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PubMed1977

[Therapy of obesity--effectiveness of calorie-reducing diets with various basic nutritional relations].

Within the research project nutrition and performance on altogether 80 test persons during a three weeks' test period the problem was examined, whether the relations of the basal nutrients have an influence on the effectiveness of a diet with an only moderate reduction of calories. The relations of the nutrients were tested in four variants. The daily caloric intake orients at the optimum weight of the test person: 30 kcal/kg optimum weight a day. It was shown that the so-called optimum weight caloric diet led to a sufficient continuous decrease of weight without disturbances of the general state of health. The variation of the relations of the basal nutrients had no decisive influence on the effectiveness.

Adult↗
PubMed1997

Expression, purification, characterization, and X-ray analysis of selenomethionine 215 variant of leukocyte collagenase.

Matrix metalloproteinases belong to the superfamily of metzincins containing, besides a similar topology and a strictly conserved zinc environment, a 1,4-tight turn with a strictly conserved methionine residue at position three (the so called Met-turn [Bode et. al. (1993) FEBS 331, 134-140; Stöcker et al. (1995) Protein Sci. 4, 823-840]. The distal S-CH3 moiety of this methionine residue forms the hydrophobic basement of the three His residues liganding the catalytic zinc ion. To assess the importance of this methionine, we have expressed the catalytic domain of neutrophil collagenase (rHNC, residues Met80-Gly242) in the methionine auxotrophic Escherichia coli strain B834[DE3](hsd metB), with the two methionine residues replaced by selenomethionine. Complete replacement was confirmed by amino acid analysis and electrospray mass spectrometry. The folded and purified enzyme retained its catalytic activity, but showed modifications which are reflected in change kinetic parameters. The Met215SeMet substitution caused a decrease in conformational stability upon area denaturation. The X-ray crystal structure of this selenomethionine rHNC was virtually identical to that of the wild-type catalytic domain except for a very faint local disturbance around the sulfur-seleno substitution site.

Cloning, Molecular↗
PubMed1994

Biochemical and genetic analysis of variant C of caprine alpha s2-casein (Capra hircus).

Two alleles, A and B, were previously described at the goat alpha s2-casein locus. Isoelectric focusing allowed us to subdivide the former one in two new alleles, called A and C. Although alpha s2-casein C cannot actually be distinguished from its A counterpart by starch or polyacrylamide gel electrophoresis, it differs from the previous allele by a single substitution Lys (A)/Ile (C) at position 167, which was confirmed at the nucleotide level. The frequencies of the three alpha s2-casein alleles A, B and C were estimated to be 0.85, 0.04 and 0.11 in the French dairy breeds 'Alpine' and 'Saanen'.

Alleles↗
PubMed1997

[A new variant of Creutzfeldt-Jakob disease. Do we want development of a new epidemic?].

Human spongiform encephalopathies have been receiving a lot of attention lately, because of a new variant of Creutzfeldt-Jakob disease and its possible connection to bovine spongiform encephalopathy which has reached epidemic proportions in Great Britain during the last ten years. Four different human spongiform encephalopathies have been described, the most common being Creutzfeldt-Jakob disease, which can occur in a sporadic, familial or transmissible form. The infectious agent is mainly, possibly solely, composed of a pathogenous isoform of a normal membrane-bound glycoprotein, called a prion. In animals, spongiform encephalopathies occur most frequently in sheep, as scrapie, and in cattle, as bovine spongiform encephalopathy, also known as mad cow disease. There is substantial evidence to suggest that this disease in cattle is the source of the new variant of Creutzfeldt-Jakob disease, although this has yet to be proven. An important question is whether the cases of the new variant of Creutzfeldt-Jakob disease registered so far are the start of an epidemic, as in the case of bovine spongiform encephalopathy.

Animals↗
PubMed2006

Exploration, normalization, and genotype calls of high-density oligonucleotide SNP array data.

