Phylogenetic and molecular characteristics of Eurasian H9 avian influenza viruses and their detection by two different H9-specific RealTime reverse transcriptase polymerase chain reaction tests.

Avian influenza viruses (AIVs) of the H9 haemagglutinin subtype are endemic in many Asian and Middle-East countries, causing mortality and morbidity in poultry. Consequently there is a need for accurate and sensitive detection of Eurasian H9 subtype viruses. Two H9 RealTime reverse transcriptase polymerase chain reaction (RRT-PCR) tests, developed by Monne et al. (2008) and Ben Shabat et al. (2010), were originally validated with a limited number of H9 specimens. In the present study, the two tests have been assessed using 66 diverse H9 isolates and 139 clinical specimens from six H9 poultry outbreaks in four geographically disparate Eurasian countries. The Monne et al. (2008) test was modified and successfully detected all H9 viruses from all three Eurasian H9 lineages. Bayesian analysis of the clinical specimens' results revealed this test to be more sensitive (97%) than the Ben Shabat et al. (2010) test (31%). The latter test detected most H9 isolates of the G1 lineage, but no isolates from other H9 lineages. Mismatches in the primer/probe binding sequences accounted for sensitivity differences between the two H9 RRT-PCRs. Genetic analysis of 34 sequenced H9 haemagglutinin genes showed the South Asian and Middle-East H9 isolates to belong to the H9 G1 lineage, and possessed residues that appear to preferably bind alpha 2,6-linked sialic acid receptors which indicate a potential for human infection. European H9s clustered phylogenetically in a broader geographical group that includes recent North American H9 wild bird isolates and contemporary Asian viruses in the Y439 H9 lineage.

Myopodin, a synaptopodin homologue, is frequently deleted in invasive prostate cancers.

Prostate cancer is one of the leading causes of cancer-related deaths for men in the United States. Like other malignancies, prostate cancer is underscored by a variety of aberrant genetic alterations during its development. Although loss of heterozygosity or allelic loss is frequently identified among prostate cancers, few genes have been identified thus far as critical to the development of invasive prostate cancers. In this report, we used the recently developed technology, the "differential subtraction chain," to perform a genome-wide search for sequences that are deleted in an aggressive prostate cancer. Among the deleted sequences, we found that one sequence was deleted in >50% of prostate cancers we tested. We mapped this sequence to chromosome 4q25 by screening the Genebridge 4 hamster radiation panel with primers specific to this probe, and subsequently identify a 54-kb minimal common deletion region that contains the sequence encoding myopodin. Sequence analysis indicates that myopodin shares significant homology with synaptopodin, a protein closely associated with podocyte and neuron differentiation. Further study shows that frequent complete or partial deletions of the myopodin gene occurred among invasive prostate cancer cases (25 of 31 cases, or 80%). Statistical analysis indicates that deletion of myopodin is highly correlated with the invasiveness of prostate cancers, and thus may hold promise as an important prognostic marker for prostate cancers.

E-cadherin expression and PSA secretion in human prostate epithelial cells.

Prostate-specific antigen (PSA) is the most widely used marker for the diagnosis of prostate cancer and is an independent predictor of prostatic capsular invasion. A number of studies have identified E-cadherin, a cell adhesion protein, as a potential invasion suppressor which is decreased in prostate adenocarcinoma. Our goal in the present study was to evaluate E-cadherin expression in primary cultures and determine the relationship between E-cadherin expression and PSA secretion in both primary cultures and the prostate tumor cell line, LNCaP. Immunohistochemical studies and Western blot analysis confirmed greater expression of E-cadherin in normal epithelial cells than tumor-derived prostate cells. This is the first report that the incubation of normal prostate epithelial cells with E-cadherin antibody increases the amount of PSA detected in the media of normal cells as well as in LNCaP. Since E-cadherin may function as an invasion suppressor, an understanding of the decreased expression of this adhesion factor and the impact on PSA secretion may aid in understanding epithelial tumorigenesis.

Expression patterns of vitamin D receptor in human prostate.

UV exposure and serum levels of vitamin D have been linked in several studies with prostate cancer risk. At the cellular level, the principal action of vitamin D is mediated though vitamin D receptors (VDR). Since prostate cancer is a disease strongly associated with age, we examined the presence of VDR in normal prostate from donors of various ages to determine if the VDR expression pattern changed with age. We also compared the VDR expression in the peripheral and central zones of the prostate to determine if the expression pattern varied by location. Immunohistochemical studies were performed on paraffin-embedded tissue from cases selected by the following age decades; 10-19, 20-29, 30-39, 40-49, 50-59, and 60-69. Both the central and peripheral zones were examined for VDR expression. The intensity of VDR expression in prostate was compared with expression in different types of human tissues. Mean VDR expression was lowest in the 10-19 years of age group. The intensity of the nuclear VDR was higher though the fifth decade, and then declined in cases of ages 60-70. When multiple sections of the same donor prostate were compared, VDR expression was greater in the peripheral zone compared to the central zone.

