Family physician and endocrinologist coordination as the basis for diabetes care in clinical practice

Background

The role of estrogens in male physiology has become evident. However, clinically useful normative data for estradiol secretion in boys has not previously been established due to the insensitivity of current methods used in clinical routine. By use of a validated ultra-sensitive extraction RIA, our aim was to establish normative data from a group consisting of healthy boys in prepuberty and during pubertal development.

Methods

Sixty-two 24-hours serum profiles (6 samples/24 hours) were obtained from 44 healthy boys (ages; 7.2–18.6 years) during their pubertal development, classified into five stages: prepuberty (testis, 1–2 mL), early (testis, 3–6 mL), mid (testis, 8–12 mL), late-1 (testis,15–25 mL, not reached final height) and late-2 (testis,15–25 mL, reached final height). Serum estradiol was determined by an ultra- sensitive extraction radioimmunoassay with detection limit 4 pmol/L and functional sensitivity 6 pmol/L.

Results

Mean estradiol concentrations during 24-hours secretion increased from prepuberty (median: <4 (5–95 percentiles: <4 – 7) pmol/L) to early puberty (6 (<4 – 12 pmol/L) but then remained relatively constant until a marked increase between mid-puberty (8 (4 – 17) pmol/L) and late-1 (21 (12 – 37) pmol/L) puberty, followed by a slower increase until late-2 puberty (32 (20 – 47) pmol/L). The diurnal rhythm of serum estradiol was non-measurable in pre- and early puberty, but discerned in mid-puberty, and become evident in late pubertal stages with peak values at 0600 to 1000 h.

Conclusion

With the use of an ultra-sensitive extraction RIA, we have provided clinically useful normative data for estradiol secretion in boys.     

Rapid and accurate quantification of different HCV genotypes by LightCycler Real Time PCR and direct sequencing of HCV amplicons

Follow-up of chronically infected HCV patients is the primary clinical goal in therapy administration. In the absence of an HCV vaccine, the timely monitoring of HCV viral load combined with the information of the viral genotype could contribute to patient disease management. A LightCycler Real Time RT-PCR assay was developed and optimized allowing rapid and accurate quantification of HCV RNA over an extended dynamic range using a single human reference standard. A total of 5,096 plasma samples, collected over almost 5 years, were tested and HCV RNA was quantified in 2,435 samples with levels ranging from 5.7x10(1) to 2.52x10(9) IU/ml. The precision and reproducibility of the test are documented by various inter-assay parameters of the reference standard obtained in 409 RT-PCR runs. This Real Time RT-PCR protocol uses the LightCycler cDNA amplicons for direct sequence analysis and reduces the sequencing time to approximately 3 hours. Nearly all HCV genotypes were identified. Viral sequences showed a similarity level close to 100%, independently from the viral load, while the LightCycler melting temperature analysis did not correlate with HCV genotypes. All this makes the LightCycler Real Time RT-PCR protocol a suitable tool for the diagnosis and monitoring of HCV infections.     

Molecular cloning, expression and characterization of the first three genes in the mevalonate-independent isoprenoid pathway in Streptomyces coelicolor.

The mevalonate-independent biosynthetic pathway to isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors to the isoprenoids, operates in eubacteria, including Escherichia coli, in algae, and in the plastids of higher plants. A search of the Sanger Centre Streptomyces coelicolor genome database revealed open reading frames with ca. 40--50% identity at the deduced amino acid level to the first three E. coli enzymes of this pathway, corresponding to deoxyxylulose phosphate synthase, deoxyxylulose phosphate reductoisomerase and 2-C-methyl erythritol 4-phosphate cytidylyltransferase. The S. coelicolor genes have been cloned and expressed in E. coli, and the recombinant proteins characterized physically and kinetically. The presence of the corresponding enzyme activities in extracts of S. coelicolor CH999 further supports the operation of the mevalonate-independent pathway in this organism.     

Proteomic analysis of mature melanosomes from the retinal pigmented epithelium

The protein content of melanosomes in the retinal pigment epithelium (RPE) was analyzed by mass spectrometry. More than 100 proteins were found to be common to two out of three variations of sample preparation. Some proteins normally associated with other organelles were detected. Several lysosomal enzymes were detected, with the presence of cathepsin D confirmed by immunoelectron microscopy, thus supporting the previously suggested notion that melanosomes may contribute to the degradation of ingested photoreceptor outer segment disks.     

