Neuronal ceroid lipofuscinosis in a Schapendoes dog is caused by a missense variant in CLN6.

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative disorders that occur in humans, dogs, and several other species. NCL is characterised clinically by progressive deterioration of cognitive and motor function, epileptic seizures, and visual impairment. Most forms present early in life and eventually lead to premature death. Typical pathological changes include neuronal accumulation of autofluorescent, periodic acid-Schiff- and Sudan black B-positive lipopigments, as well as marked loss of neurons in the central nervous system. Here, we describe a 19-month-old Schapendoes dog, where clinical signs were indicative of lysosomal storage disease, which was corroborated by pathological findings consistent with NCL. Whole genome sequencing of the affected dog and both parents, followed by variant calling and visual inspection of known NCL genes, identified a missense variant in CLN6 (c.386T>C). The variant is located in a highly conserved region of the gene and predicted to be harmful, which supports a causal relationship. The identification of this novel CLN6 variant enables pre-breeding DNA-testing to prevent future cases of NCL6 in the Schapendoes breed, and presents a potential natural model for NCL6 in humans.

Cataracts in Havanese: genome wide association study reveals two loci associated with posterior polar cataract.

BACKGROUND: Cataract is considered an important health issue in Havanese, and studies indicate a breed predisposition. Possible consequences of cataracts include lens induced uveitis, reduced eyesight, and blindness in severe cases. Reducing the prevalence of cataracts could therefore improve health and welfare significantly. The most frequently diagnosed forms of cataract in Havanese are cortical- and anterior suture line cataract, but cases of posterior polar cataract are also regularly reported. Out of the three, posterior polar- and cortical cataracts are considered the most clinically relevant. RESULTS: We performed a genome wide association study that included 57 controls and 27 + 23 + 7 cases of cortical-, anterior suture line- and posterior polar cataract, respectively. An association analysis using a mixed linear model, revealed two SNPs on CFA20 (BICF2S23632983, p = 7.2e-09) and CFA21 (BICF2G630640490, p = 3.3e-09), that were significantly associated with posterior polar cataract, both of which are linked to relevant candidate genes. The results suggest that the two variants are linked to alleles with large effects on posterior polar cataract formation, possibly in a dominant fashion, and identifies regions that should be subject to further sequencing. Promising regions on CFA4 and CF30 were also identified in the association analysis of cortical cataract. The top SNPs on each chromosome, chr4_12164500 (p = 4.3e-06) and chr30_28836339 (p = 5.6e-06), are located within, or in immediate proximity to, potential cataract candidate genes. The study shows that age at examination is strongly associated with sensitivity of cataract screening. Havanese in Norway are on average 3.4 years old when eye examinations are performed: an age where most dogs that are genetically at risk have not yet developed clinically observable changes. Increasing the average age of breeding animals could increase accuracy of selection, leading to improved health. CONCLUSIONS: The study identified two loci, on CFA20 and CFA21, that were significantly associated with posterior polar cataract in Havanese. SNPs that showed putative association with cortical cataracts, were observed on CFA4 and CFA30. All the top SNPs are located in close proximity to cataract candidate genes. The study also show that sensitivity of cataract screening is highly dependent on age at examination.

A 1 bp deletion in HACE1 causes ataxia in Norwegian elkhound, black.

A number of inherited ataxias is known in humans, with more than 250 loci implicated, most of which are included in human ataxia screening panels. Anecdotally, cases of ataxia in the Norwegian elkhound black have been known for the last 40 years. Affected puppies from three litters were clinically and neurologically examined, and postmortem samples were collected for morphological studies, including ultrastructural analyses. The puppies displayed vestibulocerebellar neurological signs and had degenerative histopathological alterations in cerebellum and brain stem. Three affected dogs, each from different litters, as well as both parents and one healthy littermate from each litter, were whole genome sequenced. Through variant calling we discovered a disease-associated 1 bp deletion in HACE1 (CFA12), resulting in a frameshift at codon 333 and a premature stop codon at codon 366. The perfect association combined with the predicted significant molecular effect, strongly suggest that we have found the causative mutation for Norwegian elkhound black ataxia. We have identified a novel candidate gene for ataxia where dogs can serve as a spontaneous model for improved understanding of ataxia, also in human.