Identification of a novel gene with increasing rate of suppression in high grade prostate cancers.

Prostate cancer is the second leading cause of cancer-related deaths in the United States. However, the underlying molecular events for prostate cancer development are not clear. In this study, we applied the recently developed technology known as differential subtraction chain (DSC) to identify a novel gene whose expression is inactivated in high grade prostate cancer. This gene, designated as SAPC, is expressed in normal prostate acinar cells. Its expression is dramatically down-regulated in high grade prostate cancers (4/4) but is unaltered in low grade prostate cancers. It encodes a 7.7-kd protein. Its sequence shares some homology with the cysteine-rich domain of 2-5A-dependent RNase L, which is a critical component of the interferon-induced apoptosis cascade. The selective inactivation in the more aggressive prostate cancers holds promise for SAPC as a potential prognostic marker for high grade prostate cancer.

Expression of cadherins and catenins in paired tumor and non-neoplastic primary prostate cultures and corresponding prostatectomy specimens.

Cadherins are a family of transmembrane proteins that play a crucial role in cell differentiation, cell migration, and intercellular adhesion. Cadherins are associated with catenins through their highly conserved cytoplasmic domain. Down-regulation of E-cadherin protein has been shown in various human cancers. This study examined the expression of cadherins and associated catenins at the mRNA level. Paired tumor and nonneoplastic primary prostate cultures were obtained from surgical specimens. Quantitative multiplex fluorescence reverse transcriptase-polymerase chain reaction (QMF RT-PCR) and quantitative analysis were performed and correlated with immunostain results. Six of seven cases of neoplastic cultures showed moderately-to-markedly decreased levels of E-cadherin and P-cadherin mRNA. Similar losses of alpha-catenin and beta-catenin mRNA were also observed. The results of QMF RT-PCR showed good correlation with the results of immunohistochemical studies based on corresponding formalin-fixed sections. In conclusion, this paper presents a coordinated down-regulation in the expression of E-cadherin and associated catenins at the mRNA and protein level in most of the cases studied. This down-regulation may play an important role in the pathogenesis of prostate cancer.

Differential effects of vitamin D on normal human prostate epithelial and stromal cells in primary culture.

OBJECTIVES: Because epidemiologic evidence has demonstrated that vitamin D may play a role in the etiology of prostate cancer, we tested the inhibitory effect of the biologically active form of vitamin D (1,25-D) on the cell proliferation of human prostate epithelial and stromal cells in a chemically defined situation in the presence and absence of dihydrotestosterone (DHT). We also tested the effect of 1,25-D in castrated rats in the presence and absence of flutamide, an androgen receptor blocker. METHODS: Prostate stromal and epithelial cells were isolated from freshly collected human prostatectomy specimens, and cell proliferation was measured with the MTT assay. Immunohistochemistry was performed to detect the presence of 1,25-D receptors, androgen receptors, smooth muscle actin, and E-cadherin. For in vivo analysis of 1,25-D, male Sprague-Dawley rats were castrated, then treated with either 1,25-D, 1,25-D with flutamide, or vehicle control. RESULTS: Incubation of primary cultures of prostate epithelial cells with 1,25-D at a concentration of 10(-8) M reduced cell proliferation by 40% of controls. The inhibition of growth by 1,25-D was maintained in the presence of DHT. Conversely, the effect of a similar dose of 1,25-D on stromal cell exposure was increased proliferation. In vivo, 1,25-D increased the prostatic weight of castrated rats that had serum testosterone levels below the detectable limit. The addition of flutamide did not alter this effect. CONCLUSIONS: These results confirm that vitamin D may be an effective antiproliferative agent of epithelial cells in prostate cancer therapy and support in vivo studies performed in the normal rat prostate.

Methylation of the CD44 metastasis suppressor gene in human prostate cancer.