First breeding record of a one-year-old female Iberian lynx

According to literature, female Iberian lynx (Lynx pardinus) start reproducing at 3 years of age. In more recent years, females breeding at 2 years have been recorded in wild populations. Here, however, we describe the first breeding record for a one-year-old female Iberian lynx. This finding was obtained by camera-trapping data, provided by the yearly monitorization of the species that is carried out across its distribution area. A litter of two lynx kittens was found at the south-east limit of the Andujar-Cardeña population. Two camera-traps were set up near the den to identify the breeding female. Photographs obtained confirmed that the breeding female had already been identified the previous year at less than 3 months of age. The case reported here of a yearling female breeding is the first ever from historical distribution range of the species, and as such, is an exceptional event in the reproduction of the Iberian lynx.     

A new bioassay method reveals pathogenicity of Metarhizium anisopliae and Beauveria bassiana against early stages of Capnodis tenebrionis (Coleoptera; Buprestidae)

We used a newly developed bioassay method to demonstrate for the first time the potential of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to be used for the control of neonate larvae of Capnodis tenebrionis, a major threat to stone-fruit orchards in several countries. Four B. bassiana and four M. anisopliae isolates were all pathogenic for neonate larvae of C. tenebrionis; mortality rates 10 days after inoculation by dipping in a suspension with 10(8)conidia/ml varied from 23.5% to 100%. Three of the four M. anisopliae isolates caused 100% mortality. In most cases, postmortem hyphal growth and sporulation of M. anisopliae or B. bassiana was observed covering the larvae in their galleries. The eight isolates were also evaluated for pathogenicity to C. tenebrionis eggs at the same dosage. Only two B. bassiana isolates caused significant egg hatching reduction of 84.5% and 94.5%. Our results indicate that M. anisopliae and B. bassiana may be considered as promising for a new approach to prevent larval infestations by C. tenebrionis.     

Inflammation may modulate IL-6 and C-reactive protein gene expression in the adipose tissue: the role of IL-6 cell membrane receptor

Only few studies have been addressed to the presence and regulation of C-reactive protein (CRP) gene expression in different districts of adipose tissue, and no study has investigated the role of adipose tissue in presence of inflammation. Therefore, the aim of this study was to investigate the inflammatory involvement of either adipose tissue or adipose cells (adipocytes and stromal cells, respectively) in patients with chronic inflammatory disease, focusing on regional adipose tissue CRP gene expression. Eighteen patients with inflammatory disease and 14 healthy controls were enrolled. All subjects underwent specific surgical procedures. Inflamed and noninflamed patients provided samples of subcutaneous and/or omental adipose tissue. All samples were analyzed by RT-PCR and real-time PCR for specific gene expression. In addition, both adipocytes and stromal cells were studied by real-time PCR and immunoprecipitation to evaluate either gene or protein expression of CRP. Our results (real-time PCR) demonstrated a higher gene expression of CRP, IL-6, and both IL-6 membrane receptors in subcutaneous samples of inflamed patients than in healthy controls. Furthermore, in omental fragments of inflamed patients, an enhanced mRNA abundance of the same genes, compared with subcutaneous, was observed. The results obtained at cellular level did not provide evidence of any difference between adipocytes and stromal cell CRP gene expression, whereas immunoprecipitation demonstrated the presence of CRP in inflamed subjects. These results provide first-time evidence of the involvement of adipose tissue in the course of chronic inflammatory diseases, with a different degree of participation of the different adipose tissue districts.     

Effects of Culture Conditions on Poly(β-Hydroxybutyric Acid) Production by Haloferax mediterranei

The halobacterium Haloferax mediterranei accumulates poly(β-hydroxybutyrate) (PHB) as intracellular granules. The conditions for PHB production in batch and continuous cultures have been studied and optimized. Phosphate limitation is essential for PHB accumulation in large quantities. Glucose and starch are the best carbon sources. With 2% starch, 0.00375% KH₂PO₄, and 0.2% NH₄Cl in batch culture, a production of ca. 6 g of PHB per liter was reached, being 60% of the total biomass dry weight, and giving a yield over the carbon source of 0.33 g/g. The PHB production in continuous cultures was stable over a 3-month period. Our results demonstrate that H. mediterranei is an interesting candidate for industrial production of biological polyesters.