Astrocytes perform a variety of functions in the adult central nervous system (CNS) that contribute to the survival of neurons. Thus, it is likely that the activities of astrocytes affect the extent of brain damage after ischemic stroke. The authors tested this hypothesis by using a mouse ischemia model to compare the infarct volume produced in wild-type mice with that produced in mice lacking glial fibrillary acidic protein (GFAP), an astrocyte specific intermediate filament component. Astrocytes lacking GFAP have been shown to have defects in process formation, induction of the blood-brain barrier. and volume regulation; therefore, they might be compromised in their ability to protect the CNS after injury. The authors reported here that 48 hours after combined permanent middle cerebral artery occlusion (MCAO) and 15 minutes transient carotid artery occlusion (CAO) GFAP-null mice had a significantly (P < 0.001) larger cortical infarct volume (16.7 +/- 2.2 mm3) than their wild-type littermates (10.1 +/- 3.9 mm3). Laser-Doppler flowmetry revealed that the GFAP-null mice had a more extensive and profound decrease in cortical cerebral blood flow within 2 minutes after MCAO with CAO. These results indicated a high susceptibility to cerebral ischemia in GFAP-null mice and suggested an important role for astrocytes and GFAP in the progress of ischemic brain damage after focal cerebral ischemia with partial reperfusion.

We have determined the intermediate filament proteins present in normal and malignant mesothelium in vivo. Pure sheets of normal lung mesothelium were obtained by transfer to agarcoated slides or by gentle scraping and cytocentrifugation. Cytoplasmic filament networks in the mesothelium were labeled via indirect immunofluorescence both by anti-Mr 40,000 keratin and anti-vimentin antisera. Two-dimensional gel electrophoresis of the Triton:high-salt-insoluble proteins of normal lung mesothelium disclosed the presence of vimentin and all but the largest (Mr 55,000) of the four simple epithelial keratins synthesized by mesothelial cells in culture. Samples of three peritoneal and three pleural mesotheliomas were found to contain either all four simple epithelial keratins or all but the Mr 55,000 keratin. Notably, none of the keratins characteristic of stratified and many glandular epithelia and their malignant forms was present in these mesotheliomas. Two mesothelioma samples from which the tumor cells could be obtained free of other cell types were found to contain vimentin as well as simple epithelial keratins and to synthesize these same proteins during short-term culture. None of the mesotheliomas placed in culture grew progressively in medium optimal for the growth of normal mesothelial cells. These data demonstrate that malignant mesothelial cells preserve the normal pattern of intermediate filament protein synthesis, including coexpression of simple epithelial keratins and vimentin, and suggest the use of this characteristic as an aid in the identification of cells of mesothelial origin.

A panel of 60 human tumor cell lines is currently being used in the U.S. National Cancer Institute's in vitro anticancer drug screen. The panel is organized into 7 subpanels; 6 leukemia/lymphoma lines comprise one subpanel, and 54 other lines are organized into subpanels representing solid tumors of the central nervous system (CNS), colon, lung, ovaries, kidneys and melanomas. In the present study, the leukemia and lymphoma cell lines were analyzed by flow cytometry for appropriate CD antigens; all but 1 line showed patterns of expression consistent with their reported derivations. The solid tumor lines were characterized individually using morphological and immunocytochemical techniques to determine their relative degrees of representativity for the subpanels within which they are currently grouped. Histological, histochemical and ultrastructural examinations were performed on cell lines grown under identical conventional culture conditions and as xenografts in nude mice. Immunocytochemistry using panels of antibodies raised against 6 types of intermediate filaments, 7 adenocarcinoma-associated antigens, 7 melanoma/neuro-ectodermal-associated antigens, 3 neuroendocrine-associated antigens, 9 urinary tract associated antigens, and 4 markers of muscle differentiation was done on cells grown in monolayer culture. Central nervous system (CNS) cell lines lacked expression of glial fibrillary acidic protein, but all had other features consistent with derivation from glioblastoma. Lines derived from adenocarcinomas of the colon, lung and ovary, for the most part, expressed adenocarcinoma-associated antigens and showed histological and/or ultrastructural evidence of gland formation and other adenomatous features. Most of these lines were poorly differentiated. Lines derived from large-cell and squamous-cell cancers also showed some characteristics consistent with their reported origins, except for one line which showed immunocytochemical and morphologic characteristics consistent with rhabdomyosarcoma. The 2 lines derived from small cell lung cancer (SCLC) lacked neurosecretory granules and 3 other SCLC markers but showed morphologic features consistent with SCLC. Most melanoma cell lines strongly expressed melanoma-associated antigens and were morphologically similar to human melanoma. Five lines produced premelanosomes, melanosomes or melanin. Most of the renal cancer cell lines showed morphologic or immunocytochemical features consistent with renal clear cell carcinoma. Collectively, these morphological and immunocytochemical analyses provide information concerning tissue of origin, tumor type, degree of differentiation and other biologic features essential to the use of these lines in a disease-oriented in vitro antitumor drug screen and to the interpretation of data derived therefrom.

