Molecular and cytogenetic organization of the 5S ribosomal DNA array in chicken (Gallus gallus)

The 5S ribosomal (r) RNA genes encode a small (120-bp) highly-conserved component of the large ribosomal subunit. The objective of the present research was to study the molecular and cytogenetic organization of the chicken 5S rDNA. A predominant 2.2-kb gene (5S) consisting of a coding and intergenic spacer (IGS) region was identified in ten research and commercial populations. A variant gene repeat of 0.6kb (5S) was observed in some of the populations. Genetic linkage analysis and cytogenetic localization by fluorescence in-situ hybridization assigned the 5S rDNA to chromosome 9. The 5S rDNA array was determined to be 80.2±7.0kb upon electrophoretic sizing following EcoRV digestion. Sequence analysis of 5S IGS regions revealed considerable conservation between chicken subspecies (98.4% identity) as well as homology with vertebrate Pol III promoter and regulatory sequence motifs. Minor intraindividual sequence variation within 1000bp of IGS was observed in four cloned Red Jungle Fowl (Gallus gallus gallus) 5S repeats (95.5% identity in this region). Sequence comparisons between IGS regions of 5S and 5S genes indicated two short continuous (>20bp) and many short non-continuous homologous regions as well as other conserved features such as promoter and termination motifs.     

Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.)

Procedures for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) have been developed for chickpea (Cicer arietinum). Suspensions of intact chromosomes were prepared from root tips treated to achieve a high degree of metaphase synchrony. The optimal protocol consisted of a treatment of roots with 2mmol/L hydroxyurea for 18h, a 4.5-h recovery in hydroxyurea-free medium, 2h incubation with 10µmol/L oryzalin, and ice-water treatment overnight. This procedure resulted in an average metaphase index of 47%. Synchronized root tips were fixed in 2% formaldehyde for 20min, and chromosome suspensions prepared by mechanical homogenization of fixed root tips. More than 4×10⁵ morphologically intact chromosomes could be isolated from 15 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing eight peaks, representing individual chromosomes and/or groups of chromosomes with a similar relative DNA content. Five peaks could be assigned to individual chromosomes (A, B, C, G, H). The purity of sorted chromosome fractions was high, and chromosomes B and H could be sorted with 100% purity. PCR on flow-sorted chromosome fractions with primers for sequence-tagged microsatellite site (STMS) markers permitted assignment of the genetic linkage group LG8 to the smallest chickpea chromosome H. This study extends the number of legume species for which flow cytogenetics is available, and demonstrates the potential of flow cytogenetics for genome mapping in chickpea.     

Molecular Analysis of Fiji Disease Fijivirus Genome Segments 1 and 3

Fiji disease fijivirus (FDV) genomic segments 1 (S1) and 3 (S3) were completely sequenced. FDV S1 comprised 4532nt and was predicted to encode a 170.6kDa protein. FDV S3 comprised 3623nt and was predicted to encode a 135.5kDa protein. The terminal sequences of S1 and S3 were 5 AAGUUUUU......CAGCUAGCGUC 3 and 5 AAGUUUUU......CAGCAGAUGUC 3, respectively, and located immediately adjacent to these sequences were 12bp imperfect inverted repeats. The predicted translation product of FDV S1 showed highest similarity to Rice black-streaked dwarf virus (RBSDV) S1 and is thought to encode the viral RNA-dependent RNA polymerase (RdRp). The predicted translation product of FDV S3 was found to be most similar to RBSDV S4 which is thought to encode the 'B-spike' protein. The FDV sequence contained an ATP/GTP binding motif and a leucine zipper motif, but these motifs were not found in the RBSDV sequence. Phylogenetic analysis based on the amino acid sequences of the RdRp of FDV S1 and other reoviruses revealed that the fijiviruses form a cluster close to the oryzaviruses. The RdRp sequences were grouped into genera that were consistent with the current reovirus classification scheme that is based on physico-chemical and biological properties.     

