The repeated 36 amino acid motif of Chlamydia trachomatis Hc2 protein binds to the major groove of DNA.

The gram-negative, obligate intracellular human pathogen, Chlamydia trachomatis has a bi-phasic developmental cycle. The histone H1-like C. trachomatis DNA binding protein, Hc2, is produced late during the developmental cycle when the dividing reticulate body transforms into the smaller, metabolically inactive elementary body. Together with Hc1, the two proteins compact the chlamydial chromosome and arrest replication and transcription. Hc2 is heterogeneous in length due to variation in the number of lysine rich pentamers. Six pentamers and one hexamer constitute a 36 amino acid long repetitive unit that, in spite of variations, is unique for Chlamydiaceae. Using synthetic peptides, the DNA-binding capacity of the 36 amino acid peptide and that of a randomized peptide was analyzed. Both peptides bound and compacted plasmid DNA, however, electron microscopy of peptide/DNA complexes showed major differences in the resulting aggregated structures. Fluorescence spectroscopy was used to analyze the binding. After complexing plasmid DNA with each of three different intercalating dyes, increasing amounts of peptides were added and fluorescence spectroscopy performed. The major groove binder, methyl green, was displaced by both peptides at low concentrations, while the minor groove binder, Hoechts, and the intercalating dye, Ethidium Bromide, were displaced only at high concentrations of peptides.

Ossification of ungular cartilages in front feet of cold-blooded trotters--a clinical radiographic evaluation of development over time.

BACKGROUND: It has not yet been shown that ossification of ungular cartilages (OUC) is a pathological condition. Beside heredity, factors such as sex, age, repeated concussion, local trauma, hoof and body size have been suggested as contributing factors for OUC development. By comparing radiographs of front hooves from cold-blooded trotters with different age we wanted to evaluate when development of OUC in cold-blooded trotters occurs and if and when it stabilizes in relation to age and workload. Diagnosis and grading of OUC were based on radiological field examinations of 649 Swedish and Norwegian cold-blooded trotters' front hooves. A hundred and forty-seven of the horses were re-examined 3-13 years (mean age 9, median 8 years) after the first occasion. All radiographs were evaluated blind, using two different grading systems for OUC. Work load, in form of number of races completed, and body size score were collected from official data. Four statistical ordinal regression models were used, compared and evaluated. RESULTS: We identified a breakpoint at 2.8 ± 0.38 years of age when ossification ends and proposed a simpler grading system with more consistent results. There was no significant correlation between body size and grade of OUC. Comparison of different statistical methods for evaluation of ordinal data revealed a piecewise linear regression model as most suitable. CONCLUSIONS: Individuals with OUC developed this condition during the stage of life when their hooves develop in size. Results from this study can assist equine practitioners when examining and for understanding this condition in their clinical work and is also beneficial for the Scandinavian equine industry when devising breeding programs.

A feasibility study of a telephone-based screening service for mild cognitive impairment and its uptake by elderly people.

The risk of developing mild cognitive impairment (MCI), and subsequently dementia, increases with age. Early detection requires a comprehensive clinical examination, which is time consuming and expensive; a face-to-face examination can also be problematic for people living in rural areas which may result in unequal access to services. Telephone-based screening may provide a feasible method of identifying people who would benefit from a full diagnostic workup. We conducted a pilot study in which we offered telephone screening to all patients aged over 60 years at a health clinic in rural northern Norway (n = 259). Fifteen percent of them volunteered (n = 39). Screening identified a number of suspicious cases and we recommended to their general practitioner that 7 patients (18%) be offered a follow-up appointment. Surveys showed that the volunteers were generally positive towards the service, as was the general practitioner who found it helpful to be provided with such information about the elderly patients in his care. In addition, we surveyed the opinions of all general practitioners (n = 480) in the three northernmost counties of Norway concerning a potential service. There was a response rate of 40% (n = 190). Almost half of respondents (45%) would like to make use of such a service if it existed, and 34% believed that their patients would make use of it if available. The pilot study demonstrates the feasibility of telephone screening for clinically significant memory decline, and that users (general practitioners and the elderly) are positive towards such a service.

