Differential binding of mannose-specific lectins to the carbohydrate chains of fibrinogen domains D and E

The interaction of fibrinogen with the mannose-specific lectins concanavalin A (ConA), its acetyl derivative (Ac-ConA) and Lens culinaris agglutinin (LcH) was studied. Both ConA and LcH interact specifically with individual fibrinogen B beta and gamma chains and with denatured fragments D and E. However, analysis of the binding data shows that four moles of Ac-ConA are bound per mole of fibrinogen with two sets of binding sites (Kd1 = 2.4 microM and Kd2 = 16.6 microM; n1 = n2 = 2) while only two moles of LcH are bound per mole of fibrinogen (Kd = 2.6 microM). Ultracentrifugation studies are also in agreement with the presence in the fibrinogen molecule of two and four binding sites for LcH and Ac-ConA, respectively. No aggregates of fibrinogen formed through LcH or Ac-ConA linkages are observed. The use of a crosslinking reagent and ultracentrifugal analysis of the lectin-fibrinogen fragments D1 and E complexes indicated that ConA, as well as Ac-ConA, interact with both fragments D and E while LcH interacts only with fragment D. Furthermore, the binding of ConA to both D and E domains in the intact fibrinogen molecule is clearly demonstrated by using a bifunctional reagent. The bivalent character of ConA tetramers may be misinterpreted as a lack of accessibility of the lectin to two of the four carbohydrate chains of fibrinogen. The differential binding of LcH and ConA to the carbohydrate chains of fibrinogen can be related to a different exposure of the oligosaccharide in D and E fragments and domains and to the different requirements of both lectins for their binding to glycoproteins.     

Effect of lectin-binding to fibrinogen D and E domains on coagulation and fibrinolysis

The effect on fibrinogen coagulation and fibrinolysis of the mannose-specific lectins concanavalin A, its acetyl derivative and Lens culinaris agglutinin was studied. Concanavalin A and acetyl-concanavalin A, which bind to the four carbohydrate chains of fibrinogen, and L. culinaris agglutinin, which only binds to the carbohydrate present in fibrinogen D domains, has the same effect on the coagulation rate: an inhibition at low lectin concentrations and an increase at high concentrations. On the other hand, L. culinaris agglutinin does not alter fibrin crosslinking while acetyl-concanavalin A produces a slight inhibition of both gamma-gamma and alpha-polymer formation. However, this effect is very small when compared with the clear inhibitory effect produced by concanavalin A. Concanavalin A and acetyl-concanavalin A have an inhibitory effect on the rate of fibrin clot lysis proportional to the lectin concentration. Nearly 100% inhibition was obtained when two lectin-binding sites were occupied by either concanavalin A or acetyl-concanavalin A. However, L. culinaris agglutinin has a clearly weaker effect and more than 50% inhibition was not observed. The comparative study of the effect of the three lectins on fibrinolysis as well as on the formation of fibrinogen aggregates suggests that the inhibitory effect of concanavalin A and acetyl-concanavalin A is primarily due to their binding to the carbohydrate chains of fibrinogen E domain.     

Purification of plasma membranes from mouse parotid gland and membrane reorganization in response to isoproterenol

Two highly purified plasma membrane fractions have been obtained from mouse parotid glands by a combination of differential centrifugation and isopycnic centrifugation in discontinuous sucrose gradients. The membranes were characterized by enzymic, chemical and morphological criteria. The effect of isoproterenol, which induces parotid acinar cells to proliferate, upon sialic acid and five different enzyme activities located in the plasma membrane phosphodiesterase (EC 3.1.4.1), Mg2+-ATPase (EC 3.6.1.4), leucine aminopeptidase (EC 3.4.1.1), protein kinase (EC 2.7.1.37) and sialyltransferase (EC 2.4.99.1), were quantified along the cell cycle. Plasma membrane sialic acid content falls 30% within 30 min and remains depressed for at least 6 h with the major restoration towards normal levels occurring between 12 and 16 h later. In contrast multiple daily isoproterenol injections lead to a more than 2-fold elevation of sialic acid content. Sialyltransferase activity rises 2-fold by 12 h after isoproterenol treatment and then rapidly falls. This enzyme has a pH optimum of 6.5, requires a divalent cation for activity and is inhibited by Triton X-100. Other enzyme activities showed markedly different changes after isoproterenol stimulation, either increasing, decreasing or remaining unaltered. These continuous functional modifications suggest an active role of the plasma membrane in the control of the proliferative cycle.     

