Confluent Growth Research Articles

Cistadenofibroma endometrioide borderline de ovario: un tumor infrecuente

Summary Endometrioid cystadenofibromatous tumours of the ovary with a low malignant potential are infrequently described in literature. There is currently no agreement on the criteria for distinguishing them. The presence of a confluent growth pattern with stromal invasion distinguishes malignant from borderline tumours. Some of these neoplasms will be associated with endometrial hyperplasia or carcinomas and rarely with extra ovarian manifestations. Conservative surgery may be proposed and have an excellent prognosis. We describe a case fdetected during ultrasound screening. At the moment, the biological behaviour of this type of pathology cannot be evaluated because of the few cases published.

Cancer and Pre‐cancer of the Cervix

Luesley DM, Barasso R editors . Cancer and Pre-cancer of the Cervix . 437 pages. Illustrations. Tables. London : Chapman and Hall , 1998 . Price GBP 85.00 . During the last decade there has been substantial progress in our understanding of cervical cancer. Modern molecular biological techniques have shown that human papilloma virus is the cause of this disease. Considerable knowledge has accumulated on the oncogenic potential of different HPV subtypes and how oncogenicity is expressed and regulated in the infected cells. Recent experience in the laboratory is currently transferred to the clinic and new follow-up and treatment strategies are being introduced: Cost-effective and safe conization by loop excision, chemotherapy or combined chemo-radiotherapy as primary treatment modalities in advanced stages of cervical cancer, the use of CT and MR to image the spread of cervical cancer, the use of laparoscopy to dissect lymph nodes have all changed the practical handling of the disease. In the present volume 42 experts contribute to illustrate these matters in 26 chapters. The authors have written their chapters independently of each other, which may explain some overlapping of presented data. The normal histology of the cervix and the colposcopic and histologic features of the transformation zone are presented in the introducing chapters. Fox, discussing histopathology of cervical cancer, stresses other factors than just cancer invasion to be evaluated, ie, vascular space invasion, confluent growth pattern, estimation of horizontal growth as well as the use of mucin staining to diagnose adenocarcinoma. The use of new techniques, flow cytometric DNA analyses, the expression of oncogenes and suppressor genes and cell proliferation markers to detect early stage cancer with bad prognosis are discussed. Although, worldwide, the reduction in cervical cancer is regarded as a benefit of screening programs Skrabanek claims that running national programs are without clear benefits and exaggeration of their benefits is unethical. The editors, however, do not appear to agree with his arguments. Epidemiological and molecular biological data of HPV as the single main etiological factor for cervical dysplasia and cancer are presented. Techniques to detect HPV in cervical smears and tissue specimens are described. Positive arguments to include HPV screening in conjunction with Pap smear screening are presented. Cervical immunology, immunosuppression and the role of lymphokines and cytokines are discussed in relation to HPV infection and progression of disease. The diagnosis, treatment and follow up of dysplasia is thoroughly described. The importance of colposcopy and careful case selection in a screening program is stressed. Conventional radical as well as minimal invasive techniques for the treatment of invasive cancer are described in detail. Experiences on laparoscopy in lymphadenectomy in conjunction with radical vaginal hysterectomy or trachelectomy or as a staging procedure before radical operation or exenteration are presented by Dargent. Criteria for exenterative procedures are discussed by Heintz. One chapter is devoted to current techniques and indications for radiotherapy in primary or postoperative treatment. A comprehensive review on the role of chemotherapy, especially in neoadjuvant treatment in advanced cancer, is given. Aspects of quality of life and the psychosocial impacts of preinvasive and invasive disease are discussed in two chapters. Generally, many of the authors focus on the importance of individualizing the treatment for one single woman, avoiding overtreatment. The present volume nicely summarizes experts’ views of today's knowledge on preinvasive and invasive cervical cancer and can be recommended to people in this field: molecular biologists, epidemiologists, gynecologists, oncologists and cyto-pathologists.