Identification of the shortest Aß-peptide generating highly specific antibodies against the C-terminal end of amyloid-ß42.

Alzheimer's disease (AD) is characterized by neurofibrillary tangles, consisting of hyperphosphorylated tau protein and senile plaques, which are consisting mainly of amyloid-ß (Aß). Attempts to generate a safe vaccine against Aß rely on both B- and T-cell epitopes within the neurotoxic peptide Aß1-42. This, however, poses a risk for an inflammatory and/or autoimmune response against Aß-peptides in the brain. To overcome such risks we wanted to identify the shortest C-terminal Aß-peptide that would induce antibodies selectively recognizing the C-terminal end of Aß42. Immunization with this antigen should result in a non-inflammatory Th2 immune response and the T-cell response should be against a T-cell epitope covalently attached to the small Aß-peptide. Antigen constructs were made by the ligand presenting assembly (LPA) technology, comprising dimeric presentation of short Aß-peptides ending at amino acid 42 in connection with potent T-cell epitopes. Mice were immunized with antigen constructs using different adjuvants, and sera from mice were tested to characterize the generated immune response. Immunization with Keyhole limpet hemocyanin (KLH)-Aß(37-42) resulted in generation of IgG1 antibodies specific for the Aß42 C-terminal end, indicating a Th2-response. The T-cell mediated response was predominantly against T-cell epitopes in KLH. The antibodies stained senile plaques specifically in brain tissue from AD patients. Thus, KLH-Aß(37-42) was able to induce a non-inflammatory and highly specific antibody response against Aß42.

Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody.

The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope in human AD tissue and stains plaques with high specificity. Therefore the monoclonal antibody can thus be useful in the histological investigations of the AD pathology.

Identification of NCAM-binding peptides promoting neurite outgrowth via a heterotrimeric G-protein-coupled pathway.

A combinatorial library of undecapeptides was produced and utilized for the isolation of peptide binding to the fibronectin type 3 modules (F3I-F3II) of the neural cell adhesion molecule (NCAM). The isolated peptides were sequenced and produced as dendrimers. Two of the peptides (denoted ENFIN2 and ENFIN11) were confirmed to bind to F3I-F3II of NCAM by surface plasmon resonance. The peptides induced neurite outgrowth in primary cerebellar neurons and PC12E2 cells, but had no apparent neuroprotective properties. NCAM is known to activate different intracellular pathways, including signaling through the fibroblast growth factor receptor, the Src-related non-receptor tyrosine kinase Fyn, and heterotrimeric G-proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin, an inhibitor of certain heterotrimeric G-proteins. Neurite outgrowth induced by trans-homophilic NCAM was unaffected by the peptides, whereas knockdown of NCAM completely abrogated ENFIN2- and ENFIN11-induced neuritogenesis. These observations suggest that ENFIN2 and ENFIN11 induce neurite outgrowth in an NCAM-dependent manner through G-protein-coupled signal transduction pathways. Thus, ENFIN2 and ENFIN11 may be valuable for exploring this particular type of NCAM-mediated signaling.

The course of C-reactive protein response in untreated upper respiratory tract infection.

