Essential oils of young and mature rhizomes, air-dried and steamed rhizomes, and seed rhizomes of Zingiber officinale Roscoe (ginger) were prepared, and their inhibition of filamentation by Candida albicans was examined. Ginger essential oils, and particularly those from seed and air-dried rhizomes, had potent inhibitory activity compared to ginger oleoresins obtained by ethanol and hypercritical carbon dioxide extraction and essential oils of 5 other plants in the family Zingiberaceae. Of the constituents,[6]-shogaol was most active against filament formation and growth of C. albicans, followed by citral and [6]-gingerol. Ginger oleoresin, and especially that obtained by ethanol extraction, with a high [6]-gingerol content exhibited potent scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radicals in comparison to essential oils of ginger and other Zingiberaceae plants.

In adults, the lateral pterygoid muscle (LPM) is usually divided into the upper and lower heads, between which the buccal nerve passes. Using sagittal or horizontal sections of 14 fetuses and seven embryos (five specimens at approximately 20-25 weeks; five at 14-16 weeks; four at 8 weeks; seven at 6-7 weeks), we examined the topographical relationship between the LPM and the buccal nerve. In large fetuses later than 15 weeks, the upper head of the LPM was clearly discriminated from the lower head. However, the upper head was much smaller than the lower head in the smaller fetuses. Thus, in the latter, the upper head was better described as an 'anterior slip' extending from the lower head or the major muscle mass to the anterior side of the buccal nerve. The postero-anterior nerve course seemed to be determined by a branch to the temporalis muscle (i.e. the anterior deep temporal nerve). At 8 weeks, the buccal nerve passed through the roof of the small, fan-like LPM. At 6-7 weeks, the LPM anlage was embedded between the temporobuccal nerve trunk and the inferior alveolar nerve. Therefore, parts of the LPM were likely to 'leak' out of slits between the origins of the mandibular nerve branches at 7-8 weeks, and seemed to grow in size during weeks 14-20 and extend anterosuperiorly along the infratemporal surface of the prominently developing greater wing of the sphenoid bone. Consequently, the topographical relationship between the LPM and the buccal nerve appeared to 'change' during fetal development due to delayed development of the upper head.

Complicating (secondary) leukemias in 20 patients, 18 of whom had various forms of neoplasia treated with chemotherapy and/or radiation, have been examined in regard to the karyotypic findings present in the leukemic cells of the marrow. In addition, the published cases of complicating leukemia have been tabulated. Based on the results of the present study and those in the literature it appears that chromosomes #3 and #17 should be stressed as being nonrandomly involved in complicating leukemia in addition to the previously stressed chromosomes #5 and #7. The results of the present study are discussed in relation to those reported in the literature, and stress put on the significance of cytogenetic changes in relation to possible causative factors of the complicating leukemia. The hypothesis is advanced that these nonrandom chromosome changes may reflect causative specificity of the complicating leukemia and that a concerted effort must be made to obtain more cogent information on the role of noxious agents in the causation of complicating leukemia.

The specificity of human skin collagenase and of an enzyme from an invasive tumor were studied by using types I, II, III, IV, and V (AB) collagen as substrates. Human skin collagenase degraded types I, II, and III collagen, producing the characteristic 3/4 and 1/4 cleavage products, but failed to degrade type IV or V collagen. Collagenase prepared from the invasive tumors showed maximal activity after trypsin treatment. The tumor enzyme degraded type IV (basement membrane) collagen, producing fragments consistent with a single cleavage site but did not attack types I, II, III, and V collagen. Because type IV collagen prepared by pepsinization of placenta was also digested, it is likely that cleavage of type IV collagen by the tumor collagenase occurs within a largely helical domain. A type IV collagenase could play a significant role in tumor metastases and in normal tissues where basement membrane turnover takes place.

Normal somatic cells are endowed with limited doubling potential in culture, and the process of immortalization is an inevitable step in neoplastic transformation of the cells. To examine the roles of p53 in this process, the cells of p53-deficient mice were examined for doubling potential. Fibroblast-like cells from a variety of tissues of these mice proliferated continuously without showing aging or crisis. The aneuploid cells overcome the population with passage, but cloning experiment indicated that chromosomal changes were not essential to this process. The enhanced proliferative potential in culture of cells from the p53-deficient mice was also observed in epithelial cells of lens, mammary glands and seminal vesicles and in neural precursor cells. Proliferation of bone marrow cells in response to stem cell factor was enhanced in long term culture, but not in in vitro colony assay; no permanent cell lines could be obtained. No effects of p53-deficiency were found in proliferation of cardiac muscle cells or hepatocytes.

