Extraction and quantification of amino acids in human stratum corneum in vivo.

BACKGROUND: Amino acid (AA) levels in stratum corneum (SC) are potential biomarkers of skin health while their systemic levels may be used to diagnose inherited metabolic diseases. OBJECTIVES: To examine reverse iontophoresis, in human volunteers, as a minimally invasive tool to analyse AAs within the skin and subdermally. METHODS: In four volunteers, the amounts of iontophoretically extracted AAs were compared with those determined in the SC following repetitive tape stripping and with the plasma concentrations. Glucose levels, evaluated in the different compartments, were used as a control. RESULTS: SC concentrations of 13 essentially zwitterionic AAs were ∼100-fold higher than the respective plasma levels. Passive and reverse iontophoretic extraction for 4 h did not deplete the SC depot of AAs, a fact reinforced by postextraction tape stripping, which revealed that AAs remained in the SC at this time. In contrast, glucose was much less abundant in the SC and was fully and relatively quickly extracted by reverse iontophoresis. CONCLUSIONS: It follows that reverse iontophoresis is useful for quantifying AAs in the SC and these data are highly correlated with levels obtained by tape stripping. However, reverse iontophoresis is impractical for the routine monitoring of AA plasma concentrations (unlike the situation for glucose, the skin reservoir of which is much smaller).

The influence of surfactant on PLGA microsphere glass transition and water sorption: remodeling the surface morphology to attenuate the burst release.

PURPOSE: The stability of protein unloaded and loaded poly(lactic-co-glycolic acid) (PLGA) microspheres fabricated with surfactant was challenged through exposure to environmental conditions of different relative humidity. METHODS: Polyvinyl alcohol (PVA) or Triton X-100 was added to the primary emulsion of the double-emulsion solvent evaporation technique. After storage at ambient humidity and 75% relative humidity, the mechanical stability of the polymer was tested to reveal PLGA chain mobility using differential scanning calorimetry. Subsequent surface modifications were examined by atomic force microscopy (AFM), and protein release profiles were collected. RESULTS: Residual amounts of PVA and particularly Triton X-100 raised the hydrophilicity of the microspheres. When exposed to ambient humidity or 75% relative humidity, PVA and Triton X-100 had, respectively, an antiplasticizing and a plasticizing effect upon PLGA, and both led to physical aging. The high-resolution AFM imaging of microspheres containing model protein and Triton X-100 showed that the depth of the surface pores was reduced when exposed to 75% relative humidity, and the initial burst release subsequently decreased. CONCLUSION: These studies suggested that the mechanical stability of PLGA was influenced by the addition of surfactants, which, depending on the formulation, led to surface pore remodeling under high humidity, reducing the initial burst release while maintaining the spherical integrity of the microsphere.

Microsatellite variation in an introduced mouflon population.

As previous studies of genetic polymorphism in the mouflon (Ovis gmelini) have not provided any valuable markers for population studies, we tested the capacity of microsatellites to index the genetic diversity of a recently introduced mouflon population. Six pairs of bovine primer amplified microsatellites in mouflon, and all six were polymorphic. Furthermore, despite the low number of founders, five loci had a high gene diversity in this introduced population. Unlike other genetic markers, microsatellites could be powerful to study the genetic structure of mouflon populations.

A precise meiotic map in the class I region of the human major histocompatibility complex.

Human families in which recombinant meiotic event(s) are known to have occurred are powerful tools with which to analyze more precisely the structures of defined genomic regions, especially unstable areas. Such families allow the determination of the haplotypes of each member and, taking into account the recombinant event, it is possible to localize very precisely the point of crossover. Using families in which crossovers between the genes HLA-A and -B have occurred, we have constructed a meiotic map localizing the meiotic breakpoint events with respect to both anonymous markers and the principal genes of the region. Such mapping, which depends on the direct analysis of genomic DNA, is essential for fine structural analysis and is a powerful means of verification of the order and the localization of markers: physical mapping alone, using yeast artificial chromosomes, presents some uncertainties due to the numerous chimeras and inversions that can be produced. The establishment of this map will allow us to determine efficiently the precise location for new markers already localized to the map region. Three microsatellites (D6S265, D6S276, and D6S306), localized in the HLA region by linkage analysis, have been precisely located with respect to the points of recombination in the class I region. The sites of meiotic recombination in the MHC class I region seem to be not randomly distributed but in the majority of cases occurred between HLA-C and the microsatellite D6S265. This study also shows two cases of abnormal segregation of alleles. We discuss how these mutations correspond to a spontaneous mutation event at the somatic or germinal level.

Localization of the OTF3 gene within the human MHC class I region by physical and meiotic mapping.

OTF3 (octamer transcription factor 3) is a transcription factor containing a POU-specific domain and a homeodomain that could play a role in early development. In situ hybridization and pairwise linkage analysis showed that OTF3 gene maps close to the human MHC (major histocompatibility complex). In this paper, we define its localization within the MHC, around 100 kb telomeric to HLA-C, using a combination of physical and genetic analyses.

