Evidence for a dinuclear active site in the metallo-beta-lactamase BcII with substoichiometric Co(II). A new model for metal uptake

Metallo-beta-lactamases are zinc-dependent enzymes that constitute one of the main resistance mechanisms to beta-lactam antibiotics. Metallo-beta-lactamases have been characterized both in mono- and dimetallic forms. Despite many studies, the role of each metal binding site in substrate binding and catalysis is still unclear. This is mostly due to the difficulties in assessing the metal content and site occupancy in solution. For this reason, Co(II) has been utilized as a useful probe of the active site structure. We have employed UV-visible, EPR, and NMR spectroscopy to study Co(II) binding to the metallo-beta-lactamase BcII from Bacillus cereus. The spectroscopic features were attributed to the two canonical metal binding sites, the 3H (His(116), His(118), and His(196)) and DCH (Asp(120), Cys(221), and His(263)) sites. These data clearly reveal the coexistence of mononuclear and dinuclear Co(II)-loaded forms at Co(II)/enzyme ratios as low as 0.6. This picture is consistent with the macroscopic dissociation constants here determined from competition binding experiments. A spectral feature previously assigned to the DCH site in the dinuclear species corresponds to a third, weakly bound Co(II) site. The present work emphasizes the importance of using different spectroscopic techniques to follow the metal content and localization during metallo-beta-lactamase turnover.     

Temporal genetic differentiation in cultured and natural beds of the brown mussel Perna perna (Mytilidae)

Perna perna is the most important cultivated mussel of Santa Catarina, Brazil, sustaining an important economic input for many local families. Natural stocks of P. perna are depleted by the extraction of adults and seeds for consumption and culture. The aim of the present study was to use the microsatellite locus pms-2 to study the variation of the genetic composition and diversity between natural and cultured stocks in samples of 2001 and 2005 from Penha, Santa Catarina. DNA was extracted from adductor muscle by Chelex/proteinase-K and phenol/chloroform protocols. Amplification by polymerase chain reaction was performed using specific primers for analyzing the pms-2 locus. Polymerase chain reaction products were submitted to vertical denatured 6% polyacrylamide gel electrophoresis and horizontal 2% agarose gel electrophoresis, and visualized by silver staining and ethidium bromide, respectively. Allele diversity and heterozygote deficiency were higher for samples of 2005 than for those of 2001. No significant genetic differentiation was found between natural and cultured stocks of 2001 by the chi(2) test, but G(2) (likelihood ratio) detected slight differences (I = 0.949; chi(2), P = 0.147; G(2), P = 0.046), while cultured and natural stocks of 2005 were very different (I = 0.798, P = 0.006). Between the years of 2001 and 2005, a large change in genetic composition was observed (I = 0.582; P < 0.001). Although nothing is known about natural changes in the genetic composition of this species with time, the results suggest a strong impact of human activities on natural stocks of P. perna, which is expected to be related to heavy extraction and farming.     

Leber congenital amaurosis and retinitis pigmentosa with Coats-like exudative vasculopathy are associated with mutations in the crumbs homologue 1 (CRB1) gene

Mutations in the crumbs homologue 1 (CRB1) gene cause a specific form of retinitis pigmentosa (RP) that is designated "RP12" and is characterized by a preserved para-arteriolar retinal pigment epithelium (PPRPE) and by severe loss of vision at age <20 years. Because of the early onset of disease in patients who have RP with PPRPE, we considered CRB1 to be a good candidate gene for Leber congenital amaurosis (LCA). Mutations were detected in 7 (13%) of 52 patients with LCA from the Netherlands, Germany, and the United States. In addition, CRB1 mutations were detected in five of nine patients who had RP with Coats-like exudative vasculopathy, a relatively rare complication of RP that may progress to partial or total retinal detachment. Given that four of five patients had developed the complication in one eye and that not all siblings with RP have the complication, CRB1 mutations should be considered an important risk factor for the Coats-like reaction, although its development may require additional genetic or environmental factors. Although no clear-cut genotype-phenotype correlation could be established, patients with LCA, which is the most severe retinal dystrophy, carry null alleles more frequently than do patients with RP. Our findings suggest that CRB1 mutations are a frequent cause of LCA and are strongly associated with the development of Coats-like exudative vasculopathy in patients with RP.     

Electric and magnetic fields of the closed depolarization front in the anisotropic myocardium

On the basis of the bidomain model of anisotropic myocardium, mathematical relationships for calculating the characteristics of the extracellular electric and magnetic fields outside a closed or open depolarization front were derived on the assumption that the generator and fields are axially symmetric with respect to the longitudinal direction of the fibres making up the excitable tissue. The spatial configuration of the fields was determined and quantitative estimates were obtained for the electric potential and magnetic induction caused solely by the macroscopic anisotropy of the myocardium tissue as a volume conductor.     

Rhythmical sporogenesis in Streptomyces colonies. A one-dimensional model

A one-dimensional model imitating the growth and zone formation of a colony of the radiant fungus streptomycete on a synthetic agar nutrient medium with pyruvate, glucose, or succinate as a limiting source of energy and carbon was considered. The key parameters were determined whose values depend on the composition of nutrient medium and environmental conditions.     

Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival

Matrix Gla protein (MGP) is a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. Studies of MGP-deficient mice suggest that MGP is an inhibitor of extracellular matrix calcification in arteries and the epiphyseal growth plate. In the mammalian growth plate, MGP is expressed by proliferative and late hypertrophic chondrocytes, but not by the intervening chondrocytes. To investigate the functional significance of this biphasic expression pattern, we used the ATDC5 mouse chondrogenic cell line. We found that after induction of the cell line with insulin, the differentiating chondrocytes express MGP in a stage-specific biphasic manner as in vivo. Treatment of the ATDC5 cultures with MGP antiserum during the proliferative phase leads to their apoptosis before maturation, whereas treatment during the hypertrophic phase has no effect on chondrocyte viability or mineralization. After stable transfection of ATDC5 cells with inducible sense or antisense MGP cDNA constructs, we found that overexpression of MGP in maturing chondrocytes and underexpression of MGP in proliferative and hypertrophic chondrocytes induced apoptosis. However, overexpression of MGP during the hypertrophic phase has no effect on chondrocyte viability, but it does reduce mineralization. This work suggests that coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization.