Unexpected smoking-linked high MMP-9 in induced sputum of hazardous dust-exposed workers

We investigated whether metalloproteinase-9 (MMP-9) values in induced sputum (IS) and pulmonary function tests can serve as a marker of damage and intensity of exposure to hazardous dust. Thirty-nine factory employees (28 workers exposed to metal particles and 11 supposedly nonexposed office workers) underwent IS induction. Samples were processed by conventional methods within 2 hours. The proportion of particles with diameters of 0-2 mu in IS samples was significantly higher in nonexposed than exposed workers and in smoker compared to nonsmoker workers. MMP-9 and TIMP-1 levels were similar for both groups. A linear regression model for MMP-9 based on exposure, smoking habits, and proportion of particles < 5 mu revealed a positive correlation between each of the explanatory variables and MMP-9 values. MMP-9 may serve as a marker for pulmonary injury.     

Sample extraction techniques for enhanced proteomic analysis of plant tissues

Major improvements in proteomic techniques in recent years have led to an increase in their application in all biological fields, including plant sciences. For all proteomic approaches, protein extraction and sample preparation are of utmost importance for optimal results; however, extraction of proteins from plant tissues represents a great challenge. Plant tissues usually contain relatively low amounts of proteins and high concentrations of proteases and compounds that potentially can limit tissue disintegration and interfere with subsequent protein separation and identification. An effective protein extraction protocol must also be adaptable to the great variation in the sets of secondary metabolites and potentially contaminating compounds that occurs between tissues (e.g., leaves, roots, fruit, seeds and stems) and between species. Here we present two basic protein extraction protocols that have successfully been used with diverse plant tissues, including recalcitrant tissues. The first method is based on phenol extraction coupled with ammonium acetate precipitation, and the second is based on trichloroacetic acid (TCA) precipitation. Both extraction protocols can be completed within 2 d.     

Intracellular domains interactions and gated motions of IKS potassium channel subunits

Voltage‐gated K⁺ channels co‐assemble with auxiliary β subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore‐forming subunits with KCNE1 β subunits generates the repolarizing K⁺ current I_KS. However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life‐threatening long or short QT syndromes. Here, we studied the interactions and voltage‐dependent motions of I_KS channel intracellular domains, using fluorescence resonance energy transfer combined with voltage‐clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C‐terminus interacts with the coiled‐coil helix C of the Kv7.1 tetramerization domain. This association is important for I_KS channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant‐negative C‐terminal domain. On channel opening, the C‐termini of Kv7.1 and KCNE1 come close together. Co‐expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K⁺ currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C‐termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation.     

Peculiarities of Activation of Muscles of the Shoulder Belt in Voluntary Two-Joint Movements of the Upper Limb

We studied coordination of central motor commands (СMCs) coming to muscles of the shoulder and shoulder belt in the course of single-joint and two-joint movements including flexion and extension of the elbow and shoulder joints. Characteristics of rectified and averaged EMGs recorded from a few muscles of the upper limb were considered correlates of the CMC parameters. Special attention was paid to coordination of CMCs coming to two-joint muscles that are able to function as common flexors (m. biceps brachii, caput breve, BBcb) and common extensors (m. triceps brachii, caput longum, TBcl) of the elbow and shoulder joints. Upper limb movements used in the tests included planar shifts of the arm from one spatial point to another resulting from either simultaneous changes in the angles of the shoulder and elbow joints or isolated sequential (two-stage) changes in these joint angles. As was found, shoulder muscles providing movements of the elbow with changes in the angle of the elbow joint, i.e., BBcb and TBcl, were also intensely involved in the performance of single-joint movements in the shoulder joint. The CMCs coming to two-joint muscles in the course of two-joint movements appeared, in the first approximation, as sums of the commands received by these muscles in the course of corresponding single-joint movements in the elbow and shoulder joints. Therefore, if we interpret the isolated forearm movement performed due to a change in the angle of the elbow joint as the main motor event, while the shoulder movement is considered the accessory one, we can conclude that realization of a two-joint movement of the upper-limb distal part is based on superposition of CMCs related to basic movements (main and accessory). Neirofiziologiya/Neurophysiology, Vol. 41, No. 1, pp. 48–56, January–February, 2009.     

