The human plasma glutathione peroxidase-encoding gene: organization, sequence and localization to chromosome 5q32.

A genomic clone encoding the human plasma glutathione peroxidase (PGPx), a major enzyme in reducing lipid hydroperoxide and hydrogen peroxide in plasma, was isolated and 5618 nucleotides (nt) were determined. The nt sequence data revealed that the PGPx gene is composed of five exons spanning approx. 10 kb. Primer extension experiments mapped the transcription start point at 298 nt upstream from the predicted start codon. Twenty nt upstream from the polyadenylation site of the gene, an uncanonical polyadenylation signal, AGTAAA, was found. Human PGPx was localized on chromosome 5 band q32 by fluorescence in situ hybridization.

Preferential expression of long form prolactin receptor mRNA in the rat brain during the oestrous cycle, pregnancy and lactation: hormones involved in its gene expression.

The mRNA species for prolactin receptor (PRL-R) isoforms, long and short form PRL-Rs, were estimated by the reverse transcription-polymerase chain reaction method in the rat brain (cerebrum) during the oestrous cycle, pregnancy and lactation. The levels of long form PRL-R mRNA increased at pro-oestrus and oestrus, at the same time as serum prolactin levels increased, whereas the mRNA level of short form PRL-R was relatively unchanged. Long form PRL-R mRNA expression was also markedly increased in the brain at mid- and late gestation, and this elevated mRNA level was maintained during the period of lactation. In contrast, basal levels of short form PRL-R mRNA were also maintained throughout these periods of gestation and lactation. Ovariectomy moderately reduced brain mRNA levels of both long and short form PRL-R from the levels of those in control dioestrous rats, and hypophysectomy further suppressed them to the lowest levels. Administration of oestradiol valerate (E2V) or 17 alpha-hydroxyprogesterone caproate (17OHPC) to ovariectomized rats resulted in dramatic increases in long form PRL-R mRNA levels in the brain, whereas no significant increase in short form PRL-R mRNA was observed. In rats which were ovariectomized and hypophysectomized, the administration of 17OHPC, rat prolactin or rat GH partially restored the brain level of long form PRL-R mRNA but not short form PRL-R mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

The failure of oral tolerance induction is functionally coupled to the absence of T cells in Peyer's patches under germfree conditions.

Although intestinal bacterial flora has been thought to play a role in the induction of oral tolerance, the mechanism has yet to be elucidated. We therefore examined the bacterial flora-dependent acquisition of susceptibility to oral tolerance induction using a gnotobiotic murine model. Germ-free (GF) mice exhibited a significant shortage of T cells in the PPs in comparison to SPF mice. A recovery in the number of such T cells was accomplished in the gnotobiotic mice associated with Bifidobacterium infantis or Escherichia coli but not in the gnotobiotic mice with Clostridium perfringens or Staphylococcus aureus. To examine the susceptibility to oral tolerance induction, these mice were orally given ovalbumin (OVA) as a tolerogen and then injected i.p. with the Ag. The Ag-specific IgG1 in the serum remained at a low level in both SPF and those gnotobiotic mice groups containing a sufficient number of T cells in the PPs. However, no such unresponsiveness in the Ab response was observed in GF or the other gnotobiotic mice groups containing only a few T cells in the tissues. Adoptive cell transfer analysis clearly showed that a sufficient number of T cells in the PPs is required for the induction of oral tolerance. Furthermore, the reduced expression of SLC (secondary lymphoid-tissue chemokine), which is responsible for T-cell migration to lymphoid organs, was observed in the PPs of GF mice, resulting in a shortage of T cells in the tissues. However, the reduced expression of SLC was restored even in the GF mice after conventionalization, thus suggesting that the failure of oral tolerance induction is functionally coupled to the innate absence of T cells under the GF condition.

Genotoxicity in vivo of phenazine and aminophenazines assayed in the wing spot test and the DNA-repair test with Drosophila melanogaster.

