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Milk proteins are frequently used as supplements in fortified foods. However, processing produces chemical changes which likely affect the nutritional advantage. This study was intended to explore the possible difference in digestibility between extruded and non-extruded caseins and how the dietary N (ε) -carboxymethyllysine (CML) is metabolised. Normal rats were randomized into either an extruded protein diet (EP) or the same with unextruded proteins (UEP), for two periods of 2 weeks at 7 to 9 and 11 to 13 weeks of age. However, no difference in protein digestibility was detected between the two diets, either in young or in adult animals, despite a 9.4-fold higher level of CML and an 8.5-fold higher level of lysinoalanine in the EP than in the UEP. No diet-related changes were observed in plasma CML, either protein bound or free. Amounts of 38 and 48 % of the orally absorbed CML were excreted in urine and faeces, respectively, in UEP-fed rats. Lower rates of excretion were found in the EP-fed rats (23 and 37 %, respectively). A second animal study using a single oral dose of free CML (400 μg/rat) was set up to measure the systemic concentration of CML every hour from 0 to 4 h. It revealed that protein-bound CML was not affected by the oral dose of CML, and the highest free CML level found in the circulation was 600 ng/mL. Extruded proteins, therefore, appear to be well digested, and CML rapidly eliminated. Since its elimination is, however, incomplete, the question of its biodistribution and metabolism remains open.

The interaction between lysine (Lys) and arginine (Arg) in the proximal intestinal region of Pacific bluefin tuna (Thunnus orientalis) was evaluated using the everted intestine method. This in vitro intestinal system has been shown to be an effective tool for studying the nutrient absorption without the need to handle the tuna fish in marine cages as needed for digestibility and amino acid (AA) absorption. We used a factorial design with two sets of variables: low and high Lys concentration (10 and 75 mM) and four different Arg concentrations (3, 10, 20, and 30 mM). Both amino acids were dissolved in marine Ringer solution with a basal amino acidic composition consisting of a tryptone solution (9 mg mL(-1)). No interaction was observed between the absorption of Lys and Arg during the first 10 min of the experiment when low concentration of Lys and Arg was used in the hydrolyzate solution. However, there seemed to be a positive effect on Lys absorption when both amino acids were at high concentrations (30 and 75 mM, respectively). This type of studies will led us to test different formulations and/or additives to better understand the efficiency of AA supplementation as an alternative to in situ studies that are difficult to follow to design with the Pacific Bluefin Tuna.

Background: N(ε)-carboxymethyl-lysine (CML) is a major advanced glycation end-product (AGEs) widely found in foods. The aim of our study was to evaluate how exogenous CML-peptide is dynamically absorbed from the gastrointestinal tract and eliminated by renal tubular secretion using microPET imaging., Methods: The present study consisted of three investigations. In study I, we synthesized the imaging tracer (18)F-CML by reacting N-succinimidyl 4-(18)F-fluorobenzoate ((18)F-SFB) with CML. In study II, the biological activity of (18)F-CML was evaluated in RAW264.7 cells and HepG2 cells. In study III, the biodistribution and elimination of AGEs in ICR mice were studied in vivo following tail vein injection and intragastric administration of (18)F-CML., Result: The formation of (18)F-CML was confirmed by comparing its retention time with the corresponding reference compound (19)F-CML. The radiochemical purity (RCP) of (18)F-CML was >95%, and it showed a stable character in vitro and in vivo. Uptake of (18)F-CML by RAW264.7 cells and HepG2 cells could be inhibited by unmodified CML. (18)F-CML was quickly distributed via the blood, and it was rapidly excreted through the kidneys 20 min after tail vein injection. However, (18)F-CML was only slightly absorbed following intragastric administration. After administration of (18)F-CML via a stomach tube, the radioactivity was completely localized in the stomach for the first 15 min. At 150 min post intragastric administration, intense accumulation of radioactivity in the intestines was still observed., Conclusions: PET technology is a powerful tool for the in vivo analysis of the gastrointestinal absorption of orally administered drugs. (18)F-CML is hardly absorbed by the gastrointestinal tract. It is rapidly distributed and eliminated from blood following intravenous administration. Thus, it may not be harmful to healthy bodies. Our study showed the feasibility of noninvasively imaging (18)F-labeled AGEs and was the first to describe CML-peptide gastrointestinal absorption by means of PET.

Experiments were conducted to evaluate the availability to ruminants of lysine from hydroxymethyl lysine, a product potentially resistant to ruminal degradation yet able to release free lysine when subjected to the acidic environment of the abomasum. An in vitro ruminal fermentation assay that led to ammonia production from free lysine was used for initial assessments, but the hydroxymethyl lysine was inhibitory to lysine degradation at the concentrations tested in vitro; therefore, an in vivo assay with sheep, using plasma lysine concentrations as the response criterion, was used for assessment. twelve mature sheep were fed graded amounts of lysine from either a commercially available ruminally protected lysine product with known availability or from hydroxymethyl lysine. the protected lysine product provided 3 or 6 g/d of metabolizable lysine, whereas the hydroxymethyl lysine provided 3 or 6 g/d of total lysine. Plasma lysine concentrations increased linearly in response to both the ruminally protected lysine product and hydroxymethyl lysine. by slope ratio analysis, the bioavailability of lysine in hydroxymethyl lysine was estimated to be 94% of that for the commercially available product. We concluded that hydroxymethyl lysine may be used as an effective means of supplementing lysine to ruminants.

Objective: The present investigation was aimed at optimizing of estradiol (E2) loaded l-amino acid derivatives organogel formulations resulting in improved the high initial release problems and sustained release of E2., Methods: The visco-elastic properties of blank organogels were measured by rheometer. The E2 organogel formulations were optimized using a central composite design. Also, the effect of gelator structure and composition of the gel formulations on release behavior (in vitro and in vivo) had been studied., Results: The change of the gelator structure could affect significantly the stiffness of the implants. The release behavior of gel without N-Methyl-2-pyrrolidinone (NMP) was controlled by gel corrosion only. While the drug release of the gel with NMP was controlled by both corrosion and diffusion. The high initial release problems of the organogels were improved by optimizing the formulations. The system consisting by N-Lauroyl L-lysine methyl ester (LLM) derivative in the oil indicated the lowest initial drug release, showed a much lower blood drug level and maintained a steady state for nearly 1 month., Conclusion: Organogels based on L-lysine methyl ester derivative were ideal carriers for long-term parenteral administration of E2.

It is of urgent need to identify the exact physico-chemical characteristics which allow maximum uptake and accumulation in secondary target organs of nanoparticulate drug delivery systems after oral ingestion. We administered radiolabelled gold nanoparticles in different sizes (1.4-200 nm) with negative surface charge and 2.8 nm nanoparticles with opposite surface charges by intra-oesophageal instillation into healthy adult female rats. The quantitative amount of the particles in organs, tissues and excrements was measured after 24 h by gamma-spectroscopy. The highest accumulation in secondary organs was mostly found for 1.4 nm particles; the negatively charged particles were accumulated mostly more than positively charged particles. Importantly, 18 nm particles show a higher accumulation in brain and heart compared to other sized particles. No general rule accumulation can be made so far. Therefore, specialized drug delivery systems via the oral route have to be individually designed, depending on the respective target organ.

Palifosfamide, the DNA-alkylating metabolite of ifosfamide (IFOS), has been synthesized as a stabilized tris or lysine salt and found to have preclinical and clinical antitumor activity. Stabilized palifosfamide overcomes limitations of IFOS because of patient-to-patient variability in response resulting from variable prodrug activation, resistance and toxicities of metabolic byproducts, acrolein and chloroacetaldehyde. Palifosfamide represents an effective alternative to IFOS and other DNA-alkylating prodrugs. The antitumor activities of stabilized palifosfamide were investigated in vivo. Dose response, route and schedule of administration, and interaction with docetaxel or doxorubicin were investigated in NCr-nu/nu mice bearing established orthotopic mammary MX-1 tumor xenografts. Oral activity was investigated in P388-1 leukemia in CD2F1 mice. Oral and intraperitoneal bioavailabilities were compared in Sprague-Dawley rats. Stabilized palifosfamide administered by optimized regimens suppressed MX-1 tumor growth (P<0.05) by greater than 80% with 17% complete antitumor responses and up to three-fold increase in time to three tumor doublings over controls. Median survival in the P388-1 (P<0.001) model was increased by 9 days over controls. Oral bioavailability in rats was 48-73% of parenteral administration, and antitumor activity in mice was equivalent by both routes. Treatment with palifosfamide-tris combined with docetaxel or doxorubicin at optimal regimens resulted in complete tumor regression in 62-75% of mice. These studies support investigation of stabilized palifosfamide in human cancers by parenteral or oral administration as a single agent and in combination with other approved drugs. The potential for clinical translation of the cooperative interaction of palifosfamide-tris with doxorubicin by intravenous administration is supported by results from a recent randomized Phase-II study in unresectable or metastatic soft-tissue sarcoma.