In most microarray technologies, a number of critical steps are required to convert raw intensity measurements into the data relied upon by data analysts, biologists, and clinicians. These data manipulations, referred to as preprocessing, can influence the quality of the ultimate measurements. In the last few years, the high-throughput measurement of gene expression is the most popular application of microarray technology. For this application, various groups have demonstrated that the use of modern statistical methodology can substantially improve accuracy and precision of the gene expression measurements, relative to ad hoc procedures introduced by designers and manufacturers of the technology. Currently, other applications of microarrays are becoming more and more popular. In this paper, we describe a preprocessing methodology for a technology designed for the identification of DNA sequence variants in specific genes or regions of the human genome that are associated with phenotypes of interest such as disease. In particular, we describe a methodology useful for preprocessing Affymetrix single-nucleotide polymorphism chips and obtaining genotype calls with the preprocessed data. We demonstrate how our procedure improves existing approaches using data from 3 relatively large studies including the one in which large numbers of independent calls are available. The proposed methods are implemented in the package oligo available from Bioconductor.

Algorithms↗
PubMed2000

Cloning and functional characterization of a novel c-mpl variant expressed in human CD34 cells and platelets.

The thrombopoietin receptor, c-mpl, is a crucial element not only in thrombopoietin (TPO)-initiated signaling pathways but also in the regulation of the circulating amount of TPO. We have identified a new c-mpl isoform, called c-mpl-del, that lacks 72 bp (24 amino acids) in the extracellular region of c-mpl and arises as a consequence of alternative RNA splicing between exons 8 and 9. c-mpl-del is expressed along with c-mpl-wt in blood mononuclear cells, CD34(+)cells, megakaryocytes, and platelets prepared from either normal donors or ET patients, although its relative expression appears to increase with megakaryocyte differentiation. The c-mpl-del-transfected cells expressed greater amounts of c-mpl-del RNA and protein than the comparable c-mpl-wt-transfected cells, however flow cytometry analysis could not detect any c-mpl receptor on the surface of the c-mpl-del-transfected cells. Further evidence for the absence of surface c-mpl-del was that in contrast to cells transfected with c-mpl-wt, those transfected with c-mpl-del did not grow in response to TPO, failed to undergo tyrosine phosphorylation of TPO-specific signal molecules, and did not bind(125)I-rHuTPO. Taken together, these results demonstrate that c-mpl-del, a naturally occurring variant of c-mpl, fails to be incorporated into the cell membrane but might serve as a mechanism to decrease the overall expression of functional c-mpl late in megakaryocyte differentiation.

Alternative Splicing↗
PubMed1995

AFAP-120. A variant form of the Src SH2/SH3-binding partner AFAP-110 is detected in brain and contains a novel internal sequence which binds to a 67-kDa protein.

SH2 and SH3 domains have been characterized as functional domains that mediate protein-protein interactions in signal transduction. Recently, the cDNA sequence of a novel Src- and Fyn-binding protein called AFAP-110, for Actin-Filament Associated Protein-110 kDa, was reported. This protein was distinctive in that it is both an SH2 and SH3 binding partner for the non-receptor tyrosine kinases Src and Fyn. Here, we report the characterization of an alternatively processed form of AFAP-110 that encodes an additional 258 base pair (bp) of open reading frame. Transient expression of this full-length clone reveals a molecular mass of 120 kDa. Western blot analysis indicate that a larger 120-kDa variant of AFAP-110 can be detected in brain and is not detectable in any other tissues examined. Northern blot analysis indicate that the novel 258-bp insert can be detected in brain RNA but not chick embryo fibroblast RNA. We propose the name AFAP-120, for Actin Filament-Associated Protein-120 kDa. Expression of the 258-bp novel insert (NINS) as a glutathione S-transferase-encoded fusion protein permits adsorption of a 67-kDa protein from tissue lysates. Deletion analysis of the NINS indicates that the interaction with p67 can be attributed to a proline-rich motif that resembles an SH3-binding motif. We hypothesize that AFAP-120 facilitates interactions in brain between SH2/SH3 signaling proteins and actin filaments and that a proline-rich motif in the NINS may exist to facilitate additional interactions between cellular proteins in brain and actin filaments.