Heritability of distichiasis in Havanese dogs in Norway.

BACKGROUND: Distichiasis is a presumed inherited eyelid disease, characterized by misplaced eyelashes. The effect on eye health and animal welfare varies between individuals; most mild cases show no clinical signs, but some affected animals develop painful corneal disease. In this study, we investigated the prevalence and heritability of distichiasis in the Norwegian population of Havanese dogs. RESULTS: A total of 1156 Havanese were included in the study. Out of these, 168 were affected with distichiasis, making the prevalence in our sample 14.5% (95% CI 12.5-16.6%). There was no sex predisposition. Most affected individuals were graded "mildly affected". The estimates generally showed high heritabilities, which varied between 0.276 (linear model) and 0.720 (Bayesian threshold model). The linear estimates, after conversion to the underlying scale (h2l = 0.664-0.674), corresponds well to the results of the Bayesian models. CONCLUSIONS: The estimated heritability of distichiasis in Havanese is high and the prevalence is moderate. The high heritability indicate that a significant selection response could be obtained by simple mass selection. To secure good animal welfare, it's important to control the number of affected individuals and especially the severely affected.

Distichiasis is an eye condition, characterized by misplaced eyelashes, that is frequently seen in dogs. Some dog breeds appear to be more at risk than others. The degree of clinical signs in affected dogs varies a lot. Many mild cases appear to be completely asymptomatic, while others suffer pain and damage to the eye, which necessitates removal of the hairs.In this study, we investigate both how common distichiasis is in the Havanese dog breed and estimate the degree of genetic influence on the trait. We find that 14.5% of eye screened Havanese, registered in the Norwegian Kennel Club, are affected with distichiasis. Most cases are graded "mild". There is no significant difference in how many males and females are affected.We find high heritability estimates of distichiasis in Havanese (≈0.28 calculated by linear models and 0.59-0.72 calculated by Bayesian threshold models), showing a high genetic influence on the trait. The high estimated heritability mean that it should be possible to reduce the prevalence of the condition, and contribute to improved animal welfare, though systematic breeding.We recommend that all Havanese are eye screened prior to breeding, to control the prevalence of distichiasis, as well as other eye conditions that are relevant in the breed, like cataracts. Dogs with severe distichiasis or ectopic cilia should not be bred. Dogs with mild or moderate distichiasis may be bred to an unaffected partner.

Short and sweet: foreleg abnormalities in Havanese and the role of the FGF4 retrogene.

BACKGROUND: Cases of foreleg deformities, characterized by varying degrees of shortened and bowed forelegs, have been reported in the Havanese breed. Because the health and welfare implications are severe in some of the affected dogs, further efforts should be made to investigate the genetic background of the trait. A FGF4-retrogene on CFA18 is known to cause chondrodystrophy in dogs. In most breeds, either the wild type allele or the mutant allele is fixed. However, the large degree of genetic diversity reported in Havanese, could entail that both the wild type and the mutant allele segregate in this breed. We hypothesize that the shortened and bowed forelegs seen in some Havanese could be a consequence of FGF4RG-associated chondrodystrophy. Here we study the population prevalence of the wild type and mutant allele, as well as effect on phenotype. We also investigate how the prevalence of the allele associated with chondrodystrophy have changed over time. We hypothesize that recent selection, may have led to a gradual decline in the population frequency of the lower-risk, wild type allele. RESULTS: We studied the FGF4-retrogene on CFA18 in 355 Havanese and found variation in the presence/absence of the retrogene. The prevalence of the non-chondrodystrophic wild type is low, with allele frequencies of 0.025 and 0.975 for the wild type and mutant allele, respectively (linked marker). We found that carriers of the beneficial wild type allele were significantly taller at the shoulder than mutant allele homozygotes, with average heights of 31.3 cm and 26.4 cm, respectively. We further found that wild type carriers were born on average 4.7 years earlier than mutant allele homozygotes and that there has been a gradual decline in the population frequency of the wild type allele during the past two decades. CONCLUSIONS: Our results indicate that FGF4RG-associated chondrodystrophy may contribute to the shortened forelegs found in some Havanese and that both the wild type and mutant allele segregate in the breed. The population frequency of the wild type allele is low and appear to be decreasing. Efforts should be made to preserve the healthier wild type in the population, increase the prevalence of a more moderate phenotype and possibly reduce the risk of foreleg pathology.

