Proteasome inhibitor (MG132) rescues Nav1.5 protein content and the cardiac sodium current in dystrophin-deficient mdx (5cv) mice

The cardiac voltage-gated sodium channel, Nav1.5, plays a central role in cardiac excitability and impulse propagation and associates with the dystrophin multiprotein complex at the lateral membrane of cardiomyocytes. It was previously shown that Nav1.5 protein content and the sodium current (l Na) were both decreased in cardiomyocytes of dystrophin-deficient mdx (5cv) mice. In this study, wild-type and mdx (5cv) mice were treated for 7 days with the proteasome inhibitor MG132 (10 μg/Kg/24 h) using implanted osmotic mini pumps. MG132 rescued both the total amount of Nav1.5 protein and l Na but, unlike in previous studies, de novo expression of dystrophin was not observed in skeletal or cardiac muscle. This study suggests that the reduced expression of Nav1.5 in dystrophin-deficient cells is dependent on proteasomal degradation.

Histopathological alterations, EROD activity, CYP1A protein and biliary metabolites in gilthead seabream Sparus aurata exposed to benzo(a)pyrene

This study compared for seabream, Sparus aurata exposed to benzo(a)pyrene-B(a)P-, the response of molecular cytochrome P450 1A (CYP1A) and cellular histopathology biomarkers. Male gilthead seabream, Sparus aurata specimens were exposed for 20 days via water to a series of high B(a)P concentrations. CYP1A was assessed by measuring enzymatic activity (EROD) and CYP1A protein content, and cellular responses were evaluated by routine histopathological methods. In addition, biliary metabolites were measured in order to verify that B(a)P was absorbed and metabolised. Histological lesions, both in liver and gills, increased in parallel to B(a)P concentrations, with the majority of changes representing rather non-specific alterations. Hepatic EROD and CYP1A proteins data showed a concentration-dependent induction, while in the gills, EROD activity but not CYP1A proteins showed a non-monotonous dose response, with a maximum induction level at 200 microg B(a)P.L-1 and decreasing levels thereafter. The findings provide evidence that short-term, high dose exposure of fish can result in significant uptake and metabolism of the lipophilic B(a)P, and in pronounced pathological damage of absorptive epithelia and internal organs.

Nitric oxide synthase protein levels, not the mRNA, are downregulated in olfactory bulb interneurons of reeler mice

Homozygous mutations in the Reelin gene result in severe disruption of brain development. The histogenesis of layered regions, like the neocortex, hippocampus and the cerebellum, is most notably affected in mouse reeler mutants and similar traits are also present in mice lacking molecular components of the Reelin signalling pathway. Moreover, there is evidence for an additional role of Reelin in sustaining synaptic plasticity in adult networks. Nitric oxide is an important gaseous messenger that can modulate neuronal plasticity both in developing and mature synaptic networks and has been shown to facilitate synaptic changes in the hippocampus, cerebellum and olfactory bulb. We studied the distribution and content of neuronal nitric oxide synthase in the olfactory bulbs of reeler and wildtype mice. Immunocytochemistry reveals that Reelin and neuronal nitric oxide synthase containing interneurons are two distinct, non overlapping cell populations of the olfactory bulb. We show by in situ hybridization that both nitrergic and Reelin expressing cells represent only a subset of olfactory bulb GABAergic neurons. Immunoblots show that neuronal nitric oxide synthase protein content is decreased by two thirds in reeler mice causing a detectable loss of immunolabelled cells throughout the olfactory bulb of this strain. However, neuronal nitric oxide synthase mRNA levels, essayed by quantitative real-time RT-PCR, are unaffected in the reeler olfactory bulb. Thus, disruption of the Reelin signalling pathway may modify the turnover of neuronal nitric oxide synthase in the olfactory bulb and possibly affects nitric oxide functions in reeler mice.

