Kinesin's tail domain is an inhibitory regulator of the motor domain.

When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.

Organelle transport and sorting in axons.

Recent advances of the past year in the field of organelle transport have documented the existence of numerous kinesin-related proteins and the presence of multiple conventional kinesins within neurons. Biochemical and genetic mutant analyses indicate that different kinesin superfamily members transport different organelles. In addition to microtubule-based systems, actin filaments and myosin motors are associated with organelle transport in axons. The great diversity of motor proteins suggests that they may play a role in sorting, in addition to transport.

Microbial mineralization of diisopropanolamine.

Diisopropanolamine (DIPA) is a "sweetening agent" used to remove hydrogen sulfide from sour natural gas, and it is a contaminant at some sour gas treatment facilities in western Canada. To investigate the biodegradation of this alkanolamine, 14C-DIPA was used in anaerobic and aerobic mineralization studies. Between 3 and 78% of the radioactivity from this compound was released as 14CO2 in sediment-enrichment cultures incubated under nitrate-reducing conditions. Similarly, 12-78% of the label was converted to 14CO2 in sediment-enrichment cultures incubated under Mn(IV)-reducing conditions. These activities were observed at 8 degrees C, a typical groundwater temperature in western Canada, and at 28 degrees C. In contrast, DIPA-degrading activity was difficult to sustain under Fe(III)-reducing conditions, and < 25% of the radioactive label from 14C-DIPA was liberated as 14CO2. Two mixed cultures and two isolates (both irregular, non-sporeforming, Gram-positive rods) were used to assess aerobic mineralization of 14C-DIPA. The aerobic mixed cultures released 73 and 79% of the radioactive label as 14CO2, whereas the pure cultures liberated only 39 and 47% as 14CO2. Between one-third and one-half of the nitrogen from DIPA was found as ammonium-N in aerobic batch cultures. These results clearly demonstrate that DIPA is mineralized under a variety of incubation conditions.

Kinesin takes one 8-nm step for each ATP that it hydrolyzes.

Conventional kinesin is a motor protein that moves stepwise along microtubules carrying membrane-bound organelles toward the periphery of cells. The steps are of amplitude 8.1 nm, the distance between adjacent tubulin binding sites, and are powered by the hydrolysis of ATP. We have asked: how many steps does kinesin take for each molecule of ATP that it hydrolyzes? To answer this question, the motility and ATP hydrolysis of recombinant, heterotetrameric and homodimeric conventional Drosophila kinesins adsorbed to 200-nm-diameter casein-coated silica beads were assayed under identical, single-molecule conditions. Division of the speed by the maximum microtubule-activated ATPase rate gave a stoichiometry of 1. 08 +/- 0.09 steps for each ATP hydrolyzed at 1 mM ATP. Therefore, under low loads in which the drag force << 1 pN, coupling between the chemical and mechanical cycles of kinesin is tight, consistent with conventional power stroke models. Our results rule out models that require two or more ATPs/step, such as some thermal ratchet models, or that propose multiple steps powered by single ATPs.

Postoperative course after portacaval anastomosis in rats is determined by the portacaval pressure gradient.

Variability in experimental results have led to criticism regarding the validity of the rat after portacaval anastomosis (PCA) as a model of changes induced by portal-systemic shunting (PSS). A nonsuture technique using cyanoacrylate glue has been reported to yield a better experimental preparation. To investigate if variations in splanchnic hemodynamics could explain different outcomes after the procedure, male rats received either an end-to-side PCA or sham operations (16 rats each). The PCA was constructed using the "suture" or "glue" technique (8 rats each). Beginning on postoperative day 24 under methoxyflurane and ketamine anesthesia, pressures were recorded from the portal vein, inferior vena cava, and femoral artery. Blood flow to the splanchnic organs and the percent PSS were assessed using the microsphere technique. The rate of delivery of NH3 from the portal vein to the systemic circulation and the concentration of glutamine in the cerebrospinal fluid were determined. In PCA rats, weight gain was significantly impaired, and all animals had evidence of liver atrophy (in both suture and glue groups) when compared with sham animals; a trend toward greater weight gain was seen in glue rats. Portal vein inflow, PSS, NH3 delivery, and CSF glutamine were significantly increased in both PCA-suture and PCA-glue animals compared with sham rats, although no significant differences were seen between the two PCA techniques. When PCA rats from either technique were grouped according to the pressure gradient between portal vein and inferior vena cava, striking differences between animals were now evident.(ABSTRACT TRUNCATED AT 250 WORDS)

A low-protein diet ameliorates disrupted diurnal locomotor activity in rats after portacaval anastomosis.

In previous studies we noted a disruption of the circadian locomotor activity rhythm in rats after portacaval anastomosis (PCA). To examine whether this abnormality is related to factors that aggravate hepatic encephalopathy in humans, we studied the effect of dietary formulation and protein content on body weight, locomotor activity, and entrainment to the light-dark cycle in rats after PCA or sham operation. Postoperative weight loss was prevented by pair-feeding with a purified liquid diet. However, the behavioral abnormalities persisted in PCA rats fed a high-protein diet, with a reduction in total activity and entrainment to the light-dark cycle. These were ameliorated by a low dietary protein content. Since this treatment reduces the load of gut-derived nitrogenous substances that might alter brain metabolism, our data strengthen the hypothesis that the abnormal circadian activity patterns in PCA rats may be part of the spectrum of hepatic encephalopathy.

Portal-systemic shunting and the disruption of circadian locomotor activity in the rat.

To determine if the extent of portal-systemic shunting (PSS) influences the disruption of circadian function in chronic liver disease, locomotor activity was examined in two rat models with varying degrees of PSS, i.e., portal vein ligation (PVL) and end-to-side portacaval anastomosis (PCA). Animals were housed in individual activity cages under conditions of 12 hour light/12 hour darkness (weeks 0-3), then under conditions of constant dim light (weeks 4-7). Cages were equipped with running wheels connected to a continuous recorder, and daily tracings of running activity were recorded for 7 weeks. Computer analysis of wheel revolutions per hour with a chi 2 periodogram was used to calculate Qp, a measure of the amplitude of a circadian rhythm. The degree of PSS was measured by means of radioactive microspheres injected into the ileocolic vein and spleen. PVL rats were found to have PSS from the splenic and mesenteric territories of 88% and 27%, respectively; circadian periodicity was maintained in all PVL rats. PCA rats had complete shunting (greater than 99%) and showed a range of disrupted circadian rhythms from blunting of the amplitude to complete absence of the locomotor activity rhythm. This spectrum of disorganization occurred in spite of similar degrees of liver atrophy and weight gain. Whereas PCA in rats markedly disturbs the circadian rhythm of locomotor activity, animals with considerably less PSS from PVL exhibit normal behavior. The extent of PSS could be a variable affecting the expression of circadian rhythms in liver disease.