A case report of acute claudication due to vena cava thromboembolism in a dog naturally infected with Leishmania Infantum.

Canine leishmaniosis is a vector-borne disease caused by Leishmania infantum, and clinical manifestations of infection range from absent or severe to fatal and result from immune-mediated mechanisms. In dogs, the most common clinical signs of leishmaniosis include skin lesions and lymphadenomegaly. However, the presence of other nontypical signs has been described, and diagnosing these cases can be challenging. The aim of the present short communication was to describe the impact of the formation of circulating immunocomplexes due to L. infantum in a dog with leishmaniosis affected by a massive venous thrombus of the caudal vena cava and external iliac veins. On admission, the dog presented bilateral cutaneous vasculopathy of the thigh and renal disease due to L. infantum infection. Two weeks after starting anti-Leishmania treatment based on meglumine antimoniate and allopurinol administration, the animal developed acute claudication of the hind limbs with the presence of a thrombus in the caudal vena cava and the external iliac veins and a high level of circulating immunocomplexes detected by enzyme-linked immunosorbent assay. Exacerbation of the humoral immune response, along with deposition of circulating immune complexes in the tissues and the concurrent presence of kidney and liver damage, might have contributed to an imbalance in haemostasis in this patient. Future studies should evaluate and analyse the pathological mechanisms contributing to thrombosis in dogs with leishmaniosis.

Corrugated intimal surface of the ovine aorta: when physiology resembles pathology.

The aortic lumen in healthy animals is characterized by a smooth, whitish surface, but sheep have macroscopic corrugation of the intimal surface in the thoracic aorta (TA). Our aim was to determine if this finding was pathological or physiological. Thirteen sheep aortas were included in this work together with aortas from cattle (n = 3), a goat (n = 1), horses (n = 4), dogs (n = 2), rabbits (n = 2) and a pig (n = 1). A corrugated intimal surface in the TA was seen in all the sheep and the goat but was less evident in the cattle. Histologically, in sheep the TA intimal surface was seen to have multifocal bulging areas that protruded into the lumen. The outer half of the tunica media had numerous, randomly distributed muscle islands that disrupted the arrangement of the elastic lamella, displacing them towards the lumen. We conclude that the intimal corrugation of the TA in sheep is physiological and must not be misinterpreted as pathological.

Mannheimia haemolytica-associated fibrinonecrotizing abomasitis in lambs.

Mannheimia haemolytica-associated abomasitis has been clinically described as a cause of sudden death in lambs, but it is poorly characterized. We describe the pathological features of a severe fibrinonecrotizing abomasitis in 3 lambs that died suddenly. All 3 abomasums had a thickened submucosa due to edema and necrotic areas delimited by bands of degenerate neutrophils with slender nuclei (oat cells) and angiocentric distributions. The overlying mucosa was congested. Myriads of gram-negative coccobacilli were observed within the oat cell bands. M. haemolytica was isolated from the abomasum in all 3 animals and was serotyped as A2 in one of them. Pericarditis and pleuritis were observed in 2 of the lambs. Clostridium spp. were isolated in 1 lamb and detected by immunohistochemistry in the 3 animals, suggesting clostridial co-infection. M. haemolytica should be considered among the differential diagnoses of necrotizing abomasitis in lambs.

A case of canine intestinal malakoplakia.

Malakoplakia is a rare chronic granulomatous disease usually affecting the urinary bladder and other locations. In humans, the gastrointestinal tract is the second most common location but there are no reports of intestinal malakoplakia in animals. A 10-month-old female French Bulldog was presented with chronic haemorrhagic diarrhoea and anorexia with normochromic-normocytic anaemia and hypoalbuminaemia. Grossly, there was mucosal thickening and ulceration of the caecum, colon and rectum. Microscopically, transmural sheets of foamy macrophages were seen in these tissues. Macrophages were periodic acid-Schiff, vimentin and ionized calcium-binding adaptor molecule 1 positive and contained von Kossa- and Prussian blue-positive Michaelis-Gutmann bodies. Giemsa staining revealed rod-shaped bacterial colonies and fluorescence in-situ hybridization demonstrated Escherichia coli within macrophages. This is the first reported case of intestinal malakoplakia in domestic animals. Pathological features of intestinal malakoplakia share many similarities with ulcerative histiocytic colitis in dogs but it is unclear if they are different forms of the same pathological process or distinct entities.

