An amino-acid substitution in the influenza-B NB protein affects ion-channel gating.

The effects of site-directed mutations in NB, a protein encoded by the influenza B virus that has been shown to form cation-selective ion channels at pH 6.0, were studied on ion channel characteristics in artificial lipid bilayers. It was thought that the residues in the hydrophobic region of NB we selected for mutation might be involved in the transport of cations across the channel and that changes in these residues might affect channel properties such as gating and ion-selectivity. Serine residues at positions 20 and 28, threonine at position 24 and cysteine at position 26 were replaced by alanine. We found that the mutation S20A gave channels that did not gate and that remained open most of the time. Proton permeability of NB channels, as detected by fluorescence quenching, was also altered by the mutation S20A: channels were no longer proton-permeable. The other mutations, S28A, T24A and C26A, did not have any detectable effect on the activity or proton permeability of channels formed by NB. The results indicate that serine 20 may have an important role in normal function of NB channels.

Characterization of PitA and PitB from Escherichia coli.

Escherichia coli contains two major systems for transporting inorganic phosphate (P(i)). The low-affinity P(i) transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the high-affinity Pst system (pstSCAB) is induced at low external P(i) concentrations by the pho regulon and is an ABC transporter. We isolated a third putative P(i) transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional P(i) transporter that may be repressed at low P(i) levels by the pho regulon. While a pitB(+) cosmid clone allowed growth on medium containing 500 microM P(i), E. coli with wild-type genomic pitB (pitA Delta pstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB Delta pstC345 triple mutant. The mutation Delta pstC345 constitutively activates the pho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting the phoB-phoR operon allowed the pitB(+) pitA Delta pstC345 strain to utilize P(i), with P(i) uptake rates significantly higher than background levels. In addition, the apparent K(m) of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitA mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.

Attrition prevention with individuals awaiting publicly funded drug treatment.

AIMS: To evaluate the effectiveness of a motivational intervention to reduce attrition from a waiting list for substance abusers seeking publicly funded treatment. DESIGN: Randomized clinical trial comparing an "attrition prevention" condition to standard care while awaiting treatment admission. SETTING: A centralized substance abuse assessment and referral center in Seattle, Washington. PARTICIPANTS: Substance abusers (n = 654) eligible for publicly funded drug abuse treatment. MEASUREMENTS: Alcohol and drug use, substance-related negative consequences, areas in need of help, perceived need for help, emotional status, readiness to change, reasons for seeking and perceived barriers to entering treatment. FINDINGS: Overall, approximately 70% of clients entered treatment, and of these approximately 70% completed their assigned treatment. Those who entered treatment showed significant reductions in substance use and improved psychosocial function at a short-term 3-month follow-up. However, the attrition prevention intervention had no differential effect on treatment entry, completion or outcome compared to the standard waiting list. Further, there were no differences across therapists on these outcome measures. CONCLUSIONS: A motivational attrition prevention intervention did not enhance treatment entry, completion or outcome among treatment-seeking substance abusers. It is suggested that alternative strategies, such as contingency management and case management, may help facilitate treatment entry for individuals seeking publicly funded treatment.

GABA concentration sets the conductance of delayed GABAA channels in outside-out patches from rat hippocampal neurons.

GABAA channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches). The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased with GABA concentration from less than 10 pS (0.5 microm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC50 value of 33 microm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of 1 microm diazepam, the GABA EC50 decreased to 0.2 microm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 microm GABA the bicuculline IC50 was 19 microm. The effect of GABA on channel conductance shows that the role of the ligand in GABAA receptor channel function is more complex than previously thought.

Utilization of services by persons discharged from involuntary chemical dependency treatment.

This report compares services utilization pre-admission and post-discharge in 735 consecutive persons involuntarily committed to a chemical dependency treatment program in Washington State. Patients entering treatment were in their late 30s, had multiple health problems, previous arrests for misdemeanors or felonies, and minimal structured daily activities. Post discharge, there were decreases in the use of costly acute care services including detox, psychiatric hospitalization, and mental health crisis services. Patients who completed the program were less likely to use acute care services and were more likely to participate in outpatient treatment after discharge. The overall death rate of 29.4 per 1000 persons per year was 4 times greater than the age adjusted death rate for the US adult population. Further studies of other involuntary chemical dependency treatment programs are needed to evaluate the results of this report.