Ramipril increases the protein level of skeletal muscle IRS-1 and alters protein tyrosine phosphatase activity in spontaneously hypertensive rats

To investigate mechanisms by which angiotensin converting enzyme (ACE)-inhibition increases insulin sensitivity, spontaneously hypertensive (SH) rats were treated with or without ramipril (1 mg/kg per day) for 12 weeks. Insulin binding and protein levels of insulin receptor substrate-1 (IRS-1), p85-subunit of phosphatidylinositol 3'-kinase (p85) and Src homology 2 domain-containing phosphatase-2 (SHP2) were then determined in hindlimb muscle and liver. Additionally, protein tyrosine phosphatase (PTPase) activities towards immobilized phosphorylated insulin receptor or phosphorylated IRS-1 of membrane (MF) and cytosolic fractions (CF) of these tissues were measured. Ramipril treatment increased IRS-1-protein content in muscle by 31+/-9% (P<0.05). No effects were observed on IRS-1 content in liver or on insulin binding or protein expression of p85 or SHP2 in both tissues. Ramipril treatment also increased dephosphorylation of insulin receptor by muscle CF (22.0+/-1.0%/60 min compared to 16.8+/-1.5%/60 min; P<0.05), and of IRS-1 by liver MF (37.2+/-1.7%/7.5 min compared to 33.8+/-1.7%/7.5 min; P<0.05) and CF (36.8+/-1.0%/7.5 min compared to 33.2+/-1.0%/7.5 min; P<0.05). We conclude that the observed effects of ACE-inhibition by ramipril on the protein expression of IRS-1 and on PTPase activity might contribute to its effect on insulin sensitivity.

Protein changes and proteolytic degradation in red and white clover plants subjected to waterlogging

Red (Trifolium pratense L., cv. “Start”) and white clover varieties (Trifolium repens L., cv. “Debut” and cv. “Haifa”) were waterlogged for 14 days and subsequently recovered for the period of 21 days. Physiological and biochemical responses of the clover varieties were distinctive, which suggested different sensitivity toward flooding. The comparative study of morphological and biochemical parameters such as stem length, leaflet area, dry weight, protein content, protein pattern and proteolytic degradation revealed prominent changes under waterlogging conditions. Protease activity in the stressed plants increased significantly, especially in red clover cv. “Start”, which exhibited eightfold higher azocaseinolytic activity compared to the control. Changes in the protein profiles were detected by SDS-PAGE electrophoresis. The specific response of some proteins (Rubisco, Rubisco-binding protein, Rubisco activase, ClpA and ClpP protease subunits) toward the applied stress was assessed by immunoblotting. The results characterized the red clover cultivar “Start” as the most sensitive toward waterlogging, expressing reduced levels of Rubisco large and small subunits, high content of ClpP protease subunits and increased activity of protease isoforms.

Focal cystic high-attenuation lesions: characterization in renal phantom by using photon-counting spectral CT--improved differentiation of lesion composition

To evaluate the capability of spectral computed tomography (CT) to improve the characterization of cystic high-attenuation lesions in a renal phantom and to test the hypothesis that spectral CT will improve the differentiation of cystic renal lesions with high protein content and those that have undergone hemorrhage or malignant contrast-enhancing transformation.

Comparison of three strip-type tests and two laboratory methods for salivary buffering analysis

This study evaluated the correlation between three strip-type, colorimetric tests and two laboratory methods with respect to the analysis of salivary buffering. The strip-type tests were saliva-check buffer, Dentobuff strip and CRT(®) Buffer test. The laboratory methods included Ericsson's laboratory method and a monotone acid/base titration to create a reference scale for the salivary titratable acidity. Additionally, defined buffer solutions were prepared and tested to simulate the carbonate, phosphate and protein buffer systems of saliva. The correlation between the methods was analysed by the Spearman's rank test. Disagreement was detected between buffering capacity values obtained with three strip-type tests that was more pronounced in case of saliva samples with medium and low buffering capacities. All strip-type tests were able to assign the hydrogencarbonate, di-hydrogenphosphate and 0.1% protein buffer solutions to the correct buffer categories. However, at 0.6% total protein concentrations, none of the test systems worked accurately. Improvements are necessary for strip-type tests because of certain disagreement with the Ericsson's laboratory method and dependence on the protein content of saliva.

