Technological difficulties in ghrelin and obestatin assays

In recent years we have performed more than 1,000 radioimmunoassays of ghrelin and obestatin. In these assays, we have encountered several technological obstacles. Another difficulty was the enormous discrepancy of plasma ghrelin results published by different authors. The aim of this article is to comment on these problems. Not all peptides of the hypothalamus and intestines are present in blood circulation. Several neuropeptides do not cross the blood-brain barrier, and several gastrointestinal peptides are present in extremely low concentrations in the blood. That requires time-consuming and laborious extraction. In these procedures, considerable amounts of peptides may be lost. In addition, these peptides are very unstable and prone to enzymatic degradation. This makes it mandatory to add enzymatic inhibitors to plasma samples. The peptides are also unstable in elevated temperatures, hence the assays should be performed in air-conditioned laboratories and the kits should be transported in proper low temperature conditions. Peptides may appear in several isoforms of different biological activity, but antibodies routinely used in these assays are polyclonal and do not differentiate between these forms. This complicates clinical evaluation of the results. To date, there are no international standards of ghrelin, obestatin or other active peptides, probably because of their extreme instability. Because of technological difficulties, the results of peptide assays performed in different scientific research institutions vary greatly and cannot be compared to each other. This disadvantage may be partially diminished by including samples of healthy subjects in each assay run to check whether the peptide concentrations of the patients differ significantly from that of control subjects.     

The binding specificity of the marker antibodies Tra-1-60 and Tra-1-81 reveals a novel pluripotency-associated type 1 lactosamine epitope

The expression of the epitopes recognized by the monoclonal antibodies Tra-1-60 and Tra-1-81 is routinely used to assess the pluripotency status of human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells. Although it is known that the epitopes recognized by Tra-1-60 and Tra-1-81 are carbohydrates, the exact molecular identity of these epitopes has been unclear. Glycan array analysis with more than 500 oligosaccharide structures revealed specific binding of Tra-1-60 and Tra-1-81 to two molecules containing terminal type 1 lactosamine: Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc and Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3GlcNAcβ1-3)Galβ1-4Glc. The type 1 disaccharide in itself was not sufficient for binding, indicating that the complete epitope requires an extended tetrasaccharide structure where the type 1 disaccharide is β1,3-linked to type 2 lactosamine. Our mass spectrometric analysis complemented with glycosidase digestions of hESC O-glycans indicated the presence of the extended tetrasaccharide epitope on an O-glycan with the likely structure Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAc. Thus, the present data indicate that the pluripotency marker antibodies Tra-1-60 and Tra-1-81 recognize the minimal epitope Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc, which is present in hESCs as a part of a mucin-type O-glycan structure. The exact molecular identity of Tra-1-60 and Tra-1-81 is important for the development of improved tools to characterize the pluripotent phenotype.     

Phospholipase D controls Dictyostelium development by regulating G protein signaling

Dictyostelium discoideum cells normally exist as individual amoebae, but will enter a period of multicellular development upon starvation. The initial stages of development involve the aggregation of individual cells, using cAMP as a chemoattractant. Chemotaxis is initiated when cAMP binds to its receptor, cAR1, and activates the associated G protein, Gα2βγ. However, chemotaxis will not occur unless there is a high density of starving cells present, as measured by high levels of the secreted quorum sensing molecule, CMF. We previously demonstrated that cells lacking PldB bypass the need for CMF and can aggregate at low cell density, whereas cells overexpressing pldB do not aggregate even at high cell density. Here, we found that PldB controlled both cAMP chemotaxis and cell sorting. PldB was also required by CMF to regulate G protein signaling. Specifically, CMF used PldB, to regulate the dissociation of Gα2 from Gβγ. Using fluorescence resonance energy transfer (FRET), we found that along with cAMP, CMF increased the dissociation of the G protein. In fact, CMF augmented the dissociation induced by cAMP. This augmentation was lost in cells lacking PldB. PldB appears to mediate the CMF signal through the production of phosphatidic acid, as exogenously added phosphatidic acid phenocopies overexpression of pldB. These results suggest that phospholipase D activity is required for CMF to alter the kinetics of cAMP-induced G protein signaling.     

Adaptive secondary sex ratio adjustments via sex-specific infanticide in a bird

Infanticide is easiest to understand when it involves killing the offspring of others [1], but a parent may also kill its own offspring if the sacrifice of currently dependent young leads to higher survival of brood mates [2] or an improvement in the parent's likely future reproduction [3]. However, sex-specific infanticide by parents of their own offspring, although occurring in some human societies [4], is rare across species. Its rarity may be because killing one sex combines wasted parental effort with consequent biases in population sex ratios that are detrimental for the fitness of the overproduced sex [5-7]. We show that killing male offspring can be advantageous to Eclectus parrot (Eclectus roratus) mothers even though frequency-dependent selection then elevates the reproductive value of sons above that of daughters. In poorer-quality nest hollows, broods with a single female nestling had higher reproductive value than broods in which the female had a younger brother. Our data demonstrate frequent targeted removal of male nestlings within 3 days of hatching in these specific brood types and nesting conditions. The ability of Eclectus parrots to perceive the sex of their offspring relatively early may favor decisions to kill one sex before further investment in parental care.     

