Analysis of the NADH-dependent retinaldehyde reductase activity of amphioxus retinol dehydrogenase enzymes enhances our understanding of the evolution of the retinol dehydrogenase family

In vertebrates, multiple microsomal retinol dehydrogenases are involved in reversible retinol/retinal interconversion, thereby controlling retinoid metabolism and retinoic acid availability. The physiologic functions of these enzymes are not, however, fully understood, as each vertebrate form has several, usually overlapping, biochemical roles. Within this context, amphioxus, a group of chordates that are simpler, at both the functional and genomic levels, than vertebrates, provides a suitable evolutionary model for comparative studies of retinol dehydrogenase enzymes. In a previous study, we identified two amphioxus enzymes, Branchiostoma floridae retinol dehydrogenase 1 and retinol dehydrogenase 2, both candidates to be the cephalochordate orthologs of the vertebrate retinol dehydrogenase enzymes. We have now proceeded to characterize these amphioxus enzymes. Kinetic studies have revealed that retinol dehydrogenase 1 and retinol dehydrogenase 2 are microsomal proteins that catalyze the reduction of all-trans-retinaldehyde using NADH as cofactor, a remarkable combination of substrate and cofactor preferences. Moreover, evolutionary analysis, including the amphioxus sequences, indicates that Rdh genes were extensively duplicated after cephalochordate divergence, leading to the gene cluster organization found in several mammalian species. Overall, our data provide an evolutionary reference with which to better understand the origin, activity and evolution of retinol dehydrogenase enzymes.     

Leptin values in placental cord blood of human newborns with normal intrauterine growth after 30-42 weeks of gestation

OBJECTIVE: To evaluate leptin values in placental cord blood of newborns with normal intrauterine growth after 30-42 weeks of gestation. DESIGN: Leptin, a protein encoded by the ob gene, plays an important role in the regulation of feeding behaviour and energy balance in rodents, primates and humans. The presence of leptin in human amniotic fluid and cord blood has recently been reported in human gestations at term and the possible role of leptin in human fetal growth suggested. However, little is known of leptin synthesis during human foetal development. Thus, the aim of our work was to measure leptin (RIA, Linco Research, Inc.) in placental cord blood of human newborns at different fetal ages. PATIENTS: One hundred and twenty-six healthy newborns with normal intrauterine growth were studied. Twenty-nine were preterm (15 males and 14 females; gestational age: 30-36 weeks) and 99 were at term (49 males and 48 females; gestational age: 37-42 weeks). RESULTS: Leptin values increase progressively throughout gestation from 1.30 +/- 0.53 ng/ml at 30 weeks of gestation to 7.98 +/- 4.96 ng/ml (mean +/- SD) at term, and correlate positively with birth weight (r = 0.56, p < 0. 005, n = 126), length (r = 0.37, p < 0.005, n = 126), BMI (r = 0.57, p < 0.005, n = 126), head circumference (r = 0.37, p < 0.005, n = 126), gestational age (r = 0.48, p < 0.005, n = 126) and placental weight (r = 0.38, p < 0.003, n = 59). Leptin values are statistically significantly lower (p < 0.005) preterm (median: 2.05 ng/ml; range: 0.7-8.3 ng/ml) than at term (median: 7.0 ng/ml; range: 1.1-28.1 ng/ml). Leptin values are also significantly (p < 0.005) higher in females (median: 7.2 ng/ml; range: 0.9-23.6 ng/ml, n = 62) than in males (median: 4.8 ng/ml; range: 0.7-28.1 ng/ml, n = 64), although there are no differences in weight (2,864 +/- 536 g in females vs. 2,937 +/- 744 g in males). Multiple regression analysis shows weight to be a positive sex-independent predictor of serum leptin values (p < 0.0005). Sex also proves to be a predictor of leptin, independently of weight and is higher in females than in males (p < 0.003). CONCLUSION: Leptin is present in placental human cord blood after 30-42 weeks of gestation. Newborn weight and sex are independent predictors of leptin values.     

Preliminary studies of the 2D crystallization of Omp1 of Serratia marcescens: observation by atomic force microscopy in native membranes environment and reconstituted in proteolipid sheets

In this work the porin Omp1 of Serratia marcescens was expressed in a porin deficient mutant (Escherichia coli UH302) and its functionality studied following the accumulation of ciprofloxacin in bacteria. The protein was extracted, purified and reconstituted in proteoliposomes of different composition (lipopolysaccharide (LPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)). Maximum extraction of the detergent was achieved applying different steps of dialysis and centrifugation. Proteolipid sheets with different composition were spread onto mica and observed by atomic force microscopy. Two-dimensional crystal of Omp1 was not observed in any case due to low resolution achieved. Judging from the images features POPC is the most suitable phospholipid to enhance 2D lattice formation for Omp1.     