Performance and metabolic profile of dairy cows during a lactational and deliberately induced negative energy balance with subsequent realimentation

Homeorhetic and homeostatic controls in dairy cows are essential for adapting to alterations in physiological and environmental conditions. To study the different mechanisms during adaptation processes, effects of a deliberately induced negative energy balance (NEB) by feed restriction near 100 d in milk (DIM) on performance and metabolic measures were compared with lactation energy deficiency after parturition. Fifty multiparous cows were studied in 3 periods (1=early lactation up to 12 wk postpartum; 2=feed restriction for 3 wk beginning at 98+/-7 DIM with a feed-restricted and control group; and 3=a subsequent realimentation period for the feed-restricted group for 8 wk). In period 1, despite NEB in early lactation [-42 MJ of net energy for lactation (NE(L))/d, wk 1 to 3] up to wk 9, milk yield increased from 27.5+/-0.7 kg to a maximum of 39.5+/-0.8 kg (wk 6). For period 2, the NEB was induced by individual limitation of feed quantity and reduction of dietary energy density. Feed-restricted cows experienced a greater NEB (-63 MJ of NEL/d) than did cows in early lactation. Feed-restricted cows in period 2 showed only a small decline in milk yield of -3.1+/-1.1 kg and milk protein content of -0.2+/-0.1% compared with control cows (30.5+/-1.1 kg and 3.8+/-0.1%, respectively). In feed-restricted cows (period 2), plasma glucose was lower (-0.2+/-0.0 mmol/L) and nonesterified fatty acids higher (+0.1+/-0.1 mmol/L) compared with control cows. Compared with the NEB in period 1, the decreases in body weight due to the deliberately induced NEB (period 2) were greater (56+/-4 vs. 23+/-3 kg), but decreases in body condition score (0.16+/-0.03 vs. 0.34+/-0.04) and muscle diameter (2.0+/-0.4 vs. 3.5+/-0.4 mm) were lesser. The changes in metabolic measures in period 2 were marginal compared with the adjustments directly after parturition in period 1. Despite the greater induced energy deficiency at 100 DIM than the early lactation NEB, the metabolic load experienced by the dairy cows was not as high as that observed in early lactation. The different effects of energy deficiency at the 2 stages in lactation show that metabolic problems in early lactating dairy cows are not due only to the NEB, but mainly to the specific metabolic regulation during this period.

Mutations in the bovine ABCG2 and the ovine MSTN gene added to the few quantitative trait nucleotides identified in farm animals: a mini-review

The progress in molecular genetics in animal breeding is moderately effective as compared to traditional animal breeding using quantitative genetic approaches. There is an extensive disparity between the number of reported quantitative trait loci (QTLs) and their linked genetic variations in cattle, pig, and chicken. The identification of causative mutations affecting quantitative traits is still very challenging and hampered by the cloudy relationship between genotype and phenotype. There are relatively few reports in which a successful identification of a causative mutation for an animal production trait was demonstrated. The examples that have attracted considerable attention from the animal breeding community are briefly summarized and presented in a table. In this mini-review, the recent progress in mapping quantitative trait nucleotides (QTNs) are reviewed, including the ABCG2 gene mutation that underlies a QTL for fat and protein content and the ovine MSTN gene mutation that causes muscular hypertrophy in Texel sheep. It is concluded that the progress in molecular genetics might facilitate the elucidation of the genetic architecture of QTLs, so that also the high-hanging fruits can be harvested in order to contribute to efficient and sustainable animal production.

Differences in milk production, glucose metabolism, and carcass composition of 2 Charolais x Holstein F2 families derived from reciprocal paternal and maternal grandsire crosses

Two F(2) Charolais x German Holstein families comprising full and half sibs share identical but reciprocal paternal and maternal Charolais grandfathers differ in milk production. We hypothesized that differences in milk production were related to differences in nutritional partitioning revealed by glucose metabolism and carcass composition. In 18F(2) cows originating from mating Charolais bulls to German Holstein cows and a following intercross of the F(1) individuals (n=9 each for family Ab and Ba; capital letters indicate the paternal and lowercase letter the maternal grandsire), glucose tolerance tests were performed at 10 d before calving and 30 and 93 d in milk (DIM) during second lactation. Glucose half-time as well as areas under the concentration curve for plasma glucose and insulin were calculated. At 94 DIM cows were infused intravenously with 18.3 micromol of d-[U-(13)C(6)]glucose/kg(0.75) of BW, and blood samples were taken to measure rate of glucose appearance and glucose oxidation as well as plasma concentrations of metabolites and hormones. Cows were slaughtered at 100 DIM and carcass size and composition was evaluated. Liver samples were taken to measure glycogen and fat content, gene expression levels, and enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase as well as gene expression of glucose transporter 2. Milk yield was higher and milk protein content at 30 DIM was lower in Ba than in Ab cows. Glucose half-life was higher but insulin secretion after glucose challenge was lower in Ba than in Ab cows. Cows of Ab showed higher glucose oxidation, and plasma concentrations at 94 DIM were lower for glucose and insulin, whereas beta-hydroxybutyrate was higher in Ba cows. Hepatic gene expression of pyruvate carboxylase, glucose 6-phosphatase, and glucose transporter 2 were higher whereas phosphoenolpyruvate carboxykinase activities were lower in Ba than in Ab cows. Carcass weight as well as fat content of the carcass were higher in Ab than in Ba cows, whereas mammary gland mass was lower in Ab than in Ba cows. Fat classification indicated leaner carcass composition in Ba than in Ab cows. In conclusion, the 2 families showed remarkable differences in milk production that were accompanied by changes in glucose metabolism and body composition, indicating capacity for milk production as main metabolic driving force. Sex chromosomal effects provide an important regulatory mechanism for milk performance and nutrient partitioning that requires further investigation.

