Carboxypeptidase II Research Articles

Autoradiographic characterization of [18 F]PSMA-1007 binding in rat brain.

Carboxypeptidase II (CBPII) in brain metabolizes the neuroactive substance N-acetyl-L-aspartyl-L-glutamate (NAGG) to yield the elements of glutamate and N-acetyl-aspartate (NAA). In peripheral organs, CBPII is known as prostrate specific membrane antigen (PSMA), which presents an important target for nuclear medicine imaging in prostate cancer. Available PSMA ligands for PET imaging do not cross the blood-brain barrier, and there is scant knowledge of the neurobiology of CBPII, despite its implication in the regulation of glutamatergic neurotransmission. In this study we used the clinical PET tracer [18 F]-PSMA-1007 ([18 F]PSMA) for an autoradiographic characterization of CGPII in rat brain. Ligand binding and displacement curves indicated a single site in brain, with KD of about 0.5nM, and Bmax ranging from 9nM in cortex to 19nM in white matter (corpus callosum and fimbria) and 24nM in hypothalamus. The binding properties of [18 F]PSMA in vitro should enable its use for autoradiographic investigations of CBPII expression in animal models of human neuropsychiatric conditions.

Effects of intrathecal 2-PMPA on chronic inflammatory pain in rats

Objective To evaluate the effects of intrathecal 2-PMPA on chronic inflammatory pain in rats . Methods Twenty four male Sprague-Dawley rats ,aged 4-6 months ,weighing 200-250 g ,were randomly divided into 3 groups （n=8 each） using a random number table ：normal saline （NS） group ,complete Freund′s adjuvant （CFA ） group and N-acetylaspartylglutamate peptidase inhibitor 2-PMPA group （group 2-PMPA ） . Chronic inflammatory pain was induced by injecting 100μl of CFA into the plantar surface of the left hindpaw .Immediately after injection of CFA ,2-PMPA 100 μg was injected intrathecally once a day for 3 consecutive days in group 2-PMPA ,while the equal volume of NS was given instead of 2-PMPA in NS and CFA groups .The paw withdrawal latency to thermal nociceptive stimulus （TWL ） and paw withdrawal threshold （PWT ） to von Frey filament stimulation were measured before injection of CFA （baseline ,T1 ） and after the last injection of CFA （T2 ） .Then the rats were sacrificed and the L4 ,5 segments of the spinal cord were removed for determination of NR2B expression by Western blot .Results Compared with group NS ,TWL and PWT were significantly decreased at T2 and the expression of NR2B was up-regulated in CFA and 2-PMPA groups （ P〈0.05 ） .Compared with group CFA ,TWL and PWT were significantly increased at T2 and the expression of NR2B was down-regulated in group 2-PMPA （ P〈0.05） .Conclusion Intrathecal 2-PMPA can alleviate CFA-induced chronic inflammatory pain in rats ,and inhibition of NR2B expression in the spinal cord is involved in the mechanism . Key words: Glutamate carboxypeptidase Ⅱ; Pain; Inflammation; Injections,spinal

Bacterial Genes in the Aphid Genome: Absence of Functional Gene Transfer from Buchnera to Its Host

Genome reduction is typical of obligate symbionts. In cellular organelles, this reduction partly reflects transfer of ancestral bacterial genes to the host genome, but little is known about gene transfer in other obligate symbioses. Aphids harbor anciently acquired obligate mutualists, Buchnera aphidicola (Gammaproteobacteria), which have highly reduced genomes (420–650 kb), raising the possibility of gene transfer from ancestral Buchnera to the aphid genome. In addition, aphids often harbor other bacteria that also are potential sources of transferred genes. Previous limited sampling of genes expressed in bacteriocytes, the specialized cells that harbor Buchnera, revealed that aphids acquired at least two genes from bacteria. The newly sequenced genome of the pea aphid, Acyrthosiphon pisum, presents the first opportunity for a complete inventory of genes transferred from bacteria to the host genome in the context of an ancient obligate symbiosis. Computational screening of the entire A. pisum genome, followed by phylogenetic and experimental analyses, provided strong support for the transfer of 12 genes or gene fragments from bacteria to the aphid genome: three LD–carboxypeptidases (LdcA1, LdcA2,ψLdcA), five rare lipoprotein As (RlpA1-5), N-acetylmuramoyl-L-alanine amidase (AmiD), 1,4-beta-N-acetylmuramidase (bLys), DNA polymerase III alpha chain (ψDnaE), and ATP synthase delta chain (ψAtpH). Buchnera was the apparent source of two highly truncated pseudogenes (ψDnaE and ψAtpH). Most other transferred genes were closely related to genes from relatives of Wolbachia (Alphaproteobacteria). At least eight of the transferred genes (LdcA1, AmiD, RlpA1-5, bLys) appear to be functional, and expression of seven (LdcA1, AmiD, RlpA1-5) are highly upregulated in bacteriocytes. The LdcAs and RlpAs appear to have been duplicated after transfer. Our results excluded the hypothesis that genome reduction in Buchnera has been accompanied by gene transfer to the host nuclear genome, but suggest that aphids utilize a set of duplicated genes acquired from other bacteria in the context of the Buchnera–aphid mutualism.