Purpose: We have synthesized and evaluated in vivo 2-(3-{1-carboxy-5-[(6-[(18)F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid, [(18)F]DCFPyL, as a potential imaging agent for the prostate-specific membrane antigen (PSMA). PSMA is upregulated in prostate cancer epithelia and in the neovasculature of most solid tumors., Experimental Design: [(18)F]DCFPyL was synthesized in two steps from the p-methoxybenzyl (PMB) protected lys-C(O)-glu urea precursor using 6-[(18)F]fluoronicotinic acid tetrafluorophenyl ester ([(18)F]F-Py-TFP) for introduction of (18)F. Radiochemical synthesis was followed by biodistribution and imaging with PET in immunocompromised mice using isogenic PSMA PC3 PIP and PSMA- PC3 flu xenograft models. Human radiation dosimetry estimates were calculated using OLINDA/EXM 1.0., Results: DCFPyL displays a K(i) value of 1.1 ± 0.1 nmol/L for PSMA. [(18)F]DCFPyL was produced in radiochemical yields of 36%-53% (decay corrected) and specific radioactivities of 340-480 Ci/mmol (12.6-17.8 GBq/μmol, n = 3). In an immunocompromised mouse model [(18)F]DCFPyL clearly delineated PSMA+ PC3 PIP prostate tumor xenografts on imaging with PET. At 2 hours postinjection, 39.4 ± 5.4 percent injected dose per gram of tissue (%ID/g) was evident within the PSMA+ PC3 PIP tumor, with a ratio of 358:1 of uptake within PSMA+ PC3 PIP to PSMA- PC3 flu tumor placed in the opposite flank. At or after 1 hour postinjection, minimal nontarget tissue uptake of [(18)F]DCFPyL was observed. The bladder wall is the dose-limiting organ., Conclusions: These data suggest [(18)F]DCFPyL as a viable, new positron-emitting imaging agent for PSMA-expressing tissues., (©2011 AACR.)

Purpose: Avidin-coupled monoclonal antibody MX35 (avidin-MX35) and astatine-211-labeled, biotinylated, succinylated poly-l-lysine ((211)At-B-PL(suc)) were administered in mice to assess potential efficacy as an intraperitoneal (i.p.) therapy for microscopic tumors. We aimed to establish a timeline for pretargeted radioimmunotherapy using these substances, and estimate the maximum tolerable activity., Methods: (125)I-avidin-MX35 and (211)At-B-PL(suc) were administered i.p. in nude mice. Tissue distributions were studied at various time points and mean absorbed doses were estimated from organ uptake of (211)At-B-PL(suc). Studies of myelotoxicity were performed after administration of different activities of (211)At-B-PL(suc)., Results: We observed low blood content of both (125)I-avidin-MX35 and (211)At-B-PL(suc), indicating fast clearance. After sodium perchlorate blocking, the highest (211)At uptake was found in kidneys. Red bone marrow (RBM) accumulated some (211)At activity. Mean absorbed doses of special interest were 2.3 Gy/MBq for kidneys, 0.4 Gy/MBq for blood, and 0.9 Gy/MBq for RBM. An absorbed dose of 0.9 Gy to the RBM was found to be safe. These values suggested that RBM would be the key dose-limiting organ in the proposed pretargeting scheme, and that blood data alone was not sufficient for predicting its absorbed dose., Conclusions: To attain a favorable distribution of activity and avoid major toxicities, at least 1.0 MBq of (211)At-B-PL(suc) can be administered 24 hours after an i.p. injection of avidin-MX35. These results provide a basis for future i.p. therapy studies in mice of microscopic ovarian cancer.

Small interfering RNA (siRNA) has great therapeutic potential for the suppression of proteins associated with disease, but delivery methods are needed for improved efficacy. Here, we investigated the properties of micellar siRNA delivery vehicles prepared with poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLL) comprising lysine amines modified to contain amidine and thiol functionality. Lysine modification was achieved using 2-iminothiolane (2-IT) [yielding PEG-b-PLL(N2IM-IM)] or dimethyl 3,3'-dithiobispropionimidate (DTBP) [yielding PEG-b-PLL(MPA)], with modifications aimed to impart disulfide cross-linking ability without compromising cationic charge. These two lysine modification reagents resulted in vastly different chemistry contained in the reacted block copolymer, which affected micelle formation behavior and stability along with in vitro and in vivo performance. Amidines formed with 2-IT were unstable and rearranged into a noncharged ring structure lacking free thiol functionality, whereas amidines generated with DTBP were stable. Micelles formed with siRNA and PEG-b-PLL(N2IM-IM) at higher molar ratios of polymer/siRNA, while PEG-b-PLL(MPA) produced micelles only near stoichiometric molar ratios. In vitro gene silencing was highest for PEG-b-PLL(MPA)/siRNA micelles, which were also more sensitive to disruption under disulfide-reducing conditions. Blood circulation was most improved for PEG-b-PLL(N2IM-IM)/siRNA micelles, with a circulation half-life 3× longer than naked siRNA. Both micelle formulations are promising for siRNA delivery applications in vitro and in vivo.

The aims of the present study were to elucidate the potential mechanism of propofol emulsion destabilization following the addition of lidocaine, and to evaluate the effects of various electrokinetic stabilizers on the physicochemical properties of lidocaine-propofol emulsions. The assessments included pH observations and determination of the maximum globule diameter (MGD) and zeta potential (ZP). The MGD of propofol emulsions increased up to several tens mum following the addition of 50 mg of lidocaine to 200mg of propofol, and the proposed destabilization mechanism involves localization of protonated lidocaine molecules between lecithin molecules in propofol emulsions, which consequently leads to increased ZP. The ZP of propofol emulsions containing acidic amino acid or neutral amino acid increased following the addition of lidocaine, and a charge reversal occurred. Therefore, the MGD of emulsions increased to several tens (m. However, the MGD of emulsions that contained basic amino acids or basic compounds remained below 5 (m, despite the addition of large amounts lidocaine (50 mg), and the ZP did not pass through the point of zero charge. In conclusion, our results provide not only further insight into the physical stability of propofol emulsions containing lidocaine, but also a better understanding of the administration of propofol in existing applications., (Copyright 2010 Elsevier B.V. All rights reserved.)

Growth assays were performed to determine lysine bioavailability for kittens and rats in untreated and heated casein; these values were compared with estimates obtained with an in vitro method. Body weight, food intake, nitrogen and dry matter digestibility, and plasma lysine were determined during an 80-day growth trial using kittens (n = 16). Body weight and food intake were determined during a 21-day growth trial using weanling rats (n = 80). The growth data showed bioavailable lysine to be 102.4% and 100.2% (for untreated casein) and 66.1% and 51.7% (for heated casein) for kittens and rats, respectively. There was no relationship between plasma lysine and dietary lysine concentrations for kittens. There were no significant differences in nitrogen or dry matter digestibility among diets for kittens. The chemically reactive lysine content of untreated casein was 99.6%, and of heated casein was 67.1%. Heat treatment of casein resulted in significantly decreased lysine bioavailability as estimated by all methods. For untreated casein, both growth assays showed good agreement with the in vitro method for available lysine. For heated casein, the rat growth assay significantly underestimated bioavailable lysine as determined in kittens while the in vitro method closely approximated this value for the cat., (© 2010 Blackwell Verlag GmbH.)

