Monoclonal antibodies for the detection of desialylation of erythrocyte membranes during haemolytic disease and haemolytic uraemic syndrome caused by the in vivo action of microbial neuraminidase

Especially in childhood, the in vivo action of microbial neuraminidase may cause haemolytic anaemia or life-threatening haemolytic uraemic syndrome. The exposure of the Thomsen-Friedenreich (T) crypto-antigen and T-antigen polyagglutinability of erythrocytes has been described as the first sign of toxic cleavage of N-acetylneuraminic acid (Neu5Ac) from sialoglycoproteins of cell membranes. This phenomenon may, however, be too unspecific to initiate treatment for toxin elimination. The present study investigated the diagnostic effectiveness of a panel of three monoclonal antibodies (mcabs) for the estimation of the clinical significance of neuraminidase action in vivo. Depending on the amount of Neu5Ac released, the mcabs I-C4, II-Q9 and III-Y12 recognized different epitopes on erythrocyte asialoglycophorin. In 1345 patients, the mcab II-Q9 detected cleavage of Neu5Ac in 32 children who had T-antigen polyagglutinability and mild to moderate haemolytic anaemia. However, only 10 patients, whose erythrocytes were agglutinated by the mcabs III-Y12 or I-C4, developed severe haemolysis, thrombocytopenia, and finally the life-threatening haemolytic uraemic syndrome (p<0.0002). In conclusion, these mcabs provided an early marker of the in vivo action of neuraminidase. Two different degrees of erythrocyte desialylation, as defined by these mcabs, are suggested to reflect the severity of toxin-associated disease. This revised version was published online in November 2006 with corrections to the Cover Date.     

SVS II--an androgen-dependent actin-binding glycoprotein in rat semen.

Rat seminal vesicles and the lateral prostate secrete a glycoprotein designated as SVS II in an androgen-dependent manner. SVS II, which has a M(r) of 49,000 and a pI of 10.5, is an actin-binding protein. G- and F-actins cosediment with SVS II at a ratio of 2:1 (actin:SVS II). SVS II affects the kinetics of actin polymerization in the same way as do barbed end capping proteins. Interaction with actin is specific for the skeletal and cardiac muscle isoforms and there is no corresponding interaction with cytoplasmic actins. The binding site is close to the C-terminus of actin. Monospecific polyclonal antibodies directed against the N-terminus of actin cross-react with SVS II, but there is no cross-reaction by a monoclonal antibody directed against a C-terminal epitope on actin. Recent sequence analysis of SVS II shows a sequence of about 14 residues that is repeated 13 times between residues 86 and 298. The consensus sequence based on these repeats is homologous to residues 10 to 25 of actin; this may account for the immunological cross-reactivity. Like actin, SVS II binds and inhibits the activity of DNase I, but SVS II has no effect on the ATPase activity of myosin subfragment 1. Thus, SVS II is an actin-binding protein which retains some properties of actin itself.     

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA‐based protocols. Glyoxal acted faster than PFA, cross‐linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA‐based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.     

The Rad9 Gene of Coprinus Cinereus Encodes a Proline-Rich Protein Required for Meiotic Chromosome Condensation and Synapsis

The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stage of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus.     

Synthesis, crystal structure and magnetic properties of the first structurally characterized 1,2-dithiocroconato-containing Cu(II) complex, [Cu(bpca)(H2O)]2[Cu(1,2-dtcr)2]·2H2O

