Characterisation of an industrial affinity process used in the manufacturing of digoxin-specific polyclonal Fab fragments

This paper describes the effect of several variables on the affinity process for the production of the FDA approved biotherapeutic product Digoxin Immune Fab (Ovine) (DigiFab, Protherics Inc., TN, USA). The study considers the effects of column re-use on matrix capacity and on the subsequent recovery of the antibody product, and the impact of varying column loading on matrix performance. The methodology used could be equally applied to assess the feasibility of using an affinity matrix for commercial scale purification of alternative antibody derived biotherapeutics. The capacity and specific Fab recovery were calculated through 24h equilibrium and mass balance studies. Results were assessed against data obtained through confocal scanning laser microscopy. Scale-down experiments produced specific Fab recoveries and purities that were comparable with those at production scale. The matrix capacity was found to be 45+/-15 mg of Fab/ml of matrix. Through the use of fluorescent DigiFab and confocal scanning techniques, Fab uptake onto single affinity bead was evaluated. Average intensity values calculated for each sample provided direct real-time, measure of Fab binding and matrix capacity. The results suggest that the affinity matrix had a limited reuse life as a drop in recovery is observed following the completion of a small number of process cycles (30% after three runs). The findings support that which is seen at the current manufacturing scale, where the affinity column is used for a limited number of runs. Results from this study can be used as a basis for future optimisation of this purification process.     

Integrative Approach Uncovers New Patterns of Ecomorphological Convergence in Slow Arboreal Xenarthrans

Journal of Mammalian Evolution - Identifying ecomorphological convergence examples is a central focus in evolutionary biology. In xenarthrans, slow arboreality independently arose at least three...     

Individuality,kin similarity and experimental playback of contact calls in cooperatively breeding riflemen

Riflemen/tītipounamu (Acanthisitta chloris) are kin-based cooperatively breeding birds, which appear able to recognise their relatives. Here, we investigate the potential for vocalisations to act as recognition cues in riflemen. We identified an appropriate contact call and recorded it at the nest from 19 adult riflemen. Measurements of call characteristics were individually repeatable. In addition, call similarity was significantly correlated with relatedness among all birds and among males. Thus, in principle, these contact calls contain sufficient information for individual recognition of familiar kin, and some assessment of relatedness between unfamiliar birds. To test whether riflemen responded differently to calls of kin, we broadcast calls of relatives and non-relatives as separate treatments in a playback experiment. Focal birds rarely responded aggressively or affiliatively, and their tendency to do so was unrelated to treatment. We conclude that zip calls are suitable kin recognition cues, but whether they are used as such remains unknown.     

Longevity and life history of cave bears – a review and novel data from tooth cementum and relative emergence of permanent dentition

Abstract

Longevity and other life history variables are key to understanding evolutionary processes and the biology of extinct animals. For the past 20 years, the lifespan of cave bears received an increased interest. Studies focusing on incremental lines of tooth cementum resulted in detailed mortality patterns from different localities. In this review, we summarise literature on age estimation as well as mortality of different European cave bear localities and present novel data on longevity from 94 teeth originating from 20 European localities. Additionally, the relative tooth emergence pattern of the permanent dentition is investigated under the Schultz’s rule framework of possible life history implications. For this, the known sequences of extant bear species are compared with the one of cave bears. Our results suggest that the typical duration of the life of cave bears was 19 years but data from literature show that in rare cases ages of up to 30–32 years were achieved. Additionally, we present the oldest known age for the Middle Pleistocene cave bear Ursus deningeri, 29 years. The tooth eruption pattern of cave bears exhibits a heterochronic shift that implies, under the assumption of Schulz’ rule, a slightly faster life history than closely related species.     

