Characterisation of Lactobacillus amylovorus DSM 16698 S-layer Proteins and Isolation of Putative S-layer Protein Receptors from Porcine Epithelial Cells

Surface (S-) layers, structural entities that surround the cell envelope of various bacteria, are comprised of a porous lattice of identical protein or glycoprotein subunits. Interestingly, the S-layer is able to promote adherence to host epithelial cells in a variety of Lactobacillus species. L. amylovorus DSM 16698, a strain of porcine origin, encodes at least three putative types of S-layer proteins in its genome sequence. In this study the surface structure of L. amylovorus DSM 16698 strain and the adhesion properties of its S-layer proteins to porcine intestinal epithelial IPEC-1 cell line were examined based on preliminary results. In addition, host receptors potentially specific for S-layer proteins were isolated from IPEC-1 cells. Cloned recombinant S-layer proteins rSlpA and rSlpB of DSM 16698 were reassembled onto fluorescent-labeled L. amylovorus cell wall extracts as a means to mimic the native S-layer lattice structure. Adhesion between the reassembled recombinant S-layer complexes and IPEC-1 cells was assessed qualitatively by microscopy and quantitatively by measuring fluorescence intensity. Results from in vitro adhesion assays indicate that the rSlpA and rSlpB proteins both mediated the adherence of the L. amylovorus DSM 16698 strain to porcine intestinal epithelial cells. Antibodymediated adhesion inhibition experiments were also performed, in which the two rSlps were pretreated with their specific anti-rSlp serum, and showed that adhesion between the rSlps and IPEC-1 cells could be inhibited by the antibody treatment. Moreover, by using fluorescent-labeled rSlp-specific antibody, the surface structure of L. amylovorus cells was microscopically examined. With this immunofluorescent technique, the SlpA and SlpB proteins were both observed to localize on the cell surface and exhibit a similar distribution pattern. Putative S-layer host cell receptors were isolated from the interaction between the reassembled rSlp/cell wall complexes and IPEC-1 derived membrane proteins using a SDS-PAGE-based system. Receptor isolation experiments resulted in repeatedly the same protein profile. It has previously been shown that L. amylovorus DSM 16698 attaches to IPEC-1 cells, but the identities of surface-localized components that mediate this microbe-host interaction had yet to be determined. In this present study, S-layer proteins were found to be an important mediator in the interaction between L. amylovorus DSM 16698 and a porcine epithelial cell line. Additionally, it was shown how S-layer proteins are localized on the surface of L. amylovorus DSM 16698 cells.Prokaryooteilla tavattava proteiinipintakerros (eng. S-layer) on identtisistä proteiini- tai glykoproteiiniyksiköistä koostuva huokoinen ja verkkomainen solun pintarakenne, jonka on havaittu monilla Lactobacillus-suvun bakteereilla välittävän bakteerin kiinnittymistä isäntänsä epiteelisoluihin. Porsaille probioottisen L. amylovorus DSM 16698 -bakteerin genomisekvenssistä on löydetty ainakin kolme mahdollista pintakerrosproteiinigeeniä. Tässä tutkimuksessa selvitettiin L. amylovorus DSM 16698 -bakteerin ulkopintarakennetta ja bakteerin pintakerrosproteiinien kiinnittymisominaisuuksia porsaan IPEC-1 suolistoepiteelisolulinjan soluihin alustavien tuloksien pohjalta. Lisäksi eristettiin mahdollisia pintakerrosproteiinireseptoreja IPEC-1 -soluista. L. amylovorus DSM 16698 -bakteerin kloonattuja rekombinanttipintakerrosproteiineja rSlpA ja rSlpB kiinnitettiin bakteerista eristettyihin, fluoresenssileimattuihin soluseiniin natiivin pintakerrosrakenteen mimikoimiseksi ja muodostuneiden kompleksien kiinnittymistä IPEC-1 -soluihin tutkittiin sekä kvalitatiivisesti mikroskopoiden että kvantitatiivisesti fluoresenssimittauksilla. Kokeiden perusteella molemmat Slp-proteiinit välittävät L. amylovorus DSM 16698 -bakteerin adheesiota porsaan suolistoepiteelisoluihin in vitro. Kiinnittymistä tutkittiin myös inhibitiokokeilla käsittelemällä rSlp-proteiinit ennen adheesiota proteiineja vastaan tuotetuilla vasta-aineseerumeilla. Seerumikäsittelyn jälkeen proteiinien kiinnittyminen IPEC-1 -soluihin heikkeni. L. amylovorus DSM 16698 -bakteerin ulkopintarakennetta tutkittiin immunofluoresenssikokeilla rSlp-proteiinien vasta-aineiden avulla. Molemmat Slp-proteiinit esiintyvät L. amylovorus DSM 16698 -bakteerisolun pinnalla ja jakautuvat keskenään samalla tavoin ympäri solun pintaa. IPEC-1 -soluista eristettiin mahdollisia pintakerrosproteiinireseptoreja kokeessa, jossa rSlp-proteiinit soluseinään kiinnitettyinä reagoivat IPEC-1 -soluista eristettyjen membraaniproteiinien kanssa ja mahdolliset reseptorit yhdessä rSlp-proteiinin kanssa havaittiin SDS-PAGE -ajossa. Reseptorin eristyksessä saatiin toistettavasti sama proteiiniprofiili. Aiemmin on osoitettu, että L. amylovorus DSM 16698 -bakteerikanta kiinnittyy IPEC-1 - epiteelisolulinjaan, mutta bakteerin adheesiotekijöitä ei ole tutkittu tarkemmin. Tämä tutkimus osoitti L. amylovorus DSM 16698 -bakteerikannan pintakerrosproteiinien lokalisaation solun pinnalla ja pintakerrosproteiinien roolin bakteerin kiinnittymisessä porsaan epiteelisolulinjan soluihin

Inter-fingercoordinated DC motor driven grasping robotic hand

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2009.Cataloged from PDF version of thesis.Includes bibliographical references (p. 16).Many robotic hands have individually driven joints, which is both energy inefficient and complicated to control. An alternative method includes employing only one motor to control multiple joints. Investigating humanoid hand movements used in everyday tasks, graduate students working with Professor Asada discovered through Principal Component Analysis that a majority of these motions can be approximated by a grasping motion. Implementing PCA results on a humanoid robotic hand yields an inter-finger coordinated motion in which each of the four fingers closes towards the palm and each individual joint's velocity is linearly proportional to the others. The coordination is achieved through a system of pulleys with different radii. The cables used in conjunction with these pulleys, emulating tendons, will be driven by a DC motor for simple control of a complex hand mechanism. The thumb is excluded from this system as it functions differently from the remaining digits.by Sarah Jane Wikman.S.B