Expression of the type VI intermediate filament proteins CP49 and filensin in the mouse lens epithelium.

PURPOSE: The differentiated lens fiber cell assembles a filamentous cytoskeletal structure referred to as the beaded filament (BF). The BF requires CP49 (bfsp2) and filensin (bfsp1) for assembly, both of which are highly divergent members of the large intermediate filament (IF) family of proteins. Thus far, these two proteins have been reported only in the differentiated lens fiber cell. For this reason, both proteins have been considered robust markers of fiber cell differentiation. We report here that both proteins are also expressed in the mouse lens epithelium, but only after 5 weeks of age. METHODS: Localization of CP49 was achieved with immunocytochemical probing of wild-type, CP49 knockout, filensin knockout, and vimentin knockout mice, in sections and in the explanted lens epithelium, at the light microscope and electron microscope levels. The relationship between CP49 and other cytoskeletal elements was probed using fluorescent phalloidin, as well as with antibodies to vimentin, GFAP, and α-tubulin. The relationship between CP49 and the aggresome was probed with antibodies to γ-tubulin, ubiquitin, and HDAC6. RESULTS: CP49 and filensin were expressed in the mouse lens epithelium, but only after 5 weeks of age. At the light microscope level, these two proteins colocalize to a large tubular structure, approximately 7 × 1 µm, which was typically present at one to two copies per cell. This structure is found in the anterior and anterolateral lens epithelium, including the zone where mitosis occurs. The structure becomes smaller and largely undetectable closer to the equator where the cell exits the cell cycle and commits to fiber cell differentiation. This structure bears some resemblance to the aggresome and is reactive with antibodies to HDAC6, a marker for the aggresome. However, the structure does not colocalize with antibodies to γ-tubulin or ubiquitin, also markers for the aggresome. The structure also colocalizes with actin but appears to largely exclude vimentin and α-tubulin. In the CP49 and filensin knockouts, this structure is absent, confirming the identity of CP49 and filensin in this structure, and suggesting a requirement for the physiologic coassembly of CP49 and filensin. CONCLUSIONS: CP49 and filensin have been considered robust markers for mouse lens fiber cell differentiation. The data reported here, however, document both proteins in the mouse lens epithelium, but only after 5 weeks of age, when lens epithelial growth and mitotic activity have slowed. Because of this, CP49 and filensin must be considered markers of differentiation for both fiber cells and the lens epithelium in the mouse. In addition, to our knowledge, no other protein has been shown to emerge so late in the development of the mouse lens epithelium, suggesting that lens epithelial differentiation may continue well into post-natal life. If this structure is related to the aggresome, it is a rare, or perhaps unique example of a large, stable aggresome in wild-type tissue.

An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies.

PURPOSE: Several properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques. METHODS: A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed. RESULTS: An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence. CONCLUSIONS: A simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential shrinkage, autofluorescence, and tissue immunoreactivity have been problematic.

The structure of vimentin linker 1 and rod 1B domains characterized by site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) and X-ray crystallography.

Despite the passage of ∼30 years since the complete primary sequence of the intermediate filament (IF) protein vimentin was reported, the structure remains unknown for both an individual protomer and the assembled filament. In this report, we present data describing the structure of vimentin linker 1 (L1) and rod 1B. Electron paramagnetic resonance spectra collected from samples bearing site-directed spin labels demonstrate that L1 is not a flexible segment between coiled-coils (CCs) but instead forms a rigid, tightly packed structure. An x-ray crystal structure of a construct containing L1 and rod 1B shows that it forms a tetramer comprising two equivalent parallel CC dimers that interact with one another in the form of a symmetrical anti-parallel dimer. Remarkably, the parallel CC dimers are themselves asymmetrical, which enables them to tetramerize rather than undergoing higher order oligomerization. This functionally vital asymmetry in the CC structure, encoded in the primary sequence of rod 1B, provides a striking example of evolutionary exploitation of the structural plasticity of proteins. EPR and crystallographic data consistently suggest that a very short region within L1 represents a minor local distortion in what is likely to be a continuous CC from the end of rod 1A through the entirety of rod 1B. The concordance of this structural model with previously published cross-linking and spectral data supports the conclusion that the crystallographic oligomer represents a native biological structure.

Site-directed spin labeling and electron paramagnetic resonance determination of vimentin head domain structure.

