Uroporphyrinogen decarboxylase: complete human gene sequence and molecular study of three families with hepatoerythropoietic porphyria.

A deficiency in uroporphyrinogen decarboxylase (UROD) enzyme activity, the fifth enzyme of the heme biosynthetic pathway, is found in patients with sporadic porphyria cutanea tarda (s-PCT), familial porphyria cutanea tarda (f-PCT), and hepatoerythropoietic porphyria (HEP). Subnormal UROD activity is due to mutations of the UROD gene in both f-PCT and HEP, but no mutations have been found in s-PCT. Genetic analysis has determined that f-PCT is transmitted as an autosomal dominant trait. In contrast, HEP, a severe form of cutaneous porphyria, is transmitted as an autosomal recessive trait. HEP is characterized by a profound deficiency of UROD activity, and the disease is usually manifest in childhood. In this study, a strategy was designed to identify alleles responsible for the HEP phenotype in three unrelated families. Mutations of UROD were identified by direct sequencing of four amplified fragments that contained the entire coding sequence of the UROD gene. Two new missense mutations were observed at the homoallelic state: P62L (proline-to-leucine substitution at codon 62) in a Portuguese family and Y311C (tyrosine-to-cysteine substitution at codon 311) in an Italian family. A third mutation, G281E, was observed in a Spanish family. This mutation has been previously described in three families from Spain and one from Tunisia. In the Spanish family described in this report, a paternal uncle of the proband developed clinically overt PCT as an adult and proved to be heterozygous for the G281E mutation. Mutant cDNAs corresponding to the P62L and Y311C changes detected in these families were created by site-directed mutagenesis. Recombinant proteins proved to have subnormal enzyme activity, and the Y311C mutant was thermolabile.     

Human embryonic stem cells: biology and clinical implications

Embryonic stem cells (ESCs) are derived from the inner cell mass of the preimplantation stage embryo and are capable of prolonged symmetrical self-renewal (both daughter cells remain escs) as well as differentiation into derivatives of all three embryonic germ layers. ESCs therefore have the potential to provide an unlimited supply of transplantable cells to replace or regenerate damaged or diseased tissues. However, several barriers must be overcome before successful clinical trials are possible: for example, pure populations of the desired cell type need to be selected and expanded in clinically relevant numbers, and a method for preventing immunological rejection of the transplanted cells without long-term immunosuppressive therapy is also required. In this review, we highlight recent developments in human ESC derivation and expansion, outline current understanding of the signalling pathways underlying stem cell renewal, and discuss challenging problems related to the selective differentiation and immune properties of human ESCs.     

Heterospecific aggression bias towards a rarer colour morph

Colour polymorphisms are a striking example of phenotypic diversity, yet the sources of selection that allow different morphs to persist within populations remain poorly understood. In particular, despite the importance of aggression in mediating social dominance, few studies have considered how heterospecific aggression might contribute to the maintenance or divergence of different colour morphs. To redress this gap, we carried out a field-based study in a Nicaraguan crater lake to investigate patterns of heterospecific aggression directed by the cichlid fish, Hypsophrys nicaraguensis, towards colour polymorphic cichlids in the genus Amphilophus. We found that H. nicaraguensis was the most frequent territorial neighbour of the colour polymorphic A. sagittae. Furthermore, when manipulating territorial intrusions using models, H. nicaraguensis were more aggressive towards the gold than dark colour morph of the sympatric Amphilophus species, including A. sagittae. Such a pattern of heterospecific aggression should be costly to the gold colour morph, potentially accounting for its lower than expected frequency and, more generally, highlighting the importance of considering heterospecific aggression in the context of morph frequencies and coexistence in the wild.     