Feline osteochondromatosis in a 12-year-old feline leukaemia virus-negative cat.

Feline osteochondromatosis is a spontaneous osteocartilaginous exostosis associated with feline leukaemia virus (FeLV) infection or due to a frameshift variant in the exostosin glycosyltransferase 1 (EXT1) gene. Osteochondromatosis was diagnosed in an indoor-only, 12-year-old, neutered female, Russian Blue cat. Radiographs revealed bilateral calcified proliferations in the elbow, costochondral and sternochondral joints, which distorted the normal skeletal structure. Grossly, the proliferated joints presented with consistent, rounded masses, causing complete ankylosis. The main histopathological finding was an osteocartilaginous proliferation composed of multiple irregular islands of well-differentiated hyaline cartilage surrounded and delimited by osteoid tissue. Immunohistochemistry of the osteochondromas, bone marrow and mediastinal lymph nodes, using a primary anti-FeLV gp70 antibody, and FeLV proviral DNA real-time polymerase chain reaction on bone marrow were negative. Sequencing of exon 6 of the EXT1 gene was performed and nucleotide BLAST analysis demonstrated the absence of a frameshift variant. This study reports the only case of spontaneous feline osteochondromatosis in an animal more than 10 years old.

Transfer of photothermal nanoparticles using stem cell derived small extracellular vesicles for in vivo treatment of primary and multinodular tumours.

The main current challenges in oncology are (1) avoiding systemic side effects in therapy, and (2) developing alternative treatment strategies for metastatic tumours. Nanomedicine was assumed to provide answers to these issues, but delivering enough therapeutic nanoparticles (NPs) to tumours still remains a huge challenge in nanomaterials-based treatments. Extracellular vesicles (EVs) play a key role in cell communication processes and can be combined with nanomaterials to improve their targeting capabilities. In this work, we leverage the ability of EVs derived from stem cells to reach tumour areas successfully, being used as delivery vehicles for nanoparticles acting as hyperthermia agents. Once small extracellular vesicles (sEVs) loaded with NIR-sensitive hollow gold NPs reached primary subcutaneous solid tumours, they were irradiated with a NIR laser and almost complete tumour remission was obtained. More interestingly, those sEV vehicles were also able to reach multinodular areas similar to those on advanced metastatic phases, eradicating most tumour growth regions in multiple cancerous nodules located in the pancreas region.

Worldwide Prevalence of Small Ruminant Lentiviruses in Sheep: A Systematic Review and Meta-Analysis.

Small Ruminant Lentiviruses (SRLV) are highly prevalent retroviruses with significant genetic diversity and antigenic heterogeneity that cause a progressive wasting disease of sheep called Maedi-visna. This work provides a systematic review and meta-analysis of the last 40 years (1981-2020) of scientific publications on SRLV individual and flock prevalence. Fifty-eight publications and 314 studies were included. Most articles used a single diagnostic test to estimate prevalence (77.6%), whereas articles using three or more tests were scarce (6.9%). Serological tests are more frequently used than direct methods and ELISA has progressively replaced AGID over the last decades. SRLV infection in sheep is widespread across the world, with Europe showing the highest individual prevalence (40.9%) and being the geographical area in which most studies have been performed. Africa, Asia, and North America show values between 16.7% to 21.8% at the individual level. South and Central America show the lowest individual SRLV prevalence (1.7%). There was a strong positive correlation between individual and flock prevalence (ρ = 0.728; p ≤ 0.001). Despite the global importance of small ruminants, the coverage of knowledge on SRLV prevalence is patchy and inconsistent. There is a lack of a gold standard method and a defined sampling strategy among countries and continents.

Metabolic syndrome and endocrine status in HIV-infected transwomen.

