Uptake inhibitors do not change the effect of imidazoline α2-adrenoceptor agonists on transmitter release evoked by single pulse stimulation in mouse vas deferens

Summary The present study was undertaken to compare the presynaptic interaction of neuronal noradrenaline uptake inhibitors with imidazoline and phenylethylamine ₂adrenoceptor agonists under two different conditions: at low and high noradrenaline concentrations in the biophase.Isolated mouse vasa deferentia were stimulated with trains of 7 pulses given at 0.2 Hz and the inhibition by the ₂-adrenoceptor agonists clonidine, -methylnoradrenaline, and UK-14,304 of twitch responses was measured in the absence and in the presence of either cocaine (12 mol/l) or desipramine (40 nmol/l). The effects were determined for the first (equivalent to single pulse stimulation) and the last stimulus of each train. Both uptake inhibitors antagonized the presynaptic inhibitory effects of imidazolines (clonidine and UK-14,304) on the last twitch; the effects on the first twitch remained unchanged. In contrast, the uptake inhibitors potentiated the inhibitory effect of the phenylethylamine (-methylnoradrenaline) on both the first and the last twitches.These results support the view that the concentration of noradrenaline in the biophase plays a decisive role in the inhibition by a₂-adrenoceptor agonists of the electrically evoked release of noradrenaline. Agonists that are not substrates of neuronal uptake (i.e., clonidine, UK-14,304) become less effective when noradrenaline is present in the biophase while substrates of neuronal uptake (i. e., -methylnoradrenaline) do not. The results argue against the hypothesis that uptake inhibitors interact directly with presynaptic ₂-adrenoceptors or act at some link between uptake and receptor sites. Send offprint requests to S. Guimarães at the above address     

Determination of Red Blood Cell Velocity by Video Shuttering and Image Analysis

A novel modification of conventional video imaging techniques has been developed to determine the velocity of red blood cells (RBCs), which offers compatibility with existing video-based methods for determining blood oxygenation and hemoglobin concentration. Traditional frame-by-frame analysis of video recordings limits the maximum velocity that can be measured for individual cells in vivo to about 2 mm/s. We have extended this range to about 20 mm/s, by electronic shuttering of an intensified charge-coupled device camera to produce multiple images of a single RBC in the same video frame. RBCs were labeled with fluorescein isothiocyanate and the labeled cells (FRBCs) were used as probes to determine RBC velocities in microvessels of the hamster retractor muscle. Velocity was computed as the product of the distance between centroids of two consecutive image positions of a FRBC and the shuttering frequency of the camera intensifier. In vitro calibrations of the system using FRBC and Sephadex beads coated onto a rotating disk yielded an average coefficient of variation of about 6%. Flow conservation studies at bifurcations indicated that the maximum diameter of microvessels below which all the FRBCs in the lumen could be detected was 50 m. The technique was used to estimate mean-FRBC velocity distributions in vessels with diameters ranging from 8 to 50 m. The mean-FRBC velocity profiles were found to be blunter than would be expected for Poiseuille flow. Single FRBCs tracked along an unbranched arteriole exhibited significant temporal variations in velocity. © 1999 Biomedical Engineering Society. PAC99: 8719Tt, 8717Jj, 4279Pw, 8780Tq, 8719Ff, 4230Va, 0705Pj     

