MiR-181a confers resistance of cervical cancer to radiation therapy through targeting the pro-apoptotic PRKCD gene.

The purpose of this study was to define the roles of miR-181a in determining sensitivity of cervical cancer to radiation therapy, to explore the underlying mechanism and to evaluate the potential of miR-181a as a biomarker for predicting radio-sensitivity. Tumor specimens from 18 patients with a histological diagnosis of squamous cervical carcinoma (stage IIIB) were used in the micro-RNA profiling and comparison. These patients never received any chemotherapy before radiation therapy. Human cervical cancer cell lines, SiHa and Me180, were used in vitro (cell culture) and in vivo (animal) studies. Transfection of tumor cells with the mimic or inhibitor of miR-181a, and reporter gene assay, were performed to investigate the role of miR-181a in determining radio-sensitivity and the target gene. Higher expression of miR-181a was observed in human cervical cancer specimens and cell lines that were insensitive to radiation therapy, as compared with sensitive cancer specimens and the cell lines. We also found that miR-181a negatively regulated the expression of PRKCD, a pro-apoptotic protein kinase, via targeting its 3'-untranslated region (UTR), thereby inhibiting irradiation-induced apoptosis and decreasing G2/M block. The role of miR-181a in conferring cellular resistance to radiation treatment was validated both in cell culture models and in mouse tumor xenograft models. The effect of miR-181a on radio-resistance was mediated through targeting the 3'-UTR of PRKCD gene. Thus, the expression level of miR-181a in cervical cancer may serve as a biomarker for sensitivity to radiation therapy, and targeting miR-181a may represent a new approach to sensitizing cervical cancer to radiation treatment.

Molecular characterization of Newcastle disease viruses isolated from recent outbreaks in Taiwan.

A reverse transcription-polymerase chain reaction (RT-PCR) was described which amplified a portion of the F and HN genes of Newcastle disease virus (NDV) isolated from recent outbreaks in Taiwan. The F protein plays an important role in determining the virulence of NDV strains. Sequencing of a region specifying the F protein cleavage site was therefore undertaken and this verified the correlation between deduced amino sequences and pathogenicity. Analysis of the deduced amino acid sequences of the F protein cleavage site showed that all recent Taiwanese isolates in 1999 were velogenic viruses. All the virulent viruses have the amino acid sequence 112RRQKR116 for the C-terminus of the F2 protein and phenylanine (F) at the N-terminus of the F1 protein, residue 117. A phylogenetic tree based on the nucleotide sequences of the F gene revealed that recent Taiwanese NDV isolates responsible for recent outbreaks were classified into two distant genotypes (VI and VII). Genotype VI virus is the first finding in Taiwan and has a highly genetic similarity to European isolates, suggesting that they might have originated from a common ancestor.

Detection of Mycobacterium bovis lymphocyte stimulating antigens in culture filtrates of a recombinant Mycobacterium smegmatis cosmid library.

Culture filtrates derived from a Mycobacterium bovis cosmid library in Mycobacterium smegmatis were screened for bovine lymphocyte stimulatory antigens using peripheral blood mononuclear cells (PBMC) from cattle vaccinated with a low dose of Mycobacterium bovis BCG. Lymphocyte proliferation and interferon-gamma (IFN-gamma) production were used as cellular response markers for antigen recognition. In the primary screen, approximately 28% of all culture filtrates (CF) stimulated responses by PBMC from at least two out of four vaccinated cattle. In one of these CF, the M. bovis Ag85-B antigen was detected by Western-blot analysis. Despite heterogeneous lymphocyte responses of the animals, twenty-four of the culture filtrates stimulated lymphocyte proliferation and IFN-gamma production from at least six out of eight vaccinated animals in a secondary screen. Analysis of the cosmid DNA associated with these positive CF demonstrated that several contained homologous DNA sequences. It appears that the lymphocyte screening has detected M. bovis antigens that are immuno-dominant in cattle vaccinated with M. bovis BCG.

[Long-term results of surgical treatment of intractable Menière's disease for control of vertigo].

