Biexponential Kinetics of (R)-α-[3H]Methylhistamine Binding to the Rat Brain H3 Histamine Receptor

The H3 histamine receptor is a high-affinity receptor reported to mediate inhibition of CNS histidine decarboxylase activity and depolarization-induced histamine release. We have used (R)-alpha-[3H]methylhistamine, a specific, high-affinity agonist, to characterize ligand binding to this receptor. Saturation binding studies with rat brain membranes disclosed a single class of sites (KD = 0.68 nM; Bmax = 78 fmol/mg of protein). Competition binding assays also yielded an apparently single class of sites with a rank order of potency for ligands characteristic of an H3 histamine receptor: N alpha-methylhistamine, (R)-alpha-methylhistamine greater than histamine, thioperamide greater than impromidine greater than burimamide greater than dimaprit. In contrast, kinetic studies disclosed two classes of sites, one with fast, the other with slow on-and-off rates. Density of (R)-alpha-[3H]methylhistamine binding followed the order: caudate, midbrain (thalamus and hippocampus), cortex greater than hypothalamus greater than brainstem greater than cerebellum. These data are consistent with an H3 histamine receptor, distinct from H1 and H2 receptors, that occurs in two conformations with respect to agonist association and dissociation or with multiple H3 receptor subtypes that are at present pharmacologically undifferentiated.     

Comparison of different cationic probes for electron microscopic visualization of bacterial polyanionic capsular material

We have isolated from the ovine rumen eight bacterial strains belonging to the speciesButyrivibrio fibrisolvens. DNA hybridization studies showed that the eight strains could be divided into four homology groups, of which none was closely related to the type strain ATCC 19171. Measurement of cross-hybridization between selected pairs of bacterial strains showed that DNA types which produced low, but significant, cross-hybridization on dot-blots were able to form heteroduplexes with between 8.4% and 32.9% of the efficiency of homoduplex formation. Thermal denaturation of the same heteroduplexes resulted in Tm values 6.4–7.5°C lower than those of the homologous duplexes formed under the same conditions. In some cases, hybridization between strains was below the level of reliable measurement. Similar experiments with ten recently isolated strains ofBacteroides ruminicola sub-sp.brevis revealed a similar degree of genetic divergence between isolates.     

Pure human inactive renin. Evidence that native inactive renin is prorenin

To clarify contradicting observations on the identity of inactive renin and prorenin, inactive renin was completely purified from native human chorion laeve and the culture medium of human chorion cells. A 720,000-fold purification with 14% recovery was achieved from chorion laeve in 6 steps, including immunoaffinity chromatography on a monoclonal antibody to human renin coupled to Protein A-Sepharose CL-4B. A 3,100-fold purification with 40% recovery was achieved from chorion culture medium in 4 steps, including immunoaffinity chromatography. Inactive renin purified from the two different sources migrated as a single protein band with the same molecular weight of 47,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of multiple components that could be resolved by isoelectric focusing. Both had the same pI values which shifted downward upon activation by trypsin; however, relative peak heights were different between the two preparations. The purified inactive renin from chorion laeve was completely inactive and did not bind to pepstatin-aminohexyl-Sepharose; however, that from chorion culture medium was partially active and completely bound to the pepstatin gel, indicating that each molecule is partially activated. Trypsin-activated inactive renins from both sources were identical with human renal renin in terms of pH optimum and Km. Specific activities of trypsin-activated inactive renin from chorion laeve and chorion culture medium were 529 Goldblatt units/mg of protein and 449 Goldblatt units/mg of protein, respectively. Amino acid sequence analysis of both of the purified inactive renin preparations demonstrated a leucine residue at the amino terminus. The sequence of 11 additional amino acids was identical in both and agreed with that predicted from the base sequence of the renin gene. These findings indicate that preprorenin is converted to prorenin following removal of a 23-amino acid signal peptide and that the native inactive renin, whose amino acid sequence commences with Leu-Pro-Thr..., is prorenin.     

Operant conditioning of scratching in lemurs

An adult femaleLemur catta and an adult femaleEulemur fulvus were given edible rewards for scratching. Both subjects learned to scratch in order to obtain the rewards, showed diminished rates of scratching during periods of extinction, and learned to scratch preferentially with one foot when required. TheLemur catta subject was more responsive to the changing experimental conditions than theEulemur fulvus. The conditionability of scratching in primates does not appear to be directly related to the widespread occurrence of scratching in simian social contexts.     

