[Molecular classification of bladder cancer. Possible similarities to breast cancer]. / Molekulare Klassifikation des Harnblasenkarzinoms. Mögliche Ähnlichkeit zum Mammakarzinom.

Therapeutic decisions for breast cancer are increasingly becoming based on subtype-specific gene expression tests. For bladder cancer very similar subtypes have been identified by genome-wide mRNA analysis, which as for breast cancer differ with respect to the prognosis and response to therapy on the basis of their hormone dependency. At the DNA level, however, the type of mutations and their frequencies within the subtypes are strikingly different between bladder and breast cancers. It will be interesting to see whether possible driver mutations can serve as therapeutic targets in both indications. In contrast, the apparent hormone dependency of a substantial number of bladder carcinomas suggests that hormonal and anti-hormonal treatment can be valid therapy options similar to breast cancer. Moreover, gender-specific differences with respect to the incidence and aggressiveness of male compared to female bladder cancers can be explained by hormonal effects. Together with forthcoming immunomodulatory therapies these multiple therapy options raise and give new hope to efficiently combat this aggressive disease.

Bone-metastatic prostate carcinoma favors mesenchymal stem cell differentiation toward osteoblasts and reduces their osteoclastogenic potential.

Bone homeostasis is achieved by the balance between osteoclast-dependent bone resorption and osteoblastic events involving differentiation of adult mesenchymal stem cells (MSCs). Prostate carcinoma (PC) cells display the propensity to metastasize to bone marrow where they disrupt bone homeostasis as a result of mixed osteolytic and osteoblastic lesions. The PC-dependent activation of osteoclasts represents the initial step of tumor engraftment into bone, followed by an accelerated osteoblastic activity and exaggerated bone formation. However, the interactions between PC cells and MSCs and their participation in the disease progression remain as yet unclear. In this study, we show that bone metastatic PC-3 carcinoma cells release factors that increase the expression by human (h)MSCs of several known pro-osteoblastic commitment factors, such as α5/ß1 integrins, fibronectin, and osteoprotegerin. As a consequence, as shown in an osteogenesis assay, hMSCs treated with conditioned medium (C(ed) M) derived from PC-3 cells have an enhanced potential to differentiate into osteoblasts, as compared to hMSCs treated with control medium or with C(ed) M from non-metastatic 22RV1 cells. We demonstrate that FGF-9, one of the factors produced by PC-3 cells, is involved in this process. Furthermore, we show that PC-3 C(ed) M decreases the pro-osteoclastic activity of hMSCs. Altogether, these findings allow us to propose clues to understand the mechanisms by which PC favors bone synthesis by regulating MSC outcome and properties.

Chinese hamster ovary cells lacking GM1 and GD1a synthesize gangliosides upon transfection with human GM2 synthase.

GM3-positive Chinese hamster ovary cells (CHO-K1 cells) lack the ability to synthesize GM2 and the complex gangliosides GM1 and GD1a from [3H]Gal added to the culture medium. However, they acquire the ability to synthesize GM2 and to synthesize and immunoexpress complex gangliosides upon transient transfection with a cDNA encoding the human GM3:N-acetylgalactosaminyl transferase (GM2 synthase). The activities of endogenous GM1- and GD1a-synthases in the parental cell line and in cells transfected with the plasmid with or without the GM2 synthase cDNA were essentially identical and comparable in terms of specific activity with the endogenous GM3 synthase. Results indicate that glycosyltransferases acting on GM2 to produce GM1 and GD1a are constitutively present in CHO-K1 cells, and that the expression of their activities depend on the supply of the acceptor GM2. In addition, these results lend support to the notion that GM2 synthase is a key regulatory enzyme influencing the balance between simple and complex gangliosides.

Immunohistochemical localization of two proteinases in skeletal muscle.

Immunohistochemical localizations of cytosolic and myofibrillar proteinases revealed a different myofiber locale for each enzyme in the rat. Although the cytosolic proteinase was most pronounced in mast cells within soleus and extensor digitorum longus (EDL) muscles, certain fibers of the EDL were also positive. The myofibrillar proteinase, on the other hand, appeared to be present in interstitial spaces between muscle fibers in the EDL but conversely present in some fibers of the soleus muscle.