Acquisition of Pseudomonas aeruginosa, the most prevalent organism isolated from cystic fibrosis (CF) airways, is associated with an accelerated clinical deterioration and reduced survival. Strategies to chronically suppress P. aeruginosa infections in individuals with CF have evolved over the last four decades and now largely focus on regular administration of aerosolized antibiotics. Aztreonam lysine (AZLI; Cayston, Gilead Pharmaceuticals [Foster City, CA, USA]), a novel formulation of the monobactam aztreonam suitable for aerosol delivery has recently been developed. AZLI is administered as 75 mg three-times daily for 28 days in 'on/off' cycles using the Altera/eFlow electronic nebulizer (PARI Innovative Manufacturers [Midlothian, VA, USA]). In individuals with CF chronically infected with P. aeruginosa, AZLI improved healthcare-associated quality-of-life scores, pulmonary function and weight, prolonged time to requirement of antibacterial therapy for symptoms of pulmonary exacerbation and reduced P. aeruginosa sputum burdens. These outcomes were durable over 18 months of cycled use. AZLI has been demonstrated to be safe and effective, and expands available chronic maintenance therapies in CF.

Paramagnetic nanomaterials for use as magnetic resonance imaging (MRI) contrast agents have higher relaxivity than conventional low molecular weight MRI agents. However, the biocompatibility and pharmacokinetics of such nanomaterials will strongly affect the likelihood of clinical approval. We synthesized MRI contrast agents based on biocompatible lysine-dendri-grafts: Gd-BzDTPA-lysineG2 and Gd-BzDTPA-lysineG3. The relaxivity of Gd-BzDTPA-lysineG2 and Gd-BzDTPA-lysineG3 increased with sample temperature, while the relaxivity of Gd-BzDTPA-PAMAMG4 decreased with increasing sample temperature. The increase in relaxivity with increasing temperature may be attributed to accessibility of water to the internal Gd chelates with lysine-dendri-grafts, which does not occur with PAMAM dendrimers. Gd-BzDTPA-lysineG3 had a long intravascular half-life but were largely excreted by the kidneys. Therefore, Gd-BzDTPA-lysineG3 enhanced the blood vessels for longer periods than Gd-BzDTPA-PAMAMG4, but was still excreted through the kidney. Because of their biocompatibility, desirable magneto-physical characteristics and favorable pharmacokinetics, which are derived from different interior structures rather than the physical size, lysine-dendri-graft MR contrast agents may be feasible for clinical use.

Atlantic salmon (Salmo salar L.) is one of the most economically important cultured fish and also a key model species in fish nutrition. During digestion, dietary proteins are enzymatically cleaved and a fraction of degradation products in the form of di- and tripeptides translocates from the intestinal lumen into the enterocyte via the Peptide Transporter 1 (PepT1). With this in mind, a full-length cDNA encoding the Atlantic salmon PepT1 (asPepT1) was cloned and functionally characterized. When overexpressed in Xenopus laevis oocytes, asPepT1 operated as a low-affinity/high-capacity transport system, and its maximal transport activity slightly increased as external proton concentration decreased (varying extracellular pH from 6.5 to 8.5). A total of 19 tested di- and tripeptides, some with acknowledged bioactive properties, some containing lysine, which is conditionally growth limiting in fish, were identified as well transported substrates, with affinities ranging between approximately 0.5 and approximately 1.5 mmol/L. Analysis of body tissue distribution showed the highest levels of asPepT1 mRNA in the digestive tract. In particular, asPepT1 mRNA was present in all segments after the stomach, with higher levels in the pyloric caeca and midgut region and lower levels in the hindgut. Depriving salmon of food for 6 d resulted in a approximately 70% reduction of intestinal PepT1 mRNA levels. asPepT1 will allow systematic in vitro analysis of transport of selected di- and tripeptides that may be generated in Atlantic salmon intestine during gastrointestinal transit. Also, asPepT1 will be useful as a marker to estimate protein absorption function along the intestine under various physiological and pathological conditions.

Objective: To evaluate the bioequivalence between two 250 mg-tablets of lysine clonixinate, Dorixina Forte (Siegfried Rhein, México) as reference product, and Prestodol (Farmaceúticos Rayere, S.A., México) as test formulation., Methods: 26 healthy adult female Mexican volunteers received a single oral dose of 250-mg lysine clonixinate under fasting conditions. The drug was administered following a randomized, two-period, two-sequence, cross-over design. Twelve serial blood samples were collected up to 8 h after dosing, and clonixin (CLX) was measured by ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry. Decimal logarithm values of Cmax and area under the curve (AUC) were used to construct a classic confidence interval at 90% (90% CI). Bioequivalence was established if 90% CI of mean ratios (test/reference) fall within the 0.8-1.25 range., Results: Volunteers formed a homogeneous population in terms of age (27.2 +/- 6.3 years), weight (55.9 +/- 6.5 kg), height (1.6 +/- 0.04 m), and body mass index (BMI) (22.91 +/- 2.03 kg/m(2)). Reference formulation exhibited the following pharmacokinetics: C(max) (32.39 +/- 8.32 microg/ml); t(max) (0.64 +/- 0.2 h); AUC0-8h (48.92 +/- 16.51 microg x h/ml); t1/2 (1.3 +/- 0.24 h); CLapp (5.64 +/- 1.99 l/h), and Vdapp (10.22 +/- 2.9 l). Concerning bioequivalence, 90% CI were: C(max) (82.32 - 98.79), AUC0-t (94.59-106.29), and AUC(0-inf) (94.61-106.42), with a statistical power of > 0.90 at every tested interval., Conclusions: This single-dose study found that both 250-mg immediate-release tablets of lysine clonixinate met the Mexican regulatory criteria for bioequivalence in these volunteers.

Empirically, 3-6 samples at each sampling time point have been used for most preclinical one-point sampling experiments without any theoretical justification. The purpose of the present study is to propose a practical approach to determine the minimum sample number (N(min)) based on Monte Carlo simulation and a bootstrap resampling. A computer program MOMENT(BS), in which a bootstrap resampling algorithm is used to estimate mean and standard deviations of pharmacokinetic parameters, such as area under the curve and mean residence time, was applied to estimate N(min). A new simulation program, MONTE1, was developed to generate simulated data for bootstrap resampling using the model parameters including inter- and/or intra-individual variations. Then, an index, S(2)CV calculated as the sum of the squared coefficient of variation is proposed to determine the N(min). The proposed approach was applied to the actual data in preclinical experiments, and the usefulness of the approach was suggested. An issue that one-point sampling data cannot separately assess inter- and intra-individual variability is discussed., (2009 Wiley-Liss, Inc. and the American Pharmacists Association)

Previous reports on milk protein responses to AA supplementation focused on Lys and Met concentrations expressed as a percentage of metabolizable protein, not the amounts of AA supplied. The objective of this study was to quantify the milk protein yield (MPY; g/d) response in studies in which Met or Lys was supplied either by postruminal infusion or in a rumen-protected form. A meta-analysis using a logistic regression model fitted using nonlinear mixed model procedures was performed on results from 23 published studies involving postruminal supplementation of Lys (18 experiments) and Met (35 experiments) in lactating dairy cows. Variance caused by study effect was removed by designation of individual study as subject within the random component within the nonlinear model. Milk protein responses to supplemental Met decreased from 16 to 4 g of milk protein per gram of metabolizable Met intake as Met intake varied from 25 to 70 g per cow per day. Similarly, milk protein responses to supplemental Lys decreased from 5.0 to 3.2 g of milk protein per gram of metabolizable Lys intake as Lys intake varied from 80 to 203 g per cow per day. Assuming Met and Lys concentrations of 2.76 and 7.63 g/100 g of milk protein, respectively, the implied marginal efficiencies of metabolizable AA use for MPY decreased from 44 to 12% for Met and from 39 to 25% for Lys over the range of metabolizable AA intakes. Although the estimated efficiencies were low compared with previous estimates, a low marginal efficiency of amino acid utilization would be expected when amino acid supply is at or near to the animal's requirement, as was the case in these experiments. This suggests that current models that assume both a constant MPY response and constant AA utilization efficiency are inadequate. Models that assume a constant efficiency of AA use will overestimate production responses to individual AA supply, especially when high amounts of metabolizable AA are fed.

Seahorses, sea dragons and pipefishes of the teleost family Syngnathidae are unique in that embryos develop within specialized brooding structures of the male. We enriched brooding Syngnathus fuscus and Syngnathus floridae males with injections of L-lysine-[(15)N(2)] and 16:0-palmitic acid 1-[(13)C] to demonstrate embryonic uptake of paternally-derived nutrients. While all embryos demonstrated amino acid enrichment, late stages showed significantly higher [(15)N], indicating greater utilization of paternal resources as yolk reserves diminished and embryonic energy demands increased. Limited embryonic [(13)C] uptake, defined as less than 10% of adult enrichment, in 75 and 81% of S. floridae and S. fuscus respectively signified rapid lipid metabolism and thus the need for greater enrichment. Interspecific differences in embryonic uptake of paternally-derived nutrients were not demonstrated. However, interspecific differences in egg nutrient reserves and fry size but comparable fry nutrient levels along with data from a published paternal exposure study indicate paternal transfer in S. fuscus most likely compensates for the comparative egg nutrient deficiency. This study is the first to our knowledge to provide direct evidence for the functional significance of the brood pouch in nutrient provisioning. These results add comparative information on the diversity of Syngnathid paternal care and further our understanding of paternal influence on development.

