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The interaction of CD95 with its ligand CD95L is important for negative selection of B cells during the germinal center (GC) reaction. Recently, mutations conferring resistance to CD95-induced apoptosis have been described for human GC B cells. Hence, as has been demonstrated for CD95-deficient mice, also GC-derived autoreactive B cells carrying somatic CD95 gene mutations may potentially service negative selection and participate in the development of autoimmune diseases. Here, single plasmablasts (PB) which are implicated in the production of autoantibodies in systemic lupus erythematosus (SLE) patients as well as ten human B cell lines producing autoantibodies were analyzed for destructive somatic CD95 gene mutations. However, inactivating CD95 gene mutations were very rare in PB and not detected in the cell lines. Sequence analysis of V gene rearrangements amplified from single PB confirmed that the cells are (post) GC B cells and additionally demonstrated massive clonal expansion of these cells in two of four SLE patients. We conclude that CD95 gene mutations play little if any role in the generation of the pool of PB in SLE patients and that mutations in the CD95 gene are rare among autoantibody-producing B cells in SLE and rheumatoid arthritis.

Quantification of the expression levels of nuclear genes encoding plastid proteins under different genetic or environmental conditions can contribute to the genetic dissection of plastid functions. To facilitate such measurements, a set of 1,827 Arabidopsis thaliana genes coding for plastid proteins was PCR-amplified from genomic DNA and spotted on nylon membranes to generate an array of chloroplast-specific gene-sequence-tags (GSTs). The sensitivity and reliability of the experimental system was evaluated and a procedure was developed for detecting differential gene expression. The GST array was found to serve as a reliable monitor of changes in gene expression induced by environmental and genetic alteration of chloroplast functions. Based on comparisons of dark- versus light-grown seedlings, and wild-type versus prpl11-1 plants, lists of differentially expressed genes are provided which include 193/7 and 25/42 up/down-regulated genes, respectively. The cut-off values for differential expression were 2.5-times (up) and 0.40 (down). Additional up-regulated genes with relatively low expression ratios (from 1.5- to 2.5-times) or down-regulated with relatively high ratios (0.4-0.67) can be accessed at the website: http://www.mpiz-koeln.mpg.de/~richly/GST-array.html. A sample of genes analysed by quantitative reverse transcription PCR confirmed the expression profiles monitored by the GST array. Differential hybridisation experiments with the prpl11-1 mutant revealed the existence of regulatory networks sensing the protein state of the chloroplast and transmitting the signal to the nucleus.

The NBS-LRR (nucleotide-binding site plus leucine-rich repeat) genes represent the major class of disease resistance genes in flowering plants and comprise 166 genes in the ecotype Col-0 of Arabidopsis thaliana. NBS-LRR genes are organized in single-gene loci, clusters, and superclusters. Phylogenetic analysis reveals nine monophyletic clades and a few phylogenetic orphans. Most clusters contain only genes from the same phylogenetic lineage, reflecting their origin from the exchange of sequence blocks as a result of intralocus recombination. Multiple duplications increased the number of NBS-LRR genes in the progenitors of Arabidopsis, suggesting that the present complexity in Col-0 may derive from as few as 17 progenitors. The combination of physical and phylogenetic analyses of the NBS-LRR genes makes it possible to detect relatively recent gene rearrangements, which increased the number of NBS-LRR genes by about 50, but which are almost never associated with large segmental duplications. The identification of 10 heterogeneous clusters containing members from different clades demonstrates that sequence sampling between different resistance gene loci and clades has occurred. Such events may have taken place early during flowering plant evolution, but they generated modules that have been duplicated and remobilized also more recently.

The purposes of this study were to determine the prevalence of posttreatment open gingival embrasures in adult orthodontic patients and to examine the association of pretreatment maxillary incisor malalignment, posttreatment alveolar bone height, interproximal contact position, root angulation, crown shape, and embrasure area with open gingival embrasures. Posttreatment intraoral photographs of 337 adult orthodontic patients were evaluated to determine the prevalence of open gingival embrasures. A subsample of 119 patients was identified for measurement and divided into 2 groups: normal gingival embrasures and open gingival embrasures. Digital images of the pretreatment maxillary models and posttreatment maxillary central incisor periapical radiographs were made to measure the pretreatment and posttreatment variables. The prevalence of posttreatment open gingival embrasures in adult orthodontic patients was 38%. Pretreatment maxillary central incisor rotation and overlap were not statistically associated with posttreatment open gingival embrasures. A posttreatment alveolar bone-interproximal contact distance greater than 5.5 mm was associated with open gingival embrasures. Short and more incisally positioned posttreatment interproximal contacts were associated with open gingival embrasures. Open gingival embrasures were found to have more divergent root angulations and more divergent or triangular-shaped crown forms than normal gingival embrasures. Embrasure areas larger than 5.09 mm(2) were also correlated with open gingival embrasures. Increased alveolar bone-interproximal contact distance and increased root angulation demonstrated the greatest increase in the odds of an association with an open gingival embrasure. This investigation indicates that open gingival embrasures are common in adults who have undergone orthodontic treatment and that posttreatment variables are significant factors in open gingival embrasures.