Genome Organization and Expression of the Penicillium stoloniferum Virus S

The complete sequences of two double-stranded RNAs (dsRNAs) (referred to S1 and S2) of Penicillium stoloniferum virus S (PsV-S) were established. The S1 dsRNA was 1,690bp in length, and it contained a unique open reading frame (ORF) of 539 amino acids (molecular weight of 62kDa, referred to P62). The S2 dsRNA was 1,523bp in length, and also it contained one ORF of 434 amino acids (molecular weight of 47kDa, referred to P47). Both S1 and S2 ORFs were identified only on the positive strand of each dsRNA segment. A sequence motif of (5-CUG-3) was found at the 3-termini of the positive strands of PsV-S1 and S2 dsRNAs. The predicted amino acid sequences of S1 dsRNA showed high sequence homology with the putative RNA-dependent RNA polymerases of RNA viruses. Near full-length and positive-sense single-stranded RNAs derived from the S1 and S2 dsRNAs were detected from the PsV-infected host cell. The expressed proteins of P62 and P47 showed a positive reaction against PsV-S antiserum in Western blot analysis. Phylogenetic analysis using the RDRP sequences and the capsid proteins of the various partitiviruses revealed that PsV-S is a definite member of the partitivirus, the family Partitiviridae, and especially clusters well along with D. destructiva virus 1 and 2.     

Some properties of the UL-FS 49 block of the hyperpolarization-activated current (i f) in sino-atrial node myocytes

Block of the hyperpolarization-activated pacemaker current (i _f) by the bradycardic agent UL-FS 49 was studied in isolated sino-atrial (SA) node myocytes. Using repetitive activation/deactivation protocols, micromolar concentrations of UL-FS 49 blockedi _f in a dose-dependent fashion. Block development was slow, with time constants decreasing with drug concentration and ranging from 25.8 s at 10 M to 75.5 s at 1 M UL-FS 49. Block did not develop in cells held at –35 mV, at which voltagei _f channels are closed, indicating that channels must open before blocking occurs. Apparently in contrast with the requirement of negative voltages for block development, block was relieved by hyperpolarization with a time course slower than current kinetics. Due to the hyperpolarization-induced block relief, current/voltage (I/V) relations in the presence of UL-FS 49 displayed inward-going rectification. Experimental data fitted the hypothesis that UL-FS 49 behaves as an open channel blocker of single-ioni _f channels. Block occurs within the pore, at a distance of about 39% of the membrane thickness from its internal side.     

The complete nucleotide sequence of the genome RNA of Lily symptomless virus and its comparison with that of other carlaviruses

Summary. The complete genomic nucleotide sequence and genome structure of Lily symptomless virus (LSV), a lily-infecting carlavirus, have been obtained. The genome of the Korean strain of LSV, LSV-Kr, was 8,394 nucleotides long and contained six open reading frames (ORFs) coding for proteins of Mr 220kDa (1,948aa), 25kDa (228aa), 12kDa (106aa), 7kDa (64aa), 32kDa (291aa) and 16kDa (140aa) from the 5 to 3 end, respectively, which is typical of carlaviruses. Genetic heterogeneity was observed in the ORF1 gene. A total of 221 of 5,847 nucleotides (nt) were heterologous in the ORF1 of replicase; 162nt portions were silent and 59nt resulted in amino acid changes. This heterogeneity indicates that the LSV-infecting lily plants contained a genetically heterogeneous population of LSV (quasispecies). Overall similarities to those of other carlaviruses for the six ORFs of LSV were from 76.1% to 31.6% and from 87.3% to 13.7%, at nucleotide and amino acid levels, respectively. The ORF1 replicase gene of LSV shares 40.9% to 56.8% and 48.9% and 58.6% identities with that of 5 other carlaviruses at the amino acid and nucleotide levels, respectively. LSV was closest to Blueberry scorch virus (BlScV) in this ORF, among the carlaviruses for which sequence information is available. The three triple gene blocks (ORF2-4), ORF5 (coat protein) and 3-proximal 16kDa ORF6 genes were further analyzed, and phylogenetic trees for the coding regions indicate that the LSV was the most closely related to Kalanchoe latent virus and BlScV. This is the first report of the complete nucleotide sequence and genome structure of LSV.Received December 13, 2002; accepted May 14, 2003 Published online July 17, 2003     

Mother-Baby psychiatric units (MBUs): National data collection in France and in Belgium (1999–2000)

