Isomerization in the CDR2 of a monoclonal antibody: Binding analysis and factors that influence the isomerization rate

Isomerization of a monoclonal antibody is one of the common routes of protein degradation. An isomerization in the complementarity‐determining region (CDR) was found previously and is investigated in depth in this work. Affinity analysis proves that the antibody with one isomerized heavy chain has lower binding. Binding constants were determined, and exhibited a slower on‐rate in conjunction with a faster off‐rate for this isomerization. To determine the role of the buffer on the rate of isomerization, this antibody was incubated in various matrices and the amount of isomerized antibody was determined by hydrophobic interaction chromatography (HIC). The rate was found to be dependent on the pH as well as the net negative charge of the buffer components that can act as proton acceptors. An Arrhenius plot was performed to predict the levels of isomerization and a comparison of real samples proved the model was correct. This work affirms that isomerization in the CDR of a therapeutic antibody is important to monitor and the formulation buffer plays a significant role in the rate of the isomerization. Biotechnol. Bioeng. 2010; 105: 515–523. © 2009 Wiley Periodicals, Inc.     

Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase

The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.     

Zoospore encystment and pathogenicity of Phytophthora and Pythium species on plant roots

Seven plant species (lucerne, maize, oat, sugarbeet, sorghum, tomato, wheat) and 12 Pythium and Phytophthora species were used in a comparative study designed to investigate the effects of plant and oomycete inter-specific variation on zoospore encystment density and pathogenicity. Zoospores showed differential encystment behaviour and they encysted more on dicotyledonous than on monocotyledonous plants. Pythium aphanidermatum, P. deliense, and Phytophthora nicotianae were the most aggressive species. Sugarbeet was the most severely attacked plant species followed by tomato while oat plants were relatively unaffected. The relationship between zoospore encystment on roots and disease severity depended on the oomycete-plant combination. Correlation analysis between zoospore encystment density and disease severity indicated low and no significant levels (p.05) of association for most plant-oomycete combinations.     

Trichinella spiralis infections of inbred mice: genetics of the host response following infection with different Trichinella isolates

The immune response of inbred strains of mice was studied following infection with isolates of Trichinella from a pig (P1), an arctic fox (AF1), and T. spiralis var. pseudospiralis (TP). Strains of mice previously characterized as highly resistant to a separate pig isolate of T. spiralis responded to the P1 and AF1 isolates by expelling over 80% of the worms by day 10 postinfection (PI), and by suppressing the in vitro release of newborn larvae by female worms. However, the response induced by AF1 worms was expressed more quickly when compared to responses induced by the P1 and TP isolates. The host response to TP was less as recovery was always higher at day 10 PI and antifecundity effects were not induced in TP worms even in highly resistant strains of mice. Strains of mice previously characterized as susceptible to T. spiralis infection were slow to develop resistance when compared to the resistant mouse strains, but even among the susceptible strains, infection with AF1 induced a more rapid response. The mouse strains used in these experiments allowed us to assess the role of the major histocompatibility complex (MHC) and/or non-MHC genes in influencing the responses observed. As previously reported for a pig isolate of T. spiralis, both MHC and non-MHC genes influenced the rate at which worms were expelled from the gut and the host response that limits the fecundity of adult female worms.     

Time‐lapse cameras reveal latitude and season influence breeding phenology durations in penguins

Variation in the phenology of avian taxa has long been studied to understand how a species reacts to environmental changes over both space and time. Penguins (Sphenicidae) serve as an important example of how biotic and abiotic factors influence certain stages of seabird phenology because of their large ranges and the extreme, dynamic conditions present in their Southern Ocean habitats. Here, we examined the phenology of gentoo (Pygoscelis papua) and chinstrap penguins (Pygoscelis antarctica) at 17 sites across the Scotia arc, including the first documented monitoring of phenology on the South Sandwich Islands, to determine which breeding phases are intrinsic, or rather vary across a species range and between years. We used a novel method to measure seabird breeding phenology and egg and chick survival: time‐lapse cameras. Contrary to the long‐standing theory that these phases are consistent between colonies, we found that latitude and season had a predominant influence on the length of the nest establishment, incubation, and guard durations. We observe a trend toward longer incubation times occurring farther south, where ambient temperatures are colder, which may indicate that exposure to cold slows embryo growth. Across species, in colonies located farther south, parents abandoned nests later when eggs were lost or chicks died and the latest record of eggs or chicks in the nest occurred earlier during the breeding period. The variation in both space and time observed in penguin phenology provides evidence that the duration of phases within the annual cycle of birds is not fundamental, or genetic, as previously understood. Additionally, the recorded phenology dates should inform field researchers on the best timing to count colonies at the peak of breeding, which is poorly understood.     

