The molecular basis of long QT syndrome and prospects for therapy

Long QT syndrome (LQT) is a cardiac disorder that causes sudden death from ventricular tachyarrhythmias, specifically torsade de pointes. Two types of LQT have been reported, autosomal-dominant LQT (Romano-Ward syndrome) and autosomal-recessive LQT (Jervell and Lange-Nielsen syndrome); Jervell and Lange-Nielsen syndrome is also associated with deafness. Four LQT genes have been identified for autosomal-dominant LQT: K+ channel genes KVLQT1 on chromosome 11p15.5, HERG on 7q35-36 and minK on 21q22, and the cardiac Na+ channel gene SCN5A on chromosome 3p21-24. Two genes, KVLQT1 and minK, have been identified for Jervell and Lange-Nielsen syndrome. Genetic testing and gene-specific therapies are available for some LQT patients.     

Biochemical and molecular basis of Bernard-Soulier syndrome: a review

Bernard-Soulier syndrome (BSS) is a rare hereditary recessive autosomal bleeding disorder characterized by a prolonged bleeding time, giant platelets, thrombocytopenia, normal platelet aggregation in response to ADP and no agglutination in response to ristocetin. This disease is due to absence or abnormality of the platelet membrane glycoprotein GPIb-IX-V, the receptor for von Willebrand factor. All four genes encoding the complex have been cloned and 17 forms of BSS have to date been characterized at the functional, immunological and molecular levels. The mutations can be divided into two main groups. Firstly, mutations located in leucine rich repeats (LRR), responsible for conformational modifications of the molecule, in some cases higher sensitivity to proteases and loss of adhesive function of the receptor, which is expressed at lower than normal levels at the platelet membrane. When mutations affect the LRR of GPIbalpha, the presence of the other chains varies from normal to residual amounts. When mutations affect the LRR of GPIX, expression of the other chains is strongly diminished, suggesting that GPIX plays a major role in the stability of the complex. A second type of mutations leads to synthesis of a truncated molecule lacking the transmembrane domain and absence of its expression at the platelet surface, while the other chains are present in residual amounts. Expression of recombinant proteins in eukaryotic cells has recently confirmed the results derived from studies of natural mutations. Separate expression of each chain can be obtained, although the presence of all subunits is required for full expression.     

The structural basis of molecular adaptation

Expression levels of fast-twitch (SERCA1), slow-twitch (SERCA2a) and "housekeeping" (SERCA2b) isoforms of the sarcoplasmic reticulum Ca(2+)-transport ATPase were monitored during regeneration of rat soleus muscles following necrosis induced by the toxin notexin at the tissue level by Western blot analysis and at the cellular level by immunocytochemical analysis. Due to necrosis, levels of muscle-specific SERCA1 and SERCA2a isoforms dropped to low levels on the third day after injection of the toxin. Subsequently, during regeneration both isoforms recovered but with a different time course. Expression of the fast type SERCA1 increased first. This type showed its most pronounced increase between day 3 and 10. Expression of the slow type SERCA2a was biphasic. After an increase to approximately one third of the control value on days 5-10, it showed its main increase up to the control level between day 10 and 21. Expression levels of the house-keeping SERCA2b isoform remained relatively constant throughout the 4 weeks of regeneration. Between day 10 and 28, when new innervation is established, SERCA2a expression spread gradually over almost all fibers whereas the number of SERCA1-expressing fibers decreased and only a limited number of fibers co-expressed SERCA1 and SERCA2a. At 4 weeks of regeneration, expression of the fast isoform was found only in 12% of the fibers, whereas the slow form was found in 98% of the fibers. In the contralateral untreated soleus muscles, 26% SERCA1-positive and 81% SERCA2a-positive fibers were observed. Immunocytochemical analysis showed that SERCA1 and SERCA2a were co-expressed with fast and slow myosin isoforms in fibers of normal muscles but in regenerated muscle only slow myosin and slow SERCA isoforms correlated. The results show that during regeneration levels of fast and slow SERCA proteins change in a similar way as their mRNAs do. However, in regenerated soleus, unlike in normal muscle, expression of slow SERCA is coregulated only with the slow myosin isoform. This finding is in agreement with the fact that the number of slow type fibers is increased in regenerated soleus.     