Summary The French version of the Marcé checklist was used to collect data for 176 joint admissions to 11 psychiatric mother-baby units in 1999 and 2000. Mean age of the babies at admission ranged from 4 to 16 weeks. Two units also admitted older children. Mothers admitted were diagnosed with schizophrenia or chronic delusional disorders (n=44), acute transitory psychosis Bouffée délirante (n=20), bipolar disorders (n=20), depressive illness (n=38), personality disorders or intellectual disability (n=39), and other disorders (n=15). The mean duration of hospitalisation was 11 weeks. Units that also offered day-care admission in the same or a near-by unit had shorter mean admissions. More than half the womens partners (or babies fathers) had mental health problems. Women with schizophrenia or chronic delusional disorders and personality disorders or intellectual disability remained hospitalised longer, improved less, and were more often separated from their babies, or discharged with supervision, than women admitted with other diagnoses.     

Effects of Age and Experience on Mating Activity in the Sibling Species Drosophila pavani and Drosophila gaucha

The effects of age and experience on sexual activity and on intra- and interspecific discrimination were studied in two sibling species of the mesophragmatica group, Drosophila pavani and Drosophila gaucha. Sexual activity of a total of 2970 individual couples of the same or of both species was observed at two ages: 10 days, (young inexperienced) and 18-20 days (old, either inexperienced or experienced, if either the male or the female had copulated previously). In the 1186 (39.97%) pairs that mated, the latency to copula and duration of copula were registered. Age has a different effect in both species: young Drosophila pavani and old Drosophila gaucha females are less receptive to males of either species of the corresponding age. The receptivity of females is also reflected in heterospecific matings, as Drosophila gaucha males increase their mating activity with age. In both species, female receptivity decreases with experience, whereas mating activity of males increases with experience, especially that of Drosophila gaucha toward heterospecific females. Drosophila pavani females take longer to mate than those of Drosophila gaucha. In both species old males tend to mate faster, whereas experience increases the latency to mating in females and decreases it in males. Both species differ significantly in the duration of copula. It is longer in Drosophila pavani than in Drosophila gaucha and is determined mainly by the male. The duration of copula increases with age, especially in Drosophila pavani females, whereas it is reduced in males of the same species.     

Sequence analysis of the fragment of the phosphoprotein gene of Polish distemper virus isolates

Summary. The nucleotide sequence analysis of the 429bp fragment of the P gene of 11 Polish field isolates of Canine distemper virus (CDV), reference strains and other virus isolates available in the GenBank was the aim of the studies. High homology between all dog strains from east-southern region of Poland and reference strains of CDV was demonstrated. It was estimated as 97–100% for CDV-OND; 96.7–99.8% for CDV-Rock; 96.7–99.8% for CDV-LED and 96.3–97.9% for A75-17. The 100% homology of the nucleotide sequence was observed between CDV Pulawy 92, CDV Pulawy 97 and the reference CDV-OND. The homology between CDV-OND and viruses isolated from the mink and ferret was estimated as 97.7% and 98.4%, respectively. Virus strains isolated from blue foxes demonstrated the highest homology to CDV-OND – equal to 97.7% for DV 79 and 99.5% for DV 92. The fox isolate from 1992 had higher level of homology to dog isolates (96.5–99.5%) than the strain isolated from the fox in 1979 (97.2–98.8%). The phylogenetic tree has two main lineages representing two separated genetic groups: I containing PDV and II containing all distemper virus strains isolated from terrestrial carnivores. CDV strains isolated from dogs from Pulawy region between 1992–1998 and from the fox (DV 92) formed the separate lineage containing also reference strains. They differed from the native isolates from the mink and ferret as well as from Japanese strains of CDV.Received October 29, 2002; accepted April 2, 2003 Published online June 11, 2003     

The relationship between HPV16 and expression of CD44v6, nm23H1 in esophageal squamous cell carcinoma

