Magnesium deficiency results in an increased formation of osteoclasts

Magnesium (Mg²⁺) deficiency is a frequently occurring disorder that leads to loss of bone mass, abnormal bone growth and skeletal weakness. It is not clear whether Mg²⁺ deficiency affects the formation and/or activity of osteoclasts. We evaluated the effect of Mg²⁺ restriction on these parameters. Bone marrow cells from long bone and jaw of mice were seeded on plastic and on bone in medium containing different concentrations of Mg²⁺ (0.8 mM which is 100% of the normal value, 0.4, 0.08 and 0 mM). The effect of Mg²⁺ deficiency was evaluated on osteoclast precursors for their viability after 3 days and proliferation rate after 3 and 6 days, as was mRNA expression of osteoclastogenesis-related genes and Mg²⁺-related genes. After 6 days of incubation, the number of tartrate resistant acid phosphatase-positive (TRACP⁺) multinucleated cells was determined, and the TRACP activity of the medium was measured. Osteoclastic activity was assessed at 8 days by resorption pit analysis. Mg²⁺ deficiency resulted in increased numbers of osteoclast-like cells, a phenomenon found for both types of marrow. Mg²⁺ deficiency had no effect on cell viability and proliferation. Increased osteoclastogenesis due to Mg²⁺ deficiency was reflected in higher expression of osteoclast-related genes. However, resorption per osteoclast and TRACP activity were lower in the absence of Mg²⁺. In conclusion, Mg²⁺ deficiency augmented osteoclastogenesis but appeared to inhibit the activity of these cells. Together, our in vitro data suggest that altered osteoclast numbers and activity may contribute to the skeletal phenotype as seen in Mg²⁺ deficient patients.     

Novel thiol- and thioether-containing amino acids: cystathionine and homocysteine families

Natural l-homocysteine and l,l-cystathionine, along with a series of unnatural analogues, have been prepared from l-aspartic and l-glutamic acid. Manipulation of the protected derivatives provided ω-iodoamino acids, which were used in thioalkylation reactions of sulfur nucleophiles, such as the ester of l-cysteine and potassium thioacetate.     

Acute creatine administration improves mitochondrial membrane potential and protects against pentylenetetrazol-induced seizures

A growing body of evidence indicates that creatine (Cr) exerts beneficial effects on a variety of pathologies where energy metabolism and oxidative stress play an etiological role. However, the benefits of Cr treatment for epileptics are still shrouded in controversy. In the present study, we found that acute Cr treatment (300 mg/kg, p.o.) prevented the increase in electroencephalographic wave amplitude typically elicited by PTZ (30, 45 or 60 mg/kg, i.p.). Cr treatment also increased the latency periods of first myoclonic jerks, lengthened the latency periods of the generalized tonic–clonic seizures and reduced the time spent in the generalized tonic–clonic seizures induced by PTZ (60 mg/kg). Administration of PTZ (all doses) decreased Na⁺, K⁺-ATPase activity as well as adenosine triphosphate (ATP) and adenosine diphosphate levels in the cerebral cortex, but Cr treatment prevented these effects. Cr administration also prevented increases in xanthine oxidase activity, adenosine monophosphate levels, adenosine levels, inosine levels and uric acid levels that normally occur after PTZ treatment (60 mg/kg, i.p.). We also showed that Cr treatment increased the total Cr (Cr + PCr) content, creatine kinase activity and the mitochondrial membrane potential (ΔΨ) in the cerebral cortex. In addition, Cr prevented PTZ-induced mitochondrial dysfunction characterized by decreasing ΔΨ, increasing thiobarbituric acid-reactive substance levels and increasing protein carbonylation. These experimental findings reinforce the idea that mitochondrial dysfunction plays a critical role in models of epileptic seizures and suggest that buffering brain energy levels through Cr treatment may be a promising therapeutic approach for the treatment of this neurological disease.     

