Mycobacterial hypersensitivity pneumonitis requires TLR9-MyD88 in lung CD11b+ CD11c+ cells.

Mycobacteria are among the most common causes of hypersensitivity pneumonitis (HP), but controversy persists with regard to the involvement of the infectious potency of the organism in mycobacterial HP (hot tub lung). This study aimed to establish a mouse model of hot tub lung to clarify its pathophysiology. Mice were exposed intranasally to formalin-killed Mycobacterium avium from a patient with hot tub lung (HP strain) or chronic pulmonary infection (non-HP strain), and bronchoalveolar lavage fluids and lung tissues were evaluated for allergic inflammation. Dead M. avium HP strain, but not non-HP strain, elicited marked HP-like pulmonary inflammation in wild-type mice. Although the inflammation was induced in mice lacking CD4 or CD8, the induction of HP-like responses was prevented in mice lacking myeloid differentiation factor (MyD)88 or Toll-like receptor (TLR)9. Cultured lung CD11c+ cells responded to M. avium in a TLR9-dependent manner, and reconstitution of TLR9-/- mice with lung CD11c+ cells from wild-type mice restored the inflammatory responses. Further investigation revealed that pulmonary exposure to M. avium HP strain increased the number of lung CD11b+ CD11c+ cells (dendritic cells) through TLR9 signalling. Our results provide evidence that hot tub lung develops via the mycobacterial engagement of TLR9-MyD88 signalling in lung CD11b+ dendritic cells independent of the mycobacterial infectious capacity.

Association between mycobacterial genotypes and disease progression in Mycobacterium avium pulmonary infection.

BACKGROUND: Non-tuberculous mycobacterial lung disease, most commonly caused by Mycobacterium avium infection, tends to show variable disease progression, and significant disease predictors have not been adequately established. METHODS: Variable numbers of tandem repeats (VNTR) were evaluated in 16 mycobacterial interspersed repetitive unit (MIRU) loci from M avium isolates cultured from respiratory specimens obtained from 2005 to 2007. Specifically, the association between VNTR profiles and disease progression was assessed. RESULTS: Among the 37 subjects who provided positive respiratory cultures for M avium during the 2005-6 period, 15 subjects were treated within 10 months following a microbiological diagnosis of progressive M avium lung disease. Nine subjects underwent long-term follow-up (>24 months) without treatment for stable M avium lung disease. Based on a neighbour-joining cluster analysis used to classify M avium-positive subjects according to the VNTR profile, subjects with progressive versus stable lung disease were found to be grouped together in distinct clusters. Further analysis using logistic regression modelling showed that disease progression was significantly associated with the genetic distance of the M avium isolate from an appropriately selected reference (age-adjusted odds ratio 1.95; 95% confidence interval 1.16 to 3.30; p = 0.01 for the most significant model). A best-fit model could be used to predict the progression of M avium lung disease when subjects from the 2005-6 period were combined with those from 2007 (p = 0.003). CONCLUSION: Progressive lung disease due to M avium infection is associated with specific VNTR genotypes of M avium.

PGE2 signal via EP2 receptors evoked by a selective agonist enhances regeneration of injured articular cartilage.

OBJECTIVE: The effect of the prostaglandin E2 (PGE2) signal through prostaglandin E receptor 2 (EP2) receptors on the repair of injured articular cartilage was investigated using a selective agonist for EP2. METHODS: Chondral and osteochondral defects were prepared on the rabbit femoral concave in both knee joints, and gelatin containing polylactic-co-glycolic acid microspheres conjugated with or without the EP2 agonist was placed nearby. Animals were sacrificed at 4 or 12 weeks post-operation, and regenerated cartilage tissues and subchondral structure remodeling were evaluated by histological scoring. The quality of regenerated tissues was also evaluated by the immunohistochemical staining of EP2, type II collagen, and proliferating cell nuclear antigen (PCNA). As an evaluation of side effects, the inflammatory reaction of the synovial membrane was analyzed based on histology and the mRNA expression of matrix metalloproteinase3 (MMP3), tissue inhibitor of metalloproteinase 3 (TIMP3), and interleukin-1 beta (IL-1 beta). Also, the activity of MMP3 and the amount of tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein in joint fluid were measured. RESULTS: In both models, the EP2 agonist enhanced the regeneration of the type II collagen-positive tissues containing EP2- and PCNA-positive chondrocytes, and the histological scale of regenerated tissue and subchondral bone was better than that of on the control side, particularly at 12 weeks post-operation. No inflammatory reaction in the synovial membrane was observed, and no induction of pro-inflammatory cytokines was found in joint fluid. CONCLUSION: Selective stimulation of the PGE2 signal through EP2 receptors by a specific agonist promoted regeneration of cartilage tissues with a physiological osteochondral boundary, suggesting the potential usefulness of this small molecule for the treatment of injured articular cartilages.