OBJECTIVE: To acquire the long-term (an average of over 10 years follow-up) results of surgical treatment of patients with Meniere's disease for control of vertigo. METHOD: Endolymphatic sac surgery was done in 6 cases (1 case of endolymphatic sac decompression and 5 cases of sac-mastoid shunting) and translabyrinth vestibular neurotomy was operated on 6 cases. RESULT: Complete relief from symptoms of vertigo occurred in the 6 cases through vestibular neurotomy and there were no recurrence in 11-13 years follow-up. Three of 6 cases with endolymphatic sac surgery had complete control of vertigo and were cured clinically. Symptoms of vertigo of the rest 3 cases were effectively controlled. CONCLUSION: Endolymphatic sac surgery and translabyrinth vestibular neurotomy are effective way for control of vertigo of patients with intractable Meniere's disease. Translabyrinth vestibular neurotomy are more effective for control of vertigo for appropriately selected patients.

[Transantral orbit decompression to malignant ophthalmopathy].

OBJECTIVE: To recognize the indication and the value of transantral orbit decompression to malignant ophthalmopathy. METHOD: 9 eyes of 5 cases undergone transantral orbital decompression from November 1986 to October 1998 were reviewed. RESULT: After 3 months-2 years of follow-up. We found this approach successfully improved visual function of all 5 cases. Average proptosis reduction was 5 mm. The eye lids could be closed in 4 of the 5 cases and the palpebral fissures was decreased 1-2 mm in 1 of the 5 cases. Preoperative exposure keratitis resolved in both of the 2 cases. Diplopia was cured in both of the 2 cases. Secondary glucorna also recovered in both the 2 cases. Disturbance of ocular motility in both of the 2 cases improved postoperatively and preoperative visual acurity worsen also improved in both of the 2 cases. CONCLUSION: It has been suggested that the transantral decompression of the orbit provided a safe, effective therapeutic modality for patients with vision-threatening or major cosmetic problem.

Leishmania mexicana: extracellular proton concentration is a key regulator of cysteine proteinase CPb expression.

The purpose of this work was to determine which parameters trigger expression of proteins that are potentially important for the differentiation of Leishmania mexicana from the promastigote to the amastigote stage. To this effect, a protein-free axenic incubation system was used that supported the differentiation of L. mexicana promastigotes into amastigotes at 33 degreesC and at acidic pH. The predominant modification detected in SDS-PAGE patterns of extracted soluble proteins was the appearance in parasites cultured for 4 days of a strong 28-kDa protein band that displayed the same position and intensity as seen in amastigotes extracted from a mouse lesion. These molecules exhibited in gelatin gels the typical lytic pattern of cysteine proteinases (CPs) and were shown to belong to the CPb family, as further demonstrated by N-terminal amino acid sequencing. The expression of these enzymes was quantified by their lytic activity on the fluorogenic Z-F-R-AMC CP substrate. When the parasites were incubated at 33 degreesC for 3 days at various initial pHs, CPb started to be induced when the pH dropped below 5. When comparing cultures maintained at 26 or 33 degreesC for 3 days, it was seen that a rise in extracellular proton concentration (to pH 4.2-4.6) resulted in production of CPb at both temperatures (around 20-fold over the concentration measured in promastigotes cultured at 26 degreesC, pH >6). These results demonstrate that extracellular proton concentration is a key regulator of cysteine proteinase CPb synthesis and that an increase in temperature is neither necessary nor sufficient for the expression of this enzyme.

DDC-4, an apoptosis-associated gene, is a secreted frizzled relative.

The differential display method has been used in our laboratory as a coincidence analysis to isolate genes expressed in common in each of three different rat tissues undergoing physiological apoptosis: mammary gland, ovarian corpus luteum and ventral prostate. The most interesting of these isolates, DDC-4, shows a clear association with apoptosis, its expression being confined to these three organs, and only during their involution. Using DDC-4 as probe, we screened a rat ovarian cDNA library to obtain full-length isolates. One isolate, Y81 clone 40, gives rise to a protein of approximately 40 kDa with coupled in vitro transcription/translation. Sequencing of this clone indicates an open reading frame of 1044 nucleotides encoding a protein of 39.7 kDa with a putative signal sequence. This clone exhibits a high homology with the cysteine-rich domain, i.e. the ligand-binding domain, of the fizzled gene family originally defined as tissue polarity genes in Drosophila. The homology of Y81 clone 40 is most extensive with the newly described secreted frizzled relatives, the frzb subfamily.