Mapping of ferroelectric domain structure using angle-resolved piezoresponse force microscopy.

Angle-resolved piezoresponse force microscopy (AR-PFM) was used in conjunction with electron backscatter diffraction (EBSD) to study ferroelectric domain structure in polycrystalline near-morphotropic lead zirconate titanate (PZT). We introduce the details of AR-PFM including experimental method, the process to generate AR-PFM maps, and the interpretation of AR-PFM map, using domain patterns observed in bulk PZT. The spatial distortion caused by scanner creep and non-linearity in scanning probe microscopy was corrected through image registration, taking advantage of the features present in topography images. Domain structures were mapped using AR-PFM data, and the maps consistently show alternating piezoresponse axes in a lamellar pattern of non-180° domain structure. Comparison of AR-PFM and EBSD data showed a discrepancy between the direction of lateral surface displacement and the in-plane polarization direction. Additionally, using suitable domain patterns, AR-PFM enabled discrimination between the tetragonal and rhombohedral phases at the sub-grain scale.

Investigation of thermal and electrical stabilities of a GdBCO coil using grease as an insulation material for practical superconducting applications.

This paper presents the effects of thermal grease on the electrical and thermal characteristics of GdBCO pancake coils, observed through charge-discharge, sudden discharge, over-current, and thermal quench testing. In charge-discharge and sudden discharge tests, a coil using thermal grease as an insulation material demonstrated faster charging/discharging rates compared to a coil without turn-to-turn insulation. In the case of over-current tests, the coil using thermal grease exhibited the highest electrical stability. Furthermore, thermal quench testing showed the coil employing thermal grease to possess superior thermal characteristics, with rapid cooling and low temperature rise. Overall, the use of thermal grease as an insulation material may be a potential solution to the problems observed with the existing insulation materials, possessing fast charging/discharging rates with superior thermal and electrical stabilities.

Predictors for the progression of geographic atrophy in patients with age-related macular degeneration: fundus autofluorescence study with modified fundus camera.

PURPOSE: We examined the association between abnormal fundus autofluorescence (FAF) features on images obtained by a modified fundus camera (mFC) and geographic atrophy (GA) progression in patients with age-related macular degeneration (AMD). METHODS: Serial FAF images of 131 eyes from 131 patients with GA were included in the study. All FAF images were obtained with an mFC (excitation, ∼ 500-610 nm; emission, ∼ 675-715 nm). The GA area was quantified at baseline and 1 year later using a customized segmentation program. The yearly GA enlargement rate was then calculated. Abnormal FAF patterns in the junctional zone of GA were classified as None or Minimal change, Focal, Patchy, Banded, or Diffuse according to previously published classification based on confocal scanning laser ophthalmoscopy (cSLO). The relationship between GA enlargement and abnormal FAF was evaluated. RESULTS: The mean rate of GA enlargement was the fastest in eyes with Diffuse pattern (1.74 mm(2) per year), followed by eyes with the Banded pattern (1.69 mm(2) per year). Binary logistic regression analysis revealed that eyes with the Banded and Diffuse pattern had significantly higher risk for GA enlargement compared with eyes with the other patterns. CONCLUSIONS: FAF image obtained by mFC appears to be acceptable for evaluating GA in accordance with an established cSLO-based classification. Eyes with the Banded or the Diffuse patterns of abnormal FAF at baseline indicate a high risk for GA progression. Identifying patients at high risk for GA progression using an mFC is broadly available method that can provide additional information to help predict disease course.