The molecular basis of haemophilia B

PURPOSE OF THE STUDY: High tibial osteotomy (HTO) is a routine procedure for medial gonarthrosis. Mid-term results are known to be satisfactory, but they deteriorate with longer follow-up. The authors present a long term survival analysis of 109 out of 111 consecutive HTO with a minimal potential follow-up of ten years. MATERIAL AND METHODS: 111 patients were consecutively operated on for isolated primary varus gonarthrosis between 1977 and 1985: 57 men and 54 women, with a mean age of 53 years (range, 27 to 79 years). X-ray measurements were done on stance, hip-ankle view. Global axial deformation was defined as the angle between mechanical axes of femur and tibia. The respective part of congenital and degenerative tibial deformation was assessed according to Dejour. The angle between femoral and tibial bicondylar lines, representing lateral instability, was added to the tibial degenerative deformation to represent the total degenerative deformation. The goal of correction was a 3 to 7 degree mechanical valgus angulation. At the time of bone healing, 82 patients (74 per cent) had an optimal correction. Two patients were excluded from the follow-up study because of a severe complication (1 bacterial arthritis and 1 tibia non union) which could interfere with the long term result. The 109 remaining patients were followed for a minimal period of 1 year (mean: 8.4 years). GUEPAR pain grading and the occurrence of a revision were prospectively analyzed. 57 non reoperated patients could be re-examined at a mean maximal follow-up of 13.5 years (range, 10 to 18 years). Failure was defined as either the occurrence of a grade 2 or 3. GUEPAR pain during the whole follow-up, or a clinical or functional Knee Society score < 80 points at final follow-up, or revision. Failure and revision rates were calculated according to Kaplan and Meier. RESULTS: 11 patients were reoperated on before final examination (10 per cent): 2 medial unicondylar and 9 total knee prostheses. At final follow-up, the mean clinical and functional scores were respectively 87.0 points (range, 24 to 100 points) and 86.3 (range, 45 to 100 points). The cumulative failure rate was 33 per cent after 10 years and 54 per cent after 15 years; the respective revision rates were 9 per cent and 19 per cent. A pre-operative total degenerative deformation superior to 3 degrees led to a 3.5 fold increased failure rate (p < 0.000,1). A pre-operative medial joint space narrowing over the half of the normal, lateral one led to a 2.2 fold increased failure rate (p = 0.014). An optimal post-operative correction led to a 3.2 fold decreased failure rate (p = 0.000,1). For a given total degenerative deformation, patients with a congenital deformation superior to 5 degrees had a significant lower failure rate (p < 0.000,1). No factor significantly influenced the revision rate. DISCUSSION: Ideal patients for HTO, with an expected survival rate of 100 per cent after 13 years, have moderate degenerative changes and a congenital deformation superior to 5 degrees. Patients with advanced degenerative changes and no congenital deformation experienced a 35 per cent failure rate after 10 years. In this population, unicondylar replacement should be considered as a valuable alternative.     

The molecular basis of myelodysplastic syndromes

Rabbit lenses expressing spontaneous oscillations in translens short-circuit current (Isc) are obtained somewhat frequently, with this phenomenon observed in approximately 30% of isolated lenses as described earlier (Exp. Eye Res. 61, 129-140, 1995). Since pharmacological protocols to consistently elicit Isc oscillations were not found, characterizations of the underlying transport processes have been limited to the application of various inhibitors on the spontaneous phenomenon. The present report extends the initial observations by confirming that oscillations are immediately inhibited upon the anterior addition of the Ca2+ channel blocker nifedipine (10 microM), and by demonstrating that other treatments which should affect epithelial Ca2+ homeostasis are also inhibitory (e.g., Bay K 8644 (10 microM), diltiazem (10 microM), EGTA (2 mm), and Ca2+-free media). Furthermore, Isc oscillations are immediately inhibited by the K+ channel blocker, Ba2+, but not by the Na+-K+ pump inhibitor, ouabain. The intracellular Ca2+ mobilizing agents thapsigargin (0.1 microM) or acetylcholine (1 microM) modified but did not permanently inhibit the oscillations, confirming earlier observations. At 50 microM, however, acetylcholine addition was inhibitory, but reversible, for oscillations restarted upon its subsequent removal. In addition, lens oscillations were also characterized under open-circuit conditions with microelectrodes inserted in the superficial cells near the equator of lenses isolated in a divided chamber. The potential difference (PD) across each lens face was recorded, as was the translens PD (PDt), which equals the difference between the PDs across each lens surface. Oscillations in PDt were obtained in 7 of 26 lenses. The oscillations arose only from an oscillation in the PD across the anterior face (PDa). While PDa and PDt oscillated with the same amplitude (approximately 12 mV) and period (approximately 70 sec), the PD across the posterior surface remained stable. During these oscillations the conductance of the anterior surface was maximal at the most positive voltage of the anterior bath with respect to the lens interior (46 mV), whereas, minimal conductance occurred at the least positive PDa (34 mV). Overall, these observations are consistent with the likely presence of voltage-operated Ca2+ channels in parallel with various Ca2+-sensitive K+ channels in the epithelial basolateral membrane. A model to explain the oscillatory pattern across the anterior face while the PD across the posterior face remains unaltered is presented.     