This study was conducted to compare the concentration of standardized digestible (SDD) Lys and relative bioavailable Lys in 7 sources of corn distillers dried grains with solubles (DDGS). A second objective was to evaluate 2 in vitro methods, reactive Lys and color score, to predict the concentration of SDD Lys and bioavailable Lys in DDGS. Seven sources of DDGS were fed to cecectomized roosters, and digestibility of amino acids was measured using the total excreta collection method. To measure the relative bioavailable Lys in DDGS, a standard curve (r(2) = 0.96, P < 0.01) was constructed from 9-d weight gain of young chicks fed a Lys-deficient basal diet or diets containing increasing concentrations of l-Lys-HCl. Seven additional diets were formulated by adding each of the 7 sources of DDGS to the basal diet, and total weight gain of chicks was measured. Weight gain of chicks fed each DDGS-containing diet was then compared with the standard curve to calculate the bioavailable Lys and bioavailability of Lys in each source of DDGS. All DDGS sources were analyzed for reactive Lys using the guanidination procedure, and a Hunterlab color score was used to measure the degree of lightness (L), redness (a), and yellowness (b). Results showed that the mean SDD Lys values and the mean relative bioavailability of Lys were 61.4 and 69.0%, respectively. Differences between the concentration of SDD Lys and the concentration of bio-available Lys were not observed in 5 of 7 sources of DDGS. The concentration of SDD Lys was correlated (r(2) = 0.84, P < 0.05) with the concentration of reactive Lys in DDGS. Greater Hunterlab L scores were associated with a greater (r(2) = 0.90, P < 0.05) concentration of bioavailable Lys in DDGS. In conclusion, the concentration of SDD Lys in DDGS does not overestimate the concentration of bioavailable Lys for poultry. Values for reactive Lys may be used to estimate the concentration of SDD Lys, whereas Hunterlab L may be used to estimate the concentration of bioavailable Lys in DDGS.

Amino acids in enterocytes are thought to be absorbed in the intestinal epithelium via various types of amino acid transport, although the regulation of these amino acid transport systems has not been elucidated. We examined in the present study the effect of several inhibitors involved in mRNA and protein synthesis, and of protein translocation on the L-leucine (Leu) uptake in human intestinal epithelial-like Caco-2 cells. Culturing Caco-2 cells with cycloheximide (CHX) enabled the L-Leu uptake to be significantly increased in a dose- and time-dependent manner. The uptake of L-lysine (Lys) was also increased by the CHX treatment, whereas the uptake of L-glutamate, taurine, and Gly-Gln was not changed. Among the two transport systems, b(0,+) and y+, which are known to be involved in L-Lys uptake by Caco-2, the system b(0,+) component was greatly increased by the CHX treatment, suggesting that system b(0,+) was mainly responsible for the increase in L-Leu and L-Lys uptake. The mRNA levels of rBAT and b(0,+) AT, whose molecules comprise system b(0,+), were both significantly increased by the CHX treatment in a time-dependent manner. These results strongly suggest that the CHX treatment increased the Leu and Lys uptake by activating system b(0,+) and inducing rBAT and b(0,+) AT mRNA expression in human intestinal epithelial Caco-2 cells.

Background: Diastolic dysfunction is a frequent cause of heart failure, particularly in dialysis patients. Advanced glycation end-products (AGEs) are increased in dialysis patients and are suggested to play a role in the development of diastolic dysfunction. The aim of our study was to assess whether AGE accumulation in dialysis patients is related to the presence of diastolic dysfunction., Methods and Results: Data were analyzed from 43 dialysis patients, age 58 +/- 15 years, of whom 65% were male. Diastolic function was assessed using tissue velocity imaging (TVI) on echocardiography. Tissue AGE accumulation was measured using a validated skin-autofluorescence (skin-AF) reader. Plasma N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine (CEL) were measured by stable-isotope-dilution tandem mass spectrometry. Plasma pentosidine was measured by high-performance liquid chromatography. Skin-AF correlated with mean E' (r = -0.51, P < .001), E/A ratio (r = -0.39, P = .014), and E/E' (r = 0.38, P = .019). Plasma AGEs were not significantly associated with diastolic function. Multivariable linear regression analysis revealed that 54% of the variance of average E' was explained by age (P = .007), dialysis type (P = 0.016), and skin-AF (P = .013)., Conclusions: Tissue AGEs measured as skin-AF, but not plasma AGE levels, were related to diastolic function in dialysis patients. Although this may support the concept that tissue AGEs explain part of the increased prevalence of diastolic dysfunction in these patients, the ambiguous relation between plasma and tissue AGEs needs further exploring.

What Is Already Known About This Subject: Ibuprofen is a nonsteroidal anti-inflammatory agent that induces closure of the patent ductus arteriosus in neonates. Few studies of ibuprofen pharmacokinetics have been performed and were limited to small groups of preterm infants, showing a large intersubject variability and an increase in clearance with either postnatal or gestational age., What This Study Adds: A population pharmacokinetic study was performed on 66 neonates to characterize the concentration-time courses of ibuprofen. Ibuprofen clearance significantly increased from postnatal age day 1 to day 8, but not with gestational age. A relationship was shown between ibuprofen area under the curve (AUC) and patent ductus arteriosus closure rate, and an effective threshold AUC was evidenced. Dosing schemes were proposed as a function of postnatal age, to achieve this AUC and to improve the efficacy of treatment for patent ductus arteriosus in neonates. AIMS To describe ibuprofen pharmacokinetics in preterm neonates with patent ductus arteriosus (PDA) and to establish relationships between doses, plasma concentrations and ibuprofen efficacy and safety., Methods: Sixty-six neonates were treated with median daily doses of 10, 5 and 5 mg kg(-1) of ibuprofen-lysine by intravenous infusion on 3 consecutive days. A population pharmacokinetic model was developed with NONMEM. Bayesian individual pharmacokinetic estimates were used to calculate areas under the curve (AUC) and to simulate doses. A logistic regression was performed on PDA closure., Results: Ibuprofen pharmacokinetics were described by a one-compartment model with linear elimination. Mean population pharmacokinetic estimates with corresponding intersubject variabilities (%) were: elimination clearance CL = 9.49 ml h(-1) (62%) and volume of distribution V = 375 ml (72%). Ibuprofen CL significantly increased with postnatal age (PNA): CL = 9.49*(PNA/96.3)(1.49). AUC after the first dose (AUC1D), the sum of AUC after the three doses (AUC3D) and gestational age were significantly higher in 57 neonates with closing PDA than in nine neonates without PDA closure (P = 0.02). PDA closure was observed in 50% of the neonates when AUC1D < 600 mg l(-1) h (or AUC3D < 900 mg l(-1) h) and in 91% when AUC1D > 600 mg l(-1) h (or AUC3D > 900 mg l(-1) h) (P = 0.006). No correlation between AUC and side-effects could be demonstrated., Conclusions: To achieve these optimal AUCs, irrespective of gestational age, three administrations at 24 h intervals are recommended of 10, 5, 5 mg kg(-1) for neonates younger than 70 h, 14, 7, 7 mg kg(-1) for neonates between 70 and 108 h and 18, 9, 9 mg kg(-1) for neonates between 108 and 180 h.