The effect of ganglioside GQ1b on the NMDA receptor signaling pathway in H19-7 cells and rat hippocampus.

Gangliosides, sialic acid-containing glycosphingolipids, are related to various synaptic functions in the rat brain. Previously, we investigated the behavioral effects of the ganglioside GQ1b on learning and memory using the Y-maze and Morris water maze test. GQ1b-treated rats showed highly increased memory performance on the Y-maze and the Morris water maze test. In this study, we determined the role of GQ1b on the activation of the N-methyl-d-aspartate (NMDA) receptor signaling pathway in H19-7 rat hippocampal cells and the hippocampus of rats. After 12 h of treatment with GQ1b, the expression levels of NMDA receptor subunit 2A and 2B were increased in H19-7 cells and the hippocampus of rats. In addition, treatment of GQ1b increased the tyrosine phosphorylation of NR2B that may enhance NMDA receptor synaptic activation and enhancement of NMDA receptors. Also, following GQ1b treatment, the phosphorylation of extracellular signal-regulated kinases (ERK1/2) and protein kinase A, a cAMP activated protein kinase (PKA) increased in H19-7 cells and the hippocampus of rats. These increases resulted in an increase in the phosphorylation of cAMP response element binding protein (CREB). These results suggest that GQ1b might facilitate the activation of the NMDA receptor signaling pathway in the hippocampus of rats, an effect which is dependent on ERK1/2, PKA and CREB phosphorylation. Also, these data support our previous result that GQ1b improves the learning and memory of rats.

A novel chimeric promoter that is highly responsive to hypoxia and metals.

To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 x HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS-MRE-3 x HRE (E-M-H) gave a hypoxia induction ratio of 69. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1alpha, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E-M-H chimeric promoter. E-M-H was also induced by hypoxia mimetics such as Co2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E-M-H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E-M-H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E-M-H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.

Targeted destruction of the polymerized human serum albumin binding site within the preS2 region of the HBV surface antigen while retaining full immunogenicity for this epitope.

The 55-amino acid (a.a.) preS2 region of the hepatitis B virus (HBV) envelope protein is highly immunogenic, and antibodies against this epitope confer seroprotection against HBV infections. Accordingly, various experimental and clinical studies for developing and evaluating HBV vaccines that include this particular epitope have been reported. However, a pitfall in using preS2 epitopes as part of a vaccinating antigen is that polymerized human serum albumin (pHSA), which is a normal constituent of the human serum, binds to and makes complexes with this particular region. Consequently, it is most likely that the antigen epitope is masked by serum pHSA and subsequently not detected by the immune system. To overcome these limitations, a novel single a.a substitute of the preS2 region was designed that corresponds to a tyrosine to serine exchange at position 140 of preS2. Competitive enzyme-linked immunosorbent assay showed that this substitution completely abolishes pHSA-binding activities in the mutated preS2 peptide, and CD spectra analysis revealed that this property might have been induced by slight conformational changes in its secondary structure. Nevertheless, the original B-cell epitope was still preserved in the mutated preS2 as determined by experimental immunization in mice. In this regard, the preS2(120-145/Y140S) sequence may be an HBV vaccine where epitopes, with intrinsic properties have been deleted without affecting the immunogenicity of the epitope itself. It is expected that the inclusion of this point mutated preS2 epitope will improve the efficacy of conventional preS2-containing HBV vaccines.

HLA-DR expression in B-lymphocytes in vitro is not suppressed by the absence of exogenous antigens.