The molecular basis of haemophilia A

1H-NMR spectroscopy was performed on a 6-month-old boy with pachygyria. A MRI study demonstrated an abnormally thick cortex localized in the right occipital lobe. 1H-NMR spectrums were collected from the lesion and the contralateral cortex that appeared normal on MRI. The N-acetylaspartate (NAA)/Cre (creatine) ratio was markedly lower in the abnormal cortex than in the contralateral cortex and the occipital cortex. NAA localizes to neurons, axons, dendrites and synaptic connections and increases with maturation of neurons. Its decrease is considered to represent the decrease in the number of these structures and/or disturbance of neuronal maturation. We conclude that NAA/Cre can be an important index that reflects the pathogenesis of pachygyria.     

The molecular basis of chronic granulomatous disease

CGD is a rare inherited immunodeficiency syndrome, caused by the phagocytes' inability to produce (sufficient) reactive oxygen metabolites. This dysfunction is due to a defect in the NADPH oxidase, the enzyme responsible for the production of superoxide. It is composed of several subunits, two of which, gp91phox and p22phox, form the membrane-bound cytochrome b558, while its three cytosolic components, p47phox, p67phox and p40phox, have to translocate to the membrane upon activation. This is a tightly and intricately controlled process that involves, among others, several low-molecular weight GTP-binding proteins. Gp91phox is encoded on the X-chromosome and p22phox, p47phox and p67phox on different autosomal chromosomes, and a defect in one of these components leads to CGD. This explains the variable mode of inheritance seen in this syndrome. Clinically CGD manifests itself typically already at a very young age with recurrent and serious infections, most often caused by catalase-positive pathogens. Modern treatment options, including prophylaxis with trimethoprim-sulfamethoxazole and rIFN-gamma as well as early and aggressive anti-infection therapy, have improved the prognosis of this disease dramatically. CGD, as a very well-characterized inherited affection of the hematopoietic stem cells, is predestined to be among the first diseases to profit from the advances in cutting-edge therapeutics, such as gene therapy and in utero stem cell transplantation.     

The molecular basis of nephrogenic diabetes insipidus

Nephrogenic diabetes insipidus (NDI) is characterized by resistance of the kidney to the action of arginine-vasopressin (AVP); it may be due to genetic or acquired causes. Recent advances in molecular genetics have allowed the identification of the genes involved in congenital NDI. While inactivating mutations of the vasopressin V2 receptor are responsible for X-linked NDI, autosomal recessive NDI is caused by inactivating mutations of the vasopressin-regulated water channel aquaporin-2 (AQP-2). About 70 different mutations of the V2 receptor have been reported, most of them missense mutations. The functionally characterized mutants show a loss of function due to defects in their synthesis, processing, intracellular transport, AVP binding, or interaction with the G protein/adenylyl cyclase system. Thirteen different mutations of the AQP-2 gene have been reported. Functional studies of three AQP-2 mutations reveal impaired cellular routing as the main defect. The great number of different mutations with various functional defects hinders the development of a specific therapy. Gene therapy may, however, eventually become applicable to the congenital forms of NDI. At present all gene-therapeutic approaches lack safety and efficiency, which is of particular relevance in a disease that is treatable by an adequate water intake. The progress with regard to the molecular basis of antidiuresis contributes to the understanding of acquired forms of NDI on a molecular level. Recent data show that lithium dramatically reduces the expression of AQP-2. Likewise, hypokalemia reduces the expression of this water channel. The exact mechanisms leading to this reduced expression of AQP-2 remain to be determined.     

The molecular basis of intractable diarrhoea of infancy

The intractable diarrhoeas of infancy present very major problems of clinical management. However, the conceptual importance of these conditions lies in the information that they may provide about normal small-intestinal function in humans: among such infants will be found the human equivalents of the 'knock-out' mice, in which targeted gene disruption allows sometimes unexpected insight into the regulation of intestinal function. The challenge posed by the intractable diarrhoeal syndromes, of working backwards from an apparently common phenotype to probably multiple genotypes, is, however, immense. Very few of these conditions have been described at the genetic level, although the molecular basis of pathogenesis has been better explored in recent years. The two major groups of intractable diarrhoea are due to (1) primary epithelial abnormalities (which usually present within the first few days of life) and (2) immunologically mediated (which generally present after the first few weeks). The high prevalence of autoimmune enteropathy among infantile autoimmune disease, in contrast to adult autoimmunity, is intriguing and may reflect constitutive abnormality of extrathymic lymphocyte maturation. The use of potent immunosuppressive drugs and increasing expertise with parenteral nutrition are improving the outlook of these previously fatal conditions. Viewed globally, however, the pressing problem is to treat effectively the millions of infants who die from severe persistent diarrhoea and wasting, which would certainly not be considered intractable in wealthy countries.     

