Germline mosaicism for an alanine to valine substitution at residue β 140 in hemoglobin Puttelange,a new variant with high oxygen affinity

Hb Puttelange [140(H18)AlaVal] was found as a de novo mutation in two siblings of a French family suffering from polycythemia. Both parents were phenotypically normal and exclusion of paternity has been ruled out by the study of several polymorphic markers located on different chromosomes. The structural modification of Hb Puttelange was established by reversed-phase HPLC analysis of the tryptic digest of the abnormal chain. The amino acid composition of an abnormal T14 peptide revealed that one of the four residues of Ala was replaced by a Val. Tandem mass spectrometry demonstrated that the substitution concerned position 140 (H18). This hemoglobin displays an increased oxygen affinity that is responsible for the polycythemia. De novo mutations, as demonstrated again in the case of this variant, have the highest probabilities of detection when they lead to pathological manifestations. They may result either from a somatic mutation in a very early stage of the embryological development of the propositus or may have a parental origin with occurrence of a germline mosaicism. The study of the -globin gene indicated that this case of Hb Puttelange probably arose from a mutation affecting a part of the germline of the father, therefore leading to a true recurrence risk.     

A monozygotic twin pair with β-thalassemia carrier status in a Dudh Kharia tribal family of Orissa

BACKGROUND:

The β-thalassemia syndrome is a genetically inherited commonly encountered hematological disorder in the state of Orissa. It causes high degree of morbidity, mortality and fetal wastage in the poor vulnerable people.

AIMS AND OBJECTIVES:

There is an equal probability (50% chance) in every singleton pregnancy that a carrier parent of β-thalassemia major would either bear normal or carrier offspring, but not two offspring with carrier of β-thalassemia major genotype together. For the first time, a carrier parent of β-thalassemia major gene has born progeny (three daughters and a twin male offspring) with a carrier status of β-thalassemia major in Dudh Kharia tribal family studied from Sundargarh district of Orissa.

MATERIALS AND METHODS:

We screened randomly selected population of Dudh Kharia tribe from Sundargarh district of Orissa for hemoglobinopathies to assess the extent of the problem, design possible interventions and provide genetic counseling to them. A family with twin children was identified during screening in Lata Gaon in Bargaon block of Sundargarh district of Orissa for the above-mentioned study. Background information for this family such as name, age, sex, tribe, native place, reproductive history, family pedigree and clinical signs and symptoms were also recorded. Standardized genetic and hematological procedures and techniques were followed for analysis.

RESULTS:

Laboratory investigations for alkaline electrophoresis of blood lysate on cellulose acetate membrane showed raised hemoglobin A₂ level in mother (Hb A₂ = 5.3%), in three daughters (Hb A₂ =6.5, 5.9, 5.5% in chronological and birth order), in two twin sons (Hb A₂ =5.9% and 6.0%) and normal (Hb A₂ = 3.3%) for father. Hence, all the children i.e., three daughters and two twin sons, including the mother were β-thalassemia carriers. Since all the hematological parameters i.e., red cell indices, G-6-PD enzyme activity, ABO and Rhesus blood groups and identical β-thalassemia (trait) genotypes with identical clinical manifestations and hematological profile of the twin sons under similar environmental conditions, hence they were labeled as identical monozygotic twins.

CONCLUSIONS:

It is a rare occasion when a single pregnancy carries either one or two abnormal genotypes at a time in a womb in human beings. Monozygotic twins are genetically alike and provide appraisal of the expression of identical genotype under the different environmental conditions.     

Expression of the gene coding for bacterial hemoglobin improves β-galactosidase production in a recombinant Pichia pastoris

Genes coding for Vitreoscilla hemoglobin (VHb) with peroxisome targeting signal (PTS1) tag and -galactosidase were co-expressed in Pichia pastoris under the alcohol oxidase1 (AOX1) promoter. The expression of VHb-PTS1 had no positive effect on cell growth but significantly enhanced the whole cell -galactosidase activity to 4-fold higher than that of VHb-free cell in yeast extract/peptone/methanol medium under aerobic cultivation.     