Poliovirus-sensitive transgenic mice were produced by introducing the human gene encoding cellular receptors for poliovirus into the mouse genome. Expression of the receptor mRNAs in tissues of the transgenic mice was analyzed by using RNA blot hybridization and the polymerase chain reaction. The human gene is expressed in many tissues of the transgenic mice just as in tissues of humans. The transgenic mice are susceptible to all three poliovirus serotypes, and the mice inoculated with poliovirus show clinical symptoms similar to those observed in humans and monkeys. Rabbit antipoliovirus serum detects the antigens mainly in motor neurons in the anterior horn of the spinal cord and in nerve cells in the medulla oblongata and pons of the paralyzed transgenic mice. Therefore, cell types sensitive to poliovirus in the central nervous system of the transgenic mice appear to be identical to those of humans and monkeys. Furthermore, many more doses of oral poliovirus vaccine strains than of the virulent strains are required to cause paralysis in the transgenic mice. This may reflect the observation that the virulent strain multiplies more efficiently in the central nervous system than the attenuated strain. Thus, the transgenic mice may become an excellent new animal model to study molecular mechanisms of pathogenesis of poliovirus and to assess oral poliovirus vaccines.

Relapsing polychondritis is a disorder of unknown cause characterized by the destruction of cartilage. To test the hypothesis that immunologic mechanisms are involved in the pathogenesis of relapsing polychondritis, we analyzed the serum of 15 patients for the presence of antibodies to cartilage. Antibodies to Type II (cartilage) collagen were found in the serum of five patients at the time of acute symptoms. No antibodies were detected either to cartilage proteoglycan or to other collagen types. The antibodies were detected at the onset of the disease and their titers appeared to correlate with severity of disease. Circulating immune complexes were also detected in the serum of these patients. Our findings support an immunologic involvement in this condition.

A neutral protease has been extracted from the media of cultured metastatic tumor cells and purified approximately 1000 times after sequential ammonium sulfate fractionization, concanavalin A column chromatography, and molecular sieve chromatography. The protease has an apparent molecular weight of 70 000--80 000, is inactive at acid pH, requires trypsin activation, and is inhibited by ethylene-diaminetetraacetic acid but not by phenylmethanesulfonyl fluoride, N-ethylmaleimide, or soybean trypsin inhibitor. The enzyme produces specific cleavage products for both chains of pro type IV collagen isolated without pepsinization and apparently cleaves at one point in a major pepsin-extracted chain of placenta type IV collagen. The partially purified enzyme fails to significantly degrade other collagens or fibronectin under digestion conditions in which specific reaction products are produced for type IV collagen. The existence of this enzyme is significant since previously described animal collagenases fail to degrade type IV collagen. Such a type IV specific collagenase could play a role in tumor invasion and may be secreted by other cells such as endothelial cells, epithelial cells, and immune cells.

BACKGROUND. Hematogenous metastasis requires angiogenesis within the tumor. Previous studies have shown that microvessel counts in histologic sections of the primary tumor, which reflect angiogenesis, are correlated with metastasis in breast, prostate and Stage I nonsmall cell lung carcinoma. In this study, the authors investigated the association between angiogenesis, hematogenous metastasis and lymph node metastasis in all stages of lung adenocarcinoma. METHODS. Microvessels were highlighted by immunostaining endothelial cells for factor VIII. We counted microvessels within the tumors of 42 patients who had surgical resection (25 with relapse and 11 without relapse more than 5 years after surgical resection). Without knowledge of patient outcome, microvessels were counted on a 200x field (0.723 mm2) in the most active areas of neovascularization. RESULTS. The microvessel counts from patients with relapse after surgical resection (mean +/- standard deviation, 75.4 +/- 64.3) were significantly higher than those without relapse more than 5 years after surgical resection (42.6 +/- 26.0)(P = 0.027). Analysis of regional lymph node metastases (factor N) revealed that the microvessel counts were 62.6 +/- 35.1 for N0 (no regional lymph node metastasis), 51.7 +/- 22.2 for N1 (metastasis in ipsilateral, peribronchial and/or ipsilateral hilar lymph nodes, including direct extension), 75.4 +/- 75.3 for N2 (metastasis in ipsilateral mediastinal and/or subcarinal lymph nodes), and 74.0 for N3 (metastasis in contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene or supraclavicular lymph node[s]), and these values were not significantly different from each other. CONCLUSIONS. Angiogenesis assessed by microvessel counts, correlated positively with relapse after surgical resection and hematogenous metastasis in all stages of lung adenocarcinoma; there was no correlation with lymph node metastasis in lung adenocarcinoma.

