Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood-brain barrier.

There is an increasing need to develop improved systems for predicting the safety of xenobiotics. However, to move beyond hazard identification the available concentration of the test compounds needs to be incorporated. In this study cyclosporine A (CsA) was used as a model compound to assess the kinetic profiles in two rodent brain cell cultures after single and repeated exposures. CsA induced-cyclophilin B (Cyp-B) secretion was also determined as CsA-specific pharmacodynamic endpoint. Since CsA is a potent p-glycoprotein substrate, the ability of this compound to cross the blood-brain barrier (BBB) was also investigated using an in vitro bovine model with repeated exposures up to 14 days. Finally, CsA uptake mechanisms were studied using a parallel artificial membrane assay (PAMPA) in combination with a Caco-2 model. Kinetic results indicate a low intracellular CsA uptake, with no marked bioaccumulation or biotransformation. In addition, only low CsA amounts crossed the BBB. PAMPA and Caco-2 experiments revealed that CsA is mostly trapped to lipophilic compartments and exits the cell apically via active transport. Thus, although CsA is unlikely to enter the brain at cytotoxic concentrations, it may cause alterations in electrical activity and is likely to increase the CNS concentration of other compounds by occupying the BBBs extrusion capacity. Such an integrated testing system, incorporating BBB, brain culture models and kinetics could be applied for assessing neurotoxicity potential of compounds.

Dynamic phase microscopy reveals periodic oscillations of endoplasmic reticulum during network formation.

Dynamic phase microscopy was used to study the dynamic events of formation of the endoplasmic reticulum (ER) in interphase-arrested Xenopus egg extract. We have shown that the ER periodically oscillated in an ATP-dependent manner in the frequency range of 1.6-2.2 Hz, while the tubular membrane network formed in vitro. The spectral density, i.e. the pattern of a given frequency component in the Fourier spectrum, was strongly correlated with the dynamic events during microtubule-dependent and microtubule-independent ER network formation observed by video-enhanced contrast differential interference contrast and fluorescence microscopy. Because the 1.6-2.2 Hz frequency of oscillation during the network formation was detected both in the presence and absence of microtubules, it appears to be an intrinsic ATP-dependent ER membrane property. Several characteristic active and inactive stages of ER network formation were observed both in the presence and absence of microtubules. However, data analysis of these stages indicated that microtubules and dynein motor activity have a strong influence and a cooperative effect on the kinetics of ER formation by controlled fusion reaction.

Radiosensitizing effect of epothilone B on human epithelial cancer cells.

BACKGROUND: A combined modality treatment employing radiation and chemotherapy plays a central role in the management of solid tumors. In our study, we examined the cytotoxic and radiosensitive effect of the microtubule stabilizer epothilone B on two human epithelial tumor cell lines in vitro and its influence on the microtubule assembly. METHODS: Cancer cells were treated with epothilone B in proliferation assays and in combination with radiation in colony-forming assays. For the analysis of ionizing radiation-induced DNA damage and the influence of the drug on its repair a γH2AX foci assay was used. To determine the effect of epothilone B on the microtubule assembly in cells and on purified tubulin, immunofluorescence staining and tubulin polymerization assay, respectively, were conducted. RESULTS: Epothilone B induced a concentration- and application-dependent antiproliferative effect on the cells, with IC(50) values in the low nanomolar range. Colony forming assays showed a synergistic radiosensitive effect on both cell lines which was dependent on incubation time and applied concentration of epothilone B. The γH2AX assays demonstrated that ionizing radiation combined with the drug resulted in a concentration-dependent increase in the number of double-strand breaks and suggested a reduction in DNA repair capacity. Epothilone B produced enhanced microtubule bundling and abnormal spindle formation as revealed by immunofluorescence microscopy and caused microtubule formation from purified tubulin. CONCLUSION: The results of this study showed that epothilone B displays cytotoxic antitumor activity at low nanomolar concentrations and also enhances the radiation response in the tumor cells tested; this may be induced by a reduced DNA repair capacity triggered by epothilone B. It was also demonstrated that epothilone B in fact targets microtubules in a more effective manner than paclitaxel.

