A single packet of transmitter does not saturate postsynaptic glutamate receptors

Neurotransmitter is stored in synaptic vesicles and released by exocytosis into the synaptic cleft. One of the fundamental questions in central synaptic transmission is whether a quantal packet of transmitter saturates postsynaptic receptors. To address this question, we loaded the excitatory transmitter L-glutamate via whole-cell recording pipettes into the giant nerve terminal, the calyx of Held, in rat brainstem slices. This caused marked potentiations of both quantal and action potential-evoked EPSCs mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. These results directly demonstrate that neither AMPA nor NMDA receptors are saturated by a single packet of transmitter, and indicate that vesicular transmitter content is an important determinant of synaptic efficacy.     

PCR detection of DNAs of animal origin in feed by primers based on sequences of short and long interspersed repetitive elements

PCR primers for the detection of materials derived from ruminants, pigs, and chickens were newly designed on the basis of sequences of the Art2 short interspersed repetitive element (SINE), PRE-1 SINE, and CR1 long interspersed repetitive element (LINE), respectively. These primers amplified the SINE or LINE from total DNA extracted from the target animals and from test feed containing commercial meat and bone meal (MBM). With the primers, detection of Art2, PRE-1, or CR1 in test feed at concentrations of 0.01% MBM or less was possible. This method was suitable for the detection of microcontamination of feed by animal materials.     

Cloning and sequencing of the genes encoding cyclic tetrasaccharide-synthesizing enzymes from Bacillus globisporus C11

The genes for isomaltosyltransferase (CtsY) and 6-glucosyltransferase (CtsZ), involved in synthesis of a cyclic tetrasaccharide from alpha-glucan, have been cloned from the genome of Bacillus globisporus C11. The amino-acid sequence deduced from the ctsY gene is composed of 1093 residues having a signal sequence of 29 residues in its N-terminus. The ctsZ gene encodes a protein consisting of 1284 residues with a signal sequence of 35 residues. Both of the gene products show similarities to alpha-glucosidases belonging to glycoside hydrolase family 31 and conserve two aspartic acids corresponding to the putative catalytic residues of these enzymes. The two genes are linked together, forming ctsYZ. The DNA sequence of 16,515 bp analyzed in this study contains four open reading frames (ORFs) upstream of ctsYZ and one ORF downstream. The first six ORFs, including ctsYZ, form a gene cluster, ctsUVWXYZ. The amino-acid sequences deduced from ctsUV are similar in to a sequence permease and a sugar-binding protein for the sugar transport system from Thermococcus sp. B1001. The third ctsW encodes a protein similar to CtsY, suggested to be another isomaltosyltransferase preferring panose to high-molecular-mass substrates.     

Feeding of Ginkgo biloba extract (GBE) enhances gene expression of hepatic cytochrome P-450 and attenuates the hypotensive effect of nicardipine in rats

Ginkgo biloba extract (GBE) has been used clinically for improving peripheral vascular diseases in France and Germany and is ingested widely as a herbal medicine in some countries. However, accurate information about its safety as an herbal medicine has not been sufficiently established. To address this issue, we examined the effect of GBE on hepatic drug metabolizing enzymes and their influence on hypotensive drug in rats. Male rats were fed either a control diet or diet containing GBE (0.5% w/w) for 4 weeks. The feeding of a GBE diet did not change the serum transaminase activity, but increased the liver weight and the phospholipid concentration in the liver. In addition, the GBE diet markedly increased the content of cytochrome P-450 (CYP), and the activity of glutathione S-transferase in the liver. Furthermore, the GBE diet markedly induced levels of CYP2B1/2, CYP3A1 and CYP3A2 mRNA in the liver. The levels of CYP1A1, CYP1A2, CYP2E1, CYP2C11 and CYP4A1 were unchanged. The feeding of GBE for 4 weeks significantly reduced the hypotensive effect of nicardipine that was reported to be metabolized by CYP3A2 in rats. These findings suggest that GBE reduces the therapeutic potency of the Ca2+ channel blocker, nicardipine, via enhancement of cytochrome P-450 expression.     