Mutating the highly conserved second membrane-spanning region 9' leucine residue in the alpha(1) or beta(1) subunit produces subunit-specific changes in the function of human alpha(1)beta(1) gamma-aminobutyric Acid(A) receptors.

The properties of the human alpha(1)beta(1) gamma-aminobutyric acid (GABA)(A) receptors were investigated after mutation of a highly conserved leucine residue at the 9' position in the second membrane-spanning region (TM2). The role of this residue in alpha(1) and beta(1) subunits was examined by mutating the 9' leucine to phenylalanine, tyrosine, or alanine. The mutations were in either the alpha(1) subunit (alpha*beta), the beta(1) subunit (alphabeta*), or in both subunits (alpha*beta*), and the receptors were expressed in Sf9 cells. Our results show that the rate of desensitization is increased as the size and hydrophobicity of the 9' residue in the alpha(1) subunit is increased: Y, F > L > A, T. Mutation of L9' in only the beta(1) subunit (alphabeta*) to either phenylalanine or tyrosine increased the EC(50) value for GABA at least 100 times, but the EC(50) was unchanged in alphabeta* alanine mutants. In the 9' alpha(1) mutants (alpha*beta, alpha*beta*) the GABA EC(50) was minimally affected. In alpha*beta and alpha*beta*, but not alphabeta*, the peak currents evoked by millimolar concentrations of GABA were greatly reduced. The reduction in currents could only be partially accounted for by decreased expression of the receptors These findings suggest different roles for the two types of subunits in GABA activation and later desensitization of alpha(1)beta(1) receptors. In addition, an increase in the resting membrane conductance was recorded in alanine but not in phenylalanine and tyrosine mutants, indicating that the side chain size at the 9' position is a major determinant of current flow in the closed conformation.

Sub-cellular localisation of the white/scarlet ABC transporter to pigment granule membranes within the compound eye of Drosophila melanogaster.

The white, scarlet, and brown genes of Drosophila melanogaster encode ABC transporters involved with the uptake and storage of metabolic precursors to the red and brown eye colour pigments. It has generally been assumed that these proteins are localised in the plasma membrane and transport precursor molecules from the heamolymph into the eye pigment cells. However, the immuno-electron microscopy experiments in this study reveal that the White and Scarlet proteins are located in the membranes of pigment granules within pigment cells and retinula cells of the compound eye. No evidence of their presence in the plasma membrane was observed. This result suggests that, rather than tranporting tryptophan into the cell across the plasma membrane, the White/Scarlet complex transports a metabolic intermediate (such as 3-hydroxy kynurenine) from the cytoplasm into the pigment granules. Other functional implications of this new finding are discussed.

Mutant human alpha(1)beta(1)(T262Q) GABA(A) receptors are directly activated but not modulated by pentobarbital.

Pentobarbital activates GABA(A) receptors and enhances GABA-activated currents. A threonine residue (262) in the second membrane spanning region at the 12' position in the beta(1) subunit, alpha(1)beta(1)(T12'Q), is necessary for the potentiating action of pentobarbital. We examined whether T12'Q-mutated receptors expressed in Spodoptera frugipedra (Sf 9) cells responded to direct activation by pentobarbital. In both mutant and wild type receptors, pentobarbital (100 microM to 1 mM) evoked a current response. The pentobarbital EC(50) values were similar; 119 and 158 microM for alpha(1)beta(1) and alpha(1)beta(1)(T12'Q) receptors, respectively. The results show it is possible to discriminate between agonistic and potentiating effects of pentobarbital, suggesting these actions involve separate mechanisms.

Mutations in the white gene of Drosophila melanogaster affecting ABC transporters that determine eye colouration.

The white, brown and scarlet genes of Drosophila melanogaster encode proteins which transport guanine or tryptophan (precursors of the red and brown eye colour pigments) and belong to the ABC transporter superfamily. Current models envisage that the white and brown gene products interact to form a guanine specific transporter, while white and scarlet gene products interact to form a tryptophan transporter. In this study, we report the nucleotide sequence of the coding regions of five white alleles isolated from flies with partially pigmented eyes. In all cases, single amino acid changes were identified, highlighting residues with roles in structure and/or function of the transporters. Mutations in w(cf) (G589E) and w(sat) (F590G) occur at the extracellular end of predicted transmembrane helix 5 and correlate with a major decrease in red pigments in the eyes, while brown pigments are near wild-type levels. Therefore, those residues have a more significant role in the guanine transporter than the tryptophan transporter. Mutations identified in w(crr) (H298N) and w(101) (G243S) affect amino acids which are highly conserved among the ABC transporter superfamily within the nucleotide binding domain. Both cause substantial and similar decreases of red and brown pigments indicating that both tryptophan and guanine transport are impaired. The mutation identified in w(Et87) alters an amino acid within an intracellular loop between transmembrane helices 2 and 3 of the predicted structure. Red and brown pigments are reduced to very low levels by this mutation indicating this loop region is important for the function of both guanine and tryptophan transporters.

