Mammalian Urine Research Articles

Hydrothermally synthesized nitrogen-doped hydrochar from sawdust biomass for supercapacitor electrodes

Porous carbon generated from biomass is among the most promising electrode materials for supercapacitor applications. In this research work, sawdust was used as a carbon source to synthesize N-doped hydrochars via the hydrothermal method in the presence of nitrogen-containing compounds including ammonium chloride, urea agar, ammonium molybdate tetrahydrate, and mammalian urine, followed by chemical activation by potassium hydroxide. Using the hydrothermal technique, it was possible to dope 7.06 wt% of nitrogen into the hydrochar. The as synthesized materials were characterized by TGA/DTA, FTIR, BET, XRD, SEM/EDS, XPS, and proximate analysis. Furthermore, EIS, CV, and GCD were used to assess electrochemical performance. All the N-doped hydrochars synthesized samples possess crystalline and mesoporous structures with hydroxyl and amide functional groups. The KOH activation improved the specific surface area and pore volume by 8.5 % and 21 %, respectively. The maximum specific surface area and pore volume were found to be 560.72 m2/g and 0.2246 cc/g, respectively. The CV findings demonstrate the battery-like characteristics of the electrocatalysts made with molybdenum (VI) oxide (MoO3) embedded in these N-doped hydrochars, yielding 35.16 F/g specific capacitance at 10 mV/s. In contrast, the GCD's specific capacitance displays 80 F/g at 0.5 Ag−1.

Effect of functional groups on the adsorption of urea on activated carbon

The recovery of urea, present in the urine of mammals, has the potential of converting a current wastewater pollutant into an energy source due to its high hydrogen content, in alignment with the waste-to-energy principles. Carbon-based materials enable this approach due to their relatively high urea adsorption capacities. This work reveals that the adsorption of urea from aqueous solutions on activated carbon is a combination of weak van der Waals interactions such hydrogen bonds with the aromatic structure of the pristine carbon surface and also with carboxyl groups on the functionalised carbon, showing no appreciable interaction with hydroxyl or lactone groups. The selective introduction and quantification of different oxygen functional groups on activated carbon reveals their independent effect on the urea adsorption capacity, bringing an end to previous discrepancies in the literature. It also demonstrates that the optimisation of carbon materials must balance the increase of carboxyl groups and the decrease of the surface area associated to functionalisation treatments, providing guidance for future developments of carbon-based materials for circular economy applications.

Epidemiology, Clinical and Laboratory Findings of Pediatric Leptospirosis in Southeastern Turkey

Background: Leptospirosis is a significant zoonotic infection caused by Leptospira spirochetes, which are distributed globally. Infection typically occurs through exposure to water or soil contaminated by the urine of mammals, including dogs, cattle, pigs, or rodents. Objectives: The objective of this study was to elucidate the epidemiological, clinical, and laboratory characteristics of leptospirosis cases in Turkey. Methods: In a retrospective review, we analyzed the clinical and laboratory data of all individuals under 18 years diagnosed with leptospirosis at a secondary care center in Southeast Turkey from the beginning of 2020 to the end of 2022. Results: Over the two-year period, 36 confirmed cases of leptospirosis in individuals under 18 were identified. All patients had been engaged in activities at wells in cornfields, assisting their families with irrigation or related to well operations. Of these patients, 3 (8%) resided in urban areas, while 33 (92%) lived in rural settings. None required intensive care, and there were no fatalities reported. The most common symptoms at presentation were fatigue (83%), fever (75%), and nausea/vomiting (75%). Laboratory tests revealed that all patients had positive C-reactive protein (CRP) levels, with a significant portion (61%) showing highly positive results (CRP > 100). Conclusions: In conclusion, leptospirosis should be considered by clinicians when evaluating patients, especially those with risk factors like occupational exposure. Prompt testing for the disease is advisable under these circumstances.