The genotoxicity and DNA-damaging activity of 6 phenazine and aminophenazine derivatives were assayed in the wing spot and DNA-repair tests in Drosophila melanogaster. Phenazine (Pz), and all aminophenazines tested, namely, 1-aminophenazine (APz), 2-APz, 2,3-diaminophenazine (DAPz), 2,7-DAPz and 2,7-diamino-3,8-dimethylphenazine (DADMPz), exhibited mutagenicity significantly in the wing spot test. The activities in the wing spot test were ranked in a sequence DADMPz > (2,7-DAPz, 2,3-DAPz) > (2-APz, 1-APz, Pz). In the DNA-repair test, 2,3-DAPz, 2,7-DAPz, and DADMPz clearly showed DNA-damaging activity, but Pz, 1-APz and 2-APz were inactive. Based on these results, we predict that DADMPz, 2,3-DAPz and 2,7-DAPz are likely to be more carcinogenic than 2-APz, 1-APz or Pz.

Pure laparoscopic hepatectomy for hepatocellular carcinoma patients with severe liver cirrhosis.

Hepatocellular carcinoma often arises in cirrhotic livers. Patients with severe liver cirrhosis who undergo hepatectomy often develop postoperative liver failure, even if the hepatectomy is limited. Here, we report six patients with severe liver cirrhosis (Child-Pugh B/C and indocyanine green retention rate at 15 min ≥ 40%) who underwent pure laparoscopic hepatectomy. Their perioperative course was favorable and comparable to that of other hepatocellular carcinoma patients with mild-moderate liver cirrhosis. In patients with severe liver cirrhosis, pure laparoscopic hepatectomy minimizes the disturbance in collateral blood and lymphatic flow caused by laparotomy and liver mobilization, as well as the mesenchymal injury caused by compression of the liver. It limits complications such as massive ascites, which can lead to severe postoperative liver failure. Good candidates for the procedure include patients with severe liver cirrhosis who have tumors on the liver surface and in whom adaptation to ablation therapy is difficult and/or who experience local recurrence after repeat treatments.

The repair of full-thickness articular cartilage defects. Immune responses to reparative tissue formed by allogeneic growth plate chondrocyte implants.

Growth plate cartilage cultivated in vitro was attached with a fibrin clot to a full-thickness articular cartilage defect on knee joints in allogeneic New Zealand rabbits. The healing of the defects was assessed by gross examination, light microscopy, and immunologic analysis for 24 weeks. Immunologic assessment of cell-mediated immunity, cytotoxicity of a humoral antibody by a 51chromium release assay, and immunofluorescence studies were carried out. During the first two weeks following grafting, healing was excellent in 11 of the 17 defects. From three to 24 weeks, 11 of 42 defects examined had good results. Host lymphocytes had accumulated around the allograft at two to 12 weeks. Most of the implanted cartilage grown in vitro died and was replaced by fibrous tissue. The immunologic studies suggested that the implanted cartilage began to degenerate two to three weeks after implantation partially because of a humoral immune response but more importantly because of cell-mediated cytotoxicity.

Cryopreservation of cartilage.

We have investigated the viability and function of cells in cartilage slices after various methods of preservation, and have examined the viability of cells by measuring the incorporation of Na2(35)SO4 at different concentrations, temperatures and times of exposure to cryopreservatives. We have assessed the viability of cells when exposed to pre-freezing temperatures, and after preservation at -80 degrees C. The best survival rate was found to be with a concentration of cryopreservatives of approximately 10%, with pre-freeze exposure for four hours at 4 degrees C. In the stage cooling technique, the best initial cooling was at -30 degrees C for 30 minutes, followed by rapid cooling of the cartilage to -80 degrees C. The best survival rate for cryopreserved cartilage in 10% DMSO was, on average, 19% in intact slices and 34% when holes had been made in the slices. Proteoglycan synthesis after thawing appeared normal, and proteoglycan labelled after 48 hours in culture also showed a normal pattern.

Treatment of complete acromioclavicular separation by coracoacromial ligament transfer.

There is still discussion concerning the methods for treating Type 3 dislocations of the acromioclavicular joints. Since 1969 the authors have treated 41 patients with Type 3 dislocations by transfer of the coracoacromial ligament, with favorable results. The advantages of the method are as follows: (1) The replaced coracoacromial ligaments substitute for the strong coracoclavicular ligaments, reproducing a construction that closely resembles a physiologically and anatomically normal state. (2) There is no fear of rupture if transplanted tendon or wire is used. A concentrated force will not occur in this system because of dispersion in the tendon, and no injury is produced in the clavicle. (3) The operation can be performed under the same visual field, and the technique is relatively simple.