Alkaline proteinase localization in myoblasts.

Recent interest in elucidating the role of non-lysosomal proteases in intracellular protein catabolism in muscle has led to various investigations with three alkaline proteases: a trypsin-like, a chymotrypsin-like, and a high molecular weight cysteine proteinase. Although in vitro biochemical assays have revealed the catabolic potential of at least two of these proteases, confirmation of their presence in muscle cells has been difficult. In this study immunohistochemical techniques were employed to localize each of these proteases in rat myoblasts. Antisera against the trypsin-like and chymotrypsin-like proteinase (both serine proteinases) showed strong localization in the cytoplasm immediately around the nucleus. Both also stained chromatin material in the nucleus of these cells. Fluorescent localization of the high molecular weight cysteine proteinase (Proteinase I) also appeared to be cell-associated in the myoblasts. The use of myoblasts in cell culture sections of whole muscle was advantageous, since localization of the proteases could be assessed in the absence of other cell types.

Expression of beta 1-4 N-acetylgalactosaminyltransferase gene in the developing rat brain and retina: mRNA, protein immunoreactivity and enzyme activity.

The developmental pattern of expression of the UDP-GalNAc:GM3 N-acetylgalactosaminyltransferase (GalNAc-T) gene was examined in the rat brain and retina. A GalNAc-T cDNA cloned from a rat olfactory bulb cDNA library was used as a probe for Northern blot and in situ hybridization experiments and a rabbit polyclonal antibody to rat GalNAc-T peptide was used for Western blot analysis. In Northern blot experiments, a single approximately 3 kb transcript was detected both in brain and retina. In brain, the abundance of this transcript increased from E15 to PN1-5 and then declined while, in retina, it increased steadily from PN1 to PN13-24. The developmental trends of GalNAc-T mRNA expression, GalNAc-T immunoreactive protein and GalNAc-T activity were comparable in brain. In retina, however, GalNAc-T activity and GalNAc-T peptide immunoreactivity followed developmental patterns that were similar between them and different from that of the specific mRNA. Results suggest that post-transcriptional controls of the GalNAc-T gene expression operate in the rat CNS, which are particularly evident in retina. The expression of the GalNAc-T gene in glial and neuronal cells was examined in rat retina cell cultures by in situ hybridization. The GalNAc-T mRNA was abundant in GM1+/GD3+ neurons and almost absent in the flat, GM1-/GD3+ Müller glia-derived cells.

Extracellular matrix disruption and pain after eccentric muscle action.

Pain, stiffness, and indicators of muscle damage occur at different times after eccentric muscle action. After a single bout of maximal resisted lengthening of the elbow flexors, elbow position, pain perception, and indicators of cellular damage were measured. Immediately postexercise, a significant decrease in resting muscle length was observed that continued to 48 h. At this time, an increase in perceived muscle soreness was noted (P less than 0.05), and a biopsy of the biceps brachii revealed mast cell degranulation, separations of the extracellular matrix from myofibers, and increased plasma constituents in the extracellular space. It is proposed that myofiber disruption allows intracellular proteins to escape and extracellular proteins and ions to enter, causing swelling, whereas the disrupted extracellular matrix initiates the inflammatory response, which includes the release of mast cell granules seen at 48 h postexercise. Thus the delayed sensation of pain (soreness) after repeated eccentric muscle actions probably results from inflammation in response to extracellular matrix disruption.

Effect of spaceflight on the extracellular matrix of skeletal muscle after a crush injury.