The synaptonemal complex protein SCP3 is part of the lateral element of the synaptonemal complex, a meiosis-specific protein structure essential for synapsis of homologous chromosomes. We have investigated the fiber-forming properties of SCP3 to elucidate its role in the synaptonemal complex. By synthesis of SCP3 in cultured somatic cells, it has been shown that SCP3 can self-assemble into thick fibers and that this process requires the COOH-terminal coiled coil domain of SCP3, as well as the NH2-terminal nonhelical domain. We have further analyzed the thick SCP3 fibers by transmission electron microscopy and immunoelectron microscopy. We found that the fibers display a transversal striation with a periodicity of approximately 20 nm and consist of a large number of closely associated, thin fibers, 5-10 nm in diameter. These features suggest that the SCP3 fibers are structurally related to intermediate filaments. It is known that in some species the lateral elements of the synaptonemal complex show a highly ordered striated structure resembling that of the SCP3 fibers. We propose that SCP3 fibers constitute the core of the lateral elements of the synaptonemal complex and function as a molecular framework to which other proteins attach, regulating DNA binding to the chromatid axis, sister chromatid cohesion, synapsis, and recombination.

Providing a stable physical connection between the nucleus and the cytoskeleton is essential for a wide range of cellular functions and it could also participate in mechanosensing by transmitting intra- and extra-cellular mechanical stimuli via the cytoskeleton to the nucleus. Nesprins and SUN proteins, located at the nuclear envelope, form the LINC (linker of nucleoskeleton and cytoskeleton) complex that connects the nucleus to the cytoskeleton; underlying nuclear lamins contribute to anchoring LINC complex components at the nuclear envelope. Disruption of the LINC complex or loss of lamins can result in disturbed perinuclear actin and intermediate filament networks and causes severe functional defects, including impaired nuclear positioning, cell polarization and cell motility. Recent studies have identified the LINC complex as the major force-transmitting element at the nuclear envelope and suggest that many of the aforementioned defects can be attributed to disturbed force transmission between the nucleus and the cytoskeleton. Thus mutations in nesprins, SUN proteins or lamins, which have been linked to muscular dystrophies and cardiomyopathies, may weaken or completely eliminate LINC complex function at the nuclear envelope and result in impaired intracellular force transmission, thereby disrupting critical cellular functions.

Amplification of the N-myc proto-oncogene signifies aggressive behavior in human neuroblastoma. Likewise, overexpression of the intermediate filament nestin, a neuroectodermal stem cell marker, is linked to increased aggressiveness in several nervous system tumors. We investigated the interaction of these two proteins in human neuroblastoma cells. Neuroblastic cell variants with high levels of N-Myc protein have significantly higher nestin protein levels than non-amplified cell lines, suggesting that the transcription factor N-Myc may regulate nestin expression. Stable transfection of a nestin antisense sequence into neuroblastic, N-myc-amplified, LA1-55n cells results in a 2-fold reduction in nestin protein without altering N-Myc expression. However, cell functions attributed to N-Myc (growth rate, anchorage-independent growth, and motility) all decrease significantly. Transfection studies that modulate N-Myc levels also result in commensurate changes in nestin mRNA and protein amounts as well as in cell proliferation and motility. Thus, nestin appears to be downstream of and regulated by N-Myc. Gel mobility shift assays show that N-Myc binds specifically to E-box sequences in the regulatory second intron of the nestin gene and nuclear run-off studies show that increases in N-Myc protein up-regulate nestin transcription rate. Subcellular fractionation and immunoblot studies indicate that nestin is present in the nucleus as well as in the cytoplasm of neuroblastoma cell lines. Finally, DNA cross-linking experiments show that nestin binds DNA in N-myc-amplified N-type cell lines. Thus, nestin may be one mediator of N-myc-associated tumor aggressiveness of human neuroblastoma.

