Effect of a Low-Protein Diet Supplemented with Ketoacids on Skeletal Muscle Atrophy and Autophagy in Rats with Type 2 Diabetic Nephropathy

A low-protein diet supplemented with ketoacids maintains nutritional status in patients with diabetic nephropathy. The activation of autophagy has been shown in the skeletal muscle of diabetic and uremic rats. This study aimed to determine whether a low-protein diet supplemented with ketoacids improves muscle atrophy and decreases the increased autophagy observed in rats with type 2 diabetic nephropathy. In this study, 24-week-old Goto-Kakizaki male rats were randomly divided into groups that received either a normal protein diet (NPD group), a low-protein diet (LPD group) or a low-protein diet supplemented with ketoacids (LPD+KA group) for 24 weeks. Age- and weight-matched Wistar rats served as control animals and received a normal protein diet (control group). We found that protein restriction attenuated proteinuria and decreased blood urea nitrogen and serum creatinine levels. Compared with the NPD and LPD groups, the LPD+KA group showed a delay in body weight loss, an attenuation in soleus muscle mass loss and a decrease of the mean cross-sectional area of soleus muscle fibers. The mRNA and protein expression of autophagy-related genes, such as Beclin-1, LC3B, Bnip3, p62 and Cathepsin L, were increased in the soleus muscle of GK rats fed with NPD compared to Wistar rats. Importantly, LPD resulted in a slight reduction in the expression of autophagy-related genes; however, these differences were not statistically significant. In addition, LPD+KA abolished the upregulation of autophagy-related gene expression. Furthermore, the activation of autophagy in the NPD and LPD groups was confirmed by the appearance of autophagosomes or autolysosomes using electron microscopy, when compared with the Control and LPD+KA groups. Our results showed that LPD+KA abolished the activation of autophagy in skeletal muscle and decreased muscle loss in rats with type 2 diabetic nephropathy.     

Selective Vulnerability of Spinal and Cortical Motor Neuron Subpopulations in delta7 SMA Mice

Loss of the survival motor neuron gene (SMN1) is responsible for spinal muscular atrophy (SMA), the most common inherited cause of infant mortality. Even though the SMA phenotype is traditionally considered as related to spinal motor neuron loss, it remains debated whether the specific targeting of motor neurons could represent the best therapeutic option for the disease. We here investigated, using stereological quantification methods, the spinal cord and cerebral motor cortex of ∆7 SMA mice during development, to verify extent and selectivity of motor neuron loss. We found progressive post-natal loss of spinal motor neurons, already at pre-symptomatic stages, and a higher vulnerability of motor neurons innervating proximal and axial muscles. Larger motor neurons decreased in the course of disease, either for selective loss or specific developmental impairment. We also found a selective reduction of layer V pyramidal neurons associated with layer V gliosis in the cerebral motor cortex. Our data indicate that in the ∆7 SMA model SMN loss is critical for the spinal cord, particularly for specific motor neuron pools. Neuronal loss, however, is not selective for lower motor neurons. These data further suggest that SMA pathogenesis is likely more complex than previously anticipated. The better knowledge of SMA models might be instrumental in shaping better therapeutic options for affected patients.     

Dissipative particle dynamics simulation of a gold nanoparticle system

Dissipative particle dynamics (DPD) was carried out to study systems containing gold atoms, organic ether (oligohydroquinonyl ether terminated with a thiol group) and organic solvents. The components in the simulated system are very different in size and chemical nature. Our simulation showed that the reproduction of the macroscopic experimental phase separation, properly dividing the polymeric molecule into beads, selecting the size of gold bead, and choosing the appropriate interaction parameters between beads are crucial. In addition, the solvent effect was the dominant factor for the formation of spherical aggregates of Au atoms and organic ether molecules. We report the interaction strengths between the solvent and gold clusters. Our work has demonstrated that DPD methods can be applied to the study of complex meso-scale systems.     

Horseradish peroxidase: a reliable or a misleading tool for the investigations on the origin of the proteins of the aqueous humor?

The concept of the blood-aqueous barrier is largely based on the use of horseradish peroxidase (HRP). The present investigation was designed to check its reliability as a macromolecular tracer, especially with regard to the transport of plasma proteins. Rabbits were killed 5 min to 24 h after being intravenously injected with HRP. The tracer diffused rapidly, reaching the aqueous humor of the eye in 3 min or less and was detected at high concentration in the narrow space between the outer epithelial layer of the ciliary epithelium and the wall of the pervious capillaries in the stroma of the processes. HRP appeared to migrate from the blood to the posterior chamber, permeating the tight junctions, viz., the anatomical basis of the blood-aqueous barrier. It was detected at higher concentration at the anterior surface of the iris, at short time intervals; this was interpreted as penetration of the tracer from the aqueous humor of the anterior chamber. The choroid was also labeled in continuation with the reaction in the stroma of the pars plana of the ciliary body which, in turn, sometimes reached the iris root. Therefore, the pervious blood vessels of the choroid could be a source of macromolecules for the iris root. HRP also induced the formation of lysosomes in the ciliary epithelium. This can hardly be accepted as the way in which plasma proteins are physiologically transported to the aqueous humor. However, the pathway of HRP migration over short time intervals seems to be in agreement with previous research indicating that the entrance of serum albumin into the posterior chamber is the first step of its incorporation into the aqueous humor. Received: 7 June 1996 / Accepted: 15 January 1997     

