The role of orotic acid measurement in routine newborn screening for urea cycle disorders.

Urea cycle disorders (UCDs), including OTC deficiency (OTCD), are life-threatening diseases with a broad clinical spectrum. Early diagnosis and initiation of treatment based on a newborn screening (NBS) test for OTCD with high specificity and sensitivity may contribute to reduction of the significant complications and high mortality. The efficacy of incorporating orotic acid determination into routine NBS was evaluated. Combined measurement of orotic acid and citrulline in archived dried blood spots from newborns with urea cycle disorders and normal controls was used to develop an algorithm for routine NBS for OTCD in Israel. Clinical information and genetic confirmation results were obtained from the follow-up care providers. About 1147986 newborns underwent routine NBS including orotic acid determination, 25 of whom were ultimately diagnosed with a UCD. Of 11 newborns with OTCD, orotate was elevated in seven but normal in two males with early-onset and two males with late-onset disease. Orotate was also elevated in archived dried blood spots of all seven retrospectively tested historical OTCD patients, only three of whom had originally been identified by NBS with low citrulline and elevated glutamine. Among the other UCDs emerge, three CPS1D cases and additional three retrospective CPS1D cases otherwise reported as a very rare condition. Combined levels of orotic acid and citrulline in routine NBS can enhance the detection of UCD, especially increasing the screening sensitivity for OTCD and differentiate it from CPS1D. Our data and the negligible extra cost for orotic acid determination might contribute to the discussion on screening for proximal UCDs in routine NBS.

Age-dependent effects of chronic stress on brain plasticity and depressive behavior.

Exposure to chronic mild stress (CMS) is known to induce anhedonia in adult animals, and is associated with induction of depression in humans. However, the behavioral effects of CMS in young animals have not yet been characterized, and little is known about the long-term neurochemical effects of CMS in either young or adult animals. Here, we found that CMS induces anhedonia in adult but not in young animals, as measured by a set of behavioral paradigms. Furthermore, while CMS decreased neurogenesis and levels of brain-derived neurotrophic factor (BDNF) in the hippocampus of adult animals, it increased these parameters in young animals. We also found that CMS altered alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor GluR1 subunit levels in the hippocampus and the nucleus accumbens of adult, but not young animals. Finally, no significant differences were observed between the effects of CMS on circadian corticosterone levels in the different age groups. The substantially different neurochemical effects chronic stress exerts in young and adult animals may explain the behavioral resilience to such stress young animals possess.

Atherogenicity of amino acids in the lipid-laden macrophage model system in vitro and in atherosclerotic mice: a key role for triglyceride metabolism.

Atherosclerosis-related research has focused mainly on the effects of lipids on macrophage foam cell formation and atherogenesis, whereas the role of amino acids (AAs) was understudied. The current study aimed to identify anti- or pro-atherogenic AA in the macrophage model system and to elucidate the underlying metabolic and molecular mechanisms. J774A.1 cultured macrophages were treated with increasing concentrations of each 1 of the 20 AAs. Macrophage atherogenicity was assessed in terms of cellular toxicity, generation of reactive oxygen species (ROS) and cellular cholesterol or triglyceride content. At nontoxic concentrations (up to 1 mM), modest effects on ROS generation or cholesterol content were noted, but six specific AAs significantly affected macrophage triglyceride content. Glycine, cysteine, alanine and leucine significantly decreased macrophage triglyceride content (by 24%-38%), through attenuated uptake of triglyceride-rich very low-density lipoprotein (VLDL) by macrophages. In contrast, glutamate and glutamine caused a marked triglyceride accumulation in macrophages (by 107% and 129%, respectively), via a diacylglycerol acyltransferase-1 (DGAT1)-dependent increase in triglyceride biosynthesis rate with a concurrent maturation of the sterol regulatory element-binding protein-1 (SREBP1). Supplementation of apolipoprotein E-deficient (apoE-/-) mice with glycine for 40 days significantly decreased the triglyceride levels in serum and in peritoneal macrophages (MPMs) isolated from the mice (by 19%). In contrast, glutamine supplementation significantly increased MPM ROS generation and the accumulation of cholesterol and that of triglycerides (by 48%), via enhanced uptake of LDL and VLDL. Altogether, the present findings reveal some novel roles for specific AA in macrophage atherogenicity, mainly through modulation of cellular triglyceride metabolism.

Dopamine uptake and cocaine binding mechanisms: the involvement of charged amino acids from the transmembrane domains of the human dopamine transporter.

The wild type human dopamine transporter (DAT) and five DAT mutants were transfected into COS-7 cells and their ability to uptake dopamine or to bind cocaine was examine three days later. In each mutant, a single charged amino acid, located in areas that initial hydrophobic analysis had indicated were DAT transmembrane domains was substituted by alanine. Mutants used in this study were lysines 257 and 525 (termed K257A and K525A), arginines 283 and 521 (termed R283A and R521A), and glutamate 491 (termed E491A). Dopamine affinity was significantly enhanced in the K257A and R283A mutants, and the IC(50) for displacement of the radioactive cocaine analog 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT) by cocaine was significantly elevated in the E491A mutant. All mutants displayed a reduction or complete loss of the maximal velocity (V(m)) of dopamine transport.

The interaction of methylphenidate and benztropine with the dopamine transporter is different than other substrates and ligands.