Neurogenesis in the adult mammalian nervous system is now well established in the subventricular zone of the anterolateral ventricle and subgranular zone of the hippocampus. In these regions, neurons are thought to arise from neural stem cells, identified by their expression of specific intermediate filament proteins (nestin, vimentin, GFAP) and transcription factors (Sox2). In the present study, we show that in adult rat and mouse, the circumventricular organs (CVOs) are rich in nestin+, GFAP+, vimentin+ cells which express Sox2 and the cell cycle-regulating protein Ki67. In culture, these cells proliferate as neurospheres and express neuronal (doublecortin+, beta-tubulin III+) and glial (S100beta+, GFAP+, RIP+) phenotypic traits. Further, our in vivo studies using bromodeoxyuridine show that CVO cells proliferate and undergo constitutive neurogenesis and gliogenesis. These findings suggest that CVOs may constitute a heretofore unknown source of stem/progenitor cells, capable of giving rise to new neurons and/or glia in the adult brain.

We report here the cloning and sequencing of matrin 3, an acidic internal matrix protein, from a rat insuloma cDNA library. The nucleotide sequence has a single open reading frame encoding a polypeptide of 845 amino acids. The Genbank and National Biomedical Research Foundation databases did not contain any sequences similar to that of matrin 3. The primary structure consists of 33% charged residues and is generally hydrophilic. The amino-terminal region (residues 1-120) is positively charged and contains a large number of amino acids with free hydroxyl groups (26 of the first 100 residues) as in the lamins and several non-lamin intermediate filament proteins. A highly acidic domain (approximately 170 amino acids) near the carboxyl terminus, in which 32% of the amino acid residues are acidic (Glu or Asp), is a characteristic found in other nuclear proteins (Earnshaw, W. C. (1987) J. Cell Biol. 105, 1479-1482). A putative nuclear targeting signal sequence (Ser-Lys-Lys-Lys-Leu-Lys-Lys-Val-Glu) is located in the middle of the highly acidic domain. The corresponding human deduced partial amino acid sequence is 96% identical to the rat sequence, indicating that matrin 3 is a highly conserved protein.

Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, alpha1-syntrophin, and the sarcoglycan complex. The precise role of alpha-dystrobrevin in skeletal muscle has not yet been determined. To study alpha-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and alpha-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.

Vimentin is an intermediate filament protein normally expressed in mesenchymal cells, but evidence is accumulating in the literature which suggests that the aberrant expression of vimentin in epithelial cancer cells might be related to local invasiveness and metastatic potential. Vimentin expression has previously been associated with invasive properties in an in vitro model consisting of a set of HPV-33-transformed cervical keratinocyte cell lines. In the present study, in order to emphasize those in vitro findings, the expression of vimentin has been investigated in cervical neoplasms of different grades, using immunohistochemistry. A clear association is reported between vimentin expression and metastatic progression, since vimentin was detected in all invasive carcinomas and lymph node metastases, but not in CIN III lesions. These in vivo results are compared with present and previous data obtained in vitro on cervical keratinocyte cell lines, where vimentin expression also correlated with in vitro invasiveness.

One cellular function of intermediate filaments is to provide cells with compliance to small deformations while strengthening them when large stresses are applied. How IFs accomplish this mechanical role is revealed by recent studies of the elastic properties of single IF protein polymers and by viscoelastic characterization of the networks they form. IFs are unique among cytoskeletal filaments in withstanding large deformations. Single filaments can stretch to more than 3 times their initial length before breaking, and gels of IF withstand strains greater than 100% without damage. Even after mechanical disruption of gels formed by crossbridged neurofilaments, the elastic modulus of these gels rapidly recovers under conditions where gels formed by actin filaments are irreversibly ruptured. The polyelectrolyte properties of IFs may enable crossbridging by multivalent counterions, but identifying the mechanisms by which IFs link into bundles and networks in vivo remains a challenge.

