Dopaminergic Immunofluorescence Studies in Kidney Tissue.

The kidney is a highly integrated system of specialized differentiated cells that are responsible for fluid and electrolyte balance in the body. While much of today's research focuses on isolated nephron segments or cells from nephron segments grown in tissue culture, an often overlooked technique that can provide a unique view of many cell types in the kidney is slice culture. Here, we describe techniques that use freshly excised kidney tissue from rats to perform a variety of experiments shortly after isolating the tissue. By slicing the rat kidney in a "bread loaf" format, multiple studies can be performed on slices from the same tissue in parallel. Cryosectioning and staining of the tissue allow for the evaluation of physiological or biochemical responses in a wide variety of specific nephron segments. The procedures described within this chapter can also be extended to human or mouse kidney tissue.

Aromatase overexpression induces malignant changes in estrogen receptor α negative MCF-10A cells.

Estrogen is a risk factor of breast cancer. Elevated expression of aromatase (estrogen synthase) in breast tissues increases local estradiol concentrations and is associated with breast cancer development, but the causal relationship between aromatase and breast cancer has not been identified. Accumulating data suggest that both estrogen receptor (ER)-dependent and -independent effects are involved in estrogen carcinogenesis. We established a model by expressing aromatase in ERα- MCF-10A human breast epithelial cells to investigate ERα-independent effects of estrogen in the process of malignant transformation. Overexpression of aromatase significantly increased anchorage-independent growth. Parental- or vector-expressing MCF-10A cells did not form colonies under the same conditions. The anchorage-independent growth of MCF-10A(arom) cells can be completely abolished by pre-treatment with the aromatase inhibitor, letrozole. Neither MCF-10A(arom) nor MCF-10A(vector) cells grown in monolayer were affected by short-term exposure to estradiol. Enhanced motility is another characteristic of cellular transformation. Motility of MCF-10A(arom) cells was increased, which could be inhibited by letrozole. Increases in stem cell population in breast cancer tissues are associated with tumor recurrence and metastasis. CD44(high)/CD24(low) is a stem cell marker. We found that CD24 mRNA levels were reduced in MCF-10A(arom) cells compared with those in parental- and vector-transfected cells. By examining individual clones of MCF-10A(arom) with various aromatase activities, we found that the CD24 mRNA levels were inversely correlated with aromatase activity. The ability of MCF-10A(arom) cells to form mammospheres in the absence of serum was increased. Our results suggest that overexpression of aromatase in MCF-10A cells causes malignant transformation. Estrogen metabolite-mediated genotoxicity and induction of a stem cell/progenitor cell population are possible mechanisms. These studies provide additional evidence for ERα-independent mechanism(s) in estrogen carcinogenesis and implicate superiority of aromatase inhibitors to antiestrogens for breast cancer prevention.

Benign cylindroma causing transcalvarial invasion in a patient with familial cylindromatosis.

OBJECTIVE: To describe a case of scalp cylindroma without features of malignancy invading through the skull and dura, and producing massive intracranial extension. Tumors of epidermis and epidermal appendages rarely show bony invasion, but invasive tendency in some tumor types has been associated with increased TP53 expression. PATIENT AND METHODS: Patient with familial cyindromatosis (Brooke-Spiegler syndrome) who had undergone numerous previous surgical excisions over the past 30 years of his scalp cylindromas. Light microscopic and immunohistochemical characterization of resected tumor, with TP53 immunostaining in the invasive tumor was compared with that seen in five other cutaneous, non-invasive cylindromas. RESULTS: Tumor showed no increase in mitotic rate or increased immunostaining for TP53. CONCLUSION: Multiple previous surgeries down to pericranium may have contributed to local weakening of tissues and facilitated transcalvarial invasion. While an uncommon occurrence, both benign and malignant cylindromas have the capacity to invade bone, particularly in patients with the familial syndrome.

Cell density mediated pericellular hypoxia leads to induction of HIF-1alpha via nitric oxide and Ras/MAP kinase mediated signaling pathways.

