Marcelo Lazzaron Lamers

Segregation and activation of myosin IIB creates a rear in migrating cells

We have found that MLC-dependent activation of myosin IIB in migrating cells is required to form an extended rear, which coincides with increased directional migration. Activated myosin IIB localizes prominently at the cell rear and produces large, stable actin filament bundles and adhesions, which locally inhibit protrusion and define the morphology of the tail. Myosin IIA forms de novo filaments away from the myosin IIB–enriched center and back to form regions that support protrusion. The positioning and dynamics of myosin IIA and IIB depend on the self-assembly regions in their coiled-coil C terminus. COS7 and B16 melanoma cells lack myosin IIA and IIB, respectively; and show isoform-specific front-back polarity in migrating cells. These studies demonstrate the role of MLC activation and myosin isoforms in creating a cell rear, the segregation of isoforms during filament assembly and their differential effects on adhesion and protrusion, and a key role for the noncontractile region of the isoforms in determining their localization and function.

High Glucose-Mediated Oxidative Stress Impairs Cell Migration

Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we evaluate the hypothesis that high glucose concentrations inhibit cell migration. Using CHO.K1 cells, NIH-3T3 fibroblasts, mouse embryonic fibroblasts and primary skin fibroblasts from control and diabetic rats cultured in 5 mM D-glucose (low glucose, LG), 25 mM D-glucose (high glucose, HG) or 25 mM L-glucose medium (osmotic control - OC), we analyzed the migration speed, protrusion stability, cell polarity, adhesion maturation and the activity of the small Rho GTPase Rac1. We also analyzed the effects of reactive oxygen species by incubating cells with the antioxidant N-Acetyl-Cysteine (NAC). We observed that HG conditions inhibited cell migration when compared to LG or OC. This inhibition resulted from impaired cell polarity, protrusion destabilization and inhibition of adhesion maturation. Conversely, Rac1 activity, which promotes protrusion and blocks adhesion maturation, was increased in HG conditions, thus providing a mechanistic basis for the HG phenotype. Most of the HG effects were partially or completely rescued by treatment with NAC. These findings demonstrate that HG impairs cell migration due to an increase in oxidative stress that causes polarity loss, deficient adhesion and protrusion. These alterations arise, in large part, from increased Rac1 activity and may contribute to the poor wound healing observed in diabetic patients.

Homocysteine induces oxidative stress, inflammatory infiltration, fibrosis and reduces glycogen/glycoprotein content in liver of rats.

Hyperhomocysteinemia has been related to various diseases, including homocystinuria, neurodegenerative and hepatic diseases. In the present study we initially investigated the effect of chronic homocysteine administration on some parameters of oxidative stress, named total radical‐trapping antioxidant potential, total antioxidant reactivity, catalase activity, chemiluminescence, thiobarbituric acid‐reactive substances, and total thiol content in liver of rats. We also performed histological analysis, evaluating steatosis, inflammatory infiltration, fibrosis, and glycogen/glycoprotein content in liver tissue sections from hyperhomocysteinemic rats. Finally, we evaluated the activities of aminotransferases in liver and plasma of hyperhomocysteinemic rats. Wistar rats received daily subcutaneous injection of Hcy from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, liver and plasma were collected. Hyperhomocysteinemia decreased antioxidant defenses and total thiol content, and increased lipid peroxidation in liver of rats, characterizing a reliable oxidative stress. Histological analysis indicated the presence of inflammatory infiltrate, fibrosis and reduced content of glycogen/glycoprotein in liver tissue sections from hyperhomocysteinemic rats. Aminotransferases activities were not altered by homocysteine. Our data showed a consistent profile of liver injury elicited by homocysteine, which could contribute to explain, at least in part, the mechanisms involved in human liver diseases associated to hyperhomocysteinemia.

