Sra-1 interacts with Kette and Wasp and is required for neuronal and bristle development in Drosophila

Regulation of growth cone and cell motility involves the coordinated control of F-actin dynamics. An important regulator of F-actin formation is the Arp2/3 complex, which in turn is activated by Wasp and Wave. A complex comprising Kette/Nap1, Sra-1/Pir121/CYFIP, Abi and HSPC300 modulates the activity of Wave and Wasp. We present the characterization of Drosophila Sra-1 (specifically Rac1-associated protein 1). sra-1 and kette are spatially and temporally co-expressed, and both encoded proteins interact in vivo. During late embryonic and larval development, the Sra-1 protein is found in the neuropile. Outgrowing photoreceptor neurons express high levels of Sra-1 also in growth cones. Expression of double stranded sra-1 RNA in photoreceptor neurons leads to a stalling of axonal growth. Following knockdown of sra-1 function in motoneurons, we noted abnormal neuromuscular junctions similar to what we determined for hypomorphic kette mutations. Similar mutant phenotypes were induced after expression of membrane-bound Sra-1 that lacks the Kette-binding domain, suggesting that sra-1 function is mediated through kette. Furthermore, we could show that both proteins stabilize each other and directly control the regulation of the F-actin cytoskeleton in a Wasp-dependent manner.     

Effect of restricted feed intake and addition of vitamins B2 and B6 and folic acid on the liver concentration of zinc and copper in rats

The aim of the present study was to investigate the influence of nutritional deficiency and dietary addition of vitamins (B₂, B₆, and folate) on hepatic concentration of zinc and copper in rats. The experiment was performed on 260 growing male Wistar rats divided into 13 groups. Animals of 11 groups were fed isocaloric diets (14.7 MJ/kg) in which the 20% of energy was derived from protein. Another two groups of rats were offered diets with 9% or 4.5% of energy originating from protein. Animals of both mentioned groups and of the control group (20% of energy from protein) were offered diets ad libitum. The other 10 groups were offered 50% and 30% of the amount consumed in the control group. Eight groups, from those 10 restricted ones, were differentiated by dietary addition of vitamins B₂ and B₆ and folate (300% addition). Restricted feed intake did not affect the liver zinc concentration but significantly increased the copper concentration. The addition of vitamin B₆ decreased the liver Zn concentration. The highest liver Cu concentration was noted in rats offered restricted diets to only 30% of intake in the control group and high in vitamin B₂ and in rats supplemented with all of studied vitamins together. It suggests that vitamin B₂ had the strongest impact on liver Cu concentration in rats fed restricted diets.     

Diurnal rhythms of pinealocyte ultrastructure, pineal serotonin content and plasma melatonin level in the domestic pig

The study was conducted to investigate diurnal changes in pinealocyte ultrastructure, pineal serotonin content and plasma melatonin concentration in the domestic pig. The immature pigs (n=24) were kept under a cycle of 12 h light : 12 h dark, with a photophase between 0800 and 2000. During the photophase the animals were exposed to direct sunlight. After four weeks the gilts were slaughtered at 0900, 1400, 2100 and 0200. The pineals were removed and divided into two parts - one for quantitative ultrastructural study (by a point count method) and one for serotonin assay. Simultaneously, blood samples were taken for melatonin assay. The relative volume of mitochondria in pinealocyte perikarya was significantly higher at 1400 than at 0200 and 0900 as well as at 2100 than at 0200. The relative volume of Golgi apparatus was higher at 0900 and 1400 than at 0200. The relative volume of dense bodies of the MBB-1 type in pinealocyte perikarya was significantly lower at 1400 and 2100 than at 0900. In contrast, the relative volume of MBB-2 was higher at 1400 than at 0900 and 0200. The numerical density of DCV in perikarya was significantly higher at 0200 than at 1400. No significant differences were found in rough endoplasmic reticulum, lysosomes and multivesicular bodies. The pineal serotonin content showed a prominent rhythm with the maximum at 1400. The plasma melatonin concentration was significantly higher at 0200 than at 0900, 1400 and 2100. The obtained results demonstrate that both pinealocyte ultrastructure and pineal biochemistry in the pig undergo significant changes in the course of the diurnal rhythm.     

Simulating the embolization of blood vessels using magnetic microparticles and acupuncture needle in a magnetic field

Computer models were developed to simulate the capture and subsequent deposition of magnetic microparticles (MMPs) in a blood vessel adjacent to a ferromagnetic wire (e.g., acupuncture needle) magnetized by a uniform external magnetic field. Process parameter conditions were obtained to enable optimal capture of MMPs into the deposit. It was found that the maximum capture distance of the MMPs was within 0.5-2.0 mm when the particles were superparamagnetic and had large size (>1.0 microm) and relative large flow rates (2.5-5.0 cm/s) as in a healthy artery. It was also found that the deposits were asymmetrical and that their size was between 1.0 and 2.0 mm. For the case of lower flow rates as can be found in a tumor (<1.0 mm/s) and using small magnetite particles (0.25-2.0 microm) the maximum capture distance was larger, ranging between approximately 0.5 and 6.4 mm, depending on the blood flow rate, the radius of wire, and particle clustering. The range of embolization (deposition) in this later case was between 0.5 and 5.9 mm. The potential of this technique to generate MMPs deposits to embolize blood vessels inhibiting the blood supply and thus facilitating necrosis of tumors located deep within the patient (3-7 cm) is discussed.     

Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks

Humans and animals undergo ageing, and although their primary cells undergo cellular senescence in culture, the relationship between these two processes is unclear. Here we show that gamma-H2AX foci (gamma-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice. They colocalize with DSB repair factors, but not significantly with telomeres. These cryptogenic gamma-foci remain after repair of radiation-induced gamma-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of unrepairable DSBs may have a causal role in mammalian ageing.     

RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells

Repair of the airway epithelium after injury is critical for restoring normal lung. The reepithelialization process involves spreading and migration followed later by cell proliferation. Rho-GTPases are key components of the wound healing process in many different types of tissues, but the specific roles for RhoA and Rac1 vary and have not been identified in lung epithelial cells. We investigated whether RhoA and Rac1 regulate wound closure of bronchial epithelial cells. RhoA and Rac1 proteins were efficiently expressed in a cell line of human bronchial epithelial cells (16HBE) by adenovirus-based gene transfer. We found that both constitutively active RhoA and dominant negative RhoA inhibited wound healing, suggesting that both activation and inhibition of RhoA interfere with normal wound healing. Overexpression of wild-type Rac1 induced upregulation of RhoA, disrupted intercellular junctions, and inhibited wound closure. Dominant negative Rac1 also inhibited wound closure. Inhibition of the downstream effector of RhoA, Rho-kinase, with Y-27632 suppressed actin stress fibers and focal adhesion formation, increased Rac1 activity, and stimulated wound closure. The activity of both RhoA and Rac1 are influenced by the polymerization state of microtubules, and cell migration involves coordinated action of actin and microtubules. Microtubule depolymerization upon nocodazole treatment led to an increase in focal adhesions and decreased wound closure. We conclude that coordination of both RhoA and Rac1 activity contributes to bronchial epithelial wound repair mechanisms in vitro, that inhibition of Rho-kinase accelerates wound closure, and that efficient repair involves intact microtubules.     

Regulation of cell motility by tyrosine phosphorylated villin

Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.