Environmental signals in the cellular milieu such as hypoxia, growth factors, extracellular matrix (ECM), or cell-surface molecules on adjacent cells can activate signaling pathways that communicate the state of the environment to the nucleus. Several groups have evaluated gene expression or signaling pathways in response to increasing cell density as an in vitro surrogate for in vivo cell-cell interactions. These studies have also perhaps assumed that cells grown at various densities in standard in vitro incubator conditions do not have different pericellular oxygen levels. However, pericellular hypoxia can be induced by increasing cell density, which can exert profound influences on the target cell lines and may explain a number of findings previously attributed to normoxic cell-cell interactions. Thus, we first sought to test the hypothesis that cell-cell interactions as evaluated by the surrogate approach of increasing in vitro cell density in routine normoxic culture conditions results in pericellular hypoxia in prostate cancer cells. Second, we sought to evaluate whether such interactions affect transcription mediated by the hypoxia response element (HRE). Thirdly, we sought to elucidate the signal transduction pathways mediating the induction of HRE in response to cell density induced pericellular hypoxia in routine normoxic culture conditions. Our results indicate that paracrine cell interactions can induce nuclear localization of HIF-1a protein and this translocation is associated with strong stimulation of the HRE-reporter activity. We also make the novel observation that cell density-induced activity of the HRE is dependent on nitric oxide production, which acts as a diffusible paracrine factor secreted by densely cultured cells. These results suggest that paracrine cell interactions associated with pericellular hypoxia lead to the physiological induction of HRE activity via the cooperative action of Ras, MEK1, HIF-1a via pericellular diffusion of nitric oxide. In addition, these results highlight the importance of examining pericellular hypoxia as a possible stimulus in experiments involving in vitro cell density manipulation even in routine normoxic culture conditions.

A screen for new trithorax group genes identified little imaginal discs, the Drosophila melanogaster homologue of human retinoblastoma binding protein 2.

The proteins encoded by two groups of conserved genes, the Polycomb and trithorax groups, have been proposed to maintain, at the level of chromatin structure, the expression pattern of homeotic genes during Drosophila development. To identify new members of the trithorax group, we screened a collection of deficiencies for intergenic noncomplementation with a mutation in ash1, a trithorax group gene. Five of the noncomplementing deletions uncover genes previously classified as members of the Polycomb group. This evidence suggests that there are actually three groups of genes that maintain the expression pattern of homeotic genes during Drosophila development. The products of the third group appear to be required to maintain chromatin in both transcriptionally inactive and active states. Six of the noncomplementing deficiencies uncover previously unidentified trithorax group genes. One of these deficiencies removes 25D2-3 to 26B2-5. Within this region, there are two, allelic, lethal P-insertion mutations that identify one of these new trithorax group genes. The gene has been called little imaginal discs based on the phenotype of mutant larvae. The protein encoded by the little imaginal discs gene is the Drosophila homologue of human retinoblastoma binding protein 2.

Transmembrane motility assay of transiently transfected cells by fluorescent cell counting and luciferase measurement.

Current in vitro assays used in assessing tumor motility could be improved by the development of a simple technique that would facilitate studies of the impact of specific genes on pharmacologically altered chemotaxis. We developed a technique that improves on the classic transwell assay by using fluorescence and luminescence to assess chemotaxis. In this transient transfection system, co-transfection of a reporter construct and a gene with an unknown impact on motility are coupled with biochemical assays to quantitate the number of cells that have received a transferred gene, which subsequently crosses the membrane. This assay was found to be less variable than the conventional transwell chamber and is easily adaptable to studies of cell motility or cell invasion. We also demonstrate that this assay can detect the effect of both genetic and pharmacological inhibition of motility alone and in combination. It therefore has the potential to reveal additive or synergistic effects.

Genetic and phenotypic changes associated with the acquisition of tumorigenicity in human bladder cancer.