Rainfall-driven resuspension of pathogenic Leptospira in a leptospirosis hotspot

Leptospirosis is a zoonosis caused by Leptospira bacteria present in the urine of mammals. Leptospira is able to survive in soils and can be resuspended during rain events. Here, we analyzed the pathogenic Leptospira concentration as a function of hydrological variables in a leptospirosis hot spot. A total of 226 samples were collected at the outlet of a 3 km2 watershed degraded by ungulate mammals (deer and feral pigs) and rats which are reservoirs for leptospirosis. Water samples collected at the beginning of a rain event following a dry period contained high concentrations of pathogenic Leptospira. The concentration was generally correlated with the water level and the suspended matter concentration (SMC) during the main flood event. A secondary peak of pathogenic Leptospira was sometimes detected after the main flood and in slightly turbid waters. Lastly, the pathogenic Leptospira concentration was extremely high at the end of a wet season. The pathogenic Leptospira concentrations could not be explained by a linear combination of hydrological variables (e.g. the rainfall, water level, SMC and soil moisture). However, nonlinear machine learning models of rainfall data only provided a fair fit to the observations and explained 75 % of the variance in the log10-transformed pathogenic Leptospira concentration. A comparison of identical machine learning models for the water level, SMC and pathogenic Leptospira concentration showed that the residual error in the Leptospira concentration was due to not only the small dataset but also the intrinsic characteristics of the signal. Our results support the hypothesis whereby pathogenic Leptospira survive at different depths in soils and superficial river sediments (depending on their water saturation) and are transferred to surface water during erosion. These results might help to refine leptospirosis warnings given to the local population. Future research should be focused on larger watersheds in more densely populated areas.

Co-Infection by Leptospira montravelensis and Leptospira interrogans Serovar Pomona in Urine Samples of Donkeys and Pigs in Sardinia, Italy.

Saprophytic leptospires are spirochetes enclosed within the non-pathogenic clade of the genus Leptospira, which in turn is subdivided into two subclades S1 and S2. To date, the microorganisms included in these subclades have been isolated from the environment in various parts of the world, and are believed to have no known animal reservoirs. After a case of Leptospira interrogans serovar Pomona was notified to the owner of a farm in Sardinia, all of the farm animals (11 pigs and 3 donkeys) were examined for the presence of Leptospira. Sera of all tested animals resulted positive for antibodies to Leptospira using a microscopic agglutination test (MAT). Moreover, nine (82%) kidney samples from pigs and three urine samples collected from donkeys (100%) tested positive for Leptospira DNA after qPCR. Results obtained after MLST analysis and sequencing of rrs, rpoB, and secY genes, performed on six Leptospira strains isolated in culture, revealed the presence of the genomospecies L. interrogans serovar Pomona in the kidney samples. Conversely, whole-genome sequencing combined with mean nucleotide identity revealed the presence of the saprophytic L. montravelensis in the urine samples. Our results report, for the first time, the isolation of a saprophytic species from mammalian urine, suggesting a new ecological specialization for these bacteria, with a possible transition from free-living to a symbiotic lifestyle. Further studies will have to be conducted to understand the evolution of virulence of these bacteria, potential infectivity, and possible public health implications.

Host-microbe co-metabolism via MCAD generates circulating metabolites including hippuric acid

The human gut microbiota produces dozens of small molecules that circulate in blood, accumulate to comparable levels as pharmaceutical drugs, and influence host physiology. Despite the importance of these metabolites to human health and disease, the origin of most microbially-produced molecules and their fate in the host remains largely unknown. Here, we uncover a host-microbe co-metabolic pathway for generation of hippuric acid, one of the most abundant organic acids in mammalian urine. Combining stable isotope tracing with bacterial and host genetics, we demonstrate reduction of phenylalanine to phenylpropionic acid by gut bacteria; the host re-oxidizes phenylpropionic acid involving medium-chain acyl-CoA dehydrogenase (MCAD). Generation of germ-free male and female MCAD−/− mice enabled gnotobiotic colonization combined with untargeted metabolomics to identify additional microbial metabolites processed by MCAD in host circulation. Our findings uncover a host-microbe pathway for the abundant, non-toxic phenylalanine metabolite hippurate and identify β-oxidation via MCAD as a novel mechanism by which mammals metabolize microbiota-derived metabolites.