Intermediate filament (IF) proteins have been predicted to have a conserved tripartite domain structure consisting of a largely alpha-helical central rod domain, flanked by head and tail domains. However, crystal structures have not been reported for any IF or IF protein. Although progress has been made in determining central rod domain structure, no structural data have been reported for either the head or tail domains. We used site-directed spin labeling and electron paramagnetic resonance to analyze 45 different spin labeled mutants spanning the head domain of vimentin. The data, combined with results from a previous study, provide strong evidence that the polypeptide backbones of the head domains form a symmetric dimer of closely apposed backbones that fold back onto the rod domain, imparting an asymmetry to the dimer. By following the behavior of spin labels during the process of in vitro assembly, we show that head domain structure is dynamic, changing as a result of filament assembly. Finally, because the vimentin head domain is the major site of the phosphorylation that induces disassembly at mitosis, we studied the effects of phosphorylation on head domain structure and demonstrate that phosphorylation drives specific head domain regions apart. These data provide the first evidence-based model of IF head domain structure.

Completion of the Vimentin Rod Domain Structure Using Experimental Restraints: A New Tool for Exploring Intermediate Filament Assembly and Mutations.

Electron paramagnetic resonance (EPR) spectroscopy of full-length vimentin and X-ray crystallography of vimentin peptides has provided concordant structural data for nearly the entire central rod domain of the protein. In this report, we use a combination of EPR spectroscopy and molecular modeling to determine the structure and dynamics of the missing region and unite the separate elements into a single structure. Validation of the linker 1-2 (L1-2) modeling approach is demonstrated by the close correlation between EPR and X-ray data in the previously solved regions. Importantly, molecular dynamic (MD) simulation of the constructed model agrees with spin label motion as determined by EPR. Furthermore, MD simulation shows L1-2 heterogeneity, with a concerted switching of states among the dimer chains. These data provide the first ever experimentally driven model of a complete intermediate filament rod domain, providing research tools for further modeling and assembly studies.

Identification of phosphorylation-induced changes in vimentin intermediate filaments by site-directed spin labeling and electron paramagnetic resonance.

Phosphorylation drives the disassembly of the vimentin intermediate filament (IF) cytoskeleton at mitosis. Chromatographic analysis has suggested that phosphorylation produces a soluble vimentin tetramer, but little has been determined about the structural changes that are caused by phosphorylation or the structure of the resulting tetramer. In this study, site-directed spin labeling and electron paramagnetic resonance (SDSL-EPR) were used to examine the structural changes resulting from protein kinase A phosphorylation of vimentin IFs in vitro. EPR spectra suggest that the tetrameric species resulting from phosphorylation is the A11 configuration. EPR spectra also establish that the greatest degree of structural change was found in the linker 2 and the C-terminal half of the rod domain, despite the fact that most phosphorylation occurs in the N-terminal head domain. The phosphorylation-induced changes notably affected the proposed "trigger sequences" located in the linker 2 region, which have been hypothesized to mediate the induction of coiled-coil formation. These data are the first to document specific changes in IF structure resulting from a physiologic regulatory mechanism and provide further evidence, also generated by SDSL-EPR, that the linker regions play a key role in IF structure and regulation of assembly/disassembly.

Identifying the role of specific motifs in the lens fiber cell specific intermediate filament phakosin.

PURPOSE: Phakosin and filensin are lens fiber cell-specific intermediate filament (IF) proteins. Unlike every other cytoplasmic IF protein, they assemble into a beaded filament (BF) rather than an IF. Why the lens fiber cell requires two unique IF proteins and why and how they assemble into a structure other than an IF are unknown. In this report we test specific motifs/domains in phakosin to identify changes that that have adapted phakosin to lens-specific structure and function. METHODS: Phakosin shows the highest level of sequence identity to K18, whose natural assembly partner is K8. We therefore exchanged conserved keratin motifs between phakosin and K18 to determine whether phakosin's divergent motifs could redirect the assembly of chimeric K18 and K8. Modified proteins were bacterially expressed and purified. Assembly competence was assessed by electron microscopy. RESULTS: Substitution of the phakosin helix initiation motif (HIM) into K18 does not alter assembly with K8, establishing that the radical divergence in phakosin HIM is not by itself the mechanism by which IF assembly is redirected to BF assembly. Unexpectedly, K18 bearing phakosin HIM resulted in normal IF assembly, despite the presence of an otherwise disease-causing R-C substitution, and two helix-disrupting glycines. This disproves the widely held belief that mutation of the R is catastrophic to IF assembly. Additional data are presented that suggest normal IF assembly is dependent on sequence-specific interactions between the IF head domain and the HIM. CONCLUSIONS: In the lens fiber cell, two members of the IF family have evolved to produce BFs instead of IFs, a change that presumably adapts the IF to a fiber cell-specific function. The authors establish here that the most striking divergence seen in phakosin is not, as hypothesized, the cause of this altered assembly outcome. The authors further establish that the HIM of IFs is far more tolerant of mutations, such as those that cause some corneal dystrophies and Alexander disease, than previously hypothesized and that normal assembly involves sequence-specific interactions between the head domain and the HIM.