Porphyromonas gingivalis mutant defective in a putative extracytoplasmic function sigma factor shows a mutator phenotype.

INTRODUCTION: Porphyromonas gingivalis is implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis must possess the ability to tolerate stress signals outside the cytoplasmic membrane by transcriptional activation of genes encoding proteins involved in defense or repair processes. Some bacteria utilize a distinct subfamily of sigma factors to regulate extracytoplasmic function (hence termed the ECF subfamily). METHODS: To elucidate their role in P. gingivalis, a chromosomal mutant carrying a disruption of an ECF sigma factor PG1318-encoding gene was constructed. Hemagglutination and proteolytic activities were measured in the PG1318-defective mutant. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and southern blot analysis were used to assess transcription of kgp in the PG1318-defective mutant. Frequency of spontaneous mutation that conferred resistance to l-trifluoromethionine was measured in the PG1318-defective mutant. RESULTS: The PG1318-defective mutant formed non-pigmented colonies on blood agar plates at a relatively high frequency. Arginine-specific and lysine-specific proteinase activities of the non-pigmented variants were remarkably decreased compared with those of the parent strain and the pigmented variants. RT-PCR analysis showed that kgp was not transcribed in some non-pigmented variants and southern blot analysis revealed that there was a deletion in their kgp region. Frequency of mutation conferring resistance to l-trifluoromethionine was significantly higher in the PG1318-defective mutant than in the wild-type. CONCLUSION: These results suggest that PG1318 plays a role in the regulation of mutation frequency in the bacterium.

[Lymphangiomyomatosis pathologically diagnosed by surgery for pneumothorax].

We report a case of incipient pulmonary lymphangioleiomyomatosis (LAM) diagnosed by histopathological examination of excised lung. A 28-year-old woman was referred to our hospital because of recurrent left pneumothorax. Computed tomography showed no abnormality except for small bullae in the right middle lobe. She underwent video-assisted thoracoscopic surgery and we excised the apex of the left lung showing hypertrophic pleura. Microscopic examinations of the surgical specimen revealed multiple focal accumulations of small spindle-shaped cells stained positively with anti-HMB-45 antibody, specific for LAM. Furthermore, among these multiple lesions, vascular invasion of HMB-45 positive cells were observed, which demonstrates invasive and metastatic potential of LAM cells as previously reported. This case implicates a need for a careful pathological examination of excised specimens in female cases of surgically treated pneumothorax even though pre-operation or macroscopic examination shows no specific findings.

Regulation of taste-active components of meat by dietary branched-chain amino acids; effects of branched-chain amino acid antagonism.

1. The effects of dietary branched-chain amino acids (BCAAs) including leucine (Leu), isoleucine (Ile) and valine (Val) on taste-active components, especially free glutamate (Glu), in meat were investigated. 2. Broiler chickens (28 d old) were given varied dietary BCAA levels for 10 d before marketing. Dietary BCAA content ratios were either 100:100:100 (Low Leu group), 150:100:100 (Control group) or 150:150:150 (High Ile + Val group) for Leu:Ile:Val (% of each BCAA requirement according to NRC, 1994). Taste-related components of meat (free amino acids and ATP metabolites) and sensory scores of meat soup were estimated. 3. Free Glu content, the main taste-active component of meat, was significantly increased by dietary BCAA. Compared to the Control group, free Glu content increased by 30% in the High Ile + Val group. However, the inosine monophosphate (IMP) content in meat did not change among groups. 4. Sensory evaluation of meat soups showed that Control and High Ile + Val groups had different meat flavours. The sensory score of overall taste intensity was significantly higher in the High Ile + Val group. 5. These results suggest that dietary BCAA concentrations regulate free Glu in meat. Increasing dietary Ile + Val induces an increase in free Glu content of meat, improves meat taste and is more effective for increasing free Glu content in meat than decreasing dietary Leu level.

Origin of increased deoxycytidine excretion into urine of rats bearing Yoshida ascites sarcoma.

The metabolism of deoxycytidine (dCyd) and dCyd nucleotides in Yoshida ascites sarcoma (YS) cells and the host rat liver was investigated with reference to the increased excretion of urinary dCyd. Incorporation of [14C]orotic acid into the livers of rats at the fifth day after the transplantation of YS cells, the time when the amount of excretion of dCyd in urine was near maximal, was 2 times higher than that into the normal rat livers. After the injection of [14C]orotic acid, the ratio of the specific radioactivity of cytidylate to uridylate moieties of the host liver RNA was measured and found to be higher than that of normal rat liver RNA and to be similar to that of YS cell RNA. When [14C]orotic acid was injected into rats followed by the transplantation of YS cells, the radioactivities present in the livers disappeared more rapidly than those in the control rat livers. The activities of pyrimidine de novo synthesis enzymes, such as cytidine triphosphate synthetase (EC 6.3.4.2) and cytidine diphosphate reductase (EC 1.17.4.1), in YS were higher than those in both rat ascites hepatoma AH 7974 and Walker 256 carcinosarcoma, the transplantations of which did not induce increased excretion of dCyd into urine of the hosts. The activities of dCyd kinase (EC 2.7.1.10) and dCyd deaminase (EC 3.5.4.5) in YS cells were lower than those in the other two tumors investigated. The activities of cytidine triphosphate synthetase and cytidine diphosphate reductase in the livers of YS-bearing rats were elevated compared with those in the livers of rat ascites hepatoma AH 7974- or Walker 256 carcinosarcoma-bearing rats and normal rats, while the activities of dCyd kinase, 5'-nucleotidase (EC 3.1.3.5), and dCyd deaminase were similar between normal rat livers and tumor-bearing rat livers. These results suggest that the increased excretion of urinary dCyd in YS-bearing rats could be caused by both the stimulation of the synthesis of dCyd nucleotides and the low activity of dCyd deaminase in YS cells as well as in the host liver.

Characterization of messenger RNA for fructose 1,6-bisphosphate aldolase A isozyme of rat ascites hepatoma AH 7974 cells.

The messenger activity for fructose 1,6-bisphosphate aldolase (EC4.1.2.13) (aldolase) A isozyme has been characterized in the polysome- or the messenger RNA-directed, protein-synthesizing system using the pH 5 fraction of rat liver or wheat germ extracts, respectively. The subunit of aldolase A synthesized in vitro was detected by immunoprecipitation with anti-aldolase A antibody raised in chickens followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The synthesis of the enzyme depended on the addition of polysomes or polyadenylate-containing RNA of rat ascites hepatoma AH 7974 cells which show a complete shift of aldolase isozyme to type A, whereas polysomes of adult rat liver were inactive. The messenger activity for aldolase A was present exclusively on free polysomes but absent on membrane-bound polysomes and in the soluble supernatant fraction of AH 7974 cells. The size of aldolase A messenger RNA determined by formamide-containing sucrose density gradient centrifugation was approximately 5.8 X 10(5) daltons corresponding to 1650 nucleotides. Taking into account the number of amino acid residues in the aldolase A subunit, approximately 400 nucleotides correspond to the noncoding region of aldolase A messenger RNA.

Morphological differentiation of cultured mouse glioblastoma cells induced by dibutyryl cyclic adenosine monophosphate.

