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.

A covalent complex between horse heart cytochrome c and yeast cytochrome c peroxidase: kinetic properties.

The kinetic properties of a 1:1 covalent complex between horse-heart cytochrome c and yeast cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) have been investigated by transient-state and steady-state kinetic techniques. Evidence for heterogeneity in the complex is presented. About 50% of the complex reacts with hydrogen peroxide with a rate 20-40% faster than that of native enzyme; 20% of the complex exists in a conformation which does not react with hydrogen peroxide but converts to the reactive form at a rate of 20 +/- 5 s-1; 30% of the complex does not react with hydrogen peroxide to form the oxidized enzyme intermediate, cytochrome c peroxidase Compound I. Intramolecular electron transfer between covalently bound ferrocytochrome c and an oxidized site in cytochrome c peroxidase Compound I is too fast to measure, but a lower limit of 600 s-1 can be estimated at 5 degrees C in a 10 mM potassium phosphate buffer at pH 7.5. Free ferrocytochrome c reduces cytochrome c peroxidase Compound I covalently bound to ferricytochrome c at a rate 10(-4) to 10(-5)-times slower than for free Compound I. The transient-state ferrocytochrome c reduction rates of Compound I covalently linked to ferricytochrome c are about 70-times too slow to account for the steady-state catalytic properties of the 1:1 covalent complex. This indicates that hydrogen peroxide can interact with the 1:1 complex at sites other than the heme of cytochrome c peroxidase, generating additional species capable of oxidizing free ferrocytochrome c.

Effects of the hinge region of cecropin A(1-8)-magainin 2(1-12), a synthetic antimicrobial peptide, on liposomes, bacterial and tumor cells.

A 20-residue hybrid peptide (CA(1-8)-MA(1-12): KWKLFKKIGIGKFLHSAKKF-NH(2)) incorporating 1-8 residues of cecropin A (CA) and 1-12 residues of magainin 2 (MA) has potent antibiotic activity without hemolytic activity. In order to investigate the effects of the flexible hinge sequence, Gly-Ile-Gly of CA(1-8)-MA(1-12) (CA-MA) on antibiotic activity, CA-MA and its three analogues, CA-MA1, CA-MA2 and CA-MA3 were synthesized. The Gly-Ile-Gly sequence of CA-MA was deleted in CA-MA1 and replaced with Pro and Gly-Pro-Gly in CA-MA2 and CA-MA3, respectively. CA-MA1 and CA-MA3 caused a significant decrease in the bactericidal rate against Escherichia coli and Bacillus subtilis and the tumoricidal activity against four different tumor cells, and the PC/PS (4:1, w/w) vesicle-aggregating and disrupting activities. However, CA-MA2 showed a similar bactericidal rate and antitumor, vesicle-aggregating and disrupting activities, as compared with CA-MA. These results suggested that the flexibility or beta-turn induced by Gly-Ile-Gly or Pro in the central part of CA-MA may be important in the electrostatic interaction of the cationic short alpha-helical region in the N-terminus with the cell membrane surface and the hydrophobic interaction of amphipathic alpha-helical region in the C-terminus with the hydrophobic acyl chains in the cell membrane. CA-MA3 exhibited lower activity in antibacterial, antitumor, and vesicle-aggregating and disrupting activities than CA-MA and CA-MA2. This result suggested that the excessive beta-turn structure by Gly-Pro-Gly in CA-MA3 seems to interrupt the ion channel/pore formation on the lipid bilayer. It was concluded that the appropriate flexibility or beta-turn structure provided by the central hinge is responsible for the effective antibiotic activity of the antimicrobial peptides with the helix-hinge-helix structure.

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.

5 alpha-reductase, aromatase, and androgen receptor levels in the monkey brain during fetal development.

To elucidate the metabolic fate and possible role of androgens and their derivatives during primate fetal development, aromatase (AROM), 5 alpha-reductase (5 alpha R), and androgen receptor (AR; cytosolic) levels were assessed in the brain, heart (HRT), lung (LNG), and skeletal muscle (MUS) of fetal rhesus monkeys. Analyses were performed on tissues taken on days 100 and 160 postconception. Five male and four or five female fetuses were examined at each stage. Brain tissues analyzed included medial basal hypothalamus (MBH), amygdala (AMG), cerebellum (CB), corpus callosum (CAL; splenial region), cerebral cortex (CTX), and cingulate cortex (CNG). In the following, enzyme activities are reported as picomoles per mg protein/h, while receptor levels are femtomoles per mg protein. 5 alpha R activity was measurable in all tissues. Analysis of variance revealed significant tissue differences [P less than 0.001, combined stages and sexes; CAL (2.05) greater than MBH (1.08) greater than AMG (0.63) greater than CB (0.4)-CNG-CTX-LNG-HRT-MUS (0.02); -indicates not significantly different]. A significant age x tissue interaction (P less than 0.001) was noted which could be explained by higher MBH and CAL levels in older vs. younger fetuses and higher AMG levels in younger vs. older fetuses. There was also a significant sex x tissue interaction which was attributed to higher female values in the MBH and CAL. AROM activity was detected in all tissues. Levels varied significantly among tissues [P less than 0.001, combined stages and sexes; MBH (0.80)-AMG (0.76) greater than CAL (0.4)-CNG-CB-CTX-LNG-HRT-MUS (0.07)]. Significant age (P less than 0.001) and age x tissue (P less than 0.001) effects were noted, which were due to higher MBH and AMG levels in younger vs. older fetuses. No sex difference in AROM levels was evident in any tissue. AR was measurable in all cases. Although stage and sex differences were not significant, tissue levels varied significantly [P less than 0.001; LNG (2.8)-MUS (2.6)-MBH (2.2) greater than HRT-AMG-CB-CTX-CAL-CNG (0.9)]. These findings indicate that neural and nonneural fetal primate tissues have the potential for transforming androgens to products that could have greater or lesser biological activity. AR were also noted through which dihydrotestosterone or testosterone could effect a genomic response. Since stage, tissue, and sex differences were evident in neural tissues, metabolic and receptor activities may be important for the normal differentiation of sexually dimorphic behavioral systems in monkeys as well as for potential teratogenic changes under abnormal metabolic or physiological conditions.