Identification and characterization of complete RTX operons in Moraxella bovoculi and Moraxella ovis.

To determine if Moraxella bovoculi (M. bovoculi), a recently characterized coccoid Moraxella that was isolated from the eyes of calves affected with infectious bovine keratoconjunctivitis (IBK), and Moraxella ovis (M. ovis), originally isolated from sheep with conjunctivitis, possessed genes encoding RTX proteins, genomic DNA was amplified with oligonucleotide primers targeting RTX operon genes of Moraxella bovis (M. bovis). Complete classical RTX operons composed of RTXCABD genes closely linked to a putative secretion accessory protein encoding gene (tolC) were identified in M. bovoculi and M. ovis and were designated mbvCABDtolC and movCABDtolC, respectively. These genes were closely related to M. bovis mbxCABDtolC. Polyclonal rabbit antiserum against the carboxy terminus of M. bovoculi MbvA neutralized hemolytic activity of both M. bovoculi and M. ovis; this antiserum did not neutralize the hemolytic activity of M. bovis. M. bovoculi and M. ovis possess genes that encode proteins related to pathogenic factors of M. bovis.

Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis pilin-Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine.

To evaluate the efficacy of a recombinant Moraxella bovis pilin-M. bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye), a randomized, blinded, controlled field trial was conducted during summer 2005 in a northern California herd of beef cattle. One hundred and one steers were vaccinated with ISCOM matrix (adjuvant control), recombinant M. bovis cytotoxin carboxy terminus+ISCOM matrix (MbxA), or recombinant M. bovis pilin-cytotoxin carboxy terminus+ISCOM matrix (pilin-MbxA); calves received secondary vaccinations 21 days later. Calves were examined once weekly for 18 weeks for the development of corneal ulcers associated with IBK. Overall, the pilin-MbxA vaccinated group had the lowest overall cumulative proportion of ulcerated calves. Calves that received MbxA, whether alone or with pilin had significantly higher M. bovis cytotoxin serum neutralizing titers as compared to control calves. Results of ocular cultures suggested that vaccination with an M. bovis antigen affected organism type isolated from an ulcer: M. bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with MbxA and pilin-MbxA. In addition, vaccination of calves with MbxA and pilin-MbxA resulted in a higher prevalence of Moraxella bovoculi sp. nov. in ocular cultures. While no significant difference was observed between a cytotoxin versus pilin+cytotoxin vaccine against IBK, the reduced cumulative proportion of IBK in the pilin-cytotoxin vaccinated calves suggests it may provide an advantage over a cytotoxin vaccine alone. Efficacy of an M. bovis vaccine may be reduced in herds where IBK is associated with M. bovoculi sp. nov.

The Moraxella bovis RTX toxin locus mbx defines a pathogenicity island.

To characterize flanking regions of the mbx operon in Moraxella bovis, DNA surrounding mbxCABDtolC was sequenced in haemolytic and nonhaemolytic strains of M. bovis. In two haemolytic strains of M. bovis, the mbx operon, including the adjacent M. bovis tolC orthologue, was flanked by approximately 700 bp imperfect repeats. Nonhaemolytic strains of M. bovis had only one or no such repeats, as well as ORFs identical to those flanking the repeats from haemolytic M. bovis. Two nonhaemolytic strains also contained ORFs with deduced amino acid sequence similarity to bacterial araJ genes. The G+C content of the mbxCABDtolC gene region was lower than the flanking regions. The genetic organization and G+C content of mbxCABDtolC genes, and flanking repeats in haemolytic M. bovis, as well as the presence or absence of flanking repeats in nonhaemolytic M. bovis, suggests that this RTX operon is located on a mobile genetic element, and supports the designation of this region as a pathogenicity island, which is believed to be the first such element demonstrated in M. bovis.

Systemic and ocular immune responses in cattle following intranasal vaccination with precipitated or partially solubilized recombinant Moraxella bovis cytotoxin adjuvanted with polyacrylic acid.

