Josef P. Magyar

A TRANSGENIC RAT MODEL OF CHARCOT-MARIE-TOOTH DISEASE

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy in humans and has been associated with a partial duplication of chromosome 17 (CMT type 1A). We have generated a transgenic rat model of this disease and provide experimental evidence that CMT1A is caused by increased expression of the gene for peripheral myelin protein-22 (PMP22, gas-3). PMP22-transgenic rats develop gait abnormalities caused by a peripheral hypomyelination, Schwann cell hypertrophy (onion bulb formation), and muscle weakness. Reduced nerve conduction velocities closely resemble recordings in human patients with CMT1A. When bred to homozygosity, transgenic animals completely fail to elaborate myelin. We anticipate that the CMT rat model will facilitate the identification of a cellular disease mechanism and serve in the evaluation of potential treatment strategies.

Mass Production of Embryoid Bodies in Microbeads

Abstract: Embryonic stem cells (ESC) are totipotent cells that can differentiate into a large number of different cell types. Stem cell‐derived, differentiated cells are of increasing importance as a potential source for non‐proliferating cells (e.g., cardiomyocytes or neurons) for future tissue engineering applications. Differentiation of ESC is initiated by the formation of embryoid bodies (EB). Current protocols for the generation of EB are either of limited productivity or deliver EB with a large variation in size and differentiation state. To establish an efficient and robust EB production process, we encapsulated mouse ESC into alginate microbeads using various microencapsulation technologies. Microencapsulation and culturing of ESC in 1.1% alginate microbeads gives rise to discoid colonies, which further differentiate within the beads to cystic EB and later to EB containing spontaneously beating areas. However, if ESC are encapsulated into 1.6% alginate microbeads, differentiation is inhibited at the morula‐like stage, so that no cystic EB can be formed within the beads. ESC colonies, which are released from 1.6% alginate microbeads, can further differentiate to cystic EB with beating cardiomyocytes. Extended supplementation of the growth medium with retinoic acid promotes differentiation to smooth muscle cells.

Myelin and lymphocyte protein (MAL/MVP17/VIP17) and plasmolipin are members of an extended gene family

An increasing number of four-transmembrane proteins has been found to be associated with CNS and PNS myelin. Some of these proteins play crucial roles in the development and maintenance of the nervous system. In the CNS, proteolipid protein (PLP) is mutated in the myelin disorder Pelizaeus-Merzbacher disease and in spastic paraplegia, while in the PNS, peripheral myelin protein 22 (PMP22) and connexin32 (C x 32) are culprit genes in the most frequent forms of hereditary peripheral neuropathies. Myelin and lymphocyte protein (MAL; also called MVP17 or VIP17) and plasmolipin are additional tetraspan proteins that are highly expressed by myelinating glial cells. However, little is known about the role of these proteins in the nervous system. As a prerequisite for functional genetic approaches in the mouse, we have isolated and characterized a mouse MAL cDNA and the corresponding structural MAL gene. Computer-aided analysis and database searches revealed that MAL belongs to a larger gene family which also includes plasmolipin, BENE and the expressed sequence tag (EST) H09290. While the overall amino acid sequence identities between mouse MAL and the related proteins are relatively low (29-37%), the conserved motif -[Q/Y-G-W-V-M-F/Y-V]- which is found at the junction of the first extracellular loop and the second membrane-associated domain serves as a fingerprint for the MAL protein family. Expression analysis of the members of the MAL gene family indicates widespread expression in various tissues, suggesting a common role of these proteins in cell biology.

Progressive segregation of unmyelinated axons in peripheral nerves, myelin alterations in the CNS, and cyst formation in the kidneys of myelin and lymphocyte protein-overexpressing mice.

Abstract: Myelin and lymphocyte protein (MAL) is a putative tetraspan proteolipid that is highly expressed by Schwann cells and oligodendrocytes as a component of compact myelin. Outside of the nervous system, MAL is found in apical membranes of epithelial cells, mainly in the kidney and stomach. Because MAL is associated with glycosphingolipids, it is thought to be involved in the organization, transport, and maintenance of glycosphingolipid‐enriched membrane microdomains. In this report, we describe the generation and analysis of transgenic mice with increased MAL gene dosage. Immunohistochemical analysis revealed that the localization of MAL overexpression in the transgenic animals corresponded closely to the MAL expression pattern observed in wildtype animals, indicating correct spatial regulation of the transgene. Phenotypically, MAL overexpression led to progressive dissociation of unmyelinated axons from bundles in the PNS, a tendency to hypomyelination and aberrant myelin formation in the CNS, and the formation of large cysts in the tubular region of the kidney. Thus, increased expression of MAL appears to be deleterious to membranous structures in the affected tissues, indicating a requirement for tight control of endogenous MAL expression in Schwann cells, oligodendrocytes, and kidney epithelial cells.