A substantial body of evidence suggests that the dopamine transporter (DAT) is the principal site for cocaine-induced reward and euphoria. Interactions between the DAT and its substrates and ligands may therefore be of clinical relevance. The pharmacological characteristics of DAT compounds were compared in wild type (WT) and mutant DATs. The DAT mutants chosen for study were those with reduced binding and uptake activities (aspartic acid 79 mutated to alanine, termed D79A), reduced binding but normal uptake (tyrosine 251 mutated to alanine, termed Y251A; tyrosine 273 mutated to alanine, termed, Y273A), and normal binding but reduced uptake (a double mutation: serines 356 and 359 mutated to alanine, termed S356,359A). The WT and mutant DATs were transfected into COS-7 cells, and their pharmacological activities were examined 3 days later. Different patterns of pharmacological activity emerged. GBR 12909, cocaine, and mazindol each showed reduced affinity for the Y251A and the Y273A mutants, but their affinity for the S356,359A mutant was similar to that of the WT DAT. d-Amphetamine, MPP+, and dopamine each showed reduced affinity for the S356,359A mutant. Benztropine and methylphenidate had a different effect. Relative to the WT DAT, they both showed reduced affinity for the S356,359A mutant when displacing radioactive carboxyfluorotropane (CFT) binding, but similar affinity when inhibiting radioactive dopamine uptake. These results indicate that methylphenidate and benztropine may interact with the DAT in a different fashion then other substrates and ligands.

Behavioral analysis during the forced swimming test using a joystick device.

The behavioral test described by Porsolt in 1977 for screening potential antidepressant drugs is extensively used both in basic research and in the pharmaceutical industry. The measured behavior is the immobility time during the swimming test (preformed in rodents), which decreases upon acute antidepressant treatment. Several research groups have suggested some modifications on the original Porsolt paradigm and its analysis. Nevertheless, there are still inaccuracies resulting from either undefined intermediate behaviors or from considering the movement of the whole body as one unit without analyzing the motion of the limbs. Herein, we propose a novel and simple scoring method, based on continuous measurement of the limbs motion, using a joystick, a computer screen and simple software. We validated the method, using antidepressant drugs and studied examples of false positives and false negatives of the traditional Porsolt paradigm. The proposed method is easy to use, it accounts for all range of movements and the analysis is relatively fast. Moreover, the results obtained using this analysis method show a normal Gaussian distribution in a population of rats (while the traditional Porsolt analysis does not) which allows selective breeding of 'motivated' and 'depressed' lines of animals.

Severe traumatic brain injury and controlled hemorrhage in rats: quest for the optimal mean arterial blood pressure after whole fresh donor blood resuscitation.

Treatment of combined traumatic brain injury and hypovolemic shock poses a particular challenge due to the possible conflicting consequences. While restoring diminished volume is the treatment goal for hypovolemia, maintaining and adequate cerebral perfusion pressure and avoidance of secondary damage remain a treatment goal for the injured brain. Various treatment modalities have been proposed, but the optimal resuscitation fluid and goals have not yet been clearly defined. In this study, we investigate the physiological and neurological outcomes in a rat model of combined traumatic brain injury and hypovolemic shock, submitted to treatment with varying amounts of fresh blood. Forty-eight male Lewis rats were divided into control and treatment groups. Traumatic brain injury was inflicted by a free-falling rod on the exposed cranium. Hypovolemia was induced by controlled hemorrhage of 30% blood volume. Treatment groups were treated by fresh whole blood with varying volumes, reaching resuscitation goals of a mean arterial blood pressure (MAP) of 80, 100, and 120 mmHg at 15 min. Mean arterial blood pressure was assessed at 60 min and neurological outcomes and mortality in the subsequent 48 h. At 60 min, MAP was highest for the group resuscitated most aggressively. Neurological outcomes and mortality inversely correlated with the aggressiveness of resuscitation. In this study, we find that mild resuscitation with goals of restoring MAP to 80 mmHg (which is lower than baseline) provided best results when considering hemodynamic stability, survival, and neurological outcomes. An aggressive resuscitation may be detrimental, inducing processes that eventually cause a significant decrease in survival.

Moderate Ringer's lactate solution resuscitation yields best neurological outcome in controlled hemorrhagic shock combined with brain injury in rats.

Anesthetized rats were assigned to sham; brain injury (BI); controlled hemorrhagic shock (CHS); BI combined with CHS (combined injury [CI]); and CI groups resuscitated with 2.5 mL/kg Ringer's lactate solution (RL-2.5), 10 mL/kg RL (RL-10), or 40 mL/kg RL (RL-40). Brain injury was induced by applying 400 millibar negative pressure for 10 s through a hollow screw inserted into a 4.5-mm burr hole drilled into the left parietal region of the skull. Five minutes after BI, 30% of circulating blood volume was withdrawn for 10 min to induce CHS. One hour of fluid resuscitation commenced 20 min posthemorrhage. MAP, lactate, and base excess levels were significantly improved in the RL-40 group compared with all other hemorrhaged groups. The hematocrit level 1 h after resuscitation began was significantly lower in the RL-40 group (27.6% +/- 0.57%) than in all other groups. The RL-40 group had the worst neurological severity score 24 h postsurgery. MAP, lactate, and base excess levels were not significantly improved in the RL-2.5 group, however, the number of surviving neuronal cells in the perilesional brain region was significantly higher than in the CI or RL-40 groups. MAP, lactate, and base excess levels were significantly improved in the RL-10 group (P < 0.05). Mobility and the number of surviving neurons in the perilesional region of the brain were significantly better in the RL-10 group than in the CI or RL-40 groups (P < 0.05). Although massive fluid resuscitation yields preferable hemodynamic and metabolic outcomes, neurological outcomes are better after moderate fluid resuscitation for BI combined with controlled hemorrhagic shock.