OBJECTIVE To evaluate changes in systemic and ocular antibody responses of steers following intranasal vaccination with precipitated or partially solubilized recombinant Moraxella bovis cytotoxin (MbxA). ANIMALS 13 Angus steers with ages ranging from 318 to 389 days and weights ranging from 352 to 437 kg. PROCEDURES Steers were assigned to receive 500 µg of a precipitated (MbxA-P; n = 5) or partially solubilized (MbxA-S; 5) recombinant MbxA subunit adjuvanted with polyacrylic acid. A control group (n = 3) received the adjuvant alone. Each steer received the assigned treatment (1 mL/nostril) on days 0 and 28. Serum and tear samples were collected on days 0 (before vaccination), 14, 28, 42, and 55. Changes in MbxA-neutralizing antibody titers and MbxA-specific IgG concentrations in serum and tears and changes in MbxA-specific IgA concentrations in tears were measured. RESULTS Mean fold changes in MbxA-specific IgG concentration in serum and tears and MbxA-neutralizing antibody titer in tears for the MbxA-P group were significantly greater than those for the MbxA-S and control groups. Mean serum MbxA-neutralizing antibody titer did not differ among the 3 groups. Although the mean fold change in tear MbxA-specific IgA concentration differed significantly among the groups in the overall analysis, post hoc comparisons failed to identify any significant pairwise differences. CONCLUSIONS AND CLINICAL RELEVANCE Systemic and ocular immune responses induced by intranasal administration of the MbxA-P vaccine were superior to those induced by the MbxA-S vaccine. Additional research is necessary to determine whether the MbxA-P vaccine can prevent naturally occurring infectious bovine keratoconjunctivitis.

How to Study Intermediate Filaments in Atomic Detail.

Studies of the intermediate filament (IF) structure are a prerequisite of understanding their function. In addition, the structural information is indispensable if one wishes to gain a mechanistic view on the disease-related mutations in the IFs. Over the years, considerable progress has been made on the atomic structure of the elementary building block of all IFs, the coiled-coil dimer. Here, we discuss the approaches, methods and practices that have contributed to this advance. With abundant genetic information on hand, bioinformatics approaches give important insights into the dimer structure, including the head and tail regions poorly assessable experimentally. At the same time, the most important contribution has been provided by X-ray crystallography. Following the "divide-and-conquer" approach, many fragments from several IF proteins could be crystallized and resolved to atomic resolution. We will systematically cover the main procedures of these crystallographic studies, suggest ways to maximize their efficiency, and also discuss the possible pitfalls and limitations. In addition, electron paramagnetic resonance with site-directed spin labeling was another method providing a major impact toward the understanding of the IF structure. Upon placing the spin labels into specific positions within the full-length protein, one can evaluate the proximity of the labels and their mobility. This makes it possible to make conclusions about the dimer structure in the coiled-coil region and beyond, as well as to explore the dimer-dimer contacts.

Sequence features accurately predict genome-wide MeCP2 binding in vivo.

Methyl-CpG binding protein 2 (MeCP2) is critical for proper brain development and expressed at near-histone levels in neurons, but the mechanism of its genomic localization remains poorly understood. Using high-resolution MeCP2-binding data, we show that DNA sequence features alone can predict binding with 88% accuracy. Integrating MeCP2 binding and DNA methylation in a probabilistic graphical model, we demonstrate that previously reported genome-wide association with methylation is in part due to MeCP2's affinity to GC-rich chromatin, a result replicated using published data. Furthermore, MeCP2 co-localizes with nucleosomes. Finally, MeCP2 binding downstream of promoters correlates with increased expression in Mecp2-deficient neurons.

Isolation and characterization of the human CP49 gene promoter.

PURPOSE: This study focuses on the identification of regulatory elements that contribute to lens-specific expression of the human CP49 gene within the 5'-flanking DNA sequences. METHODS: The DNA sequence upstream of the human CP49 coding region was subcloned as a set of 5' and 3' deletion series. The constructs were transfected into lens (N/N1003A) and nonlens (NIH3T3) cell lines and chicken primary lens cultures, to test for promoter activity and specificity. To further test the specificity, a portion of the 5' flanking DNA sequence was used to drive transgene expression in mice. The flanking DNA sequence was analyzed for potential transcription factor-binding sites. RESULTS: The 5'-flanking DNA preferentially activated reporter gene expression in a lens-preferred manner when transfected into cultured cells. Transgene expression driven by the CP49 promoter region was lens specific. Analysis of the proximal promoter sequence revealed the presence of potential binding sites for the AP-1, AP-2, and OCT-1 transcription factors and the absence of TATA and CAAT boxes. CONCLUSIONS: The sequence upstream of the CP49 gene possesses promoter activity and is able to drive lens-preferred expression in both transfection and transgenic experiments. Promoter activity is dependent on the presence of the proximal 300 bp directly upstream of the coding region.

