A hydrophobic patch in a charged alpha-helix is sufficient to target proteins to dense core secretory granules

Many endocrine and neuroendocrine cells contain specialized secretory organelles called dense core secretory granules. These organelles are the repository of proteins and peptides that are secreted in a regulated manner when the cell receives a physiological stimulus. The targeting of proteins to these secretory granules is crucial for the generation of certain peptide hormones, including insulin and ACTH. Although previous work has demonstrated that proteins destined to a variety of cellular locations, including secretory granules, contain targeting sequences, no single consensus sequence for secretory granule-sorting signals has emerged. We have shown previously that alpha-helical domains in the C-terminal tail of the prohormone convertase PC1/3 play an important role in the ability of this region of the protein to direct secretory granule targeting (Jutras, I. Seidah, N. G., and Reudelhuber, T. L. (2000) J. Biol. Chem. 275, 40337-40343). In this study, we show that a variety of alpha-helical domains are capable of directing a heterologous secretory protein to granules. By testing a series of synthetic alpha-helices, we also demonstrate that the presence of charged (either positive or negative) amino acids spatially segregated from a hydrophobic patch in the alpha-helices of secretory proteins likely plays a critical role in the ability of these structures to direct secretory granule sorting.     

Mathematical and empirical proof of principle for an on-body personal lift augmentation device (PLAD)

In our laboratory, we have developed a prototype of a personal lift augmentation device (PLAD) that can be worn by workers during manual handling tasks involving lifting or lowering or static holding in symmetric and asymmetric postures. Our concept was to develop a human-speed on-body assistive device that would reduce the required lumbar moment by 20-30% without negative consequences on other joints or lifting kinematics. This paper provides mathematical proof using simplified free body diagrams and two-dimensional moment balance equations. Empirical proof is also provided based on lifting trials with nine male subjects who executed sagittal plane lifts using three lifting styles (stoop, squat, free) and three different loads (5, 15, and 25kg) under two conditions (PLAD, No-PLAD). Nine Fastrak sensors and six in-line strap force sensors were used to estimate the reduction of compressive and shear forces on L4/L5 as well as estimate the forces transferred to the shoulders and knees. Depending on lifting technique, the PLAD applied an added 23-36Nm of torque to assist the back muscles during lifting tasks. The peak pelvic girdle contact forces were estimated and their magnitudes ranged from 221.3+/-11.2N for stoop lifting, 324.3+/-17.2N for freestyle lifts to 468.47+/-23.2N for squat lifting. The PLAD was able to reduce the compression and shear forces about 23-29% and 7.9-8.5%, respectively.     

Mechanical strain increases gene transfer to skeletal muscle cells

Gene transfer techniques possess tremendous potential for treating diseases and for facilitating the study of basic physiological processes. However, further development of efficient and safe methods for gene transfer is needed. The purpose of this study was to test the hypothesis that mechanical strain increases the transfer of DNA to differentiated skeletal muscle cells. We tested this hypothesis by applying cyclic strain to cultured skeletal myotubes either prior to or immediately after the introduction of exogenous DNA complexed with lipids, with strains of varying magnitude (10%, 20% and 30%), number (1800, 3600 and 7200 strain cycles) and frequency (0.5, 1.0 and 1.5 Hz). Results demonstrated that DNA transfection was increased by exposing muscle cells to cyclic strain, and that strain magnitude, number and frequency each influenced DNA transfection. Optimal strain conditions (20% strain magnitude, 3600 cycles applied at 1 Hz) were utilized to examine the role of membrane transport systems in strain-induced increases in DNA transfection. Filipin III was used to inhibit caveolar transport and was found to inhibit strain-mediated increases in DNA transfection, whereas chlorpromazine, used to inhibit clathrin-coated vesicle transport, had no effect. These results indicate that mechanical strain may be an effective method for increasing DNA transfection in skeletal muscle through enhanced caveolar transport.     

Role of reflex gain and reflex delay in spinal stability--a dynamic simulation

The goal of this study was to investigate the role of reflex and reflex time delay in muscle recruitment and spinal stability. A dynamic biomechanical model of the musculoskeletal spine with reflex response was implemented to investigate the relationship between reflex gain, co-contraction, and stability in the spine. The first aim of the study was to investigate how reflex gain affected co-contraction predicted in the model. It was found that reflexes allowed the model to stabilize with less antagonistic co-contraction and hence lower metabolic power than when limited to intrinsic stiffness alone. In fact, without reflexes there was no feasible recruitment pattern that could maintain spinal stability when the torso was loaded with 200N external load. Reflex delay is manifest in the paraspinal muscles and represents the time from a perturbation to the onset of reflex activation. The second aim of the study was to investigate the relationship between reflex delay and the maximum tolerable reflex gain. The maximum acceptable upper bound on reflex gain decreased logarithmically with reflex delay. Thus, increased reflex delay and reduced reflex gain requires greater antagonistic co-contraction to maintain spinal stability. Results of this study may help understanding of how patients with retarded reflex delay utilize reflex for stability, and may explain why some patients preferentially recruit more intrinsic stiffness than healthy subjects.     