Receptive field organization of complex cells in cat striate cortex

Summary The receptive field organization of complex cells was studied by analyzing interaction effects between two stationary flashing light stimuli. One was placed in the most responsive part of the receptive field to produce activity against which effects of the other in different visual field positions could be determined.The receptive field was spatially organized into antagonistic center and flanks just like the fields of simple cells. However, both center and flanks were found within the receptive field area where a single slit evoked discharge. Center and flanks were elongated along the optimal stimulus orientation. The flanks were displaced from the center normal to optimal stimulus orientation.In the center, ON- and OFF-responses were usually about equal in strength and the maximum ON- and OFF-responses occurred in about the same position. This shows that complex cells are activated by input from both ON- and OFF-center cells in the lateral geniculate nucleus (LGN) where the receptive field centers of the LGN cells overlap closely. This explains most of the specific features of complex cells, e.g., the spatially overlapping ON- and OFF-zones, the large response field, the repetitive firing when a slit moves over the receptive field, and the marked non-linear spatial summation.Strong flank suppression occurred with both ON and OFF. The effects were usually stronger on one side of the center. Maximal suppression occurred on the same side with both ON and OFF. This is consistent with the interpretation that complex cells are inhibited by input from both LGN ON- and OFF-center cells with overlapping receptive field centers.A model presuming that complex cells have overlapping but acentric excitatory and inhibitory fields was tested by computer simulation and shown to fit the experimental data. This is the same model as presented for simple cells in the preceding paper (Heggelund 1980), except that the excitatory and inhibitory fields of simple cells have input from either ON- or OFF-center LGN cells, whereas in complex cells they have input from both types.The project was financially supported by the Norwegian Research Council for Science and Humanities     

Recruitment of fibre types and quadriceps muscle portions during repeated, intense knee-extensor exercise in humans

To investigate recruitment of slow-twitch (ST) and fast-twitch (FT) muscle fibres, as well as the involvement of the various quadriceps femoris muscle portions during repeated, intense, one-legged knee-extensor exercise, 12 healthy male subjects performed two 3-min exercise bouts at ~110% maximum thigh O₂ consumption (EX1 and EX2) separated by 6 min rest. Single-fibre metabolites were determined in successive muscle biopsies obtained from the vastus lateralis muscle (n=6) and intra-muscular temperatures were continuously measured at six quadriceps muscle sites (n=6). Creatine phosphate (CP) had decreased (P<0.05) by 27, 73 and 88% in ST fibres and 25, 71 and 89% in FT fibres after 15 and 180 s of EX1 and after 180 s of EX2, respectively. CP was below resting mean–1 SD in 15, 46, 84 and 100% of the ST fibres and 9, 48, 85 and 100% of the FT fibres at rest, after 15 and 180 s of EX1 and after 180 s of EX2, respectively. A significant muscle temperature increase (T_m) occurred within 2–4 s at all quadriceps muscle sites. T_m varied less than 10% between sites during EX1, but was 23% higher (P<0.05) in the vastus lateralis than in the rectus femoris muscle during EX2. T_m in the vastus lateralis was 101 and 109% of the mean quadriceps value during EX1 and EX2, respectively. We conclude that both fibre types and all quadriceps muscle portions are recruited at the onset of intense knee-extensor exercise, that essentially all quadriceps muscle fibres are activated during repeated intense exercise and that metabolic measurements in the vastus lateralis muscle provide a good indication of the whole-quadriceps muscle metabolism during repeated, intense, one-legged knee-extensor exercise.     

Distribution of vasoactive intestinal peptide-sensitive adenylate cyclase activity along the rabbit nephron

The effect of vasoactive intestinal peptide (VIP) upon adenylate cyclase (AC) activity has been determined in defined microdissected renal tubules isolated from collagenase-treated rabbit kidneys. In the presence of 10 M GTP, 1 M VIP gave marked stimulations of AC over basal values in the bright portion of the distal convoluted tubule (DCTb) (10.1-fold), and in the collecting tubule isolated from the inner stripe of the outer medulla (OMCTi, 7.8-fold). Less pronounced effects of VIP were found in the medullary collecting tubule isolated from the outer stripe (2.5-fold) and in the granular portion of the distal convoluted tubule (2.0-fold). VIP stimulation of AC activity in these segments amounted to 25 to 40% of the effect elicited by other agonists (arginine vasopressin, calcitonin or parathyroid hormone) in their respective target segments. A low response to VIP was observed in the cortical thick ascending limb (1.8-fold) which represented less than 5% of the calcitonin-stimulated AC activity. In the thin descending limb VIP produced a slight and variable stimulation of AC. VIP was without effect upon AC in the convoluted and straight portions of the proximal tubule, the medullary thick ascending limb and the cortical collecting tubule. Halfmaximal stimulation of AC by VIP was observed at 26±10 nM (n=3) in OMCTi and at 19 nM (n=2) in DCTb. Related peptides glucagon, secretin and PHI gave lower stimulations of AC compared to VIP in OMCTi. Conversely for rat OMCTi, under identical conditions, glucagon was much more effective than VIP.     

