Sex and pubertal variation in reward-related behavior and neural activation in early adolescents.

This study aimed to characterize the role of sex and pubertal markers in reward motivation behavior and neural processing in early adolescence. We used baseline and two-year follow-up data from the Adolescent Brain and Cognitive DevelopmentSM study (15844 observations; 52% from boys; age 9-13). Pubertal development was measured with parent-reported Pubertal Development Scale, and DHEA, testosterone, and estradiol levels. Reward motivation behavior and neural processing at anticipation and feedback stages were assessed with the Monetary Incentive Delay task. Boys had higher reward motivation than girls, demonstrating greater accuracy difference between reward and neutral trials and higher task earnings. Girls had lower neural activation during reward feedback than boys in the nucleus accumbens, caudate, rostral anterior cingulate, medial orbitofrontal cortex, superior frontal gyrus and posterior cingulate. Pubertal stage and testosterone levels were positively associated with reward motivation behavior, although these associations changed when controlling for age. There were no significant associations between pubertal development and neural activation during reward anticipation and feedback. Sex differences in reward-related processing exist in early adolescence, signaling the need to understand their impact on typical and atypical functioning as it unfolds into adulthood.

Sex and pubertal influences on the neurodevelopmental underpinnings of schizophrenia: A case for longitudinal research on adolescents.

Sex is a significant source of heterogeneity in schizophrenia, with more negative symptoms in males and more affective symptoms and internalizing comorbidity in females. In this narrative review, we argue that there are likely sex differences in the pathophysiological mechanisms of schizophrenia-spectrum disorders (SZ) that originate during puberty and relate to the sex-specific impacts of pubertal maturation on brain development. Pubertal maturation might also trigger underlying (genetic or other) vulnerabilities in at-risk individuals, influencing brain development trajectories that contribute to the emergence of SZ. This review is the first to integrate links between pubertal development and neural development with cognitive neuroscience research in SZ to form and evaluate these hypotheses, with a focus on the frontal-striatal and frontal-limbic networks and their hypothesized contribution to negative and mood symptoms respectively. To test these hypotheses, longitudinal research with human adolescents is needed that examines the role of sex and pubertal development using large cohorts or high risk samples. We provide recommendations for such studies, which will integrate the fields of psychiatry, developmental cognitive neuroscience, and developmental endocrinology towards a more nuanced understanding of the role of pubertal factors in the hypothesized sex-specific pathophysiological mechanisms of schizophrenia.

Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis.

A new approach for adenosine triphosphate (ATP) regeneration in a cell-free protein synthesis system is described. We first show that pyruvate can be used as a secondary energy source to replace or supplement the conventional secondary energy source, phosphoenol pyruvate (PEP). We also report that glucose-6-phosphate, an earlier intermediate of the glycolytic pathway, can be used for ATP regeneration. These new methods provide more stable maintenance of ATP concentration during protein synthesis. Because pyruvate and glucose-6-phosphate are the first and last intermediates of the glycolytic pathway, respectively, the results also suggest the possibility of using any glycolytic intermediate, or even glucose, for ATP regeneration in a cell-free protein synthesis system. As a result, the methods described provide cell-free protein synthesis with greater flexibility and cost efficiency.

Advances in Escherichia coli production of therapeutic proteins.

Escherichia coli offers a means for the rapid and economical production of recombinant proteins. These advantages, coupled with a wealth of biochemical and genetic knowledge, have enabled the production of such economically sensitive products as insulin and bovine growth hormone. Although significant progress has been made in transcription, translation and secretion, one of the major challenges is obtaining the product in a soluble and bioactive form. Recent progress in oxidative cytoplasmic folding and cell-free protein synthesis offers attractive alternatives to standard expression methods.

Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli.

We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.

Prolonging cell-free protein synthesis by selective reagent additions.

