Mitogenomic Analyses Place the Gharial (Gavialis gangeticus) on the Crocodile Tree and Provide Pre-K/T Divergence Times for Most Crocodilians

Based on morphological analyses, extant members of the order Crocodylia are divided into three families, Alligatoridae, Crocodylidae, and Gavialidae. Gavialidae includes one species, the gharial, Gavialis gangeticus. In this study we have examined crocodilian relationships in phylogenetic analyses of seven mitochondrial genomes that have been sequenced in their entirety. The analyses did not support the morphologically acknowledged separate position of the gharial in the crocodilian tree. Instead the gharial joined the false gharial (Tomistoma schlegelii) on a common branch that was shown to constitute a sister group to traditional Crocodylidae (less Tomistoma). Thus, the analyses suggest the recognition of only two Crocodylia families, Alligatoridae and Crocodylidae, with the latter encompassing traditional Crocodylidae plus Gavialis/Tomistoma. A molecular dating of the divergence between Alligatoridae and Crocodylidae suggests that this basal split among recent crocodilians took place ≈140 million years before present, at the Jurassic/Cretaceous boundary. The results suggest that at least five crocodilian lineages survived the mass extinction at the KT boundary. [Reviewing Editor: Dr. Nicolas Galtier]     

Effects of common atopy-associated amino acid substitutions in the IL-4 receptor alpha chain on IL-4 induced phenotypes

The human IL-4 receptor alpha chain gene (IL4R) is highly polymorphic and controversial reports have been published with respect to the association of different single nucleotide polymorphisms (SNPs) with atopy markers. Here we analyzed the functional and associational relevance of common IL4R coding SNPs. Transfection of B cell lines expressing the IL-4R variant V75+R576 did not result in enhanced IL-4 induced CD23 expression compared to cell lines expressing the wild type IL-4R alpha chain. Transfection of the IL-4R variant P503 into a murine T cell line did not influence IL-4 induced T-cell proliferation compared to wild type constructs. Analysis of six IL4R coding SNPs (I75V, E400A, C431R, S436L, S503P, Q576R) and common haplotypes (frequency 0.05%) in blood donors (n=300) did not indicate a significant association with elevated serum IgE level. Moreover, the most informative IL4R coding SNPs (I75V, C431R, Q576R) and related two- and three-point haplotypes (frequency 0.05%) were analyzed in a second, extended group of blood donors (n=689). Again, no significant association with elevated serum IgE was detectable. We conclude that common coding SNPs in the IL4R gene are unlikely to contribute significantly to increased IgE levels and variations outside the coding region may influence atopy susceptibility.     

Phase I and II metabolism and carbohydrate metabolism in cultured cryopreserved porcine hepatocytes

Primary porcine hepatocytes were cryopreserved using freezing boxes or a programmable freezer (PF). Upon thawing and culturing in 12-well plates cryopreserved hepatocytes were compared with their fresh controls on days 1 and 2 after plating. Cryopreserved hepatocytes attached approximately as well as fresh hepatocytes and useful cultures were obtained. In cryopreserved hepatocytes, coumarin 7-hydroxylation, 6beta-testosterone hydroxylation and p-nitrophenol glucuronidation were reduced to about 10-40, 35 and 40%, respectively, compared to their fresh counterparts. Glycogen synthesis in cryopreserved hepatocytes was reduced to about 30% on day 1 of culture and about 47% on day 2 of culture compared to the synthesis in fresh hepatocytes. Both fresh and cryopreserved hepatocytes increased the synthesis by twofold in response to stimulation with insulin. Reduced basal levels of glycogen and of glycogen synthesis could be explained by an increased energy demand in cryopreserved hepatocytes needing to repair damages caused by cryopreservation. Glycogenolysis was reduced to about 50% in cryopreserved hepatocytes and gluconeogenesis to about 40% of the glucose production in fresh hepatocytes. In both fresh and cryopreserved hepatocytes the glucose production from glycogenolysis and gluconeogenesis, respectively, was increased fourfold in response to stimulation with glucagon. Overall, the hepatocytes cryopreserved in boxes had a tendency to perform better than hepatocytes cryopreserved in a programmable freezer. In conclusion, the cryopreserved hepatocytes were metabolic active; however, to a lower extent than the fresh hepatocytes, although, the cryopreserved hepatocytes responded as well as the fresh hepatocytes to insulin and glucagon.     