Amino Acid Sequence↗
PubMedDate not supplied

Dactylitis, a term for different digit diseases.

Dorland's Illustrated Medical Dictionary gives the following definition of dactylitis (deltaalphachitauupsilonlambdaomicronsigma = digit): 'inflammation of a finger or toe'. Although any inflammatory process involving the fingers or toes may be called dactylitis, the term has entered in current use only in some well-defined entities. These differ in the involved tissue of the digit and in the type of involvement. Tuberculous dactylitis is the variant of tuberculous osteomyelitis affecting the short tubular bone of the hands and feet. Radiographs typically show a central, lytic, cystic, and expansive lesion known as spina ventosa. Syphilitic dactylitis is a manifestation of congenital syphilis. Radiological findings mimic those of tuberculous dactylitis but the involvement is bilateral and symmetric. Sarcoid dactylitis is due to typical non-caseating granulomas invading the phalanges and the adjacent soft tissue. Blistering distal dactylitis is an infection of the anterior fat pad on the volar surface of the distal portion of a single finger or more rarely a toe, mostly caused by group A beta-haemolytic streptococci. Sickle cell dactylitis, also known as 'hand-foot syndrome', is due to localized bone marrow infarction of the carpal and tarsal bones and phalanges. Spondyloarthritis dactylitis, also called 'sausage-like' digit, is a diffuse painful swelling of the fingers and toes. Recent ultrasonography (US) and magnetic resonance imaging (MRI) studies on both finger and toe dactylitis have established that dactylitis is due to flexor tenosynovitis and that the enlargement of the joint capsule is not an indispensable condition for the 'sausage-like' feature. There is no evidence of enthesitis of flexor digitorum tendons and joint capsule.

Fingers↗
PubMedDate not supplied

Macrocystic serous cystadenoma of the pancreas: CT and endosonographic features.

Recently, the term "serous cystadenoma" has been adopted in the pathological community to qualify the so-called microcystic adenoma of the pancreas. This change in terminology was based on the emergence of a new type of serous cystadenoma of the pancreas, i.e., the macrocystic variant. We report one case of pathologically proven macrocystic serous cystadenoma of the pancreas for which the diagnosis of mucinous cystadenoma was suggested preoperatively. This rare but benign macrocystic variant exhibits radiological features similar to those of mucinous cystadenoma. In addition, inflammatory changes and foci of hemorrhage within the tumor may simulate mucinous cystadenocarcinoma.

Adenoma↗
PubMed1993

Antibody engineering by parsimonious mutagenesis.

The human monoclonal antibody (humAb) problem has largely been solved with the aid of the polymerase chain reaction (PCR) [Larrick et al., Bio/Technology 7 (1989a) 934-938; Larrick et al., Biochem. Biophys. Res. Commun. 160 (1989b) 1250-1256; Chiang et al., BioTechniques 7 (1989) 360-366]. Phage display has now made it possible to recover humAb with primary response level affinities (approx. 10(6) M-1) for virtually any antigen (including self antigens) from comprehensive libraries of B-cell repertoires from non-immunized humans [Marks et al., J. Mol. Biol. 222 (1991) 581-597; Marks et al., Bio/Technology 10 (1992) 779-783; Griffiths et al., EMBO J. 12 (1993) 725-734]. This means that the goal of therapeutic humAb without immunization is within reach. However, in order to achieve the affinities generally required for therapeutic use (> or = 10(9) M-1), reliable methods will be needed to complete the affinity maturation process in vitro. Available X-ray crystallographic data and energy calculations indicate that only a fraction of the substantial contact surface between the Ab and protein antigens contribute significantly to affinity. Thus, the remaining contact surface presents multiple opportunities to develop additional high-affinity contacts, needing only a means to identify them. To this end, we have developed a computer-assisted method for oligodeoxyribonucleotide-directed scanning mutagenesis, called parsimonious mutagenesis (PM), whereby all three complementarity-determining regions (CDR) of a variable region (V-region) gene can be simultaneously and thoroughly searched for improved variants in libraries of manageable size. These libraries are made with low-redundancy 'doping' codons and biased nucleotide (nt) mixtures designed to maximize the abundance of combining sites with predetermined proportions of preselected sets of alternative amino acids (aa). This allows the library to 'probe' the surface of the antigen one or a few aa residues at a time with a wide selection of aa side chains to search out and identify new high-affinity contacts. In addition to affinity maturation in vitro, PM can also be used to remove unwanted cross-reactivities and to 'reshape' rodent mAb for human therapeutic use.

