Abstract

Water channel proteins: from their discovery in Cluj-Napoca, Romania in 1985,to the 2003 Nobel Prize in Chemistry and their implications in molecular medicine

Gheorghe Benga

Water channel proteins, later called aquaporins, are transmembrane proteins that have as their main (specific) function the water transport across biological membranes. Although the possible existence of specific pathways for water permeation across biological membranes was predicted for a long time, the first water channel protein, now called aquaporin 1, was identified or "seen" in situ (hence discovered) in the human red blood cell membrane in 1985 by Benga's group (at the Department of Cell and Molecular Biology, "Iuliu Hatieganu" University of Medicine and Pharmacy, Cluj-Napoca, Romania). This was achieved by a very selective radiolabelling of RBC membrane proteins with the water transport inhibitor ²⁰³ Hg-p- chloromercuribenzene sulfonate (PCMBS), under conditions of specific inhibition. The work was first published in 1986 in Biochemistry and Eur. J. Cell Biol. and reviewed by Benga in several articles in subsequent years. We have thus a world priority in the discovery of the first water channel protein in the RBC membrane, that was re-discovered by chance by the group of Agre (Baltimore, USA) in 1988, when they isolated a new protein from the RBC membrane, nicknamed CHIP28 (channel-forming integral membrane protein of 28 kDa). However, in addition to the 28 kDa component, this protein had a 35－60 kDa glycosylated component, the one detected by the Benga's group. Only in 1992 the Agre's group suggested that "CHIP28 is a functional unit of membrane water channels". In 1993 CHIP28 was renamed aquaporin 1. Since 1993 water channel proteins became a very hot area of research; more than 200 members of the aquaporin family have been found in bacteria, plants, animals and humans; a great diversity of physiological and pathological implications are being uncovered. If we make a comparison with the discovery of New World of America, the first man who has "seen" a part, very small indeed, of The New Land was Columbus; later, others, including Amerigo Vespucci (from whom the name derived), have better "seen" and in the subsequent years many explorers discovered the complexity of the Americas. Consequently, the initial discovery of the first water channel by Benga's group must be properly credited; the omission of Gheorghe Benga from the 2003 Nobel Prize in Chemistry (half of which was awarded to Peter Agre "for the discovery of the water channels") was a new mistake in the award of Nobel Prizes. Benga's claim is presented on the web site of the Ad Astra Association (www.ad-astra.ro/ benga). As can be seen on this site his recognition as a discoverer of the first water channel protein from the human RBC membrane is growing. Thousands of science-related professionals from hundreds of academic and research units, as well as participants in several international scientific events, have signed as supporters of Benga; his priority is also mentioned in several comments on the 2003 Nobel Prize as presented on the site.

Chemokines in cancer metastasis

Sam T. Hwang

It is clear that G-protein-coupled chemokine receptorsare often upregulated in many common humancancers, including those of the breast and colon as wellas melanoma. CXCR4 and CCR7 appear to be the mostfrequently upregulated receptors and have been reportedto be expressed in a great variety of both solid29and hematopoietic cancers. CXCR4 appears to promotecancer cell survival and may be upregulated inresponse to hypoxia and other cellular stress. Orthotopicimplantation of B16 melanoma cells transducedwith CCR7 cDNA resulted in dramatically increased metastasis to the lymph node, the most common site ofmetastasis among all cancers. Other receptors such asCCR9 and CCR10 appear to be restricted in expressionto melanoma and may contribute to metastasis to thesmall bowel and skin, respectively, through the chemokines CCL25 and CCL27. Indeed, B16 melanoma cellstransduced with CCR10 were able to avoid immunecell-mediated attack in vitro and in vivo in a CCL27-dependent manner, suggesting a role for CCR10 in theresistance of cancer cells to immune destruction. Chemokine receptors may facilitate tumor dissemination atseveral key steps of metastasis, including adherence oftumor cells to endothelium, extravasation from bloodvessels, metastatic colonization, angiogenesis, proliferation,and protection from the host response via activationof cellular pathways such as PI3K and Akt.Interestingly, many of these roles are reminiscent oftheir functions in leukocyte and stem cell trafficking.Lastly, small molecule inhibitors that block chemokinereceptor function may lead to novel therapeutic strategies for treating patients with cancer.

Embryonic genes in post-natal articular cartilage

Chisa Hidaka

Genes that regulate embryonic tissue development － 'embryonic genes' － are re-expressed during the agingrelated degeneration of several types of tissues including cartilage, muscle and blood vessels. Recently several groups have reported the up regulation of a number of genes important in limb development in cartilage affected by osteoarthritis (OA). Whereas recent advances in human and mouse genetics have elegantly elucidated the function of many of these 'embryonic genes' during development, their post-natal functions remain largely unexplored. Recently, we have examined the expression of (pre-B cell homeobox 1) Pbx1, a homeobox transcription factor that is critical for embryonic chondrocyte proliferation and joint formation, in post-natal articular cartilage. In addition we have examined the expression of other 'embryonic' genes including SRY-related homeobox factor 9 (Sox9) and pre-adipocyte factor-1 (Pref-1), among others. Changes in the expression of these and other chondrocyte-specific genes with age was examined. As we and others have shown that chondrocytes from different zones of articular cartilage have distinct phenotypes, we also examined differences in the levels of gene expression in different zones of articular cartilage. It is unclear whether the reactivation of 'embryonic genes' indicates the recruitment and/or activation of a local pool of adult progenitor/stem cells or whether it represents pathologic gene dysregulation in terminally differentiated cells. These two possibilities suggest two distinct strategies for approaching 'embryonic genes' in degenerative diseases such as OA. Our findings regarding such genes will be discussed in the context of how they may be manipulated for therapeutic purposes.

