Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration

ClC-7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H(+)-ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC-7 or the H(+)-ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC-7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC-7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron-dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC-3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H(+)-ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H(+)-ATPase and ClC-7 can underlie human osteopetrosis.     

GMI1, a structural-maintenance-of-chromosomes-hinge domain-containing protein, is involved in somatic homologous recombination in Arabidopsis

DNA double-strand breaks (DSBs) pose one of the most severe threats to genome integrity, potentially leading to cell death. After detection of a DSB, the DNA damage and repair response is initiated and the DSB is repaired by non-homologous end joining and/or homologous recombination. Many components of these processes are still unknown in Arabidopsis thaliana. In this work, we characterized γ-irradiation and mitomycin C induced 1 (GMI1), a member of the SMC-hinge domain-containing protein family. RT-PCR analysis and promoter-GUS fusion studies showed that γ-irradiation, the radio-mimetic drug bleocin, and the DNA cross-linking agent mitomycin C strongly enhance GMI1 expression particularly in meristematic tissues. The induction of GMI1 by γ-irradiation depends on the signalling kinase Ataxia telangiectasia-mutated (ATM) but not on ATM and Rad3-related (ATR). Epistasis analysis of single and double mutants demonstrated that ATM acts upstream of GMI1 while the atr gmi1-2 double mutant was more sensitive than the respective single mutants. Comet assay revealed a reduced rate of DNA double-strand break repair in gmi1 mutants during the early recovery phase after exposure to bleocin. Moreover, the rate of homologous recombination of a reporter construct was strongly reduced in gmi1 mutant plants upon exposure to bleocin or mitomycin C. GMI1 is the first member of its protein family known to be involved in DNA repair.     

Effect of mechanical stimulation on cell proliferation in mouse epidermis and on growth regulation by endogenous factors (chalones).

Mechanical stimulation of dorsal mouse skin by skin massage or removal of the horny layer results in a mutually comparable increase in DNA-labelling and mitotic activity. However, only after injury such as removal of the horny layer hyperplasia develops. This phenomenon, called "hyperplastic transformation" is characterized by a transient abolition of the epidermal G1 chalone responsiveness. There is some indication that the susceptibility to a heat labile factor, probably the epidermal G2 chalone, is not affected. Skin massage neither interferes with the responsiveness to epidermal G1 chalone nor induces "hyperplastic transformation". Mouse tail epidermis shows a "functional hyperplasia" and responds to the G1 chalone. To explain these observations, it is assumed that the epidermal stem cell population is heterogeneous consisting of G1 chalone-sensitive and G1 chalone-insensitive cells.     

The catalog of human hair keratins. II. Expression of the six type II members in the hair follicle and the combined catalog of human type I and II keratins.

The human type II hair keratin subfamily consists of six individual members and can be divided into two groups. The group A members hHb1, hHb3, and hHb6 are structurally related, whereas group C members hHb2, hHb4, and hHb5 are rather distinct. Specific antisera against the individual hair keratins were used to establish the two-dimensional catalog of human type II hair keratins. In this catalog, hHb5 showed up as a series of isoelectric variants, well separated from a lower, more acidic, and complex protein streak containing isoelectric variants of hair keratins hHb1, hHb2, hHb3, and hHb6. Both in situ hybridization and immunohistochemistry on anagen hair follicles showed that hHb5 and hHb2 defined early stages of hair differentiation in the matrix (hHb5) and cuticle (hHb5 and hHb2), respectively. Although cuticular differentiation proceeded without the expression of further type II hair keratins, cortex cells simultaneously expressed hHb1, hHb3, and hHb6 at an advanced stage of differentiation. In contrast, hHb4, which is undetectable in hair follicle extracts and sections, could be identified as the largest and most alkaline member of this subfamily in cytoskeletal extracts of dorsal tongue. This hair keratin was localized in the posterior compartment of the tongue filiform papillae. Comparative analysis of type II with the previously published type I hair keratin expression profiles suggested specific, but more likely, random keratin-pairing principles during trichocyte differentiation. Finally, by combining the previously published type I hair keratin catalog with the type II hair keratin catalog and integrating both into the existing catalog of human epithelial keratins, we present a two-dimensional compilation of the presently known human keratins.     

