Elucidation of the nature and function of the granular oyster amebocytes through histochemical studies of normal and traumatized oyster tissues

Summary A remarkable humoral component of the oyster inflammatory response was elucidated by employing the tools of the determinative histochemist. The humoral component, characterized by the release of copper and a diazotized p-nitroaniline-positive material from an acidophilic granular amebocyte, was associated with the oyster inflammatory reaction. Grossly, this humoral response was associated with the appearance of an avocado or pea green coloration in the traumatized area. A second amebocytic cell type, termed the basophilic granular amebocyte, was observed swelling in traumatized areas and may have released an additional humoral component into injured regions. Copper released in response to trauma was bound to the cells in and around the wound site and appeared to be most avidly bound by the granules of the basophilic granular amebocytes. Once incorporated into the granular matrix of these amebocytes, copper appeared to stabilize and prevent the granule from swelling.A portion of this work was excerpted from a Ph.D. thesis submitted to the Graduate School, University of Washington, Seattle.This work was supported by Public Health Service Contract No. 5 to 1 ES 00038-02. The costs of publication were defrayed in part by HSAA Award No. RR 06138 and Tumor Biology Training Grant, NIH CA 05245.     

Inhibition of mutation and combating the evolution of antibiotic resistance

The emergence of drug-resistant bacteria poses a serious threat to human health. In the case of several antibiotics, including those of the quinolone and rifamycin classes, bacteria rapidly acquire resistance through mutation of chromosomal genes during therapy. In this work, we show that preventing induction of the SOS response by interfering with the activity of the protease LexA renders pathogenic Escherichia coli unable to evolve resistance in vivo to ciprofloxacin or rifampicin, important quinolone and rifamycin antibiotics. We show in vitro that LexA cleavage is induced during RecBC-mediated repair of ciprofloxacin-mediated DNA damage and that this results in the derepression of the SOS-regulated polymerases Pol II, Pol IV and Pol V, which collaborate to induce resistance-conferring mutations. Our findings indicate that the inhibition of mutation could serve as a novel therapeutic strategy to combat the evolution of antibiotic resistance.     

Rudimentary pedal grasping in mice and implications for terminal branch arboreal quadrupedalism

We use an outbred laboratory mouse strain (ICR/CD‐1, Charles River Laboratories, Inc.) to model a type of preprimate locomotion associated with rudimentary pedal grasping. Ten male mice were assigned to either control or climbing groups (n = 5 per group). Climbing mice lived within a specialized terrarium that included ～7.5 m of thin branches (5 and 10 cm long) with a thickness of 3.3mm, arranged in a reticulated canopy. Food, water, and a nest site were placed among the branches. To discourage mice from palmigrade or digitigrade locomotion, the floor of the terrarium was flooded with a few centimeters of water. Climbing mice were placed in this setting upon weaning and reared for 3 months until they were mature in size. Litter, and age‐matched controls were also maintained for comparison with climbers. Climbing mice quickly acclimated to the requirements of the fine‐branch model using the foot and tail for grasping and balance. At maturity, climbing and control mice exhibited minor, but significant, morphological plasticity. For climbers, this includes a greater angle of the femoral neck, larger patellar groove index, relatively shorter talar neck length, and more circular talar head aspect ratio (P < 0.10). Climbers also exhibit increased curvature of the distal third metacarpal, decreased talar head angle, and relatively longer caudal vertebrae transverse processes (P < 0.05). These results in a small‐bodied eutherian mammal suggest that facultative hallucial opposability and coordinated tail use enable a kind of grasping active arboreal quadrupedality relevant to the latest stages of pre‐euarchontan evolution. In light of these data, we hypothesize that a unique advantage of mouse‐sized mammals is that they exhibit a highly flexible body plan allowing them to engage in a diverse array of anatomical positions without requiring specific limb morphologies. J. Morphol.,2011. © 2010 Wiley‐Liss, Inc.     