The gradient plate technique as a means of studying the recovery of heat‐injured Brochothrix thermosphacta

SummaryBacterial recovery from heat injury is influenced strongly by the nature of the recovery medium used to resuscitate cells. This study used agar gradient plates, in combination with image analysis, to study the synergistic effect of NaCl concentration (% w/v) and pH on the recovery of Brochothrix thermosphacta after heat treatment. Initially, exponential, early stationary and late stationary phase cultures of B. thermosphacta grown in all‐purpose tryptone (APT) broth at 25 °C were heat‐treated at 50 °C to measure thermal resistance. Late stationary phase cultures were found to be 2–3 times more heat‐resistant than the other two, with a D‐value of 14.8 min. Exponential and early stationary phase cultures were thermally treated and inoculated onto agar gradient plates (modified APT medium) and incubated at 25 °C. In the instance of the late stationary phase culture, there proved to be too low a cell concentration to obtain confluent growth. These plates had gradients of pH (4.0–7.4) and NaCl (1.5–8.1% (w/v)) running at right angles across them. After 48 h incubation, bacterial growth on these plates was measured by image analysis. In all bacterial cultures (heat‐treated or control), optimal growth was found at pH 6.8 and 1.5% NaCl (w/v) concentration. The range of salt concentrations and pH values over which growth could be observed was shown to be reduced as a consequence of heat treatment. Overall, it is suggested that the gradient plate technique, in combination with image analysis, could be useful in determining combinations of different environmental factors which are effective in preventing the recovery of heat injured bacterial cells.

A clinicopathologic analysis of atypical proliferative (borderline) tumors and well-differentiated endometrioid adenocarcinomas of the ovary.

Atypical proliferative (borderline) endometrioid tumors (APTs) and well-differentiated endometrioid carcinomas of the ovary constitute a spectrum of morphologically diverse proliferative tumors. There is currently no agreement on the criteria for distinguishing them. We report the clinicopathologic features of 56 proliferative endometrioid tumors focusing on the criteria for invasion, the clinical significance of microinvasion and cytologic atypia, and prognosis. Endometriomas, adenofibromas, adenosarcomas and moderately to poorly differentiated carcinomas were excluded, as were patients with concurrent endometrioid carcinoma of the endometrium. The tumors were classified as atypical proliferative tumor (APT) (33 tumors), APT with intraepithelial carcinoma (high-grade cytology in a tumor lacking stromal invasion) (three tumors), APT with microinvasion (invasion <5 mm) (five tumors), and invasive carcinoma (invasion > or = 5 mm) ( 15 tumors). All tumors were confined to the ovary (stage I). In 50 patients, the tumor involved one ovary, and in three patients, the tumors were bilateral. The predominant growth pattern was adenofibromatous in 29 tumors and glandular or papillary in 27 tumors. In 8 (24%) of 41 APTs, areas of benign adenofibroma were identified, and in 13 (87%) of 15 carcinomas, areas of associated APT were identified. Stromal invasion was manifested by confluent glandular growth in all 15 invasive carcinomas and all tumors with microinvasion. Destructive infiltrative growth was also present in 2 (13%) of 15 carcinomas. Confluent glandular growth was the most common manifestation of stromal invasion and therefore served as the best criterion for the diagnosis of carcinoma. Squamous differentiation was observed in 24 tumors, and mucinous differentiation was seen in 20 tumors and was most often seen in APTs. Endometriosis was present in 14 patients with APTs and one patient with carcinoma. Four patients had hyperplasia or atypical hyperplasia of the endometrium. One patient with an APT had a concurrent peritoneal serous neoplasm. Twenty-one patients had available clinical follow-up. Twenty (95%) of 21 patients, including six with invasive carcinoma, two with microinvasion, one with intraepithelial carcinoma, and 11 with APT were alive with no evidence of disease with a mean follow-up of 47 months. One patient with carcinoma had recurrent tumor after 46 months and was alive 40 months after resection of the recurrent tumor. In this large series of proliferative endometrioid tumors, all were stage I and only one patient had a recurrence. Most carcinomas contained evidence of a precursor APT, and in some APTs, an associated benign adenofibroma was identified. Microinvasion or intraepithelial carcinoma occurred in 19% of APTs. This finding likely reflects the various stages of endometrioid carcinogenesis in the ovary. For clinical management, we suggest that these tumors be divided into two categories-APTs and well-differentiated carcinoma-because based on the available data, cytologic atypia and microinvasion appear not to affect the prognosis.