Soil tillage reduces arthropod biodiversity and has lag effects within organic and conventional crop rotations

Crop rotation systems in organic and conventional farming systems differ in crop types, management and duration. However, changes in arthropod communities over the entire rotation system are poorly understood, as many studies have surveyed only single years or have not covered the entire rotation period. Here, we describe changes in arthropods in two contrasting systems at a split organic‐conventional farm: an 8‐year organically managed rotation with five crops and a 5‐year conventionally managed rotation with three crops. Arthropods were classified into three functional groups, representing epigeal predators, foliar predators/parasitoids and herbivores/pollinators. Epigeal predators were particularly reduced by soil tillage which occurred annually in the conventional rotation, but was intermittent in the organic. Arthropods were most abundant on the conventional rotation, but most taxonomically diverse on the organic. In the conventional system, all functional groups showed a cyclical change in their taxonomic composition that closely matched the crop rotation sequence, whereas in the organic rotation, the cycle was less clear. Whilst the current year's crop type was the major determinant of arthropod community composition, there was a significant “lag effect” for many taxa from the preceding year's crop. Our results suggest that both the amounts of soil tillage (e.g., in no‐till systems) and crop rotation order have major impacts on arthropods in agroecosystems. Rotations with excessive soil tillage are likely to reduce the abundance of some groups of beneficial arthropods, especially epigeal predators.     

Genotypic Differences of Candida albicans and C. dubliniensis Isolates Related to Ethnic/Racial Differences within the Same Geographic Area

Candida albicans and C. dubliniensis genotype differences among Israeli ethnic groups were assessed. Isolates from Jews (51), Arabs (35) and Druze (25) were genotyped. The distributions among ethnic groups were not different, however they differed (p = 0.002) from global populations. Therefore, C. albicans and C. dubliniensis genotype distribution differences in Israel are related to changes in all ethnic groups.     

Carbamoylphosphonate-based matrix metalloproteinase inhibitor metal complexes: solution studies and stability constants. Towards a zinc-selective binding group

Overactive matrix metalloproteinases (MMPs) are associated with a variety of disease states. Therefore, their inhibition is a highly desirable goal. Yet, more than a decade of worldwide activity has not produced even one clinically useful inhibitor. Because of the crucial role of zinc in the activity of the enzyme, the design of inhibitors is usually based upon a so-called zinc binding group (ZBG). Yet, many of the hitherto synthesized potent inhibitors failed clinically, presumably because they bind stronger to metals other than zinc. We have developed in vivo potent inhibitors based on the carbamoylphosphonic group as a putative ZBG. In this paper we report stability constants for Ca(II), Mg(II), Zn(II) and Cu(II) complexes of two potent, in vivo active, MMP inhibitors, cyclopentylcarbamoylphosphonic acid (1) and 2-(N,N-dimethylamino)ethylcarbamoylphosphonic acid (2). Precipitation prevented the determination of stability constants for iron(III) complexes of 1 and 2. For comparison with carbamoylphosphonates 1 and 2, we synthesized 2-cyclohexyl-1,1-difluoroethylphosphonic acid (3), which does not inhibit MMP, and determined the stability constants of its complexes with Mg(II), Ca(II) and Zn(II). Comparison with the values obtained from the complexes of 1 and 2 with those from 3 indicates participation of the C=O group in the metal binding of the former compounds. The complex stability orders for both 1 and 2 are Ca(II)<Mg(II)<Zn(II)<Cu(II). In addition, the results indicate that at pH>8 the dimethylamino group of compound 2 can also participate in the binding of the transition metals Cu and Zn. On the other hand, the amino group in carbamoylphosphonic acid 2 lowers the stability of the complexes with metals favoring oxygen ligands (Ca, Mg and Fe) and increases the selectivity towards Zn. These results are helpful for rationalizing the results observed on our MMP inhibitors hitherto examined, and are expected to be useful for the design of new selective inhibitors.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0524-5     