Processing and turnover of the Hedgehog protein in the endoplasmic reticulum

The Hedgehog (Hh) signaling pathway has important functions during metazoan development. The Hh ligand is generated from a precursor by self-cleavage, which requires a free cysteine in the C-terminal part of the protein and results in the production of the cholesterol-modified ligand and a C-terminal fragment. In this paper, we demonstrate that these reactions occur in the endoplasmic reticulum (ER). The catalytic cysteine needs to form a disulfide bridge with a conserved cysteine, which is subsequently reduced by protein disulfide isomerase. Generation of the C-terminal fragment is followed by its ER-associated degradation (ERAD), providing the first example of an endogenous luminal ERAD substrate that is constitutively degraded. This process requires the ubiquitin ligase Hrd1, its partner Sel1, the cytosolic adenosine triphosphatase p97, and degradation by the proteasome. Processing-defective mutants of Hh are degraded by the same ERAD components. Thus, processing of the Hh precursor competes with its rapid degradation, explaining the impaired Hh signaling of processing-defective mutants, such as those causing human holoprosencephaly.     

Chronic UVR Causes Increased Immunostaining of CD44 and Accumulation of Hyaluronan in Mouse Epidermis

Chronic intense UV radiation is the main cause of epidermal tumors. Because hyaluronan (HA), a large extracellular polysaccharide, is known to promote malignant growth, hyaluronan expression was studied in a model in which long-term UV radiation (UVR) induces epidermal tumors. Mouse back skin was exposed three times a week for 10.5 months to UVR corresponding to one minimal erythema dose, processed for histology, and stained for hyaluronan and the hyaluronan receptor CD44. This exposure protocol caused epidermal hyperplasia in most of the animals; tumors, mainly squamous cell carcinomas (SCCs), were found in ~20% of the animals. Specimens exposed to UVR showed increased hyaluronan and CD44 staining throughout the epidermal tissue. In hyperplastic areas, hyaluronan and CD44 stainings correlated positively with the degree of hyperplasia. Well-differentiated SCCs showed increased hyaluronan and CD44 staining intensities, whereas poorly differentiated tumors and dysplastic epidermis showed areas where HA and CD44 were locally reduced. The findings indicate that HA and CD44 increase in epidermal keratinocytes in the premalignant hyperplasia induced by UV irradiation and stay elevated in dysplasia and SCC, suggesting that the accumulation of hyaluronan and CD44 is an early marker for malignant transformation and may be a prerequisite for tumor formation.     

A novel minicollagen gene links cnidarians and myxozoans

Myxozoans are enigmatic endoparasitic organisms sharing morphological features with bilateria, protists and cnidarians. This, coupled with their highly divergent gene sequences, has greatly obscured their phylogenetic affinities. Here we report the sequencing and characterization of a minicollagen homologue (designated Tb-Ncol-1) in the myxozoan Tetracapsuloides bryosalmonae. Minicollagens are phylum-specific genes encoding cnidarian nematocyst proteins. Sequence analysis revealed a cysteine-rich domain (CRD) architecture and genomic organization similar to group 1 minicollagens. Homology modelling predicted similar three-dimensional structures to Hydra CRDs despite deviations from the canonical pattern of group 1 minicollagens. The discovery of this minicollagen gene strongly supports myxozoans as cnidarians that have radiated as endoparasites of freshwater, marine and terrestrial hosts. It also reveals novel protein sequence variation of relevance to understanding the evolution of nematocyst complexity, and indicates a molecular/morphological link between myxozoan polar capsules and cnidarian nematocysts. Our study is the first to illustrate the power of using genes related to a taxon-specific novelty for phylogenetic inference within the Metazoa, and it exemplifies how the evolutionary relationships of other metazoans characterized by extreme sequence divergence could be similarly resolved.     

DDTRP: Database of Drug Targets for Resistant Pathogens

Emergence of drug resistance is a major threat to public health. Many pathogens have developed resistance to most of the existing antibiotics, and multidrug-resistant and extensively drug resistant strains are extremely difficult to treat. This has resulted in an urgent need for novel drugs. We describe a database called 'Database of Drug Targets for Resistant Pathogens' (DDTRP). The database contains information on drugs with reported resistance, their respective targets, metabolic pathways involving these targets, and a list of potential alternate targets for seven pathogens. The database can be accessed freely at http://bmi.icmr.org.in/DDTRP.