Three regions of the pRB pocket domain affect its inactivation by human papillomavirus E7 proteins

A critical event in papillomavirus transformation of human cells is the inactivation of pRB by the E7 protein. E7, like many other viral oncoproteins, possesses a well-characterized LXCXE peptide motif that interacts with the pocket domain of pRB. Disruption of the LXCXE-binding cleft on pRB renders it resistant to E7 binding and inactivation. Such binding cleft mutants of pRB are capable of inducing a G(1) arrest in the human papillomavirus 18-transformed HeLa cell line. We show here that the efficient inactivation of pRB in HeLa cells does not simply depend on the integrity of the LXCXE-binding cleft. Multiple site-directed mutants that alter conserved surfaces of the pRB pocket domain cause HeLa cells to accumulate in G(1). We divide these mutants into two classes: those that can be bound by E7 and those that cannot. The E7 interacting mutants include changes in conserved residues that lie in a groove between the A and B halves of the pocket. Surprisingly, none of these mutants show a clear defect in any of the known mechanisms for pRB inactivation by E7. Analysis of mutants that are compromised for E7 binding reveals that this interaction depends on both the LXCXE-binding cleft and on a conserved group of lysines adjacent to the cleft. These basic amino acids on pRB define a discrete interaction point with E7. These residues most likely form ionic interactions with conserved acidic amino acids on E7 since a stable pRB/E7 interaction was restored when the lysine residues on pRB and the acidic residues on E7 were interchanged.     

Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development

A highly efficient and synchronousin vitro tuberization system is described. One-node stem pieces from potato (Solanum tuberosum cv. Bintje) plants grown under short day-light conditions containing an axillary bud were cultured in the dark on a tuber-inducing medium. After 5 or 6 days all axillary buds started to develop tubers. To study gene expression during tuber development, RNA isolated from tuberizing axillary buds was used for bothin vitro translation and northern blot hybridizations. The genes encoding the proteinase inhibitors I and II (PI-I and PI-II), a Kunitz-and a Bowman-Birk-type proteinase inhibitor were already expressed in uninduced axillary buds. The length of the day-light conditions differently influenced the expression level of the individual genes. In addition, the expression of each of these genes changed specifically during the development of the axillary bud to tuber. In contrast to the expression of these proteinase inhibitor genes, patatin gene expression was only detectable from the day tuberization was manifested as a radial expansion of the axillary bud.These results are discussed with respect to the regulation of the expression of the genes studied in relation to the regulation of tuber development.     

Do Treatment Quality Indicators Predict Cardiovascular Outcomes in Patients with Diabetes?

Background

Landmark clinical trials have led to optimal treatment recommendations for patients with diabetes. Whether optimal treatment is actually delivered in practice is even more important than the efficacy of the drugs tested in trials. To this end, treatment quality indicators have been developed and tested against intermediate outcomes. No studies have tested whether these treatment quality indicators also predict hard patient outcomes.

Methods

A cohort study was conducted using data collected from >10.000 diabetes patients in the Groningen Initiative to Analyze Type 2 Treatment (GIANTT) database and Dutch Hospital Data register. Included quality indicators measured glucose-, lipid-, blood pressure- and albuminuria-lowering treatment status and treatment intensification. Hard patient outcome was the composite of cardiovascular events and all-cause death. Associations were tested using Cox regression adjusting for confounding, reporting hazard ratios (HR) with 95% confidence intervals.

Results

Lipid and albuminuria treatment status, but not blood pressure lowering treatment status, were associated with the composite outcome (HR = 0.77, 0.67–0.88; HR = 0.75, 0.59–0.94). Glucose lowering treatment status was associated with the composite outcome only in patients with an elevated HbA1c level (HR = 0.72, 0.56–0.93). Treatment intensification with glucose-lowering but not with lipid-, blood pressure- and albuminuria-lowering drugs was associated with the outcome (HR = 0.73, 0.60–0.89).

Conclusion

Treatment quality indicators measuring lipid- and albuminuria-lowering treatment status are valid quality measures, since they predict a lower risk of cardiovascular events and mortality in patients with diabetes. The quality indicators for glucose-lowering treatment should only be used for restricted populations with elevated HbA1c levels. Intriguingly, the tested indicators for blood pressure-lowering treatment did not predict patient outcomes. These results question whether all treatment indicators are valid measures to judge quality of health care and its economics.     

13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h(-1) yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.