Comparative study of cell cycle kinetics and induction of apoptosis or necrosis after exposure of human Mono Mac 6 cells to radiofrequency radiation.

The possible harmful effects of radiofrequency electromagnetic fields (RF EMFs) are controversial. We have used human Mono Mac 6 cells to investigate the influence of RF EMFs in vitro on cell cycle alterations and BrdU uptake, as well as the induction of apoptosis and necrosis in human Mono Mac 6 cells, using flow cytometry after exposure to a 1,800 MHz, 2 W/kg specific absorption rate (SAR), GSM-DTX signal for 12 h. No statistically significant differences in the induction of apoptosis or necrosis, cell cycle kinetics, or BrdU uptake were detected after RF EMF exposure compared to sham or incubator controls. However, in the positive control cells treated with gliotoxin and PMA (phorbol 12 myristate-13 acetate), a significant increase in apoptotic and necrotic cells was seen. Cell cycle analysis or BrdU incorporation for 72 h showed no differences between RF EMF- or sham-exposed cells, whereas PMA treatment induced a significant accumulation of cells in G(0)/G(1)-phase and a reduction in S-phase cells. RF EMF radiation did not induce cell cycle alterations or changes in BrdU incorporation or induce apoptosis and necrosis in Mono Mac 6 cells under the exposure conditions used.

Short communication: hydroperoxides in circulating lipids from dairy cows: implications for bioactivity of endogenous-oxidized lipids.

This study was conducted to investigate the potential for increased oxidative stress of high- vs. average-producing dairy cows. Two experiments were performed using 11 and 13 Holstein cows (53 +/- 2 d postpartum). Lipohydroperoxides (LHP) were determined in serum lipids (experiment 1) and low-density lipoprotein (experiment 2) via oxidation of ferrous to ferric ions through LHP using thiocyanate as chromogen. In experiment 1, differing milk yield and milk energy output corresponded to different concentrations of LHP. In experiment 2, analysis of regression resulted in a significant relationship between milk yield and LHP. Phospholipids isolated from lipids with 6.5 microM of LHP evoked in monocytic cells a transient increase in superoxide formation, indicating inflammatory potential. The results show that high milk productivity can associate with oxidative stress indicated by oxidative modifications of circulating lipids and their changed bioactivity.

Study on cell survival, induction of apoptosis and micronucleus formation in SCL-II cells after exposure to the auger electron emitter (99m)Tc.

OBJECTIVE: To study the biological effectiveness of Auger electrons emitted by (99m)Tc on cell survival, induction of apoptosis and micronucleus (MN) formation in the human squamous cell carcinoma cell line SCL-II and compare the effects observed to those observed after exposure to external 60Co gamma radiation. MATERIAL AND METHODS: Cells were either gamma(60Co)-irradiated (0.67 Gy/min) or exposed to (99m)Tc-pertechnetate (0.95-14.3 MBq/ml) for 24 h under cell culture conditions and assayed for cell survival (colony-forming assay), micronucleus formation (cytochalasin B assay) and the frequency of apoptotic cells (fluorescence microscopy). Monte Carlo based dosimetry has been applied to derive the absorbed dose corresponding to the accumulated decays of (99m)Tc under the given geometry. RESULTS: Absorbed doses up to 0.5 Gy could be achieved after 99mTc-exposure leading to no substantial cell killing in this dose range except at one dose point (0.1 Gy) resulting in an relative biological effectiveness (RBE)SF 0.9 of 0.64 when compared to the 60Co reference radiation. MN formation was described best by a linear dose response and was consistently lower after 99mTc exposure when compared to 60Co irradiated cells resulting in an RBE of 0.37. Apoptosis induction was significantly increased after 99mTc exposure at much lower doses (0.1 Gy) when compared to the reference radiation. The (99m)Tc uptake experiments revealed an activity concentration ratio cells vs. medium of 0.07 after 24 h of exposure. CONCLUSION: No overall increased biological effectiveness due to the emitted Auger electrons of (99m)Tc, applied as sodium-pertechnetate, could be observed in the investigated cell line when compared to acute external gamma radiation. The RBEs in the range of 0.37-0.64 might be well explained by dose rate effects. The significantly increased apoptotic response after (99m)Tc-exposure at very low doses has to be further investigated.