A number of recombinants between the virulent Mahoney and attenuated Sabin strains of type 1 poliovirus were constructed by using infectious cDNA clones of the two strains. To identify a strong neurovirulence determinant(s) residing in the genome region upstream of nucleotide position 1122, these recombinant viruses were subjected to biological tests, including monkey neurovirulence tests. The results of the monkey neurovirulence tests suggested the important contribution of an adenine residue (Mahoney type) at position 480 to the expression of the neurovirulence phenotype of type 1 poliovirus. This nucleotide, however, had only a minor effect, if any, on viral temperature sensitivity. Monkey neurovirulence tests on the recombinant virus whose genome had a guanine residue (Sabin type) at position 480 and variants generated from this recombinant virus in the central nervous system of monkeys strongly suggested that only one nucleotide change, from adenine to guanine, was not sufficient for full expression of the attenuation phenotype encoded by this genome region. These results suggest that the expression of the attenuation phenotype depends on the highly ordered structure formed in the 5' noncoding sequence and that the formation of such a structure is possibly influenced by the nucleotide at position 480. Furthermore, in vitro biological tests performed on viruses recovered from the central nervous system of monkeys injected with a temperature-sensitive recombinant virus showing the small-plaque and d phenotypes revealed that most of the recovered viruses had even higher temperature sensitivities and that all of the recovered viruses that had acquired the large-plaque phenotype had lost the d phenotype to some extent. These results indicate that there may be an unknown selection pressure(s) in the central nervous system and that common determinants might be involved in the expression of the small-plaque and d phenotypes.

Pulmonary surfactant protein D (SP-D) is a hydrophilic glycoprotein with a reduced molecular mass of 43 kDa and a member of the C-type lectin superfamily, along with mannose-binding proteins and surfactant protein A (SP-A). We have recently prepared monoclonal antibodies against human SP-D and developed an enzyme-linked immunosorbent assay (ELISA). In this study, the levels of SP-D in sera and bronchoalveolar lavage (BAL) fluids of patients with lung diseases were determined by ELISA, using human recombinant SP-D as a standard. We demonstrated that the concentrations of SP-D in sera are prominently increased in patients with idiopathic pulmonary fibrosis (IPF), interstitial pneumonia with collagen disease (IPCD), and pulmonary alveolar proteinosis (PAP). Patients with IPF, IPCD, and PAP exhibited levels of serum SP-D 5.1-fold, 7.2-fold, and 7.0-fold, respectively, of those in healthy volunteers; 91.5% of the patients with IPF, 81.3% with IPCD, and 100% with PAP exhibited serum SP-D levels that exceeded the cut-off value (mean + 2 SD of control value). Serum SP-D levels appeared to reflect the disease activity of IPF and IPCD and the disease severity of PAP. High levels of SP-D in BAL fluids were shown in patients with PAP, but not with IPF and IPCD. We conclude that measurement of SP-D in sera can provide an easily identifiable and useful clinical marker for the diagnosis of IPF, IPCD, and PAP, and can predict the disease activity of IPF and IPCD and the disease severity of PAP.

The gene responsible for DNFB1 and DFNA3, connexin 26 (GJB2), was recently identified and more than 20 disease causing mutations have been reported so far. This paper presents mutation analysis for GJB2 in Japanese non-syndromic hearing loss patients compatible with recessive inheritance. It was confirmed that GJB2 mutations are an important cause of hearing loss in this population, with three mutations, 235delC, Y136X, and R143W, especially frequent. Of these three mutations, 235delC was most prevalent at 73%. Surprisingly, the 35delG mutation, which is the most common GJB2 mutation in white subjects, was not found in the present study. Our data indicated that specific combinations of GJB2 mutation exist in different populations.