Factors causing the early cessation of protein synthesis have been studied in a cell-free system from Escherichia coli. We discovered that phosphoenol pyruvate (PEP), the secondary energy source for ATP regeneration, and several amino acids are rapidly degraded during the cell-free protein synthesis reaction. The degradation of such compounds takes place even in the absence of protein synthesis. This degradation severely reduces the capacity for protein synthesis. The lost potency was completely recovered when the reaction mixture was supplied with additional PEP and amino acids. Of the 20 amino acids, only arginine, cysteine, and tryptophan were required to restore system activity. Through repeated additions of PEP, arginine, cysteine,and tryptophan, the duration of protein synthesis was greatly extended. In this fed-batch reaction, after a 2 h incubation, the level of cell-free synthesized chloramphenicol acetyl transferase (CAT) reached 350 microg/mL, which is 3.5 times the yield of the batch reaction. Addition of fresh magnesium further extended the protein synthesis. As a result, through coordinated additions of PEP, arginine, cysteine, tryptophan, and magnesium, the final concentration of cell-free synthesized CAT increased more than 4-fold compared to a batch reaction. SDS-PAGE analysis of such a fed-batch reaction produced an obvious band of CAT upon Coomassie Blue staining.

Prolonging cell-free protein synthesis with a novel ATP regeneration system.

A new approach for the regeneration of adenosine triphosphate (ATP) during cell-free protein synthesis was developed to prolong the synthesis and also to avoid the accumulation of inorganic phosphate. This approach was demonstrated in a batch system derived from Escherichia coli. Contrary to the conventional methods in which exogenous energy sources contain high-energy phosphate bonds, the new system was designed to generate continuously the required high-energy phosphate bonds within the reaction mixture, thereby recycling the phosphate released during protein synthesis. If allowed to accumulate, phosphate inhibits protein synthesis, most likely by reducing the concentration of free magnesium ion. Pediococcus sp. pyruvate oxidase, when introduced in the reaction mixture along with thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), catalyzed the generation of acetyl phosphate from pyruvate and inorganic phosphate. Acetyl kinase, already present with sufficient activity in Escherichia coli S30 extract, then catalyzed the regeneration of ATP. Oxygen is required for the generation of acetyl phosphate and the H(2)O(2) produced as a byproduct is sufficiently degraded by endogenous catalase activity. Through the continuous supply of chemical energy, and also through the prevention of inorganic phosphate accumulation, the duration of protein synthesis is extended up to 2 h. Protein accumulation levels also increase. The synthesis of human lymphotoxin receives greater benefit than than that of chloramphenicol acetyl transferase, because the former is more sensitive to phosphate inhibition. Finally, through repeated addition of pyruvate and amino acids during the reaction period, protein synthesis continued for 6 h in the new system, resulting in a final yield of 0.7 mg/mL.

Olanzapine treatment after clozapine-induced granulocytopenia in 3 patients.

BACKGROUND: How to best treat psychotic patients who have had past clozapine-induced agranulocytosis or granulocytopenia remains a problem. CASE REPORTS: We report 3 patients with chronic schizophrenia who had previously stopped clozapine due to hematologic side effects. The patients evidenced improvement with olanzapine that equated to 16- to 31-point decreases in rating scale scores during 1-year follow-up without any hematologic abnormalities. CONCLUSION: The results suggest that olanzapine may be useful in treating patients with clozapine-induced granulocytopenia without the risk of recurrence of hematologic side effects.

Expression of active human tissue-type plasminogen activator in Escherichia coli.

The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 microgram of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding.

E. coli-based in vitro transcription/translation: in vivo-specific synthesis rates and high yields in a batch system.

A highly efficient Escherichia coli-based, batch in vitro protein synthesis system using circular plasmid DNA is described. Compared to a presently available commercial kit, this improved system produced several hundredfold greater yields of the rDNA human protein thrombopoietin (ca. 450 micrograms/mL). The system is capable of obtaining specific synthesis rates similar to those in vivo, approximately a 1000-fold increase compared to the original methods previously described. It compares favorably in rates and yields to the recently published semicontinuous methods but with the convenience of a true batch system.