The SET protein regulates G2/M transition by modulating cyclin B-cyclin-dependent kinase 1 activity

The SET protein and the cell cycle inhibitor p21(Cip1) interact in vivo and in vitro. We identified here the domain (157)LIF(159) of p21(Cip1) as essential for the binding of SET. We also found that SET contains at least two domains of interaction with p21(Cip1), one located in the fragment amino acids 81-180 and the other one in the fragment including amino acids 181-277. SET and p21(Cip1) co-localize in the cell nucleus in a temporal manner. Overexpression of SET blocks the cell cycle at the G(2)/M transition in COS and HCT116 cells. Moreover, SET inhibits cyclin B-CDK1 activity both in vivo and in vitro in both cell types. This effect is specific for these complexes since SET did not inhibit either cyclin A-CDK2 or cyclin E-CDK2 complexes. SET and p21(Cip1) cooperate in the inhibition of cyclin B-CDK1 activity. The inhibitory effect of SET resides in its acidic C terminus, as demonstrated by the ability of this domain to inhibit cyclin B-CDK1 activity and by the lack of blocking G(2)/M transition when a mutated form of SET lacking this C terminus domain was overexpressed in COS cells. These results indicate that SET might regulate G(2)/M transition by modulating cyclin B-CDK1 activity.     

Plasmid maintenance and recombinant cell fitness explored in bacterial colonies

During growth of recombinant bacteria, irregular plasmid partitioning generates non-productive, plasmid-free cells whose proportion usually increases in the culture. For Escherichia coli producing engineered -galactosidases, we have shown a coincidence between plasmid stability and the extension of white/blue areas within individual colonies on X-gal plates. In this context, a good correlation between plasmid permanence in colonies and parameters accurately describing the dynamics of plasmid-free cell population in liquid cultures has been observed. Moreover, the impact of lacZ gene engineering and the metabolic burden imposed by the encoded proteins has been evaluated through plasmid stability by simple image analysis, revealing an enhanced plasmid loss rate as the cells enter into the stationary phase that is modulated by the expression of particular recombinant genes.     

The ��TuRC Revisited: A Comparative Analysis of Interphase and Mitotic Human ��TuRC Redefines the Set of Core Components and Identifies the Novel Subunit GCP8

The γ-tubulin complex is a multi-subunit protein complex that nucleates microtubule polymerization. γ-Tubulin complexes are present in all eukaryotes, but size and subunit composition vary. In Drosophila, Xenopus, and humans large γ-tubulin ring complexes (γTuRCs) have been described, which have a characteristic open ring-shaped structure and are composed of a similar set of subunits, named γ-tubulin, GCPs 2-6, and GCP-WD in humans. Despite the identification of these proteins, γTuRC function and regulation remain poorly understood. Here we establish a new method for the purification of native human γTuRC. Using mass spectrometry of whole protein mixtures we compared the composition of γTuRCs from nonsynchronized and mitotic human cells. Based on our analysis we can define core subunits as well as more transient interactors such as the augmin complex, which associates specifically with mitotic γTuRCs. We also identified GCP8/MOZART2 as a novel core subunit that is present in both interphase and mitotic γTuRCs. GCP8 depletion does not affect γTuRC assembly but interferes with γTuRC recruitment and microtubule nucleation at interphase centrosomes without disrupting general centrosome structure. GCP8-depleted cells do not display any obvious mitotic defects, suggesting that GCP8 specifically affects the organization of the interphase microtubule network.     

Hypoglycaemia-insulin test: discordant growth hormone and cortisol response in paediatric patients regarding recovery from hypoglycaemia with or without oral glucose solution

BACKGROUND: Hypoglycaemia-insulin test (HIT) is the 'gold standard' for the diagnosis of adrenal-pituitary-hypothalamic axis disorders. Controversy exists on the convenience of recovery from an insulin-induced hypoglycaemia since this test is not risk-free. OBJECTIVE: To ascertain whether recovery from insulin-induced hypoglycaemia with an oral glucose solution produces a different response of growth hormone (GH) and cortisol at different times of the study compared with spontaneous recovery from hypoglycaemia. PATIENTS AND METHODS: Prospective study of 100 children and adolescents with growth delay who underwent an HIT. Patients were consecutively assigned to two groups of 50. In one group recovery from hypoglycaemia occurred spontaneously and in the other recovery was achieved with an oral glucose solution (20 g of glucose) when glycaemia was under 30 mg/dl. The two groups did not differ in age, sex, pubertal status, weight, height and IGF-I levels. RESULTS: The response of GH at 30, 60, 90 and 120 min and cortisol at 10, 60, 90 and 120 min was lower and statistically significant in patients with recovery from hypoglycaemia with oral glucose solution. GH deficiency was diagnosed more frequently in patients recovered with glucose solutions (94%) compared to those with spontaneous recovery (68%). CONCLUSIONS: Oral glucose solution administration when glycaemia was under 30 mg/dl in HIT produced a lower GH and cortisol response to insulin stimulus and a greater frequency of GH deficit diagnosis.