The cytokeratins are the intermediate filament proteins characteristic of epithelial cells. In human cells, some 20 different cytokeratin isotypes have been identified. Epithelial cells express between two and ten cytokeratin isotypes and the consequent profile which reflects both epithelial type and differentiation status may be useful in tumour diagnosis. The transitional epithelium or urothelium of the urinary tract shows alterations in the expression and configuration of cytokeratin isotypes related to stratification and differentiation. In transitional cell carcinoma, changes in cytokeratin profile may provide information of potential diagnostic and prognostic significance. The intensification of immunolabelling with some CK8 and CK18 antibodies may underly an active role in tumour invasion and foci of CK17-positive cells may represent proliferating populations. Loss of CK13 is a marker of grade and stage and de novo expression of CK14 is indicative of squamous differentiation and an unfavourable prognosis. However, perhaps the most important recent finding is the demonstration that a normal CK20 expression pattern is predictive of tumour non-recurrence and can be used to make an objective differential diagnosis between transitional cell papilloma and carcinoma. This review will consider cytokeratin expression in urothelium and discuss the application of cytokeratin typing to the diagnosis and prognosis of patients with TCC.

The recent discoveries of prokaryotic homologs of all three major eukaryotic cytoskeletal proteins (actin, tubulin, intermediate filaments) have spurred a resurgence of activity in the field of bacterial morphology. In spirochetes, however, it has long been known that the flagellar filaments act as a cytoskeletal protein structure, contributing to their shape and conferring motility on this unique phylum of bacteria. Therefore, revisiting the spirochete cytoskeleton may lead to new paradigms for exploring general features of prokaryotic morphology. This review discusses the role that the periplasmic flagella in spirochetes play in maintaining shape and producing motility. We focus on four species of spirochetes: Borrelia burgdorferi, Treponema denticola, Treponema phagedenis and Leptonema (formerly Leptospira) illini. In spirochetes, the flagella reside in the periplasmic space. Rotation of the flagella in the above species by a flagellar motor induces changes in the cell morphology that drives motility. Mutants that do not produce flagella have a markedly different shape than wild-type cells.

In response to central nervous system (CNS) injury, and more discretely so also during aging, astrocytes become reactive and increase their expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Studies of mice deficient in astrocytic intermediate filaments have provided insights into the function of reactive gliosis. Recently we demonstrated robust integration of retinal transplants (1) and increased posttraumatic synaptic regeneration (2) in GFAP(-/-)Vim(-/-) mice, suggesting that modulation of astrocyte activity affects the permissiveness of the CNS environment for regeneration. Neurogenesis in the adult mammalian CNS is restricted to essentially two regions, the hippocampus and the subventricular zone. Here, we assessed neurogenesis in the hippocampus of 18-month-old GFAP(-/-)Vim(-/-) mice. In the granular layer of the dentate gyrus, cell proliferation/survival was 34% higher and neurogenesis 36% higher in GFAP(-/-)Vim(-/-) mice than in wildtype controls. These findings suggest that the adult hippocampal neurogenesis in healthy old mice can be increased by modulating astrocyte reactivity.

In large-scale expression profiling analyses, we have previously identified genes differentially expressed between subclones of the pancreatic cancer cell line SUIT-2. One of the genes most strongly overrepresented in the highly metastatic subclone S2-007 as compared with the rarely metastatic subclone S2-028 was the serine proteinase inhibitor SERPINE2 (protease nexin I), suggesting that this protein may play an important part in the process of metastasis. The aim of this study was to functionally characterize SERPINE2 for its potential to influence the invasive and metastatic phenotype of cancer cells in vitro and in vivo. SERPINE2 expression was weak or absent in all normal pancreas and chronic pancreatitis tissue samples examined. In contrast, it was strongly overexpressed in the majority of pancreatic carcinoma as well as gastric and colorectal cancer samples. [(3)H]Thymidine incorporation, soft agar, two chamber migration, Matrigel invasion, and zymography assays of SERPINE2-transfected S2-028 cells revealed no significant effects on metastasis-related cellular characteristics of isolated cancer cells. Although overall metastatic activity of the transfected cells in vivo was also unaltered, SERPINE2 overexpression greatly enhanced the local invasiveness of the s.c. xenograft tumors, accompanied by a massive increase in extracellular matrix (ECM) production in the invasive tumors. ECM deposits were positive for type I collagen, fibronectin, and laminin, thus resembling the desmoplastic reaction commonly observed in pancreatic cancer. Moreover, cancer cells in invasive SERPINE2-expressing tumors tended to adopt a spindle-shaped morphology and strongly expressed the mesenchymal intermediate filament marker vimentin. We propose that SERPINE2 overexpression enhances the invasive potential of pancreatic cancer cells in nude mouse xenografts by altering ECM production and organization within the tumors. Thus, our experimental system for the first time provides the opportunity to effectively model the desmoplastic reaction of pancreatic cancer and represents a valuable new tool for the study of tumor-stroma interactions.

