Untersuchungen über die Teilungsrichtungen inPollenmutterzellen und bei der Blaualge Chroococcus

Ohne Zusammenfassung     

Functional characterization of an anti-estradiol antibody by site-directed mutagenesis and molecular modelling: modulation of binding properties and prominent role of the V(L) domain in estradiol recognition

The high-affinity monoclonal anti-estradiol antibody 9D3 presents a specificity defect towards estradiol-3-sulphate and 3-glucuronide conjugates incompatible with use in direct immunoassays. The corresponding single-chain variable fragment (scFv), cloned and produced in E. coli, exhibited a 10-fold lower affinity for estradiol (K(a)=1.2 x 10(9) M (-1)) and a slightly increased specificity defect for the 3-position. Site-directed mutagenesis revealed critical residues involved in estradiol recognition and produced mutants exhibiting up to a 3-fold increase of the binding affinity for estradiol and up to a 2-fold decrease of the cross-reactivity with estradiol-3-sulphate. A comparative model of the antibody 9D3-estradiol complex was built in which the estradiol D-ring is buried into the binding pocket while the 3-, 6- and 7-positions are solvent exposed, agreeing with the lack of specificity for these three positions. Two potential alternative orientations of the A-ring, one close to CDR H3 and L2 loops, and the other one close to CDR H2 and L3 loops, have been considered for the docking of estradiol, none of which could be unambiguously privileged taking into account data from cross-reactivity measurements, photolabelling and mutagenesis studies. For both orientations, estradiol is stabilized by hydrogen bonding of the 17beta-OH group with TyrL36, His89 and GlnH35 in the first case, or TyrL36, only, in the second case and by van der Waals contacts from TyrL91 with alpha- or beta-face of estradiol, respectively, and from ValH95 and GlyH97 with the opposite face. To elucidate the molecular basis of antibody 9D3 specificity, as compared with that of another anti-estradiol antibody 15H11, single variable domains (V(H) and V(L)) and scFv hybrids have been constructed. The binding activity of V(L)9D3 as well as the specificity of the V(L)9D3/V(H)15H11 hybrid, both similar to antibody 9D3, revealed a prominent role of V(L) in estradiol recognition. These findings establish premises for antibody engineering to reduce cross-reactivity, especially with estradiol-3-conjugates.     

Mapping of cysteine genes on the chromosome of Pseudomonas aeruginosa PAO

Summary Three loci coding for different steps in the pathway of cysteine biosynthesis have been mapped by R68.45-mediated coconjugation analysis. The cysteine auxotrophic mutants could be subdivided into sulfite and sulfide-requiring mutants. Sulfide-requiring mutants (cysIV group) were localized at a single position between pyrF and pur-67, while sulfite-requiring mutants (cysI and cysII) mapped at two different regions. The cysI group was also localized between pyrF and pur-67, although more distal to pyrF than the cysIV group. This group included the cys-54 marker, which has been mapped previously. The second group of sulfite-requiring mutants, designated as cysII, was cotransducible with hisI and localized at the end of the PAO chromosomal map. This location was also confirmed for the marker cys-59.The marker cys-59 (which was cotransducible with his1) was cotransferred by R68.45-mediated conjugations with both the late marker pur-67 and the early marker ilv-226. As the late marker hisI was positioned at about 60–65 min (Herrmann and Günther, in press) the length of the PAO chromosome was estimated to be about 70 min.     

Immunocytochemistry of brain-reactive monoclonal antibodies in peripheral tissues

Among a total of 135 tissue-reactive monoclonal antibodies previously prepared, 81 were brain-selective and were classified into neuronal and non-neuronal categories. The neuronal antibodies were again subdivided into antineurofibrillar, antiperikaryonal-neurofibrillar, and antisynapse-associated groups. On the basis of morphologic, developmental, biochemical, and pathologic criteria, the antibodies in at least two of these groups were found to detect heterogeneous antigens (called "neurotypes") rather than different antigenic determinants in single antigens. On examining the distribution in peripheral organs of staining patterns of 11 antineuronal brain-reactive antibodies, we now confirm that these antibodies are, indeed, largely brain-specific. In general, non-neuronal elements in liver, lung, heart, thymus, intestine, adrenal, and spleen remained unstained. However, most of the antibodies stained peripheral neural elements. Occasional antibodies did stain selected, non-neuronal structures. Four out of five antineurofibrillar antibodies stained nerve fibers in adrenal medulla, intestine and thymus. All of three antiperikaryonal-neurofibrillar antibodies also stained nerve fibers in the adrenal medulla, but not in other organs. Two out of three antisynapse-associated antibodies stained what appear to be nerve contacts on adrenal medullary cells, but not on any other peripheral cells examined. The non-neuronal peripheral staining patterns were restricted to selective nuclear staining exhibited by two out of five antineurofibrillar antibodies and the staining of macrophage and selected cardiac muscle nuclei by two of three antisynapse-associated antibodies. However, one antineurofibrillar antibody also stained the cytoplasm of selected liver cells. Among non-neuronally reacting antibodies, two antibodies stained nuclei of all cells except neurons in brain as well as peripheral organs. An antibody staining the ciliary epithelium of choroid plexus also stained basal bodies of ciliated bronchial epithelium. The overall data suggest that the specificity of brain-reactive antibodies is high and that their cross-reactivity with epitopes in non-nervous tissue is rare. In these cases, the antibodies seem to provide specific reagents for these additional structures as well as for their specific brain antigens.     

