Molecular epidemiology of HIV-1 variants in the global AIDS pandemic: an update

The picture of HIV-1 genetic diversity in the global pandemic continues to evolve. Identification of new variants, including circulating and unique recombinant forms, recognition of new outbreaks and of changes in established epidemics, and characterization of growing numbers of full-length genomes provide a view of high dynamism and increasing complexity. The pervasive role of recombination as a major driving force in the generation of diversity in the HIV-1 pandemic is becoming evident, and is particularly visible in areas in which different genetic forms meet, referred to as "geographic recombination hotspots". The importance of superinfection and its impact on HIV-1 diversification and propagation is surfacing, although restrictions to superinfection are also apparent. Genetic diversity within subtypes is increasing over time and new geographically localized lineages deriving from point introductions are being recognized. Characterization of such variants may be of relevance to vaccine development and may allow the detection of intrasubtype recombination and superinfection. Recent studies supporting the correlation of HIV-1 clades to immune responses and to drug resistance-associated mutations lend increasing relevance to the role of molecular epidemiology as an essential tool in combating the AIDS pandemic. However, knowledge on the global HIV-1 genetic diversity and its implications is still far from adequate and a major scaling up of efforts is needed.     

Digoxin dosing in the aged: new pharmacokinetic system versus Jellife and Koup methods

To evaluate three methods for digoxin dose adjustment in aged patients, we determined the plasma digoxin levels that would be attained in 87 aged patients with doses adjusted to the kidney function by means of three separate procedures. Mean patient age was: 79.0 +/- 6.3 years; creatinine clearance (Clc): 0.70 +/- 0.23 mL/Kg of lean body weight and minute; digoxinemia/dose ratio (RCpD): 0.421 +/- 0.237 Kg/L. The dose that would attain a digoxinemia of 1.2 ng/mL, calculating the elimination constant (K) and the volume of distribution (V) as linear functions of the Clc, so that K ranges between 0.173 and 0.462 days-1 and V between 4 and 10 L/Kg of lean body weight when the Clc varies from 0 to 110 mL/minute, was 2947 ng/Kg of lean body weight, coefficient of variation (CV): 25.2%. The digoxinemia that patients would have with this D, taking into account the individual RCpD, was 1.1 ng/mL, CV: 38.0%; with figures between 0.8 y 2.0 ng/mL and above 2.0 ng/mL in the 81.6% and the 0.0% of the patients (95% confidence intervals (95% CI): 72.2% to 88.4 and 0.0% to 4.6%), respectively. The precision and the bias were 0.43 and -0.06 ng/mL (95% CI: 0.38 to 0.48 and -0.16 to 0.03 ng/mL), respectively, and with this method the digoxinemia was not associated with the Clc. We concluded that the described method would lead to good results if digoxin has not been prescribed in order to control the cardiac frequency in the setting of auricular fibrilation.     

Dystrophin and utrophin influence fiber type composition and post-synaptic membrane structure

The X-linked muscle wasting disease Duchenne muscular dystrophy is caused by the lack of dystrophin in muscle. Protein structure predictions, patient mutations, in vitro binding studies and transgenic and knockout mice suggest that dystrophin plays a mechanical role in skeletal muscle, linking the subsarcolemmal cytoskeleton with the extracellular matrix through its direct interaction with the dystrophin-associated protein complex (DAPC). Although a signaling role for dystrophin has been postulated, definitive data have been lacking. To identify potential non-mechanical roles of dystrophin, we tested the ability of various truncated dystrophin transgenes to prevent any of the skeletal muscle abnormalities associated with the double knockout mouse deficient for both dystrophin and the dystrophin-related protein utrophin. We show that restoration of the DAPC with Dp71 does not prevent the structural abnormalities of the post-synaptic membrane or the abnormal oxidative properties of utrophin/dystrophin-deficient muscle. In marked contrast, a dystrophin protein lacking the cysteine-rich domain, which is unable to prevent dystrophy in the mdx mouse, is able to ameliorate these abnormalities in utrophin/dystrophin-deficient mice. These experiments provide the first direct evidence that in addition to a mechanical role and relocalization of the DAPC, dystrophin and utrophin are able to alter both structural and biochemical properties of skeletal muscle. In addition, these mice provide unique insights into skeletal muscle fiber type composition.     

