Taxol induces concomitant hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells

Taxol, a microtubule stabilizing agent, has been extensively investigated for its antitumor activity. The cytotoxic effect of taxol is generally attributed to its antimicrotubule activity and is believed to be cell cycle dependent. Herein, we report that taxol induces hyperphosphorylation and reorganization of the vimentin intermediate filament in 9L rat brain tumor cells, in concentration- and time-dependent manner. Phosphorylation of vimentin was maximum at 10(-6) M of taxol treatment for 8 h and diminished at higher (10(-5) M) concentration. Enhanced phosphorylation of vimentin was detectable at 2 h treatment with 10(-6) M taxol and was maximum after 12 h of treatment. Taxol-induced phosphorylation of vimentin was largely abolished in cells pretreated with staurosporine and bisindolymaleimide but was unaffected by H-89, KT-5926, SB203580, genistein, and olomoucine. Thus, protein kinase C may be involved in this process. Hyperphosphorylation of vimentin was accompanied by rounding up of cells as revealed by scanning electron microscopy. Moreover, there was a concomitant reorganization of the vimentin intermediate filament in the taxol-treated cells, whereas the microtubules and the actin microfilaments were less affected. Taken together, our data demonstrate that taxol induces hyperphosphorylation of vimentin with concomitant reorganization of the vimentin intermediate filament and that this process may be mediated via a protein kinase C signaling pathway.     

Motile properties of vimentin intermediate filament networks in living cells

PURPOSE: To evaluate the clinical utility and morphologic accuracy of gadolinium-enhanced excretory magnetic resonance (MR) urography after low-dose diuretic injection and to correlate the results with those of conventional urography. MATERIALS AND METHODS: In 71 patients with urologic symptoms, excretory MR urography was performed after intravenous injection of 5-10 mg furosemide and, 30-60 seconds later, 0.1 mmol of gadopentetate dimeglumine per kilogram of body weight. The MR urograms were interpreted by three radiologists, who were blinded to the clinical outcome, and subsequently compared with conventional urograms. RESULTS: Injection of furosemide before contrast material led to rapid, uniform gadolinium distribution inside a sufficiently distended collecting system such that there was no excessive concentration of gadolinium in the urine. In patients with normal or moderately reduced excretory function, this effect allowed complete visualization of the urinary tract within 5-20 minutes of contrast material injection while minimizing gadolinium-related endoluminal T2* effects. The clinical course helped verify almost all MR urographic results. The MR urographic technique was significantly superior to conventional urography in the assessment of the ureters and bladder (P < .0001). Delineation of small caliceal abnormalities is still problematic. The best depiction of the pelvicaliceal system was obtained with fat-suppressed MR imaging, although it was still slightly inferior to conventional urography (P < .05). CONCLUSION: Gadolinium-enhanced excretory MR urography performed after low-dose diuretic injection is a promising and accurate alternative to conventional excretory urography for imaging the morphology of the urinary tract.     

Unique phosphorylation of vimentin filaments in glomerular epithelial cells in culture and in disease

Three cDNA clones for the Machado-Joseph disease gene (MJD1) were isolated, two of which have a new exon sequence and a distinct 3' terminal nucleotide sequence resulting in a new carboxyl terminal domain in the translated product. The nucleotide sequence of the other one is similar to the previously published one except for five polymorphisms, one of which is a single nucleotide substitution resulting in a change from the stop codon (TAA; allele A) to a tyrosine residue (TAC; allele C). Genetic analysis results suggest that Japanese MJD mutations are associated with allele A.     

Alpha B-crystallin is associated with intermediate filaments in astrocytoma cells

To achieve the optimal management program for the diabetic vasculopathic patient, a multidisciplinary approach incorporating the necessary major elements is required. The endocrinologist is essential in tight metabolic control of blood sugars and diet modifications. The podiatrist is indispensable in the early detection of foot ulcerations and preventive care. Visiting nurses function as a vital component in outpatient wound assessment and daily care. With this approach, the vascular surgeon is ensured of the most favorable outcome with conservative measures.     