The barley Mla locus encodes 28 characterized resistance specificities to the biotrophic fungal pathogen barley powdery mildew. We describe a single-cell transient expression assay using entire cosmid DNAs to pinpoint Mla1 within the complex 240-kb Mla locus. The MLA1 cDNA encodes a 108-kD protein containing an N-terminal coiled-coil structure, a central nucleotide binding domain, and a C-terminal leucine-rich repeat region; it also contains a second short open reading frame at the 5' end that has a possible regulatory function. Although most Mla-encoded resistance specificities require Rar1 for their function, we used the single-cell expression system to demonstrate that Mla1 triggers full resistance in the presence of the severely defective rar1-2 mutant allele. Wheat contains an ortholog of barley Mla, designated TaMla, that is tightly linked to (0.7 centimorgan) but distinct from a tested resistance specificity at the complex Pm3 locus to wheat powdery mildew. Thus, the most polymorphic powdery mildew resistance loci in barley and wheat may have evolved in parallel at two closely linked homeoloci. Barley Mla1 expressed in wheat using the single-cell transformation system failed to trigger a response to any of the wheat powdery mildew Avr genes tested, indicating that AvrMla1 is not genetically fixed in wheat mildew strains.

Epstein-Barr virus (EBV) can be detected in the tumor cells of approximately 40% of cases of classical Hodgkin disease (cHD). Clonality studies suggest that infection of the neoplastic Hodgkin and Reed/Sternberg (HRS) cells occurs before tumor clone expansion. In EBV-positive cases, variable numbers of EBER-positive small B cells are sometimes also observed that immunohistologically differ from the neoplastic cells by lack of CD30 and latent membrane protein 1 expression. To analyze the clonal relationship between these EBV(+) cells and the HRS cells, single EBV-infected CD30(-) B cells, as well as HRS cells from 3 cases of EBV-positive cHD were micromanipulated, their immunoglobulin gene rearrangements amplified and then compared with each other. In 2 cases, all small EBV-infected cells were clonally unrelated to the HRS cells. In a third case, 2 of 29 small CD30(-) cells were found to carry HRS cell-specific rearrangements. Thus, small CD30(-) EBV-infected B cells in cHD belong to the HRS tumor clone rarely, if at all. In all cases, small clones unrelated to the HRS cell clones were identified among the small EBV(+) CD30(-) cells. The vast majority of small EBV(+) CD30(-) B cells was found to carry somatically mutated V region genes, indicating that in lymph nodes of patients with HD, like in the peripheral blood of healthy individuals, EBV persists in memory B cells.

Infection of humans with Epstein-Barr virus (EBV) may cause infectious mononucleosis (IM). Analysis of single EBV-infected cells from tonsils of IM patients for rearranged immunoglobulin genes revealed two strategies of EBV for rapid and massive spread in the B cell compartment: the direct infection of many naive as well as memory and/or germinal center B cells and the expansion of the latter cells to large clones. In IM, the generation of virus-harboring memory B cells from naive B cells passing through a germinal center reaction likely plays no role. Members of clones can show distinct morphologies and likely also EBV gene expression patterns, and this ability implies a mechanism by which EBV-harboring cells can evade immune surveillance and establish a pool of persisting EBV-infected B cells.