Previous studies demonstrated that CD44 is a metastasis suppressor gene for prostate cancer and that the expression of CD44 both at mRNA and protein levels is down-regulated during prostate cancer progression, with down-regulation being correlated with higher tumor grade, aneuploidy, and distant metastasis. In this study, we evaluated DNA hypermethylation as a potential mechanism accompanying this decreased CD44 expression in human prostate cancer. Nucleotide sequence analysis revealed a CpG island in the CD44 transcriptional regulatory region. We found that cytosine methylation of CD44 promoter occurs in CD44-negative prostate cancer cell line (i.e., LNCaP) but not in prostate cancer cell lines (i.e., TSU, PC3, and DU145) expressing this gene. In addition, we examined methylation status of CD44 in 84 matched normal and cancer prostate specimens. Hypermethylation of the 5' CpG island of CD44 gene was observed in 31 of 40 primary prostate cancer specimens, 3 of 4 distant organ site metastases obtained at autopsy from men who died of prostate cancer, and 4 of the 40 matched normal tissues. These results demonstrated that methylation of the 5' CpG island of CD44 gene is closely associated with transcriptional inactivation, resulting in a decreased expression of CD44 in human prostate cancer.

The biosynthesis and secretion of prostate-specific antigen in LNCaP cells.

Prostate-specific antigen (PSA) has been demonstrated to release the active form of insulin-like growth factor I in vitro (P. Cohen et al., J. Clin. Endocrinol. & Metab., 75: 1046-1053, 1992; P. Cohen et al., J. Clin. Endocrinol. & Metab., 79: 1410-1415, 1994; P. Cohen et al., Horm. Metab. Res., 26: 81-84, 1994) and has significant mitogenic activity on osteoblast cells, fibroblasts, and other cultured cells (C. S. Killian et al., Biochem. Biophys. Res. Commun., 192: 940-947, 1993). Recently, PSA has been found not only in prostate tissues but also in breast, colon, ovarian, and other tissues (E. P. Diamandis and H. Yu, J. Clin. Endocrinol. & Metab., 80: 1515-1517, 1995; E. P. Diamandis and H. Yu, Clin. Chem., 41: 204-210, 1995; A. Clements and A. Mukhtar, J. Clin. Endocrinol. & Metab., 78: 1536-1539, 1994). Therefore, PSA has been proposed as a candidate growth factor, cytokine, or growth factor regulator. In this setting, knowing how to manipulate or block the secretion of PSA by the prostate cancer cells could be a useful approach to controlling the progression of human prostate cancers. Using metabolic labeling experiments, we have studied the biosynthesis and secretion of PSA in LNCaP cells. We have also examined the effects of DTT, tunicamycin, 1-deoxymannojirimycin, pilocarpine, and testosterone on PSA biosynthesis and secretion. The results indicate that the secretion of PSA in LNCaP cells is constitutive instead of regulated and that the disruption of intramolecular disulfide bonds affects the transport of PSA from the endoplasmic reticulum to the Golgi apparatus. The biosynthesis of PSA is potentiated by testosterone and inhibited by brefeldin A and DTT. These results will help us understand PSA biosynthesis and secretion in human prostate cancers.

A simple method for the isolation and culture of epithelial and stromal cells from benign and neoplastic prostates.

OBJECTIVES: Current primary prostate cell culture techniques use an overnight digestion or extensive media preparation. In this report, we describe a method for the culture of benign and neoplastic cells from human prostatectomy specimens that is rapid and contains no undefined factors in the medium. METHODS: Characterization of the human cultured prostate cells was performed using immunohistochemical methods and monoclonal antibodies AE1/AE3 and cytokeratin 8, as well as monoclonal antibodies against prostate-specific antigen (PSA). Polymerase chain reaction was used to measure the exclusive epithelial and stromal cell products, c-met and hepatocyte growth factor (HGF), respectively. Electron microscopy was performed to assess the cell junctions and morphologic features of epithelial cells. Optimum cell growth in different media was tested using a cell replication assay. RESULTS: Microscopic evidence revealed that the cells demonstrate typical epithelial morphology, with polyhedral cells forming tight junctions in a continuous monolayer. Desmosomes were present in electron micrographs of epithelial cells. The cultured epithelial cells described in this report also demonstrate positive cytokeratin staining. The epithelial cells reacted positively with PSA antibody, indicating that the cells retain their secretory role in cell culture for a limited period. Epithelial cells expressed the HGF receptor, c-met; stromal cells secreted HGF. Insulin, transferrin, and selenium increased the growth of cells in the chemically defined media, compared with minimum essential media (MEM) and Ham's F12. CONCLUSIONS: In summary, essentially pure cultures of prostate stromal or epithelial cells have been established using simple isolation and culture methods. These cells will be useful for the investigation of related growth factors, such as insulin-like growth factor I and insulin-like growth factor II, and in understanding the basis for stromal-epithelial cell interactions.

Association of retinol binding protein in multiple-case families with insulin-dependent diabetes.

