Micropatterning of endothelial cells by guided stimulation with angiogenic factors

Micropatterning technology holds significant promise in the development of micro/nanomedical devices. The precise control of cell position and migration is important in several applications. For example, the optimal design of implantable devices depends on the implant material's micro-and nano-texture, which influences the response of nearby tissue, including the microvessels. Therefore, we were interested in endothelial cell positioning and colonization on specific surface domains in the size range of microvasculature. To this end, endothelial cells were seeded in microfabricated grooves and exposed to vascular endothelial growth factor (VEGF), which plays a key role in the angiogenic response. Patterned silicon wafers with grooves of 50 microm width and depth and 150 microm groove spacing were used. Each patterned region had two semicircular ports at either end, one of which was used to seed human retinal endothelial cells (HREC) and the other to house VEGF embedded in Matrigel. After 1 week, cells were fixed and analyzed by laser scanning cytometry (LSC). Our results shows that we can control HREC seeding and positioning in surface grooves and that the speed of colonization of the grooves can be manipulated by local VEGF application. We were able to quantify this effect, showing that HREC relocate inside the grooves twice as fast in response to VEGF stimulation, compared to control conditions, at a speed of 3.14 +/- 0.01 and 1.55 +/- 0.01 microm/min, respectively. Our approach could be used towards the fabrication of "designer" substrates or devices that not only allow patterned cell growth, but also permit dynamic cell repositioning.     

Idiopathic Parkinsonism Treated with an Extracerebral Decarboxylase Inhibitor in Combination with Levodopa

The clinical actions of levodopa in Parkinsonism, given with and without an extracerebral decarboxylase in hibitor, L-alpha-methyldopahydrazine, were compared. Twenty-one patients were investigated in a “double-blind cross-over” study, administering levodopa in maximum tolerated dosage. L-Alpha-methyldopahydrazine failed to augment the overall therapeutic actions of levodopa but it consistently alleviated nausea. It is concluded that L-alpha-methyldopahydrazine will prove useful in the management of some Parkinsonian patients who have difficulty in taking levodopa alone.     

Podocyte-secreted angiopoietin-like-4 mediates proteinuria in glucocorticoid-sensitive nephrotic syndrome

The main manifestations of nephrotic syndrome include proteinuria, hypoalbuminemia, edema, hyperlipidemia and lipiduria. Common causes of nephrotic syndrome are diabetic nephropathy, minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) and membranous nephropathy. Among the primary glomerular diseases, MCD is usually sensitive to glucocorticoid treatment, whereas the other diseases show variable responses. Despite the identification of key structural proteins in the glomerular capillary loop which may contribute to defects in ultrafiltration, many of the disease mechanisms of nephrotic syndrome remain unresolved. In this study, we show that the glomerular expression of angiopoietin-like-4 (Angptl4), a secreted glycoprotein, is glucocorticoid sensitive and is highly upregulated in the serum and in podocytes in experimental models of MCD and in the human disease. Podocyte-specific transgenic overexpression of Angptl4 (NPHS2-Angptl4) in rats induced nephrotic-range, and selective, proteinuria (over 500-fold increase in albuminuria), loss of glomerular basement membrane (GBM) charge and foot process effacement, whereas transgenic expression specifically in the adipose tissue (aP2-Angptl4) resulted in increased circulating Angptl4, but no proteinuria. Angptl4(-/-) mice that were injected with lipopolysaccharide (LPS) or nephritogenic antisera developed markedly less proteinuria than did control mice. Angptl4 secreted from podocytes in some forms of nephrotic syndrome lacks normal sialylation. When we fed the sialic acid precursor N-acetyl-D-mannosamine (ManNAc) to NPHS2-Angptl4 transgenic rats it increased the sialylation of Angptl4 and decreased albuminuria by more than 40%. These results suggest that podocyte-secreted Angptl4 has a key role in nephrotic syndrome.     

Semen collection,cryopreservation and artificial insemination in the dromedary camel