The proper loading of exogenous peptide antigens affects the transport and cell surface expression of MHC class II molecules. In the present study, the goal was to determine to what extent this step determines the cell surface expression level of MHC class II molecules, such as the HLA-DR. EBV-transformed B-cells, were cultured either in a serum- and protein-free medium, or in a medium that contained different concentrations of exogenous antigens. Using HLA-DR-specific antibodies, the induction of the MHC class II expression was observed in cells that were cultured under serum-and protein-free conditions, when compared to those cultured with exogenous protein antigens. This upregulation was completely suppressed to the normal level by the addition of a high concentration of hen egg lysozyme to the serum- and protein-free medium. This indicates that exogenous proteins regulate the HLA-DR expression. To further examine whether this modulation is controlled at the transcription level, the expression of the HLA-DR beta-chain mRNA was analyzed by reverse transcription-PCR and Northern blots. The same levels of HLA-DRB mRNA were detectable in both culture conditions, indicating that the present observation is dependent on some regulatory mechanisms at the post-transcriptional level. This might include a different pathway for trafficking of HLA-DR molecules to the cell surface, since peptide-binding assays revealed that a high proportion of cell surface HLA-DR molecules under the serum- and protein-free condition were transported to the cell surface without associated peptide antigens.

Determination of rat leptin activity in vitro using a novel luciferase reporter assay.

Leptin is small cytokine-like protein that is involved in appetite and body weight regulation. Due to increased interest in using leptin as an anti-obesity reagent, recombinant forms of leptin have been produced for several species, including humans, mice, rats, pigs, dogs, sheep etc. The biological activities of such recombinant proteins were determined using various in vitro or in vivo systems; however so far, no specific assay system for rat leptin is available. Since rats are representative animal models in obesity research, the establishment of a biological assay system for determining rat leptin activity has been eagerly awaited. This study describes the generation of such a system using chinese hamster ovary (CHO)-cells that were transfected with the long form of the rat leptin receptor isoform, OB-Rb, whereby a signal transduces and activators of transcription-sensitive luciferase reporter system is further employed to quantify the leptin-mediated signals. This system is the first rat-specific leptin bioassay system that has been reported. It is expected that this assay will be used to further quantify and determine leptin activity from various biological fluids and sources.

Internalization of tenecin 3 by a fungal cellular process is essential for its fungicidal effect on Candida albicans.

Tenecin 3 is a glycine-rich, antifungal protein of 78 residues isolated from the insect Tenebrio molitor larva. As an initial step towards understanding the antifungal mechanism of tenecin 3, we examined how this protein interacts with the pathogenic fungus Candida albicans to exert its antifungal action. Tenecin 3 did not induce the release of a fluorescent dye trapped in the artificial membrane vesicles and it did not perturb the membrane potential of C. albicans by the initial interaction. Fluorescence confocal microscopy and flow cytometric analysis revealed that tenecin 3 is rapidly internalized into the cytoplasmic space in energy-dependent and temperature-dependent manners. This internalization is also dependent on the ionic environment and cellular metabolic states. These results suggest that the internalization of tenecin 3 into the cytoplasm of C. albicans is mediated by a fungal cellular process. The internalized tenecin 3 is dispersed in the cytoplasm, and the loss of cell viability occurs after this internalization.

Recombinant expression of biologically active rat leptin in Escherichia coli.

Leptin is a 16-kDa nonglycosylated hormone that is produced in mature adipocytes and which acts primarily in the hypothalamus to reduce food intake and body weight. While the rat is a representative laboratory animal model in obesity research, so far recombinant rat leptin was not available. In the present study, rat leptin was recombinantly expressed in Escherichia coli and purified in a bioactive form to provide a further tool for the analysis of leptin functions in rats. Leptin cDNA was cloned by RT-PCR from total RNA of SD rat adipocytes, and overexpression was achieved by subcloning the leptin cDNA into the pET-29a vector, which enabled the recombinant expression of rat leptin as an S-peptide-tagged fusion protein. Since the fusion proteins were expressed in inclusion bodies, after purification of the insoluble fraction, leptin proteins were refolded by sequential dialysis into physiological buffers. The biological activity of this recombinant protein was confirmed in proliferation assays using leptin-sensitive rat insulinoma cells as well as a newly developed leptin-sensitive luciferase assay system. The specific binding of the S-tagged leptin to leptin-receptor-expressing cells was further shown by flow cytometry using fluorescence-conjugated S-proteins.