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

Monoclonal antibody (mAb) delivery by gene transfer in vivo may be an attractive alternative to current mAb therapies for applications that require long-term therapy. This article describes a transfer system that allows inducible high-level expression of unmodified mAbs in vivo. A recombinant adeno-associated viral (rAAV) vector is used that comprises an expression cassette consisting of a dimerizer-regulated promoter that drives expression of the antibody heavy and light chains linked by a 2A self-processing peptide and a furin cleavage site. Following intravenous injection of the rAAV vector, serum mAb levels >1 mg/ml were attained by administration of the inducer, rapamycin. Antibody expression could be rapidly shut off by discontinuing treatment with rapamycin. By optimizing the furin cleavage sequence, this system generated native antibody in vivo, decreasing the likelihood of a host immune response to foreign sequences. In summary, this optimized mAb expression system allows regulated high-level expression of native full-length mAbs in vivo and may offer a new opportunity for delivery of therapeutic mAbs in the clinic.

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

Development of oral vaccine carrying GCPII gene and its role in reducing the dosage of pentobarbital in rat: a primitive research

To develop an oral vaccine carrying glutamate carboxypeptidase II (GCP II) and to explore whether it can affect the dosage of pentobarbiturate. Polymerase chain reaction, digestion of endonuclease and ligation, blue-white selection were used to construct an expression vector pcDNA3.1-GCP II. HEK293 cells were cultured. The vector pcDNA3.1-GCP II was transfected into the HEK293 cells by Ca(3)(PO(4))(2) coprecipitation method. The transfected HEK293 cells were cultured in HEM liquid culture prepared with G418. Three weeks after, positive clones, HEK293-GCP II, were identified. Reverse-transcription PCR and immunofluorescence cell staining were used to testify positive cell line; Method of CaCl(2) was used to prepare oral vaccine of attenuated Salmonella typhimurium carrying GCP II (SL-GCP II). Expression of SL-GCP II in vitro was observed by adding SL-GCP II into the primarily cultured macrophage. Fifty male SD rats were randomly divided into 2 groups of 25 rats: group A, undergoing intragastrical infusion of SL-GCP II, 600 micro l/time, in total 4 times in 4 days; and group B, as control group, undergoing intragastrical infusion of SL3261. Fifteen days after, 5 g/L pentobarbital sodium was injected intraperitoneally with the first dosage of 1.0 ml and the response was observed in 10 minutes, then 0.1 ml was added every time. The specific dosage of pentobarbital sodium was recorded when anesthesia meeting the requirement of operation was reached. Phenobarbital sodium of this dosage was used to anesthetize the rats to observe the response of the rats. Immunofluorescence method was used to detect the titer of antibody in rat circulation with HEK293 GCP II cells as target cells. An expression vector containing GCP II, pCMV-GCP II, pCDNA3.1-GCP II was constructed. The cell line, HEK 293-GCP II was established. In vitro experiment proved that primarily cultured macrophage phagocytized SL-GCP II and effectively expressed GCP II gene. After infusion of the oral vaccine 22 of the 25 SD rats of the group A produce GCP II antibodies. The dosage of pentobarbiturate used in experimental group was 36.9 mg/kg +/- 1.6 mg/kg; significantly lower than that in the control group (40.8 mg/kg +/- 1.4 mg/kg, P = 0.00). An oral vaccine carrying GCP II gene has been developed that activates the immune response of rat to produce GCP II antibodies and lower the dosage of pentobarbiturate needed.