A culture line of mouse glioblastoma cells changed morphologically to differentiated astrocyte-like cells when cultured in medium with dibutyryl cyclic adenosine monophosphate and theophylline. Morphological alteration occurred within only 5 hr when 3 mM dibutyryl cyclic adenosine monophosphate and 1 mM theophylline were used, and in 1 day when 1 mM theophylline were used. Cells showing this morphological change reverted completely to immature cells when they were transferred to medium without these two chemicals. Addition of 1 or 3 mM dibutyryl cyclic guanosine monophosphate with 1 mM theophylline to the medium also induced development of cytoplasmic processes from these cells and the cells became stellate, although the cytoplasmic processes were not as long or as numerous as those induced by dibutyryl cyclic adenosine monophosphate, and the altered cells could not be referred to as differentiated glia cells. Sodium butyrate induced morphological alterations similar to those induced by dibutyryl cyclic guanosine monophosphate, but fewer cells showed these alterations. Addition of cyclic adenosine monophosphate or cyclic guanosine monophosphate in the presence of theophylline or addition of theophylline alone did not induce morphological changes of the cells.

Experimental model for combination chemotherapy with metronidazole using human uterine cervical carcinomas transplanted into nude mice.

Human uterine cervical carcinomas (Yumoto strain) and HeLa cell tumors were transplanted into nude mice, and their transplantable strains were established. The fundamental histological features of these tumors were analyzed according to their histological construction and cytological maturation. The effect of administered drugs was examined morphologically. The Yumoto strain is a well-differentiated epidermoid-type carcinoma consisting of regularly arranged basal-type, parabasal-type, and keratinizing-type cells. The HeLa cell tumor is made up of solid medullary carcinoma cell nests in which trabecular arrangements begin to appear around the medullary areas after the third passage. This feature is maintained up to the 17th generation. The basal layer-type cancer cells of the Yumoto strain as well as trabecularly arranged cancer cells in the HeLa cell tumor were selectively influenced by administration of bleomycin and/or mitomycin and showed considerable degeneration or complete disappearance. On the contrary, metronidazole (a drug for vaginal trichomoniasis; Flagyl) displayed a cytotoxic effect on the parabasal-type and/or more mature cancer cells of the Yumoto strain as well as on the solid medullary area of the HeLa cell tumor. This result may indicate a selective affinity of drugs for malignant cells according to their histological construction, and it is conceivable that these types of carcinoma can be affected by combination administration of metronidazole and oncostatic chemicals such as bleomycin and mitomycin. This speculation was realized in this experimental animal research.

Activity of the cytosolic isozyme of thymidine kinase in human primary lung tumors with reference to malignancy.

Activity increase of the cytosolic isozyme of thymidine kinase (TK) in resected specimens of lung tumor patients would be a useful marker for tumor malignancy and prognosis. In 24 resected cases of malignant lung tumors, the whole enzyme extracts of the tumorous part of the specimens showed that the activities of TK, thymidylate synthetase, and ribonucleotide reductase increased at an average of 469 (P less than 0.001), 208 (not significant), and 193% (P less than 0.02) of the corresponding enzymes in the tumor-uninvolved lung parts, respectively. Two TK isozymes, cytosolic and mitochondrial TKs, were separated better by means of p-aminophenyl 3'-TMP:CH-Sepharose gel affinity column chromatography for precise quantitation of the activity than by polyacrylamide disc gel electrophoresis. These separated isozymes from the tumorous part of the specimens were characteristically very similar to the isozymes of cytosolic and mitochondrial fractions of the xenograft (CPX-101) of human lung tumor transplanted in athymic nude mice, respectively. The cytosolic isozyme activity isolated by this method from the tumorous part was remarkably higher and more varied than that of the tumor-uninvolved part, while that of the mitochondrial isozyme was lower and less agitated. The tumor doubling time showed a good inverse correlation to the activity of the cytosolic isozyme of TK when compared logarithmically (r = -0.798, P less than 0.01). Poorly differentiated tumors exhibited significantly higher activities of the TK cytosolic isozyme than did well-to-moderately differentiated tumors (766.0 +/- 379.1 and 308.1 +/- 119.5 pmol/mg of protein/h, mean +/- SE, respectively), a phenomenon also seen in the activities of the tumors with versus without recurrences within 12 mo after resection (803.6 +/- 278.7 and 124.1 +/- 42.1 pmol/mg of protein/h, respectively). The levels of these relationships using the cytosolic TK activity provided a clearer indication of prognosis and the state of the malignancy than those using the whole extract TK activity.

