Immunohistochemical analysis of centromere protein F expression in buccal and gingival squamous cell carcinoma

Centromere protein F (CENP-F) expression (localization and characteristics) in relation to tumor clinicopathological parameters was immunohistochemically examined and evaluated in 47 archival biopsy specimens of buccal and gingival squamous cell carcinomas (SCC). Centromere protein F expression was detected in 79% of the samples. An increase in the labeling index (LI) with WHO grading was obtained ( P  < 0.05). Correlations were obtained between the CENP-F LI and tumor size ( P  < 0.05). Immunoelectron microscopy showed CENP-F nuclear staining as punctate or fine dots. The present study shows that CENP-F expression and detection of a more specific cell subpopulation presents a theoretical advantage for the analysis of the precise cell cycle of G₂ to M cells, compared to Ki-67.     

Application of simulated annealing for estimating BAEPs in endocochlear pathologies

In this paper, we propose a new approach aimed at handling the temporal Brainstem Auditory Evoked Potentials (BAEPs) non-stationarity. It is pointed out that for some endocochlear pathologies, BAEPs could be randomly delayed from one response to another. This non-stationarity leads to smoothed BAEPs when applying ensemble averaging or any other technique based on BAEPs stationarity. In that case, waves identification is very difficult, sometimes impossible. The problem consists in estimating time delays. Knowing the distribution of delays allows subsequent study of the dynamic of the cochlea and, perhaps, identification of the nature of its pathology. The approach suggested in this paper is based on Simulated Annealing, used to minimize a non-linear criterion involving delays. This technique is advantageously compared to the non-corrected ensemble averaging method, using a set of simulated data based on a realistic model. As an illustration, results based on real signals recorded from two patients are presented and discussed at the end of the paper.     

In vitro degradation of silk fibroin

A significant need exists for long-term degradable biomaterials which can slowly and predictably transfer a load-bearing burden to developing biological tissue. In this study Bombyx mori silk fibroin yarns were incubated in 1mg/ml Protease XIV at 37 degrees C to create an in vitro model system of proteolytic degradation. Samples were harvested at designated time points up to 12 weeks and (1) prepared for scanning electron microscopy (SEM), (2) lyophilized and weighed, (3) mechanical properties determined using a servohydraulic Instron 8511, (4) dissolved and run on a SDS-PAGE gel, and (5) characterized with Fourier transform infrared spectroscopy. Control samples were incubated in phosphate-buffered saline. Fibroin was shown to proteolytically degrade with predictable rates of change in fibroin diameter, failure strength, cycles to failure, and mass. SEM indicated increasing fragmentation of individual fibroin filaments from protease-digested samples with time of exposure to the enzyme; particulate debris was present within 7 days of incubation. Gel electrophoresis indicated a decreasing amount of the silk 25 kDa light chain and a shift in the molecular weight of the heavy chain with increasing incubation time in protease. Results support that silk is a mechanically robust biomaterial with predictable long-term degradation characteristics.     

Bizarre leiomyoma of the scrotum

Scrotal leiomyomas with atypical bizarre nuclei are rare, which might be misdiagnosed as malignant tumor. We describe a case of scrotal bizarre leiomyoma in a 65-yr-old man. The tumor was a 1 cm-sized, well circumscribed, oval mass arising from the tunica dartos muscle. Histologically, it was formed by whorling bundles of fusiform cells with occasional atypical, pleomorphic nuclei and pseudo-inclusions. Mitosis was not found. Although morphologically atypical, scrotal bizarre leiomyomas take on a biologic behavior not different from that of conventional leiomyoma, they should be distinguished from leiomyosarcoma to avoid unnecessary treatment.     

