The role of kinesin and other soluble factors in organelle movement along microtubules

Kinesin is a force-generating ATPase that drives the sliding movement of microtubules on glass coverslips and the movement of plastic beads along microtubules. Although kinesin is suspected to participate in microtubule-based organelle transport, the exact role it plays in this process is unclear. To address this question, we have developed a quantitative assay that allows us to determine the ability of soluble factors to promote organelle movement. Salt-washed organelles from squid axoplasm exhibited a nearly undetectable level of movement on purified microtubules. Their frequency of movement could be increased greater than 20-fold by the addition of a high speed axoplasmic supernatant. Immunoadsorption of kinesin from this supernatant decreased the frequency of organelle movement by more than 70%; organelle movements in both directions were markedly reduced. Surprisingly, antibody purified kinesin did not promote organelle movement either by itself or when it was added back to the kinesin-depleted supernatant. This result suggested that other soluble factors necessary for organelle movement were removed along with kinesin during immunoadsorption of the supernatant. A high level of organelle motor activity was recovered in a high salt eluate of the immunoadsorbent that contained only little kinesin. On the basis of these results we propose that organelle movement on microtubules involves other soluble axoplasmic factors in addition to kinesin.     

Effects of mot gene expression on the structure of the flagellar motor

Direct freezing procedures have enabled us to visualize distinctive intramembrane particle ring structures in the cytoplasmic membranes of peritrichously flagellated bacteria by means of freeze-fracture electron microscopy. These structures were identified as flagellar motor components because their distribution matched that of flagella, and because they were absent in non-flagellated mutants of Escherichia coli. Particle rings were present in both the Gram-positive Streptococcus and the Gram-negative E. coli. In E. coli, a non-functional mocha operon produced flagellated but immotile cells lacking the particle rings. Simultaneous introduction of the motA and motB genes, led to recovery of both motility and the ring structures but neither gene alone was sufficient. The concomitant loss of the rings and motility is consistent with the ring particles having a central role in the flagellar motor.     

Periodic acid-induced blastogenesis of lymphocytes is independent of phosphoinositide turnover

The blastogenic transformation of lymphocytes by periodic acid was investigated to determine if blastogenesis induced by this mitogen was preceded by phosphoinositide turnover as previously shown for the lectins. Although periodate oxidation stimulated nucleic acid synthesis and interleukin-2 production, no changes in phosphoinositide turnover could be detected when compared to control lymphocyte cultures. These data indicate that increased phosphoinositide turnover is not an absolute prerequisite for lymphocyte proliferation.     

Characterization of the DNA-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin

The DNA-binding properties of the receptor for 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated using chromatography on DNA-cellulose columns. A maximal binding of about 40% of the total receptor complex to DNA-cellulose was observed. In order to interact with DNA, the receptor must first bind TCDD. A heat-activation step followed by gel permeation chromatography using Sephadex G-25 increased the binding of the cytosolic receptor to DNA. The DNA-binding ability of the receptor was almost lost following mild proteolysis using trypsin or alpha-chymotrypsin, although these treatments did not reduce its ligand binding capacity and had no apparent effect on its size. Furthermore, pre-treatment of the DNA-cellulose column with an intercalating drug, ethidium bromide, resulted in inhibition of the binding of the TCDD-receptor complex to DNA, indicating that not only electrostatic interactions but also the configuration of DNA are of importance in receptor-DNA interactions.     

Sugar utilization by developing wild type and shrunken-2 maize kernels

To characterize the movement of sugars during kernel development in maize, a newly devised in vitro kernel development scheme was utilized. Viable seeds of wild type maize (Zea mays L.) as well as the mutant shrunken-2 (sh2) were found to mature when grown in culture with reducing sugars or sucrose as the carbon source. However, wild type and sh2 kernels had greater germination, starch content, and seed weight when sucrose, rather than reducing sugars, was the carbon source. By the use of labeled sucrose it was shown that sucrose can move into endosperm tissue without intervening degradation and resynthesis. These results show that when grown in vitro the maize seed can utilize reducing sugars for development, but it prefers sucrose.     

Dynamic morphology of calcium-induced interactions between phosphatidylserine vesicles.

Structural changes in phosphatidylserine vesicles exposed to calcium chloride for various times have been observed by means of video-enhanced light microscopy and freeze-fracture electron microscopy. Large flat double-bilayer diaphragms form at the contacts between aggregated vesicles within milliseconds. Bilayers at and outside of diaphragms rupture and allow vesicles to collapse completely by flattening against each other within seconds. Collapse through intermediate states to a stable multilamellar phase is complete within minutes. The Ca-induced attraction energy and the resultant flattening at contacts between vesicles is far beyond that needed to stress bilayers to the point of rupture. Although the destabilizing response to this stress is preferential to the diaphragm region, 40% of adhering pairs rupture outside of the diaphragm region rather than fuse with each other. In this respect the mechanism of fusion between these vesicles may be fundamentally different from the controlled fusion process in cells.     

Quantity and quality of seston in an impounded and a free-flowing river in Virginia,U.S.A.

Seston in the impounded North Anna River (NAR) was analyzed and compared to that in the free-flowing South Anna River (SAR) in Virginia, U.S.A. A wet filtration technique was used to separate seston into five size classes. The overall quantity of organic seston was much lower in the NAR than in the SAR. The seston in the NAR was composed of more living organisms, in particular zooplankters in the medium large (234–864 µm) and small (105–234 µm) size classes and diatoms and other algae in the fine (43–105 µm) and very fine (25–43 µm) size classes. The percentage of zooplankton declined sharply before reaching the downstream study site (32 km). The seston in the NAR tended to consist of slightly larger particles, but 80–85% of the seston in both rivers was in the ultrafine (0.45–25 µm) size class, which was almost entirely composed of detritus. One indication of seston food quality, the organic/inorganic ratio, was considerably higher for the medium large and small size classes in the NAR immediately below the dam and for the fine and very fine size classes farther downstream. Another indication of food quality, the usable caloric content, showed that the seston in the NAR contained considerably less total assimilable energy as far as 32 km downstream from the dam, but that the medium large and small size classes were rich in assimilable energy immediately below the dam because of zooplankton released from the reservoir. Thus, the overall quantity and quality of seston was lower in the NAR, but the quality of the seston, for at least a short distance below the dam, was higher in a well-defined size range that can be effectively utilized by certain filter feeders.     

Analogue inhibitor of 2-5A action: effect on the interferon-mediated inhibition of encephalomyocarditis virus replication.

Chemically synthesised CH3Sp(A2'p)2A2'pp3'OCH3 has been used to assess the importance of the ppp(A2'p)nA (n greater than or equal to 2: 2-5A) system in the antiviral action of interferon against encephalomyocarditis virus (EMC). It inhibits activation of the 2-5A-dependent RNase by 2-5A in intact mouse L929 cells and cell-free systems. In interferon-treated, EMC-infected L929 cells it inhibits 2-5A-mediated rRNA cleavage and partially restores EMC RNA synthesis and virus yield. Activation of the 2-5A-dependent RNase must, therefore, play some part in interferon action against the growth of EMC virus in such cells.     

Mobilization of cloned luciferase genes into Vibrio harveyi luminescence mutants

A recombinant plasmid which carried a 5 kb fragment of Vibrio harveyi DNA containing the luxA and luxB genes was mobilized from Escherichia coli into luminescence-deficient mutants of V. harveyi. The cloned genes complemented a temperature sensitive luciferase mutation, but failed to complement lesions in two different aldehyde deficient mutants. Expression of the cloned genes was not subject to autoinduction in either E. coli or in V. harveyi.