Bacterial genetics: past achievements, present state of the field, and future challenges

Genetic tools are required to take full advantage of the wealth of information generated by genome sequencing efforts and ensuing global gene and protein expression analyses. Bacterial genetics was originally developed and refined in Escherichia coli. As a consequence, elegant plasmid, cloning, expression, and mutagenesis systems were developed over the years and a good number of them are commercially available. This is not true for other bacteria. Although the development of genetic tools has generally not kept up with the sequencing pace, substantial progress has been made in this arena with many bacterial species. This short review highlights selected topics and achievements in the field over the past 25 years and presents some strategies that may help address future challenges. BioTechniques has played an integral part in the publication of important technological advances in the field over the first 25 years of its existence and it can be anticipated that it will continue to do so in the future.     

New insights into the physiological actions of selenoproteins from genetically modified mice

Selenium (Se) is an essential trace element in mammals. Dietary Se restriction or conditions of Se malabsorption lead to deficiency syndromes or exacerbate established diseases in humans and in many animal models. It is assumed that most, if not all, physiological actions of Se are mediated by selenocysteine (Sec) containing proteins. However, the exact role of particular selenoproteins for certain molecular pathways, for the metabolism of nutrients, hormones or cellular components and for the development and adaptive responses of the organism have often remained elusive. Through the use of transgenic animals, it becomes increasingly feasible to interfere specifically with the expression of single selenoproteins in certain tissues or at certain times. While some transgenic animals exhibit phenotypes that were expected from biochemical studies, in other instances the observed effects were a surprise in view of earlier hypotheses.     

Augmentative releases of Metaphycus sp. nr flavus against citricola scale on oranges in the San Joaquin Valley of California: are early releases better than late ones?

We conducted an augmentative release trial with the encyrtid parasitoid Metaphycus sp. nr flavus (Howard) against citricola scale, Coccus pseudomagniolarum (Kuwana), in three citrus orchards in the San Joaquin Valley of California. Three parasitoid release timings (14 October-13 November, 4 January-5 February, and 26 February-16 March; hereinafter "early," "intermediate," and "late") were compared with a no-release check. Approximately 2,400 female parasitoids were released per central release tree. Late releases were associated with reduced scale survival on the release trees but no evidence of scale suppression was found on adjacent trees. Early and intermediate releases resulted in less pronounced scale suppression than late releases. Intermediate and late, but not early, releases were associated with an increase in the numbers of recovered female M. sp. nr flavus. Early releases, however, increased the numbers of recovered male Metaphycus spp. Thus, at the time of early releases, the scales may have been too small for the production of female parasitoids. A multiple regression model relating the degree of scale mortality in one orchard to the levels of recovered female Metaphycus helvolus (Compere), M. sp. nr flavus, or M. luteolus (Timberlake) (these latter two species are morphologically indistinguishable), and Coccophagus spp., suggested that the number of Coccophagus spp. females followed by M. helvolus females, were most strongly associated with scale mortality.     

The in vitro assembly of hair follicle keratins: comparison of cortex and companion layer keratins

The hair follicle consists of a complex system of multiple tissue compartments that are clearly distinguishable by their morphology and type of differentiation. We have synthesized hair follicle-specific keratins from the companion layer (K6hf, K17) and the hair cortex (Ha1, Hb3, Hb6) in Escherichia coli. The assembly of purified keratins in mixtures of K6hf/K17 and in mixtures of hair cortex keratins was compared in urea solutions, low ionic strength and physiological strength buffers, by urea melting gels, electron microscopy and analytical ultracentrifugation. Both types of keratin mixtures, keratins from the companion layer and keratins from the hair cortex, formed heterotypic complexes at 5 M urea. In low ionic strength buffers, the keratins from the companion layer were assembled to bona fide intermediate filaments. In contrast, mixtures of hair cortex keratins stayed in an oligomeric state with a mean s value of 9 as determined in sedimentation velocity experiments. Hair cortex keratins were, however, assembled into intermediate filaments at physiological salt conditions. A point mutated hair cortex keratin [Hb6(Glu402Lys)] formed no long filaments when mixed with Ha1; instead, the assembled structures showed a length distribution of 50.8 +/- 13.4 nm, comparable to the size distribution of assembly intermediates called 'unit-length' filaments.