Chaperonin-mediated assembly of wild-type and mutant subunits of human propionyl-CoA carboxylase expressed in Escherichia coli

We developed a bacterial expression system for the human alpha and beta cDNAs of propionyl-CoA carboxylase (PCC). These cDNAs (less the putative mitochondrial matrix targeting presequences) were co-expressed in Escherichia coli on one plasmid vector with each cDNA having its own IPTG-inducible promoter. Only negligible amounts of active PCC were measured despite the presence of both alpha and beta subunits as indicated by Western blot analysis and the almost complete biotinylation of the alpha subunit. Co-expression of this plasmid with a second plasmid vector over-expressing the E. coli chaperonin proteins, groES and groEL, resulted in a several hundred-fold increase in PCC specific activity, to a level comparable with that found in crude human liver extracts. PCC was partially purified on monomeric avidin affinity resin and the presence of both alpha and beta subunits was demonstrated, thereby confirming the assembly of both subunits into an active enzyme. Deficiency of either alpha PCC or beta PCC results in propionic acidemia, an autosomal recessive disorder. We used this expression system to characterize one missense mutation previously described in five Japanese alleles, namely C1283T (Thr428lle) in beta PCC. This mutation, when expressed in E.coli under the same conditions as that of wild-type PCC, had null activity, despite the presence of assembled alpha PCC and beta PCC subunits. This bacterial expression system can be useful for analysis of either alpha PCC or beta PCC mutations. Our findings indicated that the groES and groEL chaperonin proteins were essential for folding and assembly of the human PCC heteromeric subunits.      

Blood supply of the olfactory nerve

Summary The authors report the results of a series of dissections and anatomic sections of the fronto-basal region of the brain and of the anterior cranial fossa in human cadavers. The constant presence of an arachnoidal cistern above the olfactory nerve was verified. The arachnoid separates from the pial membrane and forms a bridge with the ventral part of the olfactory bulb and tract, from the lateral edge of the olfactory sulcus to the medial edge of the gyrus rectus. The cistern is wide in its anterior portion, between the gyrus rectus and the olfactory bulb, and is reduced to a virtual slit in its posterior portion where the tract is lodged in the olfactory sulcus. The olfactory nerve can be separated without damaging fronto-basal arachnoidial adhesions over several centimeters. Dissection of this region after intravascular injection of colored media shows the constant presence of an artery destined to the olfactory bulb and tract. It originates either from the lateral surface of the anterior cerebral a. (segment A2), or from the medial fronto-basal a., and consistently provides terminal branches in front of the olfactory trigone in the medial olfactory sulcus. At their ventral extremity, the olfactory structures are therefore vascularised independently for several centimeters, from the lower face of the frontal lobe. The independent vascularisation of the olfactory nerve, the tenuous and easily detachable adhesions, and the actual presence of a true arachnoidal cistern all contribute to enabling surgical techniques which conserve olfactory function during anterior approaches.

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Vascularisation du nerf olfactif. Rapports méningés et applications chirurgicales

Résumé Les auteurs rapportent les résultats d'une série de dissections et de coupes de la région fronto-basale de l'encéphale et de la fosse crânienne antérieure sur sujets cadavériques. La présence constante d'une citerne arachnoïdienne au dessus du n. olfactif a été vérifiée. L'arachnoïde se sépare du feuillet pial et passe en pont à la partie ventrale du bulbe et du tractus olfactifs, du bord latéral du sillon olfactif au bord médial du gyrus rectus. La citerne est large dans sa portion antérieure, entre le gyrus rectus et le bulbe olfactif, se réduit à une fente virtuelle postérieure lorsque le tractus se loge dans le sillon olfactif. Le n. olfactif peut être séparé sans dommage des adhérences arachnoïdiennes fronto-basales sur quelques centimètres. La dissection de cette région, après injection intravasculaire de masses colorées montre, de façon originale, la présence constante d'une artère destinée au tractus et au bulbe olfactifs. Elle naît soit de la face latérale de l'a. cérébrale antérieure (segment A2), soit de l'a. fronto-basale médiale, pour donner ses branches terminales toujours en avant du trigone olfactif dans le sillon orbitaire médial. Sur quelques centimètres à leur extrémité ventrale, les structures olfactives ont donc une vascularisation indépendante de la face inférieure du lobe frontal. L'indépendance vasculaire du n. olfactif, des adhérences ténues, facilement détachables, et la réalité vérifiée d'une véritable citerne arachnoïdienne permettent d'imaginer des techniques conservatrices de la fonction olfactive utilisées dans plusieurs indications de la chirurgie de la fosse crânienne antérieure.

     

The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Assisted reproductive technology and complex chromosomal rearrangements: the limits of ICSI

Complex chromosomal rearrangements are very rare events in the human population. According to our knowledge on the consequences of simple reciprocal translocations for male fertility, translocations involving three or more chromosomes are thought to lead to severe reproductive impairments in terms of meiotic disturbance or chromosomal imbalance of gametes. We report the case of a 48 year old man whose sperm count revealed either oligozoospermia (<10(3) spermatozoa/ml) or azoospermia. He was referred to the laboratory for in-vitro fertilization after intracytoplasmic sperm injection. Cytogenetic investigations showed a complex chromosomal rearrangement involving firstly a translocation between the short arm of chromosome 7 and the long arm of chromosome 13 and secondly a translocation between the short arm of the same chromosome 13 and the short arm of chromosome 9. Diagnosis was ascertained by fluorescence in-situ hybridization and staining of the nucleolar organizer regions. Theoretical study of the translocated chromosomes predicted a 'chain' configuration of the hexavalent at the pachytene stage of meiosis. In all, 32 modes of segregation were considered and only one resulted either in a normal or a balanced gamete karyotype. Genetic counselling and choice of appropriate artificial reproduction technique are discussed.      

