Lower-lobe shrinkage relative to total lung volume in collagen vascular diseases.

PURPOSE: It has been reported that the prognosis differs between patients who have collagen vascular diseaseassociated interstitial pneumonia (CVD-IP) and those with idiopathic IP (IIP). In this study, chest computed tomography (CT) findings were compared between patients with CVD-IP and IIP. MATERIALS AND METHODS: A retrospective analysis was performed of 47 consecutive patients (23 with CVD-IP and 24 with IIP). The lower-lobe volume (LLV), total lung volume (TLV), and their ratio (LLV/TLV) were determined by volumetry using three-dimensional computed tomography (CT). RESULTS: There was no significant difference of the LLV/TLV ratio between the CVD-IP and IIP groups. However, the LLV/TLV ratio was <0.33 in 9/23 patients with CVD-IP versus 2/24 patients with IIP, and there was a significant difference in the percentage of patients with a ratio<0.33 between the CVD-IP and IIP groups (p = 0.01). The LLV/TLV ratio was not influenced by the severity of lung disease. CONCLUSIONS: Measuring the LLV/TLV ratio by threedimensional CT can help distinguish between CVD-IP and IIP at initial diagnosis, especially in patients with CVD-IP who have pulmonary involvement before other organ diseases and symptoms caused by CVD.

K-ras and rho A mutations in malignant pleural effusion.

Mutations of the Kristen ras (K-ras) gene have been implicated in the pathogenesis of human lung cancer, especially adenocarcinoma, and have been proposed to be a prognostic factor. The K-ras mutation in codon 12 is detectable even in cell-free fluids by using the enriched polymerase chain reaction (PCR) technique. On the other hand, based on experimental results, the rho A mutation in codon 14 is also proposed to be oncogenic as observed in the K-ras mutation. Malignant pleural effusion is a common complication of lung cancer. We studied the point mutation of K-ras codon 12 and rho A codon 14 using enriched PCR in specimens of pleural effusion. Forty patients with pleural effusion were enrolled in this study. The causes of pleural effusion were non-small cell lung cancer (18 cases), small cell lung cancer (6 cases), malignant mesothelioma (2 cases), metastatic lung tumor (5 cases), thymoma (1 case), malignant lymphoma (1 case), and pleuritis tuberculosa (7 cases). The K-ras mutation was detected in 4 of 14 cases with adenocarcinoma, 1 of 3 cases with squamous cell carcinoma, 1 of 1 case with large cell carcinoma, and 1 of 5 cases with metastatic lung tumor, respectively. The rho A mutation was not detected in any pleural effusion examined in this study. Our study demonstrates the usefullness of pleural effusion as a clinical specimen for a search of point mutation of oncogenes. The K-ras codon 12 mutation is readily detected in pleural effusion, and the demonstration of this mutation has potentially important implications for the diagnosis of malignant pleural effusion.

Presence of antibodies against retinoblastoma tumor suppressor protein in patients with lung cancer.

Retinoblastoma (RB) protein and antibody against RB protein in sera from 45 lung cancer patients and 30 healthy volunteers were examined using bacterially synthesized glutathione S-transferase (GST) RB fusion protein and immunoblot analysis. RB protein was not detected in sera from any individuals with lung cancer or in any healthy volunteers. Sera from 6 patients, including 4 with non-small cell carcinoma and 2 with small cell carcinoma, reacted to a GST-RB fusion protein but not with a GST protein. Sera from 30 normal volunteers reacted to neither GST-RB fusion protein nor GST protein. The backgrounds such as age, gender, performance status, histology, stage, smoking history, and prior treatment were not significantly different between the patients with and without anti-RB antibodies. This is the first report describing the presence of anti-RB antibody in patients with malignant tumors. Further studies are needed to establish clinical significance for anti-RB antibody.

Theophylline chronotherapy of nocturnal asthma using bathyphase of circadian rhythm in peak expiratory flow rate.

We studied the efficacy of theophylline chronotherapy for nocturnal asthma using the changes of the circadian rhythms in peak expiratory flow rate (PEF). Eight patients with nocturnal asthma were evaluated for the periods with nocturnal symptoms and with an evening dose of theophylline administered daily. Patients recorded their PEF every 4 hours on one of the days (from 7:00 to 23:00 h) in each period. Circadian rhythms in PEF were examined using the single and group-mean cosinor method. Significant circadian rhythms in PEF were observed in the period with nocturnal symptoms. When nocturnal symptoms were present, the bathyphase of PEF was present between midnight and morning. A significant circadian rhythm disappeared or PEF amplitude decreased during theophylline chronotherapy. The circadian rhythm in PEF was altered according to the severity of the asthma. In patients with symptoms present between midnight and early morning, an evening dose of theophylline chronotherapy can be prophylactically used for nocturnal asthma attacks. Consideration of the circadian rhythm and bathyphase of PEF is useful in selecting appropriate chronotherapy for nocturnal asthma.

