Control of sustained attention and impulsivity by Gq-protein signalling in parvalbumin interneurons of the anterior cingulate cortex.

The anterior cingulate cortex (ACC) has been implicated in attention deficit hyperactivity disorder (ADHD). More specifically, an appropriate balance of excitatory and inhibitory activity in the ACC may be critical for the control of impulsivity, hyperactivity, and sustained attention which are centrally affected in ADHD. Hence, pharmacological augmentation of parvalbumin- (PV) or somatostatin-positive (Sst) inhibitory ACC interneurons could be a potential treatment strategy. We, therefore, tested whether stimulation of Gq-protein-coupled receptors (GqPCRs) in these interneurons could improve attention or impulsivity assessed with the 5-choice-serial reaction-time task in male mice. When challenging impulse control behaviourally or pharmacologically, activation of the chemogenetic GqPCR hM3Dq in ACC PV-cells caused a selective decrease of active erroneous-i.e. incorrect and premature-responses, indicating improved attentional and impulse control. When challenging attention, in contrast, omissions were increased, albeit without extension of reward latencies or decreases of attentional accuracy. These effects largely resembled those of the ADHD medication atomoxetine. Additionally, they were mostly independent of each other within individual animals. GqPCR activation in ACC PV-cells also reduced hyperactivity. In contrast, if hM3Dq was activated in Sst-interneurons, no improvement of impulse control was observed, and a reduction of incorrect responses was only induced at high agonist levels and accompanied by reduced motivational drive. These results suggest that the activation of GqPCRs expressed specifically in PV-cells of the ACC may be a viable strategy to improve certain aspects of sustained attention, impulsivity and hyperactivity in ADHD.

Open-MAC: A low-cost open-source motorized commutator for electro- and opto-physiological recordings in freely moving rodents.

In vivo electro- and optophysiology experiments in rodents reveal the neural mechanisms underlying behavior and brain disorders but mostly involve a cable connection between an implant in the animal and an external recording device. Standard tethers with thin cables or non-motorized commutators require constant monitoring and often manual interference to untwist the cable. Motorized commutators offer a solution, but those few that are commercially available are expensive and often not adapted to widely used connector standards of the open-source community like 12-channel SPI. Here we introduce an open-source motorized all-in-one commutator (Open-MAC): a low-cost (240-390 EUR), low-torque motorized commutator that can operate with minimal audible noise in a torque-based mode relying on dual magnetic Hall sensors. It further includes electronics to operate in a torque-free, online pose-estimation-based mode, with future developments. Operation is controlled by an onboard microcontroller (XIAO SAMD21) powered by a USB-C cable or DC power supply. The body and movable parts are 3D-printed. Different Open-MAC versions can support electrophysiology with up to 64 recording channels using the Open-Ephys / IntanTM recording systems as well as miniature endoscope (miniscope) recordings using the UCLA Miniscope v3/4, and can host a fibre for optogenetic modulation.

Chronic N-acetylcysteine treatment improves anhedonia and cognition in a mouse model of the schizophrenia prodrome.

Schizophrenia is a severe psychiatric disorder whose neurodevelopmental pathogenesis includes a prodromal phase before its diagnostically decisive-namely psychotic-symptoms are present. This prodrome is characterized by cognitive and affective deficits, and it may constitute a critical time period for an early therapeutic intervention to improve or even prevent further disease development. N-acetylcysteine (NAC) is an easily repurposable compound that has recently shown promise in improving non-psychotic symptoms in patients with established schizophrenia. Its therapeutic mechanism may involve the amelioration of circuit abnormalities like a hyper-glutamatergic state and oxidative stress in cortex which have been proposed to drive the pathogenesis of this disease. However, it is currently unknown to what extent NAC can actually improve prodromal aberrations. To investigate this preclinically, we deployed the cyclin-D2 knockout mouse model (CD2-KO) that shares physiological and behavioral abnormalities with the schizophrenia prodrome, including a hyperactive CA1 region, and cognitive and affective deficits. Applying NAC chronically in drinking water (0.9 g/l) during development (∼P22-P70), we found that excessive novelty-induced hyperlocomotion was neither ameliorated during (∼P68) nor after (∼P75) treatment; similarly, T-maze working memory (tested after treatment; ∼P84) was unaffected. However, once chronic NAC treatment was resumed (at approximately P134) in those mice that had received it before, working memory, cognitive flexibility (tested under NAC), and anhedonia (sucrose-preference, tested 1 day after NAC-treatment stopped) were improved in CD2-KO mice. This suggests that chronic NAC treatment may be a therapeutic strategy to improve some cognitive and affective dysfunctions in the schizophrenia prodrome.

