A re-evaluation of the cytology of cat Pacinian corpuscles

Summary The ultrastructure of cat mesenteric Pacinian corpuscles in cross and longitudinal sections has been examined. The terminal ends of lamellar cells of the inner core have been identified in longitudinal sections through the proximal portion of the inner core. These terminal bulbous expansions contain characteristic concentric membranes of rough endoplasmic reticulum and in some cases masses of oval membranous inclusions. The central axon as seen in cross section is oval in profile, having X-(short) and Y-(long) axes, and each axonal face is characterized by specializations of the axolemma. At the X-axis, the inner lamellae of the inner core tightly abut a smooth axolemma, with no intervening connective tissue matrix, in a manner reminiscent of a neuroepithelium. The axolemma of the Y-axis has numerous axonal spines (microspikes) that project into the cleft in the inner core. The extent of the axolemma having axonal spines can only be appreciated in longitudinal sections. The clefts contain a specialized connective tissue with elastic and collagen fibrils. The connective tissue compartment of fibers and matrix separating individual inner core lamellae is unique, in that it contains extremely thin collagen fibrils measuring approximately 15 nm in diameter. The diameter of collagen fibrils increases as the cleft is approached. Here the fibrils resemble typical endoneural collagen.     

Cloning and characterization of the rice CatA catalase gene, a homologue of the maize Cat3 gene

We isolated and sequenced a genomic clone (CatA) encoding CAT-A catalase, a homologue of the maize catalase isozyme 3 (CAT-3) from rice (Oryza sativa L.). The 5-upstream non-coding region had very low similarity with the maize Cat3 gene and possible cis elements and sequence motifs in the maize Cat3 gene were not evident, except for TATA and CAAT motifs. Several sequence motifs found in the promoters of plant seed-specific genes were identified in the 5-upstream non-coding region of the CatA gene. Northern blotting showed that the CatA gene is expressed at high levels in seeds during early development and also in young seedlings. Methyl viologen (paraquat) resulted in the 3-fold induction of the CatA gene in the leaves of young seedlings, whereas abscisic acid, wounding, salicylic acid, and hydrogen peroxide had no or only slight effects.The 1.9 kb 5-upstream fragment (–1559 to +342) of the CatA gene was fused with the Escherichia coli -glucuronidase (GUS) gene and introduced by electroporation into protoplasts prepared from rice suspension-cultured cells, then the transient expression of the GUS gene was examined. Deletion analysis of this chimeric gene suggested that a weak silencer is located in the region between –1564 to –699. Abscisic acid (ABA) at a final concentration of 10^–6 M doubled GUS activity in protoplasts electroporated with the chimeric DNAs having 1.9 to 1.2 kb 5-upstream regions. A sequence highly similar to the Sph box, a motif found in genes modulated by ABA, was found at –266 to –254. Deletion of this region however, did not eliminate the responsiveness to ABA. Expression of the chimeric gene in the protoplasts was not enhanced by stress such as low and high temperature, hydrogen peroxide, methyl viologen, salicylic acid, elicitor, and UV light.The chimeric CatA-GUS plasmid DNAs amplified in the methylation-positive strain, E. coli DH5, showed GUS gene activities, whereas all the chimeric DNAs amplified in the methylation-deficient E. coli JM 110 were completely inactive in the presence or absence of ABA in the culture medium. DNA methylation, especially of either one or both of the deoxyadenosines at the two GATC motifs (one in the first exon and the other in the first intron of the rice CatA gene), appeared to be responsible for the CatA promoter activity identified in the transient assay.author for corresondenceThe nucleotide sequence data reported will appear in the DDBJ EMBL and GenBank Nucleotide Sequence Databases under the accession number D29966.     