[The value of serum C-reactive protein as a survival determinant in patients with advanced non-small-cell lung cancer].

We retrospectively reviewed the information in our data base concerning 127 patients with advanced non-small-cell lung cancer. Seventeen pretreatment clinical variables including serum C-reactive protein (CRP) were analyzed to determine the factors related to survival using Cox's proportional hazards model. Univariate analysis revealed that eleven explanatory variables were significant. In multivariate statistical technique for those variables, six were selected as significant factors. The highest hazard ratio was observed in the serum CRP (3.82). The other factors were therapy (2.52), serum lactate dehydrogenase (2.41), serum total protein (2.20), white blood cell counts (1.98) and performance status (1.80). Median survival times estimated by the Kaplan-Meier procedures in patients with normal CRP (CRP < 0.2 mg/dl) and high positive CRP (CRP > or = 3.0 mg/dl) were 24.9 months and 3.7 months, respectively. These results suggest that serum CRP is an independent survival determinant in advanced non-small-cell lung cancer.

[A study of the minimum number of slices required for quantification of pulmonary emphysema by computed tomography].

We attempted to determine the minimum number of slices required for quantification of overall emphysema by computed tomography (CT). Forty-nine patients underwent CT scanning with a 15-mm slice interval, and 13 to 18 slices per patient were obtained. The percentage of low attenuation area (LAA%) per slice was measured with a method that we reported on previously, utilizing a CT program and NIH image. The average LAA% values for 1, 2, 3, and 6 slices evenly spaced through the lungs [LAA% (1), LAA% (2), LAA% (3), and LAA% (6)] were compared with those for all slices [LAA% (All)]. The correlation coefficients for LAA% (1), LAA% (2), LAA% (3), and LAA% (6) with LAA% (All) were 0.961, 0.981, 0.993, and 0.997, respectively. Mean differences +/- SD were -3.20 +/- 4.21%, -2.32 +/- 3.00, -0.20 +/- 1.84, and -0.16 +/- 1.26, respectively. From these results, we concluded that overall emphysema can be quantified by using at least three slices: one each of the upper, middle, and lower lung.

Divergent signaling pathways link focal adhesion kinase to mitogen-activated protein kinase cascades. Evidence for a role of paxillin in c-Jun NH(2)-terminal kinase activation.

Stimulation of a number of cell surface receptors, including integrins and G protein-coupled receptors, results in the activation of a non-receptor tyrosine kinase known as focal adhesion kinase (FAK). In turn, this kinase is believed to play a critical role in signaling to intracellular kinase cascades controlling gene expression such as extracellular signal-regulated kinases (ERKs), by a yet poorly defined mechanism. Furthermore, whether this tyrosine kinase also mediates the activation of other mitogen-activated protein kinase family members, such as c-Jun NH(2)-terminal kinases (JNKs), is still unclear. We show here that the activation of FAK by anchoring to the cell membrane is itself sufficient to stimulate potently both ERK and JNK. These effects were found to be phosphatidylinositol 3-kinase-independent, as FAK effectively stimulated Akt, and wortmannin suppressed Akt but not ERK or JNK activation. As previously reported by others, activation of ERK correlated with the ability of FAK to induce tyrosine phosphorylation of Shc. Surprisingly, however, stimulation of JNK was not dependent on the kinase activity of FAK or on the ability to induce tyrosine phosphorylation of FAK substrates. Instead, we provide evidence that FAK may stimulate JNK through a novel pathway involving the recruitment of paxillin to the plasma membrane and the subsequent activation of a biochemical route dependent on small GTP-binding proteins of the Rho family.

[Utilization of NIH image for quantification of emphysema by computed tomography].