The organization and composition of the extracellular matrix were studied in the crush-injured gastrocnemius muscle of rats subjected to 0 G. After 14 days of flight on COSMOS 2044, the gastrocnemius muscle was removed and evaluated by histochemical and immunohistochemical techniques from the five injured flight rodents and various Earth-based treatment groups. In general, the repair process was similar in all injured muscle samples with regard to the organization of the extracellular matrix and myofibers. Small and large myofibers were present within an expanded extracellular matrix, indicative of myogenesis and muscle regeneration. In the tail-suspended animals, a more complete repair was observed with no enlarged area of nonmuscle cells or matrix material visible. In contrast, the muscle samples from the flight animals were less well organized and contained more macrophages and blood vessels in the repair region, indicative of a delayed repair process, but did not demonstrate any chronic inflammation. Myofiber repair did vary in muscles from the different groups, being slowest in the flight animals and most complete in the tail-suspended ones.

Cloning, characterization and developmental expression of alpha2,8 sialyltransferase (GD3 synthase, ST8Sia I) gene in chick brain and retina.

GD3 and GM2 synthases act on ganglioside GM3 at the branching point of the pathway of synthesis of gangliosides in which the "a", "b" and "c" families are produced. The relative activities of these enzymes are important for regulating the ganglioside composition of a given tissue. In the present work, we report the cloning and characterization of a chick GD3 synthase cDNA. The cloned cDNA directed the synthesis of a functionally active enzyme in transiently transfected CHO-K1 cells and was highly homologous to mammalian GD3 synthases. In Northern blot experiments the cDNA detected a single specific GD3 synthase mRNA of about 9.0 kb both in the chicken brain and retina. The abundance of the specific mRNA transcript declined steadily from E7-E9 to very low values around PN2. The levels of enzyme activities measured at the same developmental stages roughly followed the changes of specific mRNA levels in both tissues. In situ hybridization of embryonic neural retina cells in culture showed that both glial- and neuron-like cells expressed the specific GD3 synthase mRNA, although with different intensities. Results indicate that transcription and/or stability of the specific GD3 synthase mRNA constitute a level of control of the expression of GD3 synthase and indirectly of the ganglioside composition in the developing chicken central nervous system (CNS).

Characterization of muscles injured by forced lengthening. II. Proteoglycans.

After forced muscle lengthening of rat soleus muscle, alterations in muscle connective tissues were monitored by fluorescent immunohistochemical methods. Monoclonal antibodies directed against the polysaccharide attachment region of proteoglycans were used to observe changes in localization of 4-sulfated, 6-sulfated, or unsulfated chondroitin sulfate disaccharide units covalently bound to the proteoglycan protein core after injury. Additionally, fluorescein-labeled concanavalin A lectin and polyclonal antiserum to heparan sulfate proteoglycan were also localized in muscle sections during the regenerative process over 5 days after injury. Although proteoglycan localization was absent at or near the site of myofiber damage after injury, some distinct basal lamina remained as a matrix for regenerating myofibers. By the fifth day post-injury, the localization of these matrix components had returned to that seen in uninjured soleus muscles. The physiological significance of these extracellular matrix changes appeared to center on the repair of the torn myofiber and indicate an interdependence between myofibers and the extracellular matrix in this type of regeneration.

Characterization of muscles injured by forced lengthening. I. Cellular infiltrates.

Myofiber injury-repair was studied in rat soleus muscles to elucidate the role of infiltrating cells in the injury-repair process. Muscle injury was induced by forced muscle lengthening with the contralateral muscle serving as a control. The muscles were removed for histologic, histochemical and immunohistochemical procedures at varying periods (12-120 h) post-injury. All injured muscles were severely damaged with many cells present in the interstitial spaces between myofibers. Normal appearing myofibers demonstrated elevated lysosomal proteolytic activity, but no evidence of increased activity, indicative of phagocytic cells, was found in or between damaged myofibers. The esterase stain for macrophages and immunohistochemical techniques for mast cells also provided no support for either cell type predominating in the damaged area, although mast cell degranulation could be observed in the pericapillary regions. In contrast, the use of a specific antisera for a multicatalytic protease uniquely defined most of these cells as myogenic in origin. They appeared to be most numerous between the torn ends of a myofiber. Surprisingly, the remainder of the cells appeared to be of lymphoid origin.

Some aspects of neurological presentation of patients with important cardiac arrhythmias requiring pacemaker therapy: a preliminary report.

