Intracellular calcium buffering capacity in isolated squid axons

Changes in ionized calcium were studied in axons isolated from living squid by measuring absorbance of the Ca binding dye Arsenazo III using multiwavelength differential absorption spectroscopy. Absorption changes measured in situ were calibrated in vitro with media of ionic composition similar to axoplasm containing CaEGTA buffers. Calcium loads of 50-2,500 μmol/kg axoplasm were induced by microinjection, by stimulation in 112 mM Ca seawater, or by soaking in choline saline with 1-10 mM Ca. Over this range of calcium loading of intact axoplasm, the ionized calcium in the axoplasm rose about 0.6 nM/μM load. Similar loading in axons preteated with carbonyl cyanide 4- trifluoromethoxyphenylhydrazone (FCCP) to inhibit the mitochondrial proton gradient increased ionized calcium by 5-7 percent of the imposed load, i.e. 93-95 percent of the calcium load was buffered by a process insensitive to FCCP. This FCCP- insensitive buffer system was not saturated by the largest calcium loads imposed, indicating a capacity of at least several millimolar. Treatment of previously loaded axons with FCCP or apyrase plus cyanide produced rises in ionized calcium which could be correlated with the extent of the load. Analysis of results indicated that, whereas only 6 percent of the endogenous calcium in fresh axons is stored in the FCCP-sensitive (presumably mitochondrial) buffer system, about 30 percent of an imposed exogenous load in the range of 50-2,500 μM is taken up by this system.     

安徽庐江鸭乙型肝炎病毒LJ-76转染细胞系的建立

为建立鸭乙型肝炎病毒ＬＪ－７６的转染细胞系,将ＬＪ－７６病毒ＤＮＡ插入到ｐＵＣ１９的ＥｃｏＲⅠ位点上,分离得到含有双拷贝ＬＪ－７６ＤＮＡ的重组质粒．通过磷酸钙沉淀方法,将经ＣｓＣｌ等密度离心纯化的ＬＪ－７６ＤＮＡ双体导入到人肝癌细胞ＢＥＬ７４０２中．收集转染细胞的培养液进行蔗糖密度梯度离心,所得沉淀经检测发现含有ＬＪ－７６ＤＮＡ并具有特异性ＤＨＢＶ内源性ＤＮＡ多聚酶活性;对上述样品通过ＤｏｔＥＩＡ检测ＤＨＢＶ核心抗原及表面抗原结果为阳性．Ｓｏｕｔｈｅｒｎｂｌｏｔ分析表明转染细胞内存在病毒ＤＮＡ复制中间体ｃｃｃＤＮＡ、ｓｓＤＮＡ和ｒｃＤＮＡ,而ｃｃｃＤＮＡ被认为是复制活动较为活跃的标志．电镜观察转染细胞的上清发现有病毒颗粒的存在．     

Strike‐induced chemosensory searching (SICS) in northern Pacific rattlesnakes (Crotalus viridis oreganus,Holbrook, 1840) rescued from substandard husbandry conditions. II. Complete recovery of function after two years

Six northern Pacific rattlesnakes (Crotalus viridis oreganus) rescued from substandard husbandry conditions were shipped from Woodland Park Zoological Gardens to the University of Colorado in December 1994. The snakes accepted prey and rapidly gained weight, but their searching behavior after predatory strikes was seriously depressed. We administered repeated tests of strike‐induced chemosensory searching (SICS) finding that the snakes improved steadily with each succeeding test, until performance eventually reached the level characteristic of this taxon. Although the snakes reached normal body weight within 4 months of exposure to proper husbandry conditions, normal SICS was not seen until 2 years had elapsed. Release of these snakes into natural habitat prior to recovery of SICS may well have resulted in the demise of the animals, suggesting that rehabilitation efforts must consider recovery of essential behavior patterns that can take far longer than recovery of body weight. Zoo Biol 18:141–146, 1999. © 1999 Wiley‐Liss, Inc.     

Allotypes of mouse complement component C6 in inbred strains and some wild populations

This collaborative work was undertaken to resolve discrepancies in reports of the number of forms of complement component C6 present in the circulation of mice from various inbred strains. Plasma C6 was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by isoelectric focusing (IEF), and C6 band patterns were developed by electroblotting and immunoprobing. Results of C6 allotyping of mice from 36 strains confirmed that while 20 strains (prototype strain BALB/c) possessed only one relative mass (M _r) form which typed C6A1 on IEF, the other 16 strains all possessed more than one C6 M _r form. Moreover, IEF analysis demonstrated additional polymorphic differences; among these 16 strains, 11 typed C6A l B l like the prototype strain CBA, the AKR and RF/J strains typed C6A2B2, and the Japanese MOM strain as well as the C57BR/cdJ and C57L/J strains possessed two forms with IEF mobilities intermediate between C6A1B1 and C6A2B2. These will now be referred to as C6A3B3. Thus, a total of four different mouse C6 haplotypes have been identified.Testing C6 allotypes in a limited number of wild mice revealed that haplotypes found in inbred strains of Western or Eastern origin tend to reflect haplotypes of the wild mice from Europe or Japan, respectively.     

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.     

Strike-induced chemosensory searching in venomoid pit vipers at Hogle Zoo

Six pit vipers with ligated and severed venom ducts were each observed twice. In the no-strike trial a rodent was presented for 3 sec but held (on tongs) out of striking range. The strike trial began with a 3 sec presentation, but the prey item was then moved into striking range, and all snakes immediately struck and released the rodents. Prey were removed after no-strike and strike presentations and the rate of tongue flicking was recorded for 30 min. Only strike presentations were followed by high rates of tongue flicking, which were indistinguishable from those seen in pit vipers with functioning venom apparatus. We conclude that venom injection is irrelevant for the causation of strike-induced chemosensory searching.