Isolation and expression of a rat brain cDNA encoding glutamate carboxypeptidase II

N-acetylated alpha-linked acidic dipeptidase (NAALADase) hydrolyzes acidic peptides, such as the abundant neuropeptide N-acetyl-alpha-L-aspartyl-L-glutamate (NAAG), thereby generating glutamate. Previous cDNA cloning efforts have identified a candidate rat brain NAALADase partial cDNA, and Northern analyses have identified a family of related RNA species that are found only in brain and other NAALADase-expressing cells. In this report, we describe the cloning of a set of rat brain cDNAs that describe a full-length NAALADase mRNA. Transient transfection of a full-length cDNA into the PC3 cell line confers NAAG-hydrolyzing activity that is sensitive to the NAALADase inhibitors quisqualic acid and 2-(phosphonomethyl)glutaric acid. Northern hybridization detects the expression of three similar brain RNAs approximately 3,900, 3,000, and 2,800 nucleotides in length. In situ hybridization histochemistry shows that NAALADase-related mRNAs have an uneven regional distribution in rat brain and are expressed predominantly by astrocytes as demonstrated by their colocalization with the astrocyte-specific marker glial fibrillary acidic protein.     

Cloning of zebrafish neurofilament cDNAs for plasticin and gefiltin: increased mRNA expression in ganglion cells after optic nerve injury

During retinal growth and optic axon regeneration, the differential expression of the neuronal intermediate filament proteins, plasticin and gefiltin, in the goldfish visual pathway suggests that these proteins support programmed axonal growth. To investigate plasticin and gefiltin during axonogenesis, we turned to the zebrafish, a system that is more amenable to mutational analysis. As a first step, we demonstrated that the intermediate filament compositions of goldfish and zebrafish are similar. In addition, the cDNAs for zebrafish plasticin and gefiltin were cloned and characterized. Using in situ hybridization in retina, we show increased mRNA levels for these proteins following optic nerve crush. Zebrafish plasticin and gefiltin peak and return to baseline levels of expression more rapidly than in goldfish. Furthermore, in the unoperated eye of experimental fish, there was a moderate increase in the levels of plasticin and gefiltin mRNA, suggesting that soluble factors influence the expression of these proteins. The successive expression of plasticin and gefiltin suggests that these neuronal intermediate filament proteins are integral components of axonogenesis. The cloning and characterization of cDNAs for plasticin and gefiltin permit mutational analyses of these proteins during zebrafish axonogenesis.     

Preparation of hydrogel microspheres by coacervation of aqueous polyphosphazene solutions

A new method of preparing ionically cross-linked polyphosphazene hydrogel microspheres which enables effective control over microsphere size distribution has been developed. The synthesized microspheres can be used in protein delivery and, especially, as vaccine delivery vehicles. A new technique utilizes the ability of aqueous solutions of poly[di(carboxylatophenoxy)phosphazene] (PCPP) to form coacervate microdroplets upon addition of sodium chloride. These microdroplets are then stabilized via polymer cross-linking with calcium ions. It was demonstrated that the method enables efficient microencapsulation of proteins. It was also shown that microsphere size can be controlled through the manipulation of microencapsulation conditions. The process is simple, highly reproducible and generates microspheres with a narrower microsphere size distribution, compared to the previous technologies.     

Endothelium-dependent vasodilation of the skin microcirculation in heart transplant recipients

The aim of this study was to compare the exercise intensity and rating of perceived exertion (RPE) of a high-impact (HIP) and a low-impact (LIP) university aerobic dance session. Ten women [mean (SD) age 22.9 (2.6) years] took part in the study. An incremental treadmill test was performed by each subject to determine maximum oxygen consumption (VO2max) and maximum heart rate (HRmax). The measured VO2max [mean (SD)] was 49.0 (7.5) ml x kg(-1) x min(-1). The subjects were randomly assigned to LIP and HIP sessions (i.e. five of the subjects participated in the HIP session first, and the other five participated in the LIP session first). In a laboratory, heart rate, oxygen uptake and RPE were measured throughout each session for each subject. Expired air was collected continuously throughout the sessions using Douglas bags (ten bags over a 30-min period). The sessions consisted of 20 min of aerobic exercise (bags 1-7) followed by 5 min of local muscular endurance exercise (bags 8 and 9) and 5 min of flexibility exercises (bag 10). The mean intensity of the aerobic section of the LIP and HIP sessions was 51.6% and 64.7% VO2max, respectively. Ninety-five percent confidence intervals for the average difference between the HIP and LIP sessions demonstrate that the %VO2max was between 12% and 14% higher for the HIP session. The mean %HRmax for the LIP and HIP sessions was 71.4% and 76.7%, respectively, with the %HRmax in the HIP session being between 5.4% and 7.2% higher on average than that of the LIP session. On average, the RPE for the aerobic section of the HIP session (12.1) was consistently higher than that of the LIP session (11.1). HIP activity has the potential to maintain/improve the aerobic fitness of its participants. According to the literature, the exercise intensity elicited by LIP activity may have a limited training effect for the population utilised in this study, and for some individuals may result in detraining. Conversely, LIP activities may be an appropriate mode of exercise for overweight and unfit individuals.     