Amino Acid Sequence↗
PubMed2004

H2B ubiquitylation: the end is in sight.

Historically, the first eukaryotic protein found to be modified by ubiquitin was H2A, originally isolated from HeLa cells in 1975 by Harrison Busch and coworkers as a histone-like, nonhistone chromosomal protein called A24. Ubiquitylated histones have subsequently been found in many eukaryotic species, and to date, the core histones H2A, H2B, H3, the linker histone H1, and the histone variant H2A.Z are known to carry this modification. Although first on the scene, it was only recently that studies on histone ubiquitylation have enjoyed a renaissance. Part of the reason for the relatively slow pace of research on this fascinating histone modification was the absence of a good genetic system with which to study its cellular roles. This changed in 2000, when histone H2B was found to be ubiquitylated in the budding yeast S. cerevisiae, an organism with a low histone gene copy number and highly tractable genetics. Another factor was the almost exclusive focus of research on the role of polyubiquitylation in protein turnover. Because histones are generally monoubiquitylated, a form of the modification that is not associated with protein degradation, the significance of this minimalist ubiquitin conjugation was not heavily pursued. But perhaps the key reason for the renewed interest in histone ubiquitylation was the unexpected discovery of the past year that ubiquitylated H2B plays an important role in the trans-histone methylation of histone H3, a modification with close ties to the regulation of gene expression. This review will highlight some of the recent findings on the regulation and cellular roles of H2B ubiquitylation in yeast.

Animals↗
PubMed2002

Polyetheretherketone--cytotoxicity and mutagenicity in vitro.

The results of the incubation of polyetheretherketone (PEEK) fibre material with seven different genotype variants of salmonella bacterium showed with and without an external metabolic activation system (S9) with no mutagenic or cytotoxic activity of the test material. In the so-called "plate incorporation test" in which the PEEK raw material is finely cut and applied direct to the agar plate without the addition of solvent there was, as expected, no evidence of cytotoxic or mutagenic effects. In the HPRT test there was a significant increase in the number of mutants per dish, both after addition of N-acetylaminofluorene and N-methyl-N'-nitro-N-nitrosoguanidine (with and without an external metabolic activation system = +S9), but not after treatment of the cells with PEEK-DMSO-eluate. This means that the PEEK material under study did not release any substances that cause V79 cells to mutate. The investigation of the toxic reaction on the material under study revealed that the number of surviving colonies per 10(5) surviving cells lay within the range of or below the solvent control even in the presence of high PEEK concentrations (5.0 microg/ml). Therefore, in summary, the study produced no evidence of cell damage caused by PEEK.

2-Acetylaminofluorene↗
PubMed2002

The dynamics of mutations in the YMDD motif of the hepatitis B virus polymerase gene during and after lamivudine treatment as determined by reverse hybridisation.