Effect of surgical technique on in vitro posterior capsule opacification

Purpose To compare the effect of different cataract extraction surgical techniques on residual lens epithelial cell (LEC) density and cell regrowth rates using an in vitro model of posterior capsule opacification (PCO). Setting Comparative Ophthalmology Research Laboratories, North Carolina State University, Raleigh, North Carolina, USA. Methods Lens capsule explants were prepared from freshly enucleated canine globes after extracapsular cataract extraction (ECCE), phacoemulsification, or phacoemulsification followed by capsule vacuuming. Initial cell density on the capsule and cell proliferation were determined by phase contrast microscopy. The effects of the surgical technique on time to confluent growth of the cells across the posterior lens capsule were determined. Results Residual cell density on the remaining anterior capsule immediately after lens removal was 31.6% ± 19.3%, 16.1% ± 8.9%, and 7.7% ± 5.7% in the ECCE, phacoemulsification, and phacoemulsification/capsule-vacuuming groups, respectively. Time to confluence (range 5.0 to 6.3 days) was not significantly different among the 3 groups when the lens capsules were cultured in serum-supplemented media. The confluence rate was significantly longer (by approximately 5 to 7 days) in the phacoemulsification/capsule-vacuuming group than in the other 2 groups when the capsules were cultured in serum-free media. Conclusions Phacoemulsification with and without anterior and equatorial capsular vacuuming led to less initial LEC density in the capsular bag than ECCE. However, because cell proliferation rates among the 3 groups were only marginally affected, near 100% removal of LEC at the time of cataract extraction may be necessary to prevent PCO.

CD44 acts both as a growth- and invasiveness-promoting molecule and as a tumor-suppressing cofactor.

High-molecular-weight splice variants of the CD44 transmembrane protein family have been implicated in tumorigenesis and metastasis formation. By contrast, in certain tumors--for example, Burkitt's lymphoma, neuroblastomas, and prostate cancer--loss of CD44 expression seems to accompany transformation. Here we describe two modes of action of CD44 proteins. They can bind growth factors and present them to their authentic high-affinity receptors, and thus promote proliferation and invasiveness of cells. Under these conditions the CD44 proteins recruit ERM proteins--for example, ezrin or moesin--to their cytoplasmic tails, thereby producing links to the cytoskeleton. This mode of action could account for the tumor-promoting action of CD44 proteins. The second mode of action of CD44 proteins comes into play when cells reach confluent growth conditions. Under specific conditions, binding of another ligand, the ECM component hyaluronate, leads to the activation and binding to the CD44 cytoplasmic tail of the tumor suppressor protein merlin. The activation of merlin confers growth arrest, so-called contact inhibition. This function of CD44 proteins defines them as tumor suppressors. The type of action of CD44 on a given cell will depend on the isoform pattern of CD44 expressed, on the cellular equipment with ERM protein members, on the nature of the ECM, and on yet-unknown conditions.

Granular Cell Tumors: A New Clinically Important Histologic Finding

Objective. The objective of this study was to review clinical characteristics, histological findings, and surgical treatment of patients with granular cell tumors of the vulva at Hartford Hospital and examine histologic characteristics associated with those cases that recurred.Materials and Methods. A retrospective case review of 13 patients diagnosed with granular cell tumors of the vulva at Hartford Hospital from April 1982 through May 1998 was undertaken to compare age, size and location of the lesion, histopathologic features, presenting symptoms, progression of disease, and treatment.Results. Tumor growth patterns were divided into those with confluent expansile growth (nodular) and those that infiltrated the dermis and soft tissue in a haphazard fashion interdigitating with native connective tissue and skin appendages (infiltrative). The advancing edge of the tumors was assessed as having either a “pushing” or an infiltrative border. The advancing edge of the tumor was noted to be irregular and infiltrative in 8 of the 13 cases. The tumor edge in the remaining 5 was pushing and well demarcated. Five of the eight tumors with an infiltrative edge recurred despite the fact that 3 of the 5 cases had negative surgical margins at resection. None of the 5 cases with “pushing” borders developed recurrence.Conclusions. Although rare, vulvar tumors are the most common variety of granular cell tumors encountered in the female genital tract. Histologic evaluation seems to indicate that patients are more likely to have a recurrence if an infiltrative growth pattern is present at the advancing edge of the granular cell tumor even with negative margins at resection. An appropriate management strategy may be to recommend reexcision of “infiltrative border” lesions rather than clinical observation alone.

