Evaluation of quantitative trait loci affecting intramuscular fat and reproductive traits in pigs using marker-assisted introgression.

We investigated the effects of previously identified quantitative trait loci (QTL) in an experimental backcross (BC) between Chinese Meishan pigs and commercial Duroc pigs. We performed marker-assisted introgression of two QTL for intramuscular fat (IMF) content (IMF population) and three QTL for reproductive traits (reproduction population) from a donor Meishan pig into a recipient Duroc pig. At the fourth BC generation of the IMF population and third BC generation of the reproduction population, carrier animals were selected for the production of animals homozygous for the QTL. Our previous studies have shown that the presence of a Meishan allele on the IMF QTL is associated with low IMF values, and the Meishan allele on the reproductive QTL is associated with large litters. In this study, the presence of a Duroc allele at the IMF QTL on SSC9 resulted in a 0.27% increase in IMF (additive effect = 0.27 ± 0.08), whereas the presence of a Meishan allele at the IMF QTL on SSC7 resulted in a 0.34% increase in IMF (additive effect = -0.34 ± 0.09). The presence of the Meishan allele at the IMF QTL on SSC7 thus had the opposite effect to our previous studies, that is, increased IMF. In the reproduction population, we observed no differences between the genotypes of the three QTL in regard to number of corpora lutea or litter size. Marker-assisted introgression at these QTL is thus unlikely to result in an associated increase in litter size. These results show that it is possible to introgress alleles from other breeds into a selection population using molecular markers; any unexpected results might be associated with the genetic background.

Rnx deficiency results in congenital central hypoventilation.

The genes Tlx1 (Hox11), Enx (Hox11L2, Tlx-2) and Rnx (Hox11L2, Tlx-3) constitute a family of orphan homeobox genes. In situ hybridization has revealed considerable overlap in their expression within the nervous system, but Rnx is singularly expressed in the developing dorsal and ventral region of the medulla oblongata. Tlx1-deficient and Enx-deficient mice display phenotypes in tissues where the mutated gene is singularly expressed, resulting in asplenogenesis and hyperganglionic megacolon, respectively. To determine the developmental role of Rnx, we disrupted the locus in mouse embryonic stem (ES) cells. Rnx deficient mice developed to term, but all died within 24 hours after birth from a central respiratory failure. The electromyographic activity of intercostal muscles coupled with the C4 ventral root activity assessed in a medulla-spinal cord preparation revealed a high respiratory rate with short inspiratory duration and frequent apnea. Furthermore, a coordinate pattern existed between the abnormal activity of inspiratory neurons in the ventrolateral medulla and C4 motorneuron output, indicating a central respiratory defect in Rnx mice. Thus, Rnx is critical for the development of the ventral medullary respiratory centre and its deficiency results in a syndrome resembling congenital central hypoventilation.

An approach to canine behavioural genetics employing guide dogs for the blind.

The purpose of this study was to attempt to find related variables of the canine genome with behavioural traits of dogs maintained and tested in a guide dog facility which provided a relatively uniform environment. The study involved 81 Labrador Retrievers that were being trained as guide dogs. Each dog was taken on walk-out sessions in which the trainer weekly recorded observations that were related to behavioural traits. The records were subjected to key-word analysis of 14 behaviour-related words. A factor analysis on the appearance rate of the 14 key words or phrases resulted in the extraction of six factors that accounted for 67.4% of the variance. Factor 1, referred to as aggressiveness, was significantly related to the success or failure of the dog in qualifying as a guide dog, and was also related to the variable of litter identification. Factor 2, referred to as distraction, was related to the variable of trainer. Factor 3, activity level, was related to the variable of sex, and was significantly related to the polymorphisms of c.471T>C in the solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter) member 2 gene and c.216G>A in the catechol-O-methyltransferase gene. The involvement of polymorphisms c.471T>C and c.216G>A in behavioural patterns related to activity level is similar to comparable genetic studies in other mammalian species. These results contribute to a greater understanding of the role of these genes in behaviour.

Interferon-gamma downregulates Hsp27 expression and suppresses the negative regulation of cell death in oral squamous cell carcinoma lines.

