Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development

A null mutation in the morphogen Indian hedgehog (IHH) results in an embryonic lethal phenotype characterized by the conspicuous absence of bony tissue in the extremities. We show that this ossification defect is not attributable to a permanent arrest in cartilage differentiation, since Ihh(-/-) chondrocytes undergo hypertrophy and terminal differentiation, express angiogenic markers such as Vegf, and are invaded, albeit aberrantly, by blood vessels. Subsequent steps, including vessel expansion and persistence, are impaired, and the net result is degraded cartilage matrix that is devoid of blood vessels. The absence of blood vessels is not because the Ihh(-/-) skeleton is anti-angiogenic; in fact, in an ex vivo environment, both wild-type and Ihh mutant vessels invade the Ihh(-/-) cartilage, though only wild-type vessels expand to create the marrow cavity. In the ex vivo setting, Ihh(-/-) cells differentiate into osteoblasts and deposit a bony matrix, without benefit of exogenous hedgehog in the new environment. Even more surprising is our finding that the earliest IHH-dependent skeletal defect is obvious by the time the limb mesenchyme segregates into chondrogenic and perichondrogenic condensations. Although Ihh(-/-) cells organize into chondrogenic condensations similar in size and shape to wild-type condensations, perichondrial cells surrounding the mutant condensations are clearly faulty. They fail to aggregate, elongate and flatten into a definitive, endothelial cell-rich perichondrium like their wild-type counterparts. Normally, these cells surrounding the chondrogenic condensation are exposed to IHH, as evidenced by their expression of the hedgehog target genes, patched (Ptch) and Gli1. In the mutant environment, the milieu surrounding the cartilage -- comprising osteoblast precursors and endothelial cells -- as well as the cartilage itself, develop in the absence of this important morphogen. In conclusion, the skeletal phenotype of Ihh(-/-) embryos represents the sum of disturbances in three separate cell populations, the chondrocytes, the osteoblasts and the vasculature, each of which is a direct target of hedgehog signaling.     

G-protein beta-3 subunit gene C825 T polymorphism: influence on plasma sodium and potassium concentrations in essential hypertensive patients

The C825T polymorphism of the beta-3 subunit of the protein G (GNB3) has been related to an increased activity of the Na+/H+ exchanger (NHE-1) through the synthesis of an anomalous hyperactive protein. Because of the important role of this system in essential hypertension (EH), we analysed the distribution of the different genotypes of this polymorphism in normotensive subjects (NS) and essential hypertensive patients (EHP), their relationship with the condition of salt sensitivity, plasma sodium and potassium concentrations and plasma renin activity (PRA) in EHP. 144 subjects (78 EHP and 76 NS) were studied. Salt sensitivity was assessed by the rapid protocol of Weinberger and genotype determination for GNB3 C825T polymorphism was performed by PCR. The distribution of the different genotypes was similar among EHP (CC 37.2%; CT 41.1%; TT 16.7%) and NS (CC 32.9%; CT 55.3%; TT 11.8%). In regard to general characteristics of EHP (including blood pressure levels) and the condition of salt sensitivity, there were no differences among the different genotypes. Plasma sodium concentration was higher and plasma potassium was lower in TT patients (141.0+/-1.7 and 3.7+/-0.1) than in CC patients (139.1+/-1.9 and 4.0+/-0.3) p<0.05. CT patients had intermediate values (139.9+/-1.9 and 3.9+/-0.2). PRA values were similar in the three genotypes as were the rest of analytical parameters studied. Our data demonstrate an association between the C825T polymorphism of the GNB3 and plasma sodium and potassium concentrations in EHP, as expression of an increase in NHE-1 activity, without modifications in PRA nor relationship with salt sensitivity.     

Internal iron biomineralization in Imperata cylindrica, a perennial grass: chemical composition, speciation and plant localization

* The analysis of metal distribution in Imperata cylindrica, a perennial grass isolated from the banks of Tinto River (Iberian Pyritic Belt), an extreme acidic environment with high content in metals, has shown a remarkable accumulation of iron. This property has been used to study iron speciation and its distribution among different tissues and structures of the plant. * Mossbauer (MS) and X-ray diffraction (XRD) were used to determine the iron species, scanning electron microscopy (SEM) to locate iron biominerals among plant tissue structures, and energy-dispersive X-ray microanalysis (EDAX), X-ray fluorescence (TXRF) and inductively coupled plasma emission spectroscopy (ICP-MS) to confirm their elemental composition. * The MS spectral analysis indicated that iron accumulated in this plant mainly as jarosite and ferritin. The presence of jarosite was confirmed by XRD and the distribution of both minerals in structures of different tissues was ascertained by SEM-EDAX analysis. * The convergent results obtained by complementary techniques suggest a complex iron management system in I. cylindrica, probably as a consequence of the environmental conditions of its habitat.     

Relationship between non-return rate and chromatin condensation of deep frozen bull spermatozoa

This study analyzes the relationship between chromatin condensation and field fertility, expressed as 90 days non-return rate (NRR), of bulls actively used by AI studs. Frozen-thawed semen from five bulls (six ejaculates per bull, three straws per ejaculate), that showed a non-return rate between 60 and 80%, were analyzed to assess sperm chromatin condensation and stability. The chromatin condensation was determined by flow cytometry using propidium iodide as fluorochrome, and the chromatin stability was evaluated by inducing its decondensation with SDS and EDTA. Coefficient of variation among replicates was less than 7% and 5% for chromatin condensation and stability, respectively. No correlation was present between chromatin condensation and NRR. However, significant correlation was found between chromatin stability and NRR. Chromatin stability was higher (P < 0.05) in those bulls that showed higher fertility. The results obtained in this study conclude that assessment of stability could be a valuable tool for routine evaluation and identification of ejaculates with high levels of sperm chromatin abnormalities and to detect animals of higher reproductive potential.     