There has been a general lack of human paired cell lines that both reproduce the in vivo spectrum of tumor progression of bladder cancer and have some of the genetic changes associated with progression in human tumor tissue. T24, a cell line established from an invasive human transitional cell carcinoma (TCC) of the bladder, has been used extensively in bladder cancer research. However, a significant limitation of this cell line is its lack of tumorigenicity when injected into immunocompromised mice. This characteristic was used to our advantage as we sought to characterize T24T, a highly tumorigenic variant that could then be used to elucidate the genes responsible for human bladder tumor progression. In culture, T24T has a faster doubling time, reaches a higher cell density in monolayer culture, and is more motile than T24 at higher cell densities. T24T is able to form colonies in soft agar, whereas T24 is not, and expresses HRAS, a gene associated with increased aggressiveness in human TCC, at higher levels than T24. Most importantly, T24T forms solid tumors when injected subcutaneously in SCID mice both with and without Matrigel (Sigma, St. Louis, MO), whereas T24 does not. Cytogenetically, the 2 cell lines contain at least 5 shared structural anomalies, as determined by detailed karyotyping. Interestingly, T24T has acquired 4 new structural changes, 3 of which [add(10)(p12), i(10)(q10), -15] have been observed in loss of heterozygosity (LOH) studies of tumor progression in human TCC. It appears that the T24/T24T model may be an excellent tool for the study of human TCC progression because of its relationship with known karyotypic changes associated with human bladder cancer progression. We are currently taking advantage of these paired cell lines to identify genes involved in human TCC progression. Genes Chromosomes Cancer 27:252-263, 2000.

The relationship of BRMS1 and RhoGDI2 gene expression to metastatic potential in lineage related human bladder cancer cell lines.

We have recently characterized a human bladder cancer cell line T24 and a more aggressive lineage related variant of it, T24T. To gain further insights, we have studied their metastatic ability in an in vivo model system. Results show that T24 forms significantly fewer [4/12 (1/11) mice had metastases with 1-2 lesions/mouse] metastasis in SCID/bg mice than T24T [14/14 (6/6) mice had metastases with a mean of 24-28 lesions/mouse]. To begin exploring the mechanisms underlying this difference, we evaluated the mRNA and protein expression levels of metastasis-suppressor genes, known to be important in the progression of other cancers, in our model of bladder cancer progression. A higher mRNA expression of BRMS1, a metastasis suppressor in breast cancer, was observed in T24 cells. In addition, RhoGDI2 mRNA expression was only observed in T24 when compared to T24T, suggesting that Rho activation might play a significant role in the metastatic cascade. However, a basal level mRNA expression of KISS1, described as metastasis suppressor in melanoma and breast, was observed in both the lines and had slightly higher expression in T24T. No difference of Nm23-H1, KAI1, MKK4/SEK1 and E-Cadherin protein levels were noted between these two lines. In summary, it appears that the T24/T24T paired cell lines constitute a useful model for the study of human bladder cancer metastasis that will allow both the discovery and mechanistic evaluation of genes potentially involved in this process.

Trithorax and ASH1 interact directly and associate with the trithorax group-responsive bxd region of the Ultrabithorax promoter.

Trithorax (TRX) and ASH1 belong to the trithorax group (trxG) of transcriptional activator proteins, which maintains homeotic gene expression during Drosophila development. TRX and ASH1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers. Based on genetic interactions between trx and ash1 and our previous observation that association of the TRX protein with polytene chromosomes is ash1 dependent, we investigated the possibility of a physical linkage between the two proteins. We found that the endogenous TRX and ASH1 proteins coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes. Furthermore, we demonstrated that TRX and ASH1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s). This suggests that ASH1 and TRX act on Ubx in relatively close proximity to each other. Finally, TRX and ASH1 appear to interact directly through their conserved SET domains, based on binding assays in vitro and in yeast and on coimmunoprecipitation assays with embryo extracts. Collectively, these results suggest that TRX and ASH1 are components that interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription.

Human parvovirus B19--flushed in face though healthy (fifth disease and more).

Parvovirus B19 is an ubiquitous organism. Although for many years it had been "a virus in search of a disease," it is now known to be associated with several clinical entities ranging from the benign to severe. The most significant aspect of parvovirus B19 is its effect on the fetus. Research has demonstrated the risk, although minimal, of fetal loss to be between 1.5% to 2.5%. The effect from an emotional standpoint for the pregnant exposed or infected woman is more difficult to quantify. It is imperative that nurses who care for children be well informed about the virus and able to implement a comprehensive plan.

The Drosophila ash1 gene product, which is localized at specific sites on polytene chromosomes, contains a SET domain and a PHD finger.