Urease Enzyme Biosensor for Water Pollution Detection

Urea is the most containing nitrogen substance in the Urine of mammals. It plays an important role in the metabolism of nitrogen containing compounds by animals and human. A Biosensor for Urea was made by immobilizing Urease enzyme and Bromo stain which both obtained from sigma- Aldrich company on the microparticales surface were prepared through encapsulation successively by LBL self- assembly process of polyelectrolyte used PASS & PAH Polymers of eight layers. The size of nanoparticales was (5.05 – 5.87 nm). In other case some of these microparticales were removed there cores by HCL acid and filled with Bromo stain which is sensitive to change in PH. The microparticales were prepared and determined there size, layers under the SEM microscopy. The temperature, PH buffer concentration, Urease enzyme concentration, and enzyme immobilizing procedure were investigated. Microparticales prepared with Urease enzyme gives high sensitive response time 1-2 min. This Biosensor can use to determine the pollution in water and these microparticales made possible Biosensor to carry inside many medical materials in the future.

Off-line two-dimensional LC-MS/MS determination of tryptophan enantiomers in mammalian urine and alteration of their amounts in d-amino acid oxidase deficient mice

D-Tryptophan (D-Trp) is one of the minor D-enantiomers of amino acids discovered in microbes and mollusca. In the present study, a highly-selective 2D chiral LC-MS/MS method has been designed and developed focusing on the determination of Trp enantiomers to investigate the presence and regulation of free D-Trp in mammals. The developed system consisted of a reversed-phase separation for the first dimension, an enantioselective separation for the second dimension and also the detection using a triple quadrupole mass spectrometer for the third/fourth dimensions. Using the present method, urinary D-Trp in mammals, including healthy human volunteers and mice, were successfully determined. Although only l-Trp was observed in a mixed urine sample of healthy volunteers, small amounts of D-Trp were detected in the C57BL/6J mice (n = 5, %D=6.18 ± 0.47). In B6DAO- mice lacking the activity of d-amino acid oxidase (DAO), relatively high levels of D-Trp were observed (n = 6, %d=27.43 ± 3.26). The obtained %d values of Trp in the urine of the C57BL/6J mice and B6DAO- mice were confirmed using various enantioselective columns having different separation properties. These results indicate that the urinary D-Trp level is regulated by DAO in mammals, and further investigations, such as tissue distribution and physiological significance of the intrinsic D-Trp, are expected.