An investigation of donor and culture parameters which influence epithelial outgrowths from cultured human cadaveric limbal explants

Limbal stem cell deficiency is a blinding disease which affects the cornea at the front of the eye. The definitive cure involves replacing the corneal epithelial (limbal) stem cells, for example by transplanting cultured limbal epithelial cells. One method of performing cultures is to grow a sheet of epithelial cells from a limbal explant on human amniotic membrane. The growth of limbal tissue can be variable. The aim of this study is to investigate how different donor and culture factors influence the ex vivo growth of cadaveric limbal explants. Limbal explant cultures were established from 10 different cadaveric organ cultured corneo‐scleral discs. The growth rate and the time taken for growth to be established were determined. Statistical analysis was performed to assess correlation between these factors and donor variables including donor age, sex, time from donor death to enucleation, time from enucleation to organ culture storage and duration in organ culture. Growth curves consistently showed a lag phase followed by a steeper linear growth phase. Donor age, time between death and enucleation, and time between enucleation and organ culture were not correlated to the lag time or the growth rate. Time in organ culture had a significant correlation with the duration of lag time (P = 0.003), but no relationship with the linear growth rate. This study shows that an important factor correlating with growth variation is the duration of corneo‐scleral tissue in organ culture. Interestingly, donor age was not correlated with limbal explant growth. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Autophagy in osteoblasts is involved in mineralization and bone homeostasis

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.     

Females decide whether size matters: plastic mate preferences tuned to the intensity of male-male competition

Recent studies have indicated that mating success of large malesmay improve under increasing levels of mating competition. Thisoutcome is explained 1) if male mating competition is overridingfemale preferences for male traits that are unrelated to, ornegatively correlated with, male size and dominance and, inso doing, dictates the distribution of matings or 2) if femalesalter their preferences with respect to large males when male–malecompetition is intense. Under both hypotheses, one could expectlarge, dominant males to be more successful under intense competitionthan under weak competition. However, only the first explanationpredicts that male mating success under intense competitionshould be determined by dominance; traits that are unrelatedto male dominance should be uncorrelated to mating success.In contrast, if females change their preferences (explanation2), males with traits beneficial to females independent of thecompetitive environment can maintain a high mating success underall levels of male–male competition. We tested these alternativesusing a small marine fish, the sand goby, Pomatoschistus minutus.The mating success of large males increased under conditionsallowing intense male competition, whereas females showed apreference for good nest building independent of the level ofcompetition. These findings suggest that females are in controlof their choice by altering their preference for male size inresponse to the intensity of male–male competition ratherthan female preference being overshadowed by male dominance.This plasticity of preferences implies that the strength ofsexual selection is not constant at the population level.     

Regulation of excitation-contraction coupling in mouse cardiac myocytes: integrative analysis with mathematical modelling

Background  

The cardiomyocyte is a prime example of inherently complex biological system with inter- and cross-connected feedback loops in signalling, forming the basic properties of intracellular homeostasis. Functional properties of cells and tissues have been studied e.g. with powerful tools of genetic engineering, combined with extensive experimentation. While this approach provides accurate information about the physiology at the endpoint, complementary methods, such as mathematical modelling, can provide more detailed information about the processes that have lead to the endpoint phenotype.     

Association of methylenetetrahydrofolate (MTHFR) and apolipoprotein E (apo E) genotypes with homocysteine, vitamin and lipid levels in carotid stenosis