Wound complications after median sternotomy: A study of 61 patients from a consecutive series of 9,279

Among a consecutive series of 9,279 sternotomies performed during a period of 2(1/2) years, 61 (0.66%) patients developed significant wound complications. Of these, 58 (95.1%) survived. Sternal infection occurred in 36 patients (0.39%). Predisposing factors included chronic obstructive pulmonary disease, diabetes mellitus, obesity, closed chest massage, prolonged assisted ventilation, and excessive bleeding after operation. Positive end expiratory pressure (PEEP) did not, in itself, predispose to sternal dehiscence. Intermittent positive pressure breathing (IPPB) treatments caused excessive coughing, which may have increased the likelihood of dehiscence. Disposable drapes and expeditious surgery probably contributed to the low incidence of wound infection. Early diagnosis, surgical debridement, rewiring and primary closure with substernal drainage, without continuous antibiotic irrigation, resulted in satisfactory resolution in most patients.     

New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.     

Genetic variation in Plethodon cinereus and Plethodon hubrichti from in and around a contact zone

Climate change poses several challenges to biological communities including changes in the frequency of encounters between closely related congeners as a result of range shifts. When climate change leads to increased hybridization, hybrid dysfunction or genetic swamping may increase extinction risk—particularly in range‐restricted species with low vagility. The Peaks of Otter Salamander, Plethodon hubrichti, is a fully terrestrial woodland salamander that is restricted to ~18 km of ridgeline in the mountains of southwestern Virginia, and its range is surrounded by the abundant and widespread Eastern Red‐backed Salamander, Plethodon cinereus. In order to determine whether these two species are hybridizing and how their range limits may be shifting, we assessed variation at eight microsatellite loci and a 1,008 bp region of Cytochrome B in both species at allopatric reference sites and within a contact zone. Our results show that hybridization between P. hubrichti and P. cinereus either does not occur or is very rare. However, we find that diversity and differentiation are substantially higher in the mountaintop endemic P. hubrichti than in the widespread P. cinereus, despite similar movement ability for the two species as assessed by a homing experiment. Furthermore, estimation of divergence times between reference and contact zone populations via approximate Bayesian computation is consistent with the idea that P. cinereus has expanded into the range of P. hubrichti. Given the apparent recent colonization of the contact zone by P. cinereus, future monitoring of P. cinereus range limits should be a priority for the management of P. hubrichti populations.     

How does muscle stiffness affect the internal deformations within the soft tissue layers of the buttocks under constant loading?

Mechanical loading of soft tissues covering bony prominences can cause skeletal muscle damage, ultimately resulting in a severe pressure ulcer termed deep tissue injury (DTI). Deformation plays an important role in the aetiology of DTI. Therefore, it is essential to minimise internal muscle deformations in subjects at risk of DTI. As an example, spinal cord-injured (SCI) individuals exhibit structural changes leading to a decrease in muscle thickness and stiffness, which subsequently increase the tissue deformations. In the present study, an animal-specific finite element model, where the geometry and boundary conditions were derived from magnetic resonance images, was developed. It was used to investigate the internal deformations in the muscle, fat and skin layers of the porcine buttocks during loading. The model indicated the presence of large deformations in both the muscle and the fat layers, with maximum shear strains up to 0.65 in muscle tissue and 0.63 in fat. Furthermore, a sensitivity analysis showed that the tissue deformations depend considerably on the relative stiffness values of the different tissues. For example, a change in muscle stiffness had a large effect on the muscle deformations. A 50% decrease in stiffness caused an increase in maximum shear strain from 0.65 to 0.99, whereas a 50% increase in stiffness resulted in a decrease in maximum shear strain from 0.65 to 0.49. These results indicate the importance of restoring tissue properties after SCI, with the use of, for example, electrical stimulation, to prevent the development of DTI.     

New North American Chrysauginae (Pyralidae) described by E.D. Cashatt

A Ph.D. dissertation completed by E.D. Cashatt in 1968 entitled “Revision of the Chrysauginae of North America” does not meet the criteria of publication so the new taxa described therein are not available per the International Code of Zoological Nomenclature. In order to validate the taxa proposed in that document we formally describe and illustrate the following: Arta brevivalvalis Cashatt, sp. n., Heliades lindae Cashatt, sp. n., Paragalasa Cashatt, gen. n., Paragalasa exospinalis Cashatt, sp. n., and Penthesilea sacculalis baboquivariensis Cashatt, subsp. n. We summarize other taxonomic actions proposed in the dissertation and those proposed by subsequent authors. We provide the current nomenclatural status with the literature citation of the paper in which the current status was proposed. A lectotype is designated for Clydonopteran tecomae. Adult holotypes and associated labels, and genitalia of paratypes are newly illustrated.     

The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors

The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01).