BACKGROUND

Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences.

RESULTS

We have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs) libraries, suppression subtractive hybridization (SSH) libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO) terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7 and copine 1, cytoplasmic intermediate filament (IF) protein and transcription enzymes such as elongation factor 1alpha and EF-2.

CONCLUSIONS

Production of the first cDNA microarray for profiling gene expression in B. glabrata provides a foundation for expanding our understanding of pathways and genes involved in the snail internal defence system (IDS). We demonstrate resistant strain-specific expression of genes potentially associated with the snail IDS, ranging from signalling and inflammation responses through to lysis of proteinacous products (encapsulated sporocysts or phagocytosed parasite components) and processing/degradation of these targeted products by ubiquitination.

Using antibodies raised with chlamydial fusion proteins, we have localized a protein encoded by hypothetical open reading frame CT813 in the inclusion membrane of Chlamydia trachomatis. The detection of the C. trachomatis inclusion membrane by an anti-CT813 antibody was blocked by the CT813 protein but not unrelated fusion proteins. The CT813 protein was detected as early as 12 h after chlamydial infection and was present in the inclusion membrane during the entire growth cycle. All tested serovars from C. trachomatis but not other chlamydial species expressed the CT813 protein. Exogenously expressed CT813 protein in HeLa cells displayed a cytoskeleton-like structure similar to but not overlapping with host cell intermediate filaments, suggesting that the CT813 protein is able to either polymerize or associate with host cell cytoskeletal structures. Finally, women with C. trachomatis urogenital infection developed high titers of antibodies to the CT813 protein, demonstrating that the CT813 protein is not only expressed but also immunogenic during chlamydial infection in humans. In all, the CT813 protein is an inclusion membrane protein unique to C. trachomatis species and has the potential to interact with host cells and induce host immune responses during natural infection. Thus, the CT813 protein may represent an important candidate for understanding C. trachomatis pathogenesis and developing intervention and prevention strategies for controlling C. trachomatis infection.

The gfa gene encodes glial fibrillary acidic protein, an intermediate filament protein expressed in glial cells. In vitro transcription analysis has shown that the human gfa promoter contains two initiation elements that can independently specify the transcription startpoint. One of the elements is a TATA box 25 base pairs (bp) upstream from the transcription startpoint; the other is located between 10 and 50 bp downstream from the transcription initiation site. We have now shown by transfection that both elements are required for efficient transcription in cultured cells. A partially purified natural human TATA box-binding factor (TFIID) from HeLa cells gave footprints that extended from upstream of the TATA box through the downstream initiator. Deletion of the downstream initiator inhibited both TFIID binding to the TATA box and transcription in vitro. In contrast to natural human TFIID, clone human and yeast TFIIDs expressed in bacteria gave footprints covering only the TATA box region, although hypersensitive sites were observed in the downstream region. The cloned TFIIDs also showed less dependence than natural human TFIID on the downstream initiator for both TATA box binding and in vitro transcription. These results suggest that natural human TFIID contains an additional component(s) that contribute(s) to stable TFIID binding and effective transcription by interacting with the downstream initiator.

BACKGROUND

Intermediate filament (IF) is one of the three major cytoskeletal filaments. Vimentin is the most widely expressed IF protein component. The Rho family of small GTPases, such as Cdc42, Rac and Rho, are thought to control the organization of actin filaments as well as other cytoskeletal filaments.

RESULTS

We determined if the vimentin filaments can be regulated by p21-activated kinase (PAK), one of targets downstream of Cdc42 or Rac. In vitro analyses revealed that vimentin served as an excellent substrate for PAK. This phosphorylated vimentin lost the potential to form 10 nm filaments. We identified Ser25, Ser38, Ser50, Ser65 and Ser72 in the amino-terminal head domain as the major phosphorylation sites on vimentin for PAK. The ectopic expression of constitutively active PAK in COS-7 cells induced vimentin phosphorylation. Fibre bundles or granulates of vimentin were frequent in these transfected cells. However, the kinase-inactive mutant induced neither vimentin phosphorylation nor filament reorganization.

CONCLUSIONS

Our observations suggest that PAK may regulate the reorganization of vimentin filaments through direct vimentin phosphorylation.

