Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration

To test the hypothesis that inhibition of axonal transport is sufficient to cause motor neuron degeneration such as that observed in amyotrophic lateral sclerosis (ALS), we engineered a targeted disruption of the dynein-dynactin complex in postnatal motor neurons of transgenic mice. Dynamitin overexpression was found to disassemble dynactin, a required activator of cytoplasmic dynein, resulting in an inhibition of retrograde axonal transport. Mice overexpressing dynamitin demonstrate a late-onset progressive motor neuron degenerative disease characterized by decreased strength and endurance, motor neuron degeneration and loss, and denervation of muscle. Previous transgenic mouse models of ALS have shown abnormalities in microtubule-based axonal transport. In this report, we describe a mouse model that confirms the critical role of disrupted axonal transport in the pathogenesis of motor neuron degenerative disease.     

Induction of olfactory mucosal and liver metabolism of lidocaine by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Formulation of drugs for administration via the nasal cavity is becoming increasingly common. It is of potential clinical relevance to determine whether intranasal drug administration itself, or exposure to other xenobiotics, can modulate the levels and/or activity of nasal mucosal metabolic enzymes, thereby affecting the metabolism and disposition of the drug. In these studies, we examined changes in several of the major metabolic enzymes in nasal epithelial tissues upon exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as well as the impact of these changes on the metabolism of a model intranasally administered drug, lidocaine. Results of these studies show that TCDD can induce multiple metabolic enzymes in the olfactory mucosa and that the pattern of induction in the olfactory mucosa does not necessarily parallel that which occurs in the liver. Further, increases in enzyme levels noted by Western blot analysis were associated with increased activities of several nasal mucosal enzymes as well as with enhanced conversion of lidocaine to its major metabolite, monoethyl glycine xylidide (MEGX). These results demonstrate that environmental exposures can influence the levels and activity of nasal mucosal enzymes and impact the pharmacology of drugs administered via the nasal route.     

Coexpression of oncostatin M and its receptors and evidence for STAT3 activation in human ovarian carcinomas

The expression of oncostatin M and leukemia inhibitory factor (LIF), JAK-STAT activators and members of the interleukin-6 family of cytokines, were examined in a series of primary ovarian carcinomas using immunohistochemistry. The malignant epithelial cells of all 29 ovarian carcinomas examined expressed oncostatin M; none expressed LIF. Oncostatin M can activate two related receptors, one consisting of a low-affinity LIF receptor subunit, LIFR beta, which forms a heterocomplex with the gp130 signal transducing protein and can recognize both oncostatin M and LIF, and a second heterocomplex consisting of a subunit that specifically recognizes oncostatin M, OSMR beta, and the gp130 protein. By immunohistochemistry, 25 of 25 ovarian carcinomas examined expressed the LIFR beta subunit in the malignant epithelial cells (all samples express gp130), and two-thirds the ovarian carcinomas studied expressed OSMR beta mRNA as determined by RT-PCR. Thus oncostatin M and its receptors are commonly coexpressed in malignant ovarian epithelial cells, and represent a potential autocrine loop in this tumor type. STAT3, of one the signaling proteins downstream of the oncostatin M/LIF receptors, was found in its phosphorylated, activated form (phosphotyrosine 705 STAT3) in the malignant epithelial cells of 17 of 23 ovarian carcinomas examined (74%) as determined by immunohistochemistry; this suggests that this protein is constitutively activated in most ovarian carcinomas, as it is in many other human malignancies. Recombinant human Oncostatin M (rhOSM) can induce the transient tyrosine 705 phosphorylation of STAT3 in serum-starved LIFR beta/OSMR beta expressing ovarian carcinoma cell lines, but does not alter cell growth and effects only a modest increase in the apoptotic rate in these cultured cells. Oncostatin M and its receptors may be part of a network of cytokine systems within ovarian carcinomas that may act to maintain STAT3 in its activated form, a phenomenon associated with the malignant phenotype.     

