Properties of narrow-range ampholytes isolated from wide-range ampholyte preparations

A simple procedure for obtaining useful narrow-pH-range ampholytes from inexpensive laboratory-synthesized ampholytes by preparative isoelectric focusing in Pevikon is described. The narrow range ampholytes prepared in this way are comparable to commercial ampholyte preparation as judged by conductivity, buffer capacity, pH gradient formation, and resolving power. These inexpensive narrow-range ampholytes are particularly well suited to preparative isoelectric focusing applications requiring large quantities of ampholytes.     

Effects of angiotensin II and dibutyryl cyclic adenosine monophosphate on phosphatidylinositol metabolism, 45Ca2+ fluxes, and aldosterone synthesis in bovine adrenal glomerulosa cells

Angiotensin II (AII) and N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP) both stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. AII altered 45Ca2+ fluxes and increased 32PO4 incorporation into phosphatidylinositol in these cells, whereas dibutyryl cyclic AMP did not affect either process. Neither AII nor dibutyryl cyclic AMP increased the mass of phosphatidylinositol. Both agents are known to stimulate pregnenolone synthesis. Thus, although dibutyryl cyclic AMP and AII may increase aldosterone synthesis at a common site (pregnenolone synthesis), they do so by different mechanisms. AII stimulation of phosphatidylinositol labeling by 32PO4 (the "PI effect") was blocked when cells were incubated in a medium containing both EGTA and the calcium antagonist, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxy-benzoate hydrochloride (TMB-8), suggesting a calcium requirement for the PI effect.     

Detection of beta-adrenergic receptors on rabbit mononuclear cells isolated free of significant contamination by other cell types

In order to study rabbit mononuclear cell surface receptors, it was necessary to develop a procedure to isolate mononuclear cell preparations that are free of significant contamination by other cell types, especially platelets. Centrifugation of dextran-sedimented, anti-coagulated whole blood through Hypaque (density 1.060) at 600 X g for 5 min at 22 degrees C eliminated greater than 93% of starting platelets. A second 5-min Hypaque centrifugation of Hypaque-Ficoll-isolated mononuclear cells (MNC) (approximately 80% lymphocytes) at 450 X g for 5 min at 22 degrees C reduced platelet contamination to less than one platelet per three MNC, and resulted in the overall removal of greater than 99.5% of starting platelets. These relatively pure MNC which were isolated in less than 2 hr were identified as having beta-adrenergic receptors by radioligand binding techniques using [125I]iodohydroxybenzylpindolol [( 125I]IHYP). Binding of [125I]IHYP to intact rabbit MNC was a saturable, stereospecific, and rapid process with a dissociation constant (KD) of 0.53 +/- 0.18 nM and a binding capacity of 3,461 +/- 235 sites/cell.     

Neuroendocrine response to cold in Raynaud's syndrome

Eleven patients with Raynaud's syndrome accompanied by monospecific IgG ANA, nine patients with Raynaud's syndrome in the absence of ANA, and nine normal volunteers were exposed to an ambient cold challenge during which time venous blood was continuously sampled. ANA negative patients were shown to have significantly higher levels of cortisol during a cold challenge than either ANA positive patients or normal controls, and exhibited significantly lower levels of plasma norepinephrine compared with normal controls. ANA positive patients did not differ significantly from normals in their neuroendocrine response to cold. It is suggested that the high plasma cortisol found in Raynaud's syndrome in the absence of ANA may be responsible for the vasospasticity in this group of patients.     

Effects of antianxiety and antipsychotic drugs on DRL responding for brain stimulation

Satiated rats could be trained to give stable rates of responding for rewarding stimulation of the lateral hypothalamus delivered on differential reinforcement of low rate (DRL) schedule requiring 2 to 8 sec interresponse intervals for reinforcement (DRL-2 to 8). The performance on a DRL-8 schedule was tested 30 min after the oral administration of benzodiazepines. Diazepam (5 and 10 mg/kg) and meprobamate (200 mg/kg) caused significant increases in response rates during the first 5 min of a session, but not thereafter. Bromazepam (1 and 5 mg/kg) also caused a significant increase in the rates during the first and second 5 min. On the other hand, chlorpromazine (20 mg/kg) caused no effect in the first 5 min but decrease in second and third 5 min. These results indicate that DRL schedules with a brain stimulation reward provided a useful tool for evaluation of antianxiety drugs. The advantage of the brain stimulation reward over food reward is that the possible effects of the drugs on hunger motivation need not be considered.     

