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Biomedical subjects

B Kachar

Publications and source records attributed to B Kachar.

At least 73 records · Page 4Linked to original sources
PubMed1994

Modulation of tight junction morphology and permeability by an epithelial factor.

We report evidence of a factor secreted at the apical side of epithelial monolayers which modulates tight junction structure and permeability. This activity was detected within 4-7 days of conditioning of the apical medium by MDCK, A6 or Caco-2 epithelial cell lines cultured on permeable membranes in bipartite chambers. Apical conditioned medium (ACM), applied to the basolateral surface of a confluent monolayer, increased the transepithelial electrical resistance (TER), progressively reaching values 12-22% higher than the baseline within 5-10 min. After 40-60 min, the TER returned slowly to the basal value. This phenomenon was not observed either when using preheated ACM or the ACM filtrate obtained through a 30,000 MW cutoff membrane. The ACM maintained its activity even when applied to cell lines from different organs and species, as demonstrated when ACM from MDCK monolayers promoted an increase of 22% in the TER of Caco-2 cells. The increase of TER induced by the ACM treatment is accompanied by a change in the distribution of the number of tight junction strands, from an initial pattern, dominated mostly by junctions with one or two strands, to a new pattern after treatment dominated by junctions with two or three strands. Our results suggest the existence of a mechanism in epithelial cells that could signal leakage of apically secreted components to the basolateral side, thereby modulating the junction structure and permeability.

Animals↗
PubMed1993

Inhibition of outer hair cell electromotility by sulfhydryl specific reagents.

Mammalian outer hair cells can change length at acoustic frequencies when they are electrically stimulated. It was postulated that these length changes depend on electromechanical transduction based on voltage dependent conformational changes in a membrane motor protein. In this report, we describe the effect of various sulfhydryl (SH)-specific reagents on the OHC electromotility. p-Chloromercuriphenylsulfonate (pCMPS), in addition to other mercurials that can react with well-protected SH-groups in proteins, inhibits this electromechanical transduction process. In contrast, N-ethylmaleimide and diamide, SH-reagents that only react with exposed SH-groups, showed no effect. These results suggest that one or more reactive SH-groups are present in a functionally important and protected region of the electromechanical transduction protein. Such reactivity can be utilized to identify and characterize this novel membrane motor.

4-Chloromercuribenzenesulfonate↗
PubMed1993

Synthesis of RNA probes by the direct in vitro transcription of PCR-generated DNA templates.

We describe a novel method for the generation of RNA probes based on the direct in vitro transcription of DNA templates amplified by polymerase chain reaction (PCR) using primers with sequence hybrids between the target gene and those of the T7 and T3 RNA polymerases promoters. This method circumvents the need for cloning and allows rapid generation of strand-specific RNA molecules that can be used for the identification of genes in hybridization experiments. We have successfully applied this method to the identification of DNA sequences by Southern blot analysis and library screening.

Base Sequence↗
PubMed1993

Kinesin-mediated vesicular transport in a biochemically defined assay.

Here we have described simple and reproducible methods to observe kinesin-mediated vesicle and microtubule movements under defined conditions using video microscopy. We are optimistic that this assay will provide a useful tool to study kinesin function, regulation, and dynamic physical interactions with membranous organelles and microtubules.

Animals↗
PubMed1993

The structural organization of the pathogenic protozoan Tritrichomonas foetus as seen in replicas of quick frozen, freeze-fractured and deep etched cells.

The quick-freezing and freeze-etching technique was used to analyse the cytoskeleton of Tritrichomonas foetus, a pathogenic protozoan of the urogenital tract of cattle. The cytoplasm presented a network of filamentous structures interacting with each other, with the surface of the hydrogenosomes and the nuclear membrane. Two nm wide filamentous structures were found in the luminal space of the Golgi complex, connecting the two faces of each cisterna. The microtubules of the pelta-axostyle system were connected by bridges 30-40 nm long and 10 nm wide, regularly spaced with an interval of 25 nm. The costa is a structure formed by a complex array of filaments and globous structures. It seems to be connected to the recurrent flagellum through a complex network formed by 15 and 10 nm wide filaments which emerge from the peripheral region of the costa and penetrate into the surface projections of the protozoan body to which the recurrent flagellum is attached. Other filaments were seen connecting the surface of these projections with the surface of the flagellum.

Animals↗
PubMed1992

A membrane-based force generation mechanism in auditory sensory cells.

Auditory outer hair cells can elongate and shorten at acoustic frequencies in response to changes of plasma membrane potential. We show that this fast bidirectional contractile activity consists of an electromechanical transduction process that occurs at the lateral plasma membrane and can be activated and analyzed independently in small membrane patches inside a patch electrode. Bidirectional forces are generated by increases and decreases in membrane area in response to hyperpolarization and depolarization, respectively. We suggest that the force generation mechanism is driven by voltage-dependent conformational changes within a dense array of large transmembrane proteins associated with the site of electromechanical transduction.

Animals↗
PubMed1992

Structural domains of the tight junctional intramembrane fibrils.