Interaction of Theiler's virus with intermediate filaments of infected cells

Theiler's murine encephalomyelitis virus is a neurotropic murine picornavirus which replicates permissively and causes a cytopathic effect in the BHK-21 cell line. We examined the interactions between the GDVII and DA strains of Theiler's virus and BHK-21 host cell proteins in a virus overlay assay. We observed binding of the virions to two proteins of approximately 60 kDa. These proteins were microsequenced and identified as desmin and vimentin, two main components of the intermediate filament network. The association between desmin or vimentin and virions was demonstrated by immunoprecipitation. Anti-desmin and anti-vimentin monoclonal antibodies precipitated GDVII or DA virions from extracts of infected BHK-21 cells. The intracellular distributions of virions and of the desmin and vimentin intermediate filaments of BHK-21 cells were investigated by two-color immunofluorescence confocal microscopy. Following infection, the intermediate filament network was rearranged into a shell-like structure which surrounded a viral inclusion. Finally, close contact between GDVII virus particles and 10-nm intermediate filaments was observed by electron microscopy.     

Development of vimentin filaments in the cells of the articular disc of the rat squamosomandibular joint with age

The organochlorines, dichloro-diphenyl-trichloroethane and polychlorinated biphenyl (PCB) are pervasive environmental contaminants. Results from previous studies have been conflicting regarding the relationship between the internal dose of these organochlorine residues and breast cancer risk. To determine whether these compounds are present in breast cyst fluids and whether cyst fluid and plasma concentrations are correlated, we analyzed organochlorines in paired cyst fluid and plasma samples from 24 subjects using gas chromatography and electron capture detection. All but one of the women had a history of multiple cysts, suggesting that they were at elevated risk for future breast cancer. DDE (a metabolite of dichloro-diphenyl-trichloroethane) was present in 22 of the cyst samples and PCB was detected in 19 of the cyst samples. Organochlorine levels were more concentrated in the plasma than in breast cyst fluids. Levels of DDE in plasma were significantly correlated with those in cyst fluid (r = 0.73; P < 0.001); in contrast to PCB levels in cyst and plasma (r = 0.37; P = 0.12). Congener specific analysis of the PCBs showed that some individual congeners were preferentially excluded from or concentrated in the cyst fluid. To our knowledge, this study is the first to demonstrate that PCB and DDE are present in cyst fluids and thus in contact with the ductal epithelium of the breast. These results support the use of plasma DDE as a proxy for DDE in the target tissue in research on the role of environmental factors in breast cancer.     

Evidence for direct binding of intracellularly distributed ganglioside GM2 to isolated vimentin intermediate filaments in normal and Tay-Sachs disease human fibroblasts

The insulinotropic action of meglitinide was compared to that of its analogs S 3075, A-4166, KAD-1229 and repaglinide. None of these hypoglycemic agents significantly enhanced insulin output from rat pancreatic islets incubated for 90 min in the absence of exogenous nutrient. However, all these agents, when tested at a 10 microM concentration, augmented insulin release evoked by either 7 mM D-glucose or 10 microM succinic acid monomethyl ester (SAM). In this respect, meglitinide was a less efficient secretagogue than the other non-sulfonylurea hypoglycemic agents. Moreover, in the presence of 7 mM D-glucose, the lowest concentration of the drug required to cause a significant increase in insulin output decreased from about 1.0 microM for meglitinide to 0.1 microM with A-4166, KAD-1229 or repaglinide and even close to 10 nM in the case of S 3075. The concentration-response relationship thus yielded the following hierarchy, S 3075 > KAD-1229 = repaglinide > A-4166 > meglitinide, there being a difference of more than two orders of magnitude between the weakest and most potent agent.     

Assembly and architecture of invertebrate cytoplasmic intermediate filaments reconcile features of vertebrate cytoplasmic and nuclear lamin-type intermediate filaments