A frequent Aγ-persistence of fetal hemoglobin in northern Sardinia: its molecular basis and hematologic phenotype in heterozygotes and compound heterozygotes with β-thalassemia

Summary A survey of hemoglobinopathies in northern Sardinia revealed a high frequency (0.3%) of carriers of a hematologic condition characterized by increased expression of fetal hemoglobin during adult life (hereditary persistence of fetal hemoglobin or HPFH). In spite of a normal hematologic phenotype, the heterozygous carriers for this condition display about 12% HbF, almost exclusively of the ^A type; compound heterozygotes with -thalassemia have 20%–26% HbF and run a very mild clinical course. The sequence analysis of the cloned ^A gene linked to the HPFH determinant revealed the presence of a GA substitution at position-117 of the ^A- gene promoter; the same mutation occurs also in Greek HPFH, although associated with different restriction polymorphisms. Another hereditary condition characterized by increased HbF (₂ ^A₂) level and a mild thalassemic phenotype in Sardinia is associated with the-196 CT substitution in the ^A-globin gene promoter (Sardinian -thalassemia). Population studies using oligonucleotides complementary both to the-117 GA and-196 CT mutations and the corresponding normal sequences confirm the presence of these mutations only in HPFH and -thalassemia chromosomes and exclude these changes being common DNA polymorphisms.     

Pl-nectin,a discoidin family member,is a ligand for βC integrins in the sea urchin embryo

Pl-nectin is a component of the extracellular matrix that surrounds embryos of the sea urchin Paracentrotus lividus. Pl-nectin mediates adhesion of dissociated embryonic cells to substrates and interfering with ectodermic cells contacting Pl-nectin results in defects in skeleton growth and morphogenesis. Recently, we reported that Pl-nectin is a new member of the discoidin family, in agreement with the notion that many discoidin-containing proteins are involved in cell adhesion processes as integrin ligands. To better understand the molecular basis for the interaction of Pl-nectin with ectoderm, we investigated the hypothesis that Pl-nectin is an integrin ligand in sea urchin embryos. We show that in P. lividus embryos, βC-containing integrins localize to the apical surface of ectodermic cells, which are in contact with Pl-nectin. Immunoprecipitation experiments indicate that the two proteins are part of a complex in vivo and affinity chromatography indicates that βC-containing integrin receptors bind purified Pl-nectin. These data support a model in which ectodermic integrins binding to Pl-nectin mediate cellular adhesion to the hyaline layer. Regulated adhesion of cells to the hyaline layer is a critical component of several morphogenetic processes and the identification of the receptors and ligands involved provides new opportunities to investigate the underlying molecular mechanisms of ECM adhesion and morphogenesis.     

Is there more than one way to skin a newt? Convergent toxin resistance in snakes is not due to a common genetic mechanism

Convergent evolution of tetrodotoxin (TTX) resistance, at both the phenotypic and genetic levels, characterizes coevolutionary arms races between amphibians and their snake predators around the world, and reveals remarkable predictability in the process of adaptation. Here we examine the repeatability of the evolution of TTX resistance in an undescribed predator–prey relationship between TTX-bearing Eastern Newts (Notophthalmus viridescens) and Eastern Hog-nosed Snakes (Heterodon platirhinos). We found that that local newts contain levels of TTX dangerous enough to dissuade most predators, and that Eastern Hog-nosed Snakes within newt range are highly resistant to TTX. In fact, these populations of Eastern Hog-nosed Snakes are so resistant to TTX that the potential for current reciprocal selection might be limited. Unlike all other cases of TTX resistance in vertebrates, H. platirhinos lacks the adaptive amino acid substitutions in the skeletal muscle sodium channel that reduce TTX binding, suggesting that physiological resistance in Eastern Hog-nosed Snakes is conferred by an alternate genetic mechanism. Thus, phenotypic convergence in this case is not due to parallel molecular evolution, indicating that there may be more than one way for this adaptation to arise, even among closely related species.     

Identification of β-glucosidase 1 as a biomarker and its high expression in hepatocellular carcinoma is associated with resistance to chemotherapy drugs

Context and objective: Long-term prognosis of hepatocellular carcinoma (HCC) patients is challenging, and novel biomarkers are needed to predict patient risk and serve as potential therapeutic target.

Results: We found β-glucosidase 1 is significantly overexpressed and activated in primary HCC tissue and multiple HCC cell lines. β-Glucosidase 1 expression is associated with predicting prognosis of HCC patients under chemotherapy. Silencing β-glucosidase 1 inhibits growth and survival of HCC cells, with preferential inhibitory effects on high β-glucosidase 1-expressing cells. Combination of chemo drug with β-glucosidase 1 inhibitor sensitized HCC cells to chemotherapy.