The first crystal and molecular structure of a transition metal complex containing 1,2-dithiocroconate (1,2-dtcr, dianion of 1,2-dimercaptocylopent-1-ene-3,4,5-trione), [Cu(bpca)(H₂O)]₂[Cu(1,2-dtcr)₂]·2H₂O (where bpca is the bis(2-pyrdidylcarbonyl)amide anion), has been determined by single crystal X-ray diffraction methods. The compound crystallizesin the monoclinic syste, space group P2₁/c, with a = 11.661(3), b = 20.255(6), c = 8.265(3) Å, ß = 107.26(2)° and Z = 2. The structure is formally built of [Cu(1,2-dtcr)₂]²⁻ and [Cu(bpca)(H₂O)]⁺ ions and water of hydration. The copper atom of the anion is situated at a crystallographic inversion centre, bonded to four sulfur atoms in a planar, approximately square arrangement. In the cation the copper equatorial plane is formed by the three nitrogen atoms of the bpca ligand and a water oxygen atom. In addition there is a very weak axial bond to one of the sulfur atoms of a 1,2-dtcr ligand in the anion. Through these latter weak bonds each anion is connected to, and sandwiched between, two cations, resulting in neutral, trinuclear, centrosymmetric formula units. The triple-decker molecules are arranged in stacks along the crystallographic a-axis creating close contacts between the terminal copper atoms and bpca groups of the neighbouring molecules. This intermolecular interaction is, however, too weak to define the structure as a chain compound. The distance between adjacent copper atoms within the trinuclear unit is 4.189(1) Å, while the shortest intra-stack metal-metal separation between terminal copper atoms is 5.281(1) Å. Variable-temperature magnetic susceptibility measurements in the temperature r.2–140 K reveal that a Curie law is followed; with three non-interacting copper(II) ions in the formula unit.     

Purification and Characterization of Hydroxyquinol 1,2-Dioxygenase from Azotobacter sp. Strain GP1

Hydroxyquinol 1,2-dioxygenase was purified from cells of the soil bacterium Azotobacter sp. strain GP1 grown with 2,4,6-trichlorophenol as the sole source of carbon. The presumable function of this dioxygenase enzyme in the degradative pathway of 2,4,6-trichlorophenol is discussed. The enzyme was highly specific for 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene) and hydroxyquinol (1,2,4-trihydroxybenzene) and was found to perform ortho cleavage of the hydroxyquinol compounds, yielding chloromaleylacetate and maleylacetate, respectively. With the conversion of 1 mol of 6-chlorohydroxyquinol, the consumption of 1 mol of O(inf2) and the formation of 1 mol of chloromaleylacetate were observed. Catechol was not accepted as a substrate. The enzyme has to be induced, and no activity was found in cells grown on succinate. The molecular weight of native hydroxyquinol 1,2-dioxygenase was estimated to 58,000, with a sedimentation coefficient of 4.32. The subunit molecular weight of 34,250 indicates a dimeric structure of the dioxygenase enzyme. The addition of Fe(sup2+) ions significantly activated enzyme activity, and metal-chelating agents inhibited it. Electron paramagnetic resonance data are consistent with high-spin iron(III) in a rhombic environment. The NH(inf2)-terminal amino acid sequence was determined for up to 40 amino acid residues and compared with sequences from literature data for other catechol and chlorocatechol dioxygenases.     

Efficacy and deficiencies of rapid biomonitoring in biodiversity conservation: a case study in South Africa

Rapid biomonitoring protocols, using biotic indices based on macroinvertebrate diversity to assess river ecosystem health, are widely used globally. Such quick assessment techniques are lauded for the rapid results obtained and the relatively easy protocol used to achieve an answer. However, do such quick assessments of water quality give enough information about ecosystems? Are important details being overlooked? When should a full faunal survey be used in preference? Important research programmes, including environmental impact studies, often misuse biomonitoring techniques, making influential management decisions using superficial, low-level data obtained using biomonitoring tools, inappropriate to address those management objectives. The value of using biomonitoring as a quick tool, versus a more detailed faunal assessment, is considered here. The assessment of teloganodid mayfly fauna occurring in South African rivers provides an example of the value of detailed studies versus superficial family level investigations, showing that a rapid biomonitoring approach should not be used as a shortcut when a more detailed survey is needed. Each situation should be assessed for its own merit in a given set of project circumstances. A checklist of criteria is presented, giving guidance on when rapid biomonitoring alone is valuable and when more detailed assessments would give a more relevant result.