Expression of Drosophila Ten-a,a dimeric receptor during embryonic development

Ten-a and Ten-m are the two Drosophila members of the newly discovered Ten-m family of dimeric type II transmembrane proteins. Here, we report complete cDNA cloning and protein expression patterns of Ten-a. The Ten-a protein, a dimeric receptor of about 500 kDa is mainly expressed on axons of the embryonic central nervous system and on muscle attachment sites.     

Outbreak of hind limb paralysis in young CFW Swiss Webster mice

An outbreak of paralysis among 16- to 20-week-old CFW Swiss Webster sentinel mice developed in one of our barrier facilities. Two months after arrival and over a period of four weeks, six of 400 mice purchased from an approved vendor, developed progressive hind limb paralysis without other clinical signs of disease. On the basis of the histopathologic changes and negative serologic test results, lymphoblastic lymphoma causing compression of the spinal cord was diagnosed. There were two leading features to this outbreak: its unusual epidemiologic presentation, and the localization of the lesions principally in the lumbar muscles. A presumptive diagnosis of retroviral infection with Abelson's murine leukemia virus (A-MuLV) was established on the basis of histopathologic and immunohistochemical findings. Little is known about retroviral status in many commercial colonies, and few users report presence of spontaneous lymphomas. This report points out complications derived from commercially available animals that carry endogenous retroviruses. It also emphasizes the need of diagnosing and reporting clusters of hind limb paralysis or lymphomas in mice to assess the prevalence and relevance of retroviral infections in commercial colonies.     

Functional genomics and sexual differentiation in amphibians

To succeed on land rather than in water, crabs require a suite of physiological and morphological changes, and ultimately the ability to reproduce without access open water. Some species have modified gills to assist in gas exchange but accessory gas exchange organs, usually lungs, occur in many species. In accomplished air-breathers the lung becomes larger and more vascularised with pulmonary vessels directing oxygenated haemolymph to the heart. The relative abundance of O₂ in air promotes relative hypoventilation and thus an internal hypercapnia to drive CO₂ excretion. Land crabs have a dual circulation via either lungs or gills and shunting between the two may depend on respiratory media or exercise state. During their breeding migration on Christmas Island Gecarcoidea natalis maintained arterial Po₂ by branchial O₂ uptake, while pulmonary O₂ pressure was reduced; partly because exercise doubled relative haemolymph flow through the gills. Related species rely on elevated haemocyanin concentration and affinity for O₂ to assist uptake but this compromises unloading at the tissues and thus the aerobic scope of tissues. Aquatic crabs exchange salt and ammonia with water via the gills but in land crabs this is not possible. Birgus latro has adopted uricotelism but other species excrete ammonia in either the urine or as gas. Land crabs minimise urinary salt loss using a filtration-reabsorption system analogous to the kidney. Urine is redirected across the gills where salt reabsorption occurs in systems under hormonal control, although in G. natalis this is stimulatory and in B. latro inhibitory. While crabs occupy a range of habitats from aquatic to terrestrial, these species do not comprise a physiological continuum but across the crab taxa individual species possess appropriate and specific physiological features to survive in their individual habitat.     

Dissection of the mechanism for the stringent factor RelA

During conditions of nutrient deprivation, ribosomes are blocked by uncharged tRNA at the A site. The stringent factor RelA binds to blocked ribosomes and catalyzes synthesis of (p)ppGpp, a secondary messenger that induces the stringent response. We demonstrate that binding of RelA and (p)ppGpp synthesis are inversely coupled, i.e., (p)ppGpp synthesis decreases the affinity of RelA for the ribosome. RelA binding to ribosomes is governed primarily by mRNA, but independently of ribosomal protein L11, while (p)ppGpp synthesis strictly requires uncharged tRNA at the A site and the presence of L11. A model is proposed whereby RelA hops between blocked ribosomes, providing an explanation for how low intracellular concentrations of RelA (1/200 ribosomes) can synthesize (p)ppGpp at levels that accurately reflect the starved ribosome population.     