Analysis of non-crystallin lens fiber cell gene expression in c-Maf -/- mice.

PURPOSE: To examine the role of the c-Maf transcription factor in the regulation of non-crystallin lens fiber cell specific gene expression. METHODS: Expression of lens fiber cell genes in c-Maf null mice was analyzed by immunohistochemical and RT-PCR methods. In addition, the effect of c-Maf on fiber cell gene promoter fragments was examined in co-transfection experiments. RESULTS: Examination of protein expression in c-Maf homozygous mutant mice revealed undetectable levels of CP49, CP115 and MIP protein. mRNA transcripts for these non-crystallin genes were observed at reduced levels in subsequent RT-PCR experiments. The co-transfection of a c-Maf expression construct with reporter constructs containing the proximal promoters of the CP49 and CP115 genes, the structural components of the lens fiber cell beaded filament, did not result in activation of reporter gene expression, as was observed with a crystallin promoter. CONCLUSIONS: While c-Maf has been shown to play a critical role in lens specific expression of the numerous crystallin genes, it does not appear to regulate some non-crystallin fiber cell genes in the same manner. In contrast to most crystallin genes, detectable levels of CP49, CP115, and MIP transcripts are achieved in the absence of c-Maf. In co-transfection experiments, the CP49 and CP115 proximal promoter sequences were not activated by c-Maf. This suggests that increases in transcription of non-crystallin lens fiber cell genes can be enacted in a c-Maf independent fashion.

An RTX operon in hemolytic Moraxella bovis is absent from nonhemolytic strains.

Pathogenic isolates of Moraxella bovis express a calcium-dependent transmembrane pore forming cytotoxin that is an RTX toxin encoded by mbxA. The DNA flanking mbxA was cloned and sequenced to determine if M. bovis contained a classical RTX operon. Open reading frames (ORFs) with deduced amino acid sequence homology to putative activation (RTX C) and transport (RTX B and D) proteins were identified and have been designated MbxC, MbxB, and MbxD, respectively. Thus, hemolytic M. bovis contains a typical RTX operon comprised of four genes arranged (5'-3') mbxCABD. In addition, the deduced amino acid sequences of DNA flanking mbxCABD revealed ORFs with amino acid sequence similarity to transposases (5'). At the 3' end of the mbx gene cluster, an ORF with homology to bacterial tolC genes was identified. Thus, as with the cya RTX operon of Bordetella pertussis, M. bovis appears to have a secretion accessory protein linked to RTX genes. Analysis of genomic DNA isolated from 5 nonhemolytic M. bovis strains by PCR and Southern blotting revealed the absence of mbxCABD. These strains did, however, amplify with primers specific for the 5' region flanking mbxC. M. bovis harbors a classical RTX operon that is absent in nonhemolytic strains.

A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration.

Thrombospondins are multimeric extracellular matrix glycoproteins that play important roles in development, synaptogenesis and wound healing in mammals. We previously identified four putative thrombospondins in the genome of the starlet sea anemone Nematostella vectensis. This study presents the first analysis of these thrombospondins, with the goals of understanding fundamental roles of thrombospondins in the Eumetazoa. Reverse transcriptase PCR showed that each of the N. vectensis thrombospondins (Nv85341, Nv22035, Nv168100 and Nv30790) is transcribed. Three of the four thrombospondins include an RGD or KGD motif in their thrombospondin type 3 repeats at sites equivalent to mammalian thrombospondins, suggesting ancient roles as RGD integrin ligands. Phylogenetic analysis based on the C-terminal regions demonstrated a high level of sequence diversity between N. vectensis thrombospondins. A full-length cDNA sequence was obtained for Nv168100 (NvTSP168100), which has an unusual domain organization. Immunohistochemistry with an antibody to NvTSP168100 revealed labeling of neuron-like cells in the mesoglea of the retractor muscles and the pharynx. In situ hybridization and quantitative PCR showed that NvTSP168100 is upregulated during regeneration. Immunohistochemistry of the area of regeneration identified strong immunostaining of the glycocalyx, the carbohydrate-rich matrix coating the epidermis, and electron microscopy identified changes in glycocalyx organization during regeneration. Thus, N. vectensis thrombospondins share structural features with thrombospondins from mammals and may have roles in the nervous system and in matrix reorganization during regeneration.