Successful treatment of tinea capitis with 2% ketoconazole shampoo.

Oral antifungal drugs are required for effective treatment of tinea capitis. Topical antifungal shampoo's, namely ketoconazole 2% shampoo or products with selenium sulfide or salicylic acid are recommended as adjunctive therapy. Topical antifungal monotherapy has not been successful in the treated of tinea capitis. The purpose of this open study was to evaluate ketoconazole 2% shampoo as a monotherapy for the treatment of tinea capitis. A total of 16 black children, aged 3-6, all with proven tinea capitis caused by Trichophyton tonsurans, were treated daily for 8 weeks with 2% ketoconazole shampoo for a total of 56 treatments. Clinical and mycologic examinations were performed every 2 weeks and again at 4 weeks following treatment. The number of colonies were counted on each plate after each visit. Patients with positive cultures after 8 weeks were placed on oral griseofulvin; those with negative cultures were followed monthly by culture for an additional 12 months. Marked clinical improvement occurred in all patients within 2 weeks and absence of pruritus was noted by the patients as early as 2-6 days. After 8 weeks of shampoo, 14 of the 15 (93%) children were clinically healed. Mycologically, the cultures dropped from a confluent growth of T. tonsurans to less than 100 colonies within 2 weeks; fewer than 50 at week 4 and 20 colonies or fewer after week 6. At 8 weeks of treatment the number of colonies remained at 20 or fewer. Six of the 15 children (40%) had negative cultures after 2, 4, and 6 weeks. One child relapsed at the first 4-week follow-up visit. Five of 15 (33%) of the children remained culturally negative for 12 months post-treatment. Ketoconazole 2% shampoo alone reduces the number of viable arthroconidia in children with tinea capitis thus reducing the transmissibility and contagious nature of the disease. Unexpectedly, complete cure was obtained in 5/15 (33%) of the children. The children remained clinically and mycologically clear as long as one year after treatment.

Sinusoidal CD30-Positive Large B-Cell Lymphoma: A Morphologic Mimic of Anaplastic Large Cell Lymphoma

Anaplastic large cell lymphoma (ALCL) has been recognized recently as a distinct clinicopathologic entity, restricted to a subset of CD30-positive diffuse large cell lymphomas of T/null lineage. Some of the characteristic features of ALCL, such as CD30 antigen expression and the presence of large pleomorphic lymphoid cells infiltrating lymph node sinuses, can be found rarely in diffuse large B-cell lymphomas. We collected 11 such cases, and their clinical, morphologic, and immunophenotypic features are reviewed. The age of the patients ranged from 36 to 82 years (mean, 63.2 years) with a male to female ratio of 1:1.2. All neoplasms were nodal with a sinusoidal infiltrative pattern, although four neoplasms also had foci of confluent growth. Eight tumors were composed predominantly of large pleomorphic cells with occasional Reed-Sternberg–like cells. The other three tumors had a higher proportion of large monomorphic lymphoid cells. Necrosis and admixed granulocytes were other common features. Immunophenotypically, all cases were positive for CD30 and CD20 or CD79a. All eight cases examined for anaplastic lymphoma kinase-1 immunoreactivity were negative. In situ hybridization for Epstein-Barr virus RNA was performed in eight cases; two were positive. Excluding one consultation case with no available clinical follow-up data, six patients died of the disease within 3 years and one had disease relapse within 1 year. We conclude that an unusual variant of diffuse large B-cell lymphoma can closely mimic ALCL. However, these neoplasms can be distinguished from ALCL by virtue of their B-lineage and lack of anaplastic lymphoma kinase-1 expression. Evidence of Epstein-Barr virus infection can be found in a small subset of these neoplasms.

Sanitation of wallboard colonized with Stachybotrys chartarum.