Interferon-gamma (IFN-gamma) induced cell death in five oral squamous cell carcinoma (SCC) lines. Cell death was specific to IFN-gamma treatment and did not occur with either IFN-alpha or TNF-alpha. IFN-gamma did not induce typical apoptotic phenotype in cells, such as morphological changes and DNA ladder formation. Caspase-3 was partially activated by IFN-gamma. Protein levels of molecular chaperones were examined in cells treated with IFN-gamma. Among these, levels of heat shock protein 27 (Hsp27) were specifically reduced upon IFN-gamma treatment of oral SCC cells. Recombinant clones overexpressing Hsp27 were more resistant to IFN-gamma-induced cell death than parent cells. Conversely, cells expressing a dominant-negative mutant of Hsp27, in which three serine residues (15, 78 and 82) were replaced by glycine, were hypersensitive to the effects of IFN-gamma and exhibited a typical apoptotic phenotype. Pretreatment of cells with IFN-gamma enhanced apoptotic cell death induced by cisplatin. Our data suggest that IFN-gamma suppresses Hsp27 expression in oral SCC cells and blocks the inhibitory effects of this molecular chaperone on apoptotic cell death. Moreover, IFN-gamma initiates the transition of oral SCC cells to the proapoptotic and/or aborted apoptotic state. Hsp27 plays a crucial role in the inhibition of apoptosis of oral SCC cells. Our findings highlight the importance of employing IFN-gamma in combination with certain anticancer drugs as treatments for oral cancer. We suggest that Hsp27 plays a significant role in the IFN-gamma-induced sensitization of oral SCC cells to anticancer drugs.

Prostaglandin F2 alpha promotes the inhibitory action of endothelin-1 on the bovine luteal function in vitro.

Prostaglandin F2 alpha (PGF2 alpha) is a primary luteolysin in the cow. Although the mechanisms involved in luteolysis are thought to be a complex of its direct action on luteal cells and indirect effect on luteal blood flow, the detailed mechanisms remain to be elucidated. This study focuses on the possible interaction of endothelial cells-derived endothelin-1 (ET-1) with PGF2 alpha in the rapid suppression of progesterone release from the bovine corpus luteum (CL). In in vitro microdialysis system (MDS) of CL, PGF2 alpha acutely stimulated the release of progesterone and oxytocin during infusion and ET-1 release after infusion. Moreover, PGF2 alpha induced slight decrease of progesterone release during the last period of the experiment (8-11 h after PGF2 alpha exposure). Two 1 h-perfusions of ET-1 at 3 h intervals induced only a slight decrease of progesterone release after the second perfusion. This treatment also affected the oxytocin release; the first ET-1 perfusion produced an acute stimulation, whereas the second ET-1 perfusion inhibited the release to below 50%. When the CL pieces were pre-perfused with PGF2 alpha for 2 h, the two consecutive perfusion of ET-1 at 3 h intervals induced drastic decrease in progesterone and oxytocin release only after the second ET-1 perfusion. Thus, a pre-exposure with PGF2 alpha clearly potentiated the inhibiting activity of ET-1 in the progesterone release. These results suggest a physiological impact of PGF2 alpha and ET-1 in the rapid cascade of functional luteolysis in vivo, and a possible interaction between endothelial cells and luteal cells.

Isolation of 9-hydroxy-delta-tetradecalactone from lipid A of Pseudomonas diminuta and Pseudomonas vesicularis.

A lipid component was isolated from the fatty acid fraction of acid hydrolysates of lipid A derived from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 lipopolysaccharides. By structural analysis of the lipid and its trimethylsilyl and acetyl derivatives by thin-layer chromatography, gas chromatography-mass spectrometry, mass spectrometry, infrared spectrometry and 13C-NMR, it was identified as 9-hydroxy-delta-tetradecalactone.

Epitope analysis of lipid A preparations from Pseudomonas diminuta and Pseudomonas vesicularis.

In vitro antigenic reactivity of lipid A from Pseudomonas diminuta and Pseudomonas vesicularis with homologous and heterologous lipid A antibodies including monoclonal antibodies was studied by inhibition test of enzyme-linked immunosorbent assay (ELISA). The results suggest that both Pseudomonas lipid As have very similar epitopes, including species-specific and cross-reactive epitopes as compared with enterobacterial lipid A.

Tetrahydrocannabinol treatment suppresses growth restriction of Legionella pneumophila in murine macrophage cultures.