Selective lesions of the nucleus basalis magnocellularis impair cognitive flexibility.

The authors tested the hypothesis that the cholinergic nucleus basalis magnocellularis (NBM) is involved in solving problems requiring cognitive flexibility. Rats with 192 IgG-saporin lesions of the NBM were assessed for perseveration (i.e., cognitive inflexibility) in the serial reversal of an operant discrimination and during subsequent extinction testing. It was hypothesized that the NBM lesion and control groups would not differ in the acquisition of the initial, simple discrimination, because this task does not demand cognitive flexibility. In contrast, it was hypothesized that the NBM lesion group would show perseveration during serial reversal and extinction testing. Results generally supported these hypotheses, suggesting that the NBM plays an important role in mediating cognitive flexibility. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The fibrinolytic effects of intermittent pneumatic compression: mechanism of enhanced fibrinolysis

BACKGROUND AND OBJECTIVES: Intermittent pneumatic compression (IPC) is an effective form of deep vein thrombosis prophylaxis for general surgery patients. The antithrombotic effect of IPC is thought to be the result of increased venous velocity and stimulation of endogenous fibrinolysis. However, the mechanism of enhanced fibrinolytic activity and the relative effects on normal and postthrombotic veins have not been defined. The purposes of this study are 1) to quantify changes in fibrinolytic activity with IPC; 2) to study the mechanism of fibrinolytic enhancement with IPC; and 3) to evaluate whether postthrombotic patients have the same capacity for fibrinolytic enhancement with IPC as do normal subjects. METHODS: Twelve volunteers (6 normal and 6 postthrombotic) had 5 IPC devices applied for 120 minutes in random fashion, 1 per week x 5 weeks. The devices included single-chamber, sequential, foot, calf, and long-leg compression. Subjects had an indwelling antecubital venous cannula placed for blood drawn at baseline, 60, 120, and 180 minutes after IPC devices were applied. Global fibrinolytic activity (euglobulin fraction, fibrin plate assay), tissue plasminogen activator (tPA) antigen (Ag) and activity (Act), plasminogen activator inhibitor-1 (PAI-1) Ag and Act, alpha-2-antiplasmin-plasmin complexes, and von Willebrand factor (vWF) antigen were assayed. RESULTS: A striking elevation in fibrinolytic activity was noted at 180 minutes with all devices in normal subjects and postthrombotic patients (p = 0.01-0.0001); however, baseline and stimulated fibrinolytic activity was attenuated in postthrombotic patients (<0.03). The tPA-Act increased only in normal subjects (3.8 +/- 1.9%) (p = 0.057), despite a decrease in plasma tPA-Ag, which was observed in both normal subjects (-12.4 +/- 3.8%) (p = 0.009) and patients (-17.2 +/- 3.1%) (p = 0.001). PAI-1-Ag decreased in both normal subjects (-13.4 +/- 3.8%) (p = 0.007) and patients (-12.0 +/- 3.1%) (p = 0.013) with a marked reduction in PAI-1-Act in both normal subjects (p = 0.003) and patients (p = 0.004). There were no changes in vWF, and alpha-2-antiplasmin-plasmin complexes increased only in postthrombotic patients (p = 0.021). CONCLUSIONS: Stimulation of endogenous fibrinolytic activity occurs after IPC, both in normal subjects and postthrombotic patients; however, baseline and overall fibrinolytic response in postthrombotic patients is reduced. The mechanism of increased fibrinolytic activity is likely because of a reduction in PAI-1, with a resulting increase of tPA activity.     

Primary structure of an invertebrate dihydrolipoamide dehydrogenase with phylogenetic relationship to vertebrate and bacterial disulfide oxidoreductases

Dihydrolipoamide dehydrogenase (E3) is a flavoprotein component of multi-enzyme complexes catalyzing oxidative decarboxylation of alpha-ketoacids in the Krebs' cycle. We have cloned a 2.4-kb E3 cDNA from an arthropod, Manduca sexta, that codes for 497 amino acids and translates to a 51-kDa protein in vitro. Sequences at and around the dinucleotide binding domains, disulfide active site and the C-terminal interface domain involved in substrate binding are highly conserved in Manduca E3. Phylogenetic analysis of protein sequences from the flavoprotein class of disulfide oxidoreductases family of enzymes suggests that in spite of the homologous nature of E3 and glutathione reductase (goR) in sequence and structure, E3 shares a common ancestor with mercuric reductase (merA), whereas goR is more related to trypanothione reductase (tryR) than to other members. All members, except goRs, seemed to be monophyletic. Plant goRs seemed to have arisen differently and are more closely related to tryRs than to bacterial and vertebrate goRs. Earlier speculation on the nature of origin of E3 in Pseudomonas is not supported by phylogenetic data. A possible structural relationship of Manduca E3 to other pyridine-binding proteins, such as the neurotransmitter transporters and channels, is proposed.