Increased H-2Dd expression following infection by a molecularly cloned ecotropic MuLV

The biological consequences of radiation leukemia virus (RadLV) infection include the stimulation of H-2D^d antigen expression in resistant mouse strains and thymoma induction in susceptible strains. In an effort to understand the genetic basis of these phenomena, the integrated ecotropic RadLV genome has been examined in a number of primary RadLV-induced tumors, as well as thymomas adapted to in vitro passage; considerable heterogeneity was observed. Examination of these polymorphic viral sequences should help define the viral gene(s) involved in the biological effects of RadLV infection; toward this end, integrated RadLV genomes were molecularly cloned and examined. The genomes and their flanking sequence were characterized by restriction enzyme analysis. Three unique viral genomes were obtained which represent four integration sites. The three RadLV genomes are shown to carry polymorphisms of the original tumor. Following DNA transfection, one of the three genomes replicated in and reinfected both mouse thymocytes and fibroblasts, but not mink fibroblasts in vitro. Virus encoded by the other two DNA genomes could not be recovered following transfection into any of the three cell types. One of these two apparently defective retroviruses encodes a truncated p15E molecule, while the other has elongated long terminal repeats (LTRs). The non-defective ecotropic isolate was collected from in vitro tissue culture supernatants, concentrated, and used to infect mice. Thymocytes of infected, resistant mice were shown to express elevated levels of H-2D^d antigen as early as 12 days post infection, a hallmark of RadLV infection. Offprint requests to: G. D. Brown.     

Hepatitis B virus DNA integration and expression of an erb B-like gene in human hepatocellular carcinoma.

Southern blot studies on Hepatitis B Virus (HBV) DNA integration in 13 human hepatocellular carcinomas (HCCs) patients revealed the presence of several distinct HBV integration sites in different human liver disease patients. In one HCC patient the DNA fragment containing the HBV integration also hybridized to an erb B probe. The erb B/HBV co-migrating DNA fragment was cloned and sequenced, and showed that HBV DNA is integrated next to a cellular DNA fragment which is homologous to the tyrosine protein kinase domain of the human epidermal growth factor receptor gene and other cell surface receptor genes. The virus-integrated cellular DNA sequence is expressed in this HCC patient, suggesting a possible role for this gene in hepatocarcinogenesis.     

Inhibition of common fouling organisms by marine bacterial isolates ith special reference to the role of pigmented bacteria

Two questions of relevance to the establishment of marine biofouling communities were addressed, viz (1) what is the frequency with which bacterial strains isolated from living and inanimate surfaces in the marine environment show inhibitory activity against the settlement of common fouling organisms, and (2) is the antifouling bacterium, D2, an inhabitant of different marine waters, and how unique is this bacterium, in its mode of action against different target organisms? With respect to the first question, ninety three marine bacteria isolated from various rock surfaces from the marine environment were tested against larvae of Balanus amphitrite and spores of Ulva lactuca. Settlement assays against the diatom Amphora sp. were also performed on 10 of these strains. Nine bacterial isolates were shown to be inhibitory against larval settlement and eight of these strains were also inhibitory against algal spores. Altogether 16 strains were inhibitory against the settlement of algal spores while none of the bacterial strains inhibited diatom settlement. With respect to the second question, D2, a dark green pigmented bacterium, isolated from an adult tunicate off the Swedish west coast, has been found to be a very effective inhibitor against common fouling organisms. In order to see if this bacterium can be found in other marine waters, bacteria from living surfaces of marine plants and animals from waters around Sydney, Australia, were isolated and screened for inhibitory activity against barnacle larvae. Seventy four percent of the 23 plant isolates were shown to be inhibitory against larval settlement while only 30% of the 23 isolates from marine animals reduced settlement. Twenty two of the isolates from different seaweeds were dark pigmented and 20 of these strains inhibited settlement of barnacle larvae and algal spores. Three of the strains showed the same phenotypic expression as D2, and the results indicate that these strains may be D2 or closely related strains, suggesting that D2 may be a common inhabitant in the marine environment.