Collection of semen with a bovine artificial vagina (AV) was attempted with each of 14 camels over a period of 2 years. Semen samples were evaluated, extended and cryopreserved. Frozen thawed semen, diluted cooled semen or whole semen was used to inseminate some female camels which were induced to ovulate with hCG. Males ejaculated semen into the AV in 74.6% collection attempts. The male copulated for at least 200s in 62.9% attempts. The remaining copulations were of shorter duration. Similarly, 49.3% ejaculates were at least 3ml of semen. Libido and donation of semen improved from December onwards and reached a peak after mid January with peak performance persisting until April. It declined during May. The majority of camels had lost libido and refuse to donate semen by the end of May. Camel semen is in gel form. While 35.9% of 203 semen samples exhibited no individual sperm motility, 28.5% exhibited low to fair grade individual sperm motility and only 35.4% exhibited >50% sperm motility. Differences existed between animals (P<0.01) and months (P<0.05) of collection, while effect of copulation time was not significant. Mass motility was not observed in camel semen. Individual sperm motility develops after liquefaction of semen. Addition of caffeine but not chymotrypsin improved the individual motility. The mean live percent sperm count and normal acrosome were 73.3+/-1.0 and 92.0+/-0.5, respectively. Only 51.1% of 45 semen samples with pre-freeze motility of >50% and 25% of 16 semen samples from low pre-freeze motility group with an overall success of 44.2% of 61 semen samples were successfully preserved. Wide variation was observed in the freezability of semen from different males. Attempts to impregnate female camels with liquid semen, frozen thawed semen and whole semen after hCG induced ovulation resulted in 0/10, 1/13 and 4/10 pregnancies.     

Candida bombicola cells immobilized on patterned lipid films as enzyme sources for the transformation of arachidonic acid to 20-HETE

Preparation of biocompatible surfaces for immobilization of enzymes and whole cells is an important aspect of biotechnology due to their potential applications in biocatalysis, biosensing, and immunological applications. In this report, patterned thermally evaporated octadecylamine (ODA) films are used for the immobilization of Candida bombicola cells. The attachment of the cells to the ODA film surface occurs possibly through nonspecific interactions such as hydrophobic interactions between the cell walls and the ODA molecules. The enzyme cytochrome P450 present in the immobilized yeast cells on the ODA film surface was used for the transformation of the arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The assembly of cells on the hydrophobic ODA surface was confirmed by quartz crystal microgravimetry (QCM), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). SEM images confirmed the strong binding of the yeast cells to the ODA film surface after biocatalytic reactions. Moreover, the biocomposite films could be easily separated from the reaction medium and reused.     

High glucose stimulates synthesis of fibronectin via a novel protein kinase C,Rap1b,and B-Raf signaling pathway

The molecular mechanism(s) by which high glucose induces fibronectin expression via G-protein activation in the kidney are largely unknown. This investigation describes the effect of high glucose (HG) on a small GTP-binding protein, Rap1b, expression and activation, and the relevance of protein kinase C (PKC) and Raf pathways in fibronectin synthesis in cultured renal glomerular mesangial cells (MCs). In vivo experiments revealed a dose-dependent increase in Rap1b expression in glomeruli of diabetic rat kidneys. Similarly, in vitro exposure of MCs to HG led to an up-regulation of Rap1b with concomitant increase in fibronectin (FN) mRNA and protein expression. The up-regulation of Rap1b mRNA was mitigated by the PKC inhibitors, calphostin C, and bisindolymaleimide, while also reducing HG- induced FN expression in non-transfected MCs. Overexpression of Rap1b by transfection with pcDNA 3.1/Rap1b in MCs resulted in the stimulation of FN synthesis; however, the PKC inhibitors had no significant effect in reducing FN expression in Rap1b-transfected MCs. Transfection of Rap1b mutants S17N (Ser --> Asn) or T61R (Thr --> Arg) in MCs inhibited the HG-induced increased FN synthesis. B-Raf and Raf-1 expression was investigated to assess whether Rap1b effects are mediated via the Raf pathway. B-Raf, and not Raf-1, expression was increased in MCs transfected with Rap1b. HG also caused activation of Rap1b, which was largely unaffected by anti-platelet-derived growth factor (PDGF) antibodies. HG-induced activation of Rap1b was specific, since Rap2b activation and expression of Rap2a and Rap2b were unaffected by HG. These findings indicate that hyperglycemia and HG cause an activation and up-regulation of Rap1b in renal glomeruli and in cultured MCs, which then stimulates FN synthesis. This effect appears to be PKC-dependent and PDGF-independent, but involves B-Raf, suggesting a novel PKC-Rap1b-B-Raf pathway responsible for HG-induced increased mesangial matrix synthesis, a hallmark of diabetic nephropathy.     

Use of real-time ultrasonography for control of follicular activity and pregnancy diagnosis in the one humped camel (Camelus dromedarius) during the non-breeding season

Ovaries of 16 adult pleuriparous, non-pregnant and non-lactating one humped female camels (Camelus dromedarius) belonging to National Research Centre on Camel, at Bikaner, India, were examined for the presence of follicular activity (< or = 0.5 cm diameter) using real-time ultrasonography during June-August, which is considered to be non-breeding season in India. Follicles > or = 1.0 cm diameter were found in eight females. These animals were mated with virile studs. In four out of eight camels pregnancy was confirmed by progesterone assay and ultrasonography. The study shows that pre-ovulatory follicle may develop in some female camels during June-August (non-breeding season in India) and successful pregnancies may be achieved after mating of individual animals during this period.