Freeze-fracture reveals intramembrane fibrils lying along the intermembrane contacts that characterize tight junctions. Tight junctions from a variety of species are reexamined here by rapid freezing prior to freeze-fracture. The tight junction fibril is uprooted alternatively from either the cytoplasmic or the exoplasmic hemibilayer during freeze-cleavage, exposing two distinct but complementary views of its hybrid structure within the same replica. When the transmembrane fibril is uprooted from the exoplasmic hemibilayer it appears on the P-fracture face as a smooth-surfaced cylinder which is sometimes resolved into periodic globular structures. The lack of indication that the P-face cylinder has been pulled out through the opposite membrane half indicates that this domain of the fibril is, in large part, buried in the hydrophobic interior of the membrane. However, when the transmembrane fibril is uprooted from the cytosolic hemibilayer it appears on the E-fracture face as a row of irregular intramembrane particles. The irregular particles on the E-face aspect of the fibril are interpreted as corresponding to transmembrane protein segments that may very well make projections onto the cytosolic surface of the bilayer. En face views of the outermost junction strand between adjacent epithelial cells show periodic lines on the bilayer on each side of the junction which are interpreted as periodic transmembrane protein segments arising from the core structure of the tight junction fibril. If the backbone of the tight junction strand is an inverted cylindrical micelle, it must typically include proteins, which might anchor it to structures outside the membrane bilayer.

Animals↗
PubMed1992

An improved method for the purification of kinesin from bovine adrenal medulla.

A method has been developed for the purification of bovine adrenal kinesin combining ion exchange chromatography on phosphocellulose and Mono-Q (FPLC), affinity binding to microtubules in the presence of tripolyphosphate and gel filtration on Superose 6 (FPLC). From 100 g of tissue this procedure yields 200 micrograms of a remarkably pure kinesin as assayed by SDS-PAGE and electron microscopy of rotary shadowed specimens. The enzyme has a Ca++ ATPase of 0.4 mumol/min per mg and a Mg++ ATPase of 0.03 mumol/min per mg in the absence of microtubules. The addition of microtubules (5 microM) activates the Mg++ ATPase activity by almost 70-fold to a value of 1.9 mumol/min per mg. This purification procedure results in a fairly large amount of a remarkably pure adrenal kinesin with high specific activity which is an important improvement over the method previously available.

Adrenal Medulla↗
PubMed1992

Structure of the cortical cytoskeleton in mammalian outer hair cells.

The cortical cytoskeletal lattice in outer hair cells is a two-dimensional actin-based structure, which can be labelled with rhodamine/phalloidin and disrupted by the enzyme deoxyribonuclease I. Structural information from thin sectioned, freeze-etched and negatively stained preparations shows that it is based upon two types of filament that form a cross-linked lattice of circumferential filaments. The cross-links are 70-80 nm long. Measurements of the spacing between circumferential filaments suggest that the lattice is stiffer circumferentially than it is longitudinally. Analysis of the orientation of circumferential filaments shows that it is composed of discrete domains of up to 10 microns 2. Relative movements between domains could allow substantial changes of cell shape without disrupting the unit structure of the lattice, thus allowing the cell cortex to retain its elastic responses to high-frequency deformations.

Actins↗
PubMed1991

Stretch sensitivity of the lateral wall of the auditory outer hair cell from the guinea pig.

The inner and outer hair cells of the mammalian hearing organ are mechano-transducer cells. Here we report evidence that the lateral wall of outer hair cells (OHCs) is a mechano-receptor. This mechano-sensitivity appears to complement that of the stereocilia. Patch clamping studies showed that stretching of the membrane patches by suction at the pipette activated potassium channels with 130 pS unit conductance specifically localized in the lateral wall. Application of an osmotic tension to the entire cell membrane under whole-cell recording produced a 10 mV hyperpolarization. The reversal potential and the magnitude of the macroscopic current under voltage clamp were consistent with the single-channel properties of stretch-activated potassium channels. The elongated cylindrical cell body of the OHC is optimally positioned in the cochlea to sense axial force due to the vibrations of the basilar membrane during sound stimulation. This sensitivity can explain the production of a predominantly hyperpolarizing response to sound stimuli, unique to the OHC. Coupled with voltage-dependent OHC motility, the stretch-activated channels may play an important role in producing a mechanical feedback, an indispensable element in cochlear tuning.

Animals↗
PubMed1991

Purified kinesin promotes vesicle motility and induces active sliding between microtubules in vitro.

We examined the ability of kinesin to support the movement of adrenal medullary chromaffin granules on microtubules in a defined in vitro system. We found that kinesin and ATP are all that is required to support efficient (33% vesicle motility) and rapid (0.4-0.6 micron/s) translocation of secretory granule membranes on microtubules in the presence of a low-salt motility buffer. Kinesin also induced the formation of microtubule asters in this buffer, with the plus ends of microtubules located at the center of each aster. This observation indicates that kinesin is capable of promoting active sliding between microtubules toward their respective plus ends, a movement analogous to that of anaphase b in the mitotic spindle. The fact that vesicle translocation, microtubule sliding, and microtubule-dependent kinesin ATPase activities are all enhanced in low-salt buffer establishes a functional parallel between this translocator and other motility ATPases, myosin, and dynein.