Identification of the duplicated segments in rice chromosomes 1 and 5 by linkage analysis of cDNA markers of known functions

We mapped two loci for ADP-ribosylation factor homologues (ARF1, ARF2) and two loci for cysteine proteinase inhibitors (oryzacystatin-I and -II: OCI, OCII) by linkage analysis of restriction fragment length polymorphism loci in rice (Oryza sativa L.) genomic DNAs using their cDNAs as probes.Oc-1 andArf-2 were found to be closely located to each other on chromosome 1, whileOc-2 andArf-1,both found on chromosome 5, were also located close to each other. The map distances are about 2 cM in both pairs. In each chromosome, theArf locus was located about 27 cM from that of the aldolase gene (Ald-2 in chromosome 1 andAld-1 in chromosome 5). These three genes are in the same order,Ald-Arf-Oc, but in opposite orientations relative to the distal ends of the linkage group. The presence of two sets of three linked genes on chromosomes 1 and 5 strongly suggests a structural similarity of the blocks of the two chromosomes, which probably reflects duplication of the segment. A recent investigation by other workers has shown that these rice blocks correspond to two regions in maize chromosomes 8 and 6, that have previously been shown to share many duplicated nucleotide sequences. It is therefore very likely that the duplication of the region occurred before the divergence of rice and maize during the evolution of the subfamilies of the grasses (Gramineae). In view of a recently discovered possible structural similarity between the small GTP-binding protein superfamily, which includesArf andras proteins, and the cystatin family, the close linkage ofOc andArf loci found in the present study suggests a possible cluster of genes related to the small GTP-binding proteins.     

The p15gag and p12gag regions are both necessary for the pathogenicity of the murine AIDS virus.

The defective murine AIDS (MAIDS) virus has unique sequences in its p15gag and p12gag regions. To clarify whether these sequences are responsible for the development of MAIDS, we constructed recombinant viruses by replacing various regions of the gag gene of the nonpathogenic replication-competent LP-BM5 ecotropic virus with those of the MAIDS virus. Recombinants containing both unique sequences of the MAIDS virus were replication defective and induced MAIDS. However, a recombinant containing either the p15gag or p12gag region of the MAIDS virus was also replication defective but nonpathogenic in mice. A recombinant virus containing only the p30gag region of the MAIDS virus was replication competent and nonpathogenic. These results indicate that the p15gag and p12gag regions of the MAIDS virus do not function like those of replication-competent viruses and that both of the unique sequences in the p15gag and p12gag regions are required to develop MAIDS.     

Inhibition of the proliferation of cultured immortalized schwann cells by forskolin with a decreased basal level of diacylglycerol

The repetitive passages of a Schwann cell culture results in the appearance of immortalized cells. In order to investigate the direct effects of cyclic AMP (cAMP) on Schwann cell proliferation, we used the immortalized Schwann cells because the responses of a short-term Schwann cell culture to agents increasing the intracellular cAMP are more complicated and it does not seem that all of them are due to the direct effects of cAMP. By adding up to 200 M of forskolin, an adenylate cyclase activator, to the culture medium, Schwann cell proliferation was inhibited and the intracellular 1,2-diacylglycerol (DG) level was decreased in a dose-dependent manner to 44 and 53% of the control values, respectively. The protein phosphorylation activity in the cytosol from the cell treated with 100 M forskolin, assayed with myelin basic protein as the acceptor, decreased to 78% and this inhibition was then reversed by the addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DG, to the assay mixture. The cell proliferation inhibited by forskolin was also restored by the addition of OAG. These data suggest that cAMP inhibits both the activity of protein kinase C (PKC) and consequently cell proliferation through suppression of intracellular DG level, an activator of PKC. Since the inositol 1,4,5-triphosphate level and the hydrolysis of phosphatidylcholine to DG and phosphorylcholine were not affected, forskolin therefore appears to suppress the de novo synthesis of DG.     

Modeling of protein loops by simulated annealing.

