The motility and dynamic properties of intermediate filaments and their constituent proteins

Intermediate filament (IF) proteins exist in multiple structural forms within cells including mature IF, short filaments or 'squiggles', and non-filamentous precursors called particles. These forms are interconvertible and their relative abundance is IF type, cell type- and cell cycle stage-dependent. These structures are often associated with molecular motors, such as kinesin and dynein, and are therefore capable of translocating through the cytoplasm along microtubules. The assembly of mature IF from their precursor particles is also coupled to translation. These dynamic properties of IF provide mechanisms for regulating their reorganization and assembly in response to the functional requirements of cells. The recent findings that IF and their precursors are frequently associated with signaling molecules have revealed new functions for IF beyond their more traditional roles as mechanical integrators of cells and tissues.     

Intermittent drying of bioproducts--an overview

Unlike the conventional practice of supplying energy for batch drying processes at a constant rate, newly developed intermittent drying processes employ time-varying heat input tailored to match the drying kinetics of the material being dried. The energy required may be supplied by combining different modes of heat transfer (e.g. convection coupled with conduction or radiation or dielectric heating simultaneously or in a pre-selected sequence) in a time-varying fashion so as to provide optimal drying kinetics as well as quality of the bioproduct. This is especially important for drying of heat-sensitive materials (such as foods, pharmaceutical, neutraceutical substances, herbs, spices and herbal medicines). Intermittent heat supply is beneficial only for materials which dry primarily in the falling rate period where internal diffusion of heat and moisture controls the overall drying rate. Periods when little or no heat is supplied for drying allow the tempering period needed for the moisture and heat to diffuse within the material. As the moisture content increases at the surface of the biomaterial during the tempering period, the rate of drying is higher when heat input is resumed. It is possible to control the heat input such that the surface temperature of the product does not exceed a pre-determined value beyond which thermal damage of the material may occur. This process results in reduction in the use of thermal energy as well as the mass of air used in convective drying. Thus, the thermal efficiency of such a process is higher. The quality of the product, as such color and ascorbic acid content, is also typically superior to that obtained with a continuous supply of heat. However, in some cases, there will be a nominal increase in drying time. In the case of microwave-assisted and heat pump drying, for example, the capital cost of the drying system can also be reduced by drying in the intermittent mode.

This paper provides an overview of the basic process, selected results from experiments and mathematical models for a variety of biomaterials dried in a wide assortment of dryers. It begins with a classification of intermittent drying processes that may be applied e.g. time-varying temperature, air flow rate, operating pressure as well as heat input by different modes and in different temporal variations. The beneficial effects of improving the quality of dried bioproducts by different intermittent processes are also included and discussed.     

Contrasting selection modes at the Adh1 locus in outcrossing Miscanthus sinensis vs. inbreeding Miscanthus condensatus (Poaceae)

We estimated DNA sequence variation of the Adh1 locus in the outcrossing Miscanthus sinensis (Poaceae) and its close selfing relative, M. condensatus. Tajima's test of selection is significantly negative for both overall exons and replacement sites in M. sinensis. Among its entire sample, nucleotide diversity of nonsynonymous sites is higher than that of synonymous sites. A McDonald and Kreitman test of neutrality indicates an excess of intraspecific replacement polymorphisms, suggesting possible directional selection toward advantageous mutants. However, frequent intragenic recombination suggests both purifying and positive selection is unlikely. Recent demographic expansions coupled with relaxation of purifying selection may have resulted in elevated genetic diversity at the Adh1 locus as well as the trnL-trnF intergenic spacer of cpDNA in this outcrossing species. In contrast, low levels of genetic diversity were detected at both the Adh1 locus and the cpDNA spacer in M. condensatus, consistent with bottlenecks associated with selfing in all populations. While Tajima's D and Fu and Li's F statistics did not reveal deviation from neutrality at the Adh1 locus in M. condensatus, 12 replacements vs. 10 synonymous changes were detected. Based on pairwise comparisons of the d(N)/d(S) ratio, lineages of closely related populations of the species distributed along saline habitats appeared to be under directional selection.     

Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.     