Cloning and characterization of cDNAs coding for heavy and light chains of a monoclonal antibody specific for pre-S2 antigen of hepatitis B virus.

Binding specificity of a monoclonal antibody (mAb) (kappa, gamma 2b) H8 which can react with the pre-S2 peptide of hepatitis B virus (HBV) was determined by Western blot analyses. From the hybridoma cell line secreting mAb H8, poly(A)+ RNA was prepared and used as a template for cDNA synthesis and cloning. Full-length cDNAs coding for the heavy and kappa light chains of the mAb were cloned from the cDNA library and characterized by nucleotide (nt) sequence analyses and N-terminal amino acid sequencing. The sequence analyses revealed that both heavy and light chain-specific cDNAs are functional, and the variable regions of the heavy and light chains are members of mouse heavy chain subgroup III(c) and light chain group I, respectively. Comparison of the nt sequences with mouse immunoglobulin genes listed in the GenBank data base show that the cDNAs have not been previously reported. The cDNAs will be used for the construction of a therapeutic antibody for HBV infection.

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.

Evaluation of the N-terminal role in peptide antigenicity of the preS2 region of the hepatitis B virus surface antigen.

Previously we reported that the N-terminal sequence 120-129 of the preS2 region of the hepatitis B virus (HBV) surface antigen (HBsAg) plays an important role in peptide antigenicity against an preS2 specific anti-HBsAg monoclonal antibody (H8 mAb) by affecting the B cell epitope conformation of a peptide existing within the sequence 130-145. In this study, we tried to confirm the previous results using a series of N-terminal sequentially deleted peptides and peptides substituted at position 127. This position was previously found to be important for H8 mAb binding. Peptide antigenicity of sequentially deleted peptides was gradually decreased when the residues from 125 to 129 were deleted, indicating that the residues cumulatively affect antigenicity. Peptide antigenicity relative to the substituted residues at position 127 of p123 showed an order of F > I > E > or = T > A > or = P > K and was correlated with their CD spectra. Structure-dependent peptide antigenicity was also found in many polyclonal antisera produced by immunization with peptide-protein conjugates and HBsAg. Twenty-nine out of 34 polyclonal antisera had reactivities increased 2-fold or more with p123 relative to those with p130. Among them, 10 antisera showed increased reactivities of 7 fold or more. These results confirm our previous results, which suggested the existence of the conformational B cell epitope in the preS2 region maintained by the N-terminal sequence. This experimental also suggests that N-terminal sequence 125-129 is important in maintaining a higher antigenic structure.

Downregulation of MHC class II expression by oxidant-induced apoptosis in EBV-transformed B-cells.

The expression of MHC class II molecules is actively regulated upon various cellular stimuli. Since apoptosis is an inducible cellular process, it was asked whether cells undergoing apoptosis would also modulate their expression of class II molecules. Using an EBV-transformed B-cell line, the cell surface expression of HLA-DR molecules was analyzed by fluorescence-activated flow cytometry on normal and oxidant-treated apoptotic cells. A rapid and continuous decrease in HLA-DR expression was observed in apoptotic cells. RNA analysis and semiquantitative RT-PCR of cytoplasmic beta-actin mRNA showed that apoptotic cells contain partially degraded RNA and much lower amounts of beta-actin mRNA. Nevertheless, when compared after normalization of intact mRNA amounts, the HLA-DRB mRNA signals were of similar strength in normal and apoptotic cells as determined by semiquantitative RT-PCR. Thus, the decrease in the number of class II molecules during apoptosis underlies no specific program for downregulation of HLA-DRB mRNA transcription but is due to a nonspecific degradation of RNA molecules accompanied by cell death.

Maltose binding protein (MBP) fusion proteins with low or no affinity to amylose resins can be single-step purified using a novel anti-MBP monoclonal antibody.

