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1. The effects of a surgical operation, to implant a guide cannula in the chick hypothalamus for microdialysis, on behavioural responses and neural activity in broiler chicks are described. 2. General behavioural activities (feeding, preening, sitting, drinking, cage pecking and beak wiping), open field and locomotor activity tests were conducted to evaluate the effects related to surgery in the immediate 4 d following this procedure. Perfusion of Ringer solution with high K+ after 4 d of guide cannula implantation was used to estimate the neural activity resulting from surgery through stimulation of monoamine release by in vivo brain dialysis. 3. The results of direct behavioral observations indicated that the stress provoked by surgical guide cannula insertion caused behavioural alterations that are particularly evident in the immediate days following this procedure. Open-field tests on day 4 after surgery showed that, compared to the intact control chickens, the treated chicks had a shorter latency to ambulate and defecate, with more vocalisation. Locomotor activity was less in the treated chicks than inthe controls. 4. After 4 d of guide cannula implantation, the serotonin concentration started to increase 30 min after the onset of perfusing high-K+ Ringer solution. It reached its highest value at one hour, suggesting that the 4 d after surgery is enough to alleviate some neurochemical dysfunction resulting from surgery. The results of behavioural observations, open-field and locomotor activity tests indicate that the surgical operation caused stress and fear in chicks which persisted up to 4 d. [ABSTRACT FROM AUTHOR]

It has been reported that the induction of cellular senescence through p53 activation is an effective strategy in tumor regression. Unfortunately, however, tumors including adult T-cell leukemia/lymphoma (ATL) have disadvantages such as p53 mutations and a lack of p16(INK4a) and/or p14(ARF). In this study we characterized Nutlin-3a-induced cell death in 16 leukemia/lymphoma cell lines. Eight cell lines, including six ATL-related cell lines, had wild-type p53 and Nutlin-3a-activated p53, and the cell lines underwent apoptosis or cell-cycle arrest, whereas eight cell lines with mutated p53 were resistant. Interestingly, senescence-associated-beta-galactosidase (SA-beta-gal) staining revealed that only ATL-related cell lines with wild-type p53 showed cellular senescence, although they lack both p16(INK4a) and p14(ARF). These results indicate that cellular senescence is an important event in p53-dependent cell death in ATL cells and is inducible without p16(INK4a) and p14(ARF). Furthermore, knockdown of Tp53-induced glycolysis and apoptosis regulator (TIGAR), a novel target gene of p53, by small interfering RNA(siRNA) indicated its important role in the induction of cellular senescence. As many patients with ATL carry wild-type p53, our study suggests that p53 activation by Nutlin-3a is a promising strategy in ATL. We also found synergism with a combination of Nutlin-3a and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), suggesting the application of Nutlin-3a-based therapy to be broader than expected. [ABSTRACT FROM AUTHOR]

We describe a family with progressive skeletal dysplasia and severe spinal involvement, short stature, premature arthrosis and joint contractures diagnosed as spondyloepiphyseal dysplasia Omani type. Mutation analysis in CHST3, the gene encoding for the chondroitin 6- O-sulfotransferase-1 (C6ST-1), revealed a homozygous missense mutation (T141M) in exon 3 in all three affected members of the family. Using recombinant C6ST-1, we showed that the identified missense mutation results in a reduction of C6ST-1 activity to 24–29% of the wild type protein. In addition to the previously noted skeletal features, affected members of this family also had cardiac involvement including mitral, tricuspid and/or aortic regurgitations and type E brachydactyly. [ABSTRACT FROM AUTHOR]

The K-ras oncogene is one of the most useful genetic markers in screening for the presence of cancers because it is largely involved in tumorigenesis. However, most mutation-detection techniques are generally unsuitable for routine use, especially due to their time-consuming, labor-intensive, and sensitive natures. Accordingly, we attempted to establish a new technique for analysis of K-ras alterations at codons 12 and 13 from body fluid specimens with tumor DNA using real-time polymerase chain reaction (PCR) with melting curve analysis (MCA). In this PCRMCA method, K-ras was easily genotyped by melting temperatures (Tm) that differ by 3.6 degreesC to 11.6 degreesC with an acceptable reproducibility of Tm. The shape of the melting curve gave easier interpretation. The detection sensitivity was at least 10[-[3]]. The entire process of the test was completed within 4 hours, saving 7 to 8 hours when compared to the current PCR-restriction fragment length polymorphism (RFLP) analysis. The examination of the MCA method using 38 clinical samples revealed the better detection rate (32% versus 24%) and diagnostic efficiency (100% versus 92%) compared with that of the PCR-RFLP. In conclusion, this small scale but high throughput method is acceptable for clinical use to analyze K-ras alterations from body fluids and plasma DNA, including small tumor DNA. [ABSTRACT FROM AUTHOR]