Novel single nucleotide polymorphisms of the human nuclear factor kappa-B 2 gene identified by sequencing the entire gene

The nuclear factor kappa-B 2 (NFKB2) gene is a member of the NFKB/Rel gene family, which is known to be a pivotal regulator of the acute phase of the inflammatory response and of immune responses. We identified three novel single nucleotide polymorphisms (SNPs) and determined their allelic frequencies, as determined by the sequencing of 48 alleles of the entire gene in a Japanese population sample. Two of the three polymorphisms were identified at nucleotide (nt) position 1837 (T/C) and nt position, 1867 (GG/G) in the upstream region of the gene. The other polymorphism was identified at nt position 2584 (G/T) within intron 1. These polymorphisms will be useful in genetic studies of the processes involved in inflammatory responses and in bone differentiation. Received: October 17, 2000 / Accepted: October 23, 2000     

Functional study of the different haplotypes cDNA in the coding region of CⅡTA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.     

Phylogenetic analysis of mtDNA haplogroup TJ in a Finnish population

An association between mitochondrial DNA (mtDNA) mutations 11778G>A and 14484T>C and mtDNA haplogroup J suggests that this haplogroup harbors substitutions capable of modifying the phenotype of Leber's disease. Our knowledge of the compilation of substitutions in haplogroup J is based on only a small number of complete mtDNA sequences, however. We constructed phylogenetic networks for mtDNA haplogroup TJ that were based on the sequence of the complete coding region and the hypervariable segment I, respectively, in 28 Finnish samples. The networks revealed a subdivision of the haplogroup into subclusters T1, T2, J1, and J2, while comparison of the two networks suggested nine fast evolving nucleotide sites in the hypervariable segment I. Genotypes of patients harboring 11778G>A or 14484T>C were obtained from the literature and were then placed in the network. Only four substitutions were found to be common to the patients, but none of these was unique to haplogroup J. If increased penetrance of the 11778G>A and 14484T>C mutations in patients belonging to haplogroup J is assumed, combinations of ancient substitutions must be implicated. Received: September 29, 2000 / Accepted: November 10, 2000     

抗人卵巢癌×抗人CD3×抗CD28VH单链三特异抗体的胞内可溶表达、纯化及体外活性测定

目的　研究抗人卵巢癌 (ovariancarcinoma ,oc)×抗人CD3×抗CD2 8VH 单链三特异抗体(singlechaintrispecificantibody,scTsAb)在大肠杆菌中的可溶表达与纯化及纯化后产物的活性测定 ,从而为其应用于卵巢癌治疗的临床研究打下基础。方法　将已构建的scTsAb表达载体转化大肠杆菌BL2 1(DE3)Star菌株 ,采用低温 (30℃ )、低剂量IPTG(0 .2mmol L)诱导 ,进行胞内可溶表达。根据抗卵巢癌三特异抗体 (ocTsAb)等电点较高 (pI9.0 ) ,而菌体蛋白大多为酸性蛋白的特点 ,利用DEAE弱阴离子交换层析(pH8.0 )进行一步纯化 ,并利用ELISA及FACS的方法检测纯化后抗卵巢癌三特异抗体的活性。结果　(1)SDS PAGE鉴定低温诱导时可溶比例达到 5 6 %。 (2 )绝大多数菌体蛋白被DEAE层析柱吸附 ,而抗卵巢癌三特异抗体在穿透液中流出 ,SDS PAGE检测纯度达到 90 %。 (3)ELISA结果显示纯化后的抗卵巢癌三特异抗体与重组CD2 8纯抗原 ,Jurkat(CD3 )细胞膜提取抗原 ,SKOV3细胞膜提取抗原均有特异性结合。 (4 )FACS结果证明纯化后的抗卵巢癌三特异抗体与Jurkat(CD3 )活细胞、SKOV3活细胞有特异性结合。结论　低温诱导胞内可溶表达的抗人卵巢癌×抗人CD3×抗CD2 8VH 单链三特异抗体经弱阴离子交换层析一步纯化后仍保持原有免疫学活性 ,这