Summary. The esophageal squamous cell carcinoma (ESCC) has high incidence in Shaanxi Province of China. More and more researches indicated that human papillomavirus type 16 (HPV16) might play an important role in carcinogenesis of ESCC but the relationship between HPV16 and CD44v6, nm23H1 has not been elucidated. HPV16 was detected by amplifying HPV16 E6 gene through polymerase chain reaction (PCR) method and the expression of CD44v6, nm23H1 in 40 ESCCs and fifteen normal esophageal mucosa (NEM) from Shaanxi Province was examined by Streptavidin-Peroxidase (SP) method using monoclonal antibody specific to CD44v6 and nm23H1. The positive rates of HPV16 E6 gene, CD44v6 and nm23H1 were 60% (24/40), 65% (26/40) and 45% (18/40) respectively in ESCCs and 26.67% (4/15), 33.33% (5/15) and 86.67% (13/15) respectively in NEMs. There exited statistical difference for HPV16, CD44v6 and nm23H1 between NEMs and ESCCs respectively (p<0.05). The relationship between HPV16 and the expression of CD44v6 in ESCCs was statistical significance (P=0.021), but no significant correlation was found between HPV16 and the expression of nm23H1 (P=0.436) in ESCCs. The infection rate of HPV16 had no statistical difference in all pathological features we observed, but the expression rates of CD44v6 and nm23H1 had statistical correlation with invasion (p=0.001, 0.013) and lymph nodes metastasis (p=0.014, 0.002) respectively. In different histology grade of ESSCs, the relationship between HPV16 and CD44v6 was statistical significance in grade I (p=0.044) but was not in grade II (p=0.165) and grade III (p=0.658), however as to the expression of nm23H1 there exited no statistical significance in all histology grades of ESCC (p>0.05). The expression rates of CD44v6 and nm23H1 were statistically different between grade I and II (p=0.004, 0.016) respectively and between grade I and grade III (p=0.014, 0.020), but not statistically different between grade II and III (p=0.792, 0.943) respectively. Our data firstly suggested that there existed the statistical relationship between the infection of HPV16 and the expression of CD44v6 in ESCCs and that HPV16 may be involved in invasion and metastasis of ESCC.     

<Emphasis Type="Italic">Rice stripe virus</Emphasis> 23.9 K protein aggregates and forms inclusion bodies in cultured insect cells and virus-infected plant cells

Summary. The genome of Rice stripe virus (RSV, genus Tenuivirus) contains seven open reading frames (ORFs). Little is known about the products of four of these ORFs, including the 23.9K protein encoded by the virus-sense ORF of RNA3. Western blotting revealed that the 23.9K protein was synthesized in the host plant and also in the planthopper vector of RSV. Using a baculovirus vector, the 23.9K protein was expressed, both unfused and fused with red-shifted green fluorescent protein, in Spodoptera frugiperda cells. Inclusion bodies were observed by light microscope in cells expressing fused or unfused proteins. Inclusion bodies in cells expressing the fused protein fluoresced under blue light. By immunoelectron microscopy, electron-dense inclusion bodies in cells expressing the unfused protein were specifically labeled with 23.9K protein antiserum. Moreover, electron-dense masses labeled with 23.9K protein antiserum were observed in virus-infected wheat tissue by electron microscopy. This paper thus demonstrates that RSV 23.9K protein can aggregate in vivo and form inclusion bodies in infected plant tissue.Received December 24, 2003; accepted June 11, 2003 Published online August 7, 2003     

Synthesis of Seoul virus RNA and structural proteins in cultured cells

Summary. Seoul virus is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). The virion has a tripartite (S, M, and L) negative-stranded RNA genome, which is characteristic of the family Bunyaviridae. However, the molecular basis of virus replication is not well known. We established a Northern blot hybridization (NB) procedure using digoxygenin-labeled RNA probes, to quantitate the hantaviral plus- and minus-strand RNAs separately. Virus RNA replication was analyzed in infected Vero E6 cells. When the Vero E6 cells were infected with Seoul virus strain KI-83-262 (KI) at m.o.i.=0.25, the plus-strand RNA was detected within 1h post-infection (hpi), and the minus-strand RNA was detected subsequently. Using laser confocal microscopy, the nucleocapsid protein (NP) was detected within 2hpi, and accumulated as scattered granules in the cytoplasm until 24hpi. In contrast, the G2 protein first appeared at 8hpi, was immediately transported to the Golgi, and accumulated in the Golgi until 24hpi. Infectious virus particles were released into the medium at 24hhpi. These findings indicate that hantavirus RNA replication starts with the appearance of NP at 2hpi, glycoproteins then accumulate gradually in the Golgi, and virion formation is initiated once the viral RNAs and proteins have accumulated.Received October 10, 2002; accepted April 25, 2003 Published online July 17, 2003     