Molecular phylogeny and evolution of the extinct bovid Myotragus balearicus

Myotragus balearicus was a dwarf artiodactyl endemic to the Eastern Balearic Islands, where it evolved in isolation for more than 5 million years before becoming extinct between 3640 and 2135 cal BC (calibrated years BC). Numerous unusual apomorphies obscure the relationship between Myotragus and the extant Caprinae. Therefore, genetic data for this species would significantly contribute to the clarification of its taxonomic position. In this study, we amplify, sequence, and clone a 338-base pair (bp) segment of the mitochondrial cytochrome b (cyt b) gene from a >9Kyr Myotragus subfossil from la Cova des Gorgs (Mallorca). Our results confirm the phylogenetic affinity of Myotragus with the sheep (Ovis) and the takin (Budorcas). In each tree, the Myotragus branch is long in comparison with the other taxa, which may be evidence of a local change in the rate of evolution in cyt b. This rate change may be due to in part to an early age of first reproduction and short generation time in Myotragus, factors that are potentially related to the extreme reduction in size of the adult Myotragus as compared to the other Caprinae.     

Ecology,development and pathogenicity of Buddenbrockia plumatellae Schröder, 1910 (Myxozoa,Malacosporea) (syn. Tetracapsula bryozoides) and establishment of Tetracapsuloides n. gen. for Tetracapsula bryosalmonae

Buddenbrockia plumatellae, an enigmatic worm-like myxozoan, was observed as continuously writhing free and attached 'worms' and as free mature spores in the coelom of the freshwater bryozoans Plumatella fungosa, Hyalinella punctata, and Fredericella sp. 'Worm' numbers could double every three days. 'Worms' and spores could be expelled from colonies by external pressure. Some mature 'worms' exited actively, entraining release of free spores, and gradually ceased movement outside the host. Bryozoans sealed off infected regions of the colony. Infected colonies grew slowly, produced no statoblasts, and eventually regressed and died. Transmission was not achieved and prevalence was low. Electron microscopy of 'worms' revealed a single layer of mural cells on a fibrous basal lamina overlying four longitudinal muscle blocks and an inner sheet of two types of proliferating cells, an organization indicative of the bilaterian ancestry of the Myxozoa. Primary type A cells were attached directly by striated tubules to mural cells at positions between muscle blocks. Secondary type A cells had a secretory function. Type B cells underwent meiosis and subsequently developed to typical malacosporean myxozoan spores filling the internal cavity of the 'worms'. External tubes were formed during capsulogenesis in 'worms' from Fredericella sp. Tetracapsula bryozoides is synonymised with Buddenbrockia plumatellae and a new genus is proposed for Tetracapsula bryosalmonae.     

Metallohistins: a new class of plant metal-binding proteins

Two small multimeric histidine-rich proteins, AgNt84 and Ag164, encoded by two nodule-specific cDNAs isolated from nodule cDNA libraries of the actinorhizal host plant Alnus glutinosa, represent a new class of plant metal binding proteins. This paper reports the characterization of the purified in vitro-expressed proteins by size exclusion chromatography, circular dichroism, equilibrium dialysis, metal affinity chromatography coupled with mass spectrometry, and nuclear magnetic resonance spectroscopy. These analyses reveal that each polypeptide is capable of binding multiple atoms of Zn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Cd²⁺ and Hg²⁺. A reversible shift in histidine C1 and C2 protons in NMR analysis occurred during titration of this protein with ZnCl₂ strongly suggesting that histidine residues are responsible for metal binding. AgNt84 and Ag164 are not related to metal binding metallothioneins and phytochelatins and represent a new class of plant metal binding proteins that we propose to call metallohistins. Possible biological roles in symbioses for AgNt84 and Ag164, and their potential for use in bioremediation are discussed.     

Carboxylase overexpression effects full carboxylation but poor release and secretion of factor IX: implications for the release of vitamin K-dependent proteins