Management, feeding, production, reproduction and udder health on organic and conventional Swiss dairy farms

Organic dairy farms (OP; n=60) and conventional dairy farms (integrated production, IP; n=60), matched in size, location, and agricultural zone (altitude), were studied for possible differences in management, feeding, production, reproduction and udder health. OP and IP farms were similar in size (17.7 and 16.9 ha), milk quota (65900 and 70,000 kg/year), cow number (14 and 15), cow age (5.3 and 5.2 years), housing of cows of the Simmental x Red Holstein or Holstein breeds (87 and 75%; 45 and 60%), but differed significantly with respect to loose housing systems (18 and 7%), outside paddocks (98 and 75%), energy-corrected 305-d milk yield (5,695 and 6,059 kg), milk protein content (31.8 and 32.7 g/kg), use of bucket milking systems (73 and 33%), observance of regular (12-h) milking intervals (47 and 68%), routine application of the California-Mastitis-Test (10 and 28%), teat dipping after milking (25 and 43%) and blanket dry cow treatments (0 and 45%). Milk somatic cell counts on OP and IP farms (119 000 and 117,000/mL) and reproduction data were similar and there were no significant differences between OP and IP farms as concerns available feeds, planning and management of feeding. Alternative veterinary treatments were used more often on OP than IP farms (55 and 17%). Main causes for cow replacements on OP and IP farms were fertility disorders (both 45%), age (40 and 42%), sale (30 and 37%) and udder health (35 and 13%).Between OP and IP Swiss dairy farms thus relatively few larger differences were found.

Acetyl-L-carnitine feeding to unloaded rats triggers in soleus muscle the coordinated expression of genes involved in mitochondrial biogenesis

The expressional profile of mitochondrial transcripts and of genes involved in the mitochondrial biogenesis pathway induced by ALCAR daily supplementation in soleus muscle of control and unloaded 3-month-old rats has been analyzed. It has been found that ALCAR treatment is able to upregulate the expression level of mitochondrial transcripts (COX I, ATP6, ND6, 16 S rRNA) in both control and unloaded animals. Interestingly, ALCAR feeding to unloaded rats resulted in the increase of transcript level for master factors involved in mitochondrial biogenesis (PGC-1alpha, NRF-1, TFAM). It also prevented the unloading-induced downregulation of mRNA levels for kinases able to transduce metabolic (AMPK) and neuronal stimuli (CaMKIIbeta) into mitochondrial biogenesis. No significant effect on the expressional level of such genes was found in control ALCAR-treated rats. In addition, ALCAR feeding was able to prevent the loss of mitochondrial protein content due to unloading condition. Correlation analysis revealed a strong coordination in the expression of genes involved in mitochondrial biogenesis only in ALCAR-treated suspended animals, supporting a differentiated effect of ALCAR treatment in relation to the loading state of the soleus muscle. In conclusions, we demonstrated the ability of ALCAR supplementation to promote only in soleus muscle of hindlimb suspended rats an orchestrated expression of genes involved in mitochondrial biogenesis, which might counteract the unloading-induced metabolic changes, preventing the loss of mitochondrial proteins.

