Chromosome preparations from hepatocytes and bone marrow of Chinese hamsters: A direct method

This report describes a method for the direct preparation of chromosomes from the hepatocytes and bone marrow of the same Chinese hamster (Cricetulus griseus). The technique is a modification of that described by Becker et al. (J. natl. Ca. Inst. 46: 1261–69, 1971) for rat hepatocytes, with the following significant differences: (1) a less extensive partial hepatectomy is employed to initiate hepatocyte regeneration, (2) the use of a larger initial dose of colchicine (4 mg/K) 46–48 hours after hepatectomy instead of 1 mg/K 24 hours after hepatectomy, (3) the use of 0.075 M KCl as hypotonic solution instead of fetal calf serum diluted 1 : 7 with distilled water and (4) flame or blaze drying of chromosome preparations instead of air drying. The combination of the above modifications gave abundant, clear and well-spread chromosomes.     

Balancing Selection for Pathogen Resistance Reveals an Intercontinental Signature of Red Queen Coevolution

The link between long-term host–parasite coevolution and genetic diversity is key to understanding genetic epidemiology and the evolution of resistance. The model of Red Queen host–parasite coevolution posits that high genetic diversity is maintained when rare host resistance variants have a selective advantage, which is believed to be the mechanistic basis for the extraordinarily high levels of diversity at disease-related genes such as the major histocompatibility complex in jawed vertebrates and R-genes in plants. The parasites that drive long-term coevolution are, however, often elusive. Here we present evidence for long-term balancing selection at the phenotypic (variation in resistance) and genomic (resistance locus) level in a particular host–parasite system: the planktonic crustacean Daphnia magna and the bacterium Pasteuria ramosa. The host shows widespread polymorphisms for pathogen resistance regardless of geographic distance, even though there is a clear genome-wide pattern of isolation by distance at other sites. In the genomic region of a previously identified resistance supergene, we observed consistent molecular signals of balancing selection, including higher genetic diversity, older coalescence times, and lower differentiation between populations, which set this region apart from the rest of the genome. We propose that specific long-term coevolution by negative-frequency-dependent selection drives this elevated diversity at the host''s resistance loci on an intercontinental scale and provide an example of a direct link between the host’s resistance to a virulent pathogen and the large-scale diversity of its underlying genes.     

Effects of disrupted beta1-integrin function on the skeletal response to short-term hindlimb unloading in mice.

The study was designed to determine whether beta1-integrin plays a role in mediating the acute skeletal response to mechanical unloading. Transgenic (TG) mice were generated to express a dominant negative form of beta1-integrin under the control of the osteocalcin promoter, which targets expression of the transgene to mature osteoblasts. At 63 days of age, wild-type (WT) and TG mice were subjected to hindlimb unloading by tail suspension for 1 wk. Pair-fed, normally loaded WT and TG mice served as age-matched controls. Bone samples from each mouse were processed for quantitative bone histomorphometry and biomechanical testing. The skeletal phenotype of TG mice was characterized by lower cancellous bone mass in the distal femoral metaphysis (-52%) and lumbar vertebral body (-20%), reduced curvature of the proximal tibia (-20%), and decreased bone strength (-20%) and stiffness (-23%) of the femoral diaphysis with relatively normal indexes of cancellous bone turnover. Hindlimb unloading for only 1 wk induced a 10% decline in tibial curvature and a 30% loss of cancellous bone in the distal femur due to a combination of increased bone resorption and decreased bone formation in both WT and TG mice. However, the strength and stiffness of the femoral diaphysis were unaffected by short-term hindlimb unloading in both genotypes. The observed increase in osteoclast surface was greater in unloaded TG mice (92%) than in unloaded WT mice (52%). Cancellous bone formation rate was decreased in unloaded WT (-29%) and TG (-15%) mice, but, in contrast to osteoclast surface, the genotype by loading interaction was not statistically significant. The results indicate that altered integrin function in mature osteoblasts may enhance the osteoclastic response to mechanical unloading but that it does not have a major effect on the development of cancellous osteopenia in mice during the early stages of hindlimb unloading.     

Femtosecond laser nanoaxotomy lab-on-a-chip for in vivo nerve regeneration studies

A thorough understanding of nerve regeneration in Caenorhabditis elegans requires performing femtosecond laser nanoaxotomy while minimally affecting the worm. We present a microfluidic device that fulfills such criteria and can easily be automated to enable high-throughput genetic and pharmacological screenings. Using the 'nanoaxotomy' chip, we discovered that axonal regeneration occurs much faster than previously described, and notably, the distal fragment of the severed axon regrows in the absence of anesthetics.     

Characterization of AtlL, a bifunctional autolysin of Staphylococcus lugdunensis with N-acetylglucosaminidase and N-acetylmuramoyl-l-alanine amidase activities

The nucleotide sequence of atlL , a gene encoding a putative Staphylococcus lugdunensis peptidoglycan hydrolase, was determined using degenerate consensus PCR and genome walking. This 3837-bp gene encodes a protein, AtlL, that appears as a putative bifunctional autolysin with a 29-amino acid putative signal peptide and two enzymatic putative centres ( N -acetylmuramoyl- l -alanine amidase and N -acetylglucosaminidase) interconnected with three imperfect repeated sequences displaying glycine–tryptophan motifs. In order to determine whether both lytic domains were functional, and verify their exact enzymatic activities, gene fragments harbouring both putative domains, AM ( N -acetylmuramoyl- l -alanine amidase enzymatic centre plus two repeated sequences) and GL ( N -acetylglucosaminidase enzymatic centre plus one repeated sequence), were isolated, subcloned, and expressed in Escherichia coli . Purified recombinant AM and GL protein truncations exhibited cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis , and S. lugdunensis. AtlL is expressed during the whole growth, with an overexpression in the early-exponential stage. Liquid chromatography-mass spectrometry analysis of muropeptides generated by digestion of B. subtilis cell walls demonstrated the hydrolytic bond specificities and confirmed both of the acetyl domains' activities as predicted by sequence homology data. AtlL is the first autolysin described in S. lugdunensis , with a bifunctional enzymatic activity involved in peptidoglycan hydrolysis.     

Monoclonal antibody to dengue capsid protein: Its application in dengue studies

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.Key words: monoclonal antibodies, capsid protein, dengue virus, diagnosis, immunoassays