Heterogeneous nuclear ribonucleoprotein B1 as early cancer biomarker for occult cancer of human lungs and bronchial dysplasia.

Heterogeneous nuclear ribonucleoprotein (hnRNP) B1 is a RNA-binding protein of Mr 37,000. We previously reported that hnRNP B1 was specifically overexpressed in the nuclei of human lung cancer cells, particularly in squamous cell carcinoma (E. Sueoka et al., Cancer Res., 59: 1404-1407, 1999). We extended this study to determine whether hnRNP BL was overexpressed in roentgenographically occult cancers of the lungs and premalignant lesions of squamous cell carcinomas, such as bronchial dysplasia. The additional object of our study was to examine the usefulness of hnRNP B1 as a potential diagnostic marker for squamous cell carcinoma of various organs, such as the oral cavity and esophagus in humans. Surgically resected specimens of bronchial dysplasia, lung cancers, and various human squamous cell carcinomas, collected at two hospitals in Japan, were subjected to immunohistochemical staining with anti-hnRNP B1 antibody. Overexpression of hnRNP B1 protein was observed in 100% of stage I lung cancer tissues, but it was not found in normal bronchial epithelium. Squamous cell carcinoma of the lungs showed stronger staining than other histological types, and elevation of hnRNP B1 was found in both roentgenographically occult lung cancers and bronchial dysplasia. Furthermore, cytological examination with anti-hnRNP B1 antibody detected cancer cells in sputum, suggesting the potential of hnRNP B1 protein as a new biomarker for the very early stage of lung cancer in humans. Because strong staining of hnRNP B1 was also observed in various squamous cell carcinomas of oral and esophageal tissues as shown in our recent reports, overexpression of hnRNP B1 seems to be a common event in the carcinogenic processes of squamous cell carcinoma. These results suggest that hnRNP B1 protein could be a useful diagnostic biomarker for both the very early stages of lung cancer and various squamous cell carcinomas in humans.

In vitro synthesis of beta- and gamma-isoactins by messenger RNA of rat ascites hepatoma.

Messenger RNA was extracted from polysomes of rat ascites hepatoma, AH 7974, which contains both beta- and gamma-actins, and was translated in nuclease-treated reticulocyte lysate. The isoactins, beta and gamma, synthesized in vitro were characterized by (1) a high affinity to DNAase I-agarose; (2) polymerization with actin purified from bovine brain; (3) coelectrophoresis with bovine brain actin on two-dimensional gel, and (4) peptide mapping of each isoactin by partial digestion with papain (EC 3.4.22.2) followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum conditions with respect to the concentration of RNA, Mg2+, and K+ for the synthesis of beta- and gamma-isoactins were identical: 300 micrograms/ml, 1.5 mM, and 100 mM, respectively. Aurintricarboxylic acid and 7-methyl-GMP (7MeGMP) inhibited the synthesis of beta- and gamma-actins to the same extent. These results strongly suggest that there is very little possibility of differential translational control of each isoactin gene. When polysomal RNA was separated by sucrose gradient centrifugation, both beta- and gamma-actin mRNAs appeared as a sharp peak at the region slightly heavier than 18 S RNA.

Modification of rat liver chromatin by N-methyl-N'-nitro-N-nitrosoguanidine or N-ethyl-N'-nitro-N-nitrosoguanidine and template activity for RNA synthesis by Escherichia coli RNA polymerase after reconstitution.