A novel opioid peptide, leumorphin, acts as an agonist at the kappa opiate receptor

The primary structure of the common precursor of porcine beta-neo-endorphin and dynorphin (preproenkephalin B) has shown the existence of a third leucine-enkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide, named leumorphin, was approximately 70 times more potent than leu-enkephalin in inhibiting the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. This action of leumorphin, like those of beta-neo-endorphin and dynorphin, was antagonized less effectively by naloxone than that of leu-enkephalin, but more effectively by Mr2266, an antagonist relatively specific for the kappa type opiate receptor. The inhibitory action of leumorphin or beta-neo-endorphin on the contraction of the guinea pig ileum muscle strip was reduced in a dose-dependent manner by pretreatment with dynorphin and vice versa. Leumorphin as well as beta-neo-endorphin and dynorphin inhibits the contraction of the rabbit vas deferens which is known to have only the kappa type opiate receptor. This action was also effectively antagonized by Mr2266. It is concluded that leumorphin has potent opioid activity and acts at the kappa receptor, like other opioid peptides derived from preproenkephalin B.     

Somatostatin mydriasis in mice

Administration of somatostatin intracerebroventricularly to mice produced a dose-dependent mydriasis, whereas intravenous injections were ineffective. Naloxone could prevent or abolish this effect. It is suggested that somatostatin either directly excites opiate receptors or activates endopioid pathways involved in the regulation of pupillary size.     

Synthesis and spectroscopic analysis of modified bile salts

For the study of hepatic bile acid transport in vivo, a series of modified bile salts were synthesized. The N-cholyl derivatives of L-leucine, L-alanine, D-alanine, beta-alanine, L-proline, and gamma-amino-butyric acid were prepared from cholic acid, ethyl chloroformate and the corresponding amino acid. Structural analysis of products was carried out mainly by electron impact mass spectrometry (20 eV) of the methyl ester/acetate derivatives. In all EI spectra, fragments in the lower mass region included McLafferty rearrangement ions (beta-cleavage) and product ions of gamma-cleavage in the vicinity of the amide linkage. In the upper mass region, fragmentation was characterized by consecutive eliminations of ketene and/or acetic acid from low intensity molecular ions. The purity of the products and their molecular weights were checked by a novel ionization technique in mass spectrometry, fast atom bombardment (FAB) mass spectrometry. FAB spectra were obtained from underivatized bile salts. The spectra were characterized by ions formed by attachment of a proton or an alkali ion to the bile salt to give intense M+H, M+Na, or M+K ions, which then showed little fragmentation.     

The exchange hypothesis for the formation of chromatid aberrations: an experimental test using bleomycin

The association between bleomycin-induced chromatid aberrations and BUdR-label exchange between sister chromatids was investigated in order to evaluate Revell's exchange hypothesis for the formation of chromatid aberrations. The results of this study indicate that a larger than expected proportion of chromatid breaks can be accounted for by the exchange hypothesis though not all breaks are the result of incomplete exchange.     

Strict co-linearity of genetic and protein folding domains in an intragenically duplicated rat lens γ-crystallin gene

Two complementary DNA clones pRLγ-2 and pRLγ-3 of different rat lens γ-crystallin messenger RNAs have been used to identify γ-crystallin gene sequences in rat genomic DNA. Subsequently, the DNA present in the 18,000 to 20,000 bases region of the EcoRI digest, giving rise to a strong doublet hybridization signal, was cloned in λ phage Charon-4A. One of the clones, λRCHγ-3, carrying an insert of 17,500 bases has been characterized in detail. From analysis at the restriction enzyme level with 5′-, “middle” and 3′-specific subprobes of pRLγ-3 it could be deduced that λRCHγ-3 contains only one γ-crystallin gene. The coding sequences of this gene are interrupted by intronic DNA. The primary structure of this gene and its flanking regions have been established by sequencing the relevant regions of a subclone of λRCHγ-3, designated pRCHγ-3.1. The sequence data show that the γ-crystallin gene extends over 2700 bases of rat genomic DNA. The gene is split by two introns, one of 87 base-pairs after the third translation codon and a large one of 1880 base-pairs after codon 84. The mosaic structure of the gene is strictly co-linear with the structure of the γ-crystallin polypeptide in that the large intron is positioned in a region which specifies the so-called “connecting peptide” and which links the two highly symmetrical and homologous protein domains. Although expected from the cDNA and protein sequence no introns were observed between the coding regions in the DNA specifying the two homologous folding motifs present in each protein domain. The relevance of this phenomenon in terms of the evolution of the mature γ-crystallin gene is discussed.