The aim of the study was to investigate the association between methylenetetrahydrofolate (MTHFR) genotypes and levels of homocysteine (Hcy), folate, vitamin B12 and lipids as well as the association between apolipoprotein E (apo E) genotypes and levels of lipids in a Croatian healthy control group and a group of patients with > 70% carotid stenosis (CS). The study included 98 Croats, 38 patients with > 70% carotid stenosis and 60 age- and sex-matched controls. The MTHFR and apo E genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), Hcy by enzyme immunoassay, vitamins by immunochemiluminiscence, and lipids by spectrophotometric method. There was no difference between control subjects and CS patients in the distribution of C677T MTHFR genotypes (p=0. 786) and alleles (p=0.904), however, differences in the frequencies of apo E genotypes (p=0.012) and alleles (p=0.029) were statistically significant. The odds ratio for apo E 3/4 genotype was 3.93 (95% CI 1.23-12.61). Hyperhomocysteinemia (> or =15 micromol/L) was found in 11% of CS patients and 5% of control subjects. Total cholesterol, triglycerides, vitamin B12 and folate were statistically different in "all MTHFR genotypes" (p<0.001, p<0.01, p=0.044 and p=0.036, respectively), and in TC/TT (p<0.001, p=0.003, p=0.030 and p=0.032, respectively) groups. The levels of total cholesterol, LDL cholesterol and triglycerides in the apo E 3/3, and total cholesterol in the apo E 3/4 group yielded statistical difference. An association was found of apo E 3/4 genotype but not of MTHFR genotypes with the risk of CS. MTHFR and apo E affect blood lipid levels, which was statistically confirmed. An association was also recorded between hyperhomocysteinemia and patients with CS. Vitamin status in CS showed a statistically verified association with TC/TT MTHFR genotype. In the group of patients with TC/TT MTHFR genotype, lower vitamin B12 and higher folate values were recorded. The results of multiple logistic analysis showed that there was no statistical significance of Hcy levels (OR 2.403, p=0.334) or conventional vascular risk factors such as smoking habit (OR 0.505, p=0.149), age (OR 1.048, p=0.087) or sex (OR 2.037, p=0.112) in predicting CS.     

Introduced predator elicits deficient brood defence behaviour in a crater lake fish

Introduced species represent one of the most serious global threats to biodiversity. In this field-based study, we assessed behavioural responses of brood tending cichlid fish to an invasive predator of their offspring. This was achieved by comparing parental defence responses of the endangered arrow cichlid (Amphilophus zaliosus), a fish species endemic to the crater lake Apoyo in Nicaragua, towards the bigmouth sleeper (Gobiomorus dormitor), a formidable predator of cichlid fry, and all other potential fish predators of offspring. The bigmouth sleeper was recently introduced into Apoyo but naturally co-exists with cichlids in a few other Nicaraguan lakes. Arrow cichlid parents allowed bigmouth sleepers to advance much closer to their fry than other predators before initiating aggressive brood defence behaviours. Interestingly, parents of a very closely related species, A. sagittae, which has coevolved with bigmouth sleepers in crater lake Xiloá, reacted to approaching bigmouth sleepers at comparable distances as to other predators of cichlid fry. These results provide a novel demonstration of the specific mechanism (i.e. naive parental behaviour) by which invasive predators may negatively affect species that lack the adequate behavioural repertoire.     

Kinetic Analysis of Glycoprotein–Lectin Interactions by Label-Free Internal Reflection Ellipsometry

Introduction

Glycoproteomics is undergoing rapid development, largely as a result of advances in technologies for isolating glycoproteins and analyzing glycan structures. However, given the number and diversity of glycans, there is need for new technologies that can more rapidly provide differential carbohydrate–protein structural information on a large scale. We describe a new microarray platform based on a label-free imaging ellipsometry technique, which permits simultaneous detection of multiple glycoprotein–lectin interactions without the need for reporter labels, while still providing high throughput kinetic information at much lower cost. Our results demonstrate the utility of LFIRE? (Label-Free Internal Reflection Ellipsometry) for the rapid kinetic screening of carbohydrate–lectin recognition. The technology was also used to evaluate the benefits of the lectin immobilization format using multi-lectin affinity chromatography (M-LAC) to capture glycoproteins (with enhanced binding strength or avidity) from biological samples. Using a printed panel of lectins, singly or in combination, we examined the binding characteristics of standard glycoproteins.

Results and Discussion

Using kinetic measurements, it was observed that the binding strength of lectins to carbohydrates is enhanced using a multi-lectin strategy, suggesting that improved selectivity and specificity can lead to increased functional avidity. The data presented confirm that this label-free technology can be used to effectively screen single or combinations of lectins. Furthermore, the combination of LFIRE? and M-LAC may permit more rapid and sensitive identification of novel biomarkers based on carbohydrate changes in glycoproteins, and lead to a better understanding of the connections of glycan function in cellular mechanisms of health and disease.