Overexpression of Escherichia coli oxidoreductases increases recombinant insulin-like growth factor-I accumulation.

Transient overexpression of either DsbA or DsbC can double the yield of periplasmic insulin-like growth factor (IGF)-I in Escherichia coli to 8.5 g/liter. Strikingly, most of the overexpressed DsbA or DsbC is found in the reduced form, implying that enhanced disulfide isomerization is responsible for the substantial increase in IGF-I yield. All of the accumulated IGF-I has had the signal sequence removed, underscoring the secretion capacity of this organism as well as its utility for efficient production of polypeptide with the correct amino terminus. The overexpressed IGF-I constitutes approximately 30% of the total cell protein. Overproduction of active site mutants of DsbA instead of the wild-type gene do not produce this increase in yield. With wild-type levels of DsbA and DsbC, most of the secreted IGF-I is found in disulfide-linked aggregates, although 10% is soluble and about 5% is correctly folded. Contrary to expectations, overexpression of the disulfide oxidoreductases decreased the soluble fraction. Because the aggregated protein can be efficiently solubilized and refolded, the increased yield is a significant benefit for the production of IGF-I.

Genetic approaches to the detection of contaminants in Escherichia coli fermentations.

In facilities which cultivate more than one rDNA organism, contamination by the same species is difficult to detect. Since the same species may be producing a different product in an adjacent fermentor or in the same vessel in a subsequent procedure, the possibility of cross-product contamination must be considered. Here we describe a simple, sensitive, and reliable technique for the detection of same-species contamination. The assay uses negative genetic markers such as the inability to use a carbohydrate, e.g., ribose. When the facility is managed to use the ribose marker for only one product, this culture can be plated on ribose minimal medium to allow rapid and sensitive detection of contaminants. If the facility is used for several rDNA products, multiple carbohydrate markers per strain can be used so that a limited number of markers can differentiate among larger host collections. The approach was developed and tested using Escherichia coli as the host organism. If hosts with auxotrophies, e.g., for amino acids, are used in the facility, the plate medium can be supplemented. When this technique is combined with existing methods for detecting different species and bacteriophage contamination, all three classes of biological contamination can be detected.

In vitro and in vivo redox states of the Escherichia coli periplasmic oxidoreductases DsbA and DsbC.

DsbC is a periplasmic protein of Escherichia coli that was previously identified by a genetic selection that rescued sensitivity to dithiothreitol in Tn10 mutagenized cells. The Erwinia chrysanthemi dsbC gene was identified in a previous genetic screen to restore motility in a dsbA null strain. In order to analyze the biochemical role of E. coli DsbC, the protein was overexpressed, purified, and compared with DsbA in terms of disulfide isomerization, thiol oxidation, and in vivo redox state. In vitro, DsbC and DsbA have an equivalent kcat for disulfide isomerization with the model substrate, misfolded insulin-like growth factor-1. However, DsbA is a more effective oxidant than DsbC of protein dithiols. In vivo, DsbA is found exclusively in the oxidized state in wild-type strains grown in rich media. On the other hand, in vivo DsbC has one pair of cysteines oxidized and one pair reduced. DsbD is required to maintain this reduced pair of cysteines, confirming previous genetic results. A dsbC deletion strain showed decreases in the production of some, but not all, heterologous proteins containing multiple disulfide bonds. Notably, those proteins affected by the dsbC deletion do not have the cysteines paired consecutively.

Frontotemporal dementia: treatment response to serotonin selective reuptake inhibitors.