The maltose binding protein (MBP) fusion protein expression system is a powerful tool to produce and isolate recombinant proteins in E. coli. Whereas the conventional isolation technique for MBP-fusion proteins takes advantage of the binding affinity of MBP to maltose, this method is limited insofar as the biological activity of MBP has to be fully conserved for a successful purification. In this study, a novel monoclonal antibody (mAb) specific for MBP, termed HAM-19, was generated and its application in the purification and detection of MBP-fusion proteins determined. Using anti-MBP immunoaffinity columns, even recombinant MBP fusion products with lowered or impaired binding affinity to maltose were purified in a single step procedure. In comparison to amylose resins, HAM-19 immunoaffinity columns showed a higher binding capacity and affinity to MBP-fusion proteins. Furthermore, the mAb HAM-19 also provides a technical improvement over polyclonal antisera for the detection and analysis of MBP-fusion proteins which are under use in various forms in the fields of molecular and cellular biology.

Generation of self-antigen reactive, anti-urocortin specific antibodies by immunization of recombinantly expressed urocortin fusion proteins.

Urocortin is a recently described 40-meric neuropeptide, which was originally detected in the rat mid-brain and is believed to play a key role in response to stress situations. While its function in the central nervous system is rather well established, the biological role in the periphery is still to be determined. To investigate its distribution and effect on peripheral cells and tissues, in the present study, urocortin was recombinantly expressed and specific antibodies were generated. So far, the immunological detection of urocortin in the rat was largely dependent on antisera generated in rabbits. However, the polyclonal nature of the serum and the remote species origin tend to show cross-reactivities and higher backgrounds. On the other hand, generation of mouse antibodies to rat urocortin was hampered since mouse and rat urocortin sequences are identical, and such antibodies would represent auto-reactive antibodies. Despite such restrictions, the immunization with a combination of various recombinantly expressed urocortin fusion proteins resulted in the successful generation of mouse antiurocortin antisera, whose specificities were confirmed by ELISA and Western blot analysis. To produce the recombinant proteins for immunization, a cDNA encoding the mature urocortin sequence was cloned and expressed in fusion either with the glutathione-S-transferase, the maltose-binding protein, thioredoxin, or a 6X His tag. Depending on the expression system, the solubility and yield of the recombinant proteins greatly varied. Together with the newly generated antibodies, these recombinantly expressed urocortin proteins will serve as valuable tools in further investigations of the biological function of urocortin.

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.

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.

Reassembly and reconstitution of separate alpha and beta chains of human leukocyte antigen DR4 molecule isolated from Escherichia coli.

The class II major histocompatibility complex molecules play a major role in presentation of exogenous antigenic peptides to the CD4 positive helper T cell. These are heterodimeric cell surface glycoproteins consisting of alpha- and beta-chains. In the present study, we cloned and expressed the alpha- and beta-chain of HLA-DR4 lacking the transmembrane and cytoplasmic domain separately in Escherichia coli using the pET-5a expression vector system. The expressed alpha- and beta-chains were purified in a denaturing condition by an ion exchange chromatography on Q-Sepharose and a gel filtration chromatography on Sephacryl S-200, respectively. The recombinant proteins were refolded and reassembled by removing the denaturing agent and concomitant reoxidation of the disulfide bond. The refolded heterodimeric rDR4 molecule was resolved by 12.5% SDS-PAGE in a nonreducing condition and confirmed by Western blot using polyclonal antibody against DR-alpha and the monoclonal antibody (L243) for the conformationally correct DR molecule. The rDR4 molecules were reconstituted with a 50-fold molar excess biot-HA (307-319), and the bound peptides to the heterodimer complex were determined by a microplate assay coated with L243 antibody using Extravidin-HRP conjugate.

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.

Androgen receptors are differentially distributed between right and left cerebral hemispheres of the fetal male rhesus monkey.

In humans there are apparent sex differences in verbal and spatial abilities as well as several cortical pathologies. These differences may arise as the result of prenatal androgen exposure and its effect on the development of the cerebral cortex. With this in mind, we have examined androgen receptor (AR), aromatase (AROM) and 5 alpha-reductase (5 alpha R) levels in the cerebral cortex of Day 70 male and female fetal rhesus monkeys (Macaca mulatta). Receptor and enzyme levels were evaluated in both right (Rt) and left (Lft) temporal (TMP) and frontal (FR) lobes of the cerebral cortex. AR levels in FR-Rt of male subjects were higher than levels in FR-Lft (for each and every subject, P less than 0.05), while in females, there was no consistent pattern in the distribution of the receptor between the two sides of FR. In contrast, AR values in TMP-Lft of male subjects were consistently higher than in TMP-Rt (P less than 0.05). As with the FR, females exhibited no consistent pattern in the distribution of AR between the two TMP sides. AROM and 5 alpha R levels were similar, regardless of sex, between both sides of the two cortical lobes indicating that the AR distribution pattern is not a general biochemical phenomenon. The differential cortical distribution of AR in fetal males versus females lends support to the hypothesis that prenatal androgens from the fetal testes may effect the differentiation of sexually dimorphic, side-specific cortical activity.