4E-binding protein phosphorylation and eukaryotic initiation factor-4E release are required for airway smooth muscle hypertrophy

The molecular mechanisms of airway smooth muscle hypertrophy, a feature of severe asthma, are poorly understood. We previously established a conditionally immortalized human bronchial smooth muscle cell line with a temperature-sensitive SV40 large T antigen. Temperature shift and loss of large T cause G1-phase cell cycle arrest that is accompanied by increased airway smooth muscle cell size. In the present study, we hypothesized that phosphorylation of eukaryotic initiation factor-4E (eIF4E)-binding protein (4E-BP), which subsequently releases eIF4E and initiates cap-dependent mRNA translation, was required for airway smooth muscle hypertrophy. Treatment of cells with chemical inhibitors of PI 3-kinase and mammalian target of rapamycin blocked protein synthesis and cell growth while decreasing the phosphorylation of 4E-BP and increasing the binding of 4E-BP to eIF4E, consistent with the notion that 4E-BP1 phosphorylation and eIF4E function are required for hypertrophy. To test this directly, we infected cells with a retrovirus encoding a phosphorylation site mutant of 4E-BP1 (AA-4E-BP-1) that dominantly inhibits eIF4E. Upon temperature shift, cells infected with AA-4E-BP-1, but not empty vector, failed to undergo hypertrophic growth. We conclude that phosphorylation of 4E-BP, eIF4E release, and cap-dependent protein synthesis are required for hypertrophy of human airway smooth muscle cells.     

Estrogen receptor-associated expression of keratinocyte growth factor and its possible role in the inhibition of apoptosis in human breast cancer

Although estrogen is known to play a crucial role in the pathogenesis of breast cancer, the molecular mechanisms underlying the action of estrogen remain elusive. In the present study, we focused on keratinocyte growth factor (KGF) and its receptor (KGFR) in the pathogenesis of breast cancer, as a growth factor mediating estrogen action, since significant roles of KGF were demonstrated in various steroid hormone-dependent tissues. First, using paraffin-embedded specimens from 42 breast cancer patients, we examined expression patterns of KGF and KGFR by both immunohistochemistry using newly generated antibodies and nonradioactive in situ hybridization with T-T dimerized synthetic oligonucleotide probes. We next compared the results with the expression of estrogen receptor (ER) alpha and beta, proliferative activity and apoptotic frequency (TUNEL staining). Also, the similar approaches were taken to analyze the expression and role of KGF in ER-positive (MCF7, ZR-75-1) and ER-negative (SK-BR-3, MDA-MB-231) human breast cancer cell lines in vitro. In the surgical specimens, KGF was expressed in cancer cells as well as stromal cells in 19/42 cases (45%), while KGFR was found in cancer cells in 24/42 cases (57%). The distribution of protein and mRNA in the analysis of both KGF and KGFR expression generally coincided. Moreover, KGF expression was closely associated with the expression of ER alpha, and the coexpression of KGF and KGFR significantly correlated with lower TUNEL index, but not with proliferative activity. In accordance with the in vivo findings, KGF expression was detected only in ER alpha-positive MCF7 and ZR-75-1 cells in vitro. And more importantly, we found the inhibitory effect of KGF upon the induction of apoptosis by anticancer drugs in MCF7 cells. Collectively, our results indicate that ER alpha may be involved in KGF expression, and that KGF may play antiapoptotic roles, rather than mitogenic, in human breast cancer.     

Early rehabilitation program in postmastectomy patients: a prospective clinical trial

The purpose of this study was to determine whether 20 patients who received an early postmastectomy rehabilitation treatment program showed more improvement in range of shoulder motion and functional activities than 13 patients who received instruction for exercise only. Data were obtained at preoperatively, three days after operation, at discharge and at postdischarge one month for each patient on parameters such as range of motion of the ipsilateral shoulder joint, upper extremity circumferential measurements, as well as 10 elements of shoulder function. Postoperatively, both groups showed an increased range of motion of the shoulder joint and improved functional activities, but the group that received postoperative rehabilitation management had a better range of shoulder motion and less difficulty in five items for functional assessment. This study also showed that an early rehabilitation program did not increase postoperative complications. We concluded that an early rehabilitation program or intensive instruction program only by a well-trained physical therapist or physiatrist was beneficial to postmastectomy patients in regaining the function and range of shoulder motion, and significantly better in a rehabilitation group.     

