Biochemical heterogeneity in xeroderma pigmentosum complementation group E.

Cells from two patients with xeroderma pigmentosum complementation group E (XP-E) have been shown to lack an activity which binds specifically to UV-irradiated DNA (Chu and Chang, 1988). We investigated the occurrence of this binding activity in cell strains from nine additional, unrelated XP-E patients and found that all but one of these strains contained normal levels of the binding protein. Furthermore, the binding activity from these XP-E strains was indistinguishable from that of normal controls in thermal stability, behavior on ion-exchange chromatography, and electrophoretic mobility of protein-DNA complexes, indicating that there were no gross structural alterations in the protein. The association of XP-E with a deficiency in DNA-damage binding protein in cells from 3 of 12 XP-E patients (compared to 0 of 20 non-XP-E controls) is statistically significant (p less than 0.05), but there is no obvious correlation between the biochemical defect and the clinical or cellular characteristics of individual patients. Implications of these findings for the role of the binding protein in XP-E are discussed.     

Clinical Studies of Neuropathy due to Macroglobulinemia (Waldenstr?m's Syndrome)

A case of severe neuropathy due to macro-globulinemia is reported with clinical, biochemical and electrophysiological studies. The clinical picture was that of a severe, symmetrical polyneuropathy of a sensory-motor type with increased protein in the CSF. Sedimentation rate was increased and a sharp rise was found in the globulin fractions on plasma electrophoresis. Immuno-assays of CSF showed increases of albumin, transferrin and gamma globulin. Blood chromosome studies were normal. EMG showed neuromuscular atrophy. Nerve conduction times were prolonged. These studies are compatible with a neuropathy of an allergic or metabolic nature. Macro-globulinemia of Waldenström should be considered in the differential diagnosis of obscure neuropathies.     

Regional differences in myosin heavy chain isoform expression and maximal shortening velocity of the rat vaginal wall smooth muscle

Contractility of the proximal and distal vaginal wall smooth muscle may play distinct roles in the female sexual response and pelvic support. The goal of this study was to determine whether differences in contractile characteristics of smooth muscle from these regions reside in differences in the expression of isoforms of myosin, the molecular motor for muscle contraction. Adult female Sprague-Dawley rats were killed on the day of estrus, and the vagina was dissected into proximal and distal segments. The Vmax at peak force was greater for tissue strips of the proximal vagina compared with that of distal (P < 0.01), although, at steady state, the Vmax for the muscle strips from the two regions was not different. Furthermore, at steady state, muscle stress was higher (P < 0.001) for distal vaginal strips (n = 5). Consistent with the high Vmax for the proximal vaginal strips, RT-PCR results revealed a higher %SM-B (P < 0.001) in the proximal vagina. A greater expression of SM-B protein (P < 0.001) was also detected by Western blotting (n = 4). Interestingly, there was no regional difference noted in SM-1/SM-2 isoforms (n = 6). The proximal vagina had a higher expression of myosin heavy chain protein (P < 0.01) and a greater percentage of smooth muscle bundles (P < 0.001). The results of this study are the first demonstration of a regional heterogeneity in Vmax and myosin isoform distribution in the vagina wall smooth muscle and confirm that the proximal vaginal smooth muscle exhibits phasic contractile characteristics compared with the distal vaginal smooth muscle, which is tonic.     

Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N -succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34⁺CD38⁻ fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34⁺, CD133⁺ and CD38⁻) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC–MSC interaction might further enhance cord blood expansion on MSC.     

Exploration of potential prodrug approach of the bis-thiazolium salts T3 and T4 for orally delivered antimalarials

We report here the synthesis and biological evaluation of a series of 37 compounds as precursors of potent antimalarial bis-thiazolium salts (T3 and T4). These prodrugs were either thioester, thiocarbonate or thiocarbamate type and were synthesized in one step by reaction of an alkaline solution of the parent drug with the appropriate activated acyl group. Structural variations affecting physicochemical properties were made in order to improve oral activity. Twenty-five of them exhibited potent antimalarial activity with IC₅₀ lower than 7 nM against Plasmodium falciparum in vitro. Notably, 3 and 22 showed IC₅₀ = 2.2 and 1.8 nM, respectively. After oral administration 22 was the most potent compound clearing the parasitemia in Plasmodium vinckei infected mice with a dose of 1.3 mg/kg.     