The percentage of the low attenuation area (LAA%) is understood to be the most accurate index in quantifying emphysema by computed tomography (CT). To date, CT scanners with enhanced post-processing programs have been used for measurements of LAA%. In this study, we sought develop a method of LAA% determination using a conventional density mapping program for CTs and NIH Image, an image-analysis software package for personal computers. Our results for overall LAA%, together with the correlations between overall LAA% and pulmonary function tests, agreed well with the findings of earlier reports. In reproducibility trials utilizing our method interoperator error for LAA% was within +/- 2%. A phantom experiment indicated that there was little difference between LAA% values obtained by this method and the values obtained by direct CT measurements. Therefore, we concluded that this method is sufficiently reliable for clinical use and capable of facilitating LAA% measurements with CT scanners that are not equipped with enhanced post-processing software.

A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho.

Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Galpha12 family, Galpha12 and Galpha13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Galpha12 and Galpha13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Galpha12 family which, in turn, activate an exchange factor acting on Rho.

Flavopiridol, a novel cyclin-dependent kinase inhibitor, suppresses the growth of head and neck squamous cell carcinomas by inducing apoptosis.

Flavopiridol (HMR 1275) has been identified recently as a novel antineoplastic agent in the primary screen conducted by the Developmental Therapeutics Program, National Cancer Institute. Flavopiridol inhibits most cyclin-dependent kinases (cdks) and displays unique anticancer properties. Here, we investigated whether this compound was effective against head and neck squamous cell carcinomas (HNSCC). Exposure of HNSCC cells to flavopiridol diminished cdc2 and cdk2 activity and potently inhibited cell proliferation (IC50 43-83 nM), which was concomitant with the appearance of cells with a sub-G1 DNA content. Moreover, DNA fragmentation and TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) reaction confirmed that flavopiridol induces apoptosis in all cell lines, even on certain HNSCC cells that are insensitive to apoptosis to DNA-damaging agents (gamma-irradiation and bleomycin). A tumorigenic HNSCC cell line was used to assess the effect of flavopiridol in vivo. Treatment (5 mg/kg per day, intraperitoneally) for 5 d led to the appearance of apoptotic cells in the tumor xenografts and caused a 60-70% reduction in tumor size, which was sustained over a period of 10 wk. Flavopiridol treatment also resulted in a remarkable reduction of cyclin D1 expression in HNSCC cells and tumor xenografts. Our data indicate that flavopiridol exerts antitumor activity in HNSCC, and thus it can be considered a suitable candidate drug for testing in the treatment of refractory carcinomas of the head and neck.

Tyrosine kinases of the Src family participate in signaling to MAP kinase from both Gq and Gi-coupled receptors.

Src-related kinases have been recently implicated in signaling from Gi-coupled receptors to MAP kinase. Whether Src-like kinases participate in MAP kinase activation by the large family of receptors coupled to G proteins of the Gq family is still unclear. Here, we show that a specific inhibitor for Src-like kinases, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), and dominant negative mutants of Src suppress MAP kinase activation in COS-7 cells when elicited by either m1 and m2 muscarinic receptors, which are typical Gq and Gi-coupled receptors, respectively. Furthermore, activation of MAP kinase by overexpression of beta gamma subunits, but not by stimulation with phorbol esters was also inhibited by the dominant-negative Src. In contrast, a dominant negative Pyk2 had only mild effects on m1 and m2 mediated-MAP kinase activation. We concluded that Src like kinase(s), acting downstream from beta gamma dimers, play an important role relaying signals from both Gq and Gi-coupled receptors to MAP kinase.

Signaling from the small GTP-binding proteins Rac1 and Cdc42 to the c-Jun N-terminal kinase/stress-activated protein kinase pathway. A role for mixed lineage kinase 3/protein-tyrosine kinase 1, a novel member of the mixed lineage kinase family.

Certain small GTP-binding proteins control the enzymatic activity of a family of closely related serine-threonine kinases known as mitogen-activated protein kinases (MAPKs). In turn, these MAPKs, such as p44(mapk) and p42(mapk), referred to herein as MAPKs, and stress-activated protein kinases, also termed c-Jun N-terminal kinases (JNKs), phosphorylate and regulate the activity of key molecules that ultimately control the expression of genes essential for many cellular processes. Whereas Ras controls the activation of MAPK, we and others have recently observed that two members of the Rho family of small GTP-binding proteins, Rac1 and Cdc42, regulate the activity of JNKs. The identity of molecules communicating Rac1 and Cdc42 to JNK is still poorly understood. It has been suggested that Pak1 is the most upstream kinase connecting these GTPases to JNK; however, we have observed that coexpression of Pak1 with activated forms of Cdc42 or Rac1 diminishes rather than enhances JNK activation. This prompted us to explore the possibility that kinases other than Pak might participate in signaling from GTP-binding proteins to JNK. In this regard, a computer-assisted search for proteins containing areas of homology to that in Pak1 that is involved in binding to Rac1 and Cdc42 led to the identification of mixed lineage kinase 3 (MLK3), also known as protein-tyrosine kinase 1, as a potential candidate for this function. In this study, we found that MLK3 overexpression is sufficient to activate JNK potently without affecting the phosphorylating activity of MAPK or p38. Furthermore, we present evidence that MLK3 binds the GTP-binding proteins Cdc42 and Rac1 in vivo and that MLK3 mediates activation of MEKK-SEK-JNK kinase cascade by Rac1 and Cdc42. Taken together, these findings strongly suggest that members of the novel MLK family of highly related kinases link small GTP-binding proteins to the JNK signaling pathway.