One-time screening for colorectal cancer with combined fecal occult-blood testing and examination of the distal colon.

BACKGROUND: Fecal occult-blood testing and sigmoidoscopy have been recommended for screening for colorectal cancer, but the sensitivity of such combined testing for detecting neoplasia is uncertain. At 13 Veterans Affairs medical centers, we performed colonoscopy to determine the prevalence of neoplasia and the sensitivity of one-time screening with a fecal occult-blood test plus sigmoidoscopy. METHODS: Asymptomatic subjects (age range, 50 to 75 years) provided stool specimens on cards from three consecutive days for fecal occult-blood testing, which were rehydrated for interpretation. They then underwent colonoscopy. Sigmoidoscopy was defined in this study as examination of the rectum and sigmoid colon during colonoscopy, and sensitivity was estimated by determining how many patients with advanced neoplasia had an adenoma in the rectum or sigmoid colon. Advanced colonic neoplasia was defined as an adenoma 10 mm or more in diameter, a villous adenoma, an adenoma with high-grade dysplasia, or invasive cancer. Classification of subjects according to the findings was based on the most advanced lesion. RESULTS: A total of 2885 subjects returned the three specimen cards for fecal occult-blood testing and underwent a complete colonoscopic examination. A total of 23.9 percent of subjects with advanced neoplasia had a positive test for fecal occult blood. As compared with subjects who had a negative test for fecal occult blood, the relative risk of advanced neoplasia in subjects who had a positive test was 3.47 (95 percent confidence interval, 2.76 to 4.35). Sigmoidoscopy identified 70.3 percent of all subjects with advanced neoplasia. Combined one-time screening with a fecal occult-blood test and sigmoidoscopy identified 75.8 percent of subjects with advanced neoplasia. CONCLUSIONS: One-time screening with both a fecal occult-blood test with rehydration and sigmoidoscopy fails to detect advanced colonic neoplasia in 24 percent of subjects with the condition.

Myosin Va bound to phagosomes binds to F-actin and delays microtubule-dependent motility.

We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome-F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubule-dependent retrograde phagosome movement toward the cell center. We propose an "antagonistic/cooperative mechanism" to explain the saltatory phagosome movement toward the cell center in normal macrophages.

Stimulation of phagocytosis and free radical production in murine macrophages by 50 Hz electromagnetic fields.

Effects of 50 Hz electromagnetic fields on phagocytosis and free radical production were examined in mouse bone marrow-derived macrophages. Macrophages were in vitro exposed to electromagnetic fields using different magnetic field densities (0.5-1.5 mT). Short-time exposure (45 min) to electromagnetic fields resulted in significantly increased phagocytic uptake (36.3% +/- 15.1%) as quantified by measuring the internalization rate of latex beads. Stimulation with 1 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) showed the same increased phagocytic activity as 1 mT electromagnetic fields. However, co-exposure to electromagnetic fields and TPA showed no further increase of bead uptake, and therefore we concluded that because of the absence of additive effects, the electromagnetic fields-induced stimulation of mouse bone marrow-derived macrophages does not involve the protein kinase C signal transduction pathway. Furthermore, a significant increased superoxide production after exposure to electromagnetic fields was detected.