Evaluations of 4 soybean meal (SBM) products were conducted in 3 experiments. The 4 products were 1) solvent SBM (SSBM), 2) SSBM treated with 0.05% baker's yeast and toasted at 100 degrees C (YSBM), 3) expeller SBM (ESBM), and 4) lignosulfonate-treated SBM (LSBM). Multiparous Holstein cows (n = 32; 152 +/- 63 d in milk; body weight = 708 +/- 77 kg; producing 41 +/- 7 kg/d of milk at the beginning of the study) were used in a 4 x 4 Latin square design with 28-d periods to investigate cow responsiveness to supplemental ruminally undegradable protein (RUP) from the SBM products. Dietary treatments were formulated by substituting all of the SSBM and part of the ground corn with YSBM, ESBM, or LSBM to yield isonitrogenous diets. Diets were formulated to provide adequate ruminally degradable protein, but deficient RUP and metabolizable protein supplies. No differences among dietary treatments were observed for dry matter intake, body weight gain, milk and component yields, or efficiency of milk production. The lack of response to changes in SBM source was likely due to an adequate RUP and metabolizable protein supply by all the diets. In situ ruminal degradations of YSBM and LSBM were slower than those of SSBM or ESBM; thus, RUP contents of YSBM and LSBM were greater than those of SSBM or ESBM. The RUP of all SBM products had similar small intestinal digestibility. Available Lys contents, estimated chemically or by using a chick growth assay, were less for YSBM and LSBM than for SSBM or ESBM, suggesting deleterious effects of processing on Lys availability in YSBM and LSBM.

We measured pharmacologically isolated GABAergic currents from layer II/III neurons of the rat auditory cortex using patch-clamp recording. Activation of muscarinic receptors by muscarine (1 microM) or oxotremorine (10 microM) decreased the amplitude of electrically evoked inhibitory postsynaptic currents to about one third of their control value. Neither miniature nor exogenously evoked GABAergic currents were altered by the presence of muscarinic agonists, indicating that the effect was spike-dependent and not mediated postsynaptically. The presence of the N- or P/Q-type Ca(2+) channel blockers omega-conotoxin GVIA (1 microM) or omega-AgaTx TK (200 nM) greatly blocked the muscarinic effect, suggesting that Ca(2+)-channels were target of the muscarinic modulation. The presence of the muscarinic M(2) receptor (M(2)R) antagonists methoctramine (5 muM) or AF-DX 116 (1 microM) blocked most of the muscarinic evoked inhibitory postsynaptic current (eIPSC) reduction, indicating that M(2)Rs were responsible for the effect, whereas the remaining component of the depression displayed M(1)R-like sensitivity. Tissue preincubation with the specific blockers of phosphatidyl-inositol-3-kinase (PI(3)K) wortmannin (200 nM), LY294002 (1 microM), or with the Ca(2+)-dependent PKC inhibitor Gö 6976 (200 nM) greatly impaired the muscarinic decrease of the eIPSC amplitude, whereas the remaining component was sensitive to preincubation in the phospholipase C blocker U73122 (10 microM). We conclude that acetylcholine release enhances the excitability of the auditory cortex by decreasing the release of GABA by inhibiting axonal V-dependent Ca(2+) channels, mostly through activation of presynaptic M(2)Rs/PI(3)K/Ca(2+)-independent PKC pathway and-to a smaller extent-by the activation of M(1)/PLC/Ca(2+)-dependent PKC.

The inferior colliculus (IC) is a large auditory nucleus in the midbrain, which is a nearly obligatory relay center for ascending auditory projections. We made in vivo whole cell patch-clamp recordings of IC cells in young-adult anesthetized C57/Bl6 mice and Wistar rats to characterize their membrane properties and spontaneous inputs. We observed spikelets in both rat (18%) and mouse (13%) IC neurons, suggesting that IC neurons may be connected by electrical synapses. In many cells, spontaneous postsynaptic potentials were sufficiently large to contribute to spike irregularity. Cells differed considerably in the number of simultaneous spontaneous postsynaptic potentials that would be needed to trigger an action potential. Depolarizing and hyperpolarizing current injections showed six different types of firing patterns: buildup, accelerating, burst-onset, burst-sustained, sustained, and accommodating. Their relative frequencies were similar in both species. In mice, about half of the cells showed a clear depolarizing sag, suggesting that they have the hyperpolarization-activated current I(h). This sag was observed more often in burst and in accommodating cells than in buildup, accelerating, or sustained neurons. Cells with I(h) had a significantly more depolarized resting membrane potential. They were more likely to fire rebound spikes and generally showed long-lasting afterhyperpolarizations following long depolarizations. We therefore suggest a separate functional role for I(h).

Lysine nutrition is unique among indispensable amino acids in that it can be conserved and can be fed 12 h out of phase (delayed supplement) with the other dietary amino acids. In piglets, high levels (2-6%) of L-lysine added to a 10% protein diet can be tolerated without obvious detrimental effects. In both rat and piglet liver preparations, the first enzyme in the saccharopine-dependent pathway of lysine catabolism, lysine alpha-ketoglutarate reductase (LKR), is found only in the mitochondrial matrix. For Lys catabolism to occur, Lys must first enter the matrix of the mitochondrion. LKR, saccharopine dehydrogenase, mitochondrial lysine uptake, and lysine oxidation (LOX) all increased>3-fold in rats fed high levels of dietary protein (up to 60%). The activities of mitochondrial Lys uptake and LOX were similar when expressed as mmol/(d.100 g body weight). Thus, LOX can be a proxy for mitochondrial Lys uptake. Piglet liver LKR and LOX increase 5- to 10-fold when piglets are fed high-protein (50 or 75%) diets. In both the rat and piglet, after adapting to the high protein diet, the activity of LKR is 400-500 times that of LOX, suggesting that Lys uptake by a transporter(s) is rate limiting. Quantitative 24-h dietary infusion studies in piglets revealed that>80% of the Lys infused (4% of the diet) could not be recovered in the urine or body or accounted for by calculated Lys oxidation based on liver activity of LOX. Other pathways and tissues may account for the Lys oxidation in piglets.

Arginine supplementation is used in several disease states. In arginine-deficient states, supplementation is a logical choice of therapy. However, the definition of an arginine-deficient state is complex. For example, plasma arginine levels could be within normal range but intracellular arginine levels could be reduced because of membrane transport problems. Lysine competes with arginine for transport into the cell. In these situations, arginine supplementation of higher than required levels is proposed. Arginine has several important functions in metabolism as it is a precursor of metabolically active components such as nitric oxide (NO), ornithine, creatine, and polyamines. Supplementing arginine in excess could potentially overstimulate metabolism via enhanced production of NO. NO is a reactive component that, via production of radicals, will inactivate proteins. NO is also a powerful vasodilator, which could lead to severe hemodynamic instability. A good marker for excess supplementation of arginine or lysine could be an increased or reduced production rate of NO. However, NO production is difficult to measure because NO is a very labile component and is rapidly oxidized in blood. Stable isotope-labeled arginine and citrulline are used to trace the arginine-NO route. During supplementation of arginine in septic pigs or patients in septic shock, NO production, measured with stable isotope technology, is enhanced.

In this study, we synthesized a sixth generation lysine dendrimer (KG6) and two PEGylated derivatives thereof and evaluated their biodistribution characteristics in both normal and tumor-bearing mice. The intact KG6 showed a rapid clearance from the blood stream and non-specific accumulation in the liver and kidney. In contrast, the PEGylated derivatives showed a better retention in blood and low accumulativeness in organs dependent of the rate of PEGylation. In addition, PEGylated KG6 with a high modification rate was accumulated effectively in tumor tissue via the enhanced permeability and retention (EPR) effect. Moreover, we clarified that multiple administrations did not affect the biodistribution characteristics of a second dose of PEGylated KG6. PEGylated lysine dendrimer would be a useful material for a clinically applicable tumor-targeting carrier.

Amadori products are formed in the early stage of the so-called Maillard reaction between reducing sugars and amino acids or proteins. Such nonenzymatic glycosylation may occur during the heating or storage of foods, but also under physiological conditions. N-epsilon-fructoselysine is formed via this reaction between the epsilon-amino group of peptide-bound lysine and glucose. Despite the fact that, in certain heated foods, up to 50% of lysyl moieties may be modified to such lysine derivatives, up to now, very little is known about the metabolic fate of alimentary administered Amadori compounds. In the present study, N-succinimidyl-4-[18F]fluorobenzoate was used to modify N-epsilon-fructoselysine at the alpha-amino group of the lysyl moiety. The in vitro stability of the resulting 4-[18F]fluorobenzoylated derivative was tested in different tissue homogenates. Furthermore, the 4-[18F]fluorobenzoylated N-epsilon-fructoselysine was used in positron emission tomography studies, as well as in studies concerning biodistribution and catabolism. The results show that the 4-[18F]fluorobenzoylated N-epsilon-fructoselysine is phosphorylated in vitro, as well as in vivo. This phosphorylation is caused by fructosamine 3-kinases and occurs in vivo, particularly in the kidneys. Despite the action of these enzymes, it was shown that a large part of the intravenously applied radiolabeled N-epsilon-fructoselysine was excreted nearly unchanged in the urine. Therefore, it was concluded that the predominant part of peptide-bound lysine that was fructosylated during food processing is not available for nutrition.