Cumulus-oocyte complexes (COCs) recovered from ovaries of mares killed at abattoirs or after in vivo collection have heterogeneous morphologies and meiotic competence as follicles of variable quality are used. It is thought that it should be possible to recover more uniform COCs, with respect to morphology and nuclear maturation, by repeated follicle aspiration. Therefore, the influence of repeated follicle aspiration on the number and diameter of follicles > or =5 mm in diameter, the morphology and recovery rate of COCs, and the chromatin configuration in oocytes was investigated. Repeated ultrasound-guided aspirations were performed on Warmblood mares (n=6) after either a normal cycle ('cyclic' sessions) or at 4-12 day intervals ('consecutive' sessions). In 88 follicle aspiration sessions, 1268 follicles were aspirated and 280 COCs were recovered: the mean number of follicles aspirated and the number of COCs obtained per session per mare were 14.4 and 3.2, respectively. The mean recovery rate was 22.1%; there was no significant difference in the recovery rate between cyclic and consecutive aspirations. However, the mean number of follicles aspirated was significantly different between cyclic and consecutive aspirations (15.3 versus 10.8, respectively) and, hence, fewer COCs were obtained in consecutive aspirations compared with cyclic aspirations (2.2 versus 3.5, respectively). The proportion of compact COCs was higher for consecutive than for cyclic aspirations (51.9 versus 28.8%, respectively; P < or = 0.02). Within consecutive sessions, the proportion of compact COCs decreased with increasing interval between aspirations. Moreover, the proportion of oocytes with a diffuse germinal vesicle chromatin configuration was higher in COCs collected in consecutive aspirations than in COCs collected in cyclic aspirations. Repeated follicle aspiration can be used to induce a more uniform follicular population and to provide more uniform COCs. The optimum interval between aspirations to provide the greatest number of meiotically competent oocytes must be determined.

Both genetic and environmental factors are involved in the etiology of Parkinson's disease (PD). The recent identification of specific autosomal genes that lead to variants of PD confirms that genetic factors are important. Identifying and confirming other genetic factors responsible for PD is a difficult task because PD is a complex disease, the results of multiple genetic and environmental factors leading to a final common pathology. This review will discuss how advances in human genetics will allow future unraveling of the complex interactions between genetics and environment in the etiology of PD.

Powdery mildew of barley, caused by Erysiphe graminis f. sp. hordei, is a model system for investigating the mechanism of gene-for-gene interaction between large-genome cereals and obligate-fungal pathogens. A large number of loci that confer resistance to this disease are located on the short arm of chromosome 5(1H). The Mla resistance-gene cluster is positioned near the telomeric end of this chromosome arm. AFLP-, RAPD-, and RFLP-derived markers were used to saturate the Mla region in a high-resolution recombinant population segregating for the (Mla6 + Mla14) and (Mla13 + Ml-Ru3) resistance specificities. These tightly linked genetic markers were used to identify and develop a physical contig of YAC and BAC clones spanning the Mla cluster. Three distinct NBS-LRR resistance-gene homologue (RGH) families were revealed via computational analysis of low-pass and BAC-end sequence data derived from Mla-spanning clones. Genetic and physical mapping delimited the Mla-associated, NBS-LRR gene families to a 240-kb interval. Recombination within the RGH families was at least 10-fold less frequent than between markers directly adjacent to the Mla cluster.

We have characterized amyloid beta peptide (Abeta) concentration, Abeta deposition, paired helical filament formation, cerebrovascular amyloid angiopathy, apolipoprotein E (ApoE) allotype, and synaptophysin concentration in entorhinal cortex and superior frontal gyrus of normal elderly control (ND) patients, Alzheimer's disease (AD) patients, and high pathology control (HPC) patients who meet pathological criteria for AD but show no synapse loss or overt antemortem symptoms of dementia. The measures of Abeta deposition, Abeta-immunoreactive plaques with and without cores, thioflavin histofluorescent plaques, and concentrations of insoluble Abeta, failed to distinguish HPC from AD patients and were poor correlates of synaptic change. By contrast, concentrations of soluble Abeta clearly distinguished HPC from AD patients and were a strong inverse correlate of synapse loss. Further investigation revealed that Abeta40, whether in soluble or insoluble form, was a particularly useful measure for classifying ND, HPC, and AD patients compared with Abeta42. Abeta40 is known to be elevated in cerebrovascular amyloid deposits, and Abeta40 (but not Abeta42) levels, cerebrovascular amyloid angiopathy, and ApoE4 allele frequency were all highly correlated with each other. Although paired helical filaments in the form of neurofibrillary tangles or a penumbra of neurites surrounding amyloid cores also distinguished HPC from AD patients, they were less robust predictors of synapse change compared with soluble Abeta, particularly soluble Abeta40. Previous experiments attempting to relate Abeta deposition to the neurodegeneration that underlies AD dementia may have failed because they assayed the classical, visible forms of the molecule, insoluble neuropil plaques, rather than the soluble, unseen forms of the molecule.