Sections (8 cm2) of unused, nonsterile gypsum wallboard (dry wall) were inoculated with varying densities (10(4) to approximately 10(8)/ml) of conidia from 14- to 21-day cultures of Stachybotrys chartarum grown on cellulose agar. The sections were permitted to air dry and were placed into vessels with 86% or 92% RH and incubated at 22-25 degrees C for up to 12 weeks. The moisture content of the dryboard increased from near 10% to over 35%. Selected sections with confluent surface growth, mainly of S. chartarum, were obtained within 3 weeks. Sections were cleaned with a quaternary or quaternary and chlorine dioxide or a concentrated oxygen-saline solution and treated, in some cases, with a preservative system and returned to humidity vessels. Reemergence of S. chartarum from inoculated and treated surfaces occurred within 5 weeks only with sections treated with the quaternary alone. Other fungi, mostly species of Aspergillus, Chaetomium and Penicillium, slowly colonized (between 9-12 weeks) at least some areas of most treated surfaces and most uninoculated control surfaces. Stachybotrys chartarum was also found on several sections of uninoculated controls. Sections treated with a quaternary/acrylic and placed in a dynamic challenging chamber remained visually free of colonized fungi for over 90 days. These studies indicate that control samples of uninstalled wallboard, available from local distributors, can contain a baseline bioburden, including S. chartarum, that will colonize surfaces under high humidity conditions. Sanitation and preservation treatment of the wallboard can markedly delay regrowth of these fungi, particularly of S. chartarum.

Time versus replication dependence of EMS-induced delayed mutation in Chinese hamster cells

We have previously observed in Chinese hamster cells that ethyl methane sulfonate (EMS) induces mutations which are distributed over at least 10–14 cell divisions following treatment. This delayed appearance of mutations could be explained by EMS-induced lesions which remain in DNA and have a probability that is significantly less than 1.0 of producing base mispairing errors during successive replication cycles (replication-dependent). Alternatively, delayed mutation may be a time-dependent process in which a slow acting or damage inducible error-prone repair process removes persistent DNA lesions and replaces them with an incorrect base during the course of 7–10 days of colony growth following EMS exposure. To address this question, the distribution of HGPRT delayed mutation events (fifth division or later) in cells plated immediately for exponential growth after EMS treatment was compared with the distribution in cells which remained under confluent growth conditions for 8 days and then were replated. Both the distribution and rate of accumulation of delayed mutations (mutations/cell division) were similar in the two culture conditions. In contrast, the frequency of early mutations (before the fifth division) in the confluent population was reduced more than 2-fold compared to dividing cells. A comparison of the frequency of EMS-induced DNA lesions in the two populations revealed that the density inhibited population contained one third the DNA lesions of the exponential population. These results argue against a time-dependent process since, if this mechanism applies, one would expect an increase in early mutant events and a decrease in delayed events in the confluent population. The results, however, are consistent with a replication model in which potential early mutant lesions are preferentially removed in the density inhibited culture during the 8 days of incubation while lesions producing late mutants are not removed.

Oncocytic metaplasia and carcinoma of the endometrium: an immunohistochemical and ultrastructural study.

Endometrial oncocytic carcinoma is an unusual neoplasm, with few cases reported. Endometrial curettage specimens coded as prominent oxyphilic metaplasia (N = 5) and oxyphilic or oncocytic carcinoma (N = 4) were reviewed, and hysterectomy slides from the four carcinomas were also examined. Immunohistochemical and ultrastructural analyses were performed in three of five metaplasias and in all four carcinomas. Most patients (89%) with oncocytic metaplasia and carcinoma had vaginal bleeding. Oncocytic metaplasia was characterized by a single layer of cells with abundant eosinophilic, granular cytoplasm, minimal pleomorphism, and rare mitotic activity. Carcinoma was diagnosed on the basis of an altered stroma (n = 2) and/or a confluent growth pattern (n = 4) and had a papillary (n = 4), glandular (n = 2), or solid (n = 1) morphology. Carcinomas showed a similar population of oncocytic cells as metaplasias, but with occasional nuclear stratification and greater pleomorphism and mitotic activity. Tumors were International Federation of Gynecology and Obstetrics (FIGO) grade 1 (n = 2) or 2 (n = 2) and FIGO stage Ib, Ic, IIb, and IIIc. Omental metastases developed in the patient with the stage III tumor at 13 months; the two patients with stage I tumors were alive with no evidence of disease at a mean of 29 months. All carcinomas expressed p53 and 75% and 100% were estrogen receptor (ER)- and progesterone receptor (PR)-negative, respectively, whereas all metaplasias were p53 negative- and ER- and PR-positive. Ki-67 labeling index was 1 to 3% in metaplasias and 14 to 33% in carcinomas. Oncocytic metaplasias and carcinomas contained abundant mitochondria and free ribosomes, accounting for the oncocytic appearance. Because oncocytic carcinomas frequently show deep myometrial invasion and require surgical staging, it is important to distinguish oncocytic metaplasia from carcinoma on biopsy material. Ki-67, p53, and ER and PR immunostains may assist in this potentially difficult differential.

