Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

Involvement of ADAM10 in axonal outgrowth and myelination of the peripheral nerve

The disintegrin and metalloproteinase 10 (ADAM10) is a membrane‐anchored metalloproteinase with both proteolytic and disintegrin characteristics. Here, we investigate the expression, regulation, and functional role of ADAM10 in axonal outgrowth and myelination of the peripheral nerve. Expression pattern analysis of 11 ADAM family members in co‐cultures of rat dorsal root ganglia (DRG) neurons and Schwann cells (SCs) demonstrated the most pronounced mRNA expression for ADAM10. In further studies, ADAM10 was found to be consistently upregulated in DRG‐SC co‐cultures before the induction of myelination. Neurons as well as SCs widely expressed ADAM10 at the protein level. In neurons, the expression of ADAM10 was exclusively limited to the axons before the induction of myelination. Inhibition of ADAM10 activity by the hydroxamate‐based inhibitors GI254023X and GW280264X resulted in a significant decrease in the mean axonal length. These data suggest that ADAM10 represents a prerequisite for myelination, although its activity is not required during the process of myelination itself as demonstrated by expression analysis of myelin protein zero (P0) and Sudan black staining. Hence, during the process of myelin formation, ADAM10 is highly upregulated and appears to be critically involved in axonal outgrowth that is a requirement for myelination in the peripheral nerve. © 2009 Wiley‐Liss, Inc.     

Development and evaluation of a case group concept for inpatients with mental disorders in Germany: Using self-report and expert-rated instruments

The aim of this study was to evaluate a case-mix system to classify inpatients with mental disorders in Germany by means of self-report and expert-rated instruments. The use of case-mix systems enhances the transparency of performance and cost structure and can thus improve the quality of mental health care. We analysed a consecutive sample of 1677 inpatients with mental disorders from 11 hospitals using regression tree analysis. The model assigns patients to 17 groups, accounting for 17% of the variance for duration of stay. Patients with eating disorders had a longer duration of stay than patients with anxiety disorder, duration of mental illness of less than 3–5 years, lower levels of interpersonal problems and higher occupational position. The results showed that besides diagnosis, variables such as duration of illness and interpersonal problems are important for classifying inpatients with mental disorders. The results of the study should be critically reviewed regarding the empirical results of other studies and the appropriateness of case group concepts for inpatients with mental disorders.     

Dietary fiber and bowel function in tube-fed patients.

In tube-fed patients, dietary fiber is often used to manage constipation/diarrhea. Dietary fiber consists of water-soluble and insoluble plant compounds that are resistant to digestion by small-bowel enzymes but are fermented to varying degrees by colonic bacteria. Many physiologic effects of fiber may be related to the degree of fermentation. Few controlled studies of fiber-containing tube feedings have been performed. These studies have limitations and are nondefinitive as to whether fiber prevents or controls constipation/diarrhea. Constipation in tube-fed patients has not been shown to respond to mixed soluble/insoluble fiber in the few studies performed to date. Likewise, fiber may be of only limited benefit in controlling diarrhea in acute illness because of such factors as stress or medication. Fiber does play a role in maintaining gut integrity in all patients, whether they have diarrhea or not. Fiber may be recommended as part of a standard tube-feeding regimen to help assure gut mucosal integrity but not specifically to treat constipation/diarrhea. Further studies are necessary before the role of fiber in the management of constipation/diarrhea in tube-fed patients is determined.     

The REPAIR-AMI and ASTAMI trials: cell isolation procedures: reply

It obviously escaped the notion of Egeland and Brinchman thatthe protocols additionally differ with regard to the washingsteps and buffer components used in the     

Initially Accelerated Fractionation of the R1H Tumor of the Rat Increases Local Tumor Control and Might Reduce Impact of Overall Treatment Time

PURPOSE: To study the effect of treatment time prolongation following initial dose acceleration on the response of subcutaneously growing R1H tumor. MATERIAL AND METHODS: Continuous standard fractionation (30 fractions/40 days) was compared to initially accelerated treatment (30 fractions/21 days) followed by five to two fractions per week yielding total treatment times from 40 to 72 days. Local tumor control was assessed as endpoint. RESULTS: Radiation dose to control 50% of the tumors (TCD50%) decreased statistically significant from 83.5 Gy (95% confidence interval [CI]: 78.6 .. 88.4) for standard fractionation to 74.1 Gy (95% CI: 72.7 .. 75.5) determined for all accelerated treatment arms (p = 0.003). Prolongation of treatment time after initial acceleration from 40 to 72 days led to a small but statistically not significant increase in TCD50% from 72.0 Gy (95% CI: 71.0 .. 72.9) to 76.2 Gy (95% CI: 69.9 .. 82.4) corresponding to a repopulated dose of 0.9 Gy per week. This time factor is considerably smaller than for conventional radiation treatment as determined in previous experiments. CONCLUSION: The results indicate that initially accelerated irradiation not only improves local tumor control but also minimizes the negative effect of treatment time prolongation. This might be due to changes in tumor cell repopulation kinetics.     

Contribution of Calcium Channel Subtypes to the Intracellular Calcium Signal in Sensory Neurons: The Effect of Injury

Background: Although the activation-induced intracellular Ca2+ signal is disrupted by sensory neuron injury, the contribution of specific Ca2+ channel subtypes is unknown.

Methods: Transients in dissociated rat dorsal root ganglion neurons were recorded using fura-2 microfluorometry. Neurons from control rats and from neuropathic animals after spinal nerve ligation were activated either by elevated bath K+ or by field stimulation. Transients were compared before and after application of selective blockers of voltage-activated Ca2+ channel subtypes.

Results: Transient amplitude and area were decreased by blockade of the L-type channel, particularly during sustained K+ stimulation. Significant contributions to the Ca2+ transient are attributable to the N-, P/Q-, and R-type channels, especially in small neurons. Results for T-type blockade varied widely between cells. After injury, transients lost sensitivity to N-type and R-type blockers in axotomized small neurons, whereas adjacent small neurons showed decreased responses to blockers of R-type channels. Axotomized large neurons were less sensitive to blockade of N- and P/Q-type channels. After injury, neurons adjacent to axotomy show decreased sensitivity of K+-induced transients to L-type blockade but increased sensitivity during field stimulation.