Conclusion: Our data support β-glucosidase 1 as a HCC biomarker due to its prognosis significance.     

A substitution of cytosine for thymine in codon 110 of the human β-globin gene is a novel cause of β-thalassemia phenotypes

Summary We have described a novel human globin gene mutation that produced in a Japanese family the -thalassemia phenotype through a post-translational mechanism. Substitution of proline for leucine at position 110 in the G-helix of the -globin chain greatly reduced the molecular stability of the -globin subunit, leading to total destruction of the variant globin chains by proteolysis and hence to the -thalassemia phenotype. The mutation could be identified after MspI digestion. This detection of the mutation on the gene level is valuable for diagnostic purposes.     

Targeting tumor-associated macrophages in an experimental glioma model with a recombinant immunotoxin to folate receptor β

Tumor-associated macrophages (TAMs) are frequently found in glioblastomas and a high degree of macrophage infiltration is associated with a poor prognosis for glioblastoma patients. However, it is unclear whether TAMs in glioblastomas promote tumor growth. In this study, we found that folate receptor β (FRβ) was expressed on macrophages in human glioblastomas and a rat C6 glioma implanted subcutaneously in nude mice. To target FRβ-expressing TAMs, we produced a recombinant immunotoxin consisting of immunoglobulin heavy and light chain Fv portions of an anti-mouse FRβ monoclonal antibody and Pseudomonas exotoxin A. Injection of the immunotoxin into C6 glioma xenografts in nude mice significantly depleted TAMs and reduced tumor growth. The immunotoxin targeting FRβ-expressing macrophages will provide a therapeutic tool for human glioblastomas.     

Doctor in the lab: what is it like for a doctor to work with scientists?

As clinical academic medical departments strive to improve the quality of their research, clinicians and scientists are forced into closer liaison. In many cases, clinical departments now have research laboratories directed by "basic scientists" but often staffed, in part at least, by doctors. To someone who has not worked in one, these laboratories may seem uncompromising and forbidding work environments. This article presents a "case report" written from the viewpoints of the doctor, the scientist, and the professor.     

Asp271 is critical for substrate interaction with the surface binding site in β-agarase a from Zobellia galactanivorans

In the marine environment agar degradation is assured by bacteria that contain large agarolytic systems with enzymes acting in various endo- and exo-modes. Agarase A (AgaA) is an endo-glycoside hydrolase of family 16 considered to initiate degradation of agarose. Agaro-oligosaccharide binding at a unique surface binding site (SBS) in AgaA from Zobellia galactanivorans was investigated by computational methods in conjunction with a structure/sequence guided approach of site-directed mutagenesis probed by surface plasmon resonance binding analysis of agaro-oligosaccharides of DP 4-10. The crystal structure has shown that agaro-octaose interacts via H-bonds and aromatic stacking along 7 subsites (L through R) of the SBS in the inactive catalytic nucleophile mutant AgaA-E147S. D271 is centrally located in the extended SBS where it forms H-bonds to galactose and 3,6-anhydrogalactose residues of agaro-octaose at subsites O and P. We propose D271 is a key residue in ligand binding to the SBS. Thus AgaA-E147S/D271A gave slightly decreasing K_D values from 625 ± 118 to 468 ± 13 μM for agaro-hexaose, -octaose, and -decaose, which represent 3- to 4-fold reduced affinity compared with AgaA-E147S. Molecular dynamics simulations and interaction analyses of AgaA-E147S/D271A indicated disruption of an extended H-bond network supporting that D271 is critical for the functional SBS. Notably, neither AgaA-E147S/W87A nor AgaA-E147S/W277A, designed to eliminate stacking with galactose residues at subsites O and Q, respectively, were produced in soluble form. W87 and W277 may thus control correct folding and structural integrity of AgaA.     

Indigestible dextrin is an excellent inducer for α-amylase, α-glucosidase and glucoamylase production in a submerged culture of Aspergillus oryzae

α-Amylase activities of Aspergillus oryzae grown on dextrin or indigestible dextrin were 7·8 and 27·7 U ml⁻¹, respectively. Glucoamylase activities of the cultures grown on dextrin or indigestible dextrin were 5·4 and 301 mU ml⁻¹, respectively. The specific glucoamylase production rate in indigestible dextrin batch culture reached 1·35 U g DW⁻¹ h⁻¹. In contrast, biomass concentration of A. oryzae in indigestible dextrin culture was 35% of that in dextrin culture. Thus, the culture method using indigestible dextrin has the potential to improve amylolytic enzyme production and fungal fermentation broth rheology.     