Functionality of MDCK kidney tubular cells on flat polymer membranes for biohybrid kidney.

The prerequisite for the development of a biohybrid artificial kidney, is a substrate for confluent growth of renal cells forming an epithelial monolayer without any leaks. Conventional cell culture supports cannot be adapted for this purpose, because they lack adequate mechanical properties and thermal stability. From two suitable materials, polysulfone and polyacrylonitrile, two permeable polymeric membranes have been produced that were, according to ISO 10993-5, not cytotoxic. Cloned Madin Darby Canine Kidney (MDCK) cells (an established renal cell line) were cultured on the surface of the plastic materials, and on conventional cell culture supports. With all materials, assays of mitochondrial and lactate dyhydrogenases exhibited similar proliferation and the viability of the MDCK cells. Transmission electron microscopy showed the expression of a normal morphology of kidney tubular cells. Perfect barrier function, consequent on the formation of intercellular junctions in a confluent tight epithelium, was visualized in electron micrographs, and quantified by measurement of the transepithelial resistance. The uniformity of the cells grown was demonstrated in samples by electron microscopy and in the whole epithelium by intravital impedance analysis. It was concluded that polymeric membranes produced from polysulfone or polyacrylonitrile are appropriate substrates in the design of biohybrid kidney devices.

Biochemical and Conjugation Studies of Romet-Resistant Strains ofAeromonas salmonicidafrom Salmonid Rearing Facilities in the Eastern United States

Abstract Strains of Aeromonas salmonicida (n = 585) were collected from covertly infected and diseased salmonid hosts from 12 hatcheries in the eastern United States. Strains and sites were selected because of their potential for harboring antimicrobial resistance, in particular, to Romet™. Resistance to Romet was displayed by 315 strains (53.8%), which were isolated from all six host species sampled at 10 of 12 sites. Thirty of the resistant strains (9.5%) from five sites had no zone of inhibition, whereas the other strains had either confluent growth or resistant colonies within a zone of inhibition. Fifty-one resistant strains, representing each of the three resistance phenotypes, were selected for biochemical and antimicrobial comparisons with Romet-sensitive strains. All were confirmed to be A. salmonicida, and no characteristic biochemical phenotypes were found to be associated with resistance to Romet. Differential resistances between resistant and sensitive strains were detected to the antimicrobi...