Apoptosis in the rat mammary gland and ventral prostate: detection of cell death-associated genes using a coincident-expression cloning approach.

Apoptosis plays a striking role in the hormone-dependent involution of the mammary gland, but it has proved difficult to distinguish between the 'cell death' associated genes and the 'tissue remodelling' genes which are expressed concurrently. To identify cell death-associated genes, we have established a 'coincidence analysis' based on the previously described 'RNA differential display' method of Liang and Pardee (1992). Coincidence analysis allows the detection of genes expressed during related processes in different organs and was employed here to identify transcripts in which expression patterns are seen to be associated with apoptosis during involution of both rat mammary- and the ventral prostate glands. That the coincidence analysis is a promising approach can be seen from the fact that while widely accepted apoptosis markers such as transglutaminase (Fesus et al, 1987; Strange et al, 1992) and sulfated glycoprotein-2 (Buttyan et al, 1989; Strange et al, 1992; Guenette et al 1994) exhibited similar expression in both regressing tissues, transcription of tissue remodelling enzymes was minimal in the involuting prostate. We describe here the characteristics of five clones isolated which show coincident expression during programmed cell death in mammary and prostate tissues. Partial sequence analysis revealed for three clones high homologies with previously described genes; the putative rat homolog of the growth arrest gene gas-1 (Schneider et al, 1988; Del Sal et al, 1992), an homolog of the mouse 'Integrin Associated Protein' (IAP) (Brown et al, 1990; Lindberg et al, 1993) and the sequence encoding for the 'Allograft Inflammatory Factor' AIF-1 (Autieri et al, 1995; Utans et al, 1995). One clone displayed homology with an expressed human sequence tag and one clone unrelated to any known DNA sequence was isolated. The expression of these genes in involuting rat mammary and ventral prostate, was correlated with that in other organs and in situ hybridization was applied to establish that the secretory epithelial cells which undergo programmed cell death are the site of elevated expression during the course of involution. Furthermore, we conclude that the coincidence analysis approach described here could be easily applied to facilitate the characterization of gene expression i.e. for the detection and comparison of hormonally regulated genes in different organs.

Isolation of cell death-associated cDNAs from involuting mouse mammary epithelium.

Although apoptosis is important in determining cell fate and maintaining tissue homeostasis, the initiation and control of apoptotic cell death in epithelium is not well understood. Post-lactationai involution of the mammary gland provides both an important developmental process and a normal physiological setting for studying apoptosis of epithelium. We used a differential screening strategy, based on previous studies correlating morphology with gene expression and nucleic acid integrity during mammary gland involution, to isolate genes involved in the regulation and execution of apoptotic cell death in regressing mammary epithelium. This screening strategy yielded a large number of genes the expression of which is significantly altered during mammary gland involution. These include genes associated with cell death processes, tissue remodelling and mesenchymal differentiation. In addition, a number of novel genes have been isolated. We have used Northern analysis and in situ hybridisation to study the expression of a selection of these putative death-associated genes during post-lactational mouse mammary gland involution.

Protein kinase A and AP-1 (c-Fos/JunD) are induced during apoptosis of mouse mammary epithelial cells.

At weaning the mammary gland undergoes a reductive remodelling process (involution) which is associated with the cessation of milk protein gene expression and programmed cell death of milk-producing epithelial cells. Elevated nuclear protein kinase A (PKA) activity was observed from one day post-lactation, paralleled by increased c-fos, junB, junD and to a lesser extent c-jun mRNA levels. AP-1 DNA binding activity was transiently induced and the AP-1 complex was shown to consist principally of cFos/JunD. Oct-1 DNA binding activity and Oct-1 protein were gradually lost from the gland over the first 4 days of involution, whereas Oct-1 mRNA levels remained unchanged. Comparing nuclear extracts from normal mammary glands with nuclear extracts from glands which had been cleared of all epithelial cells 3 weeks after birth, revealed that PKA activation, AP-1 induction and Oct-1 inactivation all are dependent on the presence of the epithelial compartment. The increased Fos/Jun expression and the inactivation of Oct-1 may be consequences of the increased PKA activity. A similar induction of AP-1 (cFos/JunD) was also observed in the involuting rat ventral prostate pointing to a possible role for AP-1 in programmed cell death.

