Oncogene-induced basement membrane invasiveness in human mammary epithelial cells

Expression of the intermediate filament protein vimentin, and loss of the cellular adhesion protein uvomorulin (E-cadherin) have been associated with increased invasiveness of established human breast cancer cell linesin vitro andin vivo. In the current study, we have further examined these relationships in oncogenically transformed human mammary epithelial cells. A normal human mammary epithelial strain, termed 184, was previously immortalized with benzo[a]pyrene, and two distinct sublines were derived (A1N4 and 184B5). These sublines were infected with retroviral vectors containing a single or two oncogenes of the nuclear, cytoplasmic, and plasma membrane-associated type (v-ras ^H, v-ras ^Ki, v -mos, SV40T and c -myc). All infectants have been previously shown to exhibit some aspects of phenotypic transformation. In the current study, cellular invasiveness was determinedin vitro using Matrigel, a reconstituted basement membrane extract. Lineage-specific differences were observed with respect to low constitutive invasiveness and invasive changes after infection withras, despite similarras-induced transformation of each line. Major effects on cellular invasiveness were observed after infection of the cells with two different oncogenes (v-ras ^H + SV40T and v -ras ^H + v -mos). In contrast, the effects of single oncogenes were only modest or negligible. All oncogenic infectants demonstrated increased attachment to laminin, but altered secretion of the 72 kDa and 92 kDa gelatinases was not associated with any aspect of malignant progression. Each of the two highly invasive double oncogene transformants were vimentinpositive and uvomorulin-negative, a phenotype indicative of the epithelial-mesenchymal transition (EMT) previously associated with invasiveness of established human breast cancer cell lines. Weakly invasive untransformed mammary epithelial cells in this study were positive for both vimentin and uvomorulin, suggesting that uvomorulin may over-ride the otherwise vimentin-associated invasiveness.     

Urofacial (Ochoa) syndrome

Between 1965 and 1986 we saw 36 children with enuresis and urinary tract infection in association with “inversion” of facial expression when laughing. Urologic work-up of these patients disclosed characteristic findings of mild neuropathic bladder in all cases, with severe urinary tract damage in most of them. The clear association of distortion in facial expression and neuropathic bladder with resultant damage to the genitourinary tract should prompt urological evaluation of individuals with “inversion” of facial expression. About two thirds of the patients also had moderate to severe constipation. We suggest the term urofacial syndrome for this disorder. The occurrence of the disorder in multiple sibs, normal parents, increased parental consanguinity, and equal sex ratio indicate autosomal recessive inheritance.     

Use of in vivo-induced antigen technology for identification of Escherichia coli O157:H7 proteins expressed during human infection

Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumvents the need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens.     

Levetiracetam Inhibits both ryanodine and IP3 receptor activated calcium induced calcium release in hippocampal neurons in culture

Epilepsy affects approximately 1% of the population worldwide, and there is a pressing need to develop new anti-epileptic drugs (AEDs) and understand their mechanisms of action. Levetiracetam (LEV) is a novel AED and despite its increasingly widespread clinical use, its mechanism of action is as yet undetermined. Intracellular calcium ([Ca²⁺]_i) regulation by both inositol 1,4,5-triphosphate receptors (IP3R) and ryanodine receptors (RyR) has been implicated in epileptogenesis and the maintenance of epilepsy. To this end, we investigated the effect of LEV on RyR and IP3R activated calcium-induced calcium release (CICR) in hippocampal neuronal cultures. RyR-mediated CICR was stimulated using the well-characterized RyR activator, caffeine. Caffeine (10 mM) caused a significant increase in [Ca²⁺]_i in hippocampal neurons. Treatment with LEV (33 μM) prior to stimulation of RyR-mediated CICR by caffeine led to a 61% decrease in the caffeine induced peak height of [Ca²⁺]_i when compared to the control. Bradykinin stimulates IP3R-activated CICR—to test the effect of LEV on IP3R-mediated CICR, bradykinin (1 μM) was used to stimulate cells pre-treated with LEV (100 μM). The data showed that LEV caused a 74% decrease in IP3R-mediated CICR compared to the control. In previous studies we have shown that altered Ca²⁺ homeostatic mechanisms play a role in seizure activity and the development of spontaneous recurrent epileptiform discharges (SREDs). Elevations in [Ca²⁺]_i mediated by CICR systems have been associated with neurotoxicity, changes in neuronal plasticity, and the development of AE. Thus, the ability of LEV to modulate the two major CICR systems demonstrates an important molecular effect of this agent on a major second messenger system in neurons.     