Genetic mapping of 14 short tandem repeat polymorphisms on human chromosome 22

We have constructed a linkage map of 14 short tandem repeat polymorphisms (11 with heterozygosity > 70%) on the long arm of human chromosome 22 using 23 non-CEPH pedigrees. Twelve of the markers could be positioned uniquely with a likelihood of at least 1,000:1, and distributed at an average distance of 6.62 cM (range 1.5–16.1 cM). The sex-combined map covers a total of 79.6 cM, the female map 93.2 cM and the male map 64.6 cM. Based on comparisons between physical maps and other genetic maps, we estimate that our map covers 70%–80% of the chromosome. The map integrates markers from previous genetic maps and uniquely positions one marker (D22S307). Data from physical mapping on the location of four genetic markers correlates well with our linkage map, and provides information on an additional marker (D22S315). This map will facilitate high resolution mapping of additional polymorphic loci and disease genes on chromosome 22, and act as a reference for building and verifying physical maps.     

Sequence diversity of pistil S-proteins associated with gametophytic self-incompatibility in Nicotiana alata

Summary In order to study the extent and nature of differences among various S-allele-associated proteins in N. alata, we carried out comparative studies of seven such proteins. We first isolated and sequenced cDNA clones for the S_z-, S_F11-, S₁-, and S_a-alleles, and then we compared the deduced amino acid sequences both of these four S-proteins and of three previously published S₂-, S₃-, and S₆-proteins. This comparison revealed (1) an average homology of 53.8% among the seven proteins and (2) two homology classes, with S_z and S_F11 in one class and S₁, S₂, S₃, and S₆ in the other class. There are 60 conserved residues, including 9 cysteines. Of the 144 variable residues, 50 were identified as hypervariable based on a calculation of their Similarity Indices. Although conserved, variable, and hypervariable residues are dispersed throughout the protein, some are clustered to form five conserved, five hypervariable, and a number of variable regions. Those variable sites which contain residues conserved within one class of S-proteins but different between classes might provide a clue to the evolutionary relationship of these two classes of S-proteins. The hypervariable residues, which account for sequence variability, may contribute to allelic specificity.     

Arachidonic acid-induced human platelet aggregation independent of cyclooxygenase and lipoxygenase

It is generally agreed that arachidonic acid (20:4ω6) can stimulate platelet aggregation after conversion to prostaglandin G₂ and H₂ and thence to thromboxane A₂. This action is prevented by cyclooxygenase inhibitors. Washed platelets were isolated on metrizamide gradient and resuspended in a Ca²⁺-free buffer. Their stimulation by C 20:4 6 was followed by ¹⁴C serotonin (5HT) release, thromboxane (TX) synthesis and an increase of light transmission, not dependent on aggregation, accompanied by slight lysis (14%). The addition of extrinsic Ca²⁺ suppressed lysis and allowed the formation of aggregates. Under these conditions, cyclooxygenase inhibitors such as acetyl salicylic acid, indomethacin or flurbiprofen totally suppressed TX synthesis without preventing platelet aggregation or [¹⁴C]-5HT release. Other C 20 polyunsaturated fatty acids could not substituted for C 20:4ω6 in inducing aggregation, and Ca²⁺ was found to be a prerequesite for protection of the cell against lysis as well as for aggregation in the absence or TX formation. The use fo the lipoxygenase inhibitor BW 755 C did not prevent C 20:4ω6-induced aggregation of aspirin-treated platelets, suggesting that the phenomenon was independent of this pathway also. The total suppression of oxidative metabolism with these inhibitors was verified by the analysis of icosanoids using glass capillary column gas chromatography. It is suggested that under these condition, C 20:4ω6-induced platelet aggregation might be due to an increased membrane permeability to Ca²⁺ induced by this fatty acid in the absence of oxidation.     

Baseline predictors of response and discontinuation of tumor necrosis factor-alpha blocking therapy in ankylosing spondylitis: a prospective longitudinal observational cohort study

Introduction  

Identifying ankylosing spondylitis (AS) patients who are likely to benefit from tumor necrosis factor-alpha (TNF-α) blocking therapy is important, especially in view of the costs and potential side effects of these agents. Recently, the AS Disease Activity Score (ASDAS) has been developed to assess both subjective and objective aspects of AS disease activity. However, data about the predictive value of the ASDAS with respect to clinical response to TNF-α blocking therapy are lacking. The aim of the present study was to identify baseline predictors of response and discontinuation of TNF-α blocking therapy in AS patients in daily clinical practice.