We performed a family study to investigate the heritability of reduced serum retinol levels observed in type 1 diabetes cases. Diet and serum factors, including retinol, total carotene, malondialdehyde, and retinol binding protein levels, were measured in 11 multiple-case families. The mean serum retinol level of the diabetics (46 ug/dl) was significantly less than the mean serum retinol level of the nondiabetics (60.9 ug/dl). The level of retinol binding protein was also significantly lower in diabetics (6.2 mg/dl) than in nondiabetics (7.6 mg/dl). The serum values of retinol binding protein were closely related within families, including both diabetic and nondiabetic family members. A characteristic shared between diabetics and one-third of their family members was a low ratio of serum retinol to total carotene, suggesting a low conversion of dietary carotene into retinol. Analysis of food frequency reports showed no difference between dietary retinol or total carotene level in diabetics or their relatives. This study offers evidence that heritability and the reduced conversion of carotene may play a role in the level of serum retinol in type 1 diabetes cases.

CD44 Expression in Benign and Neoplastic Human Prostates.

Background: CD44, a major cell surface receptor for hyaluronic acid, is a family of ubiquitous cell surface glycoproteins. Altered levels of CD44 expression, seen in many epithelial neoplasms, have prognostic implications. Expression of standard and variant isoforms of CD44 was assessed in normal and neoplastic human prostate tissue and culture cells to evaluate as a marker for malignant transformation. Methods and Results: Expression of CD44s, CD44R, v5, v6, v7/8 and v10 was assessed in prostate tissue (benign and malignant) and cell lines (DU-145, PC-3, LNCaP, p69) and primary cultures of normal prostates and adenocarcinoma cells obtained from prostatectomies using reverse transcriptor polymerase chain reaction, Western blotting, and immunofluorescence. No CD44 expression was seen in LNCaP cells. p69, DU-145, and PC-3 cells expressed CD44s and CD44R. p69, cells demonstrated a 1000-bp-long form of CD44 mRNA, unique to this normal cell line. Both normal and neoplastic prostatic tissue demonstrated CD44s on Western blotting. Conclusions: In agreement with previous studies, prostatic adenocarcinoma cells, except LNCaP, expressed CD44s. Different patterns of CD44 expression were seen in benign and neoplastic prostate. Benign prostate exhibited higher v5 protein levels, whereas neoplastic prostates demonstrated higher CD44s expression. CD44s expression was identified in all neoplastic prostates as compared with only 50% of the benign prostates. No significant difference in expression of the other variants assessed (v6, v7, v7/8, and v10) was observed in the benign and neoplastic prostates.

Effects of axial and radial gradients on Cooke triplets.

This study investigates the role of gradient-index materials in the design of Cooke triplets for use as 35-mm format photographic objectives. Cooke triplet designs are presented with different types of gradient-index profiles. Both linear axial and shallow radial gradients are shown to provide effective control of spherical aberration and astigmatism. In particular, a Cooke triplet with a combination of both linear axial and radial gradients attains performance comparable to a six-element double Gauss lens. In virtually all cases, the use of gradient-index components improves the Cooke triplets' performance significantly.

Arachidonate-dependent oxygen consumption in Dictyostelium discoideum.

A central feature of the processes of aggregation and differentiation in the cellular slime mould Dictyostelium discoideum is the periodic excitatory cycle. Originally thought to involve primarily fluctuations in cyclic AMP levels, this excitatory cycle has since been shown to involve changes in several other second messengers including cyclic GMP, calcium and inositol trisphosphate. Previous work from this laboratory using specific inhibitors strongly suggested a role for eicosanoids in this stimulus-response process. Production of eicosanoids from fatty acid precursors is an oxygen-consuming process. In this paper, we report on oxygen consumption measurements in intact D. discoideum cells and in cell extracts. We demonstrate the existence of an azide-insensitive component of oxygen consumption which can be stimulated by the addition of arachidonate and other polyunsaturated fatty acids, and at least partially inhibited by meclofenamate and eicosatetraynoic acid, both of which block eicosanoid biosynthesis in higher organisms. These observations provide further evidence for the existence of an eicosanoid-metabolizing system in D. discoideum.

Periodontal surgery as an adjunct to endodontics, orthodontics, and restorative dentistry.

Periodontal surgery and biologic principles can be used to treat a variety of restorative and endodontic complications. Four cases illustrated the application of orthodontic therapy and periodontal surgery in the treatment of these difficulties.

Treatment of localized juvenile periodontitis (periodontosis). A review.

Localized juvenile periodontitis is a disease of the adolescent periodontium characterized by rapid alveolar destruction around molar and incisor teeth of the permanent dentition. Early treatment methods were variable and often empirical due to lack of knowledge concerning etiology. Elucidation of factors associated with the disease has led to different therapeutic approaches. A comprehensive review of these modalities is presented.