Age-related emotionality is associated with cortical delta-opioid receptor dysfunction-dependent astrogliosis.

Multiple changes occur in the aging brain, leading to age-related emotional disorders. A growing body of recent evidence suggests that the cortical delta-opioid receptor system plays a critical role in anxiety- and depressive-like behaviors in the rodent. In this study, we show that aging mice promoted anxiety-like behaviors as characterized by both the light-dark and elevated plus-maze tests, and they exhibit an increase in astrocytes in the cingulate cortex due to the dysfunction of cortical delta-opioid receptor systems. As well as aging mice, mice with a dysfunction of the delta-opioid receptor system induced by chronic treatment with the selective delta-opioid receptor antagonist naltrindole, revealed astrogliosis in the cingulate cortex, which was associated with anxiety. We also found that the microinjection of cultured astrocytes into the cingulate cortex of young mice enhanced the expression of anxiety-like behavior. Our results indicate that the aging process promotes astrogliosis in the cingulate cortex through the dysfunction of cortical delta-opioid receptors. This phenomenon may lead to emotional disorders including aggravated anxiety during normal aging.

Phased A-tracts bind to the alpha subunit of RNA polymerase with increased affinity at low temperature.

Previously we showed that the expression of a Clostridium perfringens phospholipase C gene (plc) is activated by promoter upstream phased A-tracts in a low temperature-dependent manner. In this paper we characterize the interaction between the alpha subunit of C. perfringens RNA polymerase and the phased A-tracts. Hydroxyl radical footprinting and fluorescence polarization assaying revealed that the alpha subunit binds to the minor grooves of the phased A-tracts through its C-terminal domain with increased affinity at low temperature. The result provides a molecular mechanism underlying the activation of the plc promoter by the phased A-tracts.

Mutations that increase the activity of the promoter of the Escherichia coli melibiose operon improve the binding of MelR, a transcription activator triggered by melibiose.

MelR is an Escherichia coli transcription factor that activates expression of the melAB operon in response to the presence of melibiose in the environment. MelR stimulates transcription initiation at the melAB promoter by binding to four sites centered at positions -120.5, -100.5, -62.5, and -42.5 upstream of the transcript start point. In a previous study, we described a spontaneous mutant that exhibited increased melAB expression. Sequence analysis showed that this mutant carries five consecutive base changes at positions -49, -50, -51, -52, and -53 upstream of the melAB transcript start. Here we show that these changes improve MelR binding to the target site centered at position -42.5 at the melAB promoter and that this improvement is responsible for increased promoter activity. Thus, the activity of the melAB promoter is fixed by the occupation by MelR of a DNA site that overlaps the -35 hexamer: MelR appears to be a typical class II-type transcription activator.

Melibiose carrier of Escherichia coli: use of cysteine mutagenesis to identify the amino acids on the hydrophilic face of transmembrane helix 2.

The melibiose carrier from Escherichia coli is a galactoside-cation symporter. Based on both experimental evidence and hydropathy analysis, 12 transmembrane helices have been assigned to this integral membrane protein. Transmembrane helix 2 contains several charged and polar amino acids that have been shown to be essential for the cation-coupled transport of melibiose. Starting with the cysteine-less melibiose carrier, we have individually substituted cysteine for amino acids 39-66, which includes the proposed transmembrane helix 2. In the resulting derivative carriers, we measured the transport of melibiose, determined the effect of the hydrophilic sulfhydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), on transport in intact cells and inside out vesicles, and examined the ability of melibiose to protect the carrier from inactivation by the sulfhydryl reagent. We found a set of seven positions in which the reaction with the sulfhydryl reagent caused partial or complete loss of carrier function measured in intact cells or inside-out vesicles. The presence of melibiose protected five of these positions from reaction with PCMBS. The reaction of two additional positions with PCMBS resulted in the partial loss of transport function only in inside-out vesicles. Melibiose protected these two positions from reaction with the reagent. Together, the PCMBS-sensitive sites and charged residues assigned to helix 2 form a cluster of amino acids that map in three rows with each row comprised of every fourth residue. This is the pattern expected of residues that are part of an alpha-helical structure and thus the rows are tilted at an angle of 25 degrees to the helical axis. We suggest that these residues line the path of melibiose and its associated cation through the carrier.