Transmission of viruses by animal sera represents a considerable risk for humans and animals particularly when the serum is used for the production of pharmaceutical products such as vaccines. Procedures applicable for inactivating large numbers of different viruses, both enveloped and non-enveloped, are therefore mandatory. For this purpose we have developed and validated UVC irradiation as the virus-inactivation procedure of choice for serum to be used in an industrial setting. Spiking experiments in foetal calf serum (FCS) were performed by independent contract laboratories and revealed constantly high clearance rates for various viruses such as bovine parvovirus, parainfluenza type III virus, bovine diarrhoea virus, foot-and-mouth disease virus and different forms of mycoplasmas. UVC-treated sera maintained their growth-promoting activities for various cell types (MRC-5, Vero, CHO). Conventional growth curves generated in the presence of 10% and 1% UVC-treated FCS differed only slightly from controls, indicating the lack of significant damage during UVC exposure. Experiments using a sensitive photometric-based acid phosphatase assay (APA), which correlates well with the more tedious cell counting procedure, confirmed these findings even in the presence of minimal serum requirements. UVC treatment of animal sera appears advantageous compared to currently recommended inactivation procedures, such as Gamma irradiation, for at least three reasons: (i) it possesses a high inactivation capacity for parvoviruses, a pathogen that cannot be destroyed easily by conventional methods; (ii) it causes no noticeable impairment in cell growth and (iii) it can be performed in a controlled manner at the production site.

We used conserved domains in the major class (nucleotide binding site plus leucine-rich repeat) of dicot resistance (R) genes to isolate related gene fragments via PCR from the monocot species rice and barley. Peptide sequence comparison of dicot R genes and monocot R-like genes revealed shared motifs but provided no evidence for a monocot-specific signature. Mapping of these genes in rice and barley showed linkage to genetically characterized R genes and revealed the existence of mixed clusters, each harboring at least two highly dissimilar R-like genes. Diversity was detected intraspecifically with wide variation in copy number between varieties of a particular species. Interspecific analyses of R-like genes frequently revealed nonsyntenic map locations between the cereal species rice, barley, and foxtail millet although tight collinear gene order is a hallmark of monocot genomes. Our data suggest a dramatic rearrangement of R gene loci between related species and implies a different mechanism for nucleotide binding site plus leucine-rich repeat gene evolution compared with the rest of the monocot genome.

We had previously examined environmental, sociodemographic and clinical variables as predictors for Parkinson's disease with dementia (PD + D) and found that lower educational attainment, greater motor impairment and advanced age at disease onset were more common in PD + D than in subjects with Parkinson's disease without dementia (PD-D). We now explore the hypothesis that genetic traits coupled with nongenetic factors may raise the risk of development of PD + D. The study cohort of 43 PD + D and 51 PD-D subjects was analyzed examining environmental, sociodemographic and clinical variables along with 3 candidate gene markers: poor debrisoquine metabolizer allele (CYP 2D6 29B+), monoamine oxidase B allele 1, and apolipoprotein E epsilon 4 allele. Variables were initially entered into a multivariate model singly. Again lower education, age at onset and motor impairment appeared as predictors of PD + D while other variables (including allele status) failed to emerge as significant individual risk factors for dementia. We then examined environmental and genetic variables analyzed in tandem to look for potential variable interactions. Subjects who had pesticide exposure and at least 1 copy of the CYP 2D6 29B+ allele had 83% predicted probability of PD + D (stepwise logistic regression model: p = 0.0491). This case-control study provides preliminary evidence that a gene-toxin interaction may play an etiological role in PD + D. Further assessment of the role of these putative risk factors in incident dementia in PD is indicated.

Pharmacogenetics has been promoted as potentially providing benefits to patients, managed care organizations and pharmaceutical companies. This has not translated into products that benefit healthcare developers, providers or consumers. The reasons for this are many, but this will change as the financial incentives become clear for the pharmaceutical industry to develop products that use genetic susceptibility as part of the rationale for products, healthcare providers have increasing incentive to reduce costs, and patients demand up-to-date technologies to optimize healthcare. Recent studies have established genetic contributions that alter the response to therapy for some disease entities, and more will follow as pharmacogenetics becomes increasingly accepted as an important consideration in the therapeutic decision-making process.