Enhanced Cortical Metabolic Activity in Females and Males of a Slow Progressing Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with selective degeneration of motor neurons in the central nervous system. The pathophysiology of ALS is not well understood. We have used 1H-[13C]-NMR spectroscopy together with an administration of [1,6-13C2]glucose and [2-13C]acetate in female and male SOD1G37R mice to assess neuronal and astroglial metabolic activity, respectively, in the central nervous system in ALS condition. The female (p = 0.0008) and male (p < 0.0001) SOD1G37R mice exhibited decreased forelimb strength when compared with wild-type mice. There was a reduction in N-acetylaspartylglutamate level, and elevation in myo-inositol in the spinal cord of female and male SOD1G37R mice. The transgenic male mice exhibited increased acetate oxidation in the spinal cord (p = 0.05) and cerebral cortex (p = 0.03), while females showed an increase in the spinal cord (p = 0.02) only. As acetate is transported and preferentially metabolized in the astrocytes, the finding of increased rate of acetate oxidation in the transgenic mice is suggestive of astrocytic involvement in the pathogenesis of ALS. The rates of glucose oxidation in glutamatergic (p = 0.0004) and GABAergic neurons (p = 0.0052) were increased in the cerebral cortex of male SOD1G37R mice when compared with the controls. The female mice showed an increase in glutamatergic (p = 0.039) neurometabolic activity only. The neurometabolic activity was unperturbed in the spinal cord of either sex. These data suggest differential changes in neurometabolic activity across the central nervous system in SOD1G37R mice.

Distinct contributions of GluA1-containing AMPA receptors of different hippocampal subfields to salience processing, memory and impulse control.

Schizophrenia is associated with a broad range of severe and currently pharmacoresistant cognitive deficits. Prior evidence suggests that hypofunction of AMPA-type glutamate receptors (AMPARs) containing the subunit GLUA1, encoded by GRIA1, might be causally related to impairments of selective attention and memory in this disorder, at least in some patients. In order to clarify the roles of GluA1 in distinct cell populations, we investigated behavioural consequences of selective Gria1-knockout in excitatory neurons of subdivisions of the prefrontal cortex and the hippocampus, assessing sustained attention, impulsivity, cognitive flexibility, anxiety, sociability, hyperactivity, and various forms of short-term memory in mice. We found that virally induced reduction of GluA1 across multiple hippocampal subfields impaired spatial working memory. Transgene-mediated ablation of GluA1 from excitatory cells of CA2 impaired short-term memory for conspecifics and objects. Gria1 knockout in CA3 pyramidal cells caused mild impairments of object-related and spatial short-term memory, but appeared to partially increase social interaction and sustained attention and to reduce motor impulsivity. Our data suggest that reduced hippocampal GluA1 expression-as seen in some patients with schizophrenia-may be a central cause particularly for several short-term memory deficits. However, as impulse control and sustained attention actually appeared to improve with GluA1 ablation in CA3, strategies of enhancement of AMPAR signalling likely require a fine balance to be therapeutically effective across the broad symptom spectrum of schizophrenia.

A low-cost open-source 5-choice operant box system optimized for electrophysiology and optophysiology in mice.