Caveolae: stable membrane domains with a potential for internalization

The role of caveolae in endocytosis is hotly debated. Here, we argue that most caveolae are stable microdomains at the cell surface. Only a small fraction of caveolae is constitutively internalized, leading to a quantitatively minor uptake of ligands and receptors. In addition, we suggest that a more pronounced downregulation of caveolae from the plasma membrane can occur, presumably stimulated by receptor cross-linking and clustering in caveolae. Finally, we propose that future studies dealing with internalization of caveolae should actually document such internalization and include kinetic data.     

Genetic and environmental influences of surfactant protein D serum levels

The collectin surfactant protein D (SP-D) is an important component of the pulmonary innate immune system, but SP-D is also present on extrapulmonary epithelial surfaces and in serum, where it has been used as a biomarker for pulmonary disease states. In this study, we investigate the mechanisms defining the constitutional serum level of SP-D and determine the magnitude of the genetic contribution to serum SP-D in the adult population. Recent studies have demonstrated that serum SP-D concentrations in children are genetically determined and that a single nucleotide polymorphism (SNP) located in the NH(2)-terminal region (Met11Thr) of the mature protein is significantly associated with the serum SP-D levels. A classic twin study was performed on a twin population including 1,476 self-reported healthy adults. The serum SP-D levels increased with male sex, age, and smoking status. The intraclass correlation was significantly higher for monozygotic (MZ) twin pairs than for dizygotic (DZ) twin pairs. Serum SP-D variance was influenced by nonshared environmental effects and additive genetic effects. Multivariate analysis of MZ and DZ covariance matrixes showed significant genetic correlation among serum SP-D and metabolic variables. The Met11Thr variant explained a significant part of the heritability indicating that serum SP-D variance could be decomposed into non-shared environmental effects (e(2) = 0.19), additive genetic effects (h(2) = 0.42), and the effect of the Met11Thr variations (q(2) = 0.39).     

Effects of Lactococcus lactis on Composition of Intestinal Microbiota: Role of Nisin

This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing and the non-nisin-producing L. lactis strains significantly increased the number of Bifidobacterium cells in fecal samples during the first 8 days but decreased the number of enterococci/streptococci in duodenum, ileum, cecum, and colon samples as detected by selective cultivation. No significant changes in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two L. lactis strains were different from that of control animals dosed with saline. However, profiles of the microbiota from animals dosed with nisin did not differ from the controls. The concentrations of nisin estimated by competitive enzyme-linked immunosorbent assay (ELISA) were approximately 10-fold higher in the small intestine and 200-fold higher in feces than the corresponding concentrations estimated by a biological assay. This indicates that nisin was degraded or inactivated in the gastrointestinal tract, since fragments of this bacteriocin are detected by ELISA while an intact molecule is needed to retain biological activity.     

Photochemical internalization (PCI) of HER2-targeted toxins: Synergy is dependent on the treatment sequence

Background

Photochemical internalization (PCI) is a modality for cytosolic release of drugs trapped in endocytic vesicles. The method is based upon photosensitizers localized in the membranes of endocytic vesicles which create membrane rupture upon light exposure by generating reactive oxygen species (ROS), predominantly singlet oxygen (¹O₂).

Methods

The human epidermal growth factor receptor 2 (HER2)-targeted immunotoxin (IT), trastuzumab–saporin, was evaluated in combination with PCI using TPCS_2a (Amphinex®), a new photosensitizer approved for clinical use.

Results

PCI synergistically enhanced the cytotoxicity of trastuzumab–saporin on trastuzumab-resistant HER2⁺ Zr-75-1 cells. The PCI effect was only observed when the IT was administered prior to the photochemical treatment (“light after” strategy), while administration of a non-targeted drug may equally well be performed after light exposure. Mechanistic studies showed reduced ligand-induced HER2 phosphorylation and receptor-mediated endocytosis after TPCS_2a-PDT. Photochemical disruption of the cytoplasmic domain of HER2 was found to be induced by ¹O₂ generated both by photosensitizer located in the endocytic vesicles and in the outer leaflet of the plasma membrane.

Conclusions

Administration of the HER2-targeted toxin prior to light exposure is a prerequisite for successful PCI-mediated delivery of HER2-targeted toxins.

General significance

PCI of HER2-targeted toxins is demonstrated as a highly effective treatment modality which may overcome trastuzumab resistance. The mechanistic studies of the lack of PCI effect of the “light first” procedure is of outermost importance when designing a clinical PCI treatment protocol for delivery of HER2-targeted therapies.