BACKGROUND: Patients with frontotemporal dementia (FTD) present initially with primarily behavioral rather than cognitive symptoms. Decreased serotonin receptor binding has been reported in the frontal lobes, temporal lobes, and hypothalamus in autopsy-proven FTD cases. This study tests the hypothesis that many of the behavioral symptoms of FTD (including disinhibition, depressive symptoms, carbohydrate craving, and compulsions) will respond to serotonin selective reuptake inhibitors (SSRIs). METHOD: Eleven subjects meeting the Lund-Manchester clinical, neuropsychological, and neuroimaging criteria for FTD were treated with SSRIs (fluoxetine, sertraline, or paroxetine). After 3 months, treatment responses for disinhibition, depressive symptoms, carbohydrate craving, and compulsions were evaluated prospectively without placebo control. RESULTS: After treatment, disinhibition, depressive symptoms, carbohydrate craving, and compulsions all showed improvement in at least half the subjects in which they had been present. One subject stopped sertraline treatment because of diarrhea, while another stopped paroxetine treatment due to increased anxiety. The presence of individual behavioral symptoms and also the response of each symptom to SSRIs were unrelated to cognitive impairment as measured by baseline Mini-Mental Status Examination (.07 < or = p < or = 1.00) [corrected]. CONCLUSION: The behavioral symptoms of FTD may improve after treatment with SSRIs. Future neurochemical studies and controlled pharmacologic trials may improve available treatments.

Behavioral phenomenology in Alzheimer's disease, frontotemporal dementia, and late-life depression: a retrospective analysis.

Often patients in the early stages of Alzheimer's disease (AD), frontotemporal dementia (FTD), and late-life depression can be difficult to differentiate clinically. Although subtle cognitive distinctions exist between these disorders, noncognitive behavioral phenomenology may provide additional discriminating power. In 19 subjects with AD, 19 with FTD, 16 with late-life psychotic depression (LLPD), and 19 with late-life nonpsychotic depression (LLNPD), noncognitive behavioral symptoms were quantified retrospectively using the Schedules for Clinical Assessment in Neuropsychiatry (SCAN) and compared using both a one-way ANOVA and a multivariate stepwise discriminant analysis, which utilized a jackknife procedure. The FTD group showed the highest mean total SCAN score, while the AD group showed the lowest. ANOVA showed significant differences in the mean total SCAN scores between the four diagnostic groups (P < .0001). With the discriminant analysis, the four disorders demonstrated different clusters of behavioral abnormalities and were differentiated by these symptoms (P < .0001). A subset of 14 SCAN item group symptoms was identified that collectively classified the following percentages of subjects in each diagnostic category: AD 94.7%, FTD 100%, LLPD 87.5%, and LLNPD 100%. These results indicate that AD, FTD, LLPD, and LLNPD were distinguished retrospectively by the SCAN without using cognitive data. Better definition of the longitudinal course of noncognitive behavioral symptoms in different dementias and psychiatric disorders will be valuable both for diagnosis and to help define behavioral syndromes that are associated with selective neuroanatomic and neurochemical brain pathology.

Tardive dyskinesia and ethnicity: review of the literature.

Tardive dyskinesia (TD) is a side effect of long-term neuroleptic administration. The wide variation of 2 to 51% in its reported prevalence can be attributed to the varied definitions of TD, the use of different methods of assessment, and the lack of control of independent variables. Why only certain patients develop this side effect is an intriguing question. The occurrence of TD in family members and in those persons with a family history of Parkinson's disease (PD) is suggestive of genetic vulnerability. Further support for a genetic predisposition comes from the fact that only certain strains of monkeys, such as the Cebus apella strain, have a higher propensity to develop TD than others, such as the Macaca sepciosa strain. If genetic factors play a significant role in the development of TD, then, genetically diverse ethnic groups may have a different propensity for the development of TD. One method of evaluating such a possibility is to compare its prevalence in different countries. The current literature on ethnic differences in the prevalence rates of TD is reviewed. This area of study needs further rigorous investigation.

Compulsive behaviors as presenting symptoms of frontotemporal dementia.