The determined state of Drosophila imaginal discs depends on stable patterns of homeotic gene expression. The stability of these patterns requires the function of the ash1 gene, a member of the trithorax group. The primary translation product of the 7.5-kb ash1 transcript is predicted to be a basic protein of 2144 amino acids. The ASH1 protein contains a SET domain and a PHD finger. Both of these motifs are found in the products of some trithorax group and Polycomb group genes. We have determined the nucleotide sequence alterations in 10 ash1 mutant alleles and have examined their mutant phenotype. The best candidate for a null allele is ash1. The truncated protein product of this mutant allele is predicted to contain only 47 amino acids. The ASH1 protein is localized on polytene chromosomes of larval salivary glands at > 100 sites. The chromosomal localization of ASH1 implies that it functions at the transcriptional level to maintain the expression pattern of homeotic selector genes.

Aortic valve endocarditis. Determinants of early survival and late morbidity.

BACKGROUND: Aortic valve surgery for endocarditis remains a high-risk procedure. The objective of this study was to analyze the interaction between the various subsets of endocarditis (native, prosthetic, healed, and active), timing of surgery, and their influence on early and late outcomes. METHODS AND RESULTS: During a 20-year period starting January 1972, 200 patients underwent aortic valve replacement for infective endocarditis (age range, 13 to 88 years; median, 53 years). There were 51 (26%) females, and 109 (55%) were in New York Heart Association functional class IV before surgery. Native valve endocarditis (NVE) and prosthetic valve endocarditis (PVE) were present in 132 (66%) and 68 (34%) patients, respectively. Surgery was required in 120 (60%) during the active phase (AE) and 80 (40%) during the healed phase (HE) of endocarditis. The main indication for surgery in the healed group was progressive congestive heart failure. The indications for the active group were congestive heart failure (68%), continuing active sepsis (70%), echocardiographic vegetation (28%), peripheral emboli (30%), and arrhythmias (13%). Streptococcal infections predominated in NVE, staphylococcal in PVE and AE; culture-negative endocarditis predominated in the healed group. Isolated aortic valve surgery was performed in 68% of the patients, and concomitant procedures (32%) included mitral valve and coronary bypass procedures. The overall operative mortality (OM) was 12.5%. The OM was 7.5% and 22% for NVE and PVE, respectively (P = .004), and 7% for HE versus 15% for AE (P = .06). The OM for early PVE was 33% versus 18% for late PVE (P < .05). Multivariate logistic regression analysis identified PVE and New York Heart Association functional class IV to be independent predictors for early death. Recurrent endocarditis occurred 26 times in 24 patients (11 early, 13 late), with three operative deaths in the early group, all due to residual staphylococcal infections. Freedom from recurrent endocarditis was significantly different between HE (96 +/- 3% and 86 +/- 6% at 5 and 10 years, respectively) and AE (89 +/- 3% and 83 +/- 4%, respectively (P = .02). Long-term survival for discharged patients was 81 +/- 3% and 63 +/- 5% at 5 and 10 years, respectively, with no significant difference between NVE, PVE, AE, and HE. CONCLUSIONS: These data suggest that for active endocarditis, surgery should be delayed to achieve a healed status provided there is no pressing need for immediate surgery. Patients with staphylococcal endocarditis, particularly on a prosthesis, should be operated on sooner and should be covered with antibiotics for an extended period to prevent recurrent PVE. This study stresses the need for aggressive antibiotic prophylaxis, particularly in the presence of a prosthesis.

Single-clamp technique: an important adjunct to myocardial and cerebral protection in coronary operations.

To determine the myocardial and cerebral protective properties of the single cross-clamp (group I; n = 160) versus the partial occluding clamp (group II; n = 150) technique for construction of the proximal anastomoses, a retrospective analysis of 310 patients operated on by the same surgeon was performed. Group I patients were older (median age, 70 versus 64 years; p < or = 0.0001), with 83 (52%), versus 41 (27%) in group II, 70 years and older (p < or = 0.0001). More group I patients were in New York Heart Association functional class IV (42 [26%] versus 22 [15%]; p = 0.008); more required preoperative balloon counterpulsation (35 [22%] versus 16 [11%]; p = 0.006); and more required emergent operation (20 [13%] versus 3 [2%]; p < or = 0.0001). Antegrade crystalloid cardioplegia was used in both groups. The median cross-clamp time was 58 minutes for group I versus 44 minutes for group II (p < or = 0.0001). However, there was no significant difference between the two groups in terms of the number of bypass grafts, the use of the mammary artery, or the bypass time. The operative mortality was 2.5% (n = 4) for group I versus 5.3% (n = 8) for group II (p = 0.16), and the perioperative myocardial infarction/low cardiac output state was seen in 6 patients (3.8%) in group I versus 18 patients (12%) in group II (p = 0.006). The median creatine kinase MB release was 13 U/L for group I versus 19 U/L for group II (p = 0.0029).(ABSTRACT TRUNCATED AT 250 WORDS)