Mechanisms Involved in Governing Adherence of Vibrio cholerae to Granular Starch

Vibrio cholerae has been shown to adhere to cornstarch granules. The present work explored the mechanisms involved in this adhesion and the possibility of its occurrence in vivo. The findings suggest that both specific and nonspecific interactions are involved in the adhesion. Nonspecific hydrophobic interactions may play a role, since both V. cholerae and cornstarch granules exhibited hydrophobic properties when they were tested using a xylene-water system. In addition, the presence of bile acids reduced the adhesion. The adhesion also involves some specific carbohydrate-binding moieties on the cell surface, as reflected by reduced adhesion following pretreatment of the bacteria with proteinase K and sodium m-periodate. Further investigations supported these observations and showed that media containing low-molecular-weight carbohydrates had a significant inhibitory effect. Binding cell lysate to starch granules and removing the adhered proteins using either glucose or bile acids led to identification (by liquid chromatography-tandem mass spectrometry analysis) of several candidate V. cholerae outer membrane-associated starch-binding proteins. Different sets of proteins were isolated by removal in a glucose solution or bile acids. When the upper gastrointestinal tract conditions were simulated in vitro, both bile salts and the amylolytic activity of the pancreatic juices were found to have an inhibitory effect on the adherence of V. cholerae to starch. However, during acute diarrhea, this inhibitory effect may be significantly reduced due to dilution, suggesting that adhesion does occur in vivo. Such adhesion may contribute to the beneficial effects observed following administration of granular starch-based oral rehydration solutions to cholera patients.Cholera is a severe diarrheal disease that kills thousands of people each year and affects the lives of millions. This disease is caused by specific serogroups of Vibrio cholerae that are pathogenic to humans (20). Infection by V. cholerae usually starts after consumption of contaminated water or food. The severity of symptoms varies among patients, and in the severe form (cholera gravis) the rate of diarrhea may quickly reach 500 to 1,000 ml h⁻¹, which leads to severe dehydration and, without appropriate treatment, death (20, 40, 41). Death in cholera patients is caused by loss of fluids and salts; therefore, the key to therapy is sufficient rehydration. Furthermore, the rehydration solution should have an electrolyte composition similar to that of the lost fluids (16, 20). This understanding led to what is considered to be one of the most important medical advances in the 20th century, oral rehydration therapy (ORT) (16).The life-saving effect of oral rehydration solutions (ORS) is achieved primarily by maintaining the electrolyte balance (e.g., by stimulating absorption of sodium from the small intestine) (16, 20). However, these solutions do not prevent or reduce to any significant extent the symptoms of cholera. Although in controlled studies ORT is very effective at reducing mortality (4, 29), its use remains low in both developing and developed countries. Despite extensive health education efforts (4, 48), a common perception is that oral rehydration is not effective since it does not reduce the manifestations of diarrhea, such as loss of fluid in the feces, or the duration of the illness (12). Moreover, the glucose-based ORS recommended by the World Health Organization (WHO) may paradoxically increase fecal fluid loss. Because of these limitations, there has been a substantial impetus to develop improved ORS (48).Beneficial effects of starch-based ORS have been shown for the treatment of cholera. Clinical trials with starch-based ORS showed that there was a marked improvement in symptom manifestation, in addition to a life-saving effect (38). Ramakrishna et al. (38) hypothesized that part of the beneficial effect of ORS containing high-amylose cornstarch is due to short-chain fatty acid (SCFA) formation by the colon microbiota, which changes the fluid balance in the colon. The massive loss of fluid reported during cholera episodes (500 to 1,000 ml h⁻¹ in the severe form) has raised the question of whether significant amounts of SCFA are indeed formed under these conditions by colonic microbiota or if an alternative mechanism is responsible for the improvement in symptoms.In search of an explanation, we previously demonstrated that V. cholerae strongly adheres to starch granules (15) and suggested that this may explain, at least in part, the beneficial effect of starch-containing ORS in the treatment of cholera compared to treatment with regular ORS. This study was aimed at understanding the mechanisms involved in adhesion of V. cholerae to starch granules.     

Unexpected Population Distribution in a Microbial Mat Community: Sulfate-Reducing Bacteria Localized to the Highly Oxic Chemocline in Contrast to a Eukaryotic Preference for Anoxia

The distribution and abundance of sulfate-reducing bacteria (SRB) and eukaryotes within the upper 4 mm of a hypersaline cyanobacterial mat community were characterized at high resolution with group-specific hybridization probes to quantify 16S rRNA extracted from 100-μm depth intervals. This revealed a preferential localization of SRB within the region defined by the oxygen chemocline. Among the different groups of SRB quantified, including members of the provisional families “Desulfovibrionaceae” and “Desulfobacteriaceae,” Desulfonema-like populations dominated and accounted for up to 30% of total rRNA extracted from certain depth intervals of the chemocline. These data suggest that recognized genera of SRB are not necessarily restricted by high levels of oxygen in this mat community and the possibility of significant sulfur cycling within the chemocline. In marked contrast, eukaryotic populations in this community demonstrated a preference for regions of anoxia.