BACKGROUND: HIV-infected transwomen face multiple specific issues. Economic and social marginalization, sex work, substance abuse, hormonal consumption and silicone injection may affect the course of HIV infection and lead to metabolic and endocrine complications. METHODS: A matched case-control study was performed between 2013 and 2015 in a University Hospital and compared metabolic syndrome (MetS), thyroid and adrenal functions in HIV-infected transwomen (i.e. cases) and cisgender HIV-infected men (i.e. controls) matched for age and antiretroviral therapy. The interaction between hormonal consumption, the course of HIV infection and antiretroviral therapy was also studied. Clinical and biological data (CD4 cell count, HIV RNA load, antiretroviral plasma drug concentration, HDL, triglycerides, glucose, cortisol, thyroid stimulating hormone, free thyroxine, prolactine) were measured. RESULTS: A total of 292 HIV-infected patients (100 cases and 192 controls) were prospectively included. There was no difference between the two populations in terms of frequency of MetS, but subclinical hypothyroidism and adrenal insufficiency were more frequent in cases than in controls with, respectively, 12 vs. 3% (P < 0.002) for hypothyroidism and 20 vs. 8% (P < 0.001) for adrenal insufficiency. Prolactinemia, only performed in transwomen, was often elevated (21%) but rarely confirmed as true active hyperprolactinemia (monomeric form) (3%). Although hormonal intake was frequent among transwomen (31%), no impact on antiretroviral bioavailability and efficacy was detected. CONCLUSION: In this study, no increase in the prevalence of MetS was detected in HIV-infected transwomen patients. In contrast, adrenal and thyroid functions abnormalities were frequent and should be systematically assessed in this population. No impact of hormonal intake on antiretroviral bioavailability and efficacy was detected.

Initial Evaluation of Prospective and Parallel Assessments of Cystic Fibrosis Newborn Screening Protocols in Eastern Andalusia: IRT/IRT versus IRT/PAP/IRT.

Identifying newborns at risk for cystic fibrosis (CF) by newborn screening (NBS) using dried blood spot (DBS) specimens provides an opportunity for presymptomatic detection. All NBS strategies for CF begin with measuring immunoreactive trypsinogen (IRT). Pancreatitis-associated protein (PAP) has been suggested as second-tier testing. The main objective of this study was to evaluate the analytical performance of an IRT/PAP/IRT strategy versus the current IRT/IRT strategy over a two-year pilot study including 68,502 newborns. The design of the study, carried out in a prospective and parallel manner, allowed us to compare four different CF-NBS protocols after performing a post hoc analysis. The best PAP cutoff point and the potential sources of PAP false positive results in our non-CF newborn population were also studied. 14 CF newborns were detected, resulting in an overall CF prevalence of 1/4, 893 newborns. The IRT/IRT algorithm detected all CF cases, but the IRT/PAP/IRT algorithm failed to detect one case of CF. The IRT/PAP/IRT with an IRT-dependent safety net protocol was a good alternative to improve sensitivity to 100%. The IRT × PAP/IRT strategy clearly performed better, with a sensitivity of 100% and a positive predictive value (PPV) of 39%. Our calculated optimal cutoffs were 2.31 µg/L for PAP and 167.4 µg2/L2 for IRT × PAP. PAP levels were higher in females and newborns with low birth weight. PAP false positive results were found mainly in newborns with conditions such as prematurity, sepsis, and hypoxic-ischemic encephalopathy.

Biosafety assessment of conducting nanostructured materials by using co-cultures of neurons and astrocytes.

Neural electrode implants are made mostly of noble materials. We have synthesized a nanostructured material combining the good electrochemical properties of iridium oxide (IrOx) and carbon-nanotubes (CNT) and the properties of poly(3,4-ethylenedioxythiophene) (PEDOT). IrOx-CNT-PEDOT charge storage capacity was lower than that of IrOx and IrOx-CNT, but higher than that of other PEDOT-containing hybrids and Pt. Cyclic voltammetry, SEM, XPS and micro-Raman spectroscopy suggest that PEDOT encapsulates IrOx and CNT. In our search for a cell culture platform that could optimize modelling the in vivo environment, we determined cell viability, neuron and astrocyte functionality and the response of astrocytes to an inflammatory insult by using primary cultures of neurons, of astrocytes and co-cultures of both. The materials tested (based on IrOx, CNT and PEDOT, as well as Pt as a reference) allowed adhesion and proliferation of astrocytes and full compatibility for neurons grown in co-cultures. Functionality assays show that uptake of glutamate in neuron-astrocyte co-culture was significantly higher than the sum of the uptake in astrocytes and neurons. In co-cultures on IrOx, IrOx-CNT and IrOx-CNT-PEDOT, glutamate was released by a depolarizing stimulus and induced a significant increase in intracellular calcium, supporting the expression of functional NMDA/glutamate receptors. LPS-induced inflammatory response in astrocytes showed a decreased response in NOS2 and COX2 mRNA expression for IrOx-CNT-PEDOT. Results indicate that neuron-astrocyte co-cultures are a reliable model for assessing the biocompatibility and safety of nanostructured materials, evidencing also that hybrid IrOx-CNT-PEDOT nanocomposite materials may offer larger resistance to inflammatory insults.

Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography.

Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO2) chromatography enrichment prior to nLC-MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.

Quality of life in home-ventilated children and their families.

HMV (home mechanical ventilation) in children has increased over the last years. The aim of the study was to assess perceived quality of life (QOL) of these children and their families as well as the problems they face in their daily life.We performed a multicentric cross-sectional study using a semi-structured interview about the impact of HMV on families and an evaluation questionnaire about perceived QOL by the patient and their families (pediatric quality of life questionnaire (PedsQL4.0)). We studied 41 subjects (mean age 8.2 years). Global scores in PedsQL questionnaire for subjects (median 61.4), and their parents (median 52.2) were below those of healthy children. 24.4% received medical follow-up at home and 71.8% attended school. Mothers were the main caregivers (75.6%), 48.8% of which were fully dedicated to the care of their child. 71.1% consider economic and healthcare resources insufficient. All families were satisfied with the care they provide to their children, even though it was considered emotionally overwhelming (65.9%). Marital conflict and neglect of siblings appeared in 42.1 and 36% of families, respectively. CONCLUSIONS: Perceived QOL by children with HMV and their families is lower than that of healthy children. Parents are happy to care for their children at home, even though it negatively affects family life. What is Known: • The use of home mechanical ventilation (HMV) in children has increased over the last years. • Normal family functioning is usually disrupted by HMV. What is New: • The aim of HMV is to provide a lifestyle similar to that of healthy children, but perceived quality of life by these patients and their parents is low. • Most of the families caring for children on HMV agree that support and resources provided by national health institutions is insufficient.

High Prevalence of Diastolic Dysfunction in Incident Patients on Peritoneal Dialysis: Association with Low Thyroid Hormones.

BACKGROUND/AIMS: Diastolic dysfunction (DD) and low levels of thyroid hormones (TH) are frequent found in chronic kidney disease; both are associated with all-cause and cardiovascular mortality. However, a link between them has not yet been established. The aim of this study was to analyze DD as a surrogate marker of fibrosis and its association with TH in incident patients on peritoneal dialysis (PD). METHODS: A cross-sectional study with 183 incident patients on PD with preserved ejection fraction was performed. Clinical and demographic data were registered. Serum total and free (t/f) triiodothyronine (T3), thyroxin (T4), and thyroid stimulating hormone levels were determined by RIA kits, albumin and high-sensitivity C-reactive protein by conventional assays. Transthoracic 2D echocardiogram was performed for evaluation of left ventricular (LV) mass and ejection fraction. DD was evaluated using pulsed-wave tissue Doppler imaging. RESULTS: Patients were 43 ± 12, 42% with diabetes mellitus (DM). Some degree of DD was found in 62% of patients; 18% had grade I DD, 8% grade II DD and 36% grade III DD. Patients with grade III DD were more likely to have diabetes, older, high LV mass and low serum albumin, t/fT3 and tT4 levels. In logistic multivariate regression analysis, it was found that diabetes (B = -0.86, 95% CI 0.182-0.992, p < 0.05), hypertension (B = -0.95, 95% CI 0.184-0.817, p = 0.01) and tT3 (B = -1.94, 95% CI 0.023-0.876, p < 0.05) were associated with grade III DD. CONCLUSIONS: High prevalence of grade III DD was found in incident patients on PD. In addition to DM and hypertension, tT3 was found to be an independent risk factor for grade III DD and more studies are needed to understand the reasons as to why this association is present.

IrOx-carbon nanotube hybrids: a nanostructured material for electrodes with increased charge capacity in neural systems.

Nanostructured iridium oxide-carbon nanotube hybrids (IrOx-CNT) deposited as thin films by dynamic electrochemical methods are suggested as novel materials for neural electrodes. Single-walled carbon nanotubes (SWCNT) serve as scaffolds for growing the oxide, yielding a tridimensional structure with improved physical, chemical and electrical properties, in addition to high biocompatibility. In biological environments, SWCNT encapsulation by IrOx makes more resistant electrodes and prevents the nanotube release to the media, preventing cellular toxicity. Chemical, electrochemical, structural and surface characterization of the hybrids has been accomplished. The high performance of the material in electrochemical measurements and the significant increase in cathodal charge storage capacity obtained for the hybrid in comparison with bare IrOx represent a significant advance in electric field application in biosystems, while its cyclability is also an order of magnitude greater than pure IrOx. Moreover, experiments using in vitro neuronal cultures suggest high biocompatibility for IrOx-CNT coatings and full functionality of neurons, validating this material for use in neural electrodes.