The two major intermediate filament (IF) proteins from the esophagus epithelium of the snail Helix pomatia and the two major IF proteins from muscle tissue of the nematode Ascaris suum were investigated under a variety of assembly conditions. The lowest-order complexes from each of the four protostomic invertebrate (p-INV) IF proteins are parallel, unstaggered dimers involving two-stranded alpha-helical coiled coil formation of their approximately 350 amino acid residue central rod domain (i.e. long-rod). In the electron microscope these are readily recognized by their distinct approximately 56 nm long rod with two globular domains (i.e. representing the non-helical carboxy-terminal tail domain of the p-INV IF proteins) attached at one end, closely resembling vertebrate lamin dimers. The next-higher-order oligomers are tetramers, which are easily recognized by their two pairs of globular tail domains attached at either end of a approximately 72 nm long central rod portion. According to their size and shape, these tetramers are built from two dimers associated laterally in an antiparallel, approximately half-staggered fashion via the amino-terminal halves of their rod domains. This is similar to the NN-type tetramers found as the most abundant oligomer species in all types of vertebrate cytoplasmic IF proteins, which contain a approximately 310 amino acid residue central rod domain (i.e. short-rod). As a first step toward filament formation, the p-INV IF tetramers anneal longitudinally into protofilaments by antiparallel CC-type association of the carboxy-terminal halves of their dimer rods. The next step involves radial growth, occurring initially through lateral association of two four-chain protofilaments into octameric subfibrils, which then further associate into mature, full-width filaments. Head-to-tail polymers of dimers and paracrystalline fibers commonly observed with vertebrate lamins were only rarely seen with p-INV IF proteins. The globular domains residing at the carboxy-terminal end of p-INV IF dimers were studding the surface of the filaments at regular, approximately 24.5 nm intervals, thereby giving them a "beaded" appearance with an axial periodicity of about 24.5 nm, which is approximately 3 nm longer than the corresponding approximately 21.5 nm repeat pattern exhibited by short-rod vertebrate IFs.     

Association of calponin with desmin intermediate filaments

Our previous immunoelectron microscopy studies of chicken gizzard smooth muscle cells showed that in certain areas the distribution of anti-calponin exhibits a high degree of overlap with beta-actin, filamin, and in particular, desmin, suggesting that in situ a fraction of calponin may be associated with intermediate filaments of the cytoskeleton. In this work we further explore this idea by studying the interaction between calponin and desmin. We found that at physiological salt concentrations, calponin bound only weakly to synthetic desmin intermediate filaments. On the other hand, calponin bound strongly to nonfilamentous desmin tetramers and was incorporated into intermediate filaments when the two proteins were mixed in a buffer containing 6 M urea and dialyzed into a buffer containing 0.15 M NaCl. Anti-calponin was found to label a portion of intermediate filaments and dense bodies isolated from gizzard tissues. Our findings suggest that in chicken gizzard smooth muscle cells, calponin may be an integral component of desmin intermediate filaments in the vicinity of dense bodies. Since calponin is also known to bind actin, we hypothesize that one of the functions of calponin might be to bridge intermediate filaments with actin in dense bodies.     

Changes in the distribution of intermediate filaments in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development

BACKGROUND: Intermediate filaments (IFs) are components of the cytoskeleton. In mammalian Sertoli cell, IFs are formed by vimentin. Previous studies have shown some characteristics of its distribution in Sertoli cells, however, very little is known of its distributional changes during the seminiferous epithelium cycle and during postnatal development. METHODS: Immunohistochemical and electron microscopic methods were used to determine the distribution of vimentin-type IFs in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development. RESULTS: The distribution of IFs in adult rat Sertoli cell showed distinct cyclic changes during the seminiferous epithelium cycle. At stages I-VI, bundles of IFs extend from the perinuclear region to the supranuclear and apical regions of the Sertoli cell. These apical extensions became shorter at stage VII, and at stages VIII-X IFs were observed only in the perinuclear region. Short apical extensions reappeared at stages XI-XII; and at stages XIII-XIV, they extended again into the apical region. During this cycle, IFs were always closely associated with the heads of elongate spermatids. IFs were also shown to be in close apposition to some specialized structures on the cell membrane, such as the ectoplasmic specialization between adjacent Sertoli cells. During postnatal (p.n.) development, IFs were mainly observed at the basal nuclear region on p.n. day 7. The IFs in the supranuclear or apical regions first appeared at p.n. day 14 and gradually increased during the development. The perinuclear IFs network was fully established by p.n. day 28 and the adult distribution pattern of the IFs was established by p.n. day 42. CONCLUSIONS: Vimentin-type IFs in rat Sertoli cells are a delicate endocellular network, which is centered in the perinuclear region and extends to the apical region of the cell. During the seminiferous epithelium cycle, the distribution of IFs changes in a stage-dependent manner and is closely related to the location of the heads of elongate spermatids. During postnatal development, IFs gradually increase in numbers and the main distribution area is transferred from the basal nuclear to the perinuclear and supranuclear regions.     