Ca2+ imaging of mouse neocortical interneurone dendrites: Ia-type K+ channels control action potential backpropagation

GABAergic interneurones are essential in cortical processing, yet the functional properties of their dendrites are still poorly understood. In this first study, we combined two-photon calcium imaging with whole-cell recording and anatomical reconstructions to examine the calcium dynamics during action potential (AP) backpropagation in three types of V1 supragranular interneurones: parvalbumin-positive fast spikers (FS), calretinin-positive irregular spikers (IS), and adapting cells (AD). Somatically generated APs actively backpropagated into the dendritic tree and evoked instantaneous calcium accumulations. Although voltage-gated calcium channels were expressed throughout the dendritic arbor, calcium signals during backpropagation of both single APs and AP trains were restricted to proximal dendrites. This spatial control of AP backpropagation was mediated by Ia-type potassium currents and could be mitigated by by previous synaptic activity. Further, we observed supralinear summation of calcium signals in synaptically activated dendritic compartments. Together, these findings indicate that in interneurons, dendritic AP propagation is synaptically regulated. We propose that interneurones have a perisomatic and a distal dendritic functional compartment, with different integrative functions.     

Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism

An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform.     

Virilizing mature ovarian cystic teratomas

 Three further cases of mature benign cystic teratomas of the ovary associated with virilization are added to the three previously reported in the literature. They were found in postmenopausal, obese, diabetic women aged 52, 61, and 67 years. The patients presented with hirsutism and voice changes and clitoromegaly was present in one. Testosterone and androstenedione levels were elevated but promptly regressed after removal of the tumours. Histologically, sheets of stromal luteinized cells were found peripherally at the interface between the neoplasm and ovarian tissue. Luteinization of ovarian stroma induced by an unknown factor related to diabetes mellitus is the origin of the virilization. Received: 8 January 1997 / Accepted: 28 February 1997     

Towards cellular receptors for prions

Transmissible spongiform encephalopathies (TSE) are attributed to the conversion of the cellular prion protein (PrP(c)) into an abnormal isoform (PrP(sc)). This can be caused by the invasion of living organisms by infectious particles, or be inherited due to mutations on the PrP(c) gene. One of the most intriguing problems of prion biology is the inability to generate the infectious agent in vitro. This argues strongly that other cellular proteins besides those added in test tubes or found in cellular preparations are necessary for infection. Despite recent progress in the understanding of prion pathology, the subcellular compartments in which the interaction and conversion of PrP(c) into PrP(sc) take place are still controversial. PrP(c) interacts with various macromolecules at the cell membrane, in endocytic compartments and in the secretory pathway, all of which may play specific roles in the internalisation of PrP(sc) and conversion of PrP(c). A specific interacting protein required for the propagation of prions was originally proposed as a prion receptor, and later referred to as a ligand, a cofactor, protein X, or a partner. However, current studies indicate that PrP(c) associates with multi-molecular complexes, which mediate a variety of functions in distinct cellular compartments. It is proposed that a deeper understanding of the mechanics of such interactions, coupled to a better knowledge of the corresponding signalling pathways and ensuing cellular responses, will have a major impact on the prevention and treatment of TSE.     

Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry

We have tested Green Fluorescent Protein (GFP) expressed by a vaccinia virus recombinant as a marker for viral infection. Virus recombinants expressing either wild-type GFP, or a Ser₆₅ to Thr mutated version (GFP-S65T) were used to infect cultured cells, and the appearance of fluorescence was followed during infection by flow cytometry. Although both versions were detectable in infected cells, GFP-S65T gave up to 26-fold brighter fluorescence than wild-type GFP when excited by an argon laser beam (488 nm). In addition, GFP-S65T fluorescence appeared earlier, and infected cells could be detected above background as soon as 1 h after infection. We have used this construct to infect porcine peripheral blood lymphocytes, and show its usefulness to study virus tropism when used in combination with cell-type specific markers. Thus, GFP provides a direct, fast and convenient way to monitor infection by flow cytometry.