Mola hidatiforme parcial / Partial hydatidiform mole

Introducción: el término enfermedad trofoblástica gestacional es una anormal proliferación del trofoblasto, que desde el punto de vista histológico se clasifica en mola hidatiforme, mola invasora, coriocarcinoma y tumor del sitio de implantación del trofoblasto. Objetivo: describir las características clínicas y ultrasonográficas de una mola hidatiforme parcial o mola embrionada. Paciente: se presenta un caso de 24 sem de gestación con el diagnóstico ultrasonográfico de mola hidatidiforme parcial. Resultados: se realizó cariotipo fetal y determinación de niveles de gonadotropina coriónica en suero materno. Se hallaron defectos congénitos en el feto, por lo que se ofreció asesoramiento genético a la pareja, que optó por la interrupción de la gestación. Conclusiones: el diagnóstico prenatal fue confirmado por la anatomía patológica

Introduction: the term gestational trophoblastic disease is an abnormal proliferation of the trophoblast, which is histologically classified as hydatidiform mole, invasive mole, choriocarcinoma, and tumor trophoblast implantation site. Objective: to describe the clinical and ultrasonographic partial hydatidiform mole or mole embrionada. Patient: we report a 24- week - prenancy case with ultrasonographic diagnosis of partial hydatidiform mole. Results: fetal karyotype was performed and levels of chorionic gonadotropin in maternal serum were determined. We found congenital defects in the fetus, so genetic counseling was offered to the parents, who chose ending this pregnancy. Conclusions: the prenatal diagnosis was confirmed by pathology

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: the relevance of Glu299 and Leu460 for nucleotide binding.

A homology model of Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase (ATP + oxaloacetate right arrow over left arrow ADP + PEP + CO(2)) in complex with its substrates shows that the isobutyl group of Leu460 is in close proximity to the adenine ring of the nucleotide, while the carboxyl group of Glu299 is within hydrogen-bonding distance of the ribose 2'OH. The Leu460Ala mutation caused three-fold and seven-fold increases in the K (m) for ADPMn(-) and ATPMn(2-), respectively, while the Glu299Ala mutation had no effect. Binding studies showed losses of approximately 2 kcal mol(-1) in the nucleotide binding affinity due to the Leu460Ala mutation and no effect for the Glu299Ala mutation. PEP carboxykinase utilized 2'deoxyADP and 2'deoxyATP as substrates with kinetic and equilibrium dissociation constants very similar to those of ADP and ATP, respectively. These results show that the hydrophobic interaction between Leu460 and the adenine ring of the nucleotide significantly contributed to the nucleotide affinity of the enzyme. The 2'deoxy nucleotide studies and the lack of an effect of the Glu299Ala mutation in nucleotide binding suggest that the possible hydrogen bond contributed by Glu299 and the ribose 2'OH group may not be relevant for nucleotide binding.

Vesselplasty: a new technical approach to treat symptomatic vertebral compression fractures.

OBJECTIVE: The objective of our study was to evaluate the effectiveness and safety of vesselplasty to treat symptomatic vertebral compression fractures (VCFs). SUBJECTS AND METHODS: Twenty-nine patients undergoing vesselplasty at our institution between April 2006 and February 2008 were enrolled in the study. All patients had been undergoing medical therapy for one or more painful VCFs. Pain, mobility, and analgesic use scores were obtained, and restoration of vertebral body height was evaluated. A two-tailed paired Student's t test was used to compare differences in the mean scores for levels of pain, mobility, and analgesic use before and after the procedure and to evaluate changes in vertebral body height. We analyzed the influence of the age of the fracture and its cause in the variations in the pain, mobility, and analgesic use scores. RESULTS: Seven of the 29 patients had fractures in more than one level, for a total of 37 procedures. The cause of the vertebral collapse was osteoporosis in 27 (73%), high-impact trauma in five (13.5%), myeloma in three (8%), and metastatic fracture in two (5.4%). The average pain score before treatment was 8.72 +/- 1.25 (SD), whereas the average pain score after treatment was 3.38 +/- 2.35. The average mobility score before treatment was 2.31 +/- 1.94, whereas the average mobility score after treatment was 0.59 +/- 1.05 (p < 0.001). The average analgesic use score before treatment was 3.07 +/- 1.46, whereas it was 1.86 +/- 1.90 after treatment (p < 0.001). There was no evidence of clinical complications. CONCLUSION: Vesselplasty offers statistically significant benefits in improvements of pain, mobility, and the need for analgesia in patients with symptomatic VCFs, thus providing a safe alternative in the treatment of these fractures.

