Study of the arterial vascularisation of the medial tibial condyle in the fetus

Summary Varus deformity of the knee is common in young children who have suffered from fulminating purpura. This study was directed at the anatomic features of the vascularisation of the upper end of the tibia that might account for such deformation. It was based on the dissection of 28 anatomic specimens prepared by injection of Indian ink into the vascular trunk. 16 specimens were diaphanised for better analysis of the intracartilaginous distribution of the vessels. The study showed that the vascularisation of the medial condyle of the tibia is poor and of terminal nature, which may explain the occurrence of ischemic growth disorders following fulminating purpura.

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Etude de la vascularisation artérielle du condyle médial du tibia chez le foetus

Résumé Les déformations en varus du genou chez les jeunes enfants ayant présenté un purpura fulminans sont fréquentes. Ce travail a pour objet de rechercher les caractéristiques anatomiques de la vascularisation de l'extrémité supérieure du tibia qui peuvent expliquer ces déformations. L'étude porte sur la dissection de 28 pièces anatomiques préparées par injection de l'axe vasculaire à l'encre de Chine. Pour mieux analyser la répartition intra-cartilagineuse des vaisseaux, 16 pièces ont été diaphanisées. Cette étude montre que la vascularisation du condyle médial du tibia est pauvre, de type terminal, ce qui peut expliquer la survenue de troubles de croissance ischémiques dans les suites d'un purpura fulminans.

     

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.      

T-cell death by apoptosis in vertically human immunodeficiency virus- infected children coincides with expansion of CD8+/interleukin-2 receptor-/HLA-DR+ T cells: sign of a possible role for herpes viruses as cofactors?

One mechanism proposed to play a role in T-cell depletion in human immunodeficiency virus (HIV) infection is apoptosis (activation-induced cell death). We assessed whether apoptosis is related to activation of T cells in vivo and its possible triggers. DNA was extracted from peripheral blood mononuclear cells (PBMC) taken from 16 vertically HIV- infected children and 9 HIV-negative children born to HIV-positive mothers (controls) and tested by agarose gel electrophoresis for the presence of DNA fragments specific for apoptosis. Signs of apoptosis were found on in vitro culture of PBMC from 12 of 16 HIV-infected children, but not in PBMC from the nine controls. Eleven of the 12 HIV- infected children with apoptosis showed an elevated (> 15%) proportion of CD3+/HLA-DR+ cells. This was due to an increased proportion of CD8+/HLA-DR+ cells, as shown in 7 of 7 further tested patients. In none of the probands an increased (> 5%) proportion of IL-2 receptor expressing CD3+ cells was found. T cells undergoing apoptosis were preferentially of the CD8+ phenotype. Expansion of circulating CD8+/interleukin-2 receptor (IL-2R)-/HLA-DR+ T cells is known to occur during active infection with herpes viruses. To investigate the possible role of herpes viral coinfections for apoptosis in HIV infection, we focused on Epstein-Barr virus (EBV) as an example for a herpes virus usually acquired during childhood. In 10 of 12 patients with apoptosis, we found increased levels of EBV genome in PBMC and/or tissues, indicating active EBV replication. By contrast, no increased burden of EBV was found in the four HIV-infected patients without apoptosis or in the controls. Our data indicate that in children the occurrence of apoptosis in HIV infection is closely related to activation of CD8+ T cells. Furthermore, primoinfection with or reactivation of herpes viruses, such as EBV, may substantially contribute to such T-cell activation and the ensuing apoptosis. Additional studies are warranted to evaluate the contribution of herpes virus-triggered apoptosis to the T-cell loss leading to the acquired immunodeficiency syndrome.     

The expression of type III hyperlipoproteinemia: involvement of lipolysis genes

Type III hyperlipoproteinemia (HLP) is mainly found in homozygous apolipoprotein (APO) E2 (R158C) carriers. Genetic factors contributing to the expression of type III HLP were investigated in 113 hyper- and 52 normolipidemic E2/2 subjects, by testing for polymorphisms in APOC3, APOA5, HL (hepatic lipase) and LPL (lipoprotein lipase) genes. In addition, 188 normolipidemic Dutch control panels (NDCP) and 141 hypertriglyceridemic (HTG) patients were genotyped as well. No associations were found for four HL gene polymorphisms and two LPL gene polymorphisms and type III HLP. The frequency of the rare allele of APOC3 3238 G>C and APOA5 −1131 T>C (in linkage disequilibrium) was significantly higher in type III HLP patients when compared with normolipidemic E2/2 subjects, 15.6 vs 6.9% and 15.1 vs 5.8%, respectively, (P<0.05). Furthermore, the frequencies of the APOA5 c.56 G>C polymorphism and LPL c.27 G>A mutation were higher in type III HLP patients, though not significant. Some 58% of the type III HLP patients carried either the APOA5 −1131 T>C, c.56 G>C and/or LPL c.27 G>A mutation as compared to 27% of the normolipidemic APOE2/2 subjects (odds ratio 3.7, 95% confidence interval=1.8–7.5, P<0.0001). The HTG patients showed similar allele frequencies of the APOA5, APOC3 and LPL polymorphisms, whereas the NDCP showed similar allele frequencies as the normolipidemic APOE2/2. Patients with the APOC3 3238 G>C/APOA5 −1131 T>C polymorphism showed a more severe hyperlipidemia than patients without this polymorphism. Polymorphisms in lipolysis genes associate with the expression and severity of type III HLP in APOE2/2.     

