3-Methylglutaconic aciduria type III in a non-Iraqi-Jewish kindred: clinical and molecular findings

Type III 3-methylglutaconic aciduria (MGA) (MIM 258501) consists of early bilateral optic atrophy, later development of spasticity, extrapyramidal dysfunction and occasionally cognitive deficits, and urinary excretion of 3-methylglutaconic acid and 3-methylglutaric acid. The presence of the disorder in an Iraqi-Jewish genetic isolate led to mapping of the OPA3 gene to chromosome 19q13.2-q13.3, followed by isolation of the gene itself. OPA3 consists of two exons and codes for a peptide of 179 amino acids. Iraqi-Jewish patients with type III MGA are homozygous for a splice site founder mutation in OPA3 (IVS1-1G>C) which abolishes mRNA expression in fibroblasts. Here we report a novel mutation in OPA3 (320-337del) in a Kurdish-Turkish patient with optic atrophy and 3-methylglutaconic and 3-methylglutaric aciduria, previously carrying the diagnosis of type IV MGA. We conclude that type III MGA occurs in patients of non-Iraqi-Jewish ancestry, and should be considered in patients with type IV MGA that have optic atrophy and ataxia.     

Clear relationship between ETF/ETFDH genotype and phenotype in patients with multiple acyl-CoA dehydrogenation deficiency

Mutations in electron transfer flavoprotein (ETF) and its dehydrogenase (ETFDH) are the molecular basis of multiple acyl-CoA dehydrogenation deficiency (MADD), an autosomal recessively inherited and clinically heterogeneous disease that has been divided into three clinical forms: a neonatal-onset form with congenital anomalies (type I), a neonatal-onset form without congenital anomalies (type II), and a late-onset form (type III). To examine whether these different clinical forms could be explained by different ETF/ETFDH mutations that result in different levels of residual ETF/ETFDH enzyme activity, we have investigated the molecular genetic basis for disease development in nine patients representing the phenotypic spectrum of MADD. We report the genomic structures of the ETFA, ETFB, and ETFDH genes and the identification and characterization of seven novel and three previously reported disease-causing mutations. Our molecular genetic investigations of these nine patients are consistent with three clinical forms of MADD showing a clear relationship between the nature of the mutations and the severity of disease. Interestingly, our data suggest that homozygosity for two null mutations causes fetal development of congenital anomalies resulting in a type I disease phenotype. Even minute amounts of residual ETF/ETFDH activity seem to be sufficient to prevent embryonic development of congenital anomalies giving rise to type II disease. Overexpression studies of an ETFB-D128N missense mutation identified in a patient with type III disease showed that the residual activity of the mutant enzyme could be rescued up to 59% of that of wild-type activity when ETFB-D128N-transformed E. coli cells were grown at low temperature. This indicates that the effect of the ETF/ETFDH genotype in patients with milder forms of MADD, in whom residual enzyme activity allows modulation of the enzymatic phenotype, may be influenced by environmental factors like cellular temperature.     

Blood Polymorphonuclear Leukocyte Activation in Atherosclerosis: Effects of Aspirin

The aim of the study was to demonstrate an activation of polymorpho-nuclear leukocytes (PMNs) in chronic progressive atherosclerosis (ATH). A group of patients with ATH, and a group of ATH patients under aspirin (ASA) therapy were compared with control persons without atherosclerotic alterations (healthy controls). Each group comprised 15 male age-matched subjects. The following inflammatory parameters related to PMN activities were measured: the polymorphonuclear leukocyte (PMN) blood count; blood PMN migration and reactive oxygen species release in vitro; the blood levels of PMN elastase, malondialdehyde, antibodies to oxidized LDL and soluble ICAM-1. In ATH patients, the PMN blood counts and the share of blood PMNs migrating upon platelet activating factor and leukotriene B4 stimulation were significnatly above the values of the healthy controls, while the other parameters were not significantly altered. ASA treatment attenuated the inflammatory response and reduced the differences between ATH and the healthy controls. It can be concluded that, in patients with chronic progressive atherosclerosis, PMNs are involved in the inflammatory process underlying the disease.     

Species-specific assessment of Mycobacterium leprae in skin biopsies by in situ hybridization and polymerase chain reaction.