Stereochemistry of the carboxylation reaction catalyzed by the ATP-dependent phosphoenolpyruvate carboxykinases from Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens.

The stereochemistry of CO(2) addition to phosphoenolpyruvate (PEP) to yield oxaloacetate catalyzed by ATP-dependent Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens PEP carboxykinases was determined using (Z)-3-fluorophosphoenolpyruvate ((Z)-F-PEP) as a substrate analog. A. succiniciproducens and S. cerevisiae PEP carboxykinases utilized (Z)-F-PEP with 1/14 and 1/47 the respective K(m) values for PEP. On the other hand, in the bacterial and yeast enzymes k(cat) was reduced to 1/67 and 1/48 the value with PEP, respectively. The binding affinity of pyridoxylphosphate-labeled S. cerevisiae and A. succiniciproducens PEP carboxykinases for PEP and (Z)-F-PEP was checked and found to be of similar magnitude for both substrates, suggesting that the lowered K(m) values for the fluorine-containing PEP analog are due to kinetic effects. The lowered k(cat) values when using (Z)-F-PEP as substrate suggest that the electron withdrawing effect of fluorine affects the nucleophilic attack of the double bond of (Z)-F-PEP to CO(2). For the stereochemical analyses, the carboxylation of (Z)-F-PEP was coupled to malate dehydrogenase to yield 3-fluoromalate, which was analyzed by (19)F NMR. The fluoromalate obtained was identified as (2R, 3R)-3-fluoromalate for both the A. succiniciproducens and S. cerevisiae PEP carboxykinases, thus indicating that CO(2) addition to (Z)-F-PEP, and hence PEP, takes place through the 2-si face of the double bond. These results, together with previously published data [Rose, I.A. et al. J. Biol. Chem. 244 (1969) 6130-6133; Hwang, S.H. and Nowak, T. Biochemistry 25 (1986) 5590-5595] indicate that PEP carboxykinases, no matter their nucleotide specificity, catalyze the carboxylation of PEP from the 2-si face of the double bond.

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: relevance of arginine 70 for catalysis.

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase is a key enzyme of the gluconeogenic pathway and catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO2 in the presence of a divalent metal ion. Previous experiments indicate that mutation of amino acid residues at metal site 1 decrease the enzyme catalytic efficiency and the affinity of the protein for PEP, evidencing the relevance of hydrogen-bond interactions between PEP and water molecules of the first coordination sphere of the metal ion for catalysis [Biochemistry 41 (2002) 12763]. To further understand the function of amino acid residues located in the PEP binding site, we have now addressed the catalytic importance of Arg70, whose guanidinium group is close to the PEP carboxyl group. Arg70 mutants of PEP carboxykinase were prepared, and almost unaltered kinetic parameters were found for the Arg70Lys PEP carboxykinase, while a decrease in 4-5 orders of magnitude for the catalytic efficiency was detected for the Arg70Gln and Arg70Met altered enzymes. To evaluate the enzyme interaction with PEP, the phosphopyridoxyl-derivatives of wild type, Arg70Lys, Arg70Gln, and Arg70Met S. cerevisiae PEP carboxykinase were prepared, and the change in the fluorescence emission of the probe upon PEP binding was used to obtain the dissociation equilibrium constant of the corresponding derivatized enzyme-PEP-Mn2+ complex. The titration experiments showed that a loss in 2.1 kcal/mol in PEP binding affinity is produced in the Arg70Met and Arg70Gln mutant enzymes. It is proposed that the electrostatic interaction between the guanidinium group of Arg70 and the carboxyl group of PEP is important for PEP binding and for further steps in catalysis.