From Clinging to Digging: The Postembryonic Skeletal Ontogeny of the Indian Purple Frog,Nasikabatrachus sahyadrensis (Anura: Nasikabatrachidae)

The Indian Purple frog, Nasikabatrachus sahyadrensis, occupies a basal phylogenetic position among neobatrachian anurans and has a very unusual life history. Tadpoles have a large ventral oral sucker, which they use to cling to rocks in torrents, whereas metamorphs possess adaptations for life underground. The developmental changes that underlie these shifts in habits and habitats, and especially the internal remodeling of the cranial and postcranial skeleton, are unknown. Using a nearly complete metamorphic series from free-living larva to metamorph, we describe the postembryonic skeletal ontogeny of this ancient and unique monotypic lineage. The torrent-dwelling larva possesses a dorsoventrally flattened body and a head with tiny dorsal eyes, robust lower and upper jaw cartilages, well-developed trabecular horns, and a definable gap between the trabecular horns and the tip of the snout. Unlike tadpoles of many other frogs, those of Nasikabatrachus retain larval mouthparts into late metamorphic stages. This unusual feature enables the larvae to maintain their clinging habit until near the end of metamorphosis. The subsequent ontogenetic shift from clinging to digging is correlated with rapid morphological changes and behavioral modifications. Metamorphs are equipped with a shortened tibiafibula and ossified prehallical elements, which likely facilitate initial digging using the hind limbs. Subsequently, the frogs may shift to headfirst burrowing by using the wedge-shaped skull, anteriorly positioned pectoral girdle, well-developed humeral crests and spatula-shaped forelimbs. The transition from an aquatic life in torrents to a terrestrial life underground entails dramatic changes in skeletal morphology and function that represent an extreme in metamorphic remodeling. Our analysis enhances the scope for detailed comparative studies across anurans, a group renowned for the diversity of its life history strategies.     

The transmembrane domain of caveolin-1 exhibits a helix–break–helix structure

Caveolin is an integral membrane protein that is found in high abundance in caveolae. Both the N- and C- termini lie on the same side of the membrane, and the transmembrane domain has been postulated to form an unusual intra-membrane horseshoe configuration. To probe the structure of the transmembrane domain, we have prepared a construct of caveolin-1 that encompasses residues 96–136 (the entire intact transmembrane domain). Caveolin-1(96–136) was over-expressed and isotopically labeled in E. coli, purified to homogeneity, and incorporated into lyso-myristoylphosphatidylglycerol micelles. Circular dichroism and NMR spectroscopy reveal that the transmembrane domain of caveolin-1 is primarily α-helical (57–65%). Furthermore, chemical shift indexing reveals that the transmembrane domain has a helix–break–helix structure which could be critical for the formation of the intra-membrane horseshoe conformation predicted for caveolin-1. The break in the helix spans residues 108 to 110, and alanine scanning mutagenesis was carried out to probe the structural significance of these residues. Our results indicate that mutation of glycine 108 to alanine does not disrupt the structure, but mutation of isoleucine 109 and proline 110 to alanine dramatically alters the helix–break–helix structure. To explore the structural determinants further, additional mutagenesis was performed. Glycine 108 can be substituted with other small side chain amino acids (i.e. alanine), leucine 109 can be substituted with other β-branched amino acids (i.e. valine), and proline 110 cannot be substituted without disrupting the helix–break–helix structure.     

Increased Expression of the dsRNA-Activated Protein Kinase PKR in Breast Cancer Promotes Sensitivity to Doxorubicin

It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H₂O₂-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression/activation and eIF2α phosphorylation may be beneficial for the treatment of breast cancer.     