Conventional histopathologic diagnosis of mycobacterial infections are limited to the determination of "acid-fast bacilli". A species-specific diagnosis is thus far impossible. In addition, routine microbiologic assessments of mycobacteria suffer from the major drawback that a species-specific diagnosis is extremely time-consuming and in several cases even impossible. As Mycobacterium leprae cannot be cultured in vitro, we tried to specifically target this obligate intracellular parasite by in situ hybridization and polymerase chain reaction (PCR) techniques. For this purpose we used a 22 mer oligonucleotide probe recognizing a species-specific sequence of the 16S rRNA of Mycobacterium leprae. Using an immunoenzymatic detection method for in situ hybridization we were able to specifically assess Mycobacterium leprae (a) in long-term cultured macrophages in vitro infected with different mycobacteria species and (b) in frozen sections of skin biopsies obtained from patients suffering from lepromatous leprosy. These results could be confirmed and extended by PCR experiments in which we used conserved oligonucleotide primers for 16S rRNA to amplify bacterial DNA isolated from different eubacterial species and from fresh-frozen as well as from formalin-fixed, paraffin-embedded and routinely processed mycobacteria-infected tissues. Upon Southern blot analysis, the Mycobacterium leprae-specific oligonucleotide probe exclusively hybridized with PCR products obtained from Mycobacterium leprae-containing samples (including paraffin sections), but not with PCR products obtained from samples containing other mycobacterial species. As species-specific oligonucleotide probes targeted at rRNA are described for a variety of mycobacterial species, these methods may be generally applied for a rapid species-specific assessment of mycobacteria in histologic material.     

Isolation and subfractionation of human peripheral blood mononuclear cells (PBMC) by density gradient centrifugation on Percoll

The use of Percoll for isolation and subfractionation of PBMC and T-lymphocytes by discontinuous and continuous density gradient centrifugation is described: PBMC were isolated from human peripheral blood by discontinuous density gradient centrifugation on Percoll. The use of Percoll instead of Ficoll-Isopaque has the advantage that Percoll, in contrast to Ficoll-Isopaque, does not alter the density of monocytes. Therefore, a better separation of lymphocytes and monocytes was achieved after subsequent continuous density gradient centrifugation on Percoll. E-RFC were isolated by discontinuous density gradient centrifugation after a first low speed centrifugation step banding lymphocytes and SRBC on a Percoll-Ficoll cushion, and a subsequent high speed centrifugation step separating high density rosettes and SRBC from low density non-E-RFC. The advantage of this procedure is the short time of performance and that there is no need to resuspend the lymphocyte/SRBC pellet. PBMC, nph.PBMC T-lymphocytes were further subfractionated by continuous density gradient centrifugation on Percoll. The method described here resulted in a good separation of lymphocytes and monocytes. However, to obtain lymphocyte fractions with minute numbers of contaminating monocytes, a depletion of monocytes prior to further subfractionation of the lymphocytes by continuous density gradient centrifugation is recommended. A marker analysis of T-lymphocytes subfractionated by continuous density gradient centrifugation on Percoll shows that high density T-lymphocytes are enriched in ANAE positive lymphocytes of type 1 and depleted of ANAE positive lymphocytes of type 2. Low density T-lymphocytes are enriched in ANAE type 2 cells and depleted of ANAE type 1 cells. On the other hand, no considerable differences were found when analyzing the T-cells from different fractions for differentiation antigens by means of monoclonal antibodies (anti Lyt 3, OKT4, and OKT8). The results may indicate that subfractionation of T-lymphocytes by continuous density gradient centrifugation on Percoll provided T-cells in different functional states rather than T-cells of distinct subclasses.     

Using infertile patients in epidemiologic studies on subfecundity and embryonal loss

Subfecundity is a frequent and serious problem that may sometimes be preventable, but we need to know more about its determinants. Different epidemiologic designs are available. The best of these use prospectively collected data from the population, but they are time consuming, expensive and often hampered by low-participation rates. Most patients undergoing infertility treatment are closely monitored for clinical reasons, making it feasible to use secondary data to study the period from conception to implantation and pregnancy. In spite that infertility patients are highly selected, there are specific exposure-effect relations that can be studied in cohorts of infertility patients. These patients offer a potentially useful setting for studying exposures that operate late in fertilization, whereas the designs may be inadequate to identify exposures that cause reduced sperm counts, anovulation and total occlusion. The clinical sampling and the treatment set limitations for what can be studied. In certain situations, infertile patients can, however, provide useful epidemiologic evidence for learning about the causes of subfecundity.     

Pyramidal actions in identified radial motornuclei of the cat

This study aimed to establish the projection from the corticospinal tract (CST) to the motoneurones innervating the deep radial (DR) forelimb muscles. In the anaesthetized cat stimulation of the contralateral pyramid and intracellular recording from identified forelimb motoneurones was used. A train of pyramidal stimuli evoked disynaptic EPSPs in DR motoneurones. The effects were very similar in the different nuclei. Pyramidal IPSPs had a slightly longer latency and occurred in most cases together with disynaptic EPSPs. It is suggested that the inhibitory actions to the distal forelimb are predominantly relayed in a trisynaptic pathway, but that a disynaptic linkage seems possible as well. The disynaptic pyramidal EPSPs remained after CST transection in C5. They were abolished after CST transections in C2. It is concluded that disynaptic corticospinal excitation of distal DR motornuclei is relayed in a short midcervical propriospinal system. Transection experiments at different cervical levels suggest that the majority of the propriospinal neurones is located in C3-C4. The CST facilitated a variety of reflex pathways to motoneurones innervating distal forelimb muscles. Disynaptic excitatory and inhibitory effects from cutaneous and low threshold group I muscle afferents were common. They were present in all investigated nuclei and powerfully facilitated from the CST. It is suggested that this allows the brain to adapt the reflex mechanisms of the distal forelimb to the synergistic-antagonistic relations between the muscles, which are changing according to the performed movement.     