Interaction between the nucleotide exchange factor Mge1 and the mitochondrial Hsp70 Ssc1

Function of Hsp70s such as DnaK of the Escherichia coli cytoplasm and Ssc1 of the mitochondrial matrix of Saccharomyces cerevisiae requires the nucleotide release factors, GrpE and Mge1, respectively. A loop, which protrudes from domain IA of the DnaK ATPase domain, is one of six sites of interaction revealed in the GrpE:DnaK co-crystal structure and has been implicated as a functionally important site in both DnaK and Ssc1. Alanine substitutions for the amino acids (Lys-108 and Arg-213 of Mge1) predicted to interact with the Hsp70 loop were analyzed. Mge1 having both substitutions was able to support growth in the absence of the essential wild-type protein. K108A/R213A Mge1 was able to stimulate nucleotide release from Ssc1 and function in refolding of denatured luciferase, albeit higher concentrations of mutant protein than wild-type protein were required. In vitro and in vivo assays using K108A/R213A Mge1 and Ssc1 indicated that the disruption of contact at this site destabilized the interaction between the two proteins. We propose that the direct interaction between the loop of Ssc1 and Mge1 is not required to effect nucleotide release but plays a role in stabilization of the Mge1-Ssc1 interaction. The robust growth of the K108A/R213A MGE1 mutant suggests that the interaction between Mge1 and Ssc1 is tighter than required for function in vivo.     

A Novel Method for Coral Explant Culture and Micropropagation

We describe here a method for the micropropagation of coral that creates progeny from tissue explants derived from a single polyp or colonial corals. Coral tissue explants of various sizes (0.5?C2.5?mm in diameter) were manually microdissected from the solitary coral Fungia granulosa. Explants could be maintained in an undeveloped state or induced to develop into polyps by manipulating environmental parameters such as light and temperature regimes, as well as substrate type. Fully developed polyps were able to be maintained for a long-term in a closed sea water system. Further, we demonstrate that mature explants are also amenable to this technique with the micropropagation of second-generation explants and their development into mature polyps. We thereby experimentally have established coral clonal lines that maintain their ability to differentiate without the need for chemical induction or genetic manipulation. The versatility of this method is also demonstrated through its application to two other coral species, the colonial corals Oculina patigonica and Favia favus.     

Quelling the red menace: haem capture by bacteria

Haem is an important bacterial nutrient. As a prosthetic group of several proteins, haem functions as a cofactor mediating oxygen transport, energy generation, and mixed-function oxidation. In addition, the iron chelated in the porphyrin ring may serve as an iron substrate for growth. However, because of its propensity for oxidizing cellular constituents, haem is always associated with proteins. Therefore, the uptake and transit of haem across bacterial membranes requires the participation of protein escorts. Bacteria have evolved a diverse array of surface-exposed receptors dedicated to binding haem and haem-proteins. Following this selective recognition at the bacterial cell surface, haem is transported across the outer membrane via a TonB-dependent process. The control of receptor expression appears to be multifactorial, probably involving a number of global regulators. A model integrating this information is presented.     

LuTHy: a double‐readout bioluminescence‐based two‐hybrid technology for quantitative mapping of protein–protein interactions in mammalian cells

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ?L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.     

Histocompatibility-2 system in wild mice

Six semicongenic lines carrying differentt haplotypes on the background of strain C57BL/10Sn (B10.t strains) and a (B10 ×T/t ⁰) F₁ hybrid were tested against one another in the mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity (CML) assays. In every instance, the MLR results paralleled those of the CML typing: strain combinations giving a positive result in one assay gave a positive result in the second; combinations in which no response was observed in the MLR assay also failed to kill target cells specifically in the CML assay. Furthermore, the MLR and CML results were concordant with the results of the serological typing of these strains, as reported previously by us. The combined results suggest sharing ofH-2 hyplotypes between B10.t¹² and B10.t³², between B10.t⁶ and B10.t^w1, and between B10.t^w2 and (B10. ×T/t ⁰) F₁. These data support the conclusion, reached in our previous publication, that members of the samet-complementation group, with few exceptions, shareH-2 haplotypes.