Pathomechanism of atlantoaxial rotatory fixation in children.

The pathomechanism of atlantoaxial rotatory fixation (AARF) was studied using roentgenography and computerized tomography in affected children as well as cadaver studies of upper cervical spine anatomy. The increased incidence of AARF in children may be related to certain anatomical differences between children and adults. The dens-facet angle of the axis was steeper in children than in adults. Meniscus-like synovial folds were found in the C0/1 and C1/2 facet joints of the spines of children but not in those of adults.

Growth plate reconstruction using chondrocyte allograft transplants.

There is, as yet, no successful replacement for a completely damaged physis. In a test of the usefulness of allograft replacement of the plate, growth plate chondrocytes were transplanted to a defect in the proximal tibial growth plate. The defect remained after resection of a bony bridge that was created in rabbits. Seven of 10 rabbits at 3 weeks and six of 8 rabbits at 6 weeks after transplantation showed resumption of growth and prevented progression of the tibial deformity. Killed epiphyseal cells, on the other hand, were rapidly replaced by bone. It is not clear whether the transplanted live cells actually grew or served as a living, but inert filler.

In vivo degradation systems of the epiphyseal cartilage.

Accumulated evidence suggests that in the growth cartilage during endochondral ossification, proteoglycans in the extracellular matrix of the lower hypertrophic zone are degraded by proteases and removed before mineralization. Neutral protease activity in the human growth cartilage was demonstrated recently, with the highest levels in the hypertrophic and calcified zones. In this study, in an in vivo model, proteoglycans in the epiphyseal cartilage are cleaved at specific sites in the protein core by enzymes acting similar to neutral metallo-proteases. Prelabeled growth cartilage (35S) chondrocytes, grown in vitro, were transplanted as an allograft to fill a defect in the proximal tibial plate of immature New Zealand white rabbits and then extracted at different time intervals. Degradation of these proteoglycans was determined by gel chromatography on Sepharose 2B columns under associative and dissociative condition. This was compared to in vivo degradation patterns of flash-labeled native growth cartilage proteoglycan. Both the in vivo labeled material and the in vitro labeled transplants appear to be degraded into smaller fragments over time. This study provides further proof that the degradation of proteoglycans does occur in vivo and that this process is an important element in the preparation of bone for mineralization. Control of degradation may alter growth processes.

Inhibitory effect of TRK-530 on inflammatory cytokines in bone marrow of rats with adjuvant arthritis.

TRK-530 is a novel synthetic bisphosphonate compound which exhibits inhibitory activity in the rat adjuvant arthritis (AA) model. We found that, during AA development, the concentrations of cytokine-induced neutrophil chemoattractant-1 (CINC-1) and tumor necrosis factor alpha (TNF-alpha) in the bone marrow increased, and that administration of TRK-530 decreased the concentrations of these cytokines. The suppression of these concentration increases paralleled the inhibition of paw edema. Paw edema inhibition by TRK-530 in rat AA may be the result of decreasing CINC-1 and TNF-alpha concentrations.

[Floating knee fracture (ipsilateral fracture of the femur and tibia)--treatment by closed Ender nailing].

Fourteen cases of ipsilateral fractures of the femur and tibia (floating knee fracture) were reviewed and patients were graded according to the type of fracture and the method of treatment. Using the criteria for assessment described by Karlström and Olerud, patients were graded as excellent, good, fair and poor. Excellent results were achieved in five patients, good in three and fair in six. When the fracture extended into the knee, it caused knee problem in three out of five. In ten cases the femoral shaft fractures were nailed and in five both bones were nailed. Nailing of both femur and tibia gave good or excellent result in all five cases. As the comminution is often severe in the cases of floating knee fracture, intramedullary nailing by Küntscher may be difficult. We have introduced a closed Ender nailing for femoral and tibial shaft fractures and we have found that this method is also useful for the floating knee fracture. It is advantageous for this type of fracture, because it is technically simple, it has wide indications and it results in rapid bone union without knee disturbance.

