Enhanced reactive lysis of paroxysmal nocturnal hemoglobinuria erythrocytes by C5b-9 does not involve increased C7 binding or cell-bound C3b

The most complement (C)-sensitive type of erythrocytes (E) occurring in paroxysmal nocturnal hemoglobinuria (type III PNH E) have previously been found to exhibit approximately twofold to fourfold greater lysis than normal human E when exposed to isolated human C5b6, C7, C8, and C9 (reactive lysis), in the absence of a known source of C3- or C5-convertases or fluid-phase C3. In further studies on the mechanism of this phenomenon, we now report that C5b6-dependent binding of 125I-C7 to two samples of PNH E (greater than 95% type III) is equal to that found with normal human E at each of several C5b6 inputs tested. Lysis developed by excess C8 and C9, however, was consistently greater for the PNH E. Thus, the exaggerated sensitivity of type III PNH E to reactive lysis cannot be explained by abnormally high uptake of C5b6 or C7 from the fluid phase. Rather, the data indicate that cell-bound C5b67 sites are converted to effective hemolytic sites with greater efficiency on type III PNH E than on normal human E, assuming that the distribution of cell-bound C7 throughout both cell populations is similar. In related studies we have addressed the proposal by other investigators that C3b putatively bound to PNH E in vivo might account for their increased sensitivity to reactive lysis in vitro, by analogy to prior observations on C3b-potentiated reactive lysis of sheep E. The latter hypothesis was made more appealing by the recent discovery that type III PNH E lack an integral membrane protein, decay-accelerating factor (DAF), which in normal E accelerates the decay of membrane-bound C3 convertases. Against this hypothesis, however, is our present finding that preincubation of PNH E with four different goat or rabbit polyclonal antibodies to human C3 failed to inhibit the subsequent reactive lysis of these cells. Under these same conditions, the C3b-dependent increment in reactive lysis of sheep EAC4b3b was abrogated by pretreatment with similar dilutions of these anti-C3 antibodies, generally in association with agglutination. Furthermore, sheep EAC4b3b displayed increased 125I-C7 binding in proportion to augmented lysis, in contrast to the findings with PNH E. Therefore, deficiency of DAF in type III PNH E does not adequately explain their supranormal sensitivity to reactive lysis unless DAF can modulate the terminal lytic steps by a mechanism distinct from its effect on C3 convertase decay. Alternatively, type III PNH E could have a more general abnormality in which DAF deficiency is one manifestation and increased sensitivity to reactive lysis is another.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Effects of combined water potential and temperature stresses on Cryptosporidium parvum oocysts.

Hosts infected with the parasite Cryptosporidium parvum may excrete oocysts on soils in watersheds that supply public drinking water. Environmental stresses decrease the numbers of oocysts after deposition on soils. However, the rates and effects of combined stresses have not been well characterized, especially for the purposes of estimating decrease in numbers. We subjected oocysts to combined stresses of water potential (-4, -12, and -33 bars), above-freezing temperatures (4 and 30 degrees C), and a subfreezing temperature (-14 degrees C) for 1, 14, and 29 days and one to six freeze-thaw cycles (-14 to 10 degrees C) to estimate coefficients to characterize population degradation using multiplicative error and exponential decay models. The experiments were carried out in NaCl solutions with water potentials of -4, -12, and -33 bars, in combination with temperature stresses at levels that could be expected in natural soils. Increased water potential increased the rate of population degradation for all temperature conditions investigated. Enhanced degradation leads to estimated rates of population degradation that are greater than those that have been reported and used in previous studies conducted to assess risk of water supply contamination from sources of C. parvum.     

Generation and characterization of a unique reagent that recognizes a panel of recombinant human monoclonal antibody therapeutics in the presence of endogenous human IgG

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This “panel-specific” feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.     

Palatability and feeding preferences of Uresiphita maorialis (Lepidoptera: Crambidae) for three Sophora species

In a three-hour bioassay, we tested the palatability and feeding preferences of Uresiphita maorialis (kōwhai moth) for Sophora tetraptera, Sophora microphylla and Sophora prostrata. Palatability tests showed no differences among the Sophora species. Feeding preferences, on the other hand, showed that S. tetraptera and S. microphylla leaves are preferred over S. prostrata leaves. Our results support our field observations in Wellington city parks and gardens showing that S. tetraptera and S. microphylla plants frequently have higher densities of larvae than S. prostrata.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

The role of fish-poultry integration on fish growth performance,yields and economic benefits among smallholder farmers in sub-Saharan Africa,Tanzania

Aquaculture practices from sub-Saharan Africa are characterised by low production, owing to improper technology. Production can be increased through integrating fish farming with other existing on-farm activities, particularly livestock husbandry. We assessed the role of fish-poultry integration on all male Nile tilapia, Oreochromis niloticus growth performance, yields and economic benefits among smallholder farmers in sub-Saharan Africa, Tanzania. The study also compared phytoplankton species composition, abundance and biomass between the fish-poultry integration and non-integrated system. After 180 days of the experiment, all male O. niloticus cultured under fish-poultry integration exhibited significantly higher growth rates than those in the non-integrated system (p < 0.05). Gross fish yield (GFY), net fish yield (NFY) and net annual yields (NAY) obtained from fish-poultry integration were significantly higher than those from non-integrated system (p < 0.05). Partial enterprise budget analysis revealed that fish-poultry integration was more profitable than the non-integrated system. Moreover, fish-poultry integrated system produced significantly higher phytoplankton abundance and biomass than those from the non-integrated system. Results demonstrate that rural smallholder farmers can achieve higher growth rate, farm net yields and income by integrating all male O. niloticus with other on-farm activities than practising a stand-alone fish culture system.     

Sutural loosening and skeletal flexibility during growth: determination of drop-like shapes in sea urchins

The shape of sea urchins may be determined mechanically by patterns of force analogous to those that determine the shape of a water droplet. This mechanical analogy implies skeletal flexibility at the time of growth. Although comprised of many rigid calcite plates, sutural collagenous ligaments could confer such flexibility if the sutures between plates loosened and acted as joints at the time of growth. We present experimental evidence of such flexibility associated with weight gain and growth. Over 13-, 4-, and 2-week periods, fed urchins (Strongylocentrotus droebachiensis) gained weight and developed looser sutures than unfed urchins that maintained or lost weight. Further, skeletons of fed urchins force-relaxed more than did those of unfed urchins and urchins with loose sutures force-relaxed more than those with tight sutures. Urchins (Strongylocentrotus franciscanus) fed for two and a half weeks, gained weight, also had looser skeletons and deposited calcite at sutural margins, whereas unfed ones did not. In field populations of S. droebachiensis the percentage having loose sutures varied with urchin diameter and reflected their size-specific growth rate. The association between feeding, weight gain, calcite deposition, force relaxation and sutural looseness supports the hypothesis that urchins deform flexibly while growing, thus determining their drop-like shapes.