The N-methyl-D-aspartate (NMDA) receptor has been reported to be important in synaptic plasticity, neuronal development, normal brain function and neurologic disease. We have recently shown that PC12W cells, a subclone of rat pheochromocytoma PC12 cell line, release nitric oxide (NO), as measured by in vitro spin-trapping combined with electron paramagnetic resonance (EPR) spectroscopy, when challenged with NMDA [Norby, S.W., Weyhenmeyer, J.A. and Clarkson, R.B., Stimulation and inhibition of NO production in macrophages and neuronal cells as observed by spin trapping, Free Rad. Biol. Med., 22 (1997) 1-9]. In the present study, we provide immunochemical evidence for the expression of both the NMDAR1 and NMDAR2A/B receptor subunits in PC12W cells, that express only the angiotensin type-2 (AT2) receptor subtype, and in NG108-15 (NG108) cells, a murine neuroblastoma x glioma hybrid that expresses both the angiotensin type-1 (AT1) and AT2 receptor subtypes. We also show that treatment of PC12W cells with angiotensin (Ang II) decreases NMDA-induced NO release by 28.0 +/- 4.2%, and that this response can be attenuated by pre-treating the cells with the isoform-specific AT2 antagonist, PD 123319. Interestingly, there was no effect on cGMP accumulation in PC12W cells treated with NMDA. Similar experiments were carried out using NG108 cells since the binding properties and functional characteristics of their NMDA receptors have been previously described [Ohkuma, S., Katsura, M., Chen, D., Chen, S. and Kuriyama, K., Presence of N-methyl-D-aspartate (NMDA) receptors in neuroblastoma x glioma hybrid NG 108-15 cells-analysis using 45Ca2+ influx and [3H]MK-801 binding as functional measures, Mol. Brain Res. 22 (1994) 166-172]. Our results show that NG108 cells significantly increase cGMP levels when challenged with NMDA (21.2 +/- 5.0% over control levels), and that this response can be attenuated by the addition of angiotensin (57.1 +/- 6.2% of stimulated levels). The effect of angiotensin on NMDA-mediated changes in cGMP levels was blocked by the AT2 antagonist, PD 123319, but was not significantly changed by the addition of the AT1 antagonist, losartan. Further, Ang II action on NMDA signalling in NG108 cells was completely inhibited by the addition of both the AT1 and AT2 antagonists. Taken together, these results suggest that AngII inhibits NMDA-mediated NO and cGMP production through a mechanism involving the AT2 receptor subtype.

Cavernous malformations of the brain are vascular lesions which are present in up to 0.4% of all individuals and which are often accompanied by seizures, migraine, hemorrhage and other neurologic problems. Using linkage analysis and a set of short tandem repeat polymorphisms, a gene responsible for cavernous malformations in a large Hispanic kindred was mapped to the q11-q22 region of chromosome 7. A maximum pairwise lod score of 4.2 was obtained at zero recombination with marker PY5-18 at locus D7S804. Lod scores in excess of 3.0 were obtained with four additional markers closely linked to PY5-18. A broad chromosome 7q haplotype of 33 cM length on the sex average map was shared by all affected individuals indicating that the gene lies between loci D7S502 and D7S479.

Little is known concerning the magnitude of variability in the nucleic acid sequence of DNA at the individual level. We have collected a large set of sequence data from the human immunoglobulin kappa light-chain-locus constant region (10,444 bp) and subgroup IV variable region (18,580 bp). For the constant region, absolute conservation of sequence was observed, even in intron and coding-region silent sites, with the exception of one previously defined polymorphic site. For the variable region, 12 heterozygous positions were identified, giving a heterozygosity of 6 x 10(-4) per nucleotide site. The amount of nucleic acid sequence variation differs significantly (chi 2 = 4.88) between these two regions, and the observed variation is two orders of magnitude lower than that reported for two Drosophila melanogaster loci. These data suggest that, for at least some regions of the human genome, nucleic acid sequence may be less variable than previously estimated.

Aberrant detoxification of environmental agents may be the basis for an inherited predisposition to Parkinson's disease. A CYP2D6 genetic marker of the debrisoquine hydroxylase "poor metabolizer" phenotype was found to be significantly increased in Parkinson's disease patients compared to controls, as has been shown in previous studies. Presence of this marker gives an odds ratio of 1.86 for Parkinson's disease (95% confidence interval 1.33-2.39, P < 0.02). For comparison, a CYP1A1 polymorphism, which is not known to be associated with aberrant drug metabolism, showed no association with Parkinson's disease in our study.