Operant boxes enable the application of complex behavioural paradigms to support circuit neuroscience and drug discovery research. However, commercial operant box systems are expensive and often not optimised for combining behaviour with neurophysiology. Here we introduce a fully open-source Python-based operant-box system in a 5-choice design (pyOS-5) that enables assessment of multiple cognitive and affective functions. It is optimized for fast turn-over between animals, and for testing of tethered mice for simultaneous physiological recordings or optogenetic manipulation. For reward delivery, we developed peristaltic and syringe pumps based on a stepper motor and 3D-printed parts. Tasks are specified using a Python-based syntax implemented on custom-designed printed circuit boards that are commercially available at low cost. We developed an open-source graphical user interface (GUI) and task definition scripts to conduct assays assessing operant learning, attention, impulsivity, working memory, or cognitive flexibility, alleviating the need for programming skills of the end user. All behavioural events are recorded with millisecond resolution, and TTL-outputs and -inputs allow straightforward integration with physiological recordings and closed-loop manipulations. This combination of features realizes a cost-effective, nose-poke-based operant box system that allows reliable circuit-neuroscience experiments investigating correlates of cognition and emotion in large cohorts of subjects.

Control of impulsivity by Gi-protein signalling in layer-5 pyramidal neurons of the anterior cingulate cortex.

Pathological impulsivity is a debilitating symptom of multiple psychiatric diseases with few effective treatment options. To identify druggable receptors with anti-impulsive action we developed a systematic target discovery approach combining behavioural chemogenetics and gene expression analysis. Spatially restricted inhibition of three subdivisions of the prefrontal cortex of mice revealed that the anterior cingulate cortex (ACC) regulates premature responding, a form of motor impulsivity. Probing three G-protein cascades with designer receptors, we found that the activation of Gi-signalling in layer-5 pyramidal cells (L5-PCs) of the ACC strongly, reproducibly, and selectively decreased challenge-induced impulsivity. Differential gene expression analysis across murine ACC cell-types and 402 GPCRs revealed that - among Gi-coupled receptor-encoding genes - Grm2 is the most selectively expressed in L5-PCs while alternative targets were scarce. Validating our approach, we confirmed that mGluR2 activation reduced premature responding. These results suggest Gi-coupled receptors in ACC L5-PCs as therapeutic targets for impulse control disorders.

Delayed-matching-to-position working memory in mice relies on NMDA-receptors in prefrontal pyramidal cells.

A hypofunction of N-methyl-D-aspartate glutamate receptors (NMDARs) has been implicated in the pathogenesis of schizophrenia by clinical and rodent studies. However, to what extent NMDAR-hypofunction in distinct cell-types across the brain causes different symptoms of this disease is largely unknown. One pharmaco-resistant core symptom of schizophrenia is impaired working memory (WM). NMDARs have been suggested to mediate sustained firing in excitatory neurons of the prefrontal cortex (PFC) that might underlie WM storage. However, if NMDAR-hypofunction in prefrontal excitatory neurons may indeed entail WM impairments is unknown. We here investigated this question in mice, in which NMDARs were genetically-ablated in PFC excitatory cells. This cell type-selective NMDAR-hypofunction caused a specific deficit in a delayed-matching-to-position (DMTP) 5-choice-based operant WM task. In contrast, T-maze rewarded alternation and several psychological functions including attention, spatial short-term habituation, novelty-processing, motivation, sociability, impulsivity, and hedonic valuation remained unimpaired at the level of GluN1-hypofunction caused by our manipulation. Our data suggest that a hypofunction of NMDARs in prefrontal excitatory neurons may indeed cause WM impairments, but are possibly not accounting for most other deficits in schizophrenia.

Lack of redundancy between electrophysiological measures of long-range neuronal communication.