From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni

The gram-negative bacterium Campylobacter jejuni was recently discovered to contain a general N-linked protein glycosylation pathway. Central to this pathway is PglB, a homologue of the Stt3p subunit of the eukaryotic oligosaccharyl transferase (OT), which is involved in the transfer of an oligosaccharide from a polyisoprenyl pyrophosphate carrier to the asparagine side chain of proteins within the conserved glycosylation sites D/E-X1-N-X2-S/T, where X1 and X2 can be any amino acids except proline. Using a library of peptide substrates and a quantitative radioactivity-based in vitro assay, we assessed the amino acids at each position of the consensus glycosylation sequence for their impact on glycosylation efficiency, whereby the sequence DQNAT was found to be the optimal acceptor substrate. In the context of a full-length folded protein, the differences between variations of the glycosylation sequences were found to be consistent with the trends observed from their peptidyl counterparts, though less dramatic because of additional influences. In addition to characterizing the acceptor preferences of PglB, we also assessed the selectivity toward the glycan donor. Interestingly, despite recent reports of relaxed selectivity toward the glycan donor, PglB was not found to be capable of utilizing glycosyl donors such as dolichyl-pyrophosphate-chitobiose, which is the minimum substrate for the eukaryotic OT process.     

Early limb patterning in the direct‐developing salamander Plethodon cinereus revealed by sox9 and col2a1

Direct‐developing amphibians form limbs during early embryonic stages, as opposed to the later, often postembryonic limb formation of metamorphosing species. Limb patterning is dramatically altered in direct‐developing frogs, but little attention has been given to direct‐developing salamanders. We use expression patterns of two genes, sox9 and col2a1, to assess skeletal patterning during embryonic limb development in the direct‐developing salamander Plethodon cinereus. Limb patterning in P. cinereus partially resembles that described in other urodele species, with early formation of digit II and a generally anterior‐to‐posterior formation of preaxial digits. Unlike other salamanders described to date, differentiation of preaxial zeugopodial cartilages (radius/tibia) is not accelerated in relation to the postaxial cartilages, and there is no early differentiation of autopodial elements in relation to more proximal cartilages. Instead, digit II forms in continuity with the ulnar/fibular arch. This amniote‐like connectivity to the first digit that forms may be a consequence of the embryonic formation of limbs in this direct‐developing species. Additionally, and contrary to recent models of amphibian digit identity, there is no evidence of vestigial digits. This is the first account of gene expression in a plethodontid salamander and only the second published account of embryonic limb patterning in a direct‐developing salamander species.     

Cranial ontogeny in Philautus silus (Anura: Ranidae: Rhacophorinae) reveals few similarities with other direct-developing anurans

Direct development has evolved in rhacophorine frogs independently from other anuran lineages, thereby offering an opportunity to assess features associated with this derived life history. Using a developmental series of the direct-developing Philautus silus (Ranidae: Rhacophorinae) from Sri Lanka, we examine features of cranial morphology that are part of a suite of adaptations that facilitate feeding in free-living tadpoles, but have been changed or lost in other direct-developing lineages. Larval-specific upper jaw cartilages, which are absent from many non-rhacophorine direct-developing species (such as Eleutherodactylus coqui), develop in embryos of P. silus. Similarly, lower jaw cartilages initially assume a larval morphology, which is subsequently remodeled into the adult jaw configuration before hatching. However, the cartilaginous jaw suspension and hyobranchial skeleton never assume a typical larval morphology. The palatoquadrate, which suspends the lower jaw, lacks the posterior connections to the braincase found in many metamorphosing species. Unlike in metamorphosing species, bone formation in P. silus begins before hatching. However, the sequence of bone formation resembles that of metamorphosing anurans more than that of other direct developers. In particular, P. silus does not exhibit precocious ossification of the lower jaw, which is characteristic of some frogs and caecilians that lack a free-living tadpole. These data reveal some similarities between Philautus and other direct-developing anurans. However, the departure of Philautus embryos from the generalized tadpole skeletal morphology is less pronounced than that observed in other direct-developing taxa.     

