Temperature and adrenoceptors in the frog heart

1. Cardiac adrenergic receptors in a frog, Rana tigrina, were examined in winter and summer months using isolated atria preparation maintained at 24 degrees, 14 degrees and 6 degrees C. Treatments included an examination of the atrial responses to selective alpha and beta adrenergic agonists (phenylephrine and isoproterenol respectively) and antagonists (phentolamine and propranolol). 2. Basal atrial beating rates differed between summer and winter months and increased with temperature. 3. Phenylephrine produced dose-dependent increases in the atrial beating rate and tension in the winter frogs only at 6 degrees C. These increases were blunted by phentolamine. 4. Isoproterenol produced positive chronotropic effects of 14 degrees and 24 degrees C but not at 6 degrees C in both summer and winter frogs; these effects were abolished by propranolol. Further, at 6 degrees C, the contractile response of the atrial tissue to isoproterenol was very sensitive. 5. Data suggests that the alpha adrenoceptor might be physiologically important to the frog in the low temperature environment of the cold season, during which period the cardiac beta adrenergic activity would be minimal or even absent.     

Specific interactions of histone H1 and a 45 kilodalton nuclear protein with a putative matrix attachment site in the distal promoter region of a cell cycle-regulated human histone gene

Protein-DNA interactions within the promoter of a cell cycle-regulated human H4 histone gene were examined by binding of 5'-end-labeled DNA segments to Western blots of nuclear protein fractions. Specific protein interactions were observed with DNA segments located between -500 bp and -1,070 bp upstream of the ATG initiation codon and included a histone H1 binding segment flanked on both sides by binding sites for a 45 kD nuclear protein. This region of the gene contains a DNase I-sensitive site in the center (-720 to -820 bp), and sequence analysis revealed the presence of scaffold attachment sequences in the two flanking segments. Topoisomerase II consensus sequences and in vitro topoisomerase II cleavage sites were also detected in the two flanking segments. Our results suggest that the 45 kd nuclear protein may preferentially interact with these two segments of the H4 histone gene to mediate association with the nuclear matrix. The presence of negative regulatory elements in this putative matrix attachment region provides a basis for the speculation that such nuclear proteins are associated with alterations in gene-matrix interaction that are functionally related to gene expression.     

Tolerance to diazepam and methyl-beta-carboline-3-carboxylate measured in substantia nigra of benzodiazepine tolerant rats

The spontaneous activity of neurons in the pars reticulata of substantia nigra (SNpr) was studied in chloral hydrate anesthetized rats. As a function of dose, intravenous diazepam decreased, and methyl-beta-carboline-3-carboxylate (beta CCM) increased discharge frequency. Two days after terminating a one week treatment with flurazepam (FZP), both diazepam and beta CCM showed decreased ability to alter SNpr neuronal activity. Neither residual FZP nor down-regulation of benzodiazepine receptors can account for these results. In contrast, behavioral testing revealed no change in the ability of i.v. beta CCM to cause convulsions, suggesting that sites other than the SNpr are of prime importance in expressing the convulsant actions of systemically injected beta CCM.     

Photoaffinity labeling of [3H]flunitrazepam- and [3H]Ro15-4513-bound pellets in rat cerebral cortex and cerebellum

Irreversible incorporation of [3H]flunitrazepam and [3H]Ro15-4513 into GABA/benzodiazepine receptor subunits was studied by UV irradiation using ligand-bound membrane pellets from rat cerebral cortical and cerebellar synaptic membranes. Specific incorporation for [3H]flunitrazepam was greater in the pellet than in the suspension. The incorporation was identical for [3H]Ro15-4513 in both pellet and suspension. With the ligand-bound pellets, 50% of the available binding sites were photolabeled by both ligands in cortex and cerebellum. SDS polyacrylamide gel electrophoresis and fluorography of [3H]flunitrazepam photo-labeled receptor revealed the same number of major sites in both brain regions. In contrast, [3H]Ro15-4513 appears to label fewer sites in cortex and cerebellum. Photoaffinity labeling with [3H]flunitrazepam in ligand-bound membrane pellet provides a more selective and reliable method for studying the subunit structure of GABA/benzodiazepine receptor complex.     

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.     

Direct cloning of cDNA inserts from lambda gt11 phage DNA into a plasmid vector by a novel and simple method

Bacteriophage lambda gt11 has been used quite extensively for producing cDNA libraries. The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis. However, isolating the recombinant DNA of interest from the phage clones can be a tedious task. Since the E. coli strain Y1088 used for lambda gt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert. In this report, we describe a method in which cDNA inserts from lambda gt11 phage were cloned directly into the pBR322 plasmid vector, bypassing the time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method is likely to be extended to the cloning of DNA inserts derived from other phage lambda vectors when bacteria containing endogenous pBR322 are used as host cells.     

Visualization of beta-sheets and side-chain clusters in two-dimensional periodic arrays of streptavidin on phospholipid monolayers by electron crystallography.

The biotin-binding protein streptavidin was crystallized as two-dimensional periodic arrays on biotinylated phospholipid monolayers. Electron diffraction patterns and images of the arrays embedded in vitreous ice were recorded to near-atomic resolution. Amplitudes and phases of structure factors were computed and combined to produce a 3 A projection density map. The reliability of the map was verified by comparing it to the available x-ray atomic model of the molecule. Projection densities from beta-strands and some amino acid side chains were identified from the electron cryomicroscopy map. These results demonstrate the first near-atomic image of this type of protein periodic array by electron crystallography, which has a great potential to aid in the structural characterization of molecular arrays engineered on a monolayer for various basic or biotechnological applications.     

The species of Ceramium (Ceramiaceae,Rhodophyta) around Taiwan

A taxonomic study was recently carried out on the species of Ceramium occurring around Taiwan. One hundred and fifty-one collections were made from January 1993 through spring 1994 at 75 sites distributed around the island. A total of thirteen species were found, including three for which names cannot currently be assigned: Ceramium aduncum, C. ciliatum, C. cimbricum, C. cliftonianum, C. flaccidum, C. gracillimum var. byssoideum, C. mazatlanense, C. nakamurai, C. paniculatum, C. tenerrimum and unidentified species 1, 2 and 3. Among these are three new records for Taiwan, C. cimbricum, C. cliftonianum, and C. mazatlanense. This paper summarizes the taxonomic characters of these species and presents a key for their identification. Distribution records of all 42 species reported from the tropical western Pacific are compared. This report is the first detailed record of the genus for Taiwan.