Phylogenetic analyses and expression studies reveal two distinct groups of calreticulin isoforms in higher plants

Calreticulin (CRT) is a multifunctional protein mainly localized to the endoplasmic reticulum in eukaryotic cells. Here, we present the first analysis, to our knowledge, of evolutionary diversity and expression profiling among different plant CRT isoforms. Phylogenetic studies and expression analysis show that higher plants contain two distinct groups of CRTs: a CRT1/CRT2 group and a CRT3 group. To corroborate the existence of these isoform groups, we cloned a putative CRT3 ortholog from Brassica rapa. The CRT3 gene appears to be most closely related to the ancestral CRT gene in higher plants. Distinct tissue-dependent expression patterns and stress-related regulation were observed for the isoform groups. Furthermore, analysis of posttranslational modifications revealed differences in the glycosylation status among members within the CRT1/CRT2 isoform group. Based on evolutionary relationship, a new nomenclature for plant CRTs is suggested. The presence of two distinct CRT isoform groups, with distinct expression patterns and posttranslational modifications, supports functional specificity among plant CRTs and could account for the multiple functional roles assigned to CRTs.     

Basal metabolic rate and the evolution of the adaptive immune system

Vertebrates have evolved an adaptive immune system in addition to the ancestral innate immune system. It is often assumed that a trade-off between costs and benefits of defence governs the evolution of immunological defence, but the costs and benefits specific to the adaptive immune system are poorly known. We used genetically engineered mice lacking lymphocytes (i.e. mice without adaptive, but with innate, immunity) as a model of the ancestral state in the evolution of the vertebrate immune system. To investigate if the magnitude of adaptive defence is constrained by the energetic costs of producing lymphocytes etc., we compared the basal metabolic rate of normal and lymphocyte-deficient mice. We found that lymphocyte-deficient mice had a higher basal metabolic rate than normal mice with both innate and adaptive immune defence. This suggests that the evolution of the adaptive immune system has not been constrained by energetic costs. Rather, it should have been favoured by the energy savings associated with a combination of innate and adaptive immune defence.     

On Reciprocal Causation in the Evolutionary Process

Recent calls for a revision of standard evolutionary theory (SET) are based partly on arguments about the reciprocal causation. Reciprocal causation means that cause–effect relationships are bi-directional, as a cause could later become an effect and vice versa. Such dynamic cause-effect relationships raise questions about the distinction between proximate and ultimate causes, as originally formulated by Ernst Mayr. They have also motivated some biologists and philosophers to argue for an Extended Evolutionary Synthesis (EES). The EES will supposedly expand the scope of the Modern Synthesis (MS) and SET, which has been characterized as gene-centred, relying primarily on natural selection and largely neglecting reciprocal causation. Here, I critically examine these claims, with a special focus on the last conjecture. I conclude that reciprocal causation has long been recognized as important by naturalists, ecologists and evolutionary biologists working in the in the MS tradition, although it it could be explored even further. Numerous empirical examples of reciprocal causation in the form of positive and negative feedback are now well known from both natural and laboratory systems. Reciprocal causation have also been explicitly incorporated in mathematical models of coevolutionary arms races, frequency-dependent selection, eco-evolutionary dynamics and sexual selection. Such dynamic feedback were already recognized by Richard Levins and Richard Lewontin in their bok The Dialectical Biologist. Reciprocal causation and dynamic feedback might also be one of the few contributions of dialectical thinking and Marxist philosophy in evolutionary theory. I discuss some promising empirical and analytical tools to study reciprocal causation and the implications for the EES. Finally, I briefly discuss how quantitative genetics can be adapated to studies of reciprocal causation, constructive inheritance and phenotypic plasticity and suggest that the flexibility of this approach might have been underestimated by critics of contemporary evolutionary biology.     

Formaldehyde sensitivity of a GFAP epitope, removed by extraction of the cytoskeleton with high salt

We have used cytofluorometry to examine the formaldehyde sensitivity of the binding of a monoclonal antibody (MAB) to its epitope on glial fibrillary acidic protein in human malignant glioma cells in culture. When acetone-extracted whole cells or cytoskeletons, made by extracting with Triton in stabilizing buffer (Tsb), are fixed with formaldehyde, binding of the MAB Tp-GFAP1 to GFAP is abolished or greatly reduced. Fixation with the bifunctional protein crosslinking reagent dithiobis (succinimidyl propionate) (DTSP) has the same negative effect as formaldehyde. If cytoskeletons are further extracted with Tsb containing 250 mM ammonium sulfate (Thsb), fixation with formaldehyde or DTSP has reduced or no effect on the binding of Tp-GFAP1. The data are consistent with the hypothesis that aldehyde sensitivity of Tp-GFAP1 is caused by the crosslinking of a second protein to GFAP that blocks the binding of the MAB to its epitope. This putative blocking protein is part of the Triton-insoluble cytoskeleton, but it begins to be solubilized in 50 mM ammonium sulfate and it is largely removed in 250 mM ammonium sulfate (Thsb). SDS-PAGE shows that extraction with Thsb also removes a large number of proteins from the cytoskeleton, one of which could be the blocking protein. A second antibody to GFAP, designated Tp-GFAP3, was raised against cytoskeletons which had been fixed with DTSP and in which the epitope recognized by Tp-GFAP1 was presumably blocked. Tp-GFAP3 is not sensitive to fixation by either formaldehyde or DTSP.     