0129 Region-Specific Changes in Brain Peptidoglycan Following Sleep Deprivation

Abstract Introduction Bacterial cell wall peptidoglycan (PG) and muramyl peptides (MPs), isolated from mammalian brains and urine following sleep deprivation (SD), promote non-rapid eye movement sleep. These PG/MPs likely originate from the host microbiome and have been quantified in neonatal murine brain. PG/MP amounts and dynamics in healthy, adult murine brain remain unknown. Methods Wildtype mice acclimated to standard lab conditions were sacrificed at Zeitgeber time (ZT) 3 or ZT15 with (treatment, N=8), or without (control, N=8) 3h of SD prior to time points. Hypothalamic (HT), somatosensory cortex (Sctx) and brain stem (BS) areas were dissected, homogenized in phosphate buffered saline and centrifuged. PG/MP contents in resultant supernatants were determined using an ELISA (MyBioSource), interpolating sample PG from the standard curve, and expressed as ng peptidoglycan per mg tissue wet weight (ng/mg). Results At ZT3 and ZT15, BS PG values were significantly higher than HT or Sctx values, while HT and Sctx values did not differ from each other. At ZT3, mean PG values from control mice were: 3.6 in HT, 3.7 in Sctx, and 8.6 ng/mg in BS. After SD, corresponding values were: 3.0, 4.8 (statistically significant increase, p<0.05), and 7.5 ng/mg. Further, within all 8 individual mice after SD prior to ZT3, PG levels in Sctx were higher than corresponding values in HT (p<0.001).At ZT15, PG control values were: 4.6 in HT, 4.6 in Sctx, and 8.9 ng/mg in BS. After SD, PG level at ZT15 was not significantly changed in any brain area assayed. However, PG values after SD at ZT15 compared to ZT3 SD values were significantly higher for HT and BS (p<0.0005 and p<0.005, respectively). In an independent experiment (see Dykstra-Aiello et. al. this volume) we confirmed PG values at ZT15 in BS were significantly higher than Sctx values. Conclusion Results indicate unique PG regulation by brain area, sleep loss, and time-of-day suggesting physiological roles for brain PG guiding host behaviors such as sleep. Thus, mammalian sleep-wake regulation and its various associated cognitive states, are the product of millions of years of co-evolutionary symbioses between microbes and their hosts. Support (If Any) Supported by the W.M. Keck Foundation and NIH (NS025378).

Mathematical Modeling of Urea Reaction with Sulfuric Acid and Phosphoric Acid to Produce Ammonium Sulfate and Ammonium Dihydrogen Phosphate Respectively

Urea is the final product of protein metabolism in mammals and can be found in different biological fluids. Use of mammalian urine in agricultural production as organic fertilizer requires safe handling to avoid the formation of ammonia that will decrease the fertilizer value due to the loss of nitrogen. Safe handling is also required to minimize the decomposition of urea into condensed products such as biuret and cyanuric acid, which will also have a negative impact on the potential sustainable production of crops and sanitation technologies. The study of thermodynamics and reaction kinetics of urea stabilization plays a key role in understanding the conditions under which undesirable compounds and impurities in urea-based fertilizers and urea-based selective catalytic reduction systems are formed. For this reason, we studied the reaction of urea in acid media to achieve urea stabilization by modeling the reaction of urea with sulfuric acid and phosphoric acid, and estimating the reaction enthalpy and adiabatic heat difference for control of the heat released from the neutralization step using Ca(OH)2 or MgO for the safety of the process. Numerical and simulation analyses were performed by studying the effect of the surrounding temperature, the ratio of acid reagent to urea concentration, the rate of addition, and the reaction rate to estimate the required time to achieve an optimum value of urea conversion into ammonium dihydrogen phosphate or ammonium sulfate as potential technological opportunities for by-product valorization. Full conversion of urea was achieved in about 10 h for reaction rates in the order of 1 × 10−5s−1 when the ratio of H2SO4 to CH4N2O was 1.5. When increasing the ratio to 10, the time required for full conversion was considerably reduced to 3 h.

Cloudy with a Chance of Peptides: Accessibility, Scalability, and Reproducibility with Cloud-Hosted Environments.

Cloud-hosted environments offer known benefits when computational needs outstrip affordable local workstations, enabling high-performance computation without a physical cluster. What has been less apparent, especially to novice users, is the transformative potential for cloud-hosted environments to bridge the digital divide that exists between poorly funded and well-resourced laboratories, and to empower modern research groups with remote personnel and trainees. Using cloud-based proteomic bioinformatic pipelines is not predicated on analyzing thousands of files, but instead can be used to improve accessibility during remote work, extreme weather, or working with under-resourced remote trainees. The general benefits of cloud-hosted environments also allow for scalability and encourage reproducibility. Since one possible hurdle to adoption is awareness, this paper is written with the nonexpert in mind. The benefits and possibilities of using a cloud-hosted environment are emphasized by describing how to setup an example workflow to analyze a previously published label-free data-dependent acquisition mass spectrometry data set of mammalian urine. Cost and time of analysis are compared using different computational tiers, and important practical considerations are described. Overall, cloud-hosted environments offer the potential to solve large computational problems, but more importantly can enable and accelerate research in smaller research groups with inadequate infrastructure and suboptimal local computational resources.