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL), and leukemic cells always carry the proviral genome monoclonally integrated into their host genomes at the same sequence site, designated as the monoclonal integration. Using Southern blot hybridization (SBH) and sequenced tagged site polymerase chain reaction assays, we examined the proviral status in 558 clinical specimens from 350 patients who are suspected to have ATL. A total of 321 specimens (57.5%) from 241 patients showed positive results for the monoclonal integration according to SBH, using EcoR1 and Pst1. The 241 patients consisted of 136 patients (56.4%) with the complete provirus (C-type), 62 patients (25.7%) with a defective provirus (D-type), and 43 patients (17.8%) with multibands (M-type). The incidence of the D- and M-types were in the order of smoldering, chronic, and acute subtypes of ATL, suggesting that such an aberrant proviral status is generated on the way to multistep carcinogenesis and is subsequently clinically important for the malignant behavior of the disease. Moreover, our data showed that the partial deletion of the proviral genome is initiated first at the site of the gag region and spreads into the sites of the pol and env regions, whereas the long terminal repeats and pX regions are almost always conserved. These results suggest that analysis of the proviral status provides useful diagnostic and virologic–oncological information about ATL and HTLV-1 pathology, especially the important role of pX gene in tumorigenesis. [ABSTRACT FROM AUTHOR]

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many transformed cells, but not in normal cells, and hence TRAIL has recently emerged as a novel anti-cancer agent. Adult T-cell leukaemia lymphoma (ATLL) is a neoplasm of T-lymphocyte origin aetiologically associated with human T-lymphotropic virus type 1 (HTLV-I), and is resistant to standard anti-cancer therapy. We thus characterized the sensitivity of ATLL cells to TRAIL in this study. Although most primary ATLL cells and cell lines expressed TRAIL death receptors on their surface, they showed only restricted sensitivity to TRAIL. Among the 10 ATLL cell lines examined, one was sensitive, but two had insufficient death-receptor expression, two had an unknown resistant mechanism with abrogation of the death signal upstream of caspase-8, and the remaining five showed attenuation of the signal in both extrinsic and intrinsic pathways by X-linked inhibitor of apoptosis and Bcl-2/Bcl-xL respectively. Furthermore, the level of HTLV-I tax expression was significantly correlated to TRAIL resistance. Interestingly, ATLL cells themselves expressed TRAIL on the cell surface. Constitutive production of TRAIL may offer resistance, thus allowing the development of TRAIL-resistant ATLL cells. Consequently, the resistant mechanism in ATLL cells against TRAIL was assigned to multiple factors and was not explained by a definitive single agent. [ABSTRACT FROM AUTHOR]

Methylthioadenosine phosphorylase (MTAP) is an important enzyme used for the salvage of adenine and methionine. Cells lacking this enzyme are expected to be sensitive to purine synthesis inhibitors and/or methionine starvation. We reported previously that the MTAP gene is deleted in adult T cell leukemia (ATL) cells. In the present study, we expanded our series and used a real-time quantitative PCR assay for accurate diagnosis of the deletion and nine of 65 primary ATL samples (13.8%) were MTAP negative. In spite of this low incidence, ATL cells showed significantly higher sensitivity to L-alanosine, an inhibitor of de novo adenosine monophosphate (AMP) synthesis, than normal lymphocytes, suggesting that the MTAP gene is inactivated not only by deletion but also by other mechanisms. Indeed, a real-time quantitative RT-PCR assay disclosed that primary ATL cells had significantly lower MTAP mRNA expression than normal lymphocytes. Since MTAPnegative ATL cell lines also showed much higher sensitivity to L-alanosine than MTAP. positive ATL cell lines, we used these cell lines to investigate whether it is possible to develop selective therapy targeting MTAP deficiency. A substrate of MTAP, methylthioadenosine (MTA) or its substitutes rescued concanavalin A (Con A)-activated normal lymphocyte proliferation from L-alanosine toxicity. All the compounds except 5'-deoxyadenosine, however, also caused the undesirable rescue of MTAPnegative ATL cell lines. 5'-Deoxyadenosine had the desired ability to rescue hematopoietic progenitor cells without rescuing ATL cell lines. These results support the rationale for a chemotherapy regimen of L-alanosine combined with 5'-deoxyadenosine rescue in MTAP-deficient ATL. [ABSTRACT FROM AUTHOR]