Heterodimeric BMPs for enhancing bone formation

Chisa Hidaka

Bone morphogenetic proteins (BMPs) are growth factors in the transforming growth factor group and are critically important in limb development and post-natal bone healing. Although clinical BMP therapy is effective, required doses are very high. Previous studies have suggested that the co-expression of two different BMP genes can result in the production of heterodimeric BMPs that may be more potent than homodimers. To test the potency of BMP2/7 heterodimers, combined BMP2 and BMP7 gene transfer was performed, either by co-transfection with two genes, or by transfection with a novel 'fusion gene' encoding both BMPs. Both approaches resulted in the production of BMP2/7 heterodimers, and these heterodimers were more potent than their respective homodimers in assays of osteoblastic differentiation. Co-transfection with BMP2 and BMP7 was also shown to be more effective than single gene transfer in achieving spine fusion in an in vivo experimental model. Our findings on heterodimeric BMPs will be discussed in the context of improving BMP therapy for applications requiring bone formation.

Bortezomib: current therapeutic strategies in multiple myeloma

Teru Hideshima

Bortezomib (Velcade®) is a proteasome inhibitor which blocks the chymotryptic activity of the 26S proteasome and was first validated as a treatment strategy in multiple myeloma (MM). Our in vitro studies have confirmed that Bortezomib induces JNK/caspasedependent apoptosis in conventional drug resistant MM cell lines, as well as patient MM cells. Bortezomib also; downregulates expression of adhesion molecules on MM cells and bone marrow stromal cells and related binding; blocks constitutive and MM cell adhesion-induced NF-κB dependent cytokine secretion in BMSCs; and inhibits angiogenesis. Bortezomib also inhibits human MM cell growth, decreases tumorassociated angiogenesis, and prolongs host survival in models of human MM in SCID mice. These preclinical studies have translated to the bedside. Phase I trials in man establishing its safety profile and early evidence of anti-MM activity, were completed in 2000. In 2001 a phase II trial of Bortezomib treatment of 202 patients with refractory relapsed MM demonstrated 35% responses, including 10% complete and near complete responses and clinical benefit. Based upon these results, Bortezomib was FDA approved for treatment of relapsed refractory MM in May 2003, with bench to bedside translation and approval in 3 years. In 2005, FDA approval was extended to patients with relapsed MM, based upon an international multicenter phase III trial comparing Bortezomib versus dexamethasone which demonstrated statistically significant prolongation in time to progression and survival in the Bortezomib-treated cohort. Clinical phase I/II trials are now combining this agent with conventional as well as novel therapies for both patients with relapsed MM and those earlier in the disease course.

Objective evaluation of dysphagia

Tai Ryoon Han

Although videofluorographic swallowing study (VFSS) is a kind of golden standard method in evaluation of dysphagia, there was no trial about the quantitative evaluation of VFSS findings. For more objective evaluation of dysphagia, we tried to make a functional dysphagia scale based on the long term outcome of dysphagia. We calculated the odds ratio and p-value of each findings of initial VFSS based on the swallowing outcome of poststroke 6 months. For the result, we found that residue in pyriform sinus, subglottic aspiration, poor tongue to palate, delayed epiglottic closure were the major factors. We made this functional dysphagia scale to be 100 scales and the reference value for poor outcome was 57.35 with ROC diagram. With this reference value, the positive predictive value was 1.0 and the negative predictive value was 0.92. For another objective evaluation of dysphagia, we did the motion analysis of VFSS in stroke patients. We digitized 7 points for motion analysis-tip of food bolus, anterior and posterior margin of hyoid bone, anterior and posterior margin of vocal cord and tip and base of epiglottis. We evaluated the trajectory of hyoid bone and epiglottis and found that there were two types of abnormalities － incoordinated type and weakness type － in hyoid bone and epiglottic movement. For tentative summary, we could prove not only incoordinated motion of pharyngeal structures but also decreased movement pattern can cause the dysphagia in stroke patients.Also, we analyzed the correlation between food bolus, hyoid bone and epiglottis movement in stroke patients with this motion analysis and compared with normal person's data. For tentative conclusion, we thought that the onset latency of epiglottis and hyoid elevation were significantly delayed in stroke patients however, maximal folding angle of epiglottis and maximal amplitude of hyoid bone were relatively maintained. Also we thought that the severity of dysphagia was related with the degree of delayed onset latency of epiglottis and hyoid bone and also related with hyoid bone-food bolus-epiglottis coordinated time sequence.