Human lysosomal cathepsin G and granzyme B share a functionally conserved broad spectrum antibacterial peptide

Human neutrophil lysosomal cathepsin G (cat G) exerts broad-spectrum antibacterial action in vitro against Gram-negative and -positive bacteria independent of its serine protease activity. We recently determined that an internal peptide of cat G (HPQYNQR), obtained after digestion of cat G with clostripain, possessed broad-spectrum antibacterial action in vitro, displaying an ED50 of 5 x 10(-5) M. In order to evaluate the structure-antibacterial properties of this peptide, synthetic variants with single alanine substitutions at each position were prepared and tested for antibacterial action. We found that alanine substitution for His-1 or Tyr-4, or certain modifications of the His-1 side chain, produced nonbactericidal peptides. A hexapeptide lacking the COOH-terminal Arg-7 but not a pentapeptide lacking both Gln-6 and Arg-7 possessed in vitro bactericidal activity. Interestingly, the cat G bactericidal peptide displays similarity to sequences within other serine proteases, notably the proposed cytotoxic granzymes present in the cytolytic granules of human and mouse cytotoxic T lymphocytes. We now report that an internal peptide of one human granzyme (granzyme B) with the sequence of HPAYNPK also displays bactericidal action in vitro. Our results suggest that an internal antibacterial domain among human serine proteases cat G and granzyme B has been functionally conserved through evolution perhaps for the purpose of host defense against microbial pathogens and targets of cytotoxic T lymphocyte killing.     

Nuclear and cytoplasmic chloroplast mutants induced by chemical mutagens in Mimulus cardinalis: Genetics and ultrastructure

Summary The modes of inheritance of chemically induced chlorophyll-deficient phenotypes in Mimulus cardinalis reveal that the chloroplast is controlled by the genome and the plastome. Three of the chlorophyll-deficient mutants in M. cardinalis are inherited through nuclear recessive genes and two are inherited through plastome genes. One chlorophyll-deficient mutant was sterile and could not be analyzed genetically. Ultrastructural analysis of the six mutant types reveals that each possesses a unique defective chloroplast type(s) in comparison to the genotypically and phenotypically normal chloroplasts. Based on plastid ultrastructure it seems reasonable to assume that the mutations, genome and plastome, are non-allelic or at least significantly different forms of the same allele. The isolation of these types of mutants provide suitable material needed to study the effects of specific biochemical blocks and the elucidation of developmental pathways leading to chloroplast biogenesis. The mutants also provide valuable information concerning the interrelationship between the nucleic acid of the genome and the plastome.This study is in part based on a dissertation submitted by the first author in partial fulfillment of the requirements for a Doctor of Philosophy degree in the Department of Botany, Miami University, Oxford, Ohio.     

The cochlea of the enigmatic pygmy right whale Caperea marginata informs mysticete phylogeny

The pygmy right whale, Caperea marginata , is the least understood extant baleen whale (Cetacea, Mysticeti). Knowledge on its basic anatomy, ecology, and fossil record is limited, even though its singular position outside both balaenids (right whales) and balaenopteroids (rorquals + grey whales) gives Caperea a pivotal role in mysticete evolution. Recent investigations of the cetacean cochlea have provided new insights into sensory capabilities and phylogeny. Here, we extend this advance to Caperea by describing, for the first time, the inner ear of this enigmatic species. The cochlea is large and appears to be sensitive to low‐frequency sounds, but its hearing limit is relatively high. The presence of a well‐developed tympanal recess links Caperea with cetotheriids and balaenopteroids, rather than balaenids, contrary to the traditional morphological view of a close Caperea‐balaenid relationship. Nevertheless, a broader sample of the cetotheriid Herpetocetus demonstrates that the presence of a tympanal recess can be variable at the specific and possibly even the intraspecific level.     

Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo

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WIKI4, a Novel Inhibitor of Tankyrase and Wnt/?-Catenin Signaling

The Wnt/ß-catenin signaling pathway controls important cellular events during development and often contributes to disease when dysregulated. Using high throughput screening we have identified a new small molecule inhibitor of Wnt/ß-catenin signaling, WIKI4. WIKI4 inhibits expression of ß-catenin target genes and cellular responses to Wnt/ß-catenin signaling in cancer cell lines as well as in human embryonic stem cells. Furthermore, we demonstrate that WIKI4 mediates its effects on Wnt/ß-catenin signaling by inhibiting the enzymatic activity of TNKS2, a regulator of AXIN ubiquitylation and degradation. While TNKS has previously been shown to be the target of small molecule inhibitors of Wnt/ß-catenin signaling, WIKI4 is structurally distinct from previously identified TNKS inhibitors.     

Structures of the Bacillus subtilis Glutamine Synthetase Dodecamer Reveal Large Intersubunit Catalytic Conformational Changes Linked to a Unique Feedback Inhibition Mechanism

Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg⁶², from an adjacent subunit. Notably, Arg⁶² must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens.     

Effects of temperature and salinity on the life history of the sailfin molly (Pisces: Poeciliidae): lipid storage and reproductive allocation

While the life history traits of animals usually exhibit substantial phenotypic plasticity, such plasticity might reflect either a simple alteration in the level of energy accrual and use or a genuine shift in energy allocation tactics between environmental conditions. The latter would represent genuine plasticity in the life history itself, and thus it is important to distinguish which of these two processes underlies the observed plasticity of life history traits. We investigated this issue by examining the effects of temperature and salinity variation during ontogeny on the allocation of biomass and lipid storage in male sailfin mollies, Poecilia latipinna. We raised males from four natural populations from birth to maturity in controlled laboratory conditions. Neither distinct temperatures (23 or 29°C) nor different salinity regimes (2, 12, or 20 parts per thousand) affected body mass, although males from different populations differed substantially in body mass. However, males raised at the higher temperature had a greater allocation of biomass to testis and a lower allocation to viscera mass. The amount of stored lipid was altered by temperature variation but the direction and magnitude of the effect varied substantially among males from the different populations. Salinity variation affected neither biomass allocation nor the level of lipid storage. These results indicate that male mollies possess a flexible developmental program with respect to temperature that canalizes body size and alters the allocation of biomass among competing demands for reproductive readiness and capacity for energy storage. Received: 25 November 1996 / Accepted: 1 December 1997     

GxG epistasis in growth and condition and the maintenance of genetic polymorphism in Gambusia holbrooki

Theory on indirect genetic effects (IGEs) indicates that variation in the genetic composition of social groups can generate GxG epistasis that may promote the evolution of stable polymorphisms. Using a livebearing fish with a genetic polymorphism in coloration and associated behavioral differences, we tested whether genotypes of social partners interacted with focal individual genotypes to influence growth and condition over 16 weeks of development. We found that IGEs had a significant influence on patterns of feeding, regardless of focal fish genotype. There was no influence of social environment on juvenile length, but there was significant GxG epistasis for body condition. Each focal juvenile was in better condition when its own genotype was not present in adult social partners. These data are consistent with negative frequency‐dependent selection in which each morph performs better when it is rare. Neither variation in feeding nor activity‐related behaviors explained variation in body condition, suggesting that GxG epistasis for condition was caused by physiological differences between the two genotypes. These findings indicate that GxG epistasis in a given polymorphism can generate fitness landscapes that contribute to the maintenance of that polymorphism and to maintenance of genetic variation for additional fitness‐related traits.