BACKGROUND: High C-reactive protein (CRP) values are frequently found in patients with bacterial respiratory infection, and CRP testing has been shown to be useful in differentiating pneumonia from other respiratory infections. Raised CRP values may also be found in viral respiratory infection, and as a result there is a risk that antibiotics may be wrongly prescribed. AIMS: To describe the course of the CRP response during untreated upper respiratory tract infections and associations between the development of CRP values, erythrocyte sedimentation rate (ESR) and respiratory symptoms. DESIGN OF STUDY: Prospective study. SETTING: Seven general practices in northern Norway. METHOD: Patients with upper respiratory tract infection aged 16 years or over, who were not treated with antibiotics and who had been ill for no more than 3 days, were recruited. Microbiological examinations were undertaken, together with measurements of CRP, ESR and recording of symptoms daily during the first week of illness and on days 10, 14 and 21. RESULTS: An aetiological agent was established in 23 of the 41 included subjects. These were: influenza A, influenza B, rhinovirus, and other agents. Among the 15 patients examined on both the second and the third day of illness, the median CRP value increased from 7-10 mg/l, and the mean value was from 19-24 mg/l between day 2 and day 3. Peak CRP values were reached on days 2 to 4. Higher CRP values were found in those infected with influenza A and B than in the other subjects (P <0.001). A CRP value >10 mg/l was found in 26 subjects during the first 7 days, compared to five subjects after 1 week. Evidence of a secondary infection with group A streptococci was found in two of these five subjects. The development of the symptoms of sore throat, fatigue, clamminess, and pain from muscles and joints followed a similar course as the CRP response, while stuffy nose, cough, sputum production, and dyspnoea tended to persist after the CRP values had approached the normal range. CONCLUSION: A moderately elevated CRP value (10-60 mg/l) is a common finding in viral upper respiratory tract infection, with a peak during days 2-4 of illness. Moderately elevated CRP values cannot support a diagnosis of bacterial infection when the illness has lasted less than 7 days, but may indicate a complication of viral infection after a week.

Identification of neurite extension inducing peptides by means of soluble combinatorial peptide libraries.

To identify hexapeptides capable of inducing neurite outgrowth, we used three groups of soluble combinatorial peptide libraries each consisting of 100 mixtures of hexapeptides (each mixture consisting of 10,000 individual peptides) with partially predetermined sequences (in two out of six amino acid positions). Using this approach a number of neuritogenic peptides were identified. Three selected peptides, QSGKKF, QSGPLA and QSGKQG, were found to induce neurite outgrowth from primary hippocampal neurons with potency comparable to that of growth factors. None of the peptides protected cerebellar granule neurons from cell death induced by withdrawal of potassium chloride. The approach described here suggests the feasibility to use combinatorial peptide libraries in order to identify compounds capable of modulating a specific functional response in the nervous system, without prior knowledge of a molecular target.

A synthetic peptide ligand of neural cell adhesion molecule (NCAM), C3d, promotes neuritogenesis and synaptogenesis and modulates presynaptic function in primary cultures of rat hippocampal neurons.

The neural cell adhesion molecule (NCAM) plays a key role in morphogenesis of the nervous system and in remodeling of neuronal connections accompanying regenerative and cognitive processes. Recently, a new synthetic ligand of NCAM, the C3-peptide, which binds to the NCAM IgI module, has been identified by means of combinatorial chemistry (Rønn, L. C. B, Olsen, M., Ostergaard, S., Kiselyov, V., Berezin, V., Mortensen, M. T., Lerche, M. H., Jensen, P. H., Soroka, V., Saffell, J. L., Doherty, P., Poulsen, F. M., Bock, E., Holm, A., and Saffells, J. L. (1999) Nat. Biotechnol. 17, 1000-1005). In vitro, the dendrimeric form of C3, termed C3d, disrupts NCAM-mediated cell adhesion, induces neurite outgrowth, and triggers intracellular signaling cascades similar to those activated by homophilic NCAM binding. The peptide may therefore be expected to regulate regeneration and synaptic plasticity. Here we demonstrate that in primary cultures of hippocampal neurons: 1) C3d induces a sustained neuritogenic response, the neuritogenic activity of the compound being dependent on the dose, starting time, and duration of peptide application; 2) the peptide triggers the neuritogenic response by forming an adhesive substratum necessary for NCAM-mediated neurite formation and elongation; 3) C3d promotes synapse formation; and 4) C3d modulates the presynaptic function, causing a transient increase of the function at low (2 and 5 microm) doses and a reduction when applied at a higher concentration (10 microm). The effect of the peptide is dependent on the activation of the fibroblast growth factor receptor. We suggest that C3d may constitute a useful lead for the development of compounds for treatment of various neurodegenerative disorders.

Environmental regulation and differential production of members of the Bdr protein family of Borrelia burgdorferi.