Synthesis and biological evaluation of L-valine-amidoximeesters as double prodrugs of amidines

In general, drugs containing amidines suffer from poor oral bioavailability and are often converted into amidoxime prodrugs to overcome low uptake from the gastrointestinal tract. The esterification of amidoximes with amino acids represents a newly developed double prodrug principle creating derivatives of amidines with both improved oral availability and water solubility. N-valoxybenzamidine (1) is a model compound for this principle, which has been transferred to the antiprotozoic drug pentamidine (8). Prodrug activation depends on esterases and mARC and is thus independent from activation by P450 enzymes. Therefore, drug-drug interactions or side effects will be minimized. The synthesis of these two compounds was established, and their biotransformation was studied in vitro and in vivo. Bioactivation of N-valoxybenzamidine (1) and N,N'-bis(valoxy)pentamidine (7) via hydrolysis and reduction has been demonstrated in vitro with porcine and human subcellular enzyme preparations and the mitochondrial Amidoxime Reducing Component (mARC). Moreover, activation of N-valoxybenzamidine (1) by porcine hepatocytes was studied. In vivo, the bioavailability in rats after oral application of N-valoxybenzamidine (1) was about 88%. Similarly, N,N'-bis(valoxy)pentamidine (7) showed oral bioavailability. Analysis of tissue samples revealed high concentrations of pentamidine (8) in liver and kidney.     

Translational research and therapeutic perspectives in dysferlinopathies

Dysferlinopathies are autosomal recessive disorders caused by mutations in the dysferlin (DYSF) gene, encoding the dysferlin protein. DYSF mutations lead to a wide range of muscular phenotypes, with the most prominent being Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B) and the second most common being LGMD. Symptoms generally appear at the end of childhood and, although disease progression is typically slow, walking impairments eventually result. Dysferlin is a modular type II transmembrane protein for which numerous binding partners have been identified. Although dysferlin function is only partially elucidated, this large protein contains seven calcium sensor C2 domains, shown to play a key role in muscle membrane repair. On the basis of this major function, along with detailed clinical observations, it has been possible to design various therapeutic approaches for dysferlin-deficient patients. Among them, exon-skipping and minigene transfer strategies have been evaluated at the preclinical level and, to date, represent promising approaches for clinical trials. This review aims to summarize the pathophysiology of dysferlinopathies and to evaluate the therapeutic potential for treatments currently under development.     

Alteration in expression of myosin isoforms in detrusor smooth muscle following bladder outlet obstruction

Partial urinary bladder outlet obstruction (PBOO) in men, secondary to benign prostatic hyperplasia, induces detrusor smooth muscle (DSM) hypertrophy. However, despite DSM hypertrophy, some bladders become severely dysfunctional (decompensated). Using a rabbit model of PBOO, we found that although DSM from sham-operated bladders expressed nearly 100% of both the smooth muscle myosin heavy chain isoform SM-B and essential light chain isoform LC_17a, DSM from severely dysfunctional bladders expressed as much as 75% SM-A and 40% LC_17b (both associated with decreased maximum velocity of shortening). DSM from dysfunctional bladder also exhibited tonic-type contractions, characterized by slow force generation and high force maintenance. Immunofluorescence microscopy showed that decreased SM-B expression in dysfunctional bladders was not due to generation of a new cell population lacking SM-B. Metabolic cage monitoring revealed decreased void volume and increased voiding frequency correlated with overexpression of SM-A and LC_17b. Myosin isoform expression and bladder function returned toward normal upon removal of the obstruction, indicating that the levels of expression of these isoforms are markers of the PBOO-induced dysfunctional bladders. bladder remodeling; bladder dysfunction; SM-A; LC_17a; benign prostatic hyperplasia     

A mathematical model of the impact of novel treatments on the A beta burden in the Alzheimer's brain,CSF and plasma

With the advent of novel therapies for Alzheimer’s disease, there is a pressing need for biomarkers that are easy to monitor, such as the amyloid-beta (Aβ) levels in the cerebrospinal fluid (CSF) and plasma. To gain a better understanding of the explanatory power of these biomarkers, we formulate and analyze a compartmental mathematical model for the Aβ accumulation in the brain, CSF and plasma, throughout the course of Alzheimer’s treatment. Our analysis reveals that the total Aβ burden in the brain is dictated by a unitless quantity called the polymerization ratio, which is the product of the production and elongation rates divided by the product of the fragmentation and loss rates. In this ratio, the production rate and loss rate include a source and sink term, respectively, related to the intercompartmental transport. Our results suggest that production inhibitors are likely to reduce the Aβ levels in all three compartments. In contrast, agents that ingest monomers off of polymers, or that increase fragmentation or block elongation, may also reduce Aβ burden in the brain, but may produce little change in—or even transiently increase—CSF and plasma Aβ levels. Hence, great care must be taken when interpreting these biomarkers.