GATA3 inhibits lysyl oxidase-mediated metastases of human basal triple-negative breast cancer cells.

Discovery of mechanisms that impede the aggressive and metastatic phenotype of human basal triple-negative-type breast cancers (BTNBCs) could provide novel targets for therapy for this form of breast cancer that has a relatively poor prognosis. Previous studies have demonstrated that expression of GATA3, the master transcriptional regulator of mammary luminal differentiation, can reduce the tumorigenicity and metastatic propensity of the human BTNBC MDA-MB-231 cell line (MB231), although the mechanism for reduced metastases was not elucidated. We demonstrate through gene expression profiling that GATA3 expression in 231 cells resulted in the dramatic reduction in the expression of lysyl oxidase (LOX), a metastasis-promoting, matrix-remodeling protein, in part, through methylation of the LOX promoter. Suppression of LOX expression by GATA3 was further confirmed in the BTNBC Hs578T cell line. Conversely, reduction of GATA3 expression by small interfering RNA in luminal BT474 cells increased LOX expression. Reconstitution of LOX expression in 231-GATA3 cells restored metastatic propensity. A strong inverse association between LOX and GATA3 expression was confirmed in a panel of 51 human breast cancer cell lines. Similarly, human breast cancer microarray data demonstrated that high LOX/low GATA3 expression is associated with the BTNBC subtype of breast cancer and poor patient prognosis. Expression of GATA3 reprograms BTNBCs to a less aggressive phenotype and inhibits a major mechanism of metastasis through inhibition of LOX. Induction of GATA3 in BTNBC cells or novel approaches that inhibit LOX expression or activity could be important strategies for treating BTNBCs.

A novel fibrotic disorder associated with increased dermal fibroblast proliferation and downregulation of genes of the microfibrillar network.

Clinical evaluation of a young woman with subcutaneous fibrotic nodules, progressive distal joint contractures and marfanoid stature revealed a previously unrecognized fibrotic disorder characterized by several unique phenotypic features and some features overlapping with known disorders. Mutational analysis of the FBN1 and FBN2 genes excluded Marfan syndrome and congenital contractural arachnodactyly. MMP2 gene sequence analysis excluded multicentric osteolysis, nodulosis and arthropathy. The lack of mutations within the MAGP2 gene also excluded an MAGP2-associated disorder. In order to establish the mechanistic basis for the severe skin pathology noted in this patient, we performed transcriptional profiling of dermal fibroblasts, and candidate gene expression studies in conjunction with immunocytochemistry and cell-based and functional assays. Data from these experiments have further excluded any previously recognized fibrotic disorder and identified a unique pattern of gene expression in this patient consistent with progressive fibrosis. The pathogenic mechanisms included persistent proliferation of dermal fibroblasts in coexistence with a disarray of the microfibrillar network. Collagen accumulation, moreover, could be linked to extensive crosslinking resulting from increased activities of lysyl oxidases (LOX and LOXL), and lack of remodelling due to deficiencies in collagenolytic matrix metalloproteinases. The disorder may represent a novel syndrome in which transforming growth factor-ß1-independent dermal fibrosis, unlike known microfibrillar disorders caused by single gene deficiencies, associates with a disarray of the microfibrillar network.

Active lysyl oxidase (LOX) correlates with focal adhesion kinase (FAK)/paxillin activation and migration in invasive astrocytes.

The extracellular matrix (ECM) plays a critical role during the development and invasion of primary brain tumours. However, the function of ECM components and signalling between a permissive ECM and invasive astrocytes is not fully understood. We have recently reported the ECM enzyme, lysyl oxidase (LOX), in the central nervous system and observed up-regulation of LOX in anaplastic astrocytoma cells. While the catalytic function of LOX is essential for cross-linking of ECM proteins, we also reported that LOX induced invasive and metastatic properties in breast tumour epithelial cells through hydrogen peroxide-mediated FAK/Src activation. In this study, we tested the hypothesis that active LOX is expressed in anaplastic astrocytes and promotes FAK activation and invasive/migratory behaviour. Results demonstrate that increased expression and activity of LOX positively correlated with invasive phenotype of malignant astrocytoma cell lines. Immunohistochemistry detected increased LOX within tumour cells and ECM in grade I-IV astrocytic neoplasm compared with normal brain and coincidence of increased LOX with the loss of glial fibrillary acidic protein in higher-grade tumours. Increased active LOX in invasive astrocytes was accompanied by phosphorylation of FAK[Tyr576] and paxillin[Tyr118]; furthermore, both FAK and paxillin tyrosine phosphorylation were diminished by beta-aminopropionitrile inhibition of LOX activity and depletion of H(2)O(2) via catalase treatment. Additionally, we provide evidence that in astrocytes, LOX is likely processed by bone morphogenic protein-1 and LOX activity might be further stimulated by the expression of fibronectin in these cells. These results demonstrate an important LOX-mediated mechanism that promotes migratory/invasive behaviour of malignant astrocytes.