Immune synapse formation requires ZAP-70 recruitment by ezrin and CD43 removal by moesin

Immunological synapse (IS) formation involves receptor–ligand pair clustering and intracellular signaling molecule recruitment with a coincident removal of other membrane proteins away from the IS. As microfilament–membrane linkage is critical to this process, we investigated the involvement of ezrin and moesin, the two ezrin/radixin/moesin proteins expressed in T cells. We demonstrate that ezrin and moesin, which are generally believed to be functionally redundant, are differentially localized and have important and complementary functions in IS formation. Specifically, we find that ezrin directly interacts with and recruits the signaling kinase ZAP-70 to the IS. Furthermore, the activation of ezrin by phosphorylation is essential for this process. In contrast, moesin dephosphorylation and removal, along with CD43, are necessary to prepare a region of the cell cortex for IS. Thus, ezrin and moesin have distinct and critical functions in the T cell cortex during IS formation.     

The in vitro effect of pH on osteoclasts and bone resorption in the cat: implications for the pathogenesis of FORL

Dental disease due to osteoclast over-activity reaches epidemic proportions in older domestic cats and has also been reported in wild cats. Feline osteoclastic resorptive lesions (FORL) involve extensive resorption of the tooth leaving it liable to root fracture and subsequent tooth loss. The aetio-pathogenesis of FORL is not known. Recent work has shown that systemic acidosis causes increased osteoclast activation and that loci of infection or inflammation in cat mouth are likely to be acidotic. To investigate this, we generated osteoclasts from cat blood and found that they formed in large numbers (approximately 400) in cultures on bovine cortical bone slices. Acidosis caused an increase in the size of cells-in cultures maintained up to 14 days at basal pH 7.25, mean osteoclast area was 0.01 +/- 0.003 mm(2), whereas an 8.6-fold increase was observed in cells cultured between 11 and 14 days at pH 7.15 (0.086 +/- 0.004 mm(2)). Acidosis caused a modest increase in the number of osteoclasts. Exposure to pH 6.92 exhibited a 5-fold increase in the area of bone slices covered by resorption lacunae ( approximately 70% bone slice resorbed). In line with this finding, significant increases were observed in the expression of cathepsin K and proton pump enzymes (both approximately 3-fold) that are key enzymes reflective of resorptive activity in osteoclasts. These results demonstrate that acidosis is a major regulator of osteoclast formation and functional activation in the cat, and suggest that local pH changes may play a significant role in the pathogenesis of FORL.     

A distinctive set of genes is upregulated during the inflammation-carcinoma sequence in mouse stomach infected by Helicobacter felis.

Helicobacter pylori infects over half the population worldwide and is a leading cause of chronic gastritis and gastric cancer. However, the mechanism by which this organism induces inflammation and carcinogenesis is not fully understood. In the present study we used insulin-gastrin (INS-GAS) transgenic mice that fully develop gastric adenocarcinoma after infection of H. pylori-related Helicobacter felis. Histological examination revealed that more than half of those mice developed invasive adenocarcinoma after 8 months of infection. These carcinomas were stained by NCC-ST-439 and HECA-452 that recognize 6-sulfated and non-sulfated sialyl Lewis X. Lymphocytic infiltration predominantly to submucosa was observed in most H. felis-infected mice, and this was associated with the formation of peripheral lymph node addressin (PNAd) on high endothelial venule (HEV)-like vessels detected by MECA-79. Time-course analysis of gene expression by using gene microarray revealed upregulation of several inflammation-associated genes including chemokines, adhesion molecules, surfactant protein D (SP-D), and CD74 in the infected stomach. Immunohistochemical analysis demonstrated that SP-D is expressed in hyperplasia and adenocarcinoma whereas CD74 is expressed in adenocarcinoma in situ and invasive carcinoma. These results as a whole indicate that H. felis induces HEV-like vessels and inflammation-associated chemokines and chemokine receptors, followed by adenocarcinoma formation.     

Prostaglandin E2 is a major inhibitor of dendritic cell maturation and function in Ixodes scapularis saliva

Tick saliva is thought to contain a number of molecules that prevent host immune and inflammatory responses. In this study, the effects of Ixodes scapularis saliva on cytokine production by bone marrow-derived dendritic cells (DCs) from C57BL/6 mice stimulated by TLR-2, TLR-4, and TLR-9 ligands were studied. Saliva at remarkably diluted concentrations (<1/2000) promotes a dose-dependent inhibition of IL-12 and TNF-alpha production induced by all TLR ligands used. Using a combination of fractionation techniques (microcon filtration, molecular sieving, and reversed-phase chromatography), we unambiguously identified PGE(2) as the salivary inhibitor of IL-12 and TNF-alpha production by DCs. Moreover, we have found that I. scapularis saliva (dilution 1/200; approximately 10 nM PGE(2)) marginally inhibited LPS-induced CD40, but not CD80, CD86, or MHC class II expression. In addition, saliva significantly suppressed the ability of DCs to stimulate Ag-specific CD4(+) T cell proliferation and IL-2 production. Notably, the effect of saliva on DC maturation and function was reproduced by comparable concentrations of standard PGE(2). These findings indicate that PGE(2) accounts for most inhibition of DC function observed with saliva in vitro. The role of salivary PGE(2) in vector-host interaction and host immune modulation and inflammation in vivo is also discussed. This study is the first to identify molecularly a DC inhibitor from blood-sucking arthropods.