Protection against beta-amyloid peptide toxicity in vivo with long-term administration of ferulic acid.

1. beta-Amyloid peptide (A beta), a 39 -- 43 amino acid peptide, is believed to induce oxidative stress and inflammation in the brain, which are postulated to play important roles in the pathogenesis of Alzheimer's disease. Ferulic acid is an antioxidant and anti-inflammatory agent derived from plants; therefore, the potential protective activity of ferulic acid against A beta toxicity in vivo was examined. 2. Mice were allowed free access to drinking water (control) or water containing ferulic acid (0.006%). After 4 weeks, A beta 1-42 (410 pmol) was administered via intracerebroventricular injection. 3. Injection of control mice with A beta 1-42 impaired performance on the passive avoidance test (35% decrease in step-through latency), the Y-maze test (19% decrease in alternation behaviour), and the water maze test (32% decrease in percentage time in platform-quadrant). In contrast, mice treated with ferulic acid prior to A beta 1-42 administration were protected from these changes (9% decrease in step-through latency; no decrease in alternation behaviour; 14% decrease in percentage time in platform-quadrant). A beta 1-42 induced 31% decrease in acetylcholine level in the cortex, which was tended to be ameliorated by ferulic acid. 4. In addition, A beta 1-42 increased immunoreactivities of the astrocyte marker glial fibrillary acidic protein (GFAP) and interleukin-1 beta (IL-1 beta) in the hippocampus, effects also suppressed by pretreatment with ferulic acid. 5. Administration of ferulic acid per se unexpectedly induced a transient and slight increase in GFAP and IL-1 beta immunoreactivity in the hippocampus on day 14, which returned to basal levels on day 28. A slight (8%) decrease in alternation behaviour was observed on day 14. 6. These results demonstrate that long-term administration of ferulic acid induces resistance to A beta 1-42 toxicity in the brain, and suggest that ferulic acid may be a useful chemopreventive agent against Alzheimer's disease.

Detection of pET-vector encoded, recombinant S-tagged proteins using the monoclonal antibody ATOM-2.

The 15-meric S-tag is a truncated form of the S-peptide, which builds together with the 103 amino acid large S-protein the whole ribonuclease S-protein. Its small size and excessive solubility have made the S-tag an excellent fusion partner in the production of recombinant proteins, and a large variety of applications have been reported using the S-tag as a carrier. While S-tagged proteins were mostly detected and analyzed so far by use of their affinity to S-proteins, monoclonal antibodies (MAbs) for this tag have been not available. The generation of antibodies specific for S-tagged proteins is expected to broaden the range of applications of such S-tag fused recombinant proteins, and in this context, a novel MAb termed ATOM-2 was generated that specifically binds S-tagged proteins, which have been expressed using pET-vectors. Antigen specificity of ATOM-2 was confirmed in Western blot and enzyme-linked immunoadsorbent assay analysis, and using a series of amino acid deletion mutants, the binding epitope of ATOM-2 was precisely mapped. This showed that ATOM-2 recognizes the C-terminal part of the 15-meric S-tag in context with a few residues of vector encoded sequences. The core sequence for ATOM-2 binding epitope is "His-Met-Asp-Ser-Pro-Asp-Leu-Gly-Thr," which is present in all pET-expression vectors encoding S-tag fusion proteins. Because the ATOM-2 binding region does not overlap with the S-protein binding sequence, a convenient tool is provided for the simultaneous or alternative detection, purification, and analysis of recombinant S-tagged proteins to conventional S-proteins.