BACKGROUND: Communication between brain areas has been implicated in a wide range of cognitive and emotive functions and is impaired in numerous mental disorders. In rodent models, various metrics have been used to quantify inter-regional neuronal communication. However, in individual studies, typically, only very few measures of coupling are reported and, hence, redundancy across such indicators is implicitly assumed. RESULTS: In order to test this assumption, we here comparatively assessed a broad range of directional and non-directional metrics like coherence, Weighted Phase Lag Index (wPLI), phase-locking value (PLV), pairwise phase consistency (PPC), parametric and non-parametric Granger causality (GC), partial directed coherence (PDC), directed transfer function (DTF), spike-phase coupling (SPC), cross-regional phase-amplitude coupling, amplitude cross-correlations and others. We applied these analyses to simultaneous field recordings from the prefrontal cortex and the ventral and dorsal hippocampus in the schizophrenia-related Gria1-knockout mouse model which displays a robust novelty-induced hyperconnectivity phenotype. Using the detectability of coupling deficits in Gria1-/- mice and bivariate correlations within animals as criteria, we found that across such measures, there is a considerable lack of functional redundancy. Except for three pairwise correlations-PLV with PPC, PDC with DTF and parametric with non-parametric Granger causality-almost none of the analysed metrics consistently co-varied with any of the other measures across the three connections and two genotypes analysed. Notable exceptions to this were the correlation of coherence with PPC and PLV that was found in most cases, and partial correspondence between these three measures and Granger causality. Perhaps most surprisingly, partial directed coherence and Granger causality-sometimes regarded as equivalent measures of directed influence-diverged profoundly. Also, amplitude cross-correlation, spike-phase coupling and theta-gamma phase-amplitude coupling each yielded distinct results compared to all other metrics. CONCLUSIONS: Our analysis highlights the difficulty of quantifying real correlates of inter-regional information transfer, underscores the need to assess multiple coupling measures and provides some guidelines which metrics to choose for a comprehensive, yet non-redundant characterization of functional connectivity.

Effect of plant secondary compounds on in vitro methane, ammonia production and ruminal protozoa population.

AIMS: The objective of this study was to evaluate the potential of secondary plant metabolites from 38 sources to serve as antimethanogenic additives in ruminant diets. The effect of leaf tannins from these different plant sources on rumen fermentation, protozoal populations and methanogenesis was also studied. METHODS AND RESULTS: Samples (200 mg dry matter, DM) were incubated without and with polyethylene glycol (PEG)-6000 (400 mg DM) as a tannin binder during 24-h incubation in the in vitro Hohenheim gas system. In the leaf samples, total phenol (g kg(-1) DM) was maximum in Pimenta officinalis (312) followed by Oenothera lamarckiana (185) and Lawsonia inermis (105). Of the 38 samples, condensed tannins exceeded 4.0 g kg(-1) in only Alpinia galanga (7.50), Cinnamomum verum (4.58), Pelargonium graveolens (18.7) and Pimenta officinalis (23.2) and were not detected in seven samples. When the bioactivity of the leaf samples was assessed using the tannin bioassay, the percentage increase in the amount of gas produced during incubation of samples with the tannin-binding agent PEG-6000 over the amount produced during incubation without the tannin binder ranged from nil (zero) to 367%, with the highest being recorded with A. galanga leaves. The ratio of methane reduction per ml of total gas reduction was maximum with Rauvolfia serpentina (131.8) leaves, followed by Indigofera tinctoria (16.8) and Withania somnifera (10.2) leaves. Total and differential protozoal counts increased with added PEG in twenty-two samples, maximum being in Pimenta officinalis. Increased accumulation of total volatile fatty acids during incubation with added PEG-6000 was recorded, and the values ranged from zero to 61%. However, the increase was significant in only 11 of the 38 tannin sources tested indicating noninterference of tannin on in vitro fermentation of carbohydrates by the majority of samples tested. Conversely, in 26 of 38 plant sources, the leaf tannins reduced N-digestibility as evidenced by increased accumulation of NH3 -N with added PEG. CONCLUSIONS: Our study unequivocally demonstrated that plants containing secondary metabolites such as Rauvolfia serpentine, Indigofera tinctoria and Withania somnifera have great potential to suppress methanogenesis with minimal adverse effect of feedstuff fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: It was established that methanogenesis was not essentially related to the density of protozoa population in vitro. The tannins contained in these plants could be of interest in the development of new additives in ruminant nutrition.

Effect of medicinal and aromatic plants on rumen fermentation, protozoa population and methanogenesis in vitro.