Legionella pneumophila is a facultative intracellular pathogen which readily grows in human and guinea pig macrophages and in peritoneal exudate macrophages from A/J mice. Macrophage cultures capable of supporting the growth of Legionella can be used to test the potency of biologically active substances suspected of modulating host mechanisms of resistance to infection. Accordingly, this model was used to evaluate the influence of delta-9-tetrahydro-cannabinol (THC) on macrophage resistance to infection with an intracellular pathogen. Pretreatment of the macrophages with THC in the concentration range of 2.5 micrograms/ml (8 microM) to 5.0 micrograms/ml (16 microM) had little if any effect on the ability of the macrophages to either ingest or support the replication of Legionella. However, THC treatment of cells following Legionella infection resulted in increased numbers of bacteria recoverable from the macrophage cultures. Stimulation of the macrophage cultures with the activating agent lipopolysaccharide (LPS) was effective in reducing the ability of Legionella to grow in the cells. However, treatment of the LPS activated macrophages with THC resulted in greater growth of the Legionella in the cultures, indicating that the drug abolished the LPS induced enhanced resistance. These results demonstrate that THC treatment of macrophages following infection rather than before infection with Legionella promotes the replication of the bacteria within the macrophages. In addition, drug treatment suppresses the growth restricting potential of macrophages activated by LPS.

Risk factors for recurrence of large HCC in patients treated by combined TAE and PEI.

BACKGROUND/AIMS: In this report, risk factors of intrahepatic recurrence of a large solitary hepatocellular carcinoma after combination therapy with transcatheter arterial embolization followed by percutaneous ethanol injection were studied. METHODOLOGY: The series included 61 patients with an unresectable large solitary hepatocellular carcinoma, the largest size of which was greater than 3 cm in diameter. All patients completely responded to combination therapy and recurrence rates were determined. The following parameters; age, sex, hepatitis B virus surface antigen, hepatitis C virus antibodies, Child's classification, alcohol abuse, alanine aminotransferase, aspartate aminotransferase, alpha-fetoprotein, indocyanine green retention rate, hepatocellular carcinoma size, hepatocellular carcinoma capsule, total amount of injected ethanol and the alpha-fetoprotein 1 month after treatment were evaluated. RESULTS: The 1-, 3-, and 5-year cancer-free survival rates of all patients were calculated to be 61%, 23%, and 13%, respectively. Among pretreatment parameters, the log-rank test and subsequent Cox's proportional hazards model showed that a tumor size of more than 5 cm in diameter was independently associated with recurrence. The posttreatment parameters of total amount of injected ethanol was also shown to be significantly related to recurrence by the log-rank test. CONCLUSIONS: Lesions more than 5 cm in diameter and insufficient injected ethanol were associated with intrahepatic recurrence after this combination therapy.

[SMANCS-TAE combined with PEI in the treatment for hepatocellular carcinoma].

From January 1996 to August 1997, 24 patients with advanced hepatocellular carcinoma (HCC) equal to or more than 2 cm (mean +/- SD; 4.1 +/- 3.0 cm) in main tumor diameter were treated by SMANCS-TAE (20 cases) or SMANCS-TAI (4 cases) combined with PEI. Six cases had solitary lesion, 16 cases had multiple lesions, and 2 cases had massive lesions. After this combination therapy, 21 of 24 cases had complete tumor necrosis. During 3 to 19 months follow up period, 12 cases had cancer-free survival (SMANCS-TAI; 3 cases), and 9 cases had tumor recurrences (3 cases were local recurrences and 6 cases involved new lesions). Two cases died of hepatic infarction and cancer death, however, the remaining 22 cases were surviving. SMANCS-TAE combined with PEI is useful treatment for advanced large or multiple HCC lesions in patients who are poor surgical risks.

Gauze packing as damage control for uncontrollable haemorrhage in severe thoracic trauma.