Objective: In endothelial cells (EC) and vascular smooth muscle cells (VSMC) from atherosclerotic mice, connexin (Cx) expression becomes distorted. Lipoprotein-derived phospholipid oxidation products (OxPAPC) play a critical role in atherosclerosis, and we hypothesized that they may act as trigger molecules causing the changes in connexin expression., Methods and Results: We applied OxPAPC to murine carotid arteries in vivo and vascular cell cocultures. OxPAPC applied to carotids induced an upregulation of both Cx37 and Cx43 in the VSMC. In EC, Cx43 was upregulated and Cx37 was downregulated, whereas Cx40 in EC remained constant. In the vascular cell coculture, OxPAPC caused similar changes in Cx37 and Cx43 but caused a decrease in Cx40 in EC and an elevation of Cx40 in VSMC. In the coculture model, OxPAPC treatment led to the selective disappearance of Cx40 at the myoendothelial junction. Biocytin dye transfer between EC and VSMC coupling was dramatically reduced by OxPAPC. The decrease in dye transfer after OxPAPC treatment was correlated with an increase in tyrosine 265 phosphorylation of Cx43, especially at the in vitro myoendothelial junction., Conclusions: We conclude that OxPAPC could be responsible for the changes in connexin expression previously reported in atherosclerosis.

During the heat treatment of protein-containing foods, the amino acid lysine is most prone to undergo chemical reactions in the course of amino acid cross-linking or Maillard reactions. Among the reaction products formed, lysinoalanine (LAL), N(epsilon)-fructoselysine (FL) and N(epsilon)-carboxymethyllysine (CML) are those which serve as sensitive markers for the heat treatment applied. From a nutritional perspective, these compounds are ingested with the diet in considerable amounts but information about their metabolic transit and putative in vivo effects is scarce. In the present study, casein-linked LAL, FL and CML were administered to rats in two different doses for 10 days. Quantitation of LAL, FL and CML in plasma, tissue and faeces samples revealed that the kidneys are the predominant sites of accumulation and excretion. The maximum percent of dietary LAL, FL and CML excreted in the urine was 5.6, 5.2 and 29%, whereas the respective recoveries in the kidneys were 0.02, 26 and 1.4%. The plasma and tissue analyses revealed that the endogenous load of either compound is increased by its dietary intake. But the dose-dependent utilisation of dietary protein-linked LAL, FL and CML in rats has been demonstrated for the first time to vary substantially from each other.

We report on a short host defense-like peptide that targets and arrests the growth of aggressive and hormone-resistant primary human prostate and breast tumors and prevents their experimental and spontaneous metastases, respectively, when systemically inoculated to immunodeficient mice. These effects are correlated with increased necrosis of the tumor cells and a significant decrease in the overall tumor microvessel density, as well as newly formed capillary tubes and prostate-specific antigen secretion (in prostate tumors). Growth inhibition of orthotopic tumors derived from stably transfected highly fluorescent human breast cancer cells and prevention of their naturally occurring metastases were visualized in real time by using noninvasive whole-body optical imaging. The exclusive selectivity of the peptide towards cancer derives from its specific binding to surface phosphatidylserine and the killing of the cancer cells via cytoplasmic membrane depolarization. These data indicate that membrane disruption can provide a therapeutic means of inhibiting tumor growth and preventing metastases of various cancers.

Formation of new recurrent excitatory circuits after brain injuries has been hypothesized as a major factor contributing to epileptogenesis. Increases in total axonal length and the density of synaptic boutons are present in layer V pyramidal neurons of chronic partial isolations of rat neocortex, a model of posttraumatic epileptogenesis. To explore the functional consequences of these changes, we used laser-scanning photostimulation combined with whole-cell patch-clamp recording from neurons in layer V of somatosensory cortex to map changes in excitatory synaptic connectivity after injury. Coronal slices were submerged in artificial CSF (23 degrees C) containing 100 microM caged glutamate, APV (2-amino-5-phosphonovaleric acid), and high divalent cation concentration to block polysynaptic responses. Focal uncaging of glutamate, accomplished by switching a pulsed UV laser to give a 200-400 micros light stimulus, evoked single- or multiple-component composite EPSCs. In neurons of the partially isolated cortex, there were significant increases in the fraction of uncaging sites from which EPSCs could be evoked ("hot spots") and a decrease in the mean amplitude of individual elements in the composite EPSC. When plotted along the cortical depth, the changes in EPSCs took place mainly between 150 and 200 microm above and below the somata, suggesting a specific enhancement of recurrent excitatory connectivity among layer V pyramidal neurons of the undercut neocortex. These changes may shift the balance within cortical circuits toward increased synaptic excitation and contribute to epileptogenesis.

Background: Advanced glycation end products (AGEs) accumulate in lesions of arteriosclerosis, Alzheimer's disease, rheumatoid arthritis, diabetic retinopathy, and diabetic nephropathy. Among AGEs, chemical quantification and immunohistologic methods for pentosidine have been established. Free pentosidine-eliminated by renal excretion- is mainly affected by renal function. In this study, we measured concentrations of plasma free and total pentosidine and immunohistologically investigated kidney graft biopsy specimens in patients after renal transplantation to investigate the renal function, plasma free and total pentosidine, and its relationship with deposition in the renal tissue., Patients and Methods: In 28 patients who underwent renal transplantation from 1996 to 2003, we measured the time course of plasma concentrations of free pentosidine, total pentosidine, and serum creatinine starting right after renal transplantation. Thirty-four graft biopsy specimens were immunohistologically investigated using anti-pentosidine antibody. Plasma free and total pentosidine, and serum creatinine were measured at the same time., Results: Plasma free and total pentosidine were positively correlated with serum creatinine. Plasma free pentosidine and serum creatinine reached nadir values on day 34.2 +/- 14.2, when the blood concentrations were 5.1 +/- 1.6 pmol/mL and 1.7 +/- 0.7 mg/dL, respectively. Plasma total pentosidine reached a nadir on day 116.5 +/- 39.7 when the plasma concentration was 4.0 +/- 1.5 pmol/mg. We correlated the time required to reach the nadir of plasma free and total pentosidine concentrations. However, neither the concentration of plasma free nor plasma total pentosidine at nadir correlated with serum creatinine. The intensity of immunostaining with anti-pentosidine antibody in proximal tubular cells was graded as weakly positive, positive, or strongly positive. Significant differences were obtained among plasma free pentosidine values between the weakly positive and strongly positive groups., Conclusions: Renal transplantation improves renal function and decreases renal excretion of free pentosidine. Accordingly, total pentosidine also decreases. However, the concentrations of plasma free and total pentosidine at nadir varied among individuals; the blood concentrations were not determined by renal function alone. It was suggested that deposition of pentosidine in proximal tubular cells was more severe among patients with higher plasma free pentosidine and serum creatinine values.

To study the absorption of microbial lysine in growing rabbits, a labelled diet (supplemented with (15)NH4Cl) was administered to six animals (group ISOT); a control group (CTRL, four rabbits) received a similar, but unlabelled, diet. Diets were administered for 30 d. An additional group of six animals were fed the unlabelled diet for 20 d and then the labelled diet for 10 d while wearing a neck collar to avoid caecotrophy (group COLL), in order to discriminate it from direct intestinal absorption. At day 30 animals were slaughtered and caecal bacteria and liver samples taken. The (15)N enrichment in amino acids of caecal bacteria and liver were determined by GC-combustion/isotope ratio MS. Lysine showed a higher enrichment in caecal microflora (0.925 atom% excess, APE) than liver (0.215 APE) in group ISOT animals, confirming the double origin of body lysine: microbial and dietary. The COLL group showed a much lower enrichment in tissue lysine (0.007 (se 0.0029) APE for liver). Any enrichment in the latter animals was due to direct absorption of microbial lysine along the digestive tract, since recycling of microbial protein (caecotrophy) was avoided. In such conditions liver enrichment was low, indicating a small direct intestinal absorption. From the ratio of [(15)N]lysine enrichment between liver and bacteria the contribution of microbes to body lysine was estimated at 23 %, with 97 % of this arising through caecotrophy. Absorption of microbial lysine through caecotrophy was 119 (se 4.0) mg/d, compared with 406 (se 1.8) mg/d available from the diet. This study confirms the importance of caecotrophy in rabbit nutrition (15 % of total protein intake).

