Hyperproliferation and p53 status of lens epithelial cells derived from alphaB-crystallin knockout mice

alphaB-Crystallin, a major protein of lens fiber cells, is a stress-induced chaperone expressed at low levels in the lens epithelium and numerous other tissues, and its expression is enhanced in certain pathological conditions. However, the function of alphaB in these tissues is not known. Lenses of alphaB-/- mice develop degeneration of specific skeletal muscles but do not develop cataracts. Recent work in our laboratory indicates that primary cultures of alphaB-/- lens epithelial cells demonstrate genomic instability and undergo hyperproliferation at a frequency 4 orders of magnitude greater than that predicted by spontaneous immortalization of rodent cells. We now demonstrate that the hyperproliferative alphaB-/- lens epithelial cells undergo phenotypic changes that include the appearance of the p53 protein as shown by immunoblot analysis. Sequence analysis showed a lack of mutations in the p53 coding region of hyperproliferative alphaB-/- cells. However, the reentry of hyperproliferative alphaB-/- cells into S phase and mitosis after DNA damage by gamma-irradiation were consistent with impaired p53 checkpoint function in these cells. The results demonstrate that expression of functionally impaired p53 is one of the factors that promote immortalization of lens epithelial cells derived from alphaB-/- mice. Fluorescence in situ hybridization using probes prepared from centromere-specific mouse P1 clones of chromosomes 1 and 9 demonstrated that the hyperproliferative alphaB-/- cells were 30% diploid and 70% tetraploid, whereas wild type cells were 83% diploid. Further evidence of genomic instability was obtained when the hyperproliferative alphaB-/- cells were labeled with anti-beta-tubulin antibodies. Examination of the hyperproliferative alphaB-/- mitotic profiles revealed the presence of cells that failed to round up for mitosis, or arrested in cytokinesis, and binucleated cells in which nuclear division had occurred without cell division. These results suggest that the stress protein and molecular chaperone alphaB-crystallin protects cells from acquiring impaired p53 protein and genomic instability.     

Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons

Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinson's disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinson's disease, we generated a mouse model bearing a germline disruption in parkin. Parkin-/- mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin-/- mice. Intracellular recordings of medium-sized striatal spiny neurons showed that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin-/- mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin-/- mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinson's disease. Steady-state levels of CDCrel-1, synphilin-1, and alpha-synuclein, which were identified previously as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin-/- brains. Together these findings provide the first evidence for a novel role of parkin in dopamine regulation and nigrostriatal function, and a non-essential role of parkin in the survival of nigral neurons in mice.     

Identification of a tight junction-associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability

Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.     

Characterization of six Leuconostoc fallax bacteriophages isolated from an industrial sauerkraut fermentation

Six bacteriophages active against Leuconostoc fallax strains were isolated from industrial sauerkraut fermentation brines. These phages were characterized as to host range, morphology, structural proteins, and genome fingerprint. They were exclusively lytic against the species L. fallax and had different host ranges among the strains of this species tested. Morphologically, three of the phages were assigned to the family Siphoviridae, and the three others were assigned to the family Myovidae: Major capsid proteins detected by electrophoresis were distinct for each of the two morphotypes. Restriction fragment length polymorphism analysis and randomly amplified polymorphic DNA fingerprinting showed that all six phages were genetically distinct. These results revealed for the first time the existence of bacteriophages that are active against L. fallax and confirmed the presence and diversity of bacteriophages in a sauerkraut fermentation. Since a variety of L. fallax strains have been shown to be present in sauerkraut fermentation, bacteriophages active against L. fallax are likely to contribute to the microbial ecology of sauerkraut fermentation and could be responsible for some of the variability observed in this type of fermentation.     

Allozyme diversity and genetic structure of the leafy cactus (Pereskia guamacho [Cactaceae])

We examined levels of genetic variation and genetic structure in the leafy cactus (Pereskia guamacho) in arid and semiarid zones in Venezuela. We surveyed genetic diversity within 17 populations using 19 allozyme loci. Genetic diversity was relatively high at both the species (P(s) = 89%, A(s) = 3.26, AP(s) = 3.53, H(es) = 0.24) and population (P(p) = 63%, A(p) = 1.90, AP(p) = 2.42, H(ep) = 0.20) levels. A significant deficit of heterozygote individuals was detected within populations in the Paraguana Peninsula region (F(IS) = 0.301). Relatively low levels of population differentiation were detected at macrogeographic (G(ST) = 0.112) and regional levels (G(ST) = 0.044 for peninsula region and G(ST) = 0.074 for mainland region), suggesting substantial genetic exchange among populations; however, gene flow in this species seems to be regulated by the distance among populations. Overall, estimates of genetic diversity found in P. guamacho are concordant with the pattern observed for other cacti surveyed, namely high levels of polymorphism and genetic diversity with one common allele and several rare alleles per locus. Differences in gene dispersal systems between this species and other cacti studied were not reflected in the patterns of genetic diversity observed. The concentration of the highest estimates of genetic variation in northwestern Venezuela suggests a potential reservoir of plant genetic diversity within xerophilous ecosystems in northern South America.