Recombinant Sindbis virus allows expression and precise targeting of proteins of the contractile apparatus in cultured cardiomyocytes

Abstract Expression of epitope-tagged sarcomeric proteins in cardiomyocytes is a powerful approach for the characterization of interacting domains. Here, we report a new strategy for the study of the targeting of contractile proteins in cardiomyocytes by Sindbis virus (SIN)-mediated gene transfer. Two recombinant SIN were generated, one encoding the myosin-light chain MLC3f-eGFP fusion protein (SINrep5/MLC3f-eGFP), and the other encoding the α-actinin-DsRed fusion protein (SINrep5/α-actinin-DsRed). After infection of long-term cultured neonatal and adult rat cardiomyocytes with SINrep5/MLC3f-eGFP, the exogenous MLC3f-eGFP fusion protein localized to the sarcomeres. Freshly isolated rod-shaped ventricular cardiomyocytes infected with SINrep5/α-actinin-DsRed exhibited a correct incorporation of the newly synthesized α-actinin-DsRed fusion protein at the Z-band of the sarcomere. This allows the assumption that the exogenous protein is assembled into myofibrils in living cardiomyocytes using the same molecular interactions equally to the endogenous counterpart. It has been thus demonstrated that the SIN expression system makes possible the straightforward analysis of the localization of sarcomeric proteins in cultured cardiomyocytes and may offer new possibilities for the characterization of mutant proteins involved in hypertrophic cardiomyopathies.

Reactivation of the Mitosis-Promoting Factor in Postmitotic Cardiomyocytes

Cardiomyocytes cease to divide shortly after birth and an irreversible cell cycle arrest is evident accompanied by the downregulation of cyclin-dependent kinase activities. To get a better understanding of the cardiac cell cycle and its regulation, the effect of functional recovery of the mitosis-promoting factor (MPF) consisting of cyclin B1 and the cyclin-dependent kinase Cdc2 was assessed in primary cultures of postmitotic ventricular adult rat cardiomyocytes (ARC). Gene transfer into ARC was achieved using the adenovirus-enhanced transferrinfection system that was characterized by the absence of cytotoxic events. Simultaneous ectopic expression of wild-type versions of cyclin B1 and Cdc2 was sufficient to induce MPF activity. Reestablished MPF resulted in a mitotic phenotype, marked by an abnormal condensation of the nuclei, histone H3 phosphorylation and variable degree of decay of the contractile apparatus. Although a complete cell division was not observed, the results provided conclusive evidence that cell cycle-related events in postmitotic cardiomyocytes could be triggered by genetic intervention downstream of the G1/S checkpoint. This will be of importance to design novel strategies to overcome the proliferation arrest in adult cardiomyocytes.

Factors Involved In The Cell Cycle Arrest Of Adult Rat Cardiomyocytes

The mammalian heart consists of two pumps called the left and right ventricles. The left ventricle has to work harder as it must force the blood around the body. Therefore, it is more likely to fail despite the fact that it has more muscle. O2 carried by the blood is essential to maintain life. If the left ventricle weakens, the body suffers from O2 deficit. A failing left ventricle will also fail to keep pace with the right ventricle, which is feeding the lungs, thus causing pulmonary blood congestion. If the right ventricle weakens, it will fail to propel enough blood through the lungs to exchange O2 to meet the needs of the rest of the body. There are four major types of cardiovascular diseases — hypertension, atherosclerosis, ischemic heart disease and thrombosis — which account for most cases where the circulatory system goes wrong. Approximately 400,000 new cases of heart failure are registered in the US each year, summing up to four to five million people (Gheorghiade and Bonow, 1998). Heart attacks may lead to death or debilitation and the risk dying thereafter is considerably increased. (Cowie et al., 1997; Gheorghiade and Bonow, 1998; Sharpe and Doughty, 1998). Survivors suffer severe damage to heart muscle tissue that cannot regenerate. Frequently, the heart reacts to an increase in load caused by disease with a growth of the muscle tissue (hypertrophy) to compensate for pathological haemodynamics. This hypertrophy is solely based on an increase of the cardiac muscle cell size. Despite the facts that during the last 50 years much progress has been made in the recognition of cardiologie risk factors, in diagnosis and therapy of cardiovascular disorders, only little is known about the molecular and cellular fundamentals of these diseases.