Electron paramagnetic resonance analysis of the vimentin tail domain reveals points of order in a largely disordered region and conformational adaptation upon filament assembly.

Very little data have been reported that describe the structure of the tail domain of any cytoplasmic intermediate filament (IF) protein. We report here the results of studies using site directed spin labeling and electron paramagnetic resonance (SDSL-EPR) to explore the structure and dynamics of the tail domain of human vimentin in tetramers (protofilaments) and filaments. The data demonstrate that in contrast to the vimentin head and rod domains, the tail domains are not closely apposed in protofilaments. However, upon assembly into intact IFs, several sites, including positions 445, 446, 451, and 452, the conserved "beta-site," become closely apposed, indicating dynamic changes in tail domain structure that accompany filament elongation. No evidence is seen for coiled-coil structure within the region studied, in either protofilaments or assembled filaments. EPR analysis also establishes that more than half of the tail domain is very flexible in both the assembly intermediate and the intact IF. However, by positioning the spin label at distinct sites, EPR is able to identify both the rod proximal region and sites flanking the beta-site motif as rigid locations within the tail. The rod proximal region is well assembled at the tetramer stage with only slight changes occurring during filament elongation. In contrast, at the beta site, the polypeptide backbone transitions from flexible in the assembly intermediate to much more rigid in the intact IF. These data support a model in which the distal tail domain structure undergoes significant conformational change during filament elongation and final assembly.

Randomized controlled field trial to assess efficacy of a Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis.

OBJECTIVE: To evaluate efficacy of a recombinant Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK). ANIMALS: 107 beef steers. PROCEDURES: 2 groups of calves were inoculated SC with an immunostimulating complex (ISCOM) matrix adjuvant (control group; n = 54) or a recombinant M bovis pilin-cytotoxin-M bovoculi cytotoxin subunit antigen with the ISCOM matrix adjuvant (vaccine group; 53); calves received booster injections 21 days later. Calves were examined once weekly for 16 weeks. Investigators and herd managers were not aware of the inoculum administered to each calf throughout the trial. Primary outcome of interest was the cumulative proportion of calves that developed IBK. Serum samples were obtained before inoculation (day 0) and on days 42 and 112. Serum hemolysin-neutralizing titers against native M bovis and M bovoculi cytotoxin were determined. RESULTS: No difference was detected between groups for the cumulative proportion of calves that developed IBK at weeks 8 and 16 after inoculation. Non-IBK-affected calves in the vaccine group had a significantly higher fold change in serum hemolysin-neutralizing titer against native M bovoculi cytotoxin from day 0 to 42 compared to control calves. CONCLUSIONS AND CLINICAL RELEVANCE: The M bovis pilin-cytotoxin-M bovoculi cytotoxin subunit vaccine with the ISCOM matrix adjuvant was not effective at preventing naturally occurring IBK. It is likely that the incorporation of additional protective antigens in a recombinant Moraxella spp subunit vaccine will be required to yield a product that can be used for effective immunization of cattle against IBK.

Intermediate filaments regulate tissue size and stiffness in the murine lens.

PURPOSE: To define the contributions of the beaded filament (BF), a lens-specific intermediate filament (IF), to lens morphology and biomechanics. METHODS: Wild-type and congenic CP49 knockout (KO) mice were compared by using electrophysiological, biomechanical, and morphometric approaches, to determine changes that occurred because of the absence of this cytoskeletal structure. RESULTS: Electrophysiological assessment established that the fiber cells lacking the lens-specific IFs were indistinguishable from wild-type fiber cells. The CP49 KO mice exhibited lower stiffness, and an unexpected higher resilience than the wild-type lenses. The absence of these filaments resulted in lenses that were smaller, and exhibited a higher ratio of lens:lens nucleus size. Finally, lens shape differed as well, with the CP49 KO showing a higher ratio of axial:equatorial diameter. CONCLUSIONS: Previous work has shown that BFs are necessary in maintaining fiber cell and lens structural phenotypes with age, and that absence of these filaments results in a loss of lens clarity. This work demonstrates that several tissue-level properties that are critical to lens function are also dependent, at least in part, on the presence of these lens-specific IFs.