Rat liver chromatin was fractionated into DNA, histones and non-histone chromosomal proteins and each component was modified with N-methyl-l-N'-nitro-N-nitrosoguanidine of N-ethyl-N'-nitrosoguanidine. The radioactivity of 14C-labeled alkyl or guanidino moieties of both compounds bound significantly to both histones and non-histone chromosomal proteins and the binding of N-methyl-N'-nitro-N-nitrosoguanidine was higher than N-ethyl-N'-nitro-N-nitrosoguanidine. However the binding of both compounds to DNA was very low and its significance was hard to evaluate. All of the three components, one of which was modified, were reconstituted into chromatin, then, [3H]UMP incorporation into acid insoluble material using Escherichia coli RNA polymerase (EC 2.7.7.6) was measured. Only with the reconstituted chromatin containing histones modified either by N-methyl-N'-nitro-N-nitrosoguanidine or N-ethyl-N'-nitro-N-nitrosoguanidine, the template activity increased drastically; i.e., about 10 or 5 times higher than that with the unmodified reconstituted chromatin, respectively. However, any remarkable alteration in the electrophoretic pattern of protein fraction of the reconstituted chromatin could not be found. The results obtained in this study are discussed in the context that the modified histones could give rise to change in the mutual interaction of chromosomal components during the reconstitution of chromatin accompanied with the increase of chromatine template activity.

Purification and characterization of a non-hemorrhagic metalloprotease from Agkistrodon halys brevicaudus venom.

A non-hemorrhagic metalloprotease (protease L4) was purified from the venom of Chinese Mamushi (Agkistrodon halys brevicaudus) by gel filtration and anion-exchange chromatography. Protease L4 has the molecular weight of 22,000 and its optimum pH was 8.5. The protein was stable in the pH range of 5-9 and below 40 degrees C. The proteolytic activity was inhibited by metal-chelating agents and some metal ions. Calcium ion activated the activity dose-dependently, but had only a minor effect on the thermal and pH stability. L4 showed fibrinogenase activity, hydrolyzing only the A alpha chain of fibrinogen. The protease cleaved preferentially at the N-terminal of Leu and His residues of some peptides.

Co-purification of thymidylate kinase and cytosolic thymidine kinase from human term placenta by affinity chromatography.

It was revealed that thymidylate kinase was purified together with cytosolic thymidine kinase from human term placenta by p-aminophenyl thymidine-3'-phosphate-CH-Sepharose affinity column chromatography, which has been commonly used for purification of thymidine kinase. In addition, it was noted that mitochondrial thymidine kinase and nucleoside diphosphate kinase were concurrently eliminated. In the presence of ATP, cytosolic thymidine kinase and thymidylate kinase could be separated from each other by Ultrogel AcA 34 filtration, and their molecular weights were estimated to be 70,000 and 50,000, respectively. On SDS-polyacrylamide gel electrophoresis, thymidine kinase protein exhibited a band of 26,000, which was compatible with the molecular weight of the enzyme subunit calculated from its cDNA, while thymidylate kinase protein showed 24,000. Thymidylate kinase could utilize either ATP or dATP as an efficient phosphate donor, and showed substrate specificity for dTMP.

Prognostic value of nucleolar protein p120 in patients with resected lung adenocarcinoma.

PURPOSE: In this study we investigated the prognostic significance of proliferation-associated nucleolar protein p120 in primary resected lung adenocarcinoma because it reflects tumor growth fractions in vitro. PATIENTS AND METHODS: Expression levels of p120 in tumors were assessed by immunohistochemistry in 74 patients who underwent radical resection. With clinical follow-up data, the prognostic significance of p120 calculated by labeling indices was evaluated using the Cox proportional hazards model. RESULTS: p120 protein was clearly detected in nucleoli of adenocarcinoma cells. Its expression levels widely varied in each sample from 8.5% to 67. 2%, with a mean +/- SD of 35.2% +/- 15.1%. No significant correlation was found between expression levels of p120 and clinicopathologic factors. However, the expression levels of p120 were negatively correlated with the tumor doubling time calculated with retrospective chest roentgenograms. Using a cutoff value of 35% in the labeling index of p120, patients with high expression of p120 experienced early recurrence and shorter survival compared with those who had low expression of p120. Multivariate analysis showed that p120 served as an independent, as well as the strongest, prognostic factor for resected lung adenocarcinoma. CONCLUSION: This report provides the first evidence that expression levels of p120 in tumor tissues can be used as an independent and powerful prognostic marker for resected lung adenocarcinoma.