ABSTRACTS (pp 58–112)

Background: Gastrointestinal blood loss accounts for a significant part of chronic recurrent anemia. Conventional diagnostic work up includes esophagogastroduodenoscopy (EGD), colonoscopy and small bowel follow-through (SBF). Aim: To determine the diagnostic yield of CE in patients with chronic recurrent anemia without active gastrointestinal bleeding referred for CE in a tertiary center in Switzerland. Methods: CE is a minuscule color videocamera, light source, battery, transmitter and antenna all embedded in a swallowable capsule (M2A capsuleâ). Pictures are received through electrodes attached to the abdomen and stored on hard disk. Data are then transferred to a workstation and analysed (RAPID workstation). All pts referred to our institution for CE are enrolled in a prospective study evaluating acceptance, diagnostic yield and possible complications of the method. Pts referred for evaluation of chronic recurrent anemia were eligible. All patients had a normal SBF and no bleeding source detected in EGD and colonoscopy. Patients with known active bleeding were excluded. Other exclusion criteria were small bowel stenosis in SBF and patients with pacemakers or ICD. Results: 32 pts underwent CE from May 2002 until May 2003. Of those, 8 pts were referred for chronic anemia and included. There were 4 men (50%) and 4 women (50%). The mean age was 60 years (range 40– 78 years). The mean hemoglobin was 94 g/l (range 65–161 g/l), the mean MCV was 85 fl (range 76–93 fl). In 4 of these 8 pts pathologies were found in the small bowel accounting for the anemia. There were 2 pts with angiodysplasias, 1 pt with an ulcer in the ileum and 1 pt with multiple erosions in the distal small bowel. All of these lesions were deemed to be potential bleeding sources. The mean hemoglobin in those pts with bleeding sources detected by CE was 84 g/l compared to 105 g/l in those pts with normal CE. The diagnostic yield of CE in this group of pts with chronic recurrent anemia and a normal SBF was 50%. Conclusions: CE provides a meaningful diagnostic yield in patients with chronic recurrent anemia and normal EGD and colonoscopy and a normal SBF. The diagnostic yield seems to increase in pts with a lower hemoglobin.

S8.5 The fourth immunoglobulin-like domain of NCAM contains a carbohydrate recognition domain for oligomannosidic glycans implicated in association with LI and neurite outgrowth

In the 3-D structure of Erythrina corallodendron lectin (ECorL) in complex with a ligand (lactose), the first of its kind for a Gal/GalNAc-specific lectin (Shaanan et al., 1991, Science, 254, 862), a hydrophobic cavity is present surrounded by Tyr I°s, Pro TM and Trp 13~ not in contact with Gal of the lactose in the combining site. The cavity can accommodate bulky substituents such as acetamido or dansylamido (NDns) at C-2 of the lectin-bound Gal. Molecular modelling suggested that the Dns of MeflGalNDns, which binds the lectin ~300 times stronger than Gal, fits snugly into the cavity. Comparison of the primary sequence of ECorL with that of the homologous peanut agglutinin (PNA), which is Gal specific but does not bind GalNAc or GalNDns, showed that in PNA such a cavity is absent, being filled by two extra amino acids. We therefore constructed a mutant L2 in which Pro134-Trp 135 in ECorL was replaced by Ser-Glu-Tyr-Asn (as in PNA), a single point mutant Y108T (also as in PNA), a double mutant L2;Y108T, and another single point mutant W135A. They were expressed in Escherichia coli as done for rECorL (Arango et al., 1992, Eur. J. Biochem., 205, 575) and examined for their specificity by inhibition of hemagglutination and of binding of the tectin to asialofetuin. In the latter assay, Y108T had a specificity similar to rECorL; L2 had the same affinity for Gal as rECorL, and 8 and 15 times lower affinity for GalNAc and MeflGalNDns, respectively. L2;Y108T had a similar affinity for Gal and GalNDns as L2, but twice the affinity for GalNAc. W135A, on the other hand, bound Gal 5 times stronger, and GalNAc and GalNDns 3 and 6 times weaker, respectively, than rECorL. Our results demonstrate that (a) Trp m contributes to the strong binding of MeflGalNDns to ECorL, whereas Tyr l°s does not; (b) the region close to Trp ~35 must be free in order to allow the lectin to bind Gal derivatives with bulky substituents at C-2; (c) changing a residue such as Trp ~35, which is not in direct contact with the Gal ligand, may change the conformation of the combining site of the lectin.