Translocation and accumulation of exogeneous hepatitis B virus preS surface proteins in the cell nucleus.

Recurrent reports about protease-sensitive sites in the junction of the preS and S region of the hepatitis B virus large surface protein have raised the question about a possible biological role of S protein-depleted, independent preS protein fragments in the virus life cycle. In the present study, this question was addressed by exogenous introduction of fluorescence-labeled recombinant preS proteins into permeabilized HepG2 cells. While maltose-binding proteins (MBP) were evenly distributed throughout the cytoplasm, MBP-preS fusion proteins selectively accumulated in the nucleus. Using truncated preS proteins, the effective domain for this nuclear accumulation was localized around the preS2 region. The mode of this action differs from conventional nuclear translocation mechanism in its energy- and mediator-independency and in that it is not saturated regardless of the increase of preS protein concentration. The biological meaning of this phenomenon has to be further studied. However, in regard to hepatitis B virus infection, this observation might provide a clue for unveiling the still poorly characterized events after initial internalization of the virus, which might make use of the nuclear translocation effect of the preS2 region to facilitate the infection.

Specific detection of cell surface-displayed human melanocortin 4 receptors with antibodies generated in mice.

The melanocortin-4 receptor (MC-4R) is a 7-transmembrane protein, which is involved in the central regulation of appetite and obesity. Despite the great interest in this protein, tools for detecting this molecule (as expressed on the cell surface in its native state) have been unavailable. Radioactive- or otherwise labeled ligands showed low receptor specificity to this particular melanocortin receptor isotype. Also, the antibodies were only available for epitopes that were displayed in the cytoplasm. To produce antibodies that enable the detection of this receptor (as expressed on the cell surface without disruption of the target cells), a candidate epitope was selected from the extracellular domains by a computer-aided analysis of the IC-4R secondary structure. This particular region was then recombinantly expressed in E. coli. Immunization of BALB/c mice with the recombinant proteins induced a specific immune reaction, which resulted in the production of MC-4R-specific antibodies. Enzyme-linked immunosorbent assays confirmed the specificity of these antibodies. To examine whether this tool also reacts with native cell surface-displayed MC-4R, HEK-293 cells were transfected with the human MC-4R cDNA. They were analyzed with these antibodies using Western blot and flow cytometry. Specificity and exclusion of cross-reactivity of these antibodies to other MC receptors were further confirmed by an immunofluorescence analysis of the HEK-293 cells that were transfected with other MC receptor isotypes. It is evident that with the availability of this tool, studies on the cell- and tissue-specificity, as well as the regulation mechanism of the MC-4 receptor, will be largely facilitated.

Determination of the protective effects of neutralizing anti-hepatitis B virus (HBV) immunoglobulins by epitope mapping with recombinant HBV surface-antigen proteins.

Anti-hepatitis B virus (HBV) surface-antigen immunoglobulins prepared from human sera are clinical reagents which have been approved for prophylactic treatment in HBV-exposed persons. The passive immunoprophylaxis with immunoglobulins is meant to cross-link viral particles, which are then further cleared by the host's own immune system. While antibodies specific for both anti-S- and anti-preS proteins have been proved to serve as effective anti-viral agents, so far the fine antigen specificity of clinical immunoglobulin preparations has not been determined. Using recombinant proteins covering the hepatitis B surface antigen, in the present study, the specificity of a commercially available immunoglobulin preparation was determined and immunodominant epitopes were mapped. Here, it is shown that the major reactivity of anti-HBV immunoglobulins is directed against the S-protein, and that no reactivity to the preS2 but a weak binding activity to the preS1 region was detectable. The antigen reactivity within the preS1 region was biased to the C-terminal region, which indicates the presence of a putative B-cell epitope. The evaluation of the antigen specificity and determination of novel protective epitopes will provide valuable information for the further development and improvement of prophylactic HBV immunoglobulins.