RECK-Mediated Inhibition of Glioma Migration and Invasion

RECK is an anti‐tumoral gene whose activity has been associated with its inhibitory effects regulating MMP‐2, MMP‐9, and MT1‐MMP. RECK level decreases as gliobastoma progresses, varying from less invasive grade II gliomas to very invasive human glioblastoma multiforme (GBM). Since RECK expression and glioma invasiveness show an inverse correlation, the aim of the present study is to investigate whether RECK expression would inhibit glioma invasive behavior. We conducted this study to explore forced RECK expression in the highly invasive T98G human GBM cell line. Expression levels as well as protein levels of RECK, MMP‐2, MMP‐9, and MT1‐MMP were assessed by qPCR and immunoblotting in T98G/RECK+ cells. The invasion and migration capacity of RECK+ cells was inhibited in transwell and wound assays. Dramatic cytoskeleton modifications were observed in the T98G/RECK+ cells, when compared to control cells, such as the abundance of stress fibers (contractile actin–myosin II bundles) and alteration of lamellipodia. T98G/RECK+ cells also displayed phosphorylated focal adhesion kinase (P‐FAK) in mature focal adhesions associated with stress fibers; whereas P‐FAK in control cells was mostly associated with immature focal complexes. Interestingly, the RECK protein was predominantly localized at the leading edge of migrating cells, associated with membrane ruffles. Unexpectedly, introduced expression of RECK effectively inhibited the invasive process through rearrangement of actin filaments, promoting a decrease in migratory ability. This work has associated RECK tumor‐suppressing activity with the inhibition of motility and invasion in this GBM model, which are two glioma characteristics responsible for the inefficiency of current available treatments. J. Cell. Biochem. 110: 52–61, 2010.

Laser phototherapy effect on protein metabolism parameters of rat salivary glands

The aim of this study was to evaluate the effects of infrared diode laser phototherapy (LP) on tissues of the submandibular gland (SMG) and parotid gland (PG). Wistar rats were randomly divided into experimental (A and B) and control (C) groups. A diode laser, 808 nm wavelength, in continuous wave mode, was applied to the PG, SMG and sublingual gland in the experimental groups on two consecutive days. The doses were 4xa0J/cm2 and 8xa0J/cm2, and total energy was 7xa0J and 14xa0J, respectively. The power output (500xa0mW) and power density (277xa0mW/cm2) were the same for both experimental groups. In order to visualize the area irradiated by the infrared laser, we used a red pilot beam (650xa0nm) with 3xa0mW maximum power for the experimental groups. For the control group, the red pilot beam was the only device used. The SMG and PG were removed after 1xa0week of the first irradiation. Total protein concentration, amylase, peroxidase, catalase and lactate dehydrogenase assays were performed, as well as histological analysis. Statistical tests revealed significant increase in the total protein concentration for groupsxa0A and B in the parotid glands (Pu2009<u20090.05). Based on the results of this study, LP altered the total protein concentration in rats’ parotid glands.

Hypermethioninemia provokes oxidative damage and histological changes in liver of rats

In the present study we evaluated the effect of chronic methionine administration on oxidative stress and biochemical parameters in liver and serum of rats, respectively. We also performed histological analysis in liver. Results showed that hypermethioninemia increased chemiluminescence, carbonyl content and glutathione peroxidase activity, decreased total antioxidant potential, as well as altered catalase activity. Hypermethioninemia increased synthesis and concentration of glycogen, besides histological studies showed morphological alterations and reduction in the glycogen/glycoprotein content in liver. Serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and glucose were increased in hypermethioninemic rats. These findings suggest that oxidative damage and histological changes caused by methionine may be related to the hepatic injury observed in hypermethioninemia.

Long- and short-term diabetes mellitus type 1 modify young and elder rat salivary glands morphology

OBJECTIVEnIn this study we performed a temporal analysis of the effects of Diabetes Mellitus on morphology and laminin deposition in salivary glands of young (2 months-old) and aging (12 months-old) male Wistar rats, using immunohistochemistry.nnnMATERIALS AND METHODSnThe animals were divided in control and diabetic (Streptozotocin induced) groups and euthanized after short and long-term diabetes induction.nnnRESULTSnShort-term induction led to vacuolization of parotid acinar cells and increased laminin deposition in both animal ages. In young rats, no difference was observed between short or long-term diabetes regarding laminin deposition, but parotid acinar cells vacuolization was more discrete after long-term diabetes. A slight decrease of submandibular gland convoluted granular ducts was observed in young and elder diabetic animal ages. In diabetic aging rats was observed an increase of laminin content only in the parotid gland.nnnCONCLUSIONSnThese results suggest that some Diabetes Mellitus effects on salivary glands are not progressive over time, possibly due to the existence of adaptive mechanisms in response to chronic hyperglycemia. They also show that the duration of the disease was more relevant to the morphological effects than the age, although it is known that aging per se affects salivary gland morphology and function.