Pharmacodynamics of antibiotics in the therapy of meningitis: infection model observations

Detailed studies of pharmacodynamic principles relevant to the therapy of bacterial meningitis are difficult to perform in man, while the rabbit model of bacterial meningitis has proved to be extremely valuable and has led to insights that appear relevant for the treatment of humans. Most importantly in the light of the restricted penetration of antibiotics into the CSF, animal studies have shown that in meningitis there is a dose-response curve between the CSF concentrations achieved by antibiotics and their bactericidal activity. This appears to be true for all classes of antibiotics thus far examined, including the beta-lactams, which do not show such a dose-response behaviour in other infections. Only CSF concentrations that exceed the MBC of the infecting organism by at least 10-30-fold achieve consistent and rapid bactericidal activity. Such rapid bactericidal activity is a requirement for successful therapy with beta-lactams and can be impaired with certain antibiotics by the specific conditions in infected CSF (protein content; acidic pH; slow-growing bacteria). However, rapid antibiotic killing of the infecting organisms may not be without adverse effects either. Some antibiotics, particularly beta-lactams lead to the brisk liberation of bacterial cell wall components (e.g. endotoxin, in the case of Gram-negative organisms) which have an inflammatory effect on the host and can lead to a temporary deterioration of the disease. Dexamethasone, when administered with the antibiotic, can prevent some of the adverse effects of rapid bacterial lysis.

Pseudovacuoles--immobilized by high-pressure freezing--are associated with blebbing in walker carcinosarcoma cells

By applying high pressure freezing and freeze-substitution, we observed large inclusions of homogeneous appearance in the front of locomoting Walker carcinosarcoma cells that have not been described earlier. Live cell imaging revealed that these inclusions were poor in lipids and nucleic acids but had a high lysine (and hence protein) content. Usually one such structure 2-5 mum in size was present at the front of motile Walker cells, predominantly in the immediate vicinity of newly forming blebs. By correlating the lysine-rich areas in fixed and embedded cells with electron microscopic pictures, inclusions could be assigned to confined, faintly stained cytoplasmic areas that lacked a surrounding membrane; they were therefore called pseudovacuoles. After high-pressure freezing and freeze substitution, pseudovacuoles appeared to be filled with 20 nm large electron-transparent patches surrounded by 12 and 15 nm large particles. The heat shock protein Hsp90 was identified by peptide sequencing as a major fluorescent band on SDS-PAGE of lysine-labelled Walker cell extracts. By immunofluorescence, Hsp90 was found to be enriched in pseudovacuoles. Colocalization of the lysine with a potassium-specific dye in living cells revealed that pseudovacuoles act as K+ stores in the vicinity of forming blebs. We propose that pseudovacuoles might support blebbing by locally regulating the intracellular hydrostatic pressure.

Olfactory bulb interneurons releasing NO exhibit the Reelin receptor ApoEr2 and part of those targeted by NO express Reelin

Nitric oxide (NO) and Reelin both modulate neuronal plasticity in developing and mature synaptic networks. We recently showed a loss of neuronal nitric oxide synthase (nNOS) protein in the olfactory bulb of reeler mutants and advanced the hypothesis that the Reelin and NO signalling pathways may influence each other. We now studied the distribution of NO sensitive guanylyl cyclase (NOsGC), Reelin and its receptor Apolipoprotein E2 (ApoEr2) in the olfactory bulb by multiple fluorescence labelling and tested whether nNOS and ApoEr2 colocalize in this area. We also essayed the protein content of NOsGC in the reeler olfactory bulb and tested whether there are any changes in nNOS and NOsGC protein in other reeler brain areas. Olfactory bulb interneurons expressing ApoEr2 and nNOS are only few in the glomerular layer but represent the large majority of granule cell layer interneurons. Conversely, NOsGC interneurons are rare in the granule cell layer and abundant as periglomerular cells. Reelin containing periglomerular cells almost entirely belong to the NOsGC subset. These data further support the hypothesis of a reciprocal signalling between Reelin/NOsGC and ApoEr2/nNOS expressing neurons to affect olfactory bulb activity. We also show that a significant rise in NOsGC content accompanies the decrease of nNOS protein in the reeler olfactory bulb. The same reciprocal changes present in the cortex/striatum and the hippocampus of reeler mice. Thus, the influence that the deficit of extracellular Reelin seems to exert on nNOS and its receptor is not limited to the olfactory bulb but is a general feature of the reeler brain.