Analysis of interactions between domains of a small heat shock protein,Hsp30 of Neurospora crassa

The alpha-crystallin-related, small heat shock proteins (sHsps), despite their overall variability in sequence, have discrete regions of conserved sequence that are involved in structural organization, as well as nonconserved regions that may perform similar roles in each protein. Recent X-ray diffraction analyses of an archeal and a plant sHsp have revealed both similarities and differences in how they are organized, suggesting that there is variability, particularly in the oligomeric organization of sHsps. As an adjunct to crystallographic analysis of sHsp structure, we employed the yeast 2-hybrid system to detect interactions between peptide regions of the sHsp of Neurospora crassa, Hsp30. We found that the conserved alpha-crystallin domain can be divided into N-terminal and C-terminal subdomains that interact strongly with one another. This interaction likely represents the tertiary contacts of the monomer that were visualized in the crystallographic structures of MjHsp16.5 and wheat Hsp16.9. The conserved sHsp monomeric fold is apparently determined by these regions of conserved sequence. We found that the C-terminal portion of the alpha-crystallin domain also interacts with itself in 2-hybrid assays; however, this interaction requires peptide extension into the semiconserved carboxyl tail. This C-terminal association may represent a principal contact site between dimers that contributes to higher-order assembly, as seen for the crystallized sHsps.     

Monoamine oxidase: radiotracer development and human studies

Monoamine oxidase (MAO) is an integral protein of outer mitochondrial membranes and occurs in neuronal and nonneuronal cells in the brain and in peripheral organs. It oxidizes amines from both endogenous and exogenous sources, thereby influencing the concentration of neurotransmitter amines as well as many xenobiotics. It occurs in two subtypes, MAO A and MAO B, which are different gene products and have different substrate and inhibitor specificities. Both MAO A and B can be imaged and quantified in the living human brain using positron emission tomography (PET) and radiotracers labeled with carbon-11. PET studies have been carried out to measure the effects of age, MAO inhibitor drugs, tobacco smoke exposure, and other factors on MAO activity in the human brain.     

The effect of ICAD-S on the formation and intracellular distribution of a nucleolytically active caspase-activated DNase

We show here that co-expression of murine CAD with either ICAD-L or ICAD-S in Escherichia coli as well as mammalian cells leads to a functional DFF complex, which after caspase-3 activation releases a nucleolytically active DNase. The chaperone activity of ICAD-S is between one and two orders of magnitude less effective than that of ICAD-L, as deduced from cleavage experiments with different activated recombinant DFF complexes produced in E.coli. With nucleolytically active EGFP fusion proteins of CAD it is demonstrated that co-expression of ICAD-S, which lacks the C-terminal domain of ICAD-L, including the NLS, leads to a homogeneous intracellular distribution of the DNase in transfected cells, whereas co-expression of human or murine ICAD-L variants lacking the NLS leads to exclusion of EGFP–CAD from the nuclei in ~50% of cells. These results attribute a particular importance of the NLS in the long isoform of the inhibitor of CAD for nuclear accumulation of the DFF complex in living cells. It is concluded that ICAD-L and ICAD-S in vivo might function as tissue-specific modulators in the regulation of apoptotic DNA degradation by controlling not only the enzymatic activity but also the amount of CAD available in the nuclei of mammalian cells.     

The C-terminal region of Escherichia coli UvrC contributes to the flexibility of the UvrABC nucleotide excision repair system

Nucleotide excision repair in Escherichia coli involves formation of the UvrB–DNA complex and subsequent DNA incisions on either site of the damage by UvrC. In this paper, we studied the incision of substrates with different damages in varying sequence contexts. We show that there is not always a correlation between the incision efficiency and the stability of the UvrB–DNA complex. Both stable and unstable UvrB–DNA complexes can be efficiently incised. However some lesions that give rise to stable UvrB–DNA complexes do result in a very low incision. We present evidence that this poor incision is due to sterical hindrance of the damage itself. In its C-terminal region UvrC contains two helix–hairpin–helix (HhH) motifs. Mutational analysis shows that these motifs constitute one functional unit, probably folded as one structural unit; the (HhH)₂ domain. This (HhH)₂ domain was previously shown to be important for the 5′ incision on a substrate containing a (cis-Pt)·GG adduct, but not for 3′ incision. Here we show that, mainly depending on the sequence context of the lesion, the (HhH)₂ domain can be important for 3′ and/or 5′ incision. We propose that the (HhH)₂ domain stabilises specific DNA structures required for the two incisions, thereby contributing to the flexibility of the UvrABC repair system.