The morphological differentiation of the internal sphincter muscle of the anus in the human embryo and fetus (26.01.1990)

Conclusion The opening of the anal canal appears to be the factor which initiated the differentiation of the sphincter apparatus.The internal sphincter m. of the anus is entirely composed of smooth muscle as distinct from the striated fibers of the m. puborectalis, and the external sphincter which is a mixture of smooth and striated fibers (of skeletal type). It develops in the terminal part of the internal circular layer of the rectal m., outside which are longitudinal fibers which descend early to form the external sphincter (beginning around the third month).This study shows that the internal sphincter is scarcely evident before 12 SA. Thus continence between 10 and 12 SA (after the closure of the anal membrane) is closely related to the other components of the sphincter apparatus. On the other hand, the internal sphincter has become well formed after 28 to 30 SA and then plays a direct role in maintaining continence.     

A comparison of three months of anticoagulation with extended anticoagulation for a first episode of idiopathic venous thromboembolism

BACKGROUND: Patients who have a first episode of venous thromboembolism in the absence of known risk factors for thrombosis (idiopathic thrombosis) are often treated with anticoagulant therapy for three months. Such patients may benefit from longer treatment, however, because they appear to have an increased risk of recurrence after anticoagulant therapy is stopped. METHODS: In this double-blind study, we randomly assigned patients who had completed 3 months of anticoagulant therapy for a first episode of idiopathic venous thromboembolism to continue receiving warfarin, with the dose adjusted to achieve an international normalized ratio of 2.0 to 3.0, or to receive placebo for a further 24 months. Our goal was to determine the effects of extended anticoagulant therapy on rates of recurrent symptomatic venous thromboembolism and bleeding. RESULTS: A prespecified interim analysis of efficacy led to the early termination of the trial after 162 patients had been enrolled and followed for an average of 10 months. Of 83 patients assigned to continue to receive placebo, 17 had a recurrent episode of venous thromboembolism (27.4 percent per patient-year), as compared with 1 of 79 patients assigned to receive warfarin (1.3 percent per patient-year, P<0.001). Warfarin resulted in a 95 percent reduction in the risk of recurrent venous thromboembolism (95 percent confidence interval, 63 to 99 percent). Three patients assigned to the warfarin group had nonfatal major bleeding (two had gastrointestinal bleeding and one genitourinary bleeding), as compared with none of those assigned to the placebo group (3.8 vs. 0 percent per patient-year, P=0.09). CONCLUSIONS: Patients with a first episode of idiopathic venous thromboembolism should be treated with anticoagulant agents for longer than three months.     

Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation

The tumour suppressor gene PTEN , which maps to 10q23.3 and encodes a 403 amino acid dual specificity phosphatase (protein tyrosine phosphatase; PTPase), was shown recently to play a broad role in human malignancy. Somatic PTEN deletions and mutations were observed in sporadic breast, brain, prostate and kidney cancer cell lines and in several primary tumours such as endometrial carcinomas, malignant melanoma and thyroid tumours. In addition, PTEN was identified as the susceptibility gene for two hamartoma syndromes: Cowden disease (CD; MIM 158350) and Bannayan-Zonana (BZS) or Ruvalcaba-Riley-Smith syndrome (MIM 153480). Constitutive DNA from 37 CD families and seven BZS families was screened for germline PTEN mutations. PTEN mutations were identified in 30 of 37 (81%) CD families, including missense and nonsense point mutations, deletions, insertions, a deletion/insertion and splice site mutations. These mutations were scattered over the entire length of PTEN , with the exception of the first, fourth and last exons. A 'hot spot' for PTEN mutation in CD was identified in exon 5 that contains the PTPase core motif, with 13 of 30 (43%) CD mutations identified in this exon. Seven of 30 (23%) were within the core motif, the majority (five of seven) of which were missense mutations, possibly pointing to the functional significance of this region. Germline PTEN mutations were identified in four of seven (57%) BZS families studied. Interestingly, none of these mutations was observed in the PTPase core motif. It is also worthy of note that a single nonsense point mutation, R233X, was observed in the germline DNA from two unrelated CD families and one BZS family. Genotype-phenotype studies were not performed on this small group of BZS families. However, genotype-phenotype analysis inthe group of CD families revealed two possible associations worthy of follow-up in independent analyses. The first was an association noted in the group of CD families with breast disease. A correlation was observed between the presence/absence of a PTEN mutation and the type of breast involvement (unaffected versus benign versus malignant). Specifically and more directly, an association was also observed between the presence of a PTEN mutation and malignant breast disease. Secondly, there appeared to be an interdependent association between mutations upstream and within the PTPase core motif, the core motif containing the majority of missense mutations, and the involvement of all major organ systems (central nervous system, thyroid, breast, skin and gastrointestinal tract). However, these observations would need to be confirmed by studying a larger number of CD families.