Objectives: Ravuconazole is a broad-spectrum antifungal triazole in clinical development. We investigated the compartmental plasma pharmacokinetics and tissue distribution of ravuconazole following administration of its novel intravenous (i.v.) di-lysine phosphoester prodrug, BMS-379224., Methods: Normal catheterized rabbits received the prodrug at 1.25, 2.5, 5, 10, 20 and 40 mg/kg once daily as 5 min i.v. bolus for 8 days. Serial plasma levels were collected at days 1 and 7, and tissues were obtained 30 min after the eighth dose. Concentrations of ravuconazole were determined by a validated HPLC method. Plasma concentration data were fitted to a three-compartment pharmacokinetic model. Pharmacokinetic parameters were estimated by weighted non-linear least squares regression analysis using the WinNonlin computer program., Results: Following single dosing, ravuconazole demonstrated linear plasma pharmacokinetics across the investigated dosage range. Cmax, AUC(0-infinity), V(ss), CL and terminal half-life (means +/- SEM) ranged from 2.03 to 58.82 mg/L, 5.80 to 234.21 mg x h/L, 5.16 to 6.43 L/kg, 0.25 to 0.18 L/h/kg and 20.55 to 26.34 h, respectively. Plasma data after multiple dosing revealed non-linear disposition at the 20 and 40 mg/kg dosage levels as evidenced by a dose-dependent decrease in CL (from 0.104-0.147 to 0.030 and 0.022 L/h/kg; P = 0.1053) and an increase in the dose-normalized AUC(0-infinity) (from 2.40-3.01 up to 11.90 and 14.56 mg x h/L; P = 0.0382). Tissue concentrations 30 min after the last dose were highest in the liver (12.91-562.68 microg/g), adipose tissue (10.57-938.55 microg/g), lung (5.46-219.12 microg/g), kidney (3.95-252.44 microg/g) and brain tissue (2.37-144.85 microg/g)., Conclusions: The pharmacokinetics of ravuconazole fitted best to a three-compartment pharmacokinetic model. The compound revealed non-linear pharmacokinetics at higher dosages, indicating saturable clearance and/or protein binding. Ravuconazole displayed a long elimination half-life and achieved substantial plasma and tissue concentrations including in the brain.

Hemin and other heme derivatives, e.g. heme-L-lysinate (HLL) and heme-L-arginate, have been used extensively to upregulate expression of heme oxygenase and production of endogenous carbon monoxide. Hemin administration has been shown to markedly decrease high blood pressure in spontaneously hypertensive rats (SHR), but not in normotensive Wistar-Kyoto or Sprague Dawley (SD) rats. While methodology to measure serum heme levels has been established long ago, metabolism of the injected hemin or heme derivatives when used to lower blood pressure has not been investigated. In this study, metabolism of hemin or HLL after injected into the rat was monitored by measuring changes in circulatory heme levels. SHR (12-20 weeks old) had significantly higher blood pressure than age-matched SD rats. In both strains, serum heme level was negligible. Hemin or HLL injection (15 mg/kg/day, i.p.) for 5-13 days significantly lowered blood pressure of 12-weeks SHR. High blood pressure was not lowered in SHR older than 20 weeks until hemin or HLL injection period was beyond 5 days. This anti-hypertensive effect of hemin and HLL was synchronized with an increase in serum heme level, from undetectable to 4.3 micromol/l. On the other hand, hemin or HLL had no effect on blood pressure of age-matched SD rats, despite serum heme level rose to the same extent as in the treated SHR. There was no significant difference between hemin and HLL injections in terms of changes in blood pressure and serum heme level in all rats. Our study for the first time correlated changes in serum heme levels with blood pressure levels after injection of hemin or HLL in SHR and SD rats. Hemin and HLL had similar effects on blood pressure change and serum heme level. By determining serum heme levels following the administration of hemin or HLL, we can better understand mechanisms for the blood pressure lowering effect of hemin therapy. Application of this heme monitoring technology will also pave the way for clinical application of hemin therapy in treatment of different types of hypertension pathologies.

Five experiments were conducted to determine Lys and P bioavailabilities of distillers dried grains with solubles (DDGS), which was derived from corn fermentation in a modern nonbeverage ethanol plant. In experiment 1, we used the precision-fed cecectomized rooster assay and estimated the true digestibility of Lys in DDGS to be 75%. In experiments 2, 3, 4, and 5 the relative bioavailabilities of Lys and P were assessed using slope-ratio chick growth experiments. In experiments 2 and 3, Lys-deficient basal diets containing 0.40 or 0.60% digestible Lys respectively, were formulated. A linear growth response (P < 0.05) was observed from the addition of 0.10 and 0.20% L-Lys from L-Lys x HCl and 10 and 20% DDGS to the basal diets. Body weight gain was regressed on Lys intake from L-Lys x HCl and DDGS, and the ratio of the slopes indicated the relative bioavailable Lys in DDGS. The values as a percentage of total Lys (0.83) in DDGS yielded availability estimates of 80% for experiment 2 and 100% for experiment 3. In experiments 4 and 5, a P-deficient basal diet containing 0.12% nonphytate P was formulated. A linear growth and tibia bone ash (%) response (P < 0.05) were observed from the addition of 0.05 and 0.10% P from K2HPO4 and 2 levels of DDGS (5 and 10% for experiment 4 and 7 and 14% for experiment 5). Tibia bone ash (%) was regressed on P intake from K2HPO4 and DDGS, and the ratio of slopes indicated the relative bioavailability of P in DDGS. The values as a percentage of total P (0.74%) in DDGS yielded availability estimates of 68% for experiment 4 and 54% for experiment 5.

The aim of this paper was to explore the efficacy of lactic acid as permeation enhancer for drug molecules across the skin. Three model permeants were chosen: acetaminophen (non-ionized), buspirone hydrochloride (cationic drug) and ibuprofen lysine (anionic drug). We also explored the association of lactic acid and iontophoresis as a means of enhancing drug delivery. Permeation experiments were performed in vitro, using rabbit ear skin as barrier. The results obtained indicate that lactic acid has some effects on model drug permeation across the skin. The effect was more evident with the anionic drug ibuprofen. Cathodal intophoresis increased ibuprofen transport, but when lactic acid was associated with cathodal iontophoresis, a concentration-dependent reduction of ibuprofen iontophoretic flux was observed, probably for the competition by the co-ion. The application of electric current (anodal iontophoresis) to a solution of acetaminophen produced an increase in its transport, due to the presence of an electroosmotic contribution; however, the effect of the association of anodal iontophoresis and lactic acid produced no further enhancement.

Dorsal root ganglia (DRG) are key elements in sensory signaling under physiological and pathological conditions. Little is known about electrical coupling among cells in these ganglia. In this study, we injected the fluorescent dye Lucifer yellow (LY) into single cells to examine dye coupling in DRG. We found no dye coupling between neurons or between neurons and their attendant satellite glial cells (SGCs). In mouse DRG, we observed that in 26.2% of the cases SGCs that surround a given neuron were dye coupled. In only 3.2% of the cases SGCs that make envelopes around different neurons were coupled. The data from mouse ganglia were very similar to those from rat and guinea pig DRG. The results obtained by injection of the tracer biocytin were very similar to those observed with LY. The coupling incidence within the envelopes increased 3.1-fold by high extracellular pH (8.0), but coupling between envelopes was not affected. Acidic pH (6.8) reduced the coupling. High extracellular K+ (9.4 mM) increased the coupling 2.4-fold and 4.7-fold within and between envelopes, respectively. Low extracellular Ca2+ (0.5, 1.0 mM) partly reversed the effect of high K+ on coupling. The results showed that SGCs in mammalian sensory ganglia are connected by gap junctions. This coupling is very sensitive to changes in pH, and can therefore be modulated under various physiological and pathological conditions. The dependence of the coupling on extracellular K+ and Ca2+ suggests that the permeability of gap junctions can be altered by physiological and pharmacological stimuli.