Interspecific Introgression in Cetaceans: DNA Markers Reveal Post-F1 Status of a Pilot Whale

Visual species identification of cetacean strandings is difficult, especially when dead specimens are degraded and/or species are morphologically similar. The two recognised pilot whale species (Globicephala melas and Globicephala macrorhynchus) are sympatric in the North Atlantic Ocean. These species are very similar in external appearance and their morphometric characteristics partially overlap; thus visual identification is not always reliable. Genetic species identification ensures correct identification of specimens. Here we have employed one mitochondrial (D-Loop region) and eight nuclear loci (microsatellites) as genetic markers to identify six stranded pilot whales found in Galicia (Northwest Spain), one of them of ambiguous phenotype. DNA analyses yielded positive amplification of all loci and enabled species identification. Nuclear microsatellite DNA genotypes revealed mixed ancestry for one individual, identified as a post-F1 interspecific hybrid employing two different Bayesian methods. From the mitochondrial sequence the maternal species was Globicephala melas. This is the first hybrid documented between Globicephala melas and G. macrorhynchus, and the first post-F1 hybrid genetically identified between cetaceans, revealing interspecific genetic introgression in marine mammals. We propose to add nuclear loci to genetic databases for cetacean species identification in order to detect hybrid individuals.     

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.     

Patterns of energy metabolism and growth kinetics of Kluyveromyces marxianus in whey chemostat culture

The influence of physiological parameters such as carbon substrate flux and O₂ uptake rates on energy metabolism are reported with reference to biomass productivity in whey chemostat culture. The combined results show that oxidoreductive energy metabolism may be attained independently of the yeast reaching its maximum respiratory capacity. A novel metabolic interpretation is presented proposing that a relative imbalance between glycolysis and subsequent oxidative steps alone is sufficient to account for the observed results. By means of a mathematical model the results could be reproduced under all experimental conditions. The new interpretation provides an insight into the manner in which energy mettbolism is regulated and influences growth-related process Kluyveromyces marxianus, as well as other yeasts with similar physiological characteristics. Correspondence to: J. I. Castrillo     

Staining of Oligodendroglia with Silver Ammino Tungstate in Unembedded and Embedded Material

Specimens of brain or spinal cord fixed in formalin, Cajal's formol-bromide, or Koenig, Groat and Windle's formalin-acacia can be used to stain oligodendrocytes in frozen, in paraffin, or in celloidin sections. The sections are soaked 3-5 min in 0.02% acetic acid, pH 3.4, then rinsed 2-3 sec in 3% H₂O₂ and transferred to a silver bath prepared as follows: Mix equal parts of 10% AgNO₃ and 10% Na₂WO₄, and dissolve the precipitate with concentrated NH₄OH; avoid an excess of ammonia. Silver at room temperature for 15-20 sec, develop in 1% formalin, dehydrate, and mount. For embedded material, prepare a mixture consisting of 1 part of 10% aqueous Aerosol MA and 4 parts of 10% Aerosol OT in 95% alcohol. Add 5 drops of this mixture to each 50 ml of dilute acetic acid and 3% H₂O₂; 5 drops to each 20 ml of the silver bath.     

TBCD Links Centriologenesis,Spindle Microtubule Dynamics,and Midbody Abscission in Human Cells

Microtubule-organizing centers recruit α- and β-tubulin polypeptides for microtubule nucleation. Tubulin synthesis is complex, requiring five specific cofactors, designated tubulin cofactors (TBCs) A–E, which contribute to various aspects of microtubule dynamics in vivo. Here, we show that tubulin cofactor D (TBCD) is concentrated at the centrosome and midbody, where it participates in centriologenesis, spindle organization, and cell abscission. TBCD exhibits a cell-cycle-specific pattern, localizing on the daughter centriole at G1 and on procentrioles by S, and disappearing from older centrioles at telophase as the protein is recruited to the midbody. Our data show that TBCD overexpression results in microtubule release from the centrosome and G1 arrest, whereas its depletion produces mitotic aberrations and incomplete microtubule retraction at the midbody during cytokinesis. TBCD is recruited to the centriole replication site at the onset of the centrosome duplication cycle. A role in centriologenesis is further supported in differentiating ciliated cells, where TBCD is organized into “centriolar rosettes”. These data suggest that TBCD participates in both canonical and de novo centriolar assembly pathways.     

Hydrolysis of nucleic acids by sepharose-micrococcal endonuclease

Initial reaction rates for the hydrolysis of nucleic acids with micrococcal endonuclease (EC 3.1.31.1) insolubilized on Sepharose are strongly influenced by diffusional limitations. Although the absolute values are low, they can be increased substantially by changing particle and pore size of the support, or enzyme concentration in the insoluble derivative. As a result of steric and diffusional limitations, the course of the reaction and selectivity to hydrolysis products for the insoluble derivatives are different to those of the native enzyme; the former produces mainly large and small fragments but few of intermediate size. Because of these differences in course and selectivity of the reaction, diffusional limitations become less important when high initial reaction rates are not required.