Borrelia burgdorferi B31MI carries 18 plasmid-carried genes that form the bdr gene family. The bdr genes of B. burgdorferi encode proteins that form three distinct subfamilies, the BdrD, BdrE, and BdrF subfamilies. bdr orthologs have been demonstrated to be carried by all Borrelia species analyzed, and their widespread distribution suggests that they play an important genus-wide functional role. The biological rationale for maintaining 18 bdr alleles has not been defined. It is our hypothesis that specific paralogs function in different environments and are differentially expressed in response to environmental conditions. As a first step in testing this hypothesis, the production patterns of the Bdr proteins in spirochetes grown under a variety of conditions were assessed through immunoblot analyses. The influence of temperature, serum deprivation, tick feeding, and the mammalian environment on Bdr production was evaluated. These analyses revealed that the synthesis of some Bdr paralogs is environmentally regulated. The production of BdrF(2,) BdrF(1), BdrE(4), and BdrE(5) were upregulated in host-adapted bacteria, while the production levels of other Bdr paralogs were influenced by temperature and serum starvation. These observations suggest that different Bdr paralogs function in different biological environments and provide insight into the biological basis for maintaining multiple members of this gene family.

The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029.

BACKGROUND: Chlamydiae are obligate intracellular bacteria, which are important human pathogens. Genome sequences of C. trachomatis and C. pneumoniae have revealed the presence of a Chlamydia specific gene family encoding polymorphic outer membrane proteins, Pmps. In C. pneumoniae the family comprises twenty-one members, which are all transcribed. In the present study, the expression, processing and localisation of the sixteen full-length Pmps in C. pneumoniae strain CWL029 have been further investigated by two-dimensional gel electrophoresis and immunofluorescence microscopy. RESULTS: Ten Pmps were identified in elementary bodies (EBs). Eight of these were investigated with respect to time dependent expression and all were found to be up-regulated between 36 and 48 hours post infection. Antibodies against Pmp6, 8, 10, 11 and 21 reacted with chlamydiae when infected cells were formalin fixed. Pmp6, Pmp20 and Pmp21 were found in cleaved forms, and the cleavage sites of Pmp6 and Pmp21 were identified. CONCLUSIONS: The Pmps are heavily up-regulated at the time of conversion of RB to EB, and at least ten Pmps are present in EBs. Due to their reaction in formalin fixation it is likely that Pmp6, 8, 10, 11 and 21 are surface exposed. The identified cleavage sites of Pmp6 and Pmp21 are in agreement with the theory that the Pmps are autotransporters.

Characterization of a novel NCAM ligand with a stimulatory effect on neurite outgrowth identified by screening a combinatorial peptide library.

The neural cell adhesion molecule, NCAM, plays a key role in neural development and plasticity mediating cell adhesion and signal transduction. By screening a combinatorial library of synthetic peptides with NCAM purified from postnatal day 10 rat brains, we identified a nonapeptide, termed NCAM binding peptide 10 (NBP10) and showed by nuclear magnetic resonance analysis that it bound the NCAM IgI module of NCAM. NBP10 modulated cell aggregation as well as neurite outgrowth induced specifically by homophilic NCAM binding. Moreover, both monomeric and multimeric forms of NBP10 stimulated neurite outgrowth from primary hippocampal neurons. The neurite outgrowth response to NBP10 was inhibited by a number of compounds previously shown to inhibit neurite outgrowth induced by homophilic NCAM binding, including voltage-dependent calcium channel antagonists, suggesting that NBP10 induced neurite outgrowth by activating a signal transduction pathway similar to that activated by NCAM itself. Moreover, an inhibitor of intracellular calcium mobilization, TMB-8, prevented NBP10-induced neurite outgrowth suggesting that NCAM-dependent neurite outgrowth also requires mobilization of calcium from intracellular calcium stores in addition to calcium influx from extracellular sources. By single-cell calcium imaging we further demonstrated that NBP10 was capable of inducing an increase in intracellular calcium in PC12E2 cells. Thus, the NBP10 peptide is a new tool for the study of molecular mechanisms underlying NCAM-dependent signal transduction and neurite outgrowth, and could prove to be a useful modulator of regenerative processes in the peripheral and central nervous system.

Induction of neuronal differentiation by a peptide corresponding to the homophilic binding site of the second Ig module of the neural cell adhesion molecule.

