Mitochondria Are Linked to Calcium Stores in Striated Muscle by Developmentally Regulated Tethering Structures

Bi-directional calcium (Ca²⁺) signaling between mitochondria and intracellular stores (endoplasmic/sarcoplasmic reticulum) underlies important cellular functions, including oxidative ATP production. In striated muscle, this coupling is achieved by mitochondria being located adjacent to Ca²⁺ stores (sarcoplasmic reticulum [SR]) and in proximity of release sites (Ca²⁺ release units [CRUs]). However, limited information is available with regard to the mechanisms of mitochondrial-SR coupling. Using electron microscopy and electron tomography, we identified small bridges, or tethers, that link the outer mitochondrial membrane to the intracellular Ca²⁺ stores of muscle. This association is sufficiently strong that treatment with hypotonic solution results in stretching of the SR membrane in correspondence of tethers. We also show that the association of mitochondria to the SR is 1) developmentally regulated, 2) involves a progressive shift from a longitudinal clustering at birth to a specific CRU-coupled transversal orientation in adult, and 3) results in a change in the mitochondrial polarization state, as shown by confocal imaging after JC1 staining. Our results suggest that tethers 1) establish and maintain SR–mitochondrial association during postnatal maturation and in adult muscle and 2) likely provide a structural framework for bi-directional signaling between the two organelles in striated muscle.     

Anaesthesia of free-ranging Northern chamois (<Emphasis Type="Italic">Rupicapra rupicapra</Emphasis>) with xylazine/ketamine and reversal with atipamezole

One-hundred and fifty-five free-ranging Northern chamois (Rupicapra rupicapra) were anaesthetised in the course of a restocking programme using xylazine plus ketamine. Mean ± SD dosages for xylazine and ketamine were 1.9 ± 0.5 and 2.2 ± 0.7 mg/kg, respectively. In 57 chamois, sedation was reversed using 0.3 ± 0.1 mg/kg atipamezole. Although all the anaesthetic dosages tested immobilised free-ranging Northern chamois, shorter induction times (4.8 ± 2.6 min), deeper sedation with no reaction to handling in >90% of the animals and quick reversal (4.0 ± 2.7 min) were obtained using 2.5 mg/kg xylazine plus 3.0 mg/kg ketamine reversed with 0.25 mg/kg atipamezole. Under the conditions of this study, suggested standard doses are 63 mg/animal xylazine plus 76 mg/animal ketamine reversed by 6.3 mg/animal atipamezole. This anaesthetic protocol improves the results from the previous study of Dematteis et al. (Vet Rec 163:184–189, 2008) using xylazine alone.     

The Tnt1 family member Retrosol copy number and structure disclose retrotransposon diversification in different <Emphasis Type="Italic">Solanum</Emphasis> species

Eukaryotic genome expansion/retraction caused by LTR-retrotransposon activity is dependent on the expression of full length copies to trigger efficient transposition and recombination-driven events. The Tnt1 family of retrotransposons has served as a model to evaluate the diversity among closely related elements within Solanaceae species and found that members of the family vary mainly in their U3 region of the long terminal repeats (LTRs). Recovery of a full length genomic copy of Retrosol was performed through a PCR-based approach from wild potato, Solanum oplocense. Further characterization focusing on both LTR sequences of the amplified copy allowed estimating an approximate insertion time at 2 million years ago thus supporting the occurrence of transposition cycles after genus divergence. Copy number of Tnt1-like elements in Solanum species were determined through genomic quantitative PCR whereby results sustain that Retrosol in Solanum species is a low copy number retrotransposon (1–4 copies) while Retrolyc1 has an intermediate copy number (38 copies) in S. peruvianum. Comparative analysis of retrotransposon content revealed no correlation between genome size or ploidy level and Retrosol copy number. The tetraploid cultivated potato with a cellular genome size of 1,715 Mbp harbours similar copy number per monoploid genome than other diploid Solanum species (613–884 Mbp). Conversely, S. peruvianum genome (1,125 Mbp) has a higher copy number. These results point towards a lineage specific dynamic flux regarding the history of amplification/activity of Tnt1-like elements in the genome of Solanum species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Role of Tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase

A mutant of the thermostable NAD⁺-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp → Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD⁺ and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 Å resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.     

Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases in hypertensive vascular remodeling

Structural vascular changes in two-kidney, one-clip (2K-1C) hypertension may result from increased matrix metalloproteinase (MMP)-2 activity. MMP-2 activation is regulated by other MMPs, including transmembrane-MMPs, and by tissue inhibitors of MMPs (TIMPs). We have investigated the localization of MMP-2, -9, -14, and TIMPs 1–4 in hypertensive aortas and measured their levels by zymography/Western blotting and immunohistochemistry. Gelatinolytic activity was assayed in tissues by in situ zymography. Sham-operated and 2K-1C hypertensive rats were treated with doxycycline (or vehicle) for 8 weeks, and the systolic blood pressure was monitored weekly. Doxycycline attenuated 2K-1C hypertension (165 ± 11.7 mmHg versus 213 ± 7.9 mm Hg in hypertensive controls, P < 0.01), and completely prevented increase in the thicknesses of the media and the intima in 2K-1C animals (P < 0.01). Increased amounts of MMP-2, -9, and -14 were found in hypertensive aortas, as well as enhanced gelatinolytic activity. A gradient in the localization of MMP-2, -9, and -14 was found, with increased amounts detected in the intima, at sites with higher gelatinolytic activity. Doxycycline attenuated hypertension induced increases in all the 3 investigated MMPs in both the media and the intima (all P < 0.05), but it did not change the amounts of TIMPs 1–4 (P > 0.05). Therefore, an imbalance between increased amounts of MMPs at the tissue level without a corresponding increase in the quantities of TIMPs, particularly in the intima and inner media layers, appears to account for the increased proteolytic activity found in 2K-1C hypertension-induced maladaptive vascular remodeling.     

Biological and structural characterization of the Mycobacterium smegmatis nitroreductase NfnB,and its role in benzothiazinone resistance

Tuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl‐β‐d ‐ribose 2′‐epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro‐group to an amino‐group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB‐BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.     

Does polyamine oxidase activity influence the oxidative metabolism of children who suffer of diabetes mellitus?

Diabetes mellitus is a metabolic disease characterized by inadequate secretion of insulin. Polyamine oxidase (PAO), a FAD-containing enzyme is involved in the biodegradation of Sp and Spd, catalyzing the oxidative deamination of Sp and Spd, resulting in production of amonia (NH₃), corresponding amino aldehydes and H₂O₂. Malondialdehyde (MDA) and acrolein (CH2=CHCHO), potentially toxic agents, which induce oxidative stress in mammalian cells, are then spontaneously formed from aminoaldehydes. The main signs of oxidative stress in diabetic children were the values of HbA1c and MDA levels. Polyamines have an insulin-like action. Antiglycation property of spermine and spermidine has been recently confirmed. There are no data in the literature about plasma polyamine oxidase (PAO) activities in children with type 1 diabetes. The idea of this study was to evaluate the polyamine metabolism through the estimation of polyamine oxidase activity. We have study children with newly diagnosed type 1 diabetes mellitus (n = 35, age group of 5–16 years, as well as age-matched healthy control subjects (n = 25). The biochemical investigations were done on diabetic children who have the pathological values of glucose (9.11–17.33 mmol/l) and glycosylated Hb (7.57–14.49% HbA_1c). The children in the control group have referent values of glucose and glycated hemoglobin (4.11-5.84 mmol/L and HbA_1c 4.22-6.81% of the total Hb. Glucose levels in blood plasma and glycosylated hemoglobin in erythrocythes hemolysates (HbA1c) were measured by using standard laboratory methods. PAO activity in venous blood plasma and the amount of malondialdehyde (MDA) were measured by the spectrophotometric methods. PAO activity, glycemia, HbA1c and MDA were significantly increased in diabetic children compared to the control subjects. PAO activity in children with type 1 diabetes mellitus was very high. The findings of higher blood HbA_1C and MDA levels confirm the presence of oxidant stress in children with type 1 diabetes mellitus and demonstrate that PAO activity may participate in these circumstances.     

The molecular characterization of a novel GH38 α-mannosidase from the crenarchaeon Sulfolobus solfataricus revealed its ability in de-mannosylating glycoproteins

α-Mannosidases, important enzymes in the N-glycan processing and degradation in Eukaryotes, are frequently found in the genome of Bacteria and Archaea in which their function is still largely unknown. The α-mannosidase from the hyperthermophilic Crenarchaeon Sulfolobus solfataricus has been identified and purified from cellular extracts and its gene has been cloned and expressed in Escherichia coli. The gene, belonging to retaining GH38 mannosidases of the carbohydrate active enzyme classification, is abundantly expressed in this Archaeon. The purified α-mannosidase activity depends on a single Zn²⁺ ion per subunit is inhibited by swainsonine with an IC₅₀ of 0.2 mM. The molecular characterization of the native and recombinant enzyme, named Ssα-man, showed that it is highly specific for α-mannosides and α(1,2), α(1,3), and α(1,6)-d-mannobioses. In addition, the enzyme is able to demannosylate Man₃GlcNAc₂ and Man₇GlcNAc₂ oligosaccharides commonly found in N-glycosylated proteins. More interestingly, Ssα-man removes mannose residues from the glycosidic moiety of the bovine pancreatic ribonuclease B, suggesting that it could process mannosylated proteins also in vivo. This is the first evidence that archaeal glycosidases are involved in the direct modification of glycoproteins.