INTRODUCTION: The usefulness of thoracic damage control (DC) for trauma requiring a thoracotomy is not established. The aim of this study was to clarify the usefulness of thoracic packing as DC surgery. METHODS: This was a retrospective case series study of 12 patients with thoracic trauma suffering uncontrollable intrathoracic haemorrhage and shock who underwent intrathoracic packing. Our thoracic DC technique consisted of ligation and packing over the bleeding point or filling gauze in the bleeding spaces as well as packing for the thoracotomy wound. The success rates of intrathoracic haemostasis, changes in the circulation and the volume of discharge from the thoracic tubes were evaluated. RESULTS: Packing was undertaken for the thoracic wall in five patients, for the lung in four patients, for the vertebrae in two patients and for the descending thoracic aorta in one patient. Haemostasis was achieved successfully in seven cases. Of these, the volume of discharge from the thoracic tube exceeded 400 ml/hr within three hours after packing in three patients, decreased to less than 200 ml/hr within seven hours in six patients and decreased to 100ml/hr within eight hours in six patients. Systolic pressure could be maintained over 70 mmHg by seven hours after packing. CONCLUSIONS: Intrathoracic packing is useful for some patients, particularly in the space around the vertebrae, at the lung apex, and between the diaphragm and the thoracic wall. After packing, it is advisable to wait for three hours to see whether vital signs can be maintained and then to wait further to see if the discharge from the thoracic tube decreases to less than 200 ml/hr within five hours.

Inhibition of colony formation of NIH 3T3 cells by the expression of the small molecular weight heat shock protein HSP27: involvement of its phosphorylation and aggregation at the C-terminal region.

The ectopic expression of the small molecular weight heat shock protein HSP27 reportedly confers resistance to heat and other types of stress, but our recent findings indicated that it rendered human immortalized fibroblast cells (KMST-6) more sensitive to oxidative stress and caused irreversible growth arrest (Arata et al., 1995, J. Cell. Physiol., 163:458-465). To clarify the relationship between HSP27 and growth regulation, we investigated the effect of overexpression of HSP27 and its mutants on the growth potential of several cell lines. Mammalian expression vectors of the wild-type, hypophosphorylatable, or C-terminal deletion mutants of human HSP27 were constructed from the pRc/CMV plasmid that contained the neomycin-resistant gene. The plasmid was introduced into mouse fibroblasts (NIH 3T3), normal human fibroblasts (TIG-3), Chinese hamster ovary (CHO-K1), or mammary tumor cells (MCF-7), which were then selected in medium containing G418. The number of drug-resistant colonies was significantly decreased by transfection with the expression vector for wild-type HSP27 compared with vector alone, whereas the overexpression of HSP27 in CHO-K1 cells had essentially no effect. The expression vectors of an hypophosphorylatable mutant (pKSm, human HSP27 gene in which codons for Ser-15, -78, and -82 were converted to code for Gly by site-directed mutagenesis) as well as C-terminal deletion mutants in which 12-36 amino acid residues from the C-terminus were deleted had no significant effect on the colony-forming efficiency of NIH 3T3 cells. Cells isolated from G418-resistant colonies formed by transfection of NIH 3T3 cells with the HSP27 expression vector expressed no detectable levels of wild-type HSP27 and did not form stable clonal transformants expressing high levels of HSP27 from NIH 3T3 cells. In contrast, several clones expressing high levels of HSP27 were obtained from CHO-K1 cells transfected with the HSP27 expression vector. In KMST-6 clones expressing high levels of HSP27, the wild-type HSP27 formed aggregates with a mean molecular mass of about 200 kDa as determined by gel filtration, and the size of the oligomers changed with oxidative stress. On the other hand, the size of aggregates of HSP27 encoded by pKSm or C-terminal deletion mutants did not change. These observations indicated that the forced expression of wild-type HSP27 participates in inhibiting the growth of some cell types and that the inhibition may be associated with its phosphorylation and aggregation.

Involvement of reactive oxygen species in the induction of chemokine JE/MCP-1 gene by phorbol-12-myristate-13-acetate in Balb 3T3 cells.

The induction of JE/MCP-1 gene by TPA was transcriptionally suppressed by antioxidants such as pyrrolidine dithiocarbamate (PDTC) or trimethylthiourea (TMTU) in Balb 3T3 cells, whereas that of other early response genes, c-fos or egr-1, was not affected by these agents. Induction of the JE gene by TNF alpha or serum was not completely inhibited by these antioxidants inhibited an increase in intracellular oxidized state of cells treated with TPA. Next we examined the transcriptional regulatory region of the rat JE gene to determine the genomic target of active oxygen species. The chloramphenicol acetyltransferase (CAT) reporter gene, containing the 5'-upstream region approximately 2.6 kb DNA from the cap site, was transfected into Balb 3T3 cells. The CAT activity induced by TPA increased in parallel with the endogenous JE and mRNA level, and the increase was inhibited by the antioxidants. The essential region for this response in the upstream region was within the -2.6 to -2.0 kb region, and further defined to -2,224 to -2,069 bp which contained and NF kappa B-binding element. Gel shift analysis indicated that the nuclear factors that bound to this essential element contained NF kappa B, and that NF kappa B activity was stimulated by TPA and inhibited by PDTC. These results suggest that active oxygen species are involved in induction of the JE gene caused by TPA in Balb 3T3 cells, through NF kappa B activation.