We report the short- and long-term effects of pacing on 82 patients presenting with significant bradyarrhythmias and neurological symptoms. The 3 categories of neurological symptoms were dizziness alone, syncope with or without dizziness and focal neurological symptoms with a history of dizziness or syncope.

An injury model myopathy mimicking dystrophy: implications regarding the function of dystrophin.

The pathogenesis of dystrophin deficient myopathies remains unknown. Rat and human muscles subjected to severe injury following repeated eccentric muscle actions demonstrate histopathological alterations which mimic a dystrophic process. Immunofluorescent histochemical examination of these injured muscles demonstrates a separation of proteoglycans of the basal lamina from the muscle plasma membrane, the identical histopathological alteration observed in Duchenne muscular dystrophy. These findings are consistent with the hypothesis that dystrophin is essential for maintenance of the structural integrity of the sarcolemma.

Aphanomyces euteiches Inoculum Potential: A Rolled-Towel Bioassay Suitable for Fine-Textured Soils.

A sensitive measure of soil inoculum potential is needed to evaluate field management of common root rot (Aphanomyces euteiches) in peas (Pisum sativum). A modified rolled-towel (RT) bioassay had been proposed to measure soil inoculum potential in fine-textured soils used for pea production in Minnesota. Homogenized soil was used because organic debris containing the inoculum could not be separated by wet sieving. The poor precision prompted an evaluation of procedures to improve this modified RT bioassay. Seed treatment with a 5% solution of sodium hypochlorite before pea seed germination and plant isolation procedures during the RT bioassay preparation/incubation reduced seedborne contamination and seedling loss to less than 5%. Tests conducted with pasteurized soil that was artificially infested with oospores showed the region of the pea taproot 1 to 2 cm below the seed to be more susceptible to infection (33% compared with 15% infected seedlings) than the region 1 to 2 cm above the root tip. A soil volume of 1.0 cm3 increased inoculum potential compared with 0.5 cm3 applied to each seedling but did not influence the random error; the 40-seedling compared with the 20-seedling RT bioassay reduced random error from 18 to 12%. The modified RT bioassay conducted on soil that was artificially infested after steam treatment or without steam treatment showed superior performance when using 40 seedlings compared with 20 seedlings when evaluated for accuracy and precision. Multiple infection theory demonstrated more multiple infections in the RT bioassay with a 0.5 cm3 soil volume applied to each seedling, which shows that soil mass is a factor preventing a higher percentage of infected seedlings. These modifications to the RT bioassay improved the method enough to reduce the random error by one-half compared with using homogenized soil without the proposed modifications.

Metabolic intervention on lipid synthesis converging pathways abrogates prostate cancer growth.

One of the most conserved features of all cancers is a profound reprogramming of cellular metabolism, favoring biosynthetic processes and limiting catalytic processes. With the acquired knowledge of some of these important changes, we have designed a combination therapy in order to force cancer cells to use a particular metabolic pathway that ultimately results in the accumulation of toxic products. This innovative approach consists of blocking lipid synthesis, at the same time that we force the cell, through the inhibition of AMP-activated kinase, to accumulate toxic intermediates, such as malonyl-coenzyme A (malonyl-CoA) or nicotinamide adenine dinucleotide phosphate. This results in excess of oxidative stress and cancer cell death. Our new therapeutic strategy, based on the manipulation of metabolic pathways, will certainly set up the basis for new upcoming studies defining a new paradigm of cancer treatment.

Metabolism and proliferation share common regulatory pathways in cancer cells.

Cancer development involves major alterations in cells' metabolism. Enhanced glycolysis and de novo fatty acids synthesis are indeed characteristic features of cancer. Cell proliferation and metabolism are tightly linked cellular processes. Others and we have previously shown a close relationship between metabolic responses and proliferative stimuli. In addition to trigger proliferative and survival signaling pathways, most oncoproteins also trigger metabolic changes to transform the cell. We present herein the view that participation of cell-cycle regulators and oncogenic proteins to cancer development extend beyond the control of cell proliferation, and discuss how these new functions may be implicated in metabolic alterations concomitant to the pathogenesis of human cancers.