Tumor necrosis factor microsatellites in four European populations.

The human genome contains a large number of interspersed simple repeat sequences that vary in length among individuals and can therefore serve as highly informative polymorphic markers. Several such variable sites (microsatellites) have been described within the TNF genes within the MHC. In this study, individuals from four Caucasian populations have been typed for three TNF-associated microsatellites in order to define their haplotypes. Of the 208 possible haplotypes, eight exist at a high frequency in all populations and account for approximately 60% of the haplotypes studied, but with marked variations in their frequencies among populations. A few population/sample-specific haplotypes have been identified. The ability of alleles to define haplotypes uniquely varies not only among the loci, but also among the alleles: some alleles displaying complete gametic association (linkage disequilibrium) and others displaying very little.

Analysis of HLA-A/B recombinant families with new polymorphic markers.

The MHC in humans is a much studied region of the genome, the genes of which display a high rate of polymorphism and a high rate of linkage disequilibrium. Four families in which intra-class-I recombination has occurred have been analyzed with six polymorphic markers between HLA-A and -B in order to determine the full haplotypes of the whole families and to localize the points of crossover. The previously proposed order of the markers was confirmed by recombination mapping. In one family, the crossover was shown to have occurred in the 20-kb stretch of DNA bounded by the two markers (P3B and P5) between which no evidence of linkage disequilibrium was found, a region which constitutes of only about 1% of the distance between HLA-A and HLA-B. Although supportive of the suggestion of a hot spot of recombination in this region, based on the apparent lack of linkage disequilibrium between the markers P3B and P5, more such families need to be tested in order to confirm or refute this hypothesis.

Polymorphism of the vitamin D binding protein (DBP) among primates: an evolutionary analysis.

The distribution of the DBP (vitamin D binding protein) polymorphism is now well characterized among human populations but for primates only limited results are known. The aim of this paper is to describe the electrophoretic polymorphism of this protein among various species. Using three different electrophoretic methods, we are able to detect an unknown polymorphism and to classify the different alleles observed. These results may be used to set an international nomenclature for further comparisons. The different electrophoretic mobilities between Old and New World Monkeys show that: 1) the Cercopithecoïdea are presenting the largest genetic heterogeneity; 2) the DBP among the Galago corresponds to the lowest isoelectric points observed among Primates; 3) during the evolution from nonhuman Primates to Man, the DBP is able to keep its affinity for vitamin D derivatives despite the occurrence of significant molecular modifications; 4) among Anthropoïdea, the electrophoretic patterns of DBP are very close to the human Gc1 proteins. These results show that evolution at the DBP level can be considered as a continuous mechanism of structural modifications. A significant transition occurs during the differentiation between Cercopithecoïdea and Anthropoïdea. It is not too speculative to consider that some electrophoretic forms detected among Gorilla, Pongo, or Pan may be identical to rare variants observed among humans.

Molecular structure and early events in the replication of Tacaribe arenavirus S RNA.

Tacaribe arenavirus S RNA was cloned and analysis of its nucleotide sequence revealed two open reading frames of significant size, one in the virus-sense strand, the other in the virus-complementary strand. The predicted amino acid sequences of the two reading frames were compared with the predicted primary structures of the nucleoprotein (N) and glycoprotein precursor (GPC) of LCM, Pichinde and Lassa viruses. The results indicated a high degree of homology between the proteins of similar properties. It was also found that in Tacaribe virus-infected cells a subgenomic viral-sense GPC RNA and a subgenomic viral-complementary N RNA are synthesized in addition to the full length viral (v) RNA and viral complementary (vc) RNAs. These results support the conclusion that in Tacaribe virus--as in Pichinde and lymphocytic choriomeningitis arenavirus-S RNA encodes the viral N and GPC proteins and has an 'ambisense' coding strategy. Analysis of the S-derived RNA species at early times post-infection in cells incubated with or without inhibitors of protein synthesis indicated that for primary transcription of the N mRNA, protein synthesis is not required; whereas synthesis of the vc RNA, GPC mRNA and v RNA does require protein synthesis to take place.

Unusual sialilation of the serum DBP associated with the Gc 1 allele in alcoholic cirrhosis of the liver.

The serum level of the 'vitamin D binding protein' (DBP) or Gc ('group-specific component'), its phenotype distribution and the quantitative estimation of the different electrophoretic isoforms were determined in a sample of healthy individuals (blood donors) and in patients with alcoholic hepatitis. It is shown that the serum DBP levels and the amount of the different electrophoretic isoforms are influenced by the protein phenotypes. In the patients an increased frequency of the Gc 1 allele is noticed. For the first time, an unusual form of the apo DBP protein was detected but only in the sera of the Gc 1 allele carriers. The protein form investigated by analytical procedures presents one more sialic acid residue than the usual Gc 1 protein. This unusual metabolic transformation of the DBP is mostly observed among male patients and is often associated with a deteriorating clinical outcome.