Gain-of-function polymorphism in mouse and human Ltk: implications for the pathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus (SLE), a complex multigenic disease, is a typical antibody-mediated autoimmune disease characterized by production of autoantibodies against a variety of autoantigens and immune complex-type tissue inflammation, most prominently in the kidney. Evidence suggests that genetic factors predisposing to aberrant proliferation/maturation of self-reactive B cells initiate and propagate the disease. In SLE-prone New Zealand Black (NZB) mice and their F1 cross with New Zealand White (NZW) mice, B cell abnormalities can be ascribed mainly to self-reactive CD5+ B1 cells. Our genome-wide scans to search for susceptibility genes for aberrant activation of B1 cells in these mice showed evidence that the gene, Ltk, encoding leukocyte tyrosine kinase (LTK), is a possible candidate. LTK is a receptor-type protein tyrosine kinase, belonging to the insulin receptor superfamily, and is mainly expressed in B lymphocyte precursors and neuronal tissues. Sequence and functional analyses of the gene revealed that NZB has a gain-of-function polymorphism in the LTK kinase domain near YXXM, a binding motif of the p85 subunit of phosphatidylinositol 3-kinase (PI3K). SLE patients also had this type of Ltk polymorphism with a significantly higher frequency compared with the healthy controls. Our findings suggest that these polymorphic LTKs cause up-regulation of the PI3K pathway and possibly form one genetic component of susceptibility to abnormal proliferation of self-reactive B cells in SLE.     

重组人免疫缺陷病毒Ⅰ型逆转录酶的纯化及其动力学性质

目的纯化重组人免疫缺陷病毒Ⅰ型逆转录酶（ＨＩＶ－１ＲＴ），筛选新的ＨＩＶ－１ＲＴ抑制剂。方法在适宜的培养条件下诱导工程菌Ｅ．ｃｏｌｉＪＭ１０９（ＰＫＲＴ２）可高效表达重组人免疫缺陷病毒Ⅰ型（ＨＩＶ－１）逆转录酶（ＲＴ）。应用ＤＥＡＥ－纤维素和磷酸纤维素离子交换柱层析法从细菌裂解液中分离、纯化重组ＲＴ。结果１升细菌培养液可得到１．１ｍｇ产物。ＳＤＳ－聚丙烯酰胺凝胶电泳分析显示所纯化的重组ＲＴ为由两个分子量分别为６６ｋＤ和５１ｋＤ的亚基组成的杂二聚体。酶活性测定结果表明，经纯化的重组ＲＴ具有很高的逆转录酶活性（比活力为１．４×１０４ｕｍｇ）。结论本文通过对ＲＴ反应条件的研究，优化了ＲＴ反应系统，并测定了磷甲酸钠（ＰＦＡ）对重组ＲＴ的抑制效应，结果表明ＰＦＡ对重组ＲＴ的抑制反应动力学机制与天然ＲＴ相同，从而进一步说明用此法纯化的重组ＲＴ可直接用于抗ＨＩＶ药物的筛选与评价。     

PCR-RFLP based molecular typing of enteroviruses isolated from patients with aseptic meningitis in Korea

Summary.  We have evaluated PCR–RFLP as a practical method for rapid typing of enteroviruses causing aseptic meningitis in Korea. Through blind examination of 80 clinical isolates from patients with aseptic meningitis, we have compared the results of conventional serotyping with PCR–RFLP based genotyping, which was developed for this study. Among the 80 case isolates, which had been previously typed by routine neutralization test, only 42 cases (52.5%) were matched with typing by PCR–RFLP. The result clearly demonstrated that the enterovirus serotype does not coincide with the genotype. Therefore, the classification of enteroviruses by genotyping with PCR–RFLP, although rapid and simple, may be complicated by regional or seasonal differences. However, the PCR–RFLP method developed in this study is applicable to the epidemiological study of enteroviruses when regional or seasonal differences exist, and is useful in identifying the source of an infection. Received August 6, 2001; accepted April 15, 2002 Published online July 10, 2002