MemBrain: improving the accuracy of predicting transmembrane helices

Prediction of transmembrane helices (TMH) in alpha helical membrane proteins provides valuable information about the protein topology when the high resolution structures are not available. Many predictors have been developed based on either amino acid hydrophobicity scale or pure statistical approaches. While these predictors perform reasonably well in identifying the number of TMHs in a protein, they are generally inaccurate in predicting the ends of TMHs, or TMHs of unusual length. To improve the accuracy of TMH detection, we developed a machine-learning based predictor, MemBrain, which integrates a number of modern bioinformatics approaches including sequence representation by multiple sequence alignment matrix, the optimized evidence-theoretic K-nearest neighbor prediction algorithm, fusion of multiple prediction window sizes, and classification by dynamic threshold. MemBrain demonstrates an overall improvement of about 20% in prediction accuracy, particularly, in predicting the ends of TMHs and TMHs that are shorter than 15 residues. It also has the capability to detect N-terminal signal peptides. The MemBrain predictor is a useful sequence-based analysis tool for functional and structural characterization of helical membrane proteins; it is freely available at http://chou.med.harvard.edu/bioinf/MemBrain/.     

EGF upregulates Na+/H+ exchanger NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness

Na+/H+ exchanger 1 (NHE1) is involved in cell migration but little is known about the signal pathways that regulate NHE1 activity and that are associated with tumor cell invasiveness. This study is to investigate the mechanisms by which epidermal growth factor (EGF) regulates NHE1 expression to promote cervical cancer cell invasiveness and the clinical significance in early-stage cervical cancer. NHE1 protein was scanty in normal or noncancerous cervical tissues of all surgical specimens examined (n = 92). Tumor tissues clearly expressed NHE1 protein with different amounts. The differential expression level of NHE1 is associated with the clinical outcome. NHE1 protein was also differentially expressed between normal cervical epithelial cells and two cervical cancer cell lines. Cervical cancer cells benefit some enhanced cellular functions from NHE1 abundance, such as cell volume regulation, migration, and invasion. Interestingly, NHE1 colocalized with EGF in cervical cancer tissues. Studies in cell culture systems indicated that EGF-stimulated NHE1 abundance in a time-dependent manner by post-translational regulation. This implies a likely autocrine or paracrine EGF stimulation of NHE1 production in vivo. In addition, the phosphoinositide 3-kinase pathway is the dominant signal controlling EGF-stimulated NHE1 abundance. Pharmacological inhibition of NHE1 activity markedly inhibited the basal and EGF-stimulated cervical cancer cell migration. Image studies and immunoprecipitaion experiments suggest that EGF-induced NHE1 translocation to the leading-edge lamellipodia, where NHE1 interacted with actin-associated protein Ezrin, thereby remodeling cytoskeleton and stimulating cervical cancer cell migration. In conclusion, EGF upregulates NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness.     

Gender Difference in the Interaction Effects of Diabetes and Hypertension on Stroke among the Elderly in the Shih-Pai Study,Taiwan

Aims

To investigate the interaction effects of diabetes and hypertension on stroke, and also investigate the independent and interaction effects of parental history and environmental factors on diabetes and hypertension in a cross-sectional elderly population.

Methods

The Shih-Pai Community Medical Service Program was a community-based, fixed cohort study conducted between June 1999 and November 2002. Socio-demographic and clinical data of subjects aged 65 years and older were collected by well-trained interviewers during home visits. Interaction effects were analyzed using Rothman’s synergy index (SI).

Results

In total, 4,124 subjects were included in the study, with 2,284 males and 1,840 females. The synergistic interaction of diabetes and hypertension on stroke was statistically significant in women (SI = 3.16, 95% CI: 1.35–7.39). The synergistic interaction of parental diabetes and being overweight on diabetes was only statistically significant in men, and not in women (SI = 3.30, 95% CI: 1.00–10.83 in men, and SI = 1.15, 95% CI: 0.30–4.39 in women).

Conclusions

A synergistic interaction was found for diabetes and hypertension in both sexes when parental history and being overweight were combined. Furthermore, combining diabetes and hypertension in elderly women was significant in terms of the risk of stroke. Strategies to control risk factors in individuals at additional high risk are urgently needed.     