Molecular analysis of <Emphasis Type="Italic">Fiji disease virus</Emphasis> genome segments 5, 6, 8 and 10

Summary. The complete sequences of Fiji disease virus (FDV) genome segments 5 (S5), S6, S8 and S10 were obtained and comprised 3150nt, 2831nt, 1959nt and 1819nt, respectively. Each segment contained a single ORF which encoded putative proteins of 115kDa, 97kDa, 69kDa and 63.0kDa, respectively. The putative amino acid sequences encoded by S5 and S6 contained putative leucine zipper motifs while FDV S5 and S8 each contained an ATP-GTP-binding motif. At the amino acid level, FDV S5, S6, S8 and S10 showed most similarity to the corresponding segments of Rice black-streaked dwarf virus. Based on sequence similarities, it is predicted that FDV S8 encodes a minor core protein, while FDV S10 encodes an outer capsid protein. The evolutionary relationships of FDV to other reoviruses are discussed.The nucleotide sequence data for FDV S5, S6, S8 and S10 are available in the DDBJ/EMBL/GenBank databases under the accession numbers AY029521, AF356083, AY297693 and AY297694, respectively.     

High-level Expression of the ORF6 Gene of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Pichia pastoris

High-level expression of the ORF6 gene of porcine reproductive and respiratory syndrome virus (PRRSV) has been proved very difficult. In this work, we cloned and sequenced the ORF6 gene of PRRSV and found that it could not be expressed in Pichia pastoris strain GS115. Then, the ORF6 gene was modified and synthesized based on the codon bias, poly (A) signal of yeast expression system and secondary structure of 5-end mRNA of foreign gene. The modified gene was inserted into the yeast expression vector pPICZA, induced and expressed by the same methods. The recombinant protein with a molecular mass of approximately 23kDa was screened by SDS-PAGE and identified by Western blot with convalescent sera of animals infected with CH-1a strain of PRRSV. The results indicated that it was similar to the native protein. The expression level of the recombinant protein could attain 2.0g/L. In the meanwhile, the optimal conditions for expression were determined. It provides an additional means for studying the structural and functional characteristics of PRRSV ORF6 gene.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Detection of enteric viruses and bacterial indicators in German environmental waters

Summary. A German mining lake and the supplying surface waters, which are located downstream of a sewage plant, were examined regarding their microbiological and virological quality. Between October 2002 and September 2003, specific PCR methods were used to determine the occurrence of enteric viruses in 123 water specimens drawn at different sites downstream of the waste water treatment plant and in 9 samples from the sewage plant influent. Detection rates in sewage plant effluents and surface water samples depended on sampling sites and were: 29–76% for enterovirus (EntV), 24–42% (astrovirus, AstV), 15–53% (norovirus, NV), 3–24% (rotavirus, RoV), 5–20% (hepatitis A virus, HAV) and 20% (adenovirus, AdV). AstV genome load of selected samples was between 3.7×10³ to 1.2×10⁸ genome equivalents per liter (gen.equ./l), depending on sampling location; NV average genome load ranged from 1.8×10⁴ to 9.7×10⁵gen.equ./l. Cell culture methods showed that three out of 18 PCR positive samples contained infectious EntV. Even though microbiolical parameters such as Escherichia coli, enterococci and coliphages indicated acceptable microbiological water quality, the virological data of this study suggest the possibility that surface waters may be a source for enteric viral infections.     

Identification and Sequence Analysis of Potato yellow vein virusCapsid Protein Minor Gene

Potato yellow vein virus (PYVV) is a whitefly-transmitted (Trialeurodes vaporariorum) closterovirus (WTC) with an as yet unidentified genome composition. PYVV dsRNA preparations consist of three high molecular weight dsRNA species (dsRNAs 1, 2 and 3) 8.0, 5.5 and 4.0kbp in size respectively, as well as two low molecular weight dsRNA species of 2.0 and 1.8kbp (denoted x and y). The PYVV capsid protein minor (CPm) gene was identified on the dsRNA 3 species, and was subsequently cloned and sequenced. The PYVV CPm gene is 2022 nucleotides long and putatively encodes a protein with estimated size 77.5kDa. The PYVV CPm gene product is considerably larger than the equivalent proteins encoded by the bipartite criniviruses, Lettuce infectious yellows virus (LIYV) and Cucurbit yellow stunting disorder virus (CYSDV) (52 and 53kDa, respectively). The PYVV CPm possesses a centralized domain which is absent from both the LIYV and CYSDV CPm counterparts. Pairwise comparisons as well as phylogenetic analysis based on the available amino acid sequences of the CPm of various WTCs, showed that PYVV is closely related to LIYV, CYSDV and also Beet pseudo-yellows virus.     