Structural and functional diversity of lysyl oxidase and the LOX-like proteins.

Lysyl oxidase (LOX) and four lysyl oxidase-like proteins, LOXL, LOXL2, LOXL3 and LOXL4, each contain a copper binding site, conserved lysyl and tyrosyl residues that may contribute to quinone co-factor formation, and a cytokine receptor-like domain. Each protein differs mainly in their N-terminal sequence, which may confer individual functions. Processing of the LOX proteins by BMP-1 and possibly other mechanisms may result in multiple functional forms. Splicing, reported for LOXL3, may also generate additional variants with unique functions. Each LOX, with its individual, developmentally regulated tissue and cell-specific expression and localization, results in a complex structural and functional variation for the LOX amine oxidases. The presence of only two LOX-like proteins in Drosophila, each with distinct spatial and temporal expression, allows for the assignment of individual function to one of these amine oxidases. Comparative expression analysis of each LOX protein is presented to help determine their functional significance.

The responsibilities and activities of internal medicine clerkship directors.

PURPOSE: To characterize the responsibilities, activities, and scholarly productivity of internal medicine clerkship directors (CDs). METHODS: In 1999, internal medicine CDs from 122 U.S. medical schools and one Canadian medical school were surveyed. The instrument asked about the CDs' demo-graphics, workloads, clerkship characteristics, and scholarly productivity. RESULTS: The response rate was 89%; 72% of the respondents were men. Mean age was 45 years, mean time as CD was 6.5 years, and 58% of the CDs had completed fellowship training. The CDs spent 28% of their professional time on the clerkship, three half days weekly in clinic, and three months on inpatient services. The CDs had published a mean of 2.2 (range 0-20) articles and received a mean of 0.7 (range 0-4) grants. Similar factors were associated with publishing articles and receiving grants; gender (men), < or = three clinic half days weekly, fellowship training, having a faculty development program, teaching other courses, and discussing expectations with their department chairs. In a multivariate analysis, fellowship training, clinic half days, teaching other courses, and discussing expectations explained 22% of the variance for papers published. For grants received, a model with gender, clinic half days, a faculty development program, discussing expectations, and teaching other courses explained 35% of the variance. CONCLUSIONS: An internal medicine CD invests significant effort administering the clerkship and contributing to clinical and educational activities. The factors associated with successful scholarship may be useful for fostering CDs' academic careers.

The impact of the location and structure of an ambulatory rotation on cognitive knowledge and performance.

With the increasing shift to community-based ambulatory education, it is essential to gain a better understanding of the impact of these changes. To assess the impact of the location and structure of an ambulatory internal medicine clerkship rotation on cognitive knowledge and clinical performance, students were assigned to one of the following: (a) a multidisciplinary ambulatory clerkship (MAC), (b) the office of a community-based general internist, or (c) a university-based internal medicine ambulatory clinic. The groups were compared on the internal medicine clerkship examination and preceptor ratings controlling for introduction to clinical medicine course performance via analysis of covariance. MAC students were rated lower than the other two groups by their preceptors. There were no other statistically significant differences. The structure and location of the rotation had little impact on cognitive knowledge. The impact of the structure of the rotation on clinical performance is less clear suggesting that further research is needed.

Creating and evaluating an independent ambulatory internal medicine clerkship.

Patient care is shifting from an inpatient setting to an ambulatory setting. Despite this shift, most internal medicine clerkships provide the majority of medical student training in inpatient settings or in university tertiary care clinics, which are not representative of patient care in a community setting. We created a separate ambulatory clerkship that used volunteer community faculty at local and distant sites. The steps involved are described here, including finding time within the clerkship, reaching consensus within the department, defining the curriculum, identifying sites, and developing preceptors. Various parameters were measured to ensure quality in educational design. Comparisons of the 1-year pilot program, the full implementation program, and the inpatient program revealed that use of community sites does not affect cognitive knowledge acquisition but does influence students' satisfaction level.

Setting standards and defining quality of performance in the validation of a standardized-patient examination format.