Angiotensin-converting enzyme inhibitor captopril attenuates ventilator-induced lung injury in rats.

We hypothesized that lung inflammation and parenchymal apoptosis in ventilator-induced lung injury (VILI) are related to ANG II and assessed the ability of the angiotensin-converting enzyme inhibitor captopril to attenuate VILI in rats. Adult male Sprague-Dawley rats were randomized to receive two ventilation strategies for 2 h: 1) tidal volume of 40 ml/kg, respiratory rate of 25 breaths/min, and inspiratory O2 fraction of 0.21 [high-volume, 0 positive end-expiratory pressure (HVZP) group] and 2) injection of captopril (100 mg/kg ip) 30 min before HVZP ventilation (HVZP+CAP group). Another group, which did not receive ventilation, served as the control. Mean arterial pressure was significantly lower in the HVZP+CAP group than in the HVZP group at 2 h of ventilation. Total protein levels were significantly higher in bronchoalveolar lavage fluid (BALF) recovered from HVZP-ventilated rats than from controls. BALF macrophage inflammatory protein-2 and lung ANG II were significantly higher in the HVZP group than in the control and HVZP+CAP groups. Lung ANG II levels correlated positively with BALF protein and macrophage inflammatory protein-2. The number of apoptotic airway and alveolar wall cells was significantly higher in the HVZP and HVZP+CAP groups than in the control group and significantly lower in the HVZP+CAP group than in the HVZP group. These results suggest that the efficiency of captopril to attenuate VILI is related to reduction of inflammatory cytokines and inhibition of apoptosis and indicate that VILI is partly mediated by the local angiotensin system.     

Hydrophilic aromatic residue and in silico structure for carbohydrate binding module

Carbohydrate binding modules (CBMs) are found in polysaccharide-targeting enzymes and increase catalytic efficiency. Because only a relatively small number of CBM structures have been solved, computational modeling represents an alternative approach in conjunction with experimental assessment of CBM functionality and ligand-binding properties. An accurate target-template sequence alignment is the crucial step during homology modeling. However, low sequence identities between target/template sequences can be a major bottleneck. We therefore incorporated the predicted hydrophilic aromatic residues (HARs) and secondary structure elements into our feature-incorporated alignment (FIA) algorithm to increase CBM alignment accuracy. An alignment performance comparison for FIA and six others was made, and the greatest average sequence identities and similarities were achieved by FIA. In addition, structure models were built for 817 representative CBMs. Our models possessed the smallest average surface-potential z scores. Besides, a large true positive value for liagnd-binding aromatic residue prediction was obtained by HAR identification. Finally, the pre-simulated CBM structures have been deposited in the Database of Simulated CBM structures (DS-CBMs). The web service is publicly available at http://dscbm.life.nthu.edu.tw/ and http://dscbm.cs.ntou.edu.tw/.     

Ketoconazole-evoked [Ca2+]i rises and non-Ca2+-triggered cell death in rabbit corneal epithelial cells (SIRC)

The effect of ketoconazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and proliferation has not been explored in corneal cells. This study examined whether ketoconazole alters Ca2+ levels and causes cell death in SIRC rabbit corneal epithelial cells. [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Ketoconazole at concentrations of 5 microM and above increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The ketoconazole-induced Ca2+ influx was insensitive to L-type Ca2+ channel blockers and protein kinase C modulators. In Ca2+-free medium, after pretreatment with 50 microM ketoconazole, thapsigargin-(1 microM)-induced [Ca2+]i rises were abolished; conversely, thapsigargin pretreatment nearly abolished ketoconazole-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change ketoconazole-induced [Ca2+]i rises. At concentrations between 5 and 100 microM, ketoconazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 microM ketoconazole was not reversed by prechelating cytosolic Ca2+ with BAPTA. In summary, in corneal cells, ketoconazole-induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from unknown pathways. Furthermore, the cytotoxicity induced by ketoconazole was not caused via a preceding [Ca2+]i rise.