclease P from Escherichia coli, can under certain conditions catalytically cleave precursors to tRNA in the absence of C5, the protein moiety of RNase P. M1 RNA itself is not cleaved during the reaction, nor does it form any covalent bonds with its substrate. Only magnesium and, to a lesser extent, manganese ions can function at the catalytic center of M1 RNA. Several other ions either inhibit the binding of magnesium ion at the active site or function as structural counterions. The reaction rate of cleavage of precursors to tRNAs by M1 RNA is enhanced in the presence of poly-(ethylene glycol) or 2-methyl-2,4-pentanediol. Many aspects of the reaction catalyzed by M1 RNA are compatible with a mechanism in which phosphodiester bond cleavage is mediated by metal ion.     

Dopaminergic Regulation of Cone Retinomotor Movement in Isolated Teleost Retinas: II. Modulation by γ-Aminobutyric Acid and Serotonin

In the accompanying paper we reported that 3,4-dihydroxyphenylethylamine (dopamine) induced light-adaptive retinomotor movements in teleost photoreceptors and that this effect was mediated by D2 dopamine receptors located on the photoreceptors themselves. In this study, we investigated the effects on cone retinomotor movement of three agents that have been reported by others to modulate retinal dopamine release: gamma-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT, serotonin), and melatonin. We report here that the GABA antagonists bicuculline and picrotoxin induced light-adaptive cone contraction in dark-adapted green sunfish retinas cultured in constant darkness; thus they mimic the effect of light or exogenously applied dopamine. Since their effects were blocked by either the D2 dopamine antagonist sulpiride or by Co2+, it seems likely that these agents act by enhancing retinal dopamine release. The GABA agonist muscimol produced effects opposite to those of GABA antagonists. Muscimol inhibited light-induced cone contraction in previously dark-adapted retinas and induced dark-adaptive cone elongation in light-adapted retinas. These results suggest that in green sunfish retinas, as has been reported for other retinas, GABA inhibits dopamine release. 5-HT induced light-adaptive cone contraction in dark-adapted retinas; thus 5-HT also mimics the effect of light or exogenously applied dopamine. The effect of 5-HT was blocked by sulpiride, Co2+, or the 5-HT antagonist mianserin. These results suggest that 5-HT induces cone contraction by stimulating dopamine release. Melatonin neither inhibited dopamine-induced cone contraction in retinas cultured in the dark nor induced cone elongation in retinas cultured in the light. Our results suggest that both GABA and 5-HT (but not melatonin) affect cone retinomotor movements in green sunfish by modulating dopamine release: GABA by inhibiting and 5-HT by stimulating dopamine release. We report in the companion paper that dopamine induced contraction in isolated cone fragments. Together these observations strongly suggest that dopamine serves as the final extracellular messenger directly inducing light-adaptive cone retinomotor movement, and that GABA and 5-HT affect these movements by modulating dopamine release.