Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice

Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A(1) receptor (A(1)AR)-knockout (A(1)KO) mice and their wild-type (A(1)WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle alpha-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was approximately 91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A(1)WT and A(1)KO mice. Furthermore, the A(1)AR was characterized by Western blot analysis using an A(1)AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.     

The blood supply of the reverse temporalis muscle flap: anatomic study and clinical implications

Although the reverse temporalis muscle flap has been used clinically, the exact vascular connection between the superficial and deep temporal vessels has not been clearly defined. The purpose of this study was to investigate the vascular territory of the reverse temporalis muscle supplied by the superficial temporal vessels. Six cadaver heads were studied using a colored lead oxide injection through the superficial temporal artery. The specimens were examined macroscopically and radiographically. The reverse temporalis muscle flap was then applied to a clinical case presenting with traumatic anterior skull base defect communicating with the nasal cavity. The cadaver specimens demonstrated that the superficial temporal artery formed an average 1.3 +/- 0.2 cm in width of dense vascular zone, which was located within 1.8 cm below the superior temporal line. The dense vascular network further perfused the anterior and posterior deep temporal arteries and the muscular branch of the middle temporal artery to supply the temporalis muscle. The mean perfused area of the temporalis muscle was 83 percent, ranging from 79 to 89 percent, in five cadaver heads. One cadaver revealed only 55 percent of perfused area in the absence of the muscular branch of the middle temporal artery. The consistent area without perfusion was located in the distal third of the posterior portion of the reverse temporalis muscle. In clinical cases, the reverse temporalis muscle flap was used successfully to obliterate the anterior skull base defect without evidence of muscle flap necrosis. The exact blood supply to the distal third of the posterior portion of the reverse temporalis muscle flap needs to be investigated further in vivo. Particular attention was paid to the inclusion of the muscular branch of the middle temporal artery in this flap to augment the blood supply to the temporalis muscle.     

Language,Reading, and Math Learning Profiles in an Epidemiological Sample of School Age Children

Dyscalculia, dyslexia, and specific language impairment (SLI) are relatively specific developmental learning disabilities in math, reading, and oral language, respectively, that occur in the context of average intellectual capacity and adequate environmental opportunities. Past research has been dominated by studies focused on single impairments despite the widespread recognition that overlapping and comorbid deficits are common. The present study took an epidemiological approach to study the learning profiles of a large school age sample in language, reading, and math. Both general learning profiles reflecting good or poor performance across measures and specific learning profiles involving either weak language, weak reading, weak math, or weak math and reading were observed. These latter four profiles characterized 70% of children with some evidence of a learning disability. Low scores in phonological short-term memory characterized clusters with a language-based weakness whereas low or variable phonological awareness was associated with the reading (but not language-based) weaknesses. The low math only group did not show these phonological deficits. These findings may suggest different etiologies for language-based deficits in language, reading, and math, reading-related impairments in reading and math, and isolated math disabilities.     

Potentiation of tumour promotion by topical application of argemone oil/isolated sanguinarine alkaloid in a model of mouse skin carcinogenesis

Several incidences of adverse effects on human health have been reported in many countries, due to consumption of edible oil adulterated with argemone oil (AO). The clinical manifestation of the disease is commonly referred to as epidemic dropsy. Our prior studies have shown that AO and isolated sanguinarine alkaloid (SANG) possess genotoxic and tumour initiating activity. In this study, the effect of AO/SANG was investigated on the development of tumour formation in mice using 7,12-dimethylbenz (a) anthracene (DMBA) initiated followed by tetradecanoyl phorbol acetate (TPA)-promoted skin tumour protocol. Single application of AO (300 μl) or SANG (4.5 μmol) when used during initiation phase in DMBA/TPA group did not reveal substantial difference in tumourigenic response. However, twice weekly application of AO (100 μl) or SANG (1.5 μmol) during promotion phase (25 weeks) resulted in enhanced tumourigenic response by ≥30% in DMBA/TPA treated group along with significant decrease in dermal tyrosinase (45–49%), histidase (30–32%), superoxide dismutase (53–56%), catalase (41%), GSH reductase (37–40%) and GSH-peroxidase activity (29–33%) compared to control. Furthermore, significant decrease of epidermal GSH (64–66%) content and enhanced formation of lipid peroxides (96–121%) was noticed following AO or SANG treatment during promotion phase to DMBA/TPA induced animals indicating the modified pro-oxidant status in skin. Although dermal biochemical parameters were also altered by AO or SANG when used during initiation phase in DMBA/TPA treated animals, nonetheless, the response in these parameters were relatively more when AO or SANG were used during promotion phase in DMBA/TPA treated animals. These results clearly suggest that AO and SANG have the ability to enhance the tumourigenic response, which may have relevance to its carcinogenic potential.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Binding of the EcoRII methyltransferase to 5-fluorocytosine-containing DNA. Isolation of a bound peptide.

The properties of the interaction of 5-fluorocytosine-containing DNA with the EcoRII methyltransferase were studied. The DNA used was either a polymer synthesized in vitro, or a 20-mer containing one CCA/TGG sequence. The DNA could be methylated by the enzyme. In the process the enzyme formed a tight binding adduct with the DNA that could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzyme activity was inhibited by this interaction. The 20-mer could be used to titrate the active site of the enzyme. The DNA polymer formed a tight binding complex that could be identified following digestion of the DNA with pancreatic deoxyribonuclease or micrococcal nuclease. A peptide-DNA adduct could be isolated after digestion of the EcoRII-DNA adduct with staphylococcal protease V8 by high pressure liquid chromatography and polyacrylamide gel electrophoresis. Sequencing of the peptide indicated the DNA bound to a region of the protein that is conserved in all procaryotic DNA(cytosine-5)-methyltransferases. We have previously shown that this region contains a cysteine that can be photomethylated with adenosylmethionine. This region, in addition to forming part of, or being adjacent to, the AdoMet binding site, also forms part of the DNA binding site.