Determination of phenylalanine enantiomers in the plasma and urine of mammals and ᴅ-amino acid oxidase deficient rodents using two-dimensional high-performance liquid chromatography

A two-dimensional (2D) HPLC system focusing on the determination of phenylalanine (Phe) enantiomers in mammalian physiological fluids has been developed. ᴅ-Phe is indicated to have potential values as a disease biomarker and therapeutic molecule in several neuronal and metabolic disorders, thus the regulation of ᴅ-Phe in mammals is a matter of interest. However, the precise determination of amino acid enantiomers is difficult in complex biological samples, and the development of an analytical method with practically acceptable sensitivity, selectivity and throughput is expected. In the present study, a 2D-HPLC system equipped with a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension has been designed, following the fluorescence derivatization of the target amino acid enantiomers with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The analytical method was validated using both plasma and urine samples, and successfully applied to human, rat and mouse fluids. Trace levels of ᴅ-Phe were determined in the plasma, and the %ᴅ values were around 0.1% for all species. In the urine, relatively large amounts of ᴅ-Phe were observed, and the %ᴅ values for humans, rats and mice were 3.99, 1.76 and 5.25%, respectively. The relationships between the enzymatic activity of ᴅ-amino acid oxidase (DAO) and the amounts of intrinsic ᴅ-Phe have also been clarified, and high ᴅ-Phe amounts were observed (around 0.3% in the plasma and around 50% in the urine) in the DAO deficient rats and mice.

Mud-puddling behaviour of butterflies in the Soraya research station, district of Subulussalam, Aceh, Indonesia

Butterflies, mostly the male congregate and uptake the sodium and amino acids from the mud, dung, and urine of mammals or decaying flesh and then transferred to the female during mating. This behaviour is usually called as puddling. Hence, the objective of the present study was to identify the puddle butterflies in the Soraya river banks, their sex and age ratio. Five sampling points were observed for three hours (09:00-12:00 am) during 5 d each. The number of individual and number of species of butterflies that do puddlings were observed. A total of 25 species of butterflies belonging to four families were identified. Appias lyncida and Graphium sarpedon were the most active in puddling followed by Caleta elna, Pathysa antiphates and Appias lybithea. Generally, butterflies that do puddle were the male and the young individual.

16S rRNA gene profiling of bacterial communities mediating production of tsetse attractive phenols in mammalian urine.

Several types of odours are involved in the location of host animals by tsetse (Diptera: Glossinidae), a vector of animal African trypanosomiasis. Host animals’ ageing urine has been shown to be the source of a phenolic blend attractive to the tsetse. Nevertheless, limited research has been performed on the microbial communities’ role in the production of phenols. This study aimed at profiling bacterial communities mediating the production of tsetse attractive phenols in mammalian urine. Urine samples were collected from African buffalo (Syncerus caffer), cattle (Bos taurus) and eland (Taurotragus oryx) at Kongoni Game Valley Ranch and Kenyatta University in Kenya. Urine samples, of each animal species, were pooled and left open to age in ambient conditions. Bacteriological and phenols analyses were then carried out, at 4 days ageing intervals, for 24 days. Phenols analysis revealed nine volatile phenols: 4-cresol, ortho-cresol, 3-cresol, phenol, 3-ethylphenol, 3-propylphenol, 2-methyloxyphenol, 4-ethylphenol and 4-propylphenol. Eight out of 19 bacterial isolates from the ageing urine revealed the potential to mediate production of phenols. 16S rRNA gene characterisation of the isolates closely resembled Enterococcus faecalis KUB3006, Psychrobacter alimentarius PAMC 27887, Streptococcus agalactiae 2603V, Morganella morganii sub.sp. morganii KT, Micrococcus luteus NCTC2665, Planococcus massiliensis strain ES2, Ochrobactrum pituitosum AA2 and Enterococcus faecalis OGIRF. This study established that some of the phenols emitted from mammalian urine, which influence the tsetse‘s host-seeking behaviour, are well characterised by certain bacteria. These results may allow the development of biotechnological models in vector control that combines the use of these bacteria in the controlled release of semiochemicals.