We investigated the effect of amrinone, a phosphodiesterase III inhibitor, on rat airway smooth muscle, and thereafter, compared its activity with aminophylline and diltiazem. Amrinone produced relaxation of the acetylcholine-induced airway contraction in a dose-related manner. This bronchodilatory activity of amrinone was similar to that of aminophylline, but smaller than that of diltiazem. The 50% relaxant effect (ED50) of amrinone, aminophylline and diltiazem were 3.6 x 10(-4) M, 1.4 x 10(-4) M and 1.4 x 10(-5) M, respectively. Diltiazem was the most potent airway relaxant, and amrinone was less potent in these experiments. Taken together in its positive inotropic and chronotropic effects and anti-inflammatory activity, however, amrinone could be beneficial for treatment of patients suffering from asthma or heart failure with cardiac asthma. [ABSTRACT FROM AUTHOR]

Hypotaurine, concentrated under reduced pressure in HCl solution, partially (30–40%) degrades into taurine (about 30%), 2-aminoethyl-2-aminoethanethiolsulfonate (about 10%) and ethanolamine. The degradation products were identified using LC/APCI-MS, NMR, amino acid analysis and various chromatographics. The identities were confirmed by comparing the HPLS, MS and NMR characteristics of authentic compounds. One of the degradation processes during concentration of HCl solution of hypotaurine is therefore a disproportionation reaction which can interfere with the experimental results, when studying hypotaurine in biological systems. [ABSTRACT FROM AUTHOR]

Although alveolar reorganization after acute lung injury depends on regeneration of alveolar epithelial cells, there is little knowledge of regulation of pulmonary healing process. Transcription factors may play key roles in this regulation. To investigate whether the CCAAT enhancer binding protein (C/EBP) family, α, β, and δ, were involved in alveolar reorganization after injury, we examined expression of C/EBP proteins and mRNAs in lung injuries induced by lipopolysaccharide (LPS) or bleomycin (Bleo) and in cell proliferation by keratinocyte growth factor (KGF). By immunohistochemistry, we demonstrated that C/EBPα and C/EBPβ were expressed in alveolar type II cells and alveolar macrophages, but C/EBPδ was expressed restrictedly in some of alveolar type II cells in a spatial pattern in the control lungs. Further, these three C/EBP family members were differentially expressed in alveolar cell proliferation and in acute lung injury, in which, interestingly, C/EBPα and C/EBPδ were reciprocally expressed in alveolar type II cell proliferation and in pulmonary fibrosis. However, expressions of their mRNAs by in situ hybridization were dramatically increased in the affected lesions of the lungs by LPS and Bleo, and Northern blot analysis showed an increased abundance of the mRNA for C/EBPβ in LPS-treated lungs and for C/EBPδ in Bleo-treated lungs, compared with those in the control lungs. Thus, differential expression of the C/EBP family may be required to maintain and reorganize the basic integrity of alveolar structure during pathological states, which suggests an important role for the C/EBP family in maintaining normal alveolar architecture and function and in repairing the damaged epithelium after injury. [ABSTRACT FROM AUTHOR]

Hydroxy acids were synthesized in good yields from ketones and fatty acids by the use of lithium naphthalene in the presence of diethylamine. For example, from cyclohexanone and undecylenic acid (I), 2-(1-hydroxy-1-cyclohexyl)-10-undecenoic acid (III) was obtained in 92% yield. Hydroxy acids were treated with p-toluenesulphonic acid to give corresponding unsaturated fatty acids. From the reaction of (III), 2-(1-cyclohexenyl)-10-undecenoic acid (IV), a 3,4-unsaturated acid was obtained (yield 59%). Corrosion and lubricity tests for these products as water-based cutting fluid additives were carried out. We have found that triethanolamine salts of 2-cyclohexenyl and 2-hydroxycyclohexyl fatty acids showed effective rust-inhibiting and antiwear properties for water-based cutting fluids. [ABSTRACT FROM AUTHOR]

Intraperitoneal administration of D,L-propargylglycine to rats resulted in an increase in the cystathionine content of whole liver and liver mitochondria. Cystathionine in mitochondria was identified by amino acid analysis, thin layer chromatography, high-voltage paper electrophoresis and liquid chromatography-mass spectrometry. The cystathionine content of whole liver was 5.37 ± 1.59 µmol per g of fresh liver at 14 h after the administration of 50 mg of D,L-propargylglycine per kg of body weight, while 0.07 ± 0.02 µmol of cystathionine per g of fresh liver was detected in the control rats. The cystathionine content of liver mitochondria from both groups of rats was 9.40 ± 1.20 and 0.19 ± 0.04 nmol of cystathionine per mg of protein, respectively. The mitochondrial cystathionine increased dose-dependently with the increase of D,L-propargylglycine administered. The increase was proportional to the time after the administration up to 12 h, and then decreased. The increase of cystathionine in the liver mitochondria was linearly proportional to that in the whole liver. These results suggest that cystathionine in liver mitochondria is in an equilibrium with that in the cytosol. [ABSTRACT FROM AUTHOR]