Micronucleus induction in Syrian hamster embryo cells following exposure to 50 Hz magnetic fields, benzo(a)pyrene, and TPA in vitro.

Electromagnetic fields (EMFs) have been associated with increased incidence of cancer suggested by epidemiological studies. To test the carcinogenic potency of EMF, the in vitro micronucleus assay with SHE cells has been used as a screening method for genotoxicity. A 50Hz magnetic field (MF) of 1mT field strength was applied either alone or with the tumour initiator benzo(a)pyrene (BP) or the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). All three treatments were applied in single, double or triple treatment regimes. MF or TPA (1nM) alone did not affect the number of micronuclei (MN) in initiated and non-initiated SHE cells. Changing the schedule of the typical initiation protocol, namely applying the initiator (BP) during exposure to MF, results in an 1.8-fold increased MN formation compared to BP treatment alone. Combined experiment with BP, TPA and MF did not cause further MN formation. Since initiation during MF exposure caused a significant increased MN formation, our findings suggest that MFs enhance the initiation process of BP. We think that this MF-enhanced co-carcinogenic effect is caused by an indirect "cell activation" process. The resulting genomic instability is proposed to be due to free radicals and/or to the unscheduled "switching-on" of signal transduction pathways.

Mutational analysis of N-ras, p53, p16INK4a, p14ARF and CDK4 genes in primary human malignant mesotheliomas.

Nineteen specimens from primary human malignant mesotheliomas obtained from 19 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 by combined RFLP-PCR/SSCP analysis. In addition, all tumours were screened for deletions and point mutations in the tumour suppressor genes p53, p16INK4a (CDKN2A) and p14ARF (exon-1beta) by combined multiplex-PCR/SSCP analysis. No mutations were found in N-ras, p53 and CDK4. Three tumours displayed homozygous deletion (co-deletion of exons 1, 2 and 3) of p16INK4a. One of them displayed additional homozygous deletion of p14ARF (exon-1beta). Two silent point mutations and 2 polymorphisms were found in p16INK4a in 3 tumours. Our preliminary data indicate that disarrangement of the Rb1 pathway may be involved in mesothelioma formation.

Use of colonoscopy to screen asymptomatic adults for colorectal cancer. Veterans Affairs Cooperative Study Group 380.

BACKGROUND AND METHODS: The role of colonoscopy in screening for colorectal cancer is uncertain. At 13 Veterans Affairs Medical Centers, we performed colonoscopy to determine the prevalence and location of advanced colonic neoplasms and the risk of advanced proximal neoplasia in asymptomatic patients (age range, 50 to 75 years) with or without distal neoplasia. Advanced colonic neoplasia was defined as an adenoma that was 10 mm or more in diameter, a villous adenoma, an adenoma with high-grade dysplasia, or invasive cancer. In patients with more than one neoplastic lesion, classification was based on the most advanced lesion. RESULTS: Of 17,732 patients screened for enrollment, 3196 were enrolled; 3121 of the enrolled patients (97.7 percent) underwent complete examination of the colon. The mean age of the patients was 62.9 years, and 96.8 percent were men. Colonoscopic examination showed one or more neoplastic lesions in 37.5 percent of the patients, an adenoma with a diameter of at least 10 mm or a villous adenoma in 7.9 percent, an adenoma with high-grade dysplasia in 1.6 percent, and invasive cancer in 1.0 percent. Of the 1765 patients with no polyps in the portion of the colon that was distal to the splenic flexure, 48 (2.7 percent) had advanced proximal neoplasms. Patients with large adenomas (> or = 10 mm) or small adenomas (< 10 mm) in the distal colon were more likely to have advanced proximal neoplasia than were patients with no distal adenomas (odds ratios, 3.4 [95 percent confidence interval, 1.8 to 6.5] and 2.6 (95 percent confidence interval, 1.7 to 4.1], respectively). However, 52 percent of the 128 patients with advanced proximal neoplasia had no distal adenomas. CONCLUSIONS: Colonoscopic screening can detect advanced colonic neoplasms in asymptomatic adults. Many of these neoplasms would not be detected with sigmoidoscopy.