The potential of tannins from 21 medicinal and aromatic plant leaves as antimethanogenic additives in ruminant feeds was investigated. The effect of tannin from these leaves on rumen fermentation parameters, protozoa population and methanogenesis was studied by incubating the samples [200 mg dry matter (DM)] without and with polyethylene glycol (PEG)-6000 (400 mg DM) as a tannin binder during 24-h incubation in the in vitro Hohenheim gas method. Based on the methane percentage estimated in the total gas produced, methane production in millilitre was calculated [methane volume (ml) = methane % × total gas produced (ml) in 24 h]. In the samples, crude protein and neutral detergent fibre (g/kg DM) ranged from 113 to 172 and from 352 to 444 respectively. The total phenol (TP; g/kg DM) content was highest in Terminalia chebula (274) followed by Hemigraphis colorata (71) and Sapindus laurifolia (51) respectively. In the remaining samples, it was <43 g/kg DM. Activity of tannins, as represented by the increase in gas volume on addition of PEG, ranged from 0 to 133%, with the highest being recorded in T. chebula. The per cent increase in methane on PEG addition was 0 for Ammi majus, Aristolochia indica, Cascabela thevetia, Ipomea nil and Lantana camara, illustrating that tannins present in these samples had no effect on methane concentration. The PEG addition increased the total protozoa count by >50% in A. indica and C. thevetica. One of the important findings of our study was that of the 21 samples screened, Entodinia population increased in 12 with PEG as compared to 7 where Holotricha increased, indicating higher susceptibility of Entodinia to tannin. There was no increase in the protozoa population with PEG when incubating Cardiospermum halicacabum, Clerodendrum inerme, Dioscorea floribunda, Nerium oleander and Selastras paniculatus, which strongly suggested that methane suppression recorded in these samples was not because of a defaunating effect of their tannins per se. The fermentation pattern reflected increased total volatile fatty acid (TVFA) concentration from 0 to 28.3% with PEG addition among the leaves. Our results confirmed further observations that methanogenesis in vitro is not essentially related to density of protozoa population. Secondly, medicinal and aromatic plants such as C. inerme, Gymnema sylvestre and Sapindus laurifolia containing tannins appear to have a potential to suppress in vitro methanogenesis.

Expression and biochemical characterization of two novel feruloyl esterases derived from fecal samples of Rusa unicolor and Equus burchelli.

Two novel genes (tvms10a, tvmz2a) were identified in the metagenomic DNA of Rusa unicolor and Equus burchelli fecal samples. The amplified PCR product of tvms10a is composed of 917bp and the gene was found to encode a protein containing 165 amino acids, while the tvmz2a PCR product was 1053bp long encoding 298 amino acid proteins. The gene has 72% primary sequence identity with Clostridiales sp. These amplified PCR products which can encode FAE were cloned into pGEMT Easy TA cloning vector and then sub-cloned into the EcoRI site of pET32a expression vector to generate pET32-tvms10a and pET32-tvmz2a, which was then transformed into Escherichia coli BL21. The recombinants were grown in LB medium and gene expression was induced with IPTG for 6h. Purified recombinant Tvms10a and Tvmz2a proteins showed molecular masses of 18.6 and 31.2kDa respectively, and displayed hydrolytic activity towards substrate ethyl ferulate. The activities of Tvms10a and Tvmz2a produced in E. coli were 15 and 9U/min respectively, and their specific activities 16.6 and 10.4U/mg protein respectively. The optimal pH is between 5.0 and 8.0 and the optimal temperature is 37°C for enzyme reaction. Unusually, these proteins were found to be capable of releasing ferulic acid (FA) and diferulic acid (diFA) from untreated crude plant cell wall materials. The substrate utilization preferences and sequence similarity of these clones place it in the type-D sub-class of FAE.

Enzymatic production of xylooligosaccharides from alkali solubilized xylan of natural grass (Sehima nervosum).

In this study, a process for producing XOS from Sehima nervosum grass was developed. The grass contains 28.1% hemicellulose. NaOH and steam application yielded 98% of original xylan in contrast to 85% by KOH application. Hydrolysis of xylan with commercial xylanase caused breakdown into XOS comprising of xylobiose, xylotriose along with xylose. Response surface model (RSM) revealed highest xylobiose yield (11 g/100g xylan) at pH 5.03, temperature 45.19°C, reaction time 10.11h with enzyme dose 17.41 U. Similarly for maximizing xylotriose yield, ideal hydrolysis conditions were pH 5.11, temperature 40.33°C, reaction time 16.55 h with enzyme dose 13.20 U. A two step process encompassing xylan fractionation and enzymatic hydrolysis enabled XOS production from the S. nervosum grass.