To probe the steric requirements for deacylation, we synthesized lysine-derived small molecule substrates and examined structure-reactivity relationships with various histone deacetylases. Rat liver, human HeLa, and human recombinant class I and II histone deacetylases (HDACs) as well as human recombinant NAD(+)-dependent SIRT1 (class III enzyme) were used in these studies. A benzyloxycarbonyl substituent on the alpha-amino group yielded the highest conversion rates. Replacing the epsilon-acetyl group with larger lipophilic acyl substituents led to a pronounced decrease in conversion by class I and II enzymes; the class III enzyme displayed a greater tolerance. Incubations with recombinant FLAG-tagged human HDACs 1, 3, and 6 showed a distinct subtype selectivity among small molecule substrates. The subtype selectivity of HDAC inhibitors could be predicted with these substrates and an easily obtainable mixture of HDAC subtypes.

Here we examined quantitatively axonal projections originating from the ventral posteromedial thalamic nucleus (VPM) to layer I of the primary somatosensory cortex (SI) by extracellular and intracellular injections of biocytin as an anterograde tracer. Following the extracellular injections, two types of VPM afferents with different arborization patterns in SI were observed. The type I extended vertically, forming dense plexus in layers IV and VI, and projected collaterals to layer I. The type II rarely branched in SI, converged in the plexus formed by the type I, and projected no collaterals to the supragranular layers. The labeled fibers in layer I derived from the first type ran parallel to the brain surface, and their mean length was 339.7 +/- 87.5 microm. Intracellular injection into VPM neurons bearing both types of afferent demonstrated the full axonal arborization in both the reticular thalamic nucleus (Rt) and SI. The total length of the axon of a neuron bearing the type I was 86,968.8 microm, and the length of axonal collaterals in layer I of SI was 433.1 microm. The total axonal length of a neuron bearing the type II was very small. The present study is the first to demonstrate substantial projections from VPM to layer I of SI, and provide quantitative data on the entire extent of the axonal arborization of thalamocortical projections from single VPM neurons.

In the normal frog, axons from the peripheral retina arising at the temporal pole course superficially in the middle stream of the diencephalic optic tract. Axons from the nasal pole course in two streams running in the opposite margins of the tract, dorsonasal axons ventrally, ventronasal axons dorsally. Axons from the dorsal and ventral poles of the retina occupy the intervals between the aforementioned middle and marginal streams. Axons from more central regions of the retina tend to occupy deeper levels of the optic tract. The regenerated optic tract does not regain its normal organization, e.g., axons of peripheral nasal origin are spread out widely over the entire width of the tract. However, axons from the temporal pole of the retina do return approximately to their original location in the middle stream. The concentration of temporal axons in the middle stream of the optic tract after regeneration may now be understood in terms of the expression pattern of the ephrin-A class of receptor tyrosine kinase ligands in the cellular matrix of the optic tract. The ephrin-As, which have a repellent effect on growing temporal retinal axons, are concentrated in and along the margins of the diencephalic optic tract and essentially absent from its middle stream. It is proposed here that peripheral temporal axons may be forced into this middle region by their avoidance of the higher levels of ephrin-A expression in the tract margins. In contrast, the growth pattern of regenerating peripheral nasal axons would not be affected by the ephrin-A gradient in the optic tract.

A pig growth assay was conducted to determine the relative biological value (RBV) of lysine from L-lysine sulfate compared with feed-grade L-lysine HCl. One hundred nursery pigs with an average initial BW of 9.5 +/- 1.5 kg were blocked by BW and gender and allotted randomly to five dietary treatments in five replicates of four pigs per pen. A corn-peanut meal diet containing 0.6% total lysine (as-fed basis) was supplemented with two levels (0.1 and 0.2%) of lysine from L-lysine-HCl or L-lysine sulfate. The RBV of L-lysine sulfate was determined using multiple regression slope-ratio methodology, with ADG and G:F as the response criteria. At the tested levels, linear responses for gain and G:F were obtained from increments of lysine from the two lysine sources. When ADG was regressed on supplemental lysine intake, the RBV of lysine in L-lysine sulfate was 99% of the RBV of lysine in L-lysine HCl. When G:F was regressed on supplemental lysine intake, the RBV of lysine in L-lysine sulfate was 97% of the RBV of lysine in L-lysine-HCl. The t-test analysis revealed that the RBV of lysine in L-lysine sulfate was not significantly different from the RBV of lysine in L-lysine HCl, which was assumed to be 100% bioavailable. In conclusion, L-lysine sulfate can replace L-lysine HCl in diets for growing swine.

Biodegradable and non-toxic multi-block copolymers based on poly(L-lysine) and poly(ethylene glycol) were synthesized. Synthesized copolymers showed almost negligible cytotoxicity above 95% cell viability and transfection efficiency compared to the PLL homopolymer with molecular weight of 25,700. Biodegradation under physiological conditions revealed that the molecular weight of copolymers decreased to 20% of the initial molecular weight within 72 h. Transfection efficiencies of copolymers were not affected by the presence of serum, while that of PLL homopolymer decreased to the level of naked DNA in the presence of serum. Based on the results, the new copolymers are believed to be a potentially efficient carrier for the delivery of bioactive agents.

Net portal absorption of AA during the 6-h postprandial period was measured in eight gilts (48.5 +/- 1.6 kg BW) in a crossover design. The pigs had chronic catheters placed in the portal vein, carotid artery, and ileal vein, and were trained to consume 1.2 kg of a standard grower diet once daily. Blood samples were taken every 30 min for 4 h and then hourly until 6 h after feeding. The first set of blood samples was taken after pigs were fed a meal of the test 16% CP corn-soybean meal diet (16% CP) or the test 12% CP corn-soybean meal diet supplemented with crystalline lysine, threonine, and tryptophan (12% CP + AA) to equal the three AA levels in the 16% CP diet. Pigs were then fed the standard diet for 2 d. Following that, blood samples were again taken after the pigs were fed a meal of the test diet that was not given to them at the first sampling period. Net portal AA absorption was calculated by multiplying porto-arterial plasma AA concentration difference by portal vein plasma flow rate (PVPF), estimated by an indicator-dilution technique employing p-aminohippuric acid as the indicator infused into the ileal vein. Plasma concentrations of lysine and threonine of pigs were affected by the diet x time interaction (P < 0.01). Portal and arterial plasma lysine and threonine concentrations in pigs attained the maximal level by 1 h postprandial when the 12% CP + AA diet was fed, but reached the peak level at 2.5 h postprandial when the 16% CP diet was given. The PVPF of pigs over the 6 h postprandial was less (P < 0.01) when the 12% CP + AA diet was given than when the 16% CP diet was fed. Net portal absorptions of lysine and threonine also were affected (P < 0.05) by time x diet interaction. The peak portal absorption of both lysine and threonine in pigs appeared at 0.5 h postprandial when the 12% CP + AA diet was given, but at 2.5 h postprandial with the feeding of the 16% CP diet. The early appearance of peak portal absorption of lysine and threonine from feeding the 12% CP + AA compared with the 16% CP diet indicates that crystalline lysine and threonine are absorbed more rapidly than protein-bound lysine and threonine in pigs fed once daily.

The study was conducted to evaluate the variability of efficiency of lysine utilisation in different feed proteins for growing pigs including wheat, unprocessed soybean flakes (SF), hydrothermal processed SF, corn gluten meal (CGM), two batches of soybean protein concentrate (SPC-1, SPC-2), different batches of peas (Pisum sativum) and field beans (Vicia faba). Data about efficiency of lysine are important for further conclusions related to lysine requirement in dependence on level of daily protein deposition. In N-balance studies 161 growing barrows (40-65 kg BW) of the genotype [Piétrain x (Duroc x Landrace)] were randomly allotted into 23 experimental treatments (n = 7) with diets in which lysine was the first limiting amino acid. Data from the N-balance trials were used to calculate efficiency of lysine and consequently lysine requirement based on an exponential N-utilisation model. Results from the present studies indicate that efficiency of lysine in different feed proteins varies in a very wide range and this variation greatly affected the calculated lysine requirement. Therefore, the variation in efficiency of lysine should be taken into account in requirement calculations and consequently in diet formulation for pigs. The results of model calculation for the lysine requirement depending on daily protein deposition (130, 145 and 160 g/d) and efficiency of lysine are given for different standards for comparison (g x kg(-0.67) x d(-1), g x d(-1) and percentage of lysine in the diet). The calculated lysine requirement of growing barrows (50 kg BW) corresponding to an average lysine efficiency was 15.5, 18.0 and 21.1 g/d for daily protein deposition of 130, 145 and 160 g, respectively. The results of the model calculation for 145 or 160 g daily protein deposition are in agreement with actual studies and recommendations of NRC (1998) and DLG (2002) for lysine supply.