Evolutionary conservation of microtubule-capture mechanisms

The dynamic nature of microtubules allows them to search the three-dimensional space of the cell. But what are they looking for? During cellular morphogenesis, microtubules are captured at sites just under the plasma membrane, and this polarizes the microtubule array and associated organelles. Recent data indicate that the signalling pathways that are involved in regulating the different microtubule cortical interactions are not only conserved between species, but also that they function in diverse processes.     

Aplysia temptin - the 'glue' in the water-borne attractin pheromone complex

Temptin, a component of the complex of water-borne protein pheromones that stimulate attraction and mating behavior in the marine mollusk Aplysia, has sequence homology to the epidermal growth factor (EGF)-like domains of higher organisms that mediate protein-cell surface contact during fertilization and blood coagulation. In this work, recombinant temptin for structural and functional studies was produced in Escherichia coli using a cold shock promoter and purified by RP-HPLC. CD spectra confirmed a predominantly beta-sheet structure. Two disulfide bonds were determined via limited proteolysis and MS. One internal disulfide (Cys57-Cys77) was predicted from initial alignments with class I EGF-like domains; the second, between Cys18 and Cys103, could protect temptin against proteolysis in seawater and stabilize its interacting surface. A three-dimensional model of temptin was prepared with our MPACK suite, based on the Ca(2+)-binding, EGF-like domain of the extracellular matrix protein fibrillin. Two temptin residues, Trp52 and Trp79, which align with cysteine residues conserved in fibrillins, lie adjacent to and could stabilize the disulfide bonds and a proposed metal-binding loop. The water-borne pheromone attractin in egg cordon eluates is complexed with other proteins. Docking results with our model and the NMR structure of attractin suggest that one face of temptin interacts with the pheromone, perhaps controlling its access to the cellular receptors. Gel shifts confirmed that temptin complexes with wild-type attractin. These results indicate that temptin, analogous to the role of fibrillin in controlling transforming growth factor-beta concentration, modulates pheromone signaling by direct binding to attractin.     

A Mixed Culture Recovery Method Indicates that Enteric Bacteria Do Not Enter the Viable but Nonculturable State

A new method, called the mixed culture recovery (MCR) method, has been developed to determine whether recovery of culturable bacterial cells from a population of largely nonculturable cells is due to resuscitation of the nonculturable cells from a viable but nonculturable state or simply to growth of residual culturable cells. The MCR method addresses this issue in that it involves the mixing of two easily distinguishable strains (e.g., lactose positive and negative) in such a way that large numbers of nonculturable cells of both strains are present together with a small number of culturable cells of only one strain, performing a nutrient addition resuscitation procedure, and then plating the cells to determine whether both cell types are recoverable. In repeated experiments with strains of Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter aerogenes, and Salmonella choleraesuis, only cells of the culturable strain were recovered after application of various resuscitation techniques. These results suggest that the nonculturable cells were dead and that the apparent resuscitation was merely due to the growth of the remaining culturable cells.     

Protein-tyrosine Phosphatase 4A3 (PTP4A3) Promotes Vascular Endothelial Growth Factor Signaling and Enables Endothelial Cell Motility

Protein-tyrosine phosphatase 4A3 (PTP4A3) is highly expressed in multiple human cancers and is hypothesized to have a critical, albeit poorly defined, role in the formation of experimental tumors in mice. PTP4A3 is broadly expressed in many tissues so the cellular basis of its etiological contributions to carcinogenesis may involve both tumor and stromal cells. In particular, PTP4A3 is expressed in the tumor vasculature and has been proposed to be a direct target of vascular endothelial growth factor (VEGF) signaling in endothelial cells. We now provide the first in vivo experimental evidence that PTP4A3 participates in VEGF signaling and contributes to the process of pathological angiogenesis. Colon tumor tissue isolated from Ptp4a3-null mice revealed reduced tumor microvessel density compared with wild type controls. Additionally, vascular cells derived from Ptp4a3-null tissues exhibited decreased invasiveness in an ex vivo wound healing assay. When primary endothelial cells were isolated and cultured in vitro, Ptp4a3-null cells displayed greatly reduced migration compared with wild type cells. Exposure to VEGF led to an increase in Src phosphorylation in wild type endothelial cells, a response that was completely ablated in Ptp4a3-null cells. In loss-of-function studies, reduced VEGF-mediated migration was also observed when human endothelial cells were treated with a small molecule inhibitor of PTP4A3. VEGF-mediated in vivo vascular permeability was significantly attenuated in PTP4A3-deficient mice. These findings strongly support a role for PTP4A3 as an important contributor to endothelial cell function and as a multimodal target for cancer therapy and mitigating VEGF-regulated angiogenesis.     

Ranking the affinity of aromatic residues for carbon nanotubes by using designed surfactant peptides.