Consequences of polymorphism of 5'-GMP.Na2 in the vibrational spectra of metal complexes and isotopic derivatives

The commercial mononucleotides are frequently used to obtain metal complexes and isotopic derivatives. Normally, the spectra of these new compounds are compared with the spectra of the commercial mononucleotides. Nevertheless, important variations in the vibrational spectra of the disodium 5'-guanosine monophosphate, 5'-GMP, have been observed in this work produced by submitting the commercial salt to the same general laboratory process that the obtained compounds, i.e., solving the commercial salt in water and subsequent recrystallization. These changes have been analyzed and interpreted. The variations are not significant in disodium 5'-cytosine monophosphate, 5'-CMP. It is important to take this information into account before carrying out vibrational studies with this type of molecules, since some bands attributed to isotopic substitutions or to the metal attack may be a result of manipulation of the nucleotide (solving and recrystallization) instead of the studied effect. Thus, before any work in which the nucleotide salt is manipulated (deuteration, synthesis of other isotopic derivatives or metal-nucleotide complexes), it should be noted that the process to which the sample is submitted on its own is enough to modify the vibrational spectrum. Then, attention should be paid to the changes observed in the vibrational spectra of recrystallized mononucleotides, since recrystallization may lead to a considerable phase change, and this can notably alter the vibrational spectra.     

Effect of ejaculate, bull, and a double swim-up sperm processing method on sperm sex ratio

Offspring gender preselection has applications of considerable economic, health and ecological interest. In this study we analysed modifications of the percentages of spermatozoa bearing Y and X chromosomes when semen samples are submitted to a double swim-up technique as a possible method for producing embryos of known sex with in vitro fertilisation protocols. As an initial experiment to provide accurate evaluation of the method we determined the possible incidence of natural deviations in the primary sex ratio between bulls or ejaculates, analysing the percentage of Y-chromosome DNA bearing spermatozoa (%Y-CDBS) with a polymerase chain reaction (PCR) amplification of X- and Y-specific fragments. Ejaculates were tested by direct semiquantitative PCR sexing and by sexing blastocysts produced in vitro with these spermatozoa. Bulls and ejaculates did not have any effect on the %Y-CDBS or on the sex ratio of embryos produced in vitro using these ejaculates. However, our double swim-up sperm preparation method produced differences in %Y-CDBS in some of the sperm fractions, suggesting that there are intrinsic differences in capacitation of X- and Y-bearing spermatozoa that might be used to produce embryos of the desired sex with in vitro fertilisation.     

Characterization of monoaminergic systems in brain regions of prematurely ageing mice

We have previously shown that differences in life span among members of Swiss mouse populations appear to be related to their exploration of a T-maze, with a slow exploration ("slow mice") being linked to increased levels of emotionality/anxiety, an impaired immune function and a shorter life span. Thus, we proposed the slow mice as prematurely ageing mice (PAM). We have now compared the monoaminergic systems of the PAM and of the non-prematurely ageing mice (NPAM), in discrete brain regions. PAM had decreased noradrenaline (NA) levels in all the brain regions analysed, whereas the 3-methoxy-4-hydroxyphenyl glycol (MHPG)/NA ratios were not significantly modified. PAM also showed decreased serotonine (5-HT) levels in hypothalamus, striatum and midbrain, as well as increased 5-hydroxyindol-3-acetic acid (5-HIAA)/5-HT ratios in hypothalamus and hippocampus. The dopamine (DA) content was lower in PAM in most regions, whereas the 3,4-dihydroxyphenylacetic acid (DOPAC)/DA and homovanillic acid (HVA)/DA ratios were either increased or unchanged depending on the region analysed. In most cases, the differences between PAM and NPAM involved both sexes. One exception was the hypothalamus where the differences only affected the male mice. The neurochemical alterations found in PAM resemble some changes reported for aged animals and are related with their behavioural features.     

Changes in the superoxide production and other macrophage functions could be related to the mortality of mice with endotoxin-induced oxidative stress

Free radicals and proinflammatory cytokines from phagocytes have been implicated in the pathogenesis of endotoxic shock, a disease with high mortality caused by Gram-negative bacterial endotoxin. In the present study, male BALB/c and Swiss mice received intraperitoneally lipopolysaccharide (LPS) at 100 mg/kg and 150 mg/kg, respectively, that led to a lethal endotoxic shock (100 % of mortality before 30 h). Swiss mice injected with 100 mg/kg, that did not show lethal endotoxic shock, were also studied. Peritoneal macrophages were obtained from animals at 2, 4, 12 or 24 h after injection of LPS or saline (control) solutions. Superoxide anion and tumor necrosis factor (TNFalpha) production were determined in these cells as well as other functions such as adherence capacity, chemotaxis and phagocytosis. The increase in superoxide anion production after endotoxin injection was higher in cells from mice with lethal shock than in those with non-lethal shock. However, the enhancement of TNFalpha production was similar in all cases, although in Swiss mice the highest levels of TNFalpha were observed at 1.5 h after endotoxin injection, while in BALB/c mice they occurred at 2 h after LPS injection. This oxidative stress was also revealed by the other functions analyzed, since adherence to substrate and phagocytosis were stimulated and chemotaxis was decreased after endotoxin injection as compared to controls, the differences being even more significant in animals with lethal shock. These data suggest that these changes, mainly the increased production of free radicals even more than the TNFalpha release, could be involved in mouse mortality caused by LPS.