We have analysed the dynamics of HBV variants related to Lamivudine resistance in 22 chronically infected patients during and after the end of Lamivudine therapy. Thirteen patients had a confirmed methionine mutation in the YMDD region of the reverse transcriptase domain determined by sequence analysis. They responded to therapy having a mean reduction of HBV DNA of 4.55 log (range 2.93-8.91). Nine patients partly responded to therapy, with a small decline of HBV DNA (mean reduction of 3.39 log, range 1.72-5.12) and no indication for an YMDD variant. Samples were re-analysed with a HBV Drug Resistance Line Probe assay (InnoLipa HBV-DR), which detected as low as 10% of a variant HBV population. With this assay, changes in the YMDD region were detected with a mean of 2 weeks (range -8 to 10) earlier than by an increase of HBV DNA levels. Increase of ALT was observed with a mean of 31 weeks (range 29-51) later than the methionine changes in the YMDD motif. Indications for a methionine into valine change could be determined in only one of the partial responders. An unexpected observation was the predominant presence of variant virus populations after end of therapy. In ten patients, we detected the wild type virus with a mean of 14 weeks after end of therapy (range 1-42 weeks). In three patients, no variant virus population could be detected at 25, 36 and 37 weeks, respectively, after cessation of treatment. This observation is important for the inclusion of the so-called naive patients in clinical trials.

Amino Acid Motifs↗
PubMed1999

Growth hormone binding affinity for its receptor surpasses the requirements for cellular activity.

The human growth hormone (hGH)-receptor interaction was used to study the relationship between hormone-receptor affinity and bioactivity. hGH has two nonequivalent sites, called site 1 and site 2, that bind two molecules of receptor in a sequential fashion. We produced both site 1 and site 2 high-affinity hGH variants either by combining alanine mutants previously found to improve affinity at site 1 or by random mutagenesis of residues in site 2 followed by phage display and receptor binding selections. The two high-affinity variants, as well as one which combined them, were used in cell proliferation assays with FDC-P1 cells expressing the hGH receptor. Interestingly, none of these variants produced a change in the EC50 for cell proliferation or the levels of JAK2 tyrosine kinase phosphorylation. Next we studied the effect of a reduction in site 1 affinity on cell proliferation. A systematic series of hGH mutants were produced in which affinity for site 1 was reduced from 5- to 500-fold. Surprisingly, the EC50 for cell proliferation was unaffected until affinity was reduced about 30-fold from wild-type hGH. Thus, native hGH-receptor affinity is much higher than it needs to be for maximal JAK2 phosphorylation or cell proliferation. These studies begin to define basic functional tolerances for receptor activation that need to be considered in the design of hGH mimics.

Animals↗
PubMed2003

[Xeroderma pigmentosum: children of the moon].

Xeroderma pigmentosum is based on a genetic defect in the DNA repair system, which is diagnosed in early childhood. Xeroderma pigmentosum is a rare disorder, which is transmitted in an autosomal recessive manner. Children with xeroderma pigmentosum display hypersensitivity to ultraviolet (UV) radiation. These patients experience serious sunburns with minimal exposure and then develop poikiloderma in the sun-exposed areas. Squamous cell carcinomas, basal cell carcinomas and malignant melanomas all appear during childhood. The majority of patients do not reach adult, but die from metastatic cutaneous malignancies. Genetically, xeroderma pigmentosum is differentiated into 7 complementation groups (XP-A to XP-G) and the xeroderma pigmentosum variants (XP-V). The assignment to the specific complementation group is made by fusing of xeroderma pigmentosum fibroblasts. Xeroderma pigmentosum must be distinguished from other so-called DNA repair deficiency syndromes, including Cockayne syndrome and trichothiodystrophy. A topical DNA repair enzyme appears to be helpful. A recombinant liposomal encapsulated T4 endonuclease V repairs UV-induced cyclobutane-pyrimidine dimers. Direct curative treatment of xeroderma pigmentosum could be achieved with gene therapy in future. Transfection of an intact repair gene which specifically codes for the missing repair protein could open new possibilities in the therapy of xeroderma pigmentosum.

Child↗
PubMed2002

Immunoglobulin heavy chain constant regions regulate immunity and tolerance to idiotypes of antibody variable regions.