Plectin is a large cytoskeletal linker protein expressed as several different isoforms from a highly complex gene. This transcript diversity is mainly caused by short 5'-sequences contained in alternative first exons. To elucidate the influence of these sequence differences and to determine potential differential functionality of the resulting protein forms, we conducted a systematic investigation of plectin isoforms on transcript and protein levels. Isoform expression was highly dependent on the different 5' ends, largely due to effects of the 5'-untranslated regions. Initiation of translation downstream of the expected start site led to loss of actin- and integrin beta4-binding in some isoforms. The small alternative N-terminal sequences (5-180 residues) profoundly affected the subcelluar localization of this >500 kDa protein. Specifically, plectin 1f was concentrated at focal adhesion contacts and plectin 1b was exclusively targeted to mitochondria, providing a connection of these organelles to intermediate filaments. Thus, with plectin as a model, we demonstrate a role for 5'-untranslated regions and alternative 5'-splicing as an important regulatory mechanism of protein expression and protein function.

We have studied the expression of chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1 at the mRNA and protein levels in adult neural progenitor cells (NPCs) in neurosphere cultures using RT-PCR and immunocytochemistry methods. NPCs were isolated from the subventricular zone of adult rat brain and propagated in vitro as neurospheres. The neurospheres showed immunoactivity of nestin, an intermediate filament marker for NPCs. NPCs in the neurosphere cultures differentiated into NeuN-, GFAP-, or GalC-positive cells in vitro. Using cultured cortical microglial cells as positive control, we demonstrated the mRNA expression of CXCR4, CCR2, CCR5, and CX3CR1 in neurospheres by RT-PCR. Double immunofluorescent staining further confirmed the co-localization of nestin with either CXCR4, CCR2, CCR5, or CX3CR1 on neurospheres. These results suggest that adult NPCs in the neurosphere cultures express chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1.

Tektins are insoluble alpha-helical proteins essential for the construction of cilia and flagella and are found throughout the eukaryotes apart from higher plants. Being almost universal but still fairly free to mutate, their coding sequences have proved useful for estimating the evolutionary relationships between closely related species. Their protein molecular structure, typically consisting of four coiled-coil rod segments connected by linkers, resembles that of intermediate filament (IF) proteins and lamins. Tektins assemble into continuous rods 2 nm in diameter that are probably equivalent to subfilaments of the 10 nm diameter IFs. Tektin and IF rod sequences both have a repeating pattern of charged amino acids superimposed on the seven-amino-acid hydrophobic pattern of coiled-coil proteins. The length of the repeat segment matches that of tubulin subunits, suggesting that tektins and tubulins may have coevolved, and that lamins and IFs may have emerged later as modified forms of tektin. Unlike IFs, tektin sequences include one copy of a conserved peptide of nine amino acids that may bind tubulin. The 2 nm filaments associate closely with tubulin in doublet and triplet microtubules of axonemes and centrioles, respectively, and help to stabilize these structures. Their supply restricts the assembled lengths of cilia and flagella. In doublet microtubules, the 2 nm filaments may also help to organize the longitudinal spacing of accessory structures, such as groups of inner dynein arms and radial spokes.

A rapid one-step purification procedure for CP49, an intermediate filament protein found in the lens, is described using reverse-phase HPLC. This protein is one of the major intermediate filament proteins of the lens fibre cells and is found in both the water insoluble fraction (WIF) and the water soluble fraction (WSF) of the lens. In order to better understand the physiological role of CP49 in lens transparency we have purified CP49 from both compartments and compared the in vitro assembly characteristics of both by electron microscopy and sedimentation assays. Our studies showed that CP49, when mixed with another lens intermediate filament protein, CP115, forms 10 nm intermediate filaments. Vimentin, another intermediate filament protein found in the lens, was unable to coassemble with CP115, thus demonstrating the specificity of the interaction of CP49 with CP115. CP49 isolated from either the WIF or the WSF formed 10-nm filaments with CP115 and indicated that CP49 from both these lens cell compartments had similar in vitro assembly characteristics. This also suggested that the post-translational modifications observed for CP49 from the different compartments was of little apparent consequence to filament formation. The inability to reconstitute beaded filaments from CP49 and CP115 suggested that other lens proteins may be needed in the reconstitution assay before these lens specific cytoskeletal elements could be repolymerised from their purified protein components. CP49 and CP115 were therefore assembled in the presence of alpha-crystallins and a beaded filament structure was observed as has been seen with type III intermediate filament proteins assembled with alpha-crystallins.