Effects of the overexpression of the small heat shock protein, HSP27, on the sensitivity of human fibroblast cells exposed to oxidative stress.

The role of the human small heat shock protein (HSP27) in oxidative stress was examined using stable transformants of an immortalized human fibroblast cell line (KMST-6) isolated by transfection of HSP27 expression vectors. Several stable transformants that expressed high or low levels of HSP27 protein were obtained. Clones expressing high levels of HSP27 were more sensitive to growth inhibition by a low dose of hydrogen peroxide (0.1 mM) than those expressing low levels. Clones expressing high levels of HSP27 did not acquire obvious resistance to hyperthermy and cytotoxic agents, except for one (#13), in which resistance to cytotoxic agents was increased. The level of phosphorylated HSP27 in clones expressing high levels of this protein increased at 30 min and was sustained even 4 hours after exposing the cells to 0.1 mM of hydrogen peroxide. On the other hand, the levels in clones expressing low levels of HSP27 were reduced within 4 hours after exposure to hydrogen peroxide. Furthermore, overexpression of nonphosphorylatable mutant HSP27 did not affect sensitivity to oxidative stress. These results suggested that constitutively high expression of HSP27 in KMST-6 cells make them susceptible to oxidative stress resulting in growth arrest, and this mechanism could involve the phosphorylation of HSP27.

Enhanced growth of Legionella pneumophila in tetrahydrocannabinol-treated macrophages.

Legionella pneumophila is an opportunistic intracellular pathogen that infects macrophages, both in vivo and in vitro. Tetrahydrocannabinol is a major psychoactive component of marijuana and can affect the functional activity of macrophages. In the present study, it was found that the treatment of macrophage cultures from permissive A/J mice with THC enhanced the growth of Legionella in these cells. Legionella grew much better in macrophages treated with low doses of THC, which caused no alteration in the number or viability of macrophages, as compared with growth in untreated cells. Furthermore, lipopolysaccharide-treated A/J mouse macrophages restricted the growth of Legionella, but this growth restriction was overcome by the addition of THC to LPS-treated macrophage cultures after infection. Thus, it is apparent that THC has the ability to enhance the growth of the intracellular opportunistic pathogen Legionella that grows in A/J mouse macrophages.

Legionella pneumophila growth restriction in permissive macrophages cocultured with nonpermissive lipopolysaccharide-activated macrophages.

Macrophages can be activated by lipopolysaccharides (LPS) from gram-negative bacteria to evince a number of biological activities, including increased resistance to intracellular infection by opportunistic bacteria. In the present study, intraperitoneal injection of LPS into A/J mice activated peritoneal macrophages so that they resisted subsequent in vitro infection with Legionella pneumophila. Coculture of these macrophages with those from nontreated A/J mice converted the entire population of cells from permissive to nonpermissive. This effect did not appear to be mediated by soluble factors released from the LPS-treated macrophages, since the levels of interleukins-1 and -6 and tumor necrosis factor alpha produced by the macrophages were not found to be markedly elevated at the time when the macrophages from the LPS-treated mice were most effective in converting normal macrophages to nonpermissiveness. Furthermore, macrophages from mice injected intraperitoneally with either interferon or tumor necrosis factor alpha did not evince nonpermissiveness and also did not have the ability to convert normal spleen cells to nonpermissiveness. Polymyxin B, a known inactivator of LPS activity, did not inhibit the macrophages from the LPS-treated mice from inducing this resistance. It seemed unlikely that free LPS released from the macrophages mediated this effect. The results of this study thus showed that macrophages activated by LPS in vivo can evince nonpermissiveness for Legionella growth in vitro and also can induce macrophages from normal, permissive mice to become nonpermissive for Legionella growth in vitro.