Phenolic Composition, Fermentation Profile, Protozoa Population and Methane Production from Sheanut (Butryospermum Parkii) Byproducts In vitro.

Sheanut cake (SNC), expeller (SNE) and solvent extractions (SNSE) samples were evaluated to determine their suitability in animal feeding. The CP content was highest in SNSE (16.2%) followed by SNE (14.7%) and SNC (11.6%). However, metabolizable energy (ME, MJ/kg) was maximum in SNC (8.2) followed by SNE (7.9) and SNSE (7.0). The tannin phenol content was about 7.0 per cent and mostly in the form of hydrolyzable tannin (HT), whereas condensed tannin (CT) was less than one per cent. The in vitro gas production profiles indicated similar y max (maximum potential of gas production) among the 3 by-products. However, the rate of degradation (k) was maximum in SNC followed by SNE and SNSE. The t(1/2) (time taken for reaching half asymptote) was lowest in SNC (14.4 h) followed by SNE (18.7 h) and SNSE (21.9 h). The increment in the in vitro gas volume (ml/200 mg DM) with PEG (polyethylene glycol)-6000 (as a tannin binder) addition was 12.0 in SNC, 9.6 in SNE and 11.0 in SNSE, respectively. The highest ratio of CH4 (ml) reduction per ml of the total gas, an indicator of the potential of tannin, was recorded in SNE (0.482) followed by SNC (0.301) and SNSE (0.261). There was significant (p<0.05) reduction in entodinia population and total protozoa population. Differential protozoa counts revealed that Entodinia populations increased to a greater extent than Holotricha when PEG was added. This is the first report on the antimethanogenic property of sheanut byproducts. It could be concluded that all the three forms of SN byproducts are medium source of protein and energy for ruminants. There is a great potential for SN by-products to be incorporated in ruminant feeding not only as a source of energy and protein, but also to protect the protein from rumen degradation and suppress enteric methanogenesis.

Modeling and forecasting livestock feed resources in India using climate variables.

The availability and efficient use of the feed resources in India are the primary drivers to maximize productivity of Indian livestock. Feed security is vital to the livestock management, extent of use, conservation and productivity enhancement. Assessment and forecasting of livestock feed resources are most important for effective planning and policy making. In the present study, 40 years of data on crop production, land use pattern, rainfall, its deviation from normal, area under crop and yield of crop were collected and modeled to forecast the likely production of feed resources for the next 20 years. The higher order auto-regressive (AR) models were used to develop efficient forecasting models. Use of climatic variables (actual rainfall and its deviation from normal) in combination with non-climatic factors like area under each crop, yield of crop, lag period etc., increased the efficiency of forecasting models. From the best fitting models, the current total dry matter (DM) availability in India was estimated to be 510.6 million tonnes (mt) comprising of 47.2 mt from concentrates, 319.6 mt from crop residues and 143.8 mt from greens. The availability of DM from dry fodder, green fodder and concentrates is forecasted at 409.4, 135.6 and 61.2 mt, respectively, for 2030.

Molecular cloning, expression and characterization of a novel feruloyl esterase enzyme from the symbionts of termite(Coptotermes formosanus) gut.

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and 37oC, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.

Effect of copper- and zinc-methionine supplementation on bioavailability, mineral status and tissue concentrations of copper and zinc in ewes.

