Ko Nakajima

Isolation of kinesin from rat liver.

Kinesin is a translocator protein which mediates microtubule-based transport of organelle and vesicles. Microtubules support major roles in some intracellular transport systems in a variety of cells. However, it is not known which proteins comprise the translocator in the liver 9 In this study, we determined the molecular weight and motility of hepatic kinesin. The translocator protein fraction, isolated from rat liver by a modification of Vales method, consisted of many kind of protein species as identified by SDS-PAGE. The most prominent of these was a ll6kDa protein. The ll6kDa polypeptide had property of forming a high affinity complex with microtubules in the presence of nonhydrolyzable ATP analog, adenylyl imidodiphosphate(AMP-PNP). The ll6kDa polypeptide released from the microtubules with ATP reacts with monoclonal antibodies to sea urchin and chicken brain kinesin heavy chain (Fig.A,B). Gliding of reconstituted microtubules in the presence of ATP on the ll6kDa polypeptide treated with glass plate was visualized with DIC-video enhanced A B microscopy. The mean i ~ 3 ~ 5 6 7 s i 2 3 4 5 6 7 s v oywa0000 1 IIi o u I,, 9 116K-.,QKHC 116-97.~indicated that the ,~_ ~ == ll6kDa polypeptide ,s~,r~ 4~from rat liver was ~ __~i__ kinesin heavy chain. Figure: Electrophoretic and immunoblotting pattern obtained in the of the preparation of kinesin from rat liver extract. A: SDA-PAGE with silver staining. B: Immunoblotting using sea urchin kinesin mAb. Lanel: Supernatant after 150000xg centrifugation. 2: Microtubule containing pellet. 3: Supernatant after wash in buffer containing ATP. 4: Pellet after ATP extraction. 5: Supernatant after wash in buffer containing GTP. 6: Pellet after GTP extraction 9 7: Supernatant after wash in buffer containing ATP and AMP-PNP. 8: Pellet after ATP and AMP-PNP extraction.

Purification and characterization of cytoplasmic dynein of rabbit liver

Cytoplasmic dynein is a microtubule-dependent motor protein, which plays a role in intracellular transport. However, there have been few studies regarding the role of cytoplasmic dynein in the liver. Purification of cytoplasmic dynein from rabbit liver took advantage of the affinity of microtubule-dependent motor proteins for microtubules. Purified dynein contained heavy chain (450 kDa), intermediate chain (75 kDa), light chains (45-58 kDa) and dynactin (150 kDa). The subunit composition was consistent with previously reported data on brain cytoplasmic dynein. Microtubules prepared from bovine brain were driven by purified cytoplasmic dynein from rabbit liver, and movements of microtubules were visualized by video-enhanced differential interference contrast microscopy. The mean velocity of the motile microtubules was 1.09 +/- 0.13 microns/s. Our study provides evidence of rapid intracellular transport in hepatocytes controlled by cytoplasmic dynein.

Effects of acetaldehyde on microtubules and hepatic kinesin:a study of the pathogenesis of alcoholic liver disease

Abstract In ballooned hepatocytes associated with alcoholic liver disease, secretory proteins are abundant, possibly due to an impairment of the microtubule-dependent vesicular transport system. We studied the effects of ethanol and acetaldehyde on microtubules and hepatic kinesin function. Tubulin and microtubule-associated protein 2 was purified by phosphocellulose and hydroxyapatite column chromatography from microtubule proteins of bovine brain. Hepatic kinesin was purified from rabbit liver. The assembly of microtubules was quantified by spectro-photometer. The kinesin motility assay is done with or without various concentrations of ethanol or acetaldehyde using video-enhanced differential interference contrast microscopy. Ethanol had no effect on the assembly or disassembly of the microtubules or on hepatic kinesin function, even at high concentrations of up to 100 mM. In contrast, acetaldehyde reduced hepatic kinesin function at concentrations greater than or equal to 100 μ M and induced microtubular disassembly at concentrations greater than or equal to 200 μ M. In alcoholic liver injury, chronic reduction of hepatic kinesin function may cause retention of secretory proteins and hepatocyte swelling. Moreover, reduction of the microtubule concentrations due to acetaldehyde may further impair the vesicular transport system and reduce the cells ability to maintain morphology, in turn accelerating hepatocyte ballooning.

A Multicentre Randomized Controlled Trial of Recombinant Interferon-Alpha-2a in the Treatment of Patients with Chronic Hepatitis C

Sixty-one chronic hepatitis C patients were randomly assigned to receive either 6 x 10(6) or 9 x 10(6) U of recombinant interferon-alpha-2a (IFN alpha-2a) six days a week for the first two weeks of treatment, followed in both cases by 6 x 10(6) U three days a week for the next 22 weeks. In the low dose group, 11 patients showed a complete response maintained for at least six months, 12 responded but then relapsed and nine did not respond; the corresponding figures in the high dose group were 10, 15 and five patients, respectively. The differences between groups are not statistically significant. Thus, this study provides no evidence of therapeutic benefit from increasing the initial dose of IFN alpha-2a. In both treatment groups, complete responders had significantly lower pretreatment viral titres than nonresponders and were significantly more likely to be infected by type 2a versus type 1b virus.

Enhancement of kinesin-driven microtubule gliding by bile acids

Abstract We tested the effects of four bile acids, taurochenodeoxycholate, taurocholate, tauroursodeoxycholate and taurodeoxycholate, on kinesin-driven microtubule motility, which may have a major influence on membrane organelle transport in cholestasis. Reconstituted microtubules were incubated with kinesin purified from rabbit liver and brain. Binding and gliding of microtubules were visualized by video-enhanced differential interference contrast microscopy. Liver and brain kinesin produced similar microtubule gliding velocities. The effect of bile acids on liver and brain kinesin was also similar. Low concentrations of the four bile acids (0.25–0.5 mM) enhanced the speed of microtubule gliding by up to 152%. Higher concentrations of taurochenodeoxycholate and taurodeoxycholate inhibited attachment, but did not slow the speed of gliding. Detachment was inhibited by the addition of tauroursodeoxycholate. ATPase activity showed no changes in the presence of low concentrations of tauroursodeoxycholate and taurochenodeoxycholate. Triton X-100, a nonionic detergent, increased microtubule gliding by up to 147% at lower concentrations, while higher concentrations (up to 5%) caused a return to normal speed without detachment. Low concentrations of bile acids in hepatocytes may enhance kinesin-driven microtubule dependent organelle transport. In contrast, higher concentrations of toxic bile acids, chenodeoxycholate and deoxycholate, associated with cholestasis may inhibit kinesin-driven motility by detaching microtubules from kinesin.