Utilization of services for mentally ill chemically abusing patients discharged from residential treatment.

Little is known about outcomes of treatment for individuals with mental illness and chemical dependencies. This article compares services utilization preadmission and postdischarge in 534 patients discharged from a residential treatment program in Washington State. A number of services, including chemical dependency detoxification, mental health crisis, inpatient psychiatric, medical emergency, and general medical inpatient hospitalization, were used less frequently in the period after discharge. The total reimbursement for all Medicaid services decreased by 44% from $5 million in the year prior to discharge to $2.8 million in the year after discharge. Also, individuals (32%) who completed the program were less likely to use costly, acute care services. This study was limited by the absence of a control group and posttreatment alcohol and drug use data. In addition, other unmeasured factors could have explained the association between program completion and better outcomes.

A threonine residue in the M2 region of the beta1 subunit is needed for expression of functional alpha1beta1 GABA(A) receptors.

Although there is a high degree of homology in the M2 transmembrane segments of alpha1 and beta1 subunits, subunit-specific effects were observed in alpha1beta1 GABA(A) receptors expressed in Spodoptera frugipedra (Sf9) cells when the conserved 13' threonine residue in the M2 transmembrane region was mutated to alanine. When threonine 263 (13') was mutated to alanine in the beta1 subunit, high-affinity muscimol binding and the response to GABA were abolished. This did not occur when the threonine 263 (13') was mutated to alanine in the alpha1 subunit, but the rate of desensitisation increased and the effect of bicuculline, a competitive inhibitor, was reduced. The results show differential effects of subunits on receptor function and support a role for M2 in desensitisation.

The amino-terminal region of Vpr from human immunodeficiency virus type 1 forms ion channels and kills neurons.

We have previously reported that the accessory protein Vpr from human immunodeficiency virus type 1 forms cation-selective ion channels in planar lipid bilayers and is able to depolarize intact cultured neurons by causing an inward sodium current, resulting in cell death. In this study, we used site-directed mutagenesis and synthetic peptides to identify the structural regions responsible for the above functions. Mutations in the N-terminal region of Vpr were found to affect channel activity, whereas this activity was not affected by mutations in the hydrophobic region of Vpr (amino acids 53 to 71). Analysis of mutants containing changes in the basic C terminus confirmed previous results that this region, although not necessary for ion channel function, was responsible for the observed rectification of wild-type Vpr currents. A peptide comprising the first 40 N-terminal amino acids of Vpr (N40) was found to be sufficient to form ion channels similar to those caused by wild-type Vpr in planar lipid bilayers. Furthermore, N40 was able to cause depolarization of the plasmalemma and cell death in cultured hippocampal neurons with a time course similar to that seen with wild-type Vpr, supporting the idea that this region is responsible for Vpr ion channel function and cytotoxic effects. Since Vpr is found in the serum and cerebrospinal fluids of AIDS patients, these results may have significance for AIDS pathology.

Psychiatric hospitalization of persons with dual diagnoses: estimates from two national surveys.

Individuals with both mental illness and alcohol or drug use disorders present distinctive treatment and human service problems, including increased risk for psychiatric hospitalization in community hospitals. Using national hospital discharge abstract data for 1990 and 1994, this study compared differences in psychiatric hospitalization in community hospitals of patients with mental illness only and those with mental disorders and substance use disorders. Individuals with dual diagnoses were younger, and a greater proportion were men. Medicaid was the primary payer for a larger percentage of those with dual diagnoses. Nationally, the number of community hospitalizations for dually diagnosed patients increased 15 percent from 1990 to 1994, and total hospital charges increased from $1.9 to $2.2 billion.

Outcome of a controlled trial of the effectiveness of intensive case management for chronic public inebriates.