Expression of nucleolar protein p120 in human lung cancer: difference in histological types as a marker for proliferation.

The function of proliferation-associated nucleolar protein p120 is unclear. A recent report that a yeast protein, NOP2, 67% homologous to human p120, is up-regulated during the onset of growth and influences the morphology of the nucleolus supports the notion that this protein could serve as a marker for proliferation in neoplastic cells. Lung cancer is characteristic in that different histological types show different biological features. We attempted to evaluate the levels of p120 expression in resected human lung cancer tissues of different histological types and the relation of p120 expression and cell proliferation using human lung cancer cell lines. When 37 frozen specimens of human lung cancer and normal lung were stained with a p120 monoclonal antibody, the nucleoli of cancer cells were positively stained, whereas a few macrophages in normal lung revealed only weak staining. The labeling index of p120 in squamous cell carcinoma (67.7 +/- 12.4%) was significantly higher than that in adenocarcinoma (35.3 +/- 12.6%) or in large cell carcinoma (30.1 +/- 17.3%; P < 0.01). In six human lung cancer cell lines and one normal lung fibroblast cell line cultured in vitro, there was a significant correlation between S-phase fraction and p120 mRNA (r = 0.851, P < 0.02)/p120 protein (r = 0.869, P < 0.01) or between doubling time and p120 protein (r = -0.928, P < 0.01). In the context of the reports that indicate higher [3H]thymidine incorporation and shorter doubling time in the squamous cell carcinoma, these results indicate that p120 can be a marker for proliferation in human lung cancer cells in vivo and in vitro, and that it has an important function in the cell cycle of tumor proliferation.

Endothelin receptor antagonist prevents parathyroid cell proliferation of low calcium diet-induced hyperparathyroidism in rats.

Secondary hyperparathyroidism, one of the most frequently encountered disorders of the calcium homeostasis, is characterized by an increase in parathyroid epithelial (PT) cell number, which is crucial from a functional viewpoint. However, it is still unknown what factors are involved in PT cell proliferation. Endothelin-1 (ET-1), a vasoconstrictive peptide, has been shown to act as a mitogen in a variety of cell types. Rat PT cells are reported to synthesize ET-1 and possess its receptors. To test the hypothesis that ET-1 plays a role in PT cell proliferation, we used rat test subjects fed a low calcium diet for 8 weeks (low Ca rats). The number of the proliferating PT cells, measured by proliferating cell nuclear antigen immunostaining, was significantly increased, with striking immunoreactivity of ET-1 in the low Ca rats. An endothelin receptor antagonist, bosentan (100 mg/kg.day), prevented any increase in the proliferation of PT cells in the low Ca rats (14.3 +/- 2.7/1000 PT cells with no bosentan; 2.1 +/- 1.3 with bosentan; P < 0.01). These results indicate that ET-1 is involved in PT cell proliferation in vivo and suggest that blocking of ET receptors may become one of the important therapeutic strategies for preventing secondary hyperparathyroidism.

Specific recognition of cytosolic thymidine kinase in the human lung tumor by monoclonal antibodies raised against recombinant human thymidine kinase.

Anti-TK monoclonal antibodies (mAbs) were raised against recombinant human cytosolic thymidine kinase (rhTK) and characterized by Western immunoblotting, enzyme-linked immunosorbent assay (ELISA) and immunostaining of tumor cells. Twenty-three clones of TK mAbs were characterized to recognize specifically not only rhTK produced by Escherichia coli but also TK subunit of 25 kDa in human lung cancer. The anti-TK mAbs reacted specifically with cytosolic TK but not with mitochondrial TK. Only one clone of the mAbs inhibited the catalytic activity of TK. By solid phase sandwich enzyme immunoassay using these mAbs, we could quantitate the cytosolic TK content in tissues. Immunohistochemical staining analysis using one of the TK mAbs showed that human lung adenocarcinoma and squamous cell carcinoma exhibited much higher staining intensity than stromal cells. These mAbs are useful for biochemical studies on the regulation of human TK in proliferating cells such as tumor cells and for diagnosis of highly proliferating tumors.