Expression of glucose transporters in human peritoneal mesothelial cells

Glucose containing solutions, the basis of peritoneal dialysis fluids, affect the proliferation and regeneration of peritoneal mesothelial cells (MsC). The aim of this study was to examine mechanisms of glucose transport into MsC, that is, the expression of facilitative glucose transporters (GLUT) and the Na(+)-dependent glucose transporter (SGLT1) in human primary MsC and a transfected MsC line. Since expression of both transporters is differentiation dependent, we investigated the effects of cell differentiation induced by culturing MsC on membranes or by addition of hexamethylene bisacetamide (HMBA; 6 mM), which enhances SGLT1 expression in LLC-PK1 cells. Levels of mRNA for GLUT1 through GLUT4 and SGLT1 were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). The presence of the corresponding proteins was examined by Western blotting and localized by immunofluorescence. Active, Na(+)-dependent glucose transport was assessed by alpha-methyl-D-[14C]glucopyranoside (AMG) with and without the SGLT1-specific inhibitor phlorizin and by patch clamp experiments in NaCl or choline-chloride, For Na(+) dependent glucose uptake choline chloride instead of NaCl served as negative control. Facilitative transport was assessed using 2-fluoro-2-deoxy-[14C]-D-glucose (FDG) with and without the inhibitors cytochalasin B or phloretin. Primary and transfected MsC express GLUT1 and GLUT3 mRNA while no transcripts were found for GLUT2 and GLUT4. No SGLT1 transcript was detectable in subconfluent cells. Semiquantitative RT-PCR analysis documented that the addition of the differentiation inducer HMBA to confluent cultures or growth of MsC on membranes for seven days produced a down-regulation of mRNA for GLUT1, no change for GLUT3, and a substantial increase for SGLT1 mRNA. Under these conditions MsC express SGLT1 protein and possess a Na(+)-dependent glucose uptake as assessed by AMG. Phlorizin (1 mM) inhibits AMG uptake by 30 to 40%. In patch clamp experiments the addition of extracellular glucose depolarized the membrane potential only in the presence of sodium. These results indicate that differentiated MsC express GLUT1, GLUT3, and SGLT1. Further characterization of these transport mechanisms and their regulation may help to understand the cellular effects of glucose on MsC in peritoneal dialysis.

Inflorescence and Flower Development in the Hedychieae (Zingiberaceae): Scaphochlamys kunstleri (Baker) Holttum

Primary inflorescence bracts are initiated in a spiral phyllotactic pattern on the flanks of the inflorescence apex. A cincinnus primordium forms in the axil of each primary bract. A prophyll is initiated in the transverse plane and later extends around the periphery of the cincinnus to produce a sheathing, but not tubular, bract. The apex of the cincinnus forms a flower. Flower development begins with the enlargement and flattening of the floral apex. Sepal initiation is sequential but differs in the first two flowers of a cincinnus. The different sequences are likely the result of developmental constraints that operate at the time of sepal initiation. After initiation, the margins of the sepals become confluent and intercalary growth produces the synsepalous calyx. The periphery of the flower, interior to the sepals, now rises to form a ring primordium composed of three common petallandroecial primordia. The common primordia enlarge, flatten, and separate to produce a petal to the exterior and an androecial member to the interior. The outer androecium forms in the regions of the ring primordium left vacant by the formation of the other floral parts. The anterior outer androecial member soon ceases growth and contributes only initially to the formation of the labellum. The gynoecium shows heterochrony in its time of initiation, which occurs much earlier than in other species of Zingiberaceae. Gynoecial initiation takes place on the margins of the central flora cup, at the time of the separation of the common primordia. Three gynoecial primordia from in antipetalous positions. Septa development differs in the basal and apical portions of the ovary. At the base, the primordia grow inward and fuse to form the central axis of the ovary. At the apex, there is little radial growth of the primordia. They cease growth soon after initiation and produce a unilocular cavity. Ovules form at the junction of these two regions and project up into the cavity. Epigynous nectaries form after all other floral organs.

Dynabeads plus 3 M Petrifilm HEC versus Vitek Immunodiagnostic Assay System for detection of E. coli O157 in minced meat.

The potentially low infective dose of Escherichia coli O157 makes it necessary to be able to detect low numbers in food, and the lack of sensitivity of direct plating has led to the development of various enrichment and detection methods. Until now, the most selective procedure for detection of E. coli O157 isolates was the immunomagnetic separation (IMS) method. The number of sorbitol non-fermenting micro-organisms other than E. coli O157 that adhere non-specifically to the magnetic beads hampers the application of IMS. The use of IMS in conjunction with 3 M Petrifilm-HEC yielded EHEC O157 in 21 of 165 samples of minced meat (12.7%). Without advance application of IMS, Petrifilm plates often yield confluent growth and colonies too numerous to count. The Vitek Immunodiagnostic Assay System (VIDAS-ECO) showed good sensitivity when testing artificially contaminated beef samples, but only four of 21 naturally contaminated samples were recognized. The addition of 3 M Petrifilm to IMS resulted in less growth of contaminants and eliminated much of the need to test presumed colonies for confirmation. The combination of IMS and 3 M Petrifilm-HEC is a fast and efficient screening procedure for E. coli O157 in minced meat.