Genetic testing for inherited eye conditions in over 6,000 individuals through the eyeGENE network

Genetic testing in a multisite clinical trial network for inherited eye conditions is described in this retrospective review of data collected through eyeGENE®, the National Ophthalmic Disease Genotyping and Phenotyping Network. Participants in eyeGENE were enrolled through a network of clinical providers throughout the United States and Canada. Blood samples and clinical data were collected to establish a phenotype:genotype database, biorepository, and patient registry. Data and samples are available for research use, and participants are provided results of clinical genetic testing. eyeGENE utilized a unique, distributed clinical trial design to enroll 6,403 participants from 5,385 families diagnosed with over 30 different inherited eye conditions. The most common diagnoses given for participants were retinitis pigmentosa (RP), Stargardt disease, and choroideremia. Pathogenic variants were most frequently reported in ABCA4 (37%), USH2A (7%), RPGR (6%), CHM (5%), and PRPH2 (3%). Among the 5,552 participants with genetic testing, at least one pathogenic or likely pathogenic variant was observed in 3,448 participants (62.1%), and variants of uncertain significance in 1,712 participants (30.8%). Ten genes represent 68% of all pathogenic and likely pathogenic variants in eyeGENE. Cross‐referencing current gene therapy clinical trials, over a thousand participants may be eligible, based on pathogenic variants in genes targeted by those therapies. This article is the first summary of genetic testing from thousands of participants tested through eyeGENE, including reports from 5,552 individuals. eyeGENE provides a launching point for inherited eye research, connects researchers with potential future study participants, and provides a valuable resource to the vision community.     

ASK1 associates with troponin T and induces troponin T phosphorylation and contractile dysfunction in cardiomyocytes

There is increasing support for the idea that excessive production of proinflammatory mediators such as tumor necrosis factor (TNF) and reactive oxygen species (ROS) contribute to the pathogenesis of cardiac dysfunction. However, the mechanisms by which cytokine/ROS production mediates cardiac dysfunction have not been established. Given that apoptosis signal-regulating kinase 1 (ASK1) is highly expressed in cardiac muscle and that ASK1 is an important mediator in the signaling pathways induced by tumor necrosis factor, interleukin-1, and ROS, we used the yeast two-hybrid system with ASK1 as bait to identify ASK1 substrates from a human heart cDNA library. The cDNA encoding the cardiac troponin T (cTnT) was isolated. ASK1 specifically interacted with cTnT, but not cTnI, in vitro and in vivo via the C-terminal ASK1 domain. ASK1 specifically phosphorylated cTnT in vitro and in vivo. Mutations in cTnT (T194/S198) at an ASK1-phosphorylation consensus sequence significantly reduced phosphorylation by ASK1. ROS-induced ASK1 activation, cTnT phosphorylation, and contractile dysfunction in cardiomyocytes showed similar kinetics. Moreover, overexpression of constitutively active ASK1 induces cTnT phosphorylation and inhibits shortening and calcium transient in adult cardiomyocytes. We conclude that ASK1 plays an important role in regulation of cardiac contractile function by phosphorylating cTnT and may participate in cytokine/ROS-induced pathogenesis of cardiomyopathy and heart failure.     

Elevated Fecal Candida Counts in Patients with Antibiotic-Associated Diarrhea: Role of Soluble Fecal Substances

To assess the role of soluble fecal substances in the elevation of fecal Candida counts in patients with antibiotic-associated diarrhea (AAD), we investigated the growth of Candida albicans in vitro in serially diluted stool fluids from patients with AAD and healthy subjects. There were significantly higher Candida albicans counts in stool fluids diluted 1:10 from AAD patients than in healthy subjects and the phosphate-buffered saline growth control, which may be due to reduced soluble Candida inhibitors and increased availability of growth factors and nutrients.     

Muscularity in adult humans: Proportion of adipose tissue‐free body mass as skeletal muscle

Muscularity, or the proportion of adipose tissue‐free body mass (ATFM) as skeletal muscle (SM), provides valuable body composition information, especially for age‐related SM loss (i.e., sarcopenia). Limited data from elderly cadavers suggest a relatively constant SM/ATFM ratio, 0.540 ± 0.046 for men (mean ± SD, n = 6) and 0.489 ± 0.049 for women (n = 7). The aim of the present study was to examine the magnitude and constancy of the SM/ATFM ratio in healthy adults. Whole‐body SM and ATFM were measured using multi‐scan magnetic resonance imaging. The SM/ATFM ratio was 0.528 ± 0.036 for men (n = 139) and 0.473 ± 0.037 for women (n = 165). Multiple regression analysis indicated that the SM/ATFM ratio was significantly influenced by sex, age, body weight, and race. The four factors explained 50% of the observed between individual variation in the SM/ATFM ratio. After adjusting for age, body weight, and race, men had a larger SM/ATFM ratio than women. Both older men and women had a lower SM/ATFM ratio than younger subjects, although the relative reduction was greater in men. After adjustment for sex, age, and body weight, there were no significant differences in the SM/ATFM ratios between Asian, Caucasian, and Hispanic subjects. In contrast, African‐American subjects had a significantly greater SM/ATFM ratio than subjects in the other three groups. In addition, the SM/ATFM ratio was significantly lower in AIDS patients than corresponding values in healthy subjects. Am. J. Hum. Biol. 13:612–619, 2001. © 2001 Wiley‐Liss, Inc.