High specificity PCR screening for 22q11.2 microdeletion in three different ethnic groups

Congenital heart defects are the most common of all human birth defects. Numerous studies have shown that a deletion within chromosome 22q11 is associated with DiGeorge syndrome and certain forms of sporadic congenital cardiovascular disease. We have determined the value of a PCR assay using markers D22S941, D22S944 and D22S264 designed for the screening of 22q11.2 deletion through consecutive homozygosity in an ethnically admixed urban population. The study population comprised 149 unrelated men and women from three different ethnic groups (white, mulatto and black). Test specificity for the overall population was estimated at 98.3%. We found no significant difference when comparing heterozygosity indices and ethnicity (P value = 0.43 (D22S944), 0.22 (D22S264), and 0.58 (D22S941)). There was no significant difference regarding assay specificity between the three different ethnic groups studied. This assay could constitute a cost-effective way to screen a large number of patients at increased risk, since PCR techniques are easily available, are fast, can be automatized, and are significantly less expensive than fluorescence in situ hybridization.

The role of autolysis loop in determining the specificity of coagulation proteases

We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an ~500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved ~10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.

Fine-tuning of defensive behaviors in the dorsal periaqueductal gray by atypical neurotransmitters

This paper presents an up-to-date review of the evidence indicating that atypical neurotransmitters such as nitric oxide (NO) and endocannabinoids (eCBs) play an important role in the regulation of aversive responses in the periaqueductal gray (PAG). Among the results supporting this role, several studies have shown that inhibitors of neuronal NO synthase or cannabinoid receptor type 1 (CB1) receptor agonists cause clear anxiolytic responses when injected into this region. The nitrergic and eCB systems can regulate the activity of classical neurotransmitters such as glutamate and γ-aminobutyric acid (GABA) that control PAG activity. We propose that they exert a ‘fine-tuning’ regulatory control of defensive responses in this area. This control, however, is probably complex, which may explain the usually bell-shaped dose-response curves observed with drugs that act on NO- or CB1-mediated neurotransmission. Even if the mechanisms responsible for this complex interaction are still poorly understood, they are beginning to be recognized. For example, activation of transient receptor potential vanilloid type-1 channel (TRPV1) receptors by anandamide seems to counteract the anxiolytic effects induced by CB1 receptor activation caused by this compound. Further studies, however, are needed to identify other mechanisms responsible for this fine-tuning effect.