Geriatric medicine in the United States. The current activities of former trainees.

To improve the health care received by frail older persons, an effort has been made in the United States to increase the number of physicians trained in geriatric medicine and geropsychiatry. The goal of training has been to create leaders in education, research, and patient care. To assess the progress of this effort, we surveyed physicians (284 in geriatric medicine and 91 in geropsychiatry) who graduated from U.S. geriatrics fellowship programs. Responses were obtained from 224 medicine (79% response) and 59 psychiatry fellows (65% response). Sixty-five percent of former geriatric medicine fellows report spending 10% or less time on teaching; 44% report doing no research, and 44% report spending more than half their time in patient care. Compared to other primary care specialties, the geriatricians reported caring for larger proportions of older patients and spending more time per patient visit. However, their role in teaching, research, and long-term care is minimal.

Discriminable effects of phencyclidine analogs evaluated by multiple drug (PCP versus OTHER) discrimination training.

This study tested structural analogs of phencyclidine (PCP) using drug discrimination procedures to determine which analogs produced discriminable effects similar to those of PCP. It also tested the utility of multiple-drug discrimination training (PCP versus other drugs or saline) as a method for increasing the specificity produced by training. All discrimination training took place in two-lever operant compartments using FR-10 reinforcement of presses on the correct lever. During training, rats were required to concurrently discriminate PCP from one or more other drug conditions. Rats in group 1 discriminated PCP (lever 1) versus saline (lever 2). Rats in group 2 discriminated PCP (lever 1) versus saline, fentanyl, phenobarbital, amphetamine, or mescaline (lever 2). In both groups 1 and 2, the required discriminations were rapidly learned. The percentage of PCP choices and the ED50 doses obtained during tests for generalization did not differ significantly in groups 1 and 2. Drugs to which responding on the PCP lever generalized included 1-[1-(2-thienyl)cyclohexyl]piperidine, N-ethyl-1-phenylcyclohexylamine, 1-phenylcyclohexylamine, ketamine, 1-(1-phenylcyclohexyl)morpholine, 1-[1-(2-thienyl)cyclohexyl]morpholine, N,N-diethyl-1-phenylcyclohexylamine, N-(iso-propyl)-1-phenylcyclohexylamine, N-methyl-1-phenylcyclohexylamine, N-(n-propyl)-1-phenylcyclohexylamine, Dextrorphan, (dl)-N-allyl-N-normetazocine, N-N-dimethyl-1-phenylcyclohexylamine, N-(n-butyl)-1-phenylcyclohexylamine, 1-[1-(2-thienyl)cyclohexyl]pyrrolidine, and N-(s-butyl)-1-phenylcyclohexylamine, in agreement with previous reports. Rats in group 3 discriminated PCP (lever 1) versus saline, cyclazocine, dextrorphan, phenobarbital, or mescaline (lever 2).(ABSTRACT TRUNCATED AT 250 WORDS)

[Surgical treatment of facial palsy].

The results of surgical treatment of 46 cases of facial nerve paralysis from 1974 to 1987 were reported. Among them, facial nerve decompression comprised 36 cases and facial nerve grafting 10 cases. The causes of facial nerve paralysis were head trauma in 13, surgical injury in 22, otitis media in 4, facial neurofibroma in 2 and Bell's palsy in 5. Thirty-six cases were followed up from 0.5 to 6.5 years. Twenty-five cases (69.5%) had totally recovered, 7 cases (19.4%) partially recovered and 4 cases (11.1%) had no recovered. The result of facial nerve decompression was better than that of facial nerve grafting. The indication, the significance of nerve excitability test and the influencing factors were discussed.