We have quantitated a specific apoprotein of rat pulmonary surfactant using an electroimmunoassay and have examined its content in the lungs of alloxan-induced diabetic rats. Rat lung surfactant was purified and was used to raise antisera in rabbits. Antibodies against rat serum proteins were removed from the IgG fraction of the antiserum by means of affinity chromatography using rat serum-linked to Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of rat surfactant revealed the presence of one major protein, a molecular weight of about 16,000 dalton, and this protein was a non-serum protein and specific to the lung by immunological tests. It seems likely that the 16,000 dalton protein obtained in the present study is similar to the 11,000 dalton or to a fragment of the 34,000 dalton protein, reported by other investigators. Using the monospecific antiserum against this protein of rat surfactant, the apoprotein content of pulmonary surfactant in alloxan-induced diabetic rats was measured by means of rocket electroimmunoassay. The lungs of diabetic rats contained less of this apoprotein than the lungs of control rats. This finding suggests that diabetes mellitus may induce a disturbance of protein metabolism in the lungs, as well as of carbohydrates and phospholipids, which may cause pulmonary dysfunction in diabetic patients observed in our previous studies. [ABSTRACT FROM AUTHOR]

Proliferation and differentiation of epithelial cells are thought to be regulated by soluble factors in extracellular fluid and insoluble components of the extracellular matrix. We have examined the combined effects of soluble factors and an extracellular matrix (EHS matrix) on DNA synthesis, cell proliferation, and surfactant protein gene expression in primary cultures of alveolar type II epithelial cells. Cells on EHS matrix cultured in DMEM containing insulin, cholera toxin, EGF, aFGF, 5% rat serum, and 15-fold concentrated bronchoalveolar lavage fluid (D-GM) formed larger aggregates than cells cultured on the same substratum in DMEM containing 5% rat serum (D-5). Cells cultured in D-GM on EHS matrix incorporated more [3H]-thymidine than cells on the same substratum in D-5, with an eight-fold increase seen on day 4 of culture. This increase in [3H]-thymidine incorporation was accompanied by a labeling index of greater than 65% of the cells. Cell counts showed that exposure of type II cells on EHS matrix to D-GM resulted in increased cell number on day 4 of culture. [3H]-thymidine autoradiography combined with immunostaining with anti-cytokeratin, anti-SP-A, and anti-vimentin antibodies demonstrated that the proliferating cells were epithelial cells that contained SP-A. Type II cells cultured on plastic in D-GM also showed increased [3H]-thymidine incorporation compared to cells cultured in D-5. The level of [3H]-thymidine incorporation by cells on plastic, however, was significantly less than that seen in cells cultured in the same medium on EHS matrix. Type II cells cultured on EHS matrix in D-GM had a decreased abundance of mRNAs for SP-A and SP-C than cells cultured on EHS matrix in D-5 as determined by Northern analysis. This inhibition was reversed by switching from D-GM to D-5 on day 4 and culturing the cells for an additional 4 days. In contrast, SP-B mRNA was increased in response to D-GM. This increase was not reversed by switching from D-GM to D-5 on day 4. These results suggest that the interaction of soluble factors and extracellular matrix components has a strong influence on type II cell proliferation, which were partially associated with the reversible inhibition of lung tissue-specific protein mRNAs. Their dynamic interplay among the type II cell, the extracellular matrix, and growth factors may determine multicellular functions and play an important role in normal lung development and in the repair of the lung epithelium following injury. [ABSTRACT FROM AUTHOR]

Objective: Proliferation of alveolar type II cells is thought to be critical for the restoration of lung function after diffuse alveolar damage or pneumonectomy. However, the factors that regulate alveolar type II cell proliferation, and the mechanism that brings about compensatory lung growth are not well understood. Hepatocyte growth factor (HGF) has been shown to have hepatotrophic and nephrotrophic functions for regeneration of the liver and kidney. We have attempted to investigate the involvement of HGF in lung regeneration after lung resection.Design: A prospective, controlled study.Setting: Adult surgical patients in a university hospital.Patients and Measurements: We measured serum HGF levels in eight patients undergoing major lung resection, and in five patients undergoing mastectomy as controls, by using radioimmunoassay and an enzyme-linked immunosorbent assay.Results: In all cases of pneumonectomy, the serum HGF levels increased 3- to 5-fold at 1-3 days (with a peak at 3 days) after operation, and then decreased to nearly basal levels in 2 weeks. However, these levels in patients undergoing mastectomy did not change much over the first 2 weeks, postoperatively.Conclusions: Serum HGF levels increased in patients after major lung resection, which suggests that HGF might play an important role in lung regeneration or compensatory lung growth in humans. [ABSTRACT FROM AUTHOR]

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