The human immunoglobulin kappa light chain (IgK) locus includes multiple variable region gene segments (Vk) that can be divided into four subgroups. Oligonucleotide primers were designed to amplify specifically gene segments of the VkI, VkII, and VkIII subgroups using the polymerase chain reaction (PCR). Product sequences were subcloned, sequenced, and compared. Phylogenetic analyses of sequences within each subgroup indicate that some subgroups can be subdivided further into "sub-subgroups." The history of Vk segment duplications apparently includes at least two separate periods, the first giving rise to the subgroups and the second generating further complexity within each subgroup. Duplications of large pieces of DNA (demonstrated by others through pulsed-field gel electrophoresis) also played a role. Rates of synonymous and non-synonymous base changes between pairs of sequences suggest that natural selection has played a major role in the evolution of the Vk variable gene segments, leading to sequence conservation in some regions and to increased diversity in others.

Monoamine oxidase B (MAO-B) is implicated in the cause of Parkinson's disease (PD) because of its role in metabolizing the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and forming H2O2 during dopamine metabolism. Altered MAO-B activity has been observed in PD platelets. Polymerase chain reaction was used to amplify a portion of the MAO-B gene. Polymerase chain reaction products were screened with restriction enzymes to identify fragments useful for single-stranded conformational polymorphism analysis. A single-stranded conformational polymorphism was identified in an MAO-B polymerase chain reaction product after Hae III digestion. One hundred twenty-one control individuals were allelotyped with frequencies of 0.45 and 0.55 for alleles 1 and 2, respectively. Frequencies of 0.62 and 0.38 (1 and 2, respectively) were observed in a population of 46 patients with PD. The presence of MAO-B allele 1 is associated with a relative risk for PD of 2.03-fold (confidence interval, 1.44-2.61; p < 0.02). For comparison, a monoamine oxidase A polymorphism was used to determine allelic frequencies in these same populations and no statistically significant differences were found. These results suggest that an inherited variant of MAO-B may be involved in a genetic predisposition for PD.

The monoamine oxidase A (MAOA) and B (MAOB) genes have been localized to chromosome Xp11.3. Recently-defined polymorphisms and linkage analysis have shown tight linkage between MAOA and MAOB, with a distance of approximately 2.7 cM between them.

Polymerase chain reaction (PCR) amplification and oligonucleotide probe hybridization may be used to detect DNA polymorphisms rapidly in large samples. In this study, 475 individuals from thirteen human populations were allelotyped at the human leukocyte antigen (HLA) DQa (DQA1) locus. A 242 or 239 bp fragment was amplified from each individual's DNA. Each of six alleles was detected by hybridization to allele specific oligonucleotide probes (ASOs). Allelic frequencies varied between populations, but the measure of gene frequency variation among populations, the FST value, was relatively low. Most populations had genotypic frequencies in agreement with Hardy-Weinberg equilibrium expectations. Principal component analysis was performed on the populations, and results are presented in graphic form. The heterozygosity at this locus is high in all populations; the average (74%) is close to the theoretical maximum (83%) for a 6 allele system. It is likely that this system is affected by stabilizing selection, which makes it less than optimal for the study of random evolutionary divergence between populations.

Apolipoproteins C (apo C-II, apo C-III0, apo C-III1 and apo C-III2) from delipidated very low density lipoproteins (VLDL) of 522 normo- and hyperlipoproteinemic Caucasians were screened by analytical isoelectric focusing. The immobilized pH gradient used was pH 4.0-5.0 with 7 M urea, which raised the apparent pH range to 4.8-5.7. As identified by immunoblotting, six unrelated persons had two major isoforms of apo C-II, the normal apo C-II-1 (which focuses between apo C-III0 and apo C-III1) and a variant, designated apo C-II-v according to Huff et al., focusing between apo C-III1 and apo C-III2 due to a more acidic pI. In narrow pH gradients, apo C-II-v can readily be discriminated from the minor isoform, apo C-II-2, due to its slightly more basic pI, corresponding to a difference of 0.01 pH units. Neuraminidase treatment did not alter the pI of apo C-II-v and on two-dimensional electrophoresis the molecular weights of apo C-II-1 and apo C-II-v were indistinguishable. The frequency of apo C-II-v was 1.2%. It was the same in males and females and was independent of hypertriglyceridemia. The autosomal codominant inheritance could be demonstrated in the pedigree of one family. Electroblotting of apo C-II-1 and apo C-II-v onto activated glass fiber sheets, followed by amino acid sequence analysis of the amino terminal ends, revealed an exchange of the amino acid lysine at position 19 by threonine in apo C-II-v.(ABSTRACT TRUNCATED AT 250 WORDS)