Disorganization of microfilaments and intermediate filaments interferes with the assembly and stability of desmosomes in MDCK epithelial cells

Using the serial reaction time (SRT) task developed by Nissen and Bullemer (1987, Cognitive Psychology, 19, 1-32), implicit memory performance was examined in four groups of subjects: nondemented healthy aged individuals; nondemented Parkinson's disease individuals; very mildly demented senile dementia of the Alzheimer type (SDAT) individuals; and mildly demented SDAT individuals. The SRT task involved four blocks of a repeated 10-item keypress sequence that tapped general skill development along with a fifth block of a nonrepeated sequence that presumably reflected the impact of switching from a learned set of associations (developed during the first four blocks) to a novel sequence. The increase in response latency from the fourth repeated block to the fifth nonrepeated block was used as the reflection of implicit learning. The results revealed preserved implicit memory performance in the very mildly demented individuals compared to that of the age-matched control individuals. However, the mildly demented SDAT individuals and the nondemented Parkinson's disease individuals showed reliably less implicit learning, compared to the age-matched control individuals. Differences between the past studies using the SRT task to tap implicit memory performance in SDAT individuals and the present study are discussed in some detail. We conclude that nondemented Parkinson's disease individuals and mildly demented SDAT individuals produce some deficit in the formation of new associations in implicit memory, as measured by the SRT task.     

Subcellular localization of proteasomes in apoptotic lung tumor cells and persistence as compared to intermediate filaments

Failure to diagnose breast carcinoma by fine-needle aspiration (FNA) is a major obstacle to expanded use of that technique. While the majority of false negative diagnoses are due to inadequate sampling and insufficient specimens, a significant minority of false negative cases result from inaccurate interpretation of adequate material. Low grade carcinomas including lobular, tubular, low grade adenosquamous, and papillary carcinomas appear to account for many of these diagnostic errors. Careful attention to nuclear detail, monomorphism of cell population and the presence of neoplastic cells with retained cytoplasm should allow the recognition of the majority of these neoplasms as malignant by cytologic examination.     

A structural scaffolding of intermediate filaments in health and disease

The cytoplasm of animal cells is structured by a scaffolding composed of actin microfilaments, microtubules, and intermediate filaments. Intermediate filaments, so named because their 10-nanometer diameter is intermediate between that of microfilaments (6 nanometers) and microtubules (23 nanometers), assemble into an anastomosed network within the cytoplasm. In combination with a recently identified class of cross-linking proteins that mediate interactions between intermediate filaments and the other cytoskeletal networks, evidence is reviewed here that intermediate filaments provide a flexible intracellular scaffolding whose function is to structure cytoplasm and to resist stresses externally applied to the cell. Mutations that weaken this structural framework increase the risk of cell rupture and cause a variety of human disorders.     

Role of intermediate filaments in migration, invasion and metastasis

The expression of intermediate filament proteins is remarkably tissue-specific which suggests that the intermediate filament (IF) type(s) present in cells is somehow related to their biological function. However, in some cancers-particularly malignant melanoma and breast carcinoma, there is a strong indication that vimentin and keratin IFs are coexpressed, thus presenting as a dedifferentiated or interconverted (between epithelial and mesenchymal) phenotype. In this review, two in vitro models are presented which recapitulate the interconverted phenotype in human melanoma and breast carcinoma, and allow, for the first time, unique observations to be made with respect to the role of IFs in cancer progression. These studies have provided direct evidence linking overexpression of keratin IFs in human melanoma with increased migratory and invasive activity in vitro, which can be down-regulated by substituting dominant-negative keratin mutants. Overexpression of vimentin IFs in the breast carcinoma model leads to augmentation of motility and invasiveness in vitro, which can be transiently down-regulated by treatment with antisense oligonucleotides to vimentin. Additional experimental evidence suggests that the mechanism(s) responsible for the differential expression of metastatic properties associated with the interconverted phenotype rest(s) in the unique interaction, either direct or indirect, of IFs with specific integrins interacting with the extracellular matrix. In this review, we discuss the observations derived from the human melanoma and breast carcinoma models to address the hypothesis that the ability to coexpress vimentin and keratins confers a selective advantage to tumor cells in their interpretation of and response to signaling cues from the extracellular matrix. The ramifications of these observations are discussed with respect to the patholophysiology of the respective in situ tumors.     