Frontotemporal dementia (FTD) is a common neurodegenerative dementia syndrome. Compulsive behaviors frequently occur in FTD and may be presenting symptoms of this disorder. This study evaluated compulsive behaviors as presenting symptoms in 29 patients with FTD compared to 48 patients with Alzheimer's disease (AD) enrolled in the UCLA Alzheimer's Disease Center. The FTD patients met the Lund and Manchester criteria for FTD and had predominant frontal hypoperfusion on single-photon emission computer tomography neuroimaging. The AD patients met National Institute of Neurological and Communicative Disorders-Alzheimer's Disease and Related Disorders criteria for clinically probable AD. Compulsive behaviors occurred in 11 FTD patients (38%) versus 5 AD patients (10%) (chi2 = 6.73, P < .01). This difference persisted after controlling for the younger age of the FTD group. There was a range of compulsive behaviors, with the most frequent being repetitive checking activities. Compulsive behaviors are common presenting symptoms among FTD patients and may result from an inability to inhibit urges to perform compulsive movements from damage to frontal-striatal circuits.

Dietary changes, compulsions and sexual behavior in frontotemporal degeneration.

The occurrence of weight gain, sweet and carbohydrate craving, hyposexuality, and compulsions in frontal lobe dementia (FLD) compared to Alzheimer's disease (AD) was evaluated. FLD is a progressive dementia with a high rate of misdiagnosis and therefore better diagnostic criteria for FLD are needed. Fourteen patients meeting research criteria for AD were compared to 14 with suspected FLD. All had cerebral perfusion measured with xenon-133 and imaged with HMPAO using brain-dedicated SPECT. The FLD group showed frontotemporal and AD posterior temporoparietal hypoperfusion. The primary caregivers were queried regarding weight gain, sweet/carbohydrate preference, sexual drive, and compulsions. Differences were compared with Fisher's exact test. The following was found in FLD versus AD: Weight gain in FLD patients amounted to 64% (AD 7%), carbohydrate craving was 79% (vs. 0%) and compulsive behavior 64% (vs. 14%). The differences for these symptoms were statistically significant, whereas for the symptoms increased sexual drive (8 vs. 8%) and reduced sexual drive (54 vs. 23%) no significant difference could be found. In FLD the first symptoms were often dietary changes or hyposexuality. Compulsions were more bizarre and severely disabling in FLD than in AD. Dietary changes, hyposexuality, and disabling compulsions are prominent early symptoms in FLD but not AD. The cause of these symptoms may be due to both frontal and subcortical serotonin loss and dysfunction of the anterior temporal lobes.

Abuse of monoamine oxidase inhibitors.

Abuse of monoamine oxidase inhibitors is not common but there are a few cases of addiction in the literature. Most of these patients had an additional diagnosis, either history of past drug abuse or personality disorder and MAOI withdrawal symptoms have been reported. We encountered three patients who received MAOI under psychiatric care. They were all self medicated by increasing the doses on their own, experienced euphoria and visited various physicians to obtain MAOI prescriptions and manifested toxic states. One of our patients had a normal, another a schizoid and the third, an addictive personality. Two were addicted in the past to amphetamine. Therefore, it is important not to prescribe MAOI's to patients who have a history of amphetamine and other addictions.

Protein folding activities of Escherichia coli protein disulfide isomerase.

DsbA is an Escherichia coli periplasmic protein that mediates disulfide bond formation in newly secreted proteins in vivo. Addition of thiol reagents to purified dsbA reduces its disulfide bond and yields disulfide isomerase activity after removal of the thiol reagent. DsbA can catalyze the conversion of a stable misfolded protein, misfolded IGF-I (mis-IGF-I), to its correctly folded conformation under physiological conditions. This conversion is the result of breaking and re-forming two disulfide bonds. The uncatalyzed rate of this reaction is undetectable. Kinetic analysis of the reaction yielded a Km of 43 microM and a kcat of 0.2 min-1. The oxidized form of dsbA stimulates the oxidative folding of completely reduced IGF-I at pH 7.0. Thus, dsbA has two possible functions depending on its redox state. The reduced form of the protein is a disulfide isomerase while the oxidized protein can assist formation of disulfide bonds in reduced substrates under physiological conditions.