Conversion of temperature-sensitive to -resistant gene expression due to mutations in the promoter region of the melibiose operon in Escherichia coli.

The melibiose utilization system of Escherichia coli W3133, a derivative of K12, is nonfunctional between 37 and 42 degreesC. The reason for this temperature sensitivity was thought to be that the melibiose transporter (MelB) of W3133 cells was temperature-sensitive. A mutant W3133-2 has been isolated as a temperature-resistant strain that can utilize melibiose between 37 and 42 degreesC. However, we found that the melibiose transporter of the W3133-2 was still temperature-sensitive. Half-life activities of the melibiose transporter at 37 degreesC (or 40 degreesC) in both E. coli W3133 and W3133-2 were exactly the same. Furthermore, we found that the nucleotide sequence of coding region of the melB structural gene (the second gene of the melibiose operon) of W3133-2 was exactly the same as that of W3133. Activity of alpha-galactosidase (product of the first gene, melA, of the melibiose operon) of W3133 cells grown at 40 degreesC was very low, although that of W3133-2 cells grown at 40 degreesC was high. These observations suggested that expression of the melibiose operon in W3133 is also temperature-sensitive. In fact, we found that the expression in W3133 cells was temperature-sensitive, while that in W3133-2 cells was temperature-resistant, by analyzing mRNA levels using the Northern blot method. Furthermore, we identified mutations in the promoter region of the melibiose operon of W3133-2 that resulted in the elongation of an 18 nucleotide inverted repeat sequence to a 28-nucleotide repeat sequence present immediately upstream of the -35 region. This may stabilize a possible stem structure due to the inverted repeat at 37-42 degreesC.

Purification of UhpT, the sugar phosphate transporter of Escherichia coli.

To purify UhpT, the sugar phosphate carrier of Escherichia coli, we constructed a variant (HisUhpT) in which 10 tandem histidine residues were placed at the UhpT N terminus and then used Ni(2+)-agarose affinity chromatography of detergent-solubilized proteins. Membrane vesicles from a strain overexpressing His-UhpT were extracted at pH 7.4 with either 1.5% n-octyl-beta-D-glucopyranoside (octylglucoside) or 1.5% n-dodecyl-beta-D-maltoside (dodecylmaltoside) in 200 mM sodium chloride, 100 mM potassium phosphate, 50 mM glucose 6-phosphate, 10-20% glycerol, 0.2% E. coli phospholipid, and 5 mM beta-mercaptoethanol. After the detergent extract was applied to a Ni(2+)-agarose column, nonspecifically bound material was removed by washing at pH 7 with the same buffer also containing 50 mM imidazole. Purified HisUhpT was released subsequently, when sodium chloride was replaced with 300 mM imidazole or 100 mM EDTA, giving an overall yield of about 25 micrograms HisUhpT/mg vesicle protein. Whether eluted by imidazole or EDTA in either octylglucoside or dodecylmaltoside, purified HisUhpT showed a specific activity of 2.5-3 mumol/min per milligram of protein as monitored by [14C]glucose 6-phosphate transport by proteoliposomes loaded with 100 mM potassium phosphate. This corresponded to a calculated turnover number near 20 s-1 for the heterologous exchange of external sugar phosphate with internal phosphate. At low temperature (4 degrees C) HisUhpT retained full activity in either octylglucoside or dodecylmaltoside; however, at elevated temperature (> or = 23 degrees C), the protein displayed a marked lability in octylglucoside (t1/2 = 11 min), but not in dodecylmaltoside (t1/2 > or = 200-300 min).