Use of Fine Needle Aspiration for Fibroblast Culture

Diagnosis in certain metabolic diseases or the establishment of long-term reservoirs of cytogenetic material requires tissue culture of fibroblasts obtained by skin snip or punch biopsy. Fine needle aspiration biopsy (FNAB) was investigated as an alternative method of fibroblast collection with advantages of minimal morbidity and the potential of better patient acceptance. After flank FNAB, culture, and incubation, confluent fibroblast growth was achieved within 19 days. The number of cells produced was sufficient for potential cytogenetic or metabolic studies. Cultured fibroblasts obtained via FNAB proved to be a successful alternative to skin biopsy, with reduced trauma and better cosmetic results.

Exposure of human renal proximal tubular cells to glucose leads to accumulation of type IV collagen and fibronectin by decreased degradation

Thickening and reduplication of the tubular basement membrane has been reported as an early event in diabetic nephropathy. In the current study we examined the effects of elevated D-glucose concentrations on human proximal tubular (HPTC) type IV collagen and fibronectin turnover. Incubation of confluent growth arrested HPTC with 25 mM D-glucose led to accumulation of both type IV collagen and fibronectin. This effect was maximal at 48 hours and represented a sevenfold increase for fibronectin (N = 4, P = 0.04), and a threefold increase for type IV collagen (N = 3, P = 0.03) over cells exposed to 5 mM D-glucose controls. This increase was not dependent on new gene transcription for either protein. Tissue inhibitor of metalloproteinases (TIMP 1 + TIMP 2) were induced following addition of 25 mM D-glucose, but not when cells were exposed to 5 mM D-glucose. Twenty-four hours after the addition of 25 mM D-glucose there was an eightfold increase in TIMP 1 (P = 0.009, N = 4), and a tenfold increase in TIMP 2 levels (P = 0.003, N = 4), over the control values for both inhibitors. The increase in both TIMP 1 and TIMP 2 in response to 25 mM D-glucose was abrogated in a dose dependent manner by the aldose reductase inhibitor sorbinil. Gelatin-substrate gel zymography showed increased activity of gelatinase A, but not of gelatinase B in response to the addition of 25 mM D-glucose to HPTC. The induction of gelatinase A was accompanied by increased gelatinase A mRNA expression, which was inhibited both by protein kinase C (PKC) depletion using PMA pre-treatment, and by the addition of a PKC inhibitor. These data demonstrate that the glucose-induced accumulation of type IV collagen and fibronectin is unrelated to increased gene transcription, but may involve alterations in the degradative pathway of these basement membrane constituents. Furthermore, the data demonstrate that glucose may simultaneously activate two intracellular pathways (the polyol pathway and a PKC dependent activation pathway), which are involved in mediating separate, complementary effects on cell function.

Cca1 is required for formation of growth-arrested confluent monolayer of rat 3Y1 cells.

A novel cDNA fragment, named cca1 (confluent 3Y1 cell-associated 1), was previously isolated on the basis of preferential accumulation of the corresponding mRNA in growth-arrested confluent but not in growing subconfluent rat 3Y1 cells. The cca1 cDNA was found to consist of 5022 nucleotides with an open reading frame of 1905 nucleotides, encoding a protein of 635 amino acids. Unlike the 3Y1 cell case, cca1 mRNA was not detected in confluent 3Y1 BU, 3Y1 BU/pTK, 3Y1-16E6, or F2408 cells, whose growth patterns monitored by phalloidin staining and bromodeoxyuridine incorporation were different from those of the confluent 3Y1 cells. A restoration of the confluent 3Y1-type growth pattern was observed in the cca1 cDNA-introduced 3Y1 BU and 3Y1 BU/pTK cells after reaching confluence but not in the cDNA-introduced 3Y1-16E6 or F2408 cells. The results allow us to conclude that cca1 is required but not sufficient for formation of growth-arrested confluent monolayer of 3Y1 cells.