Expression of intermediate filaments in normal and neoplastic exocrine pancreas

The intermediate filament (IF) proteins present in the normal and pathological exocrine human pancreas were studied by immunolocalization using antibodies to cytokeratins (CKs) and vimentin. Acinar cells of normal pancreas showed a presence of simple CKs 8 and 18. Duct epithelium consistently expressed CKs 7, 8, 18 and 19 whereas centroacinar cells were rather low in CK 7. A subpopulation of CK 4 cells was detected in inter-intralobular ducts. In addition, some ducts contained individual cells or groups of cells that were positive for the stratification-related CKs (CKs 4, 5, 13, 15, 16). All pancreatic ductal adenocarcinomas regularly expressed CKs 7, 8, 18, 19 and were also positive for the 34 beta E12 antibody. Cytokeratin 4 was detected in a minor population of tumor cells. Pancreatic carcinoma also contained minor amounts of stratification-related CKs in variable combinations. Mucinous cystoadenocarcinoma showed the presence of CKs 7, 8, 18, 19 and was also positive for 34 beta E12, whereas the serous microcystic tumor presented CKs 8, 18, 19 and a variable amount of CKs 4 and 7. The duct-ductular alterations of the exocrine pancreas contained a different combination and distribution of CK isoforms similar to normal pancreatic ductal system. Mucinous hypertrophy and pyloric gland metaplasia reacted with antibodies to CKs 7, 8, 18 and 19. Vimentin was focally present both in normal and neoplastic tissue. Our results indicate that pancreatic ducts are characterized by an intrinsic "biliary-pancreatic duct type" immunoprofile (CKs 7, 8, 18 and 19), in contrast to acinar cells expressing exclusively CKs 8 and 18. We also detected a subpopulation of ducts regularly expressing CK 4. Surprisingly, several stratification-related CKs were detected both in normal and neoplastic exocrine pancreas. Moreover, the differentiation phenotypes of pancreatic tumors were reminiscent of normal cellular compartments.     

Neuropathy by n-hexanes: a generalized disorder of the intermediate filaments

BACKGROUND: Chronic inhalation of glues containing n-hexanes produces neurofilament (NF) accumulation which induces sensory-motor polyneuropathy. In vitro assays have shown this toxic substance causes intermediate filaments (IF) aggregation in non-neuronal cells. OBJECTIVE: To describe intermediate filament changes in human pathology due to n-hexanes. PATIENTS AND METHODS: Sural nerve and skin biopsy samples from 2 patients who suffered from a severe sensory-motor polyneuropathy after prolonged inhalation of glue containing n-hexane were examined with electron microscopy and vimentin and phosphorylated NF immunocytochemistry. RESULTS: Abnormal accumulations of NF and NF-immunoreactive products occurred in nerve fibers and increased numbers of fibrils were observed in endoneurial endothelial cells of the sural nerve. In addition, abnormal vimentin-immunoreactive deposition was seen in fibroblasts and capillaries of the skin. The present results suggest that high doses of n-hexane cause a diffuse IF disorder in a similar form as occurs in giant axonal neuropathy. CONCLUSION: IF aggregation can occur in non-neuronal cells in humans, as has been previously proved in in vitro experiments. The presence of IF accumulations in Schwann cells, as seen in the ultrastructural examination, together with the electrophysiological findings showing an early decrease of sensory and motor nerve conduction velocities, suggests the existence of a primary myelinic disorder associated with axonal damage.     

Dynamic organisation of intermediate filaments and associated proteins during the cell cycle

Two black yeast isolates from plants from the Canary Islands (Spain) are described and illustrated. Absence of Woronin bodies at simple septal pores, local coralloid terminal hyphal cells, indeterminate thallus maturation, the presence of budding cells and local conversion to meristematic growth all indicate a relationship to the Dothideaceae (Dothideales, Ascomycota). Morphological properties were consistent with the genus Hormonema Lagerberg & Melin, as defined by presence of percurrent conidiogenous loci alongside undifferentiated hyphae, and results of PCR-ribotyping supported this classification. The isolates were judged to belong to a hitherto undescribed species, characterized in particular by curved conidia soon developing transverse septa. The physiological profile of this species is also described.     

The relationship between vaccinia virus DNA replication and intermediate filaments

Various DNA components which were extracted with gentle cell fractionation from the HeLa cells after 4 h vaccinia virus infection were detected by dot hybridization technique. The virus DNA mainly exist in intermediate filament-lamina-nuclear matrix complex. With DGD embedment free technique and electron microscopic autoradiography, the newly synthesized virus DNA is found to be associated with intermediate filaments. The results of southwestern hybridization demonstrate that vaccinia virus DNA has specific affinity to intermediate filaments and some nuclear matrix proteins.