Phylogenetic Analysis of Algal Symbionts Associated with Four North American Amphibian Egg Masses

Egg masses of the yellow-spotted salamander Ambystoma maculatum form an association with the green alga “Oophila amblystomatis” (Lambert ex Wille), which, in addition to growing within individual egg capsules, has recently been reported to invade embryonic tissues and cells. The binomial O. amblystomatis refers to the algae that occur in A. maculatum egg capsules, but it is unknown whether this population of symbionts constitutes one or several different algal taxa. Moreover, it is unknown whether egg masses across the geographic range of A. maculatum, or other amphibians, associate with one or multiple algal taxa. To address these questions, we conducted a phylogeographic study of algae sampled from egg capsules of A. maculatum, its allopatric congener A. gracile, and two frogs: Lithobates sylvatica and L. aurora. All of these North American amphibians form associations with algae in their egg capsules. We sampled algae from egg capsules of these four amphibians from localities across North America, established representative algal cultures, and amplified and sequenced a region of 18S rDNA for phylogenetic analysis. Our combined analysis shows that symbiotic algae found in egg masses of four North American amphibians are closely related to each other, and form a well-supported clade that also contains three strains of free-living chlamydomonads. We designate this group as the ‘Oophila’ clade, within which the symbiotic algae are further divided into four distinct subclades. Phylogenies of the host amphibians and their algal symbionts are only partially congruent, suggesting that host-switching and co-speciation both play roles in their associations. We also established conditions for isolating and rearing algal symbionts from amphibian egg capsules, which should facilitate further study of these egg mass specialist algae.     

Reliable expression and purification of highly insoluble transmembrane domains

A general procedure for the reliable preparation of insoluble transmembrane domains has been developed. Improved expression schemes were developed by expressing the transmembrane domains of caveolin proteins 1, 2, and 3 as a fusion to the Trp leader protein. This construct readily formed inclusion bodies during overexpression, allowing high levels of protein to be achieved. Cleavage of the transmembrane domain away from the Trp leader carrier protein was performed with cyanogen bromide. The transmembrane domains were then purified using reverse-phase high-performance liquid chromatography with a C4 column and were eluted with a mixture of 1-butanol and acetic acid. Using this method, the 39-42 amino acid transmembrane domains from caveolin proteins 1, 2, and 3 were successfully purified to homogeneity. Further verification of this method was successfully done with Rfbp(18-51), another insoluble transmembrane domain.     

Probing the caveolin-1 P132L mutant: critical insights into its oligomeric behavior and structure

Caveolin-1 is the most important protein found in caveolae, which are cell surface invaginations of the plasma membrane that act as signaling platforms. A single point mutation in the transmembrane domain of caveolin-1 (proline 132 to leucine) has deleterious effects on caveolae formation in vivo and has been implicated in various disease states, particularly aggressive breast cancers. Using a combination of gel filtration chromatography and analytical ultracentrifugation, we found that a fully functional construct of caveolin-1 (Cav1(62-178)) was a monomer in dodecylphosphocholine micelles. In contrast, the P132L mutant of Cav1(62-178) was dimeric. To explore the dimerization of the P132L mutant further, various truncated constructs (Cav1(82-178), Cav1(96-178), Cav1(62-136), Cav1(82-136), Cav1(96-136)) were prepared which revealed that oligomerization occurs in the transmembrane domain (residues 96-136) of caveolin-1. To characterize the mutant structurally, solution-state NMR experiments in lyso-myristoylphosphatidylglycerol were undertaken of the Cav1(96-136) P132L mutant. Chemical shift analysis revealed that, compared to the wild-type, helix 2 in the transmembrane domain was lengthened by four residues (wild-type, residues 111-129; mutant, residues 111-133), which corresponds to an extra turn in helix 2 of the mutant. Lastly, point mutations at position 132 of Cav1(62-178) (P132A, P132I, P132V, P132G, P132W, P132F) revealed that no other hydrophobic amino acid can preserve the monomeric state of Cav1(62-178), which indicates that proline 132 is critical in supporting proper caveolin-1 behavior.