PURPOSE: To evaluate whether written standards increase the reproducibility of a physician-facilitated station in an objective structured clinical examination (OSCE) designed to assess history, physical-examination, and communication skills. METHOD: The OSCE examination at the University of Texas Medical Branch-Galveston consists of ten eight-minute stations. Six of these stations consist of three History, Physical-examination, Problem-solving, and Plan (HPPP) station pairs. Each existing clinical-problem HPPP station was given to two content experts to develop standards for faculty rating scales appropriate for the evaluation of third-year medical students. Three pairs of faculty members were used to determine interrater reliability by scoring videotapes of three HPPP stations' presentation and problem-solving components. Faculty pairs scored tapes of 15 students without using standards and tapes of 15 students using the standards developed. Differences between the reliabilities without and with the standards were tested for significance using Fisher's R to Z transformation. The reproducibility and standard error of measurement (SEM) were extrapolated for increasing amounts of testing time. The HPPP component scores were also correlated with the written examination scores and preceptors' ratings. Data were obtained from the three HPPP stations used in the 1995-96 internal medicine clerkship SP examination. RESULTS: In all, 196 students completed the OSCE examination. The standards developed improved interrater reliability and reached statistical significance (p < .01) for one HPPP station. Reproducibility for the presentation and problem-solving components of the HPPP stations were > .80 after five hours of testing. The problem-solving component correlated at .37 and .19 with written examinations and with ward grades, respectively. CONCLUSION: The data from this study suggest that standards increase the reproducibility of presentation and problem-solving components of an OSCE to a level as high as, or higher than, that associated with the history, physical-examination, and communication components of traditional standardized-patient examinations.

In vitro stability of ranitidine hydrochloride in enteral nutrient formulas.

OBJECTIVE: To determine the chemical stability and physical compatibility of ranitidine in enteral nutrient formulas. MEASUREMENTS: A stability-indicating HPLC assay was used to measure the recovery of ranitidine from tablet (dissolved in water) or syrup after up to 24 hours of in vitro incubation in a variety of enteral nutrient formulas. Ranitidine binding to components of the formulas was measured after ultrafiltration. RESULTS: Eight enteral nutrient formulas were studied, and more than 90% of added ranitidine was recovered from each formula after 24 hours. The amount of ranitidine bound to components of the formulas varied between 8% and 29%. No gross physical incompatibilities were seen and the pH of each formula changed by less than 0.1 pH units over 24 hours. CONCLUSIONS: Ranitidine from either tablet or syrup was stable in the enteral nutrient formulas studied. Administration of ranitidine by admixture into these enteral formulas may be feasible.

Nonantibiotic therapy and pharmacotherapy of acute infectious diarrhea.

The most important aspect of nonantibiotic treatment of diarrhea is to prevent or repair dehydration and thus prevent significant morbidity or death. Although intravenous fluid therapy is effective, it is expensive and requires personnel and equipment. ORT has saved millions of lives in emerging nations and should be more commonly used in developed countries. Although the simple concept of glucose-stimulated Na+ and fluid absorption remains the basic tenet of such ORT, grain-based solutions appear to have added efficacy and nutritional value. Mild, nondysenteric diarrheas can be safely and reasonably effectively treated either with loperamide or with bismuth subsalicylate compounds. Effective adjunctive therapy for severe secretory diarrheas has thus far escaped discovery. Somatostatin analogues such as octreotide have approximately 50% efficacy in reduction of the moderate secretory diarrheas such as those present in AIDS. There are pharmacologic leads that should be explored to develop more gut specific antisecretory forms of alpha 2-adrenergic agents, somatostatin, or phenothiazines. In addition, Cl- channel blockers still hold great theoretic promise. Effective antisecretory therapy has been sought for the past 20 years. It is disappointing that no effective new drugs have yet emerged. Undoubtedly, this speaks of our inexact understanding of the mechanisms that result in secretory diarrheas. Because of the continued loss of life from diarrhea in emerging nations, better understandings of these mechanisms and effective therapies remain appropriate goals for medical science.

"Endogenous" benzodiazepine activity in body fluids of patients with hepatic encephalopathy.

Body fluids from patients with hepatic encephalopathy and from controls with no renal or hepatic disease were assayed for benzodiazepine immunoreactivity and benzodiazepine-receptor-binding activity. The subjects had taken no synthetic benzodiazepines for at least 3 months. Benzodiazepine receptor binding in cerebrospinal fluid was significantly higher in hepatic encephalopathy patients than in controls (210 [SE 50.2] vs 40.7 [7.3] oxazepam equivalents [ng/ml]). The severity of hepatic encephalopathy was directly and significantly correlated with the level of benzodiazepine activity by radioreceptor assay or radioimmunoassay in urine and in plasma. Benzodiazepine activity equivalent to levels of more than 900 ng/ml was found in patients with advanced encephalopathy. Although the chemical identity and source of this substance (or substances) are still unknown, its properties and the estimated levels of activity suggest it may have a role in the pathogenesis of the neural inhibition seen in hepatic encephalopathy.

Nutritional therapy and liver disease.

In terms of a general approach to these patients, the initial and most important step for the clinician is to have recognition and concern for the magnitude of malnutrition in patients with chronic liver disease. It is best to assume an inadequate diet and to have trained personnel review the individual's nutritional needs and design specific dietary regimens that supply sufficient energy and protein, perhaps in the form of frequent interval feedings. The currently available data support the use of specialized formulations only in selected patients with hepatic encephalopathy who are intolerant of an amount of protein sufficient to meet their nutritional requirements.