Tamm‐Horsfall Protein and Properdin Biochemical and Functional Interactions

Tamm‐Horsfall protein (THP), also known as uromodulin, is an abundant protein in normal mammalian urine that interacts with numerous cells and molecules of the immune system, including various complement proteins such as C1q and factor H. Properdin (factor P), which potentiates complement activation by stabilizing the alternative pathway C3 convertase, previously has been shown to bind to THP. In the present study, an enzyme‐linked immunosorbent assay (ELISA) was used to examine the effect of ionic strength, pH, and heparin on the binding between these two oppositely charged proteins. Additionally, since THP has been shown to act as a cofactor, like factor H, in factor I‐mediated C3b degradation, we investigated whether properdin altered this cofactor activity of THP. All ELISA buffers contained 1 mM MgCl2 and 1 mM CaCl2 because earlier studies showed a requirement for divalent cations in this reaction. As measured by ELISA, binding between THP and properdin was similar (K~ 10−8 M) in 20 mM, 60 mM, and 90 mM NaCl solutions, but no significant binding was detected in 154 mM NaCl buffers. There was a reduction in binding capacity, but not overall affinity in 120 mM NaCl buffers. Binding of properdin to immobilized THP in 60 mM NaCl was similar at pH 6.0, 7.5, and 8.5. The THP/properdin interaction was very sensitive to heparin, with 0.008 units of heparin/ml producing a 50% inhibition of binding of 2 μg/ml properdin to immobilized THP. In vitro cofactor assays analyzed on silver‐stained SDS‐PAGE gels demonstrated that while 300 ng of properdin inhibited by more than 50% the ability of 1 ng factor H to serve as a cofactor in factor‐I mediated C3b degradation, properdin appeared to increase by about 25% the ability of THP to serve as a cofactor in factor I‐mediated C3b degradation. Properdin, by itself, was not able to act as a cofactor in factor I‐mediated C3b degradation. In conclusion, THP and properdin bind more readily in lower ionic strength buffers than in higher ionic strength solutions, but these interactions are stable over a range of pH conditions typically found in urine. Heparin is very effective at inhibiting the binding between properdin to THP. On a functional level, properdin may augment THP's ability to act as a cofactor in C3b degradation, while it inhibits factor H's cofactor activity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of a Three-Dimensional HPLC System for the Simultaneous Determination of Lactate and 3-Hydroxybutyrate Enantiomers in Mammalian Urine

Development of a Three-Dimensional HPLC System for the Simultaneous Determination of Lactate and 3-Hydroxybutyrate Enantiomers in Mammalian Urine

Collection of Untargeted Metabolomic Data for Mammalian Urine Applying HILIC and Reversed Phase Ultra Performance Liquid Chromatography Methods Coupled to a Q Exactive Mass Spectrometer.

Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) is the most frequently applied analytical platform in the untargeted metabolomic study of mammalian urine. Here we describe two complementary UPLC-MS methods for metabolomic analysis or urine, a reversed phase C18 method and a hydrophilic interaction liquid chromatography (HILIC) method. We discuss the inclusion of pooled quality control (QC) samples and a recommended analysis list construction. Up to 96 injections can be performed every 24h, and up to 2000 metabolites can be routinely detected.