A new general approach to fluorogenic enzyme assays of high sensitivity and potential high specificity,illustrated by a procedure for β-d-galactosidase estimations

A novel fluorogenic substrate (methylumbelliferyl 2-acetamido-2-deoxy-β-d-lactoside) has been prepared enzymatically. A procedure has been developed for its use as a convenient and sensitive fluorogenic substrate for β-d-galactosidase assay with a potential for high substrate specificity. The merits of this new fluorogenic substrate for β-d-galactosidase assays are discussed, together with the potential of this approach for a wider range of enzyme activities.     

How high is your hummock? The importance of Triodia height as a habitat predictor for an endangered marsupial in a fire‐prone environment

Fire and seral vegetation succession are known to influence the distribution and abundance of a wide range of arid and semi‐arid mammal species. In Triodia hummock grasslands, the gradual increase in Triodia cover after fire is a significant factor influencing mammal distribution and abundance. However, the height of fire‐adapted hummock grass species is often ignored during habitat preference studies despite the fact that taller hummocks are likely to have improved insulation properties and greater protection from predators. We tested the relative importance of a range of Triodia habitat characteristics in determining the distribution and abundance of a fire‐adapted mammal inhabiting Triodia mallee dune fields in semi‐arid Australia. We sampled 77 sites and collected information on habitat attributes including Triodia cover, height and time since fire. The 90th percentile Triodia height (>400 mm) was the most reliable predictor of sandhill dunnart abundance, and breeding, inferred through the presence of subadults. The presence of adult sandhill dunnarts was best explained by Triodia cover, increasing when cover exceeded 25%. We suggest that while Triodia cover may be an important variable for predicting the presence of adult sandhill dunnarts, the height of Triodia is important for breeding, when tall Triodia are possibly sought for nesting sites. These Triodia height and cover requirements were not recorded at sites until at least 10 years post fire but the relationship between fire and Triodia cover and height was inconsistent; after 20 years Triodia cover declined with increasing fire age while the 90th percentile Triodia height remained relatively constant. This incongruence may explain why the presence of sandhill dunnarts appears more constrained by a minimum rather than maximum time since fire and could help explain patterns of post‐fire distribution in other arid zone mammals. Importantly, the 90th percentile Triodia height highlighted the possible significance of scattered, tall Triodia hummocks for arid zone fossorial mammals.     

The hypo-osmolarity-sensitive phenotype of the Saccharomyces cerevisiae hpo2 mutant is due to a mutation in PKC1, which regulates expression of β-glucanase

To obtain more information about the cell wall organization of Saccharomyces cerevisiae, we have developed a novel screening system to obtain cell wall-defective mutants, using a density gradient centrifugation method. Nine hypo-osmolarity-sensitive mutants were classified into two complementation groups, hpo1 and hpo2. Phase contrast microscopic observation showed that mutant cells bearing lesions at either locus became abnormally large. A gene that complemented the mutant phenotype of hpo2 was cloned and sequenced. This gene turned out to be identical to PKC1, which encodes the yeast homologue of mammalian protein kinase C. Complementation tests with pkc1Δ showed that hpo2 is allelic to pkc1. To study the reason for the fragility of hpo2 cells, cell wall was isolated and the glucan was analyzed. The amount of alkali, acid-insoluble glucan, which is responsible for the rigidity of the cell wall, was reduced to about 30% that of the wild-type cell and this may be the major cause of the fragility of the hpo2 mutant cell. Analysis of total wall proteins in hpo2 mutant cells on SDS-polyacrylamide gels revealed that a 33 kDa protein was overproduced two- to threefold relative to the wild-type level. This 33 kDa protein was identified as a β-glucanase, encoded by BGL2. Disruption of BGL2 in the hpo2 mutant partially rescued the growth rate defect. This suggests that the PKC1 kinase cascade regulates BGL2 expression negatively and overproduction of the β-glucanase is partially responsible for the growth defect. Since the bgl2 disruption did not rescue the hypo-osmolarty-sensitive phenotype of the hpo2 mutant, PKC1 must negatively regulate other enzymes involved in the biosynthesis and metabolism of the cell wall.     