A method is presented to model loops of protein to be used in homology modeling of proteins. This method employs the ESAP program of Higo et al. (Higo, J., Collura, V., & Garnier, J., 1992, Biopolymers 32, 33-43) and is based on a fast Monte Carlo simulation and a simulated annealing algorithm. The method is tested on different loops or peptide segments from immunoglobulin, bovine pancreatic trypsin inhibitor, and bovine trypsin. The predicted structure is obtained from the ensemble average of the coordinates of the Monte Carlo simulation at 300 K, which exhibits the lowest internal energy. The starting conformation of the loop prior to modeling is chosen to be completely extended, and a closing harmonic potential is applied to N, CA, C, and O atoms of the terminal residues. A rigid geometry potential of Robson and Platt (1986, J. Mol. Biol. 188, 259-281) with a united atom representation is used. This we demonstrate to yield a loop structure with good hydrogen bonding and torsion angles in the allowed regions of the Ramachandran map. The average accuracy of the modeling evaluated on the eight modeled loops is 1 A root mean square deviation (rmsd) for the backbone atoms and 2.3 A rmsd for all heavy atoms.     

Lytic Enzymes toward Aniline-assimilating Rhodococcus erythro-polis AN-13: Purification and Characterization

Three lytic enzymes, C-2, C-4 and C-5, capable of lysing cells of Rhodococcus erythropolis AN-13 were purified from the cultural filtrate of Flavobacterium species SH-548 by (NH₄)₂S0₄ fractionation and column chromatographies on CM-Toyopearl and SP-Sephadex. The three purified enzymes gave single protein bands on polyacrylamide gels. C-4 and C-5 were stable between pH 3.0 and 12.5, and C-2 between pH 5.5 and 11.0. The molecular weights of C-4 and C-5 were 26,000 and that of C-2 was 36,000, as judged on sodium dodecylsulfate-polyacrylamide gel electrophoresis. C-4 and C-5 also showed proteolytic activity toward casein, but C-2 did not exhibit such activity. C-2 showed higher specific lytic activity toward cells of R. erythropolis AN-13 than C-4 and C-5.     

Frequency,Risk Factors and Survival Associated with an Intrasubsegmental Recurrence after Radiofrequency Ablation for Hepatocellular Carcinoma

Background

In the treatment of hepatocellular carcinoma (HCC), hepatic resection has the advantage over radiofrequency ablation (RFA) in terms of systematic removal of a hepatic segment.

Methods

We enrolled 303 consecutive patients of a single naïve HCC that had been treated by RFA at The University of Tokyo Hospital from 1999 to 2004. Recurrence was categorized as either intra- or extra-subsegmental as according to the Couinaud''s segment of the original nodule. To assess the relationship between the subsegments of the original and recurrent nodules, we calculated the kappa coefficient. We assessed the risk factors for intra- and extra-subsegmental recurrence independently using univariate and multivariate Cox proportional hazard regression. We also assessed the impact of the mode of recurrence on the survival outcome.

Results

During the follow-up period, 201 patients in our cohort showed tumor recurrence distributed in a total of 340 subsegments. Recurrence was categorized as exclusively intra-subsegmental, exclusively extra-subsegmental, and simultaneously intra- and extra-subsegmental in 40 (20%), 110 (55%), and 51 (25%) patients, respectively. The kappa coefficient was measured at 0.135 (95% CI, 0.079–0.190; P<0.001). Multivariate analysis revealed that of the tumor size, AFP value and platelet count were all risk factors for both intra- and extra-subsegmental recurrence. Of the patients in whom recurrent HCC was found to be exclusively intra-subsegmental, extra-subsegmental, and simultaneously intra- and extra-subsegmental, 37 (92.5%), 99 (90.8%) and 42 (82.3%), respectively, were treated using RFA. The survival outcomes after recurrence were similar between patients with an exclusively intra- or extra-subsegmental recurrence.

Conclusions

The effectiveness of systematic subsegmentectomy may be limited in the patients with both HCC and chronic liver disease who frequently undergo multi-focal tumor recurrence.