Particular syngeneic adjuvant-free monoclonal antibodies are immunogenic and elicit antibody responses against the variable region idiotypes (Ids). We here study how heavy-chain constant regions (C(H)) regulate immune responses to Ids of free, uncomplexed monoclonal antibodies. To this end, we selected two hybridomas, called Id(3) and Id(A.01), that produce immunogenic IgM(lambda)2 directed toward 2,4,6-trinitrophenyl, and subcloned rare IgG1, IgG3, IgE, or IgA class switch variants. The purified switch variants, which possessed the Ids of their IgM progenitors, were injected repeatedly without added adjuvant into BALB/c mice, and anti-Id IgG responses were determined. These repeated injections revealed that the immunogenicity of Ids was lost by switching to IgG1 and IgG3, restored when the Fc piece of IgG1 was removed, maintained by switching to IgE and monomeric IgA, and lost in polymeric IgA. Loss of immunogenicity was associated with acquisition of Id-specific tolerogenicity, as determined by immunization challenge with Id borne by IgM. An Id borne by IgG induced tolerance when injected at least 90 days before or 3-21 days after immunization with IgM Id was begun. Ids of IgG were also tolerogenic in mice deficient in Fc(gamma)RIIB or Fc(gamma)RI + III. The results suggest that Ids that have switched to IgG and pIgA negatively control immune responses to shared Ids, including the Ids of their IgM progenitors.

Animals↗
PubMed1986

Mutation affecting the expression of immunoglobulin variable regions in the rabbit.

We have found a variant of the allotype allele a2 in the rabbit, which presumably arose by mutation, that segregates as expected for an allele at the a locus. This allele is called "ali" and the corresponding rabbit strain is called "Alicia." In heterozygous animals (ali/a1 and ali/a3) the concentration of a2 molecules is lower by a factor of 1000 than in standard a2/a2 homozygotes. In homozygous ali/ali individuals the a2 concentration varies with age--i.e., very low in young rabbits and higher in older ones--but it never reaches normal levels. The low level of a2 is compensated by increased amounts of a-negative molecules. Southern blot analysis did not reveal any gross changes in the intron between JH and C mu (joining region of immunoglobulin heavy chain and constant region of immunoglobulin mu chain) or in the number of VH gene segments encoding a locus specificities. We suggest that the ali phenotype is due to a mutation in a control element.

Animals↗
PubMed1995

The requirement for DM in class II-restricted antigen presentation and SDS-stable dimer formation is allele and species dependent.

Recently several cell lines have been identified with mutations in a major histocompatibility complex (MHC)-linked protein that lead to defects in class II-restricted antigen presentation and a defect in the formation of class II SDS-stable dimers. The defect in these cells has recently been shown to result from the inability to express the MHC-encoded nonclassical class II molecule called DM. To further examine the role of DM in class II-restricted antigen presentation, we asked if this defect would equally affect different allelic and species variants of class II molecules. To investigate this, we transfected the parent cell lines T1 and 8.1.6 and their respective antigen presentation mutants T2 and 9.5.3 with the genes encoding I-Ad and examined the derived transfectants for their ability to present antigen, the conformation of I-Ad at the cell surface, association of I-Ad with invariant chain (Ii), and the ability to form I-Ad SDS-stable dimers. The lack of functional DM expression did not affect any of the anti-I-Ad monoclonal antibody (mAb) epitopes tested or the ability of I-Ad to associate and dissociate with Ii. Surprisingly, these studies also revealed that the antigen presentation defect observed for DR in the 9.5.3 cells did not compromise I-Ad-restricted antigen presentation. In addition, we found that the level of SDS-stable dimer formation did not correlate with antigen presentation capacity for I-Ad and that the amount of SDS-stable I-Ad dimer depends on the cellular context in which the class II molecule is expressed. Our results suggest that the ability to form SDS-stable dimer is not strictly correlated with class II-restricted antigen presentation. Finally, when two allelic forms of murine class II molecules were compared in the defective T2 cell line, it was found that I-Ak but not I-Ad forms SDS-stable dimers equivalent to that seen in the parental cell lines. Overall, our results suggest that DM may modulate rather than play a requisite role in I-Ad-restricted antigen presentation and SDS-stable dimer formation and that dependency on DM may be allele or species specific.

Animals↗