Identification and Characterization of the UL24 Gene Product of Herpes Simplex Virus Type 2

The UL24 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 281 amino acid protein with a molecular mass of 30.5kDa. In this study, the HSV-2 UL24 gene product has been identified by using a rabbit polyclonal antiserum produced against a recombinant protein containing the full-length UL24 gene product of HSV-2 fused to glutathione-S-transferase. The antiserum reacted specifically with a 32kDa protein in HSV-2 186-infected Vero cells and with 31 and 32kDa proteins in UL24-expressing Cos-7 cells. Accumulation of UL24 protein to detectable levels required viral DNA synthesis, indicating that the protein was regulated as a late gene. UL24 protein was found to be associated with purified HSV-2 virions and C capsids. Indirect immunofluorescence analysis demonstrated that the UL24-specific fluorescence was detected in perinuclear regions of the cytoplasm and/or in the nucleus as small discrete granules from 9h post infection (hpi). Furthermore, the UL24 protein expressed singly was detected predominantly in the nucleus and slightly in the cytoplasm at 24h after transfection, with branch-like cytoplasmic protruding structures. Strong nucleolus staining was visible in partial cells.     

Nano-scaled hydroxyapatite/polymer composite IV. Fabrication and cell adhesion properties of a three-dimensional scaffold made of composite material with a silk fibroin substrate to develop a percutaneous device

Nano-scaled sintered hydroxyapatite (HAp) particles with an a-axis length of 87 ± 23nm, a c-axis length of 236 ± 81nm, and a mean aspect ratio (c/a) of 2.72 were covalently linked onto a silk fibroin (SF) substrate chemically modified by graft polymerization with -methacryloxypropyl trimethoxysilane (MPTS). Graft polymerization with poly(MPTS) on SF was conducted by free-radical initiation in a water solvent with pentaethylene glycol dodecyl ether as a nonionic surfactant. The alkoxysilyl groups of the graft polymers avoided hydrolysis and maintained their activity in coupling with the hydroxyl groups on the HAp surface despite the use of water as the reaction solvent. The weight gain of poly(MPTS) on SF increased with increasing the reaction time, eventually reaching a plateau value of about 15wt% after 50min of reaction time. After HAp covalent coating, the particles separated or aggregated into several crystals, as shown by scanning electron microscopic observation. L929 fibroblast cells adhered more plentifully on HAp-coated SF compared to untreated SF and hydrolyzed poly(MPTS)-grafted SF during 24h or 48h of incubation. The cells adhered only on the HAp surface but not at all on the dehydrated grafted surface of SF without HAp. A button-shaped prototype for a percutaneous device was manufactured by transplantation of HAp-coated SF fibers of about 100µm in length onto silicone moldings using an adhesive, and the device showed good cell adhesiveness.     

β subunits determine the time course of desensitization in rat α3 neuronal nicotinic acetylcholine receptors

Standard two-electrode voltage-clamp techniques were used to investigate some of the pharmacological and functional properties of two types of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes after pairwise injection of 34 or 32 mRNAs. Currents of several A amplitude were elicited by fast application of micromolar concentrations of either acetylcholine (ACh) or 1,1-dimethyl-4-piperazine (DMPP). The activation of either receptor type by DMPP showed cooperativity (Hill coefficient, n1.7) with a half-maximal activation concentration (EC₅₀) of 15–30 M. In 34 receptors, ACh displayed cooperativity (n=1.8) but was less efficacious than DMPP, yet its EC₅₀ was about equal to that of DMPP. Finally, in 32 receptors, ACh was much less efficacious and had a much lower EC₅₀. Desensitization induced by either DMPP or ACh was slow in 34 nicotinic ACh receptors but was rapid and extensive in 32 receptors, causing a significant proportion of the response to wane within the first few seconds of agonist application.