Vitamin K-dependent (VKD) proteins are modified by the VKD carboxylase as they transit through the endoplasmic reticulum. In a reaction required for their activity, clusters of Glu's are converted to Gla's, and fully carboxylated VKD proteins are normally secreted. In mammalian cell lines expressing high levels of r-VKD proteins, however, under- and uncarboxylated VKD forms are observed. Overexpression of r-carboxylase does not improve carboxylation, but the lack of effect is not understood, and the intracellular events that occur during VKD protein carboxylation have not been investigated. We analyzed carboxylation in 293- and BHK cell lines expressing r-factor IX (fIX) and endogenous carboxylase or overexpressed r-carboxylase. The fIX secreted from the four cell lines was highly carboxylated, indicating fIX-carboxylase engagement during intracellular trafficking. The r-carboxylase was functional for carboxylation: overexpression resulted in a proportional increase in fIX-carboxylase complexes that yielded full fIX carboxylation. Interestingly, the carboxylated fIX product was not efficiently released from the carboxylase in r-fIX/r-carboxylase cells, resulting in decreased fIX secretion. r-Carboxylase overexpression changed the ratios of intracellular fIX to carboxylase, and we therefore developed an in vitro assay to test whether fIX levels affect release. FIX-carboxylase complexes were in vitro carboxylated with or without excess VKD substrate or propeptide. These analyses are the first to dissect the rates of release versus carboxylation and showed that release was much slower than carboxylation. In the absence of excess VKD substrate/propeptide, fIX in the fIX-carboxylase complex was fully carboxylated by 10 min, but 95% was still complexed with carboxylase after 30 min. The presence of excess VKD substrate/propeptide, however, led to a significant increase in VKD product release, possibly through a second propeptide binding site in the carboxylase. The intracellular analyses also showed that the fIX carboxylation rate was slow in vivo and was similar in r-fIX versus r-fIX/r-carboxylase cells, despite the large differences in carboxylase levels. The results suggest that the vitamin K cofactor may be limiting for carboxylation in the cell lines.     

Blood flow dynamics after photodynamic therapy with verteporfin in the RIF-1 tumor

In the present study, the effects of photodynamic therapy (PDT) with verteporfin on tumor blood flow and tumor regrowth were compared as verteporfin distributed in different compartments within the RIF-1 tumor. Tissue distribution of verteporfin was examined by fluorescence microscopy, and blood flow measurements were taken with a laser Doppler system. It was found that, at 15 min after drug administration, when verteporfin was mainly confined within the vasculature, PDT induced a complete arrest of blood flow by 6 h after treatment. PDT treatment at a longer drug-light interval (3 h), which allowed the drug to diffuse to the tumor interstitium, caused significantly less flow decrease, only to 50% of the initial flow in 6 h. A histological study and Hoechst 33342 staining of functional tumor vasculature confirmed the primary vascular damage and the decrease in tumor perfusion. The regrowth rate of tumors treated with 15-min interval PDT was 64% of that of the control group. However, when tumors were treated with 3-h interval PDT, the regrowth rate was not significantly different from that of the control, indicating that only the 15-min interval PDT caused serious damage to the tumor vascular bed. These results support the hypothesis that temporal pharmacokinetic changes in the distribution of the photosensitizer between the tumor parenchyma and blood vessels can significantly alter the tumor target of PDT.     

Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens

The mechanism of matrix vesicle (MV) mineralization was studied using MVs isolated from normal growth plate tissue, as well as several putative intermediates in the MV mineralization pathway--amorphous calcium phosphate (ACP), calcium phosphate phosphatidylserine complex (CPLX) and hydroxyapatite (HAP). Radionuclide uptake and increase in turbidity were used to monitor mineral formation during incubation in synthetic cartilage lymph (SCL). Inhibitors of phosphate (Pi) metabolism, as well as replacing Na(+) with various cations, were used to study MV Pi transport, which had been thought to be Na(+)-dependent. MVs induced rapid mineralization approximately 3 h after addition to SCL; CPLX and HAP caused almost immediate induction; ACP required approximately 1 h. Phosphonoformate (PFA), a Pi analog, potently delayed the onset and reduced the rate of mineral formation of MV and the intermediates with IC(50)'s of 3-6 microM and approximately 10 microM, respectively. PFA:Pi molar ratios required to reduce the rate of rapid mineralization by 50% were approximately 1:30 for ACP, approximately 1:20 for HAP, approximately 1:3.3 for CPLX, and approximately 1:2.0 for MVs. MV mineralization was not found to be strictly Na(+)-dependent: substitution of Li(+) or K(+) for Na(+) had minimal effect; while N-methyl D-glucamine (NMG(+)) was totally inhibitory, choline(+) was clearly stimulatory. Na(+) substitutions had minimal effect on HAP- and CPLX-seeded mineral formation. However with ACP, NMG(+) totally blocked and choline(+) stimulated, just as they did MV mineralization. Thus, kinetic analyses indicate that ACP is a key intermediate, nevertheless, formation of CPLX appears to be the rate-limiting factor in MV mineralization.     

Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes,prepared without passaging through E. coli

Background  

Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library.