The keratocyte network of human cornea: a three-dimensional study using confocal laser scanning fluorescence microscopy.

PURPOSE: Keratocytes of the living human cornea were examined to compare quantitatively spatial arrangement and cell volume of the stromal layers. This knowledge is required for further studies toward a quantitative understanding of cellular alterations in corneal pathology. METHODS: Three human corneas were stained with calcein AM and ethidium homodimer (Live/Dead Kit) directly after enucleation. The fluorescent cells were examined with confocal laser scanning fluorescence microscopy. High-resolution three-dimensional (3-D) volumes of < or =270 microm in the z-axis were reconstructed. Cell density and volume density were determined by computer-aided morphometry. RESULTS: Three keratocyte subpopulations were distinguished. Their spatial arrangement was visualized by 3-D reconstructions of the scanned volumes. Whereas cell density decreased progressively from the anterior (100%) to posterior (53.7%) stroma, volume density was highest in the posterior stroma (17.03 +/- 5.05%) and lowest in the central stroma (9.31 +/- 1.09%). In the anterior stroma, volume density was found to be 10.19 +/- 4.37%. CONCLUSION: Confocal laser scanning fluorescence microscopy allowed quantitative analysis and the visualization of the spatial arrangement of the keratocyte network in the living human corneal tissue for the first time. The results provide a basis for further studies of alterations of the normal cellular arrangements in corneal disease.

Kerosene soot genotoxicity: enhanced effect upon co-exposure with chrysotile asbestos in Syrian hamster embryo fibroblasts.

Epidemiological and experimental studies have suggested an enhancement of asbestos-induced bronchogenic carcinoma by cigarette smoke. Further, our recent experimental and epidemiological studies have indicated that besides smoking, several other compounds including kerosene soot may accelerate disease processes in asbestos-exposed animals as well as in the humans. Incomplete combustion of kerosene oil generates large volumes of soot, which contains various polycyclic aromatic hydrocarbons and aliphatic compounds. As reported earlier, exposure to kerosene soot is known to cause biochemical and pathological changes in the pulmonary tissue, which may cause cardiopulmonary disorders. In this study we investigated genotoxic effects caused by kerosene soot and chrysotile asbestos as well as co-exposure of kerosene soot and chrysotile using Syrian hamster embryo fibroblasts (SHE). The micronucleus assay revealed a significant increase of induced micronuclei (MN), (P

Mutational analysis of the N-ras, p53, p16INK4a, CDK4, and MC1R genes in human congenital melanocytic naevi.

Eighteen human congenital melanocytic naevi (CMN) from 17 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 and for sequence variants in the MC1R gene by combined RFLP-PCR/SSCP analysis. In addition, all lesions were screened for deletions and point mutations in the tumour suppressor genes p53 and p16INK4a (CDKN2A) by combined multiplex PCR/SSCP analysis. Positive screening data were specified by sequencing of the corresponding PCR product. Activating point mutations in the N-ras gene (nine CAA (Gln) to AAA (Lys) transversions and one CAA (Gln) to CGA (Arg) transition at codon 61) were detected at high frequency (56%). Furthermore, three missense mutations (V92M) and two silent mutations (CGA (Arg) to CGG (Arg), codon 213, exon 6) were found in the MC1R and p53 genes, respectively. No mutations were found in p16 or CDK4. The activated N-ras oncogene, which is also found in human cutaneous melanomas, may constitute a potential risk factor for melanoma formation within CMN.

Dynein is required for spindle assembly in cytoplasmic extracts of Spisula solidissima oocytes.

