Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.     

Variety matters: adaptive genetic diversity and parasite load in two mouse opossums from the Brazilian Atlantic forest

The adaptive potential of a species to a changing environment and in disease defence is primarily based on genetic variation. Immune genes, such as genes of the major histocompatibility complex (MHC), may thereby be of particular importance. In marsupials, however, there is very little knowledge about natural levels and functional importance of MHC polymorphism, despite their key role in the mammalian evolution. In a previous study, we discovered remarkable differences in the MHC class II diversity between two species of mouse opossums (Gracilinanus microtarsus, Marmosops incanus) from the Brazilian Atlantic forest, which is one of the most endangered hotspots for biodiversity conservation. Since the main forces in generating MHC diversity are assumed to be pathogens, we investigated in this study gastrointestinal parasite burden and functional associations between the individual MHC constitution and parasite load. We tested two contrasting scenarios, which might explain differences in MHC diversity between species. We predicted that a species with low MHC diversity would either be under relaxed selection pressure by low parasite diversity (‘Evolutionary equilibrium’ scenario), or there was a recent loss in MHC diversity leading to a lack of resistance alleles and increased parasite burden (‘Unbalanced situation’ scenario). In both species it became apparent that the MHC class II is functionally important in defence against gastrointestinal helminths, which was shown here for the first time in marsupials. On the population level, parasite diversity did not markedly differ between the two host species. However, we did observe considerable differences in the individual parasite load (parasite prevalence and infection intensity): while M. incanus revealed low MHC DAB diversity and high parasite load, G. microtarsus showed a tenfold higher population wide MHC DAB diversity and lower parasite burden. These results support the second scenario of an unbalanced situation.     

Characterization and multiplexing of EST-SSR primers in Cynodon (Poaceae) species1

? Premise of the study: Cynodon species are multiple-use grasses that display varying levels of adaptation to biotic and abiotic stress. Previously identified EST-SSR primers were characterized and multiplexed to assess the level of genetic diversity present within a collection of almost 1200 Cynodon accessions from across Australia. ? Methods and Results: Two multiplex reactions were developed comprising a total of 16 EST-SSR markers. All SSR markers amplified across different Cynodon species and different levels of ploidy. The number of alleles ranged from one to eight per locus and the total number of alleles for the germplasm collection was 79. ? Conclusions: The 16 markers show sufficient variation for the characterization of Cynodon core collections and analysis of population genetic diversity in Cynodon grasses.     

(Not) Keeping the stem straight: a proteomic analysis of maritime pine seedlings undergoing phototropism and gravitropism

Background  

Plants are subjected to continuous stimuli from the environment and have evolved an ability to respond through various growth and development processes. Phototropism and gravitropism responses enable the plant to reorient with regard to light and gravity.     

The incC korB region of RK2 repositions a mini-RK2 replicon in Escherichia coli

Analysis by fluorescence microscopy has established that plasmid RK2 in Escherichia coli and other gram-negative bacteria is present as discrete clusters that are located inside the nucleoid at the mid- or quarter-cell positions. A mini-RK2 replicon containing an array of tetO repeats was visualized in E. coli cells that express a TetR-EYFP fusion protein. Unlike intact RK2, the RK2 mini-replicon (pCV1) was localized as a cluster at the cell poles outside of the nucleoid. Insertion of the O(B1)incC korB partitioning (par) region of RK2 into pCV1 resulted in a shift of the mini-replicon to within the nucleoid region at the mid- and quarter-cell positions. Despite the repositioning of the mini-RK2 replicon to the cellular positions where intact RK2 is normally located, the insertion of the intact O(B1) incC korB region did not significantly stabilize the mini-RK2 plasmid during cell growth. Deletions within the O(B1)incC or the korB region resulted in a failure of this par region to move pCV1 out of its polar position. The insertion of the par system of plasmid F into pCV1 resulted in a similar shift in the location of pCV1 to the nucleoid region. Unlike O(B1)incC korB, the insertion of the RK2 parABC resolvase system into pCV1 did not affect the polar positioning of pCV1. This effect of O(B1)incC korB on the location of pCV1 provides additional evidence for a partitioning role of this region of plasmid RK2. However, the failure of this region to significantly increase the stability of the mini-RK2 plasmid indicates that the localization of the plasmid to the mid- and quarter cell positions in E. coli is not in itself sufficient for the stable maintenance of plasmid RK2.     

Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia

Globozoospermia is a rare (incidence <0.1% in male infertile patients) form of teratozoospermia, mainly characterized by round-headed spermatozoa that lack an acrosome. It originates from a disturbed spermiogenesis, which is expected to be induced by a genetic factor. Several family cases and recessive mouse models with the same phenotype support this expectation. In this study, we present a consanguineous family with three affected brothers, in whom we have identified a homozygous mutation in the spermatogenesis-specific gene SPATA16. This is the first example of a nonsyndromic male infertility condition in humans caused by an autosomal gene defect, and it could also mean that the identification of other partners like SPATA16 could elucidate acrosome formation.     

Kinetic evidence is consistent with the rocker-switch mechanism of membrane transport by GlpT

Secondary active transport of substrate across the cell membrane is crucial to many cellular and physiological processes. The crystal structure of one member of the secondary active transporter family, the sn-glycerol-3-phosphate (G3P) transporter (GlpT) of the inner membrane of Escherichia coli, suggests a mechanism for substrate translocation across the membrane that involves a rocker-switch-type movement of the protein. This rocker-switch mechanism makes two specific predictions with respect to kinetic behavior: the transport rate increases with the temperature, whereas the binding affinity of the transporter to a substrate is temperature-independent. In this work, we directly tested these two predictions by transport kinetics and substrate-binding experiments, integrating the data on this single system into a coherent set of observations. The transport kinetics of the physiologically relevant G3P-phosphate antiport reaction were characterized at different temperatures using both E. coli whole cells and GlpT reconstituted into proteoliposomes. Substrate-binding affinity of the transporter was measured using tryptophan fluorescence quenching in detergent solution. Indeed, the substrate transport velocity of GlpT increased dramatically with temperature. In contrast, neither the apparent Michaelis constant (Km) nor the apparent substrate-binding dissociation constant (Kd) showed temperature dependence. Moreover, GlpT-catalyzed G3P translocation exhibited a completely linear Arrhenius function with an activation energy of 35.2 kJ mol-1 for the transporter reconstituted into proteoliposomes, suggesting that the substrate-loaded transporter is delicately poised between the inward- and outward-facing conformations. When these results are taken together, they are in agreement with a rocker-switch mechanism for GlpT.