A series of surfactant peptides were created to evaluate the affinity of aromatic AAs for single-walled carbon nanotubes in the absence of complications from peptide folding or self-association. Each surfactant peptide has a lipidlike architecture, with two Lys residues at the C-terminus as a hydrophilic head, five Val residues to form a hydrophobic tail, and the testing AA at the N-terminus. Raman and CD spectroscopic studies reveal that the surfactant peptides have a large unordered structural component which is independent of peptide concentration, suggesting that the peptides undergo minimal association under experimental conditions, thus removing this interference from interpretation of the peptide/carbon nanotube interactions. A lack of peptide self-association is also indicated by sedimentation equilibrium ultracentrifugation results. Optical spectroscopy of the peptide/carbon nanotube dispersions indicate that among the three aromatic AAs, tryptophan has the highest affinity for carbon nanotubes (both bundled and individual states) when incorporated into a surfactant peptide, while the Tyr-containing peptide is more selective for individual carbon nanotubes. Phe has the lowest overall affinity for carbon nanotubes. Raman spectra of dispersions made with SPF, SPY and SPW display similar types of nanotubes dispersed, although differences in the relative nanotube populations are observed by optical spectroscopy.     

Multiple and additive functions of ALDH3A1 and ALDH1A1: cataract phenotype and ocular oxidative damage in Aldh3a1(-/-)/Aldh1a1(-/-) knock-out mice

ALDH3A1 (aldehyde dehydrogenase 3A1) is abundant in the mouse cornea but undetectable in the lens, and ALDH1A1 is present at lower (catalytic) levels in the cornea and lens. To test the hypothesis that ALDH3A1 and ALDH1A1 protect the anterior segment of the eye against environmentally induced oxidative damage, Aldh1a1(-/-)/Aldh3a1(-/-) double knock-out and Aldh1a1(-/-) and Aldh3a1(-/-) single knock-out mice were evaluated for biochemical changes and cataract formation (lens opacification). The Aldh1a1/Aldh3a1- and Aldh3a1-null mice develop cataracts in the anterior and posterior subcapsular regions as well as punctate opacities in the cortex by 1 month of age. The Aldh1a1-null mice also develop cataracts later in life (6-9 months of age). One- to three-month-old Aldh-null mice exposed to UVB exhibited accelerated anterior lens subcapsular opacification, which was more pronounced in Aldh3a1(-/-) and Aldh3a1(-/-)/Aldh1a1(-/-) mice compared with Aldh1a1(-/-) and wild type animals. Cataract formation was associated with decreased proteasomal activity, increased protein oxidation, increased GSH levels, and increased levels of 4-hydroxy-2-nonenal- and malondialdehyde-protein adducts. In conclusion, these findings support the hypothesis that corneal ALDH3A1 and lens ALDH1A1 protect the eye against cataract formation via nonenzymatic (light filtering) and enzymatic (detoxification) functions.     

The molluscan cardioactive neuropeptide FMRFamide: Subcellular localization in bivalve ganglia

Ganglia of the marine mollusk Macrocallista nimbosa were pooled, homogenized, and subjected to differential centrifugation. The neuropeptide Phe-Met-Arg-Phe-NH₂ (FMRFamide) was concentrated in the microsomal pellet. When the medium-speed supernatant was centrifuged in a discontinuous sucrose gradient, three separate peaks of activity were detected and identified as acetylcholine, 5-hydroxytryptamine, and FMRFamide. The relative concentration of FMRFamide in each fraction was determined by bioassay and by radioimmunoassay (RIA). Both determinations revealed a peak of peptide in the middle of the sucrose gradient. Electron micrographs of each of the gradient interfaces were analyzed. The interface containing the peak of biological FMRFamide activity was enriched two- to fivefold in neurosecretory granules with a mean diameter of 104 nm and various electron densities. Morphologically similar vesicles were also seen in intact ganglia. These findings support the notion that FMRFamide is a neurosecretory product. But the physiological function of the peptide in bivalve ganglia remains unknown.     

Cutaneous vascular responses to isometric handgrip exercise during local heating and hyperthermia.

The dramatic increase in skin blood flow and sweating observed during heat stress is mediated by poorly understood sympathetic cholinergic mechanisms. One theory suggests that a single sympathetic cholinergic nerve mediates cutaneous active vasodilation (AVD) and sweating via cotransmission of separate neurotransmitters, because AVD and sweating track temporally and directionally when activated during passive whole body heat stress. It has also been suggested that these responses are regulated independently, because cutaneous vascular conductance (CVC) has been shown to decrease, whereas sweat rate increases, during combined hyperthermia and isometric handgrip exercise. We tested the hypothesis that CVC decreases during isometric handgrip exercise if skin blood flow is elevated using local heating to levels similar to that induced by pronounced hyperthermia but that this does not occur at lower levels of skin blood flow. Subjects performed isometric handgrip exercise as CVC was elevated at selected sites to varying levels by local heating (which is independent of AVD) in thermoneutral and hyperthermic conditions. During thermoneutral isometric handgrip exercise, CVC decreased at sites in which blood flow was significantly elevated before exercise (-6.5 +/- 1.8% of maximal CVC at 41 degrees C and -10.5 +/- 2.0% of maximal CVC at 43 degrees C; P < 0.05 vs. preexercise). During isometric handgrip exercise in the hyperthermic condition, an observed decrease in CVC was associated with the level of CVC before exercise. Taken together, these findings argue against withdrawal of AVD to explain the decrease in CVC observed during isometric handgrip exercise in hyperthermic conditions.