Serological Diagnosis of Human Babesiosis by IgG Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies to Babesia microti antigen was developed. B. microti antigens were harvested from experimentally infected hamster blood and used as a coating antigen. The sensitivity and specificity of the IgG ELISA relative to immunofluorescent antibody assay (IFA) testing was 95.5% and 94.1%, respectively. According to the receiver operating characteristic curve analysis, the area under the curve was 0.987. No cross-reactivity of serum samples collected from patients infected with Toxoplasma gondii, Borrelia burgdorferi, Anaplasma phagocytophilum, Bartonella quintana, Dengue virus, or West Nile virus was detected. Cross-reactivity was observed with one of 35 sera from patients infected with Bartonella henselae. These results indicate that the established ELISA methods could be utilized as an accurate measure for the clinical diagnosis of human babesiosis.     

δ15N as an indicator of N2-fixation by cyanobacterial mats in tropical marshes

Cyanobacterial mats (CBM) are important components of wetland ecosystems in limestone-based regions of the Caribbean. During two sampling periods (July 1999 and January 2000) we measured N₂-fixation in samples from 23 different marshes simultaneously with measurements of relevant environmental factors. Samples were evaluated for abundance of five groups of cyanobacteria: (1) Leptolyngbya, (2) Oscillatoria, (3) Chroococcales, (4) Nostoc-& Stigonematales, and (5) dead sheaths. Differences in nitrogen fixation, expressed as nitrogenase activity in nmol C₂H₄ cm^–2 h^–1, were best explained by the proportion of heterocyst-forming cyanobacteria. The samples were analyzed for the natural abundance of ¹⁵N. ¹⁵N values ranged from –1.99 to 11.44 and were strongly negatively correlated with N₂-fixation. With all data included, ¹⁵N was also strongly correlated with nitrates in water. With the samples from Little Belize (high nitrate content marshes) excluded, the effect of nitrate became insignificant. N₂-fixation predicted from ¹⁵N measured on an independent data set from September 2000 was moderately accurate (r² = 0.68, 0.52 and 0.54 for predictions based on July 1999, January 2000 and combined data sets, respectively). When individual sample sets were divided into two groups with ¹⁵N < 2 and ¹⁵N > 2, the two groups were always highly significantly different in terms of their N₂-fixation. The presented evidence suggests that ¹⁵N can be used as a reliable indicator of N₂-fixation by CBM.     

NHE3 inhibition activates duodenal bicarbonate secretion in the rat

We examined the effect of inhibition of Na+/H+ exchange (NHE) on duodenal bicarbonate secretion (DBS) in rats to further understand DBS regulation. DBS was measured by using the pH-stat method and by using CO2-sensitive electrodes. 5-(N,N-dimethyl)-amiloride (50 microM; DMA), a concentration that selectively inhibits the NHE isoforms NHE1 and NHE2, but not NHE3, did not affect DBS. Nevertheless, 3 mM DMA, a higher concentration that inhibits NHE1, NHE2, and NHE3, significantly increased DBS. Moreover, S1611 and S3226, both specific inhibitors of NHE3 only, or perfusion with Na+-free solutions, dose dependently increased DBS, as measured by pH-stat and CO2-sensitive electrode, without affecting intracellular pH. Coperfusion with 0.1 microM indomethacin, 0.5 mM DIDS, or 1 mM methazolamide did not affect S3226-induced DBS. Nevertheless, coperfusion with 0.1 and 0.3 mM 5-nitro-2-(3-phenylpropylamino) benzoic acid, which inhibits the cystic fibrosis transmembrane conductor regulator (CFTR), dose dependently inhibited S3226-induced DBS. In conclusion, only specific apical NHE3 inhibition increased DBS, whereas prostaglandin synthesis, Na+-HCO3- cotransporter activation, or intracellular HCO3- formation by carbonic anhydrase was not involved. Because NHE3 inhibition-increased DBS was inhibited by an anion channel inhibitor and because reciprocal CFTR regulation has been previously shown between NHE3 and apical membrane anion transporters, we speculate that NHE3 inhibition increased DBS by altering anion transporter function.