Sphingomyelinase from human placenta was purified to homogeneity in five steps: concanavalin A Sepharose, butyl agarose. Blue Sepharose, sphingosylphosphocholine Sepharose chromatography and FPLC-Mono Q. This lysosomal enzyme has a pH optimum around pH 5.0-6.0. It is a glycoprotein with an approximate molecular mass of 70 kDa which is reduced to 60 kDa by enzymatic deglycosylation. Monospecific antibodies against sphingomyelinase were isolated using sphingomyelinase covalently linked to Sepharose as affinity matrix. These antibodies effectively inhibit the sphingomyelinase activity. Peptides were released from sphingomyelinase by cyanogen bromide or proteolytically by trypsin, proteinase V8 and Lys C for gas phase sequencing. Amino-acid sequences are reported which proved to be the prerequisite for antibody and oligonucleotide screening of the respective human placenta cDNA libraries for the determination of the complete amino acid sequence of human lysosomal sphingomyelinase. In situ hybridisation with a labelled antisense RNA synthesized in vitro using cloned sphingomyelinase-specific cDNA as template, which encodes the peptide sequences described here, revealed the strong expression of sphingomyelinase in human placental villi and normal fibroblasts. Fibroblasts of a Niemann-Pick patient, however, were free of mRNA expressing the sphingomyelinase described here.

The immunoglobulin kappa light chain (IgK) locus may play a significant role in the pathology of both infectious and autoimmune diseases. Most of the work on IgK genetics has been conducted using immunological techniques for allelic typing and sequence analysis. This is restricted by availability of reagents and can be both expensive and time-consuming. PCR primers were designed to amplify the kappa constant gene (Ck), and four allele-specific oligonucleotides (ASOs) were used to distinguish the alleles in the amplified PCR products. Direct sequencing of PCR products was performed to confirm that the primers specifically amplified the Ck region and the ASOs differentiated the Km alleles. Sequencing of an average of 209 nucleotides of DNA from 50 individuals revealed no variation except at codon 191, which is known to be involved in a frequent polymorphism. An analysis of 347 different individual DNAs from 10 human populations was conducted to determine Km allelic frequencies within these populations and to apply this type of data collection to population studies.

Polymerase chain reaction polymorphisms have been used to study European, Chinese, Russian and Buryat populations at the HLA-DQ alpha and IL6 loci. DNA from individuals in these populations was specifically amplified at these loci, and analyzed either with allele-specific oligonucleotides for HLA-DQ alpha or directly on agarose gels for IL6. Allelic frequencies were calculated for each of the loci in each population. Comparisons between the population frequencies show that the Russian population is more closely related to Europeans, and that the Buryat population is more closely related to Chinese. This finding is in agreement with conclusions based on the phenotypic frequencies of classical blood-group and other protein markers in these populations.

We report a new and facile extraction method of proteins and polypeptides in the range of 100 to 1 kDa previously separated by high-resolution SDS/polyacrylamide-gel electrophoresis. Proteins and polypeptides obtained by chemical or proteolytic cleavage of proteins can directly be applied to high-sensitivity N-terminal amino-acid sequence analysis by gas-phase sequencing. The Coomassie Blue-stained protein bands are eluted from the gel slices with 0.1 M sodium acetate buffer, pH 8.5, 0.1% SDS in high yield and directly applied to the filter disc of the gas-phase sequencer. The superior efficiency for the isolation of proteins and polypeptides from polyacrylamide gels for microsequencing has been documented by a quantitative comparison of the procedure described here and the favoured electroblot-transfer method using 14C-labeled marker proteins. This highly efficient isolation has been successfully reproduced and applied to the analysis of a variety of proteins and peptides with rather divergent physical properties, particularly to hydrophobic peptides isolated from SDS/polyacrylamide gels. The electrophoretic transfer onto activated glass filters. Immobilon membranes (polyvinylidene-difluoride membranes), siliconized or chemically activated glass fiber supports can be omitted. The method considerably simplifies and speeds up the isolation, and improves the sensitivity as compared to the electroblotting procedures due to the reproducibly high recoveries.