NCAM plays a key role in neural development and plasticity-mediating cell adhesion and differentiation mainly through homophilic binding. Until recently, attempts to modulate neuronal differentiation and plasticity through NCAM have been impeded by the absence of small synthetic agonists mimicking homophilic interactions of NCAM. We show here that a peptide, P2, corresponding to a 12-amino acid sequence localized in the FG loop of the second Ig module of NCAM, binds to the first Ig module, which is the natural binding partner of the second Ig module, with an apparent K(d) of 4.7 +/- 0.9 x 10(-6) m. P2 inhibits cell aggregation and induces neurite outgrowth from hippocampal neurons, maximal neuritogenic effect being obtained at a concentration of 0.8 microm. The neuritogenic effect was inhibited by preincubation of P2 with the recombinant NCAM-IgI. Both the length of P2 and the basic amino acid residues at the N and C termini are important for its neuritogenic activity. Treatment of hippocampal cultures with P2 results in induction of phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2. Thus, P2 is a potent mimetic of NCAM, and therefore, an attractive compound for the development of drugs for the treatment of neurodegenerative diseases.

Increased intracellular calcium is required for neurite outgrowth induced by a synthetic peptide ligand of NCAM.

We have recently identified a synthetic peptide, termed C3, capable of binding the first immunoglobulin-like module of neural cell adhesion molecule (NCAM) by means of combinatorial chemistry and shown that this NCAM ligand promotes neurite outgrowth. By means of single cell calcium imaging using the calcium-sensitive probe fura-2-acetomethyl ester, we here show that the C3-peptide induced an increase in intracellular calcium in primary hippocampal neurons and PC12-E2 cells, presumably requiring mobilization of calcium from both extracellular and intracellular stores. We further observed that C3-induced neurite outgrowth was inhibited by antagonists of voltage-dependent calcium channels as well as by an inhibitor of intracellular calcium mobilization, TMB-8. These findings demonstrate at the single cell level that a synthetic NCAM ligand directly can induce an increase in intracellular calcium and suggest that NCAM-dependent neurite outgrowth requires calcium mobilization from both extracellular and intracellular calcium stores. Thus, the C3-peptide may be regarded as a useful tool for the study of NCAM-dependent signal transduction. Furthermore, the peptide may be of considerable therapeutical interest for the treatment of neurodegenerative disorders.

Urokinase receptor-associated protein (uPARAP) is expressed in connection with malignant as well as benign lesions of the human breast and occurs in specific populations of stromal cells.

The urokinase-type plasminogen activator (uPA) and the uPA receptor (uPAR) are key components in the plasminogen activation system, serving to promote specific events of extracellular matrix degradation in connection with tissue remodeling and cancer invasion. We recently described a new uPAR-associated protein (uPARAP), an internalization receptor that interacts with the pro-uPA:uPAR complex. In our study, we generated a specific polyclonal peptide antibody against human uPARAP and used it for the localization of uPARAP in different breast lesions. The affinity-purified antibodies specifically recognized uPARAP in Western blotting and gave a strong signal in immunohistochemistry. The immunohistochemic localization pattern was found to be identical to that of uPARAP mRNA as determined in parallel by in situ hybridization. uPARAP expression was then studied in both benign and malignant breast lesions. Whereas the normal breast tissue was uPARAP-negative, all benign lesions and ductal carcinoma in situ lesions showed immunoreactivity in fibroblast-like cells and myoepithelial cells associated with the lesion. In invasive carcinoma, uPARAP immunoreactivity was limited to tumor-associated mesenchymal cells. Double immunofluorescence analysis of invasive ductal carcinoma using antibodies against specific cell markers showed that uPARAP was localized in myofibroblasts and macrophages. No malignant cells, no endothelial cells and no vascular smooth muscle cells showed uPARAP immunoreactivity. We conclude that expression of uPARAP is associated with the abnormal breast and that expression appears in myofibroblasts, macrophages and myoepithelium. We suggest that uPARAP is involved in the clearance of the uPA:uPAR complex as well as other possible ligands during benign and malignant tissue remodeling.

Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2.

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.