Low incidence of transplant-related complications in patients with chronic release of tumor necrosis factor-alpha before admission to bone marrow transplantation: a clinical correlate of cytokine desensitization?

To investigate the role of tumor necrosis factor-alpha (TNF-alpha) released by activated macrophages, sequential serum samples of 120 patients undergoing bone marrow transplantation (BMT) were analyzed by enzyme-linked immunosorbent assay. De novo increases in serum TNF-alpha levels were correlated with the development of acute endothelial complications as well as acute graft-versus-host disease. In addition, the analysis of time courses revealed a different capacity of TNF-alpha regulation at various phases of BMT. While patients with acute TNF-alpha release in the first 2 weeks of BMT had a significantly enhanced incidence of complications, a subgroup of 9 patients with chronic asymptomatic release of TNF-alpha before admission to BMT was observed. These patients were protected from complications in the course of the first 6 months of BMT. Our observations indicate the occurrence of desensitization for TNF-alpha, as it is also reported after repeated injections of TNF-alpha or endotoxin in experimental models.     

Telomere und Telomerase

Zusammenfassung In letzter Zeit fanden spezielle Abschnitte des menschlichen Genoms in der Tumorforschung besondere Beachtung: die Telomere. Telomere, d.h. die Enden aller linearen eukaryotischen Chromosomen, bestehen aus repetitiven DNA-Sequenzen und aus spezifischen Proteinen. Die Funktion der Telomere besteht im Schutz der Chromosomenenden vor Degradation, Fusion und Rekombination. In den meisten somatischen Zellen verkürzen sich die Telomere mit jedem Zellzyklus. Im übertragenen Sinn kann dieser Verlust an telomeren DNA-Sequenzen als eine mitotische Uhr aufgefa?t werden, mit der eine Zelle die Anzahl der Zellteilungen z?hlt und Lebensspanne und Zellalterung dirigiert. Sind die Telomere bis zu einem kritischen Punkt verkürzt, erfolgt die Einleitung des Apoptoseprozesses. Einige wenige Zelltypen entgehen der zellul?ren Seneszenz durch die Expression des Enzyms Telomerase. Dieses Ribonukleoprotein katalysiert die De-Novo-Addition von Nukleotiden an den Telomerenden. Das Enzym ist in den meisten somatischen Zellen in vivo nicht nachweisbar, wurde aber in der Mehrzahl aller Tumorgewebe gefunden. Die Aktivierung der Telomerase scheint Tumorzellen zu unbegrenzter Proliferation und zum Erreichen der Immortalit?t zu bef?higen. Aktuelle Studien zur Telomeraseaktivit?t in Tumoren unterstreichen, da? die Progression eines Tumorzellklons u.a. ma?geblich von der Aktivierung der Telomerase abh?ngt. Deshalb k?nnten Telomeraseinhibitoren neue M?glichkeiten in der Tumortherapie er?ffnen.      

Ultrastructural determinants of murine achilles tendon strength during healing

The mechanisms by which tendon strength is established during growth and development and restored following injury are not completely understood and are likely to be complex, multifactorial processes. Several studies examining the relationship between mechanical behavior and ultrastructural characteristics of tendons and ligaments during growth and maturation suggest that collagen fibril diameter is strongly correlated with tendon strength. Because of the similarities between development and repair processes of musculoskeletal tissues, increases in tendon strength during healing may be related to increases in fibril ultrastructural parameters such as fibril size, numerical density, and area fraction. In this study, we compared murine Achilles tendons at various time points after tenotomy with sham-operated controls in tensile tests to failure and examined tendons using electron microscopy to assess collagen fibril ultrastructure. We found that in the 6-week period following Achilles tenotomy, fibril mean diameter remained significantly smaller than sham-side diameter by a factor of 2-3. Despite the persistently small fibril size, increasing numerical density resulted in a gradual increase in fibril area fraction. Biomechanical strength did not reach that of intact tendons until some time between 5 and 7 weeks, approximately the same time period when fibril area fraction began to approach sham values. These data suggest that parameters other than collagen fibril size are most responsible for increased tendon strength during healing.