The postprandial use of dietary amino acids as an energy substrate is delayed after the deamination process in rats adapted for 2 weeks to a high protein diet

The aim of this study was to determine the contribution of dietary amino acids (AA) to energy metabolism under high protein (HP) diets, using a double tracer method to follow simultaneously the metabolic fate of α-amino groups and carbon skeletons. Sixty-seven male Wistar rats were fed a normal (NP) or HP diet for 14 days. Fifteen of them were equipped with a permanent catheter. On day 15, after fasting overnight, they received a 4-g meal extrinsically labeled with a mixture of 20 U-[¹⁵N]-[¹³C] AA. Energy metabolism, dietary AA deamination and oxidation and their transfer to plasma glucose were measured kinetically for 4 h in the catheterized rats. The transfer of dietary AA to liver glycogen was determined at 4 h. The digestive kinetics of dietary AA, their transfer into liver AA and proteins and the liver glycogen content were measured in the 52 other rats that were killed sequentially hourly over a 4-h period. [¹⁵N] and [¹³C] kinetics in the splanchnic protein pools were perfectly similar. Deamination increased fivefold in HP rats compared to NP rats. In the latter, all deaminated AA were oxidized. In HP rats, the oxidation rate was slower than deamination, so that half of the deaminated AA was non-oxidized within 4 h. Non-oxidized carbon skeletons were poorly sequestrated in glycogen, although there was a significant postprandial production of hepatic glycogen. Our results strongly suggest that excess dietary AA-derived carbon skeletons above the ATP production capacity, are temporarily retained in intermediate metabolic pools until the oxidative capacities of the liver are no longer overwhelmed by an excess of substrates.     

Call divergence is correlated with geographic and genetic distance in greenish warblers (Phylloscopus trochiloides): a strong role for stochasticity in signal evolution?

Divergence in signalling systems might play a central role in speciation. To assess the importance of possible causes of signal divergence, we examine two types of vocalizations within a geographically variable species complex, the greenish warblers (Phylloscopus trochiloides Sundevall). Calls, which are used by both sexes throughout the year, and songs, which are sung primarily by breeding males, differ distinctly between two distinct Siberian forms. Through a ring of southern populations that connect the northern forms, signal divergence is correlated with both geographic distance and genetic divergence. Calls and songs differ in their particular patterns of geographic variation, probably because of the larger influence of sexual selection on songs than on calls. These patterns are supportive of neither acoustic adaptation nor morphology being major drivers of divergence in vocalizations. Rather, these results support the importance of stochastic evolution of communication systems in the evolution of new species.     

Salmosan,a β-galactomannan-rich product,in combination with Lactobacillus plantarum contributes to restore intestinal epithelial barrier function by modulation of cytokine production

Mannan-oligosaccharides (MOSs) are mannose-rich substrates with several intestinal health-promoting properties. The aim of this study was to investigate the potential capacity of Salmosan (S-βGM), a β-galactomannan-rich MOS product, to restore epithelial barrier function independently from its capacity to reduce bacterial invasion. In addition, the combination of S-βGM with the proven probiotic Lactobacillus plantarum (LP) was also tested. Paracellular permeability was assessed by transepithelial electrical resistance (TER) in co-cultures of Caco-2 cells and macrophages (differentiated from THP-1 cells) stimulated with LPS of Salmonella Enteritidis and in Caco-2 cell cultures stimulated with TNF-α in the absence or presence of 500 μg/ml S-βGM, LP (MOI 10) or a combination of both. In both culture models, TER was significantly reduced up to 25% by LPS or TNF-α stimulation, and the addition of S-βGM or LP alone did not modify TER, whereas the combination of both restored TER to values of nonstimulated cells. Under LPS stimulation, TNF-α production was significantly increased by 10-fold, whereas IL-10 and IL-6 levels were not modified. The combination of S-βGM and LP reduced TNF-α production to nonstimulated cell values and significantly increased IL-10 and IL-6 levels (5- and 7.5-fold, respectively). Moreover, S-βGM has the capacity to induce an increase of fivefold in LP growth. In conclusion, we have demonstrated that S-βGM in combination with LP protects epithelial barrier function by modulation of cytokine secretion, thus giving an additional value to this MOS as a potential symbiotic.