The small GTP-binding protein rho activates c-Jun N-terminal kinases/stress-activated protein kinases in human kidney 293T cells. Evidence for a Pak-independent signaling pathway.

Work from a number of laboratories has established a role for certain small GTP-binding proteins in controlling the enzymatic activity of a family of serine-threonine kinases known as mitogen-activated protein kinases (MAPKs). MAPKs have been classified into three subfamilies: extracellular signal-regulated kinases (ERKs), also known as MAPKs; c-Jun N-terminal kinases (JNKs); and p38 kinase. Whereas Ras controls the activation of MAPKs, we and others have recently observed that in certain cells, the small GTP-binding proteins Rac1 and Cdc42 but not Rho regulate the activity of JNKs. Furthermore, because Rac1 and Cdc42 but not Rho bind and activate a kinase known as Pak1, it has been suggested that Pak1 is the most upstream component of the pathway linking these GTPases to JNK. However, in both yeast and mammalian cells, Rho1p, a Rho homologue, and RhoA, respectively, directly interact with a number of proteins, including kinases related to protein kinase C. In addition, in yeast, Rho1p controls the activity of a MAPK cascade involved in bud formation. Considering this diversity of target molecules for small GTP-binding proteins, their likely tissue specific distribution, and the potential role for Rho in signaling to a kinase cascade, we decided to extend our initial analysis, exploring the ability of Ras and Rho-related GTP-binding proteins to activate MAPK or JNK in a variety of cell lines. We found that in the human kidney epithelial cell line, 293T, Cdc42 and all Rho proteins, RhoA, RhoB, and RhoC, but not Rac or Ras can induce activation of JNK. Furthermore, we provide evidence that signaling from Rho proteins to JNK in 293T cells does not involve Pak1. Taken together these findings demonstrate that Rho signals to JNK in a cell type-specific manner and suggest the existence of a novel, Pak1-independent signaling route communicating the Rho family of small GTP-binding proteins to the JNK pathway.

[Chronic necrotizing pulmonary aspergillosis in a patient with chronic pulmonary emphysema].

A 67-year-old man with pulmonary emphysema was admitted to the hospital because of left back pain. Chest roentgenography revealed an infiltrate in the left upper lobe, with cavitation, Mycetoma-like shadows were seen in the cavities about 3 weeks later, and a test for the precipitating antibody to Aspergillus fumigatus was positive. Chronic necrotizing pulmonary aspergillosis (CNPA) was diagnosed, and fluconazole was given. A chest roentgenogram taken 4 weeks later showed resolution of both the mycetoma-like shadows and much of the infiltrate. Systemic immunosuppression was highly unlikely: the patient had not been undergoing corticosteroid therapy, and had no predisposing conditions, such as a chronic debilitating illness or diabetes mellitus. In that sense, this case is similar to another reported recently, in which CNPA was associated with chronic obstructive pulmonary disease in an immunocompentent patient.

[A case of nodular type of muscular sarcoidosis with limitation of mandibular opening].

A 68-year-old woman who complained of limited mandibular opening and left buccal swelling was admitted to our department. Histological examination of muscle biopsy specimens from the left buccal region revealed noncaseating granulomas. MR examination showed diffuse swelling and high signal intensity of the left masticatory muscles on T1- and T2-weighted images. Palpable nodules were present in the muscles of the body and the limbs. The ACE value was high and the lung biopsy specimens showed non-caseating granulomas. From these findings, the patient was diagnosed as having nodular type of muscular sarcoidosis complicated by contracture of the left masticatory muscles. There are some previous reports of nodular type of muscular sarcoidosis with flexion contracture of the fingers. Therefore, contracture of the muscles is suggested to be an important cause of muscular dysfunction in the disease.