The effect of feeding Cu- and Zn-methionine to ewes was studied in a 240d feeding trial. The plasma and tissue Cu and Zn concentrations and Cu/Zn-superoxide dismutase (Cu/Zn-SOD) activity were employed to assess the relative bioavailability from Cu- and Zn-methionine. The macro and micronutrient intake, utilization, plasma mineral status, tissue accumulation of Cu and Zn as well as wool concentration of Cu and Zn were studied in ewes (n=12) fed a corn-soybean meal based basal diet with 50% more Cu and Zn supplementation over the basal diet either from Cu- and Zn-sulfate (Cu-Sulf+Zn-Sulf group) or Cu- and Zn-methionine (Cu-Meth+Zn-Meth group). The average daily feed intake and body weight gain of ewes did not differ due to dietary supplementation of Cu- and Zn-methionine. However, dry matter intake was comparatively lower and thus resulted in better feed: gain in Cu- and Zn-methionine group as compared to ewes fed Cu- and Zn-sulfate. Supplementation of Cu and Zn over the basal diet either from methionine-chelated or sulfate sources resulted in increased plasma Cu and Zn as well as Cu/Zn-SOD activity on d-30, which indicated a positive correlation between plasma Cu and Zn and Cu/Zn-SOD activity. The gut absorption, liver concentrations of Cu and Zn, and liver Cu/Zn-SOD activity were significantly (P<0.01) higher in ewes supplemented with Cu- and Zn-methionine compared to Cu- and Zn-sulfate. Periodical analysis of wool samples indicated no significant difference in Cu and Zn content between Cu-and Zn-methionine and Cu- and Zn-sulfate groups. Feeding of Cu and Zn from methionine-chelated source resulted in reduced (P<0.01) excretion of Cu and Zn in feces indicating their better utilization, and this will have positive implication on environment. The gut absorption values, plasma and liver tissue concentrations of Cu and Zn supported the hypothesis that Cu- and Zn-methionine supplements have better bioavailability compared to Cu- and Zn-sulfate and Cu- and Zn-dependent enzyme (Cu/Zn-SOD) could be used to determine the bioavailability of Cu and Zn.

The stimulatory effect of the organic sulfur supplement, mercaptopropane sulfonic acid on cellulolytic rumen microorganisms and microbial protein synthesis in cattle fed low sulfur roughages.

Two metabolism trials (experiments 1 and 2) were conducted to examine the effect of the organic S compound, sodium 3-mercapto-1-propane sulfonic acid (MPS) on feed intake, fiber digestibility, rumen fermentation and abundance of cellulolytic rumen microorganisms in cattle fed low S (<0.11%) roughages. Urea was provided in all treatments to compensate for the N deficiency (<0.6%) in the roughages. In experiment 1, steers (333 ± 9.5 kg liveweight) were fed Angleton grass (Dicanthium aristatum) supplemented with S in equivalent amounts as either MPS (6.0 g/day) or sodium sulfate (9.56 g/day). Supplementation of Angelton grass with either sulfate or MPS resulted in an apparent increase in flow of rumen microbial protein from the rumen. Sulfur supplementation did not significantly change whole tract dry matter digestibility or intake, even though sulfate and MPS supplementation was associated with an increase in the relative abundance of the fibrolytic bacteria Fibrobacter succinogenes and anaerobic rumen fungi. Ruminal sulfide levels were significantly higher in the sulfate treatment, which indicated that the bioavailability of the two S atoms in the MPS molecule may be low in the rumen. Based on this observation, experiment 2 was conducted in which twice the amount of S was provided in the form of MPS (8.0 g/day) compared with sodium sulfate (6.6 g/day) to heifers (275 ± 9 kg liveweight) fed rice straw. Supplementation with MPS compared with sulfate in experiment 2 resulted in an increase in concentration of total volatile fatty acids, and ammonia utilization without a change in feed intake or whole tract fiber digestibility even though S and N were above requirement for growing cattle in both these treatment groups. In conclusion, supplementation of an S deficient low-quality roughage diet with either MPS or sodium sulfate, in conjunction with urea N, improved rumen fermentation, which was reflected in an increase in urinary purine excretion. However, MPS appeared to have a greater effect on stimulating short-chain fatty acid production and ammonia utilization when provided at higher concentrations than sulfate. Thus, the metabolism of MPS in the rumen needs to be investigated further in comparison with inorganic forms of S as it may prove to be more effective in stimulating fermentation of roughage diets.

Characterization of receptors for osteogenic protein-1/bone morphogenetic protein-7 (OP-1/BMP-7) in rat kidneys.