Effector mechanism and clinical response of BAK (BRM-activated killer) immuno-cell therapy for maintaining satisfactory QOL of advanced cancer patients utilizing CD56-positive NIE (neuro-immune-endocrine) cells

A new type of immuno-cell therapy called BRM-activated killer (BAK) therapy using non-MHC-restricted lymphocytes, CD56-positive cells, was devised. Peripheral blood lymphocytes were selected by immobilization with anti-CD3 monoclonal antibody and cultured for 2 weeks in the presence of IL-2. Thereafter, they were reactivated by 1,000 U/ml of IFN-alpha for 15 min. Twenty-six outpatients with cancer whose performance status were over 80% on Karnofsky scale were selected for this study. About 6 x 10(9) BAK cells were returned by intravenous drip infusion, at one month intervals at an outpatient clinic to each of 20 advanced cancer patients in whom many metastatic lesions were found postoperatively, and to 6 patients with no postoperatively detectable metastases. The proportion of CD56-positive cells increased from 20% to 50% with culture. CD56-positive cells have strong cytotoxic activity and produced 20 ng/10(9) cells of beta-endorphin, an intracerebral hormone. During the course of BAK therapy, we adopted the Face scale as a QOL indicator. The QOL of all patients remained satisfactory or improved. Beta-endorphin is thought to make patients feel well and maintains good QOL because of its potent analgesic, sedative activity. From that facts that CD56 is a neural cell adhesion molecule and a member of the Ig superfamily, and that the CD56-positive cell produces beta-endorphin, we concluded that the CD56-positive cell is a multifunctional, integrated NIE (neuro-immune-endocrine) cell. Administration of BAK cells allowed all 20 advanced cancer patients with metastases to survive for over one year. All 6 patients receiving the same therapy for prevention of postoperative metastasis have been recurrence-free for one to five years.     

A broad replication origin of Drosophila melanogaster, oriD α, consists of AT-rich multiple discrete initiation sites

This work shows that the replication origin of Drosophila melanogaster, oriDalpha, consists of multiple discrete initiation sites. We attempted to map at high resolution the initiation sites in oriDalpha with a quantitative nascent DNA abundance assay using a competitive polymerase chain reaction (PCR) method. Nascent DNA was prepared from either cells blocked in very early S-phase and then labeled with 5-bromo-2'-deoxyuridine (BrdU), or asynchronously growing cells labeled briefly with BrdU. Denatured DNA was size-fractionated in alkaline sucrose gradients. BrdU-labeled nascent DNA was immuno-affinity purified using anti-BrdU antibodies. DNA was quantified with a competitive PCR method before and after immuno-purification. The results indicated that oriDalpha, whose size was presumed to be about 10 kb from two-dimensional gel electrophoretic analysis, contained four major initiation sites in its central 2.8 kb region, and six to approximately eight sites in 8.4 kb. All initiation sites corresponded with AT-rich sequences. Detailed analysis of one major initiation site indicated that its range was restricted to 700 bp.     

The critical balance between dopamine D2 receptor and RGS for the sensitive detection of a transient decay in dopamine signal

In behavioral learning, reward-related events are encoded into phasic dopamine (DA) signals in the brain. In particular, unexpected reward omission leads to a phasic decrease in DA (DA dip) in the striatum, which triggers long-term potentiation (LTP) in DA D2 receptor (D2R)-expressing spiny-projection neurons (D2 SPNs). While this LTP is required for reward discrimination, it is unclear how such a short DA-dip signal (0.5–2 s) is transferred through intracellular signaling to the coincidence detector, adenylate cyclase (AC). In the present study, we built a computational model of D2 signaling to determine conditions for the DA-dip detection. The DA dip can be detected only if the basal DA signal sufficiently inhibits AC, and the DA-dip signal sufficiently disinhibits AC. We found that those two requirements were simultaneously satisfied only if two key molecules, D2R and regulators of G protein signaling (RGS) were balanced within a certain range; this balance has indeed been observed in experimental studies. We also found that high level of RGS was required for the detection of a 0.5-s short DA dip, and the analytical solutions for these requirements confirmed their universality. The imbalance between D2R and RGS is associated with schizophrenia and DYT1 dystonia, both of which are accompanied by abnormal striatal LTP. Our simulations suggest that D2 SPNs in patients with schizophrenia and DYT1 dystonia cannot detect short DA dips. We finally discussed that such psychiatric and movement disorders can be understood in terms of the imbalance between D2R and RGS.     

Design, synthesis and peroxidase-like activity of 3α-helix proteins covalently bound to heme

As a model of artificial peroxidase, de novo designed three-α-helix proteins, 3α-H9 and 3α-H12, covalently bound to Fe(III)-mesoporphyrin IX were synthesized and examined for a peroxidase-like activity. The activity was regulated according to the positions of His residues in the proteins, and the His residues played a role in an acid–base catalytic function.