Structure,dynamics, and stability of the globular domain of human linker histone H1.0 and the role of positive charges

Linker histone H1 (H1) is an abundant chromatin‐binding protein that acts as an epigenetic regulator binding to nucleosomes and altering chromatin structures and dynamics. Nonetheless, the mechanistic details of its function remain poorly understood. Recent work suggest that the number and position of charged side chains on the globular domain (GD) of H1 influence chromatin structure and hence gene repression. Here, we solved the solution structure of the unbound GD of human H1.0, revealing that the structure is almost completely unperturbed by complex formation, except for a loop connecting two antiparallel β‐strands. We further quantified the role of the many positive charges of the GD for its structure and conformational stability through the analysis of 11 charge variants. We find that modulating the number of charges has little effect on the structure, but the stability is affected, resulting in a difference in melting temperature of 26 K between GD of net charge +5 versus +13. This result suggests that the large number of positive charges on H1‐GDs have evolved for function rather than structure and high stability. The stabilization of the GD upon binding to DNA can thus be expected to have a pronounced electrostatic component, a contribution that is amenable to modulation by posttranslational modifications, especially acetylation and phosphorylation.     

Characterisation of the last Fe-S cluster-binding subunit of Neurospora crassa complex I

We have cloned cDNAs encoding the last iron-sulphur protein of complex I from Neurospora crassa. The cDNA sequence contains an open reading frame that codes for a precursor polypeptide of 226 amino acid residues with a molecular mass of 24972 Da. Our results indicate that the mature protein belongs probably to the peripheral arm of complex I and is rather unstable when not assembled into the enzyme. The protein is highly homologous to the PSST subunit of bovine complex I, the most likely candidate to bind iron-sulphur cluster N-2. All the amino acid residues proposed to bind such a cluster are conserved in the fungal protein.     

Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both M?ssbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.     

Total heart volume variation throughout the cardiac cycle in humans

Variations in total heart volume (atria plus ventricles) during a cardiac cycle affect efficiency of cardiac pumping. The goals of this study were to confirm the presence, extent, and contributors of total heart volume variation during the cardiac cycle in healthy volunteers with the use of MRI. Eight healthy volunteers were examined by MRI at rest. Changes in total cardiac volume throughout the cardiac cycle were calculated using the following methods: 1) planimetry derived from gradient-echo cine images and 2) flow-sensitive sequences to quantify flow in all vessels leading to and from the heart. The maximum total heart volume diminished during systole by 8.2 +/- 0.8% (SEM, range 4.8-10.6%) measured by method 1 and 8.8 +/- 1.0% (SEM, range 5.6-11.8%) by method 2 with good agreement between the methods [difference according to Bland-Altman analysis -0.6% +/- 1.0% (SD), intraclass correlation coefficient = 0.999]. This decrease in volume is predominantly explained by variation at the midcardiac level at the widest diameter of the heart with a left-sided predominance. In the short axis of the heart, the change of slice volume was proportional to the end-diastolic slice volume. The present study has confirmed the presence of total heart volume variation that predominantly occurs in the region of atrioventricular plane movement and on the left side. The total heart volume variation may relate to the efficiency of energy use by the heart to minimize displacement of surrounding tissue while accounting for the energy required to draw blood into the atria during ventricular systole.     

Competition between lithium and magnesium ions for the G-protein transducin in the guanosine 5'-diphosphate bound conformation

Li(+) is the most effective drug used to treat bipolar disorder; however, its exact mechanism of action has yet to be elucidated. One hypothesis is that Li(+) competes with Mg2+ for the Mg2+ binding sites on guanine-nucleotide binding proteins (G-proteins). Using 7Li T1 relaxation measurements and fluorescence spectroscopy with the Mg2+ fluorophore furaptra, we detected Li(+)/Mg(2+) competition in three preparations: the purified G-protein transducin (Gt), stripped rod outer segment membranes (SROS), and SROS with purified Gt reattached (ROS-T). When purified ROS-T, SROS or transducin were titrated with Li+ in the presence of fixed amounts of Mg(2+), the apparent Li(+) binding constant decreased due to Li(+)/Mg(2+) competition. Whereas for SROS the competition mechanism was monophasic, for G(t), the competition was biphasic, suggesting that in G(t), Li(+)/Mg(2+) competition occurred with different affinities for Mg(2+) in two types of Mg(2+) binding sites. Moreover, as [Li(+)] increased, the fluorescence excitation spectra of both ROS-T and G(t) were blue shifted, indicating an increase in free [Mg(2+)] compatible with Li(+) displacement of Mg(2+) from two low affinity Mg(2+) binding sites of G(t). G(t) release from ROS-T membrane was also inhibited by Li(+) addition. In summary, we found evidence of Li(+)/Mg(2+) competition in G(t)-containing preparations.     

Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana

Agrobacterium tumefaciens-mediated transformation (agro-transformation) was successfully applied to the entomogenous fungus Beauveria bassiana. Conidia of B. bassiana were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. The efficiency of transformation was up to 28 and 96 transformants per 10(4) and 10(5) target conidia, respectively, using three distinct vectors. High mitotic stability of the transformants (80-100%) was demonstrated after five successive transfers on a nonselective medium. Abortive transformants were observed for all the hph(r) vectors used. Putative transformants were analysed for the presence of the hph gene by PCR and Southern analysis. The latter analysis revealed the integration of two or more copies of the hph gene in the genome. The agro-transformation method was found to be effective for the isolation of B. bassiana hygromycin resistant transformants and may represent a useful tool for insertional mutagenesis studies in this fungus.     

Effect of free radicals on adenosine A(2A) and dopamine D2 receptors in the striatum of young adult and aged rats

Parkinson's disease (PD) is a prevalent age-related motor dysfunction resulting from the hyperactivity of the indirect striatal pathway, which is controlled in an antagonistic manner by inhibitory dopamine D2 and facilitatory adenosine A(2A) receptors. Thus, dopamine precursors like l-DOPA are the standard therapy and A(2A) antagonists are now tested as anti-parkinsonians. Increased free radicals levels occur on aging and are proposed to be a contributing factor for PD. We now tested if free radicals affected A(2A) and D2 receptors in striatal membranes of young adult (2 months) and old (24 months) rats. The A(2A) receptor antagonist [3H]SCH 58261 bound to striatal membranes with a KD of 0.9 nM and a Bmax of 953 fmol/mg protein in young rats and with a KD of 0.8 nM and a Bmax of 725 fmol/mg protein in aged rats (24% decrease). The D2 receptor antagonist [3H]raclopride bound to striatal membranes with a KD of 4.0 nM and a Bmax of 598 fmol/mg protein in young rats and with a KD of 4.3 nM and a Bmax of 368 fmol/mg protein in aged rats (38% decrease). Exposure of striatal membranes to a free radical generation system (2 mM FeSO4 and 4 mM ascorbate) caused a similar decrease of [3H]SCH 58261 (35%) and [3H]raclopride (37%) binding in young adult rats but caused a greater decrease of [3H]SCH 58261 (49%) than of [3H]raclopride (20%) binding in aged rats. Thus, in aged rats, there is an unbalance of A(2A)/D2 receptor density favouring A(2A) receptors, which is restored on exposure to free radicals. This supports the hypothesis that the effectiveness of A(2A) receptor antagonists as anti-parkinsonians, demonstrated in young adult animals, may not be affected by a modified A(2A)/D2 receptor density in aged individuals suffering from exposure to increased free radical levels, as occurs in PD.     

Cytogenetics of genetic counseling patients in Pelotas, Rio Grande do Sul, Brazil

From 1986 to 2002, we examined the chromosomal composition of 916 patients attended by two genetic counseling services in the city of Pelotas, in the Brazilian State of Rio Grande do Sul, to determine the genetic causes of their disturbances. Patterns of G-banding using trypsin and Giemsa (GTG) and C-banding using barium and Giemsa (CBG) were studied using phytohemagglutinin M-stimulated lymphocytes cultured from peripheral blood. Among the patients, 110 had Down's syndrome, 7 had Edward's syndrome, 4 had Patau's syndrome, 29 had Turner's syndrome, 5 had Klinefelter's syndrome, and 3 had "cri-du-chat" syndrome. Abnormal chromosomes were observed in 29.3% of the patients. Most of these (56.3%) were numerical abnormalities, with the remaining being structural variants.     

Patau syndrome with a long survival. A case report

Trisomy 13 is a clinically severe entity; 85% of the patients do not survive beyond one year, and most children die before completing six months of age. We report a female child, 28 months old, white, the fourth child of a non-consanguineous couple, who presented trisomy 13. The child was born at term, from a vaginal delivery, weighing 2600 g. At birth, she was cyanotic, icteric, spastic, and cried weakly. The initial clinical examination detected polydactyly in the left hand, congenital clubfoot and convex soles, ocular hypertelorism, a low nasal bridge, numerous hemangiomas distributed throughout the body, cardiomegaly, and perimembranous inter-ventricular communication. There was no cleft lip or palate. On physical examination at 18 months old, the child weighed 6,900 g, had a cephalic perimeter of 41 cm, a thoracic perimeter of 43 cm and was 76 cm tall. At 28 months, she weighed 10,760 g and was 88.5 cm tall. Neuropsychomotor development retardation was evident from birth and, according to the psychologist and the social assistant of APAE (Handicapped Parents and Friends Association) in Cangu?u, Rio Grande do Sul, there was a noticeable improvement after physiotherapy and recreational sessions.