Advanced electron paramagnetic resonance on the catalytic iron–sulfur cluster bound to the CCG domain of heterodisulfide reductase and succinate: quinone reductase

Heterodisulfide reductase (Hdr) is a key enzyme in the energy metabolism of methanogenic archaea. The enzyme catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes M (CoM-SH) and B (CoB-SH). Cleavage of CoM-S-S-CoB at an unusual FeS cluster reveals unique substrate chemistry. The cluster is fixed by cysteines of two cysteine-rich CCG domain sequence motifs (CX_31–39CCX_35–36CXXC) of subunit HdrB of the Methanothermobacter marburgensis HdrABC complex. We report on Q-band (34 GHz) ⁵⁷Fe electron-nuclear double resonance (ENDOR) spectroscopic measurements on the oxidized form of the cluster found in HdrABC and in two other CCG-domain-containing proteins, recombinant HdrB of Hdr from M. marburgensis and recombinant SdhE of succinate: quinone reductase from Sulfolobus solfataricus P2. The spectra at 34 GHz show clearly improved resolution arising from the absence of proton resonances and polarization effects. Systematic spectral simulations of 34 GHz data combined with previous 9 GHz data allowed the unambiguous assignment of four ⁵⁷Fe hyperfine couplings to the cluster in all three proteins. ¹³C Mims ENDOR spectra of labelled CoM-SH were consistent with the attachment of the substrate to the cluster in HdrABC, whereas in the other two proteins no substrate is present. ⁵⁷Fe resonances in all three systems revealed unusually large ⁵⁷Fe ENDOR hyperfine splitting as compared to known systems. The results infer that the cluster’s unique magnetic properties arise from the CCG binding motif.     

Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR

The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-¹³C] acetate does not label α carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-¹³C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA–CO coupling in the backbone should improve the resolution of NCACX spectra.     

Soybean lecithin-based extender as an alternative for goat sperm cryopreservation

The aim of this study was to evaluate the effect of different concentrations of soybean lecithin (SL) in extenders for sperm goat cryopreservation. Sexually mature male Saanen goats (n = 4) were used, and the ejaculates were obtained using an artificial vagina method. The semen samples were pooled and diluted in a skim milk-based extender (control group; CG) or Tris extender supplemented with SL at different concentrations (G1 = 0.04%, SL G2 = 0.08% SL and G3 = 0.16%) for a final concentration of 240 × 10⁶ spermatozoa/mL. The semen samples were packed in straws (0.25 mL), frozen using an automated system and stored in liquid nitrogen (?196 °C). After thawing (37 °C/30 s), the samples were evaluated for sperm quality parameters, including sperm motility, membrane integrity, acrosome integrity and mitochondrial activity. No significant difference was observed among the experimental and control groups for all of the parameters (P > 0.05). However, even though the control group presented a significantly lower mitochondrial membrane potential compared to fresh semen (P < 0.05), the same did not occur for the extender supplemented with soybean lecithin, that is, it did not differ from fresh sperm (P > 0.05). The extender containing soybean lecithin at different concentrations preserved the sperm quality parameters in a manner similar to the conventional skim milk-based extender. Thus, it is concluded that an extender containing soybean lecithin as the lipoprotein source can be used for freezing goat semen.     

Evaluating the “recovery level” of endangered species without prior information before alien invasion

For maintaining social and financial support for eradication programs of invasive species, quantitative assessment of recovery of native species or ecosystems is important because it provides a measurable parameter of success. However, setting a concrete goal for recovery is often difficult owing to lack of information prior to the introduction of invaders. Here, we present a novel approach to evaluate the achievement level of invasive predator management based on the carrying capacity of endangered species estimated using long‐term monitoring data. In Amami‐Oshima Island, Japan, where the eradication project of introduced small Indian mongoose is ongoing since 2000, we surveyed the population densities of four endangered species threatened by the mongoose (Amami rabbit, the Otton frog, Amami tip‐nosed frog, and Amami Ishikawa's frog) at four time points ranging from 2003 to 2011. We estimated the carrying capacities of these species using the logistic growth model combined with the effects of mongoose predation and environmental heterogeneity. All species showed clear tendencies toward increasing their density in line with decreased mongoose density, and they exhibited density‐dependent population growth. The estimated carrying capacities of three endangered species had small confidence intervals enough to measure recovery levels by the mongoose management. The population density of each endangered species has recovered to the level of the carrying capacity at about 20–40% of all sites, whereas no individuals were observed at more than 25% of all sites. We propose that the present approach involving appropriate monitoring data of native organism populations will be widely applicable to various eradication projects and provide unambiguous goals for management of invasive species.