TRK-530 inhibits accumulation of superoxide anions derived from human polymorphonuclear leukocytes and bone resorption induced by activated osteoclasts.

TRK-530, a newly synthesized bisphosphonate, was assessed for its effects on the accumulation of superoxide anions derived from human formyl-methionyl-leucyl-phenylalanine-stimulated polymorphonuclear leukocytes (PMN), and for its effects on bone resorption using a pit formation assay. TRK-530 concentration-dependently inhibited superoxide accumulation derived from PMN and osteoclast pit formation stimulated by 1,25-dihydroxyvitamin D3. Incadronate and risedronate had a strong inhibitory effect on pit formation, but no antioxidative activity. These data suggest that the anti-bone resorption activities of TRK-530 are possibly unrelated to its antioxidant properties. However, it is difficult to conclude at present which mechanisms play the most important role in the anti-bone resorption activities of TRK-530.

Inhibitory effects of TRK-530 on rat adjuvant arthritis.

TRK-530 is a novel bisphosphonate derivative. We examined the anti-inflammatory effects of TRK-530 on adjuvant arthritis (AA) rats. When TRK-530 at a dose of 2.5 mg/kg was administered for 2 weeks to AA rats, it inhibited destructive changes in arthritic joints such as paw edema, bone loss and joint degeneration. TRK-530 also inhibited splenomegaly and suppressed the increase in serum sialic acid which is measured as a systemic parameter of inflammation. To clarify the inhibitory mechanism of TRK-530, interleukin-1 (IL-1)-like activities of resident peritoneal macrophages in AA rats given TRK-530 were compared with those of control rats. We found that TRK-530 inhibited IL-1-like activity induced by bacterial lipopolysaccharide 6 weeks after administration when the IL-1-like activities of control rats were still at high levels. These findings suggest that TRK-530 exerts anti-inflammatory activities in AA rats.

A tactile sensor for laparoscopic cholecystectomy.

During laparoscopic surgery, surgeons observe the three-dimensional abdominal cavity on a two-dimensional TV monitor, which is a limitation. Another limitation is that surgeons are unable to estimate the softness of organs or tissues during laparoscopic surgery as they are only allowed to use instruments which touch objects and direct palpation is not permitted during the procedure. The tactile sensor which we used displays the object softness immediately as a digital score, which can then be superimposed on a TV monitor as a graph. With the tactile sensor, we were able to ascertain the presence of a gallstone in the gallbladder or cholecystic duct during laparoscopic cholecystectomy and also able to discriminate between a stone and an air bubble during intraoperative cholangiography. We were convinced that the tactile sensor would be useful in laparoscopic surgery, which does not permit surgeons to palpate objects with human fingers.

Role of nitric oxide synthase type 2 in acute infection with murine cytomegalovirus.

Whether or not NO plays a critical role in murine CMV (MCMV) infection has yet to be elucidated. In this study, we examined the role of NO in acute infection with MCMV using NO synthase type 2 (NOS2)-deficient mice. NOS2(-/-) mice were more susceptible to lethal infection with MCMV than NOS2(+/+) mice and generated a much higher peak virus titer in the salivary gland after acute infection. A moderate increase in the MCMV titer was also observed in other organs of NOS2(-/-) mice such as the spleen, lung, and liver. The immune responses to MCMV infection including NK cell cytotoxicity and CTL response in NOS2(-/-) mice were comparable with those of NOS2(+/+) mice. Moreover, the ability to produce IFN-gamma is not impaired in NOS2(-/-) mice after MCMV infection. The peritoneal macrophages from NOS2(-/-) mice, however, exhibited a lower antiviral activity than those from NOS2(+/+) mice, resulting in an enhanced viral replication in macrophages themselves. Treatment of these cells from NOS2(+/+) mice with a selective NOS2 inhibitor decreased the antiviral activity to a level below that obtained with NOS2(-/-) mice. In addition, the absence of NOS2 and NOS2-mediated antiviral activity of macrophages resulted in not only an enhanced MCMV replication and a high mortality but also a consequent risk of the latency. It was thus concluded that the NOS2-mediated antiviral activity of macrophages via NO plays a protective role against MCMV infection at an early and late stage of the infection.