BACKGROUND: Osteogenic protein-1/bone morphogenetic protein-7 (OP-1/BMP-7), a member of the transforming growth factor-beta superfamily, has been shown to prevent kidney damage from ischemia/reperfusion injury in rats. The molecular events involved in OP-1 action on kidney are not yet understood. METHODS: In this study, we evaluated the biodistribution of (125)I-labeled OP-1 in rat kidneys. Adult rats received a single intravenous injection of 250 microg (125)I-labeled OP-1 per kg body wt, a dose that was effective in protecting kidneys from ischemic injury. Tissue localization, in situ hybridization, and immunostaining with a specific receptor antibody were performed to identify OP-1 cellular targets. Also, isolated plasma membranes from kidney cortex and medulla regions were analyzed to identify and characterize receptor structural components that recognize OP-1. RESULTS: At 10 and 180 minutes following injection, the relative uptake of (125)I-labeled OP-1 was consistently higher in kidney cortex than in medulla region. Upon autoradiography, kidney tissue sections revealed that OP-1 bound to the convoluted tubule epithelium, glomeruli, and collecting ducts. Moreover, in situ hybridization and immunostaining methods have shown localization of mRNA transcripts and the protein for BMP receptor type II in the cortex and medulla in similar areas as (125)I-labeled OP-1. Bulk membranes (enriched with plasma membranes) isolated from the cortex and medulla regions of kidney each bound specifically to (125)I-OP-1, and the binding of (125)I-labeled OP-1 was inhibited by unlabeled OP-1 in a dose-dependent manner. However, platelet-derived growth factor, transforming growth factor-beta, insulin-like growth factor, fibroblast growth factors, and other members of BMP family such as BMP-2 and cartilage-derived morphogenetic protein-1/growth and differentiation factor-5 (CDMP-1/GDF-5) failed to inhibit the binding of (125)I-labeled OP-1 to receptors, suggesting a high degree of specificity with which OP-1 bound to kidney receptors. Scatchard analysis of quantitative binding data indicated that the OP-1 receptors of kidney contained a single class of high-affinity binding sites for OP-1 with an association constant (Ka) of 2.26 x 109 mol/L-1 and a binding capacity of 1.01 pmol of OP-1 per mg membrane protein. When analyzed by a ligand blot technique, plasma membranes isolated from kidney cortex and medulla each showed the presence of a prominent specific band with a relative molecular mass (Mr) of 100 kD. Further analysis by Western blotting indicated that an antibody raised against BMP type II receptor effectively recognized the 100 kD OP-1 binding component of kidney plasma membranes. CONCLUSIONS: We demonstrated, to our knowledge for the first time, the presence of membrane-bound, specific, high-affinity OP-1 receptors in rat kidney tissues, which are likely to mediate OP-1 actions in the kidney. The major OP-1-binding component of the kidney appears to be a long form of BMP type II receptor with a Mr of 100 kD. In vivo and in vitro evidence suggests that the cellular targets for OP-1 are convoluted tubule epithelium, glomeruli, and collecting ducts. OP-1 does not share receptor binding properties with other growth factors, including BMP-2 and CDMP-1, suggesting that its mode of action in kidney appears to be specific.

Osteogenic protein-1 (OP-1) blocks cartilage damage caused by fibronectin fragments and promotes repair by enhancing proteoglycan synthesis.

OBJECTIVE AND DESIGN: The abilities of osteogenic protein-1 (OP-1) and TGF-beta1 to affect cartilage damage caused by fibronectin fragments (Fn-fs) that are known to greatly enhance cartilage proteoglycan (PG) degradation were compared. MATERIAL: Articular cartilage was obtained from 18 month old bovines. TREATMENT: To test blocking of damage, cartilage was cultured with or without OP-1 or TGF-beta in the presence of 100 nM Fn-fs. To test restoration of PG, cartilage was first cultured with Fn-fs and the cartilage then treated with factors. METHODS: Cartilage PG content was measured in papain digests using the dimethylmethylene blue assay. PG synthesis was measured by incorporation of 35S labeled sulfate. RESULTS: OP-1 blocked damage and restored PG in damaged cartilage, apparently due to enhanced PG synthesis. However, TGF-beta1 alone decreased PG content. CONCLUSIONS: These results clearly demonstrate differences between OP-1 and TGF-beta1, both members of the TGF-beta superfamily and illustrate the efficacy of OP- in blocking Fn-f mediated damage.