The molecular basis of isolated 17,20 lyase deficiency

Human P450c17 catalyzes the 17alpha-hydroxylation of pregnenolone to 17OH pregnenolone and of progesterone to 17alpha-OH progesterone; the same P450c17 enzyme also catalyzes 17,20 lyase activity on the same active site, converting 17OH-pregnenolone to DHEA. Rodent and porcine P450c17 also catalyze 17,20 lyase activity with delta4 substrates, converting 17OH-progesterone to delta4 androstenedione, but human P450c17 catalyzes this reaction very inefficiently, so that virtually all human C19 sex steroids are made via 17OH pregnenolone and DHEA. P450c17 is encoded by a single gene and a single species of mRNA. Many mutations of this gene have been described, but until recently all of these either entirely eliminated both 17alpha-hydroxylase and 17,20 lyase activity, or affected each activity equivalently. We have identified and characterized the first patients with P450c17 mutations that selectively ablate 17,20 lyase activity while retaining 17alpha-hydroxylase activity. Through a combination of enyzmologic experiments in transfected mammalian cells and in genetically manipulated yeast, plus a computer model of human P450c17, we have proven that the responsible mutations, R347H and R358Q lie in the redox-partner binding site of P450c17. This site, through which P450c17 interacts with P450 oxidoreductase to receive the electrons needed for catalysis, can be allosterically influenced by cytochrome b5. These two mutations have contributed substantially to our understanding of the mechanisms by which 17alpha-hydroxylase and 17,20 lyase activities are regulated independently, and thus have contributed to the study of regulated 17,20 lyase activity in adrenarche, aging, and the polycystic ovary syndrome.     

The cellular and molecular basis of peripheral nerve regeneration

Functional recovery from peripheral nerve injury and repair depends on a multitude of factors, both intrinsic and extrinsic to neurons. Neuronal survival after axotomy is a prerequisite for regeneration and is facilitated by an array of trophic factors from multiple sources, including neurotrophins, neuropoietic cytokines, insulin-like growth factors (IGFs), and glial-cell-line-derived neurotrophic factors (GDNFs). Axotomized neurons must switch from a transmitting mode to a growth mode and express growth-associated proteins, such as GAP-43, tubulin, and actin, as well as an array of novel neuropeptides and cytokines, all of which have the potential to promote axonal regeneration. Axonal sprouts must reach the distal nerve stump at a time when its growth support is optimal. Schwann cells in the distal stump undergo proliferation and phenotypical changes to prepare the local environment to be favorable for axonal regeneration. Schwann cells play an indispensable role in promoting regeneration by increasing their synthesis of surface cell adhesion molecules (CAMs), such as N-CAM, Ng-CAM/L1, N-cadherin, and L2/HNK-1, by elaborating basement membrane that contains many extracellular matrix proteins, such as laminin, fibronectin, and tenascin, and by producing many neurotrophic factors and their receptors. However, the growth support provided by the distal nerve stump and the capacity of the axotomized neurons to regenerate axons may not be sustained indefinitely. Axonal regenerations may be facilitated by new strategies that enhance the growth potential of neurons and optimize the growth support of the distal nerve stump in combination with prompt nerve repair.     

Paraneoplastic neurologic syndromes. 2. Lambert-Eaton myasthenic syndrome. 1) The molecular structure of calcium channel and immunology

The effect of divalent cations on the inactivation of Escherichia coli by high hydrostatic pressure was investigated. The presence of 0.5 mmol l-1 of CaCl2, MgCl2, MnCl2 and FeCl2 reduced pressure inactivation of E. coli MG1655, while 0.5 mmol l-1 of ZnCl2, NiCl2, CuCl2 and CoCl2 increased inactivation. Baroprotection by Ca2+ was found to be dose-dependent up to at least 80 mmol l-1 and was studied in more detail in terms of inactivation kinetics. Logarithmic survivor plots against time deviated from first order kinetics, suggesting that MG1655 cultures were heterogeneous with regard to pressure resistance. All cultures were shown to contain a small proportion of cells that were only slowly inactivated. Addition of Ca2+ increased the proportion of these tolerant cells in the cultures up to 1000-fold at 80 mmol l-1, but did not affect their inactivation rate. The addition of EDTA resulted in the opposite effect, lowering the proportion of pressure-tolerant cells in the cultures. Three pressure-resistant mutants of E. coli MG1655 were found to be more resistant to EDTA under pressure compared with MG1655, and were unaffected by Ca2+ under pressure. In addition, these mutants had a 30-40% lower Ca2+ content than MG1655. Based on these results, it is postulated that pressure killing of E. coli MG1655 is mediated primarily by the destabilization of Ca(2+)-binding components, and that the mutations underlying pressure resistance have resulted in pressure-stable targets with reduced Ca(2+)-binding affinity.