T15 group A streptococcal Fc receptor binds to the same location on IgG as staphylococcal protein A and IgG rheumatoid factors

Previous work has shown that IgG rheumatoid factors (RF) bind to the C gamma 2-C gamma 3 interface region of human IgG in the same area that binds staphylococcal protein A (SPA). Group A, C, and G strains of Streptococci possess Fc receptors that bind to IgG but not to fragments containing only the C gamma 2 or C gamma 3 domains. This work describes the binding site location on human IgG for the binding of the isolated Fc receptor from the T15 strain of a Group A streptococcus and its relationship to the site that binds SPA and the IgG RF. The isolated T15 Fc receptor (T15) with a molecular mass of 29.5 kD inhibited the binding of IgG RF to IgG. The binding of T15 itself to IgG was strongly inhibited by SPA (42.0 kD) and its monovalent fragment D (7 kD). Human IgG fragments consisting of the C gamma 3 domains did not inhibit the binding of T15 to IgG, whereas those with both domains were effective inhibitors. T15 did not bind to rabbit IgG fragments consisting of either the C gamma 2 or C gamma 3 domains, but did bind to those with both domains. An IgG3 myeloma protein was a poor inhibitor and has been shown to bind poorly to the IgG RF. Most IgG3 myeloma proteins did not bind to SPA. The substitution of Arg and Phe for His 435 and Tyr 436 is responsible for the poor binding of IgG3 to SPA and to the IgG RF. Chemical modification of His or Tyr on IgG reduced its ability to inhibit the binding of T15 to IgG. Reversal of the chemical modifications with hydroxylamine resulted in near complete restoration of inhibitory capacity. This information, collectively, coupled with the known positions in space of the His and Tyr residues in the C gamma 2-C gamma 3 interface region, verified that both His 435 and Tyr 436, and possibly His 310 and 433, are involved. These residues are also involved in binding SPA and the IgG RF. These data therefore indicate that the T15 Group A Streptococcal Fc receptor binds to the same location on the Fc of IgG as SPA and the IgG RF. The biologic relevance of these similarities between bacterial cell wall Fc receptors and IgG RF are not yet apparent, but suggest that RF could bear the internal image of these bacterial structures.     

Synthesis of Galβ1-3GlcNAc and Galβ1-3GlcNAcβ-SEt by an enzymatic method comprising the sequential use of β-galactosidases from bovine testes andEscherichia coli

Gal1-3GlcNAc (1) and Gal1-3GlcNAc-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes -galactosidase with lactose as donor andN-acetylglucosamine and GlcNAc-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with -galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal1-6GlcNAc-SEt (3) was synthesized by the transglycosylation reaction using -galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.     

Entry of adenovirus 2 into HeLa cells

Adenovirus 2 (Ad2) uncoating was analyzed as the destabilization of virions which renders the parental genome sensitive to DNase treatment. This event demonstrated a strong temperature dependence, and an Arrhenius plot of initial uncoating rates revealed an inflection point at around 16 degrees C. Activation energies of 331 kJ/mol below and 88 kJ/mol above this temperature were obtained for the uncoating process. Penetration of Ad2 through the plasma membrane was completely inhibited by sodium azide, whereas uncoating was only slightly influenced. This indicated that uncoating had already taken place at the outside of the plasma membrane. Incubations of Ad2 with isolated plasma membranes and cell homogenates showed that intact and metabolizing cells were required for uncoating. We further suggest, based on the inhibitory patterns of EDTA, EGTA, dansylcadaverine, and dithiothreitol, that this destabilization of virions follows upon reorganization in the plasma membrane. In the electron microscope the involvement of coated vesicles was shown for the initial uptake of virions, possibly followed by the engagement of acidic vesicles as judged from the effects of lysosomotropic agents on gene expression. The vectorial transport of virions from the plasma membrane to the nucleus was not affected by reagents interfering with the cytoskeletal system. Consequently, we propose that Ad2 virions are internalized by adsorptive endocytosis.