Preliminary pharmacokinetic study of the anticancer 6BIO in mice using an UHPLC-MS/MS approach

Indirubins represent a group of natural and synthetic products with bio-activities against numerous human cancer cell lines acting by inhibiting protein kinases. The natural sources of indirubins are plants of Isatis sp., Indigofera sp., and Polygonum sp., recombinant bacteria, mammalian urine and some marine mollusks. Specifically, the halogenated derivative 6-bromo indirubin-3′-oxime (6BIO) possesses increased selectivity against GSK-3. However, to our knowledge, no analytical method to determine 6BIO in biological fluids has been developed till now. Therefore, a rapid, sensitive and high throughput UHPLC-MS/MS methods were developed and validated to evaluate the concentrations of 6BIO in mice plasma. Plasma samples were pre-treated by protein precipation using cold mixture of methanol: acetonitrile (9:1, v/v) and separations were carried out on a Hypersil Gold C18 column (50 × 2.1 mm i.d.; 1.9 μm p.s.) using 0.1% acetic acid and methanol as mobile phase at a flow rate of 500 mL/min in a gradient mode. For quantitation, a hybrid LTQ-Orbitrap MS equipped with an electro-spray ionization source was used applying a selected reaction monitoring (SRM) option. The monitored transitions were m/z 354.0 → 324.0 for 6BIO and 297.1 → 282.1 for afromorsin (used as the internal standard) in the negative mode. Following the EMA, ICH and FDA guidelines for validation of analytical procedures, the assay method was fully validated in terms of selectivity, linearity, recovery, matrix effect, accuracy, precision, stability, and robustness. The validated methods were successfully applied to the pharmacokinetic studies of 6BIO following an oral administration to mice at the dose of 50 mg/kg. The results indicated that 6BIO possesses a Tmax of 30 min, a half-life of 1 h, and low plasma bioavailability.

Structural and biochemical studies on the role of active site Thr166 and Asp236 in the catalytic function of D-Serine deaminase from Salmonella typhimurium

D-Serine deaminase (DSD) degrades D-Ser to pyruvate and ammonia. Uropathogenic bacteria survive in the toxic D-Ser containing mammalian urine because of DSD activity. The crystal structure of the apo form of Salmonella typhimurium DSD (StDSD) has been reported earlier. In the present work, we have investigated the role of two active site residues, Thr166 and Asp236 by site directed mutagenesis (T166A and D236L). The enzyme activity is lost upon mutation of these residues. The 2.7 Å resolution crystal structure of T166A DSD with bound PLP reported here represents the first structure of the holo form of StDSD. PLP binding induces small changes in the relative dispositions of the minor and major domains of the protein and this inter-domain movement becomes substantial upon interaction with the substrate. The conformational changes bring Thr166 to a position at the active site favorable for the degradation of D-Ser. Examination of the different forms of the enzyme and comparison with structures of homologous enzymes suggests that Thr166 is the most probable base abstracting proton from the Cα atom of the substrate and Asp236 is crucial for binding of the cofactor.

Mammalian urine concentration: a review of renal medullary architecture and membrane transporters.

Mammalian kidneys play an essential role in balancing internal water and salt concentrations. When water needs to be conserved, the renal medulla produces concentrated urine. Central to this process of urine concentration is an osmotic gradient that increases from the corticomedullary boundary to the inner medullary tip. How this gradient is generated and maintained has been the subject of study since the 1940s. While it is generally accepted that the outer medulla contributes to the gradient by means of an active process involving countercurrent multiplication, the source of the gradient in the inner medulla is unclear. The last two decades have witnessed advances in our understanding of the urine-concentrating mechanism. Details of medullary architecture and permeability properties of the tubules and vessels suggest that the functional and anatomic relationships of these structures may contribute to the osmotic gradient necessary to concentrate urine. Additionally, we are learning more about the membrane transporters involved and their regulatory mechanisms. The role of medullary architecture and membrane transporters in the mammalian urine-concentrating mechanism are the focus of this review.