Adenosine Triphosphatases↗
PubMed1991

The use of native thick filaments in in vitro motility assays.

Native thick filaments from the clam, Mercinaria mercinaria translocate actin filaments both toward and away from the center of the thick filament in an in vitro motility assay. The thick filaments from the adductor muscle are about 10 microns long whereas those from the catch muscle are 30-50 microns long. These thick filaments should prove useful in understanding the mechanism of myosin-dependent movement of actin filaments.

Actin Cytoskeleton↗
PubMed1990

Polarity and velocity of sliding filaments: control of direction by actin and of speed by myosin.

Myosin filaments, which are responsible for a large repertoire of motile activities in muscle and nonmuscle cells, can translocate actin filaments both toward and away from their central bare zone. This bidirectional movement suggests that there is enough flexibility in the head portion of the tightly packed myosin molecules in the native myosin filaments to move actin filaments not only in the expected direction, but also in the direction opposite to that predicted by the regular structure of muscle--away from the center of the myosin filament.

Actins↗
PubMed1990

Structural basis for mechanical transduction in the frog vestibular sensory apparatus: I. The otolithic membrane.

The mechanical coupling of the otoliths to the hair cell sensory stereocilia at the surface of the vestibular sensory epithelium is mediated by two layers of extracellular matrix, each one with a specific role in the mechanical transduction process. The first is a rigid layer in direct contact with the otolithic mass and is known as the otolithic membrane or gelatin membrane. This structure consists of a dense, randomly cross linked filament network that uniformly distributes the force of inertia of the non-uniform otolithic mass to all stereocilia bundles. The second layer formed by a columnar organization of filaments secures the otolithic membrane above the surface of the epithelium. The long columnar filaments are organized in parallel to the stereocilia bundles and are anchored to the apical surface of the supporting cells. The zonula adherens at the apical region of each supporting cell displays a thick polygonal bundle of actin filaments forming at the surface of the epithelium a transcellular honeycomb organization that provides mechanical ground support for the columnar filament layer. The dominant aspect of this columnar filament layer indicates that it may also have an important role in attenuating the force of inertia of the large otolithic mass during acceleration, screening stresses that would be directed to an effective bending of the stereocilia bundles.

Animals↗
PubMed1989

Spontaneous polymerization of the antibiotic peptide magainin 2.

We describe here the ability of the magainin 2 peptide to assemble spontaneously into characteristic 13-nm diameter filaments having a 30 nm periodic helical substructure. Optimal conditions for extensive polymerization into filaments of several hundred microns required low pH and high ionic strength. Polymerization of the magainin 2 peptide may be involved in its recently described in vitro membrane-disrupting and antibiotic activities.

Animals↗
PubMed1989

Fast in vitro movement of outer hair cells in an external electric field: effect of digitonin, a membrane permeabilizing agent.

Isolated outer hair cells from the organ of Corti show elongation and contraction in response to an externally applied ac electric field as well as to a direct current injection into these cells. This is thought to be the basis of the positive feedback mechanism for fine tuning of the mammalian hearing organ. To test whether the mechanical response depends on the intracellular electric field or on the membrane potential, we used digitonin to shunt the membrane resistance. We observed that the application of digitonin abolished the cellular response of the outer hair cells to an ac external electric field (5-30 Hz). Coinciding with the abolition of the cellular response, the nuclear matrix started to oscillate synchronous to the external field, indicating an appreciable increase of the intracellular electric field. If the intracellular electric field was the regulating factor of the motile response, the initiation of the movement of the nuclear matrix would have been accompanied by an enhancement of the cellular movement. Our observation is therefore consistent with the interpretation that the (local) membrane potential, and not the intracellular electric field, regulates the hair cell movement.

Cell Membrane Permeability↗
PubMed1988

The mechanism of cytoplasmic streaming in characean algal cells: sliding of endoplasmic reticulum along actin filaments.

Electron microscopy of directly frozen giant cells of characean algae shows a continuous, tridimensional network of anastomosing tubes and cisternae of rough endoplasmic reticulum which pervade the streaming region of their cytoplasm. Portions of this endoplasmic reticulum contact the parallel bundles of actin filaments at the interface with the stationary cortical cytoplasm. Mitochondria, glycosomes, and other small cytoplasmic organelles enmeshed in the endoplasmic reticulum network display Brownian motion while streaming. The binding and sliding of endoplasmic reticulum membranes along actin cables can also be directly visualized after the cytoplasm of these cells is dissociated in a buffer containing ATP. The shear forces produced at the interface with the dissociated actin cables move large aggregates of endoplasmic reticulum and other organelles. The combination of fast-freezing electron microscopy and video microscopy of living cells and dissociated cytoplasm demonstrates that the cytoplasmic streaming depends on endoplasmic reticulum membranes sliding along the stationary actin cables. Thus, the continuous network of endoplasmic reticulum provides a means of exerting motive forces on cytoplasm deep inside the cell distant from the cortical actin cables where the motive force is generated.

Actins↗