Streptomyces hydrogenans exhibits binding activity for 5alpha-dihydrotestosterone (17 beta-hydroxy-5alpha-androstan-3-one) in vivo. A cell free homogenate from the microorganism binds 5alpha-dihydrotestosterone in vitro as well. The association constant is 10(8) M-1 at 4 degrees C. Progesterone, but neither testerone nor cyproterone, competes with 5alpha-dihydrotestosterone for binding. The binding fraction from Streptomyces hydrogenans permits a simple assay for 5 alpha-dihydrotestosterone in biological samples.

The stability of highly purified L-amino acid oxidase from the sand viper venom remains practically unaffected by the pH-value at 4degreesC between pH 5 and 8, whereas a sharp activity fall was observed on both sides of this range. At temperatures above 30 degreesC the enzyme is stable only at pH 5.0--5.5. The inactivation pH values above 5.5 follows a first-order rate equation with characteristic changes in the absorption and emission spectra of the enzyme. The stability of the enzyme is dependent on the temperature of storage. At pH 7.5 there is a stability minimum at --10 degrees and -- 30 degreesC. At -- 72 degreesC the enzyme is stable practically for an unlimited period of time; temperatures exceeding 50 degrees C rapidly lead to complete inactivation. Also in the cold, the L-amino acid oxidase is most stable at pH 5.5. There are characteristic changes in absorption and emission spectra in the temperature-stability minimum (--15 degreesC) and at temperatures above 30degreesC. The inactivations follow a first-order rate equation. The cold inactivation is reversible. The stability of the enzyme is diminished by some anions and cations at 37 degreesC. The cold inactivation is promoted by several inorganic anions; organic anions and ammonium sulfate prevent cold inactivation.

There are different steroid binding receptors in the cytosol of Streptomyces hydrogenans. A high molecular weight component binds 5alpha-dihydrotestosterone (17beta-hydroxy-5alphaH-androstan-3-one) with high affinity. Partial purification is achieved by density gradient centrifugation and filtration on Sephadex G-200. 5alpha-Dihydrotestosterone and progesterone compete for the same binding sites on the receptor. Cyproterone and testosterone are not bound. As it is easy to isolate the binding fraction, a simple assay for 5alpha-dihydrotestosterone or progesterone in biological samples is available.

CMV-infections are a common and dreadful complication in immunosuppressed patients after kidney transplantation. This randomized, controlled clinical study investigates the influence of CMV-hyperimmune serum (HIS)-prophylaxis on infection incidence and severity of illness with regard to the type of immunosuppression employed. Though the clinical manifestation of the disease is lowered by HIS in cyclosporin-treated patients, there is a higher infection incidence with increased severity in HIS-treated ATG/Imurek patients compared to the respective control.

A method has been developed for the determination of aldehyde oxidase. Dimethylaminocinnamaldehyde was taken as substrate. It is oxidized to the corresponding acid and changes ist absorption at 398 nm. Except dissolved oxygen no additional electron acceptor is needed. This alternative assay was found to be about twice as sensitive compared to the method with acetaldehyde and dichlorophenol indophenol. Aldehyde oxidase activity was estimated in the raw extract of livers of pig, sheep and rat. Xanthine oxidase does not interfere.

Mitochondria and submitochondrial particles of the osmophilic yeast-like fungus Moniliella tomentosa may respire by means of two pathways: a normal cytochrome pathway, sensitive to cyanide and antimycin A, and an alternative pathway, which is insensitive to these inhibitors but is specifically inhibited by salicylhydroxamic acid. The affinities of both oxidases for succinate and NADH as substrates, for O(2) as terminal electron acceptor, and for AMP as stimulator of the alternative oxidase were determined. 1. Submitochondrial particles of M. tomentosa may also respire by means of a cyanide-sensitive and/or cyanide-insensitive system. 2. The activities of both oxidases as compared with the total activity are roughly the same in submitochondrial particles as in the original mitochondria. 3. The terminal oxidase of the cyanide-insensitive pathway requires a 10-fold higher O(2) concentration for saturation than does cytochrome c oxidase. 4. The apparent K(m) for succinate is about 3 times higher for the alternative than for the normal oxidase when measured in mitochondria, and 4-10 times higher when measured in submitochondrial particles. The apparent K(m) for NADH is roughly the same for both oxidases. 5. The apparent K(m) values of both oxidases for succinate are always lower in submitochondrial particles than in mitochondria. 6. The apparent K(m) for AMP, acting as a stimulator of the alternative oxidase, is the same (25mum) in mitochondria as in sub-mitochondrial particles. These results are discussed in the light of the structure and localization of the components of the alternative oxidase.