Meiosis I spindle assembly is induced in lysate-extract mixtures prepared from clam (Spisula solidissima) oocytes. Unactivated lysate prepared from unactivated oocytes contain nuclei (germinal vesicles, GVs) which house condensed chromosomes. Treatment of unactivated lysate with clarified activated extract prepared from oocytes induced to complete meiosis by treatment with KCl induces GV breakdown (GVBD) and assembly of monopolar, bipolar, and multipolar aster-chromosome complexes. The process of in vitro meiosis I spindle assembly involves the assembly of microtubule asters and the association of these asters with the surfaces of the GVs, followed by GVBD and spindle assembly. Monoclonal antibody m74-1, known to react specifically with the N terminus of the intermediate chain of cytoplasmic dynein, recognizes Spisula oocyte dynein and inhibits in vitro meiosis I spindle assembly. Control antibody has no affect on spindle assembly. A similar inhibitory effect on spindle assembly was observed in the presence of orthovanadate, a known inhibitor of dynein ATPase activity. Neither m74-1 nor orthovanadate has any obvious affect on GVBD or aster formation. We propose that dynein function is required for the association of chromosomes with astral microtubules during in vitro meiosis I spindle assembly in these lysate-extract mixtures. However, we conclude that dynein function is not required for centrosome assembly and maturation or for centrosome-dependent aster formation.

Effects of 50 Hz EMF exposure on micronucleus formation and apoptosis in transformed and nontransformed human cell lines.

Effects of applying extremely low-frequency electromagnetic fields (ELF-EMF) for different durations (24, 48, and 72 h) and different field intensities (0.1-1.0 mT) on micronucleus (MN) formation and induction of apoptosis were examined in a human squamous cell carcinoma cell line (SCL II) and in a human amniotic fluid cell line (AFC). A statistically significant increase of MN frequency and of induction of apoptosis in SCL II cells after 48-h and 72-h continuous exposure to 50 Hz magnetic field (MF) (0.8 and 1.0 mT) was found. However, exposure of AFC cells to EMF of different intensities and for different exposure times showed no statistically significant differences when compared with controls. These results demonstrate that different human cell types respond differently to EMF. Dose-dependent induction of apoptosis and genotoxic effects, resulting in increased micronucleus formation, could be demonstrated in the transformed cell line, whereas the nontransformed cell line did not show statistically significant effects. These findings suggest that EMF could be a promotor but not an initiator of carcinogenic effects.

The involvement of the intermediate chain of cytoplasmic dynein in binding the motor complex to membranous organelles of Xenopus oocytes.

Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74-1, m74-2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 micrograms/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74-1 and m74-2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column binding assay, both antibodies inhibited the IC-p150Glued interaction. Thus these findings demonstrate that direct IC-p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.

The interaction between cytoplasmic dynein and dynactin is required for fast axonal transport.

Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic dynein is necessary but not sufficient for retrograde transport directed from the synapse to the cell body. Dynactin is a heteromultimeric protein complex, enriched in neurons, that binds to both microtubules and cytoplasmic dynein. To determine whether dynactin is required for retrograde axonal transport, we examined the effects of anti-dynactin antibodies on organelle transport in extruded axoplasm. Treatment of axoplasm with antibodies to the p150(Glued) subunit of dynactin resulted in a significant decrease in the velocity of microtubule-based organelle transport, with many organelles bound along microtubules. We examined the molecular mechanism of the observed inhibition of motility, and we demonstrated that antibodies to p150(Glued) disrupted the binding of cytoplasmic dynein to dynactin and also inhibited the association of cytoplasmic dynein with organelles. In contrast, the anti-p150(Glued) antibodies had no effect on the binding of dynactin to microtubules nor on cytoplasmic dynein-driven microtubule gliding. These results indicate that the interaction between cytoplasmic dynein and the dynactin complex is required for the axonal transport of membrane-bound vesicles and support the hypothesis that dynactin may function as a link between the organelle, the microtubule, and cytoplasmic dynein during vesicle transport.