Aortic valve replacement in the elderly. Effect of gender and coronary artery disease on operative mortality.

BACKGROUND: Aortic valve replacement remains the treatment of choice for aortic valve disease, even in the extreme elderly who may present with advanced symptoms. Defining risk factors for short-term survival was the object of this study. METHODS AND RESULTS: This was a retrospective analysis of 717 patients at least 70 years of age who underwent aortic valve replacement alone or with coronary artery bypass graft between 1980 and 1992. Age range was 70 to 95 years, and mean age was 77 years; there were 529 septuagenarians (74%); 188 were octogenarians (26%); 326 were women (45%); and 386 patients (54%) had aortic valve replacement and coronary artery bypass graft. Atrial fibrillation/flutter or heart block was present in 16%, and 34% of patients were in New York Heart Association (NYHA) functional class IV. Aortic stenosis was present in 88%, and mechanical prostheses were used in 22% of patients. There were 47 deaths, giving an overall operative mortality of 6.6%, with 4.2% for aortic valve replacement and 8.8% for aortic valve replacement and coronary artery bypass graft (P = .01). The operative mortality for aortic valve replacement was 2.9% versus 10.3% for aortic valve replacement and coronary artery bypass graft in women (P = .006). The corresponding values for men were 5.6% and 7.4% (P = .31). Multivariate logistic regression showed coronary artery bypass graft and NYHA class IV to be significant predictors of operative mortality in women. The significant predictors in men were NYHA class IV, atrial fibrillation/flutter or heart block rhythm, and the use of mechanical prosthesis. Age was not a predictor of operative mortality in either sex. CONCLUSIONS: Aortic valve replacement carries an acceptable mortality rate in elderly patients. Female gender was a significant predictor of operative mortality in the concomitant coronary artery bypass graft group; however, gender was not a predictor of operative mortality in the isolated aortic valve replacement group. Advance stage of the disease process represented by NYHA class IV was a significant predictor of mortality for the whole group, stressing the need for earlier referral for surgery.

High and dry--low and wet: the key to DI and SIADH.

The pediatric critical care nurse is often faced with children who have underlying neurologic problems. Anticipating the occurrence of diabetes insipidus (DI) and syndrome of inappropriate secretion of antidiuretic hormone (SIADH) in these children is essential. There is much confusion, however, surrounding recognition and management of these distinctly different although related problems. The key to differentiating these clinical entities may be found in the simple statement, high and dry; low and wet.

When fever becomes an enemy.

Today fever is being viewed more as a friend rather than an enemy. While this may be true in the normal pediatric setting, fever cannot be considered a friend in the pediatric critical care unit. It is here that fever becomes an enemy. The pediatric critical care nurse must understand the febrile response and recognize the potential detrimental effects of fever in the already compromised child. Fever will be more aggressively managed in the critically ill child.

AIDS and the management prerogative.

As with affirmative action in the '70s, it is the big companies that have, in just the last 10 or 12 months, begun to address AIDS in the workplace. Their top executives have come to add such action to the list of management responsibilities. More employers of all sizes may join them, especially those that grasp the true role--and the non-role--of geography in the AIDS epidemic.

Toil and trouble over outpatient prospective payment.

PACs, AVGs, EDGs--the health care alphabet soup is burbling as HCFA considers a mandate for a Medicare outpatient PPS. And some third-party payers are devising their own outpatient PPSs for non-Medicare enrollees in the interest of curbing high ambulatory care costs. Here is a report on the status and timing of all these efforts from a variety of participants and observers.