Inactivation of purified human alpha 2-antiplasmin and purified human C1 inhibitor by synthetic fibrinolytic agents

3-Hydroxypropyl flufenamide (Flu-HPA) is one of a series of flufenamic acid derivatives that enhances blood clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The profibrinolytic activity of Flu-HPA in clot lysis assays was found to be dependent on plasminogen. The influence of Flu-HPA on the ability of purified alpha 2-antiplasmin to inhibit purified plasmin was studied. Plasmin activity was determined using 125I-fibrin plates or the spectrophotometric tripeptide substrate, Val-Leu-Lys-paranitroanilide. At Flu-HPA concentrations greater than 1 mM, the inhibitory activity of alpha 2-antiplasmin was abolished in a time-dependent and concentration- dependent manner. The influence of Flu-HPA on the ability of purified Cl inhibitor to inhibit purified plasma kallikrein and beta-Factor XIIa was also studied. Cl inhibitor activity was abolished by Flu-HPA at concentrations greater than 2 mM. Notably, Flu-HPA up to 60 mM did not affect the amidolytic activities of plasmin, kallikrein, or beta-Factor XIIa. Flu-HPA did not release enzyme activity from preformed complexes of either alpha 2-antiplasmin and plasmin of Cl inhibitor and kallikrein. A water-soluble derivative of flufenamic acid, N-flufenamyl- glutamic acid, also inactivated alpha 2-antiplasm and Cl inhibitor. This inactivation was shown to be reversible. These results indicate that synthetic fibrinolytic compounds such as flufenamic acid derivatives may promote fibrinolysis by directly inactivating alpha 2- antiplasmin and Cl inhibitor.     

Molecular characterization of human factor XSan Antonio

Enzymatic amplification technique was used to isolate all eight exons and sequences around the splice junctions, putative promoter, and polyadenylation sites of human factor X DNA from a patient with factor X deficiency. Two genetic changes in factor X have been observed in this patient. The patient is most likely a compound heterozygote since there is only 14% activity associated with factor X. A point mutation that resulted in the substitution of cysteine (TGC) for arginine (CGC) at amino acid 366 was found in exon VIII of one allele of the factor X gene. This mutation, which occurs in the catalytic domain, can affect the formation of a disulfide bridge and thus could result in a reduction in factor X activity. Sequencing all the regions revealed a second mutation: a deletion of one nucleotide (TCCT to TCT) in exon VII that would cause a frame shift at amino acid 272 followed by termination. We have also shown that the point mutation in exon VIII creates an ApaL1 restriction site and destroys the HinP1 site. Enzymatic DNA amplification followed by restriction digestion provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first characterization of factor X deficiency at the molecular level. We propose to name this mutation Factor XSan Antonio.     

温血心停跳液与冷晶体心停跳液对狗心率变异性的影响

为探讨体外循环（ＣＰＢ）导致心脏植物神经系统（ＣＡＳ）损伤的机理,了解温血心停跳液能否防止ＣＰＢ后心率变异性（ＨＲＶ）的降低,采用对照方法观察了温血心停跳液与冷晶体心停跳液对狗ＨＲＶ的影响。结果显示：ＣＰＢ后温血心停跳液组（ＷＢ组）和冷晶体心停跳液组（ＣＣ组）的全频谱（ＴＰ）、低频（ＬＦ）和高频（ＨＦ）均较术前明显降低（Ｐ＜０．０５）,而且ＣＣ组比ＷＢ组降低更明显（Ｐ＜０．０５）,但ＬＦ／ＨＦ在组内及组间均无明显变化（Ｐ＞０．０５）。ＣＰＢ后２４小时平均心率（ＭＨＲ）明显增加（Ｐ＜０．０５）,且ＣＣ组高于ＷＢ组（Ｐ＜０．０５）。本研究表明：采用温血心停跳液或冷晶体心停跳液的ＣＰＢ不会干扰ＣＡＳ平衡,但均能使ＨＲＶ降低,温血心停跳液不能防止ＨＲＶ损害。     

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.