OBJECTIVE: The objective of this study was to test whether an intensive case management intervention would be effective with a group of homeless chronic public inebriate clients. The primary goals of the case management were to improve the financial and residential stability of the clients and to reduce their use of alcohol. METHOD: Subjects (N = 298, 81% male) were interviewed at baseline, randomly assigned to treatment and control conditions and given follow-up interviews at 6-month intervals for 2 years. Case management services were provided for the duration of the project. Follow-up rates for the first three interviews averaged 82%. RESULTS: Repeated measures MANCOVAs showed significant group differences favoring the case-managed group in all three areas targeted by the intervention: total income from public sources, nights spent in "own place" out of the previous 60 nights and days drinking out of the previous 30 days. The results held whether the three variables were analyzed jointly or separately and for alternative measures of drinking and homelessness. Although statistically significant, the group differences are generally not large. CONCLUSIONS: The results indicate that case management had a beneficial effect on the clients receiving it. This effect may have been the result of an increase in services received by the case-managed clients.

Two threonine residues in the M2 segment of the alpha 1 beta 1 GABAA receptor are critical for ion channel function.

The role of three threonine residues in the M2 hydrophobic region of the GABAA receptor has been investigated by replacing these polar residues with alanine at the 6', 10' and 13' positions of M2 in the GABAA alpha 1, and beta 1 subunits and co-expressing the mutated subunits in the baculovirus Sf9 insect cell system. GABA did not elicit a current in cells expressing either the 6' or 13' threonine to the alanine mutants. The mutant subunits formed intact heteromeric GABAA receptors as judged by the binding of [3H] muscimol or the relative level of alpha 1 protein present in the plasma membrane. In contrast, a chloride current was generated by GABA in cells expressing the 10' mutant receptor. However, the current decayed more rapidly to baseline in the continued presence of GABA in the 10' mutant receptor than in the wild type receptor. The results are discussed in terms of the possible roles of the threonine residues in the ion conduction pathway.

Glutamate residues at positions 219 and 252 in the a-subunit of the Escherichia coli ATP synthase are not functionally equivalent.

The role of glutamate-219 in the a-subunit of the Escherichia coli F0F1-ATPase was examined using site-directed mutagenesis. The replacement of Glu-219 by lysine, alanine or glycine resulted in a partially functional F0F1-ATPase. Combining any of these mutations with the substitution of glutamate for Gln-252 did not result in any increase in function. These findings rule out a proposal that glutamate at position 252 can functionally replace glutamate at position 219 [S.B. Vik, B.J. Antonio, J. Biol. Chem. 269 (1994) 30364-30369]. All the single and double mutants grew better at 25 degrees C than at 37 degrees C, suggesting a role for Glu-219 in maintaining the structure of the F0.

Effect of radial gradient of temperature on the performance of large-diameter high-performance liquid chromatography columns. I. Analytical conditions.

The performance of a large-diameter chromatographic column can be drastically reduced when the solvent enters the column at a temperature different from that of the wall of the column. A radial temperature gradient inside the column will affect the physical properties of the solvent and the chromatographic behaviour of the solute resulting in a deformation of the band profile. A mathematical model is proposed to take into account the effect of a radial gradient of temperature in a large-diameter column on the chromatographic peak shape under linear conditions. The model is then compared with experimental results in preparative columns of different sizes. A good agreement between experiment and theory has been found showing a serious effect of thermal conditions on separation quality, depending on the column size. It is also demonstrated that a small difference of temperature can be helpful to compensate practical effect of frits and distribution of the sample at the extremities of the packed bed. Finally, it demonstrates the necessity to perform efficiency measurement under different thermal conditions to have a good comparison between columns.

Crystal structure of the epsilon subunit of the proton-translocating ATP synthase from Escherichia coli.

BACKGROUND: Proton-translocating ATP synthases convert the energy generated from photosynthesis or respiration into ATP. These enzymes, termed F0F1-ATPases, are structurally highly conserved. In Escherichia coli, F0F1-ATPase consists of a membrane portion, F0, made up of three different polypeptides (a, b and c) and an F1 portion comprising five different polypeptides in the stoichiometry alpha 3 beta 3 gamma delta epsilon. The minor subunits gamma, delta and epsilon are required for the coupling of proton translocation with ATP synthesis; the epsilon subunit is in close contact with the alpha, beta, gamma and c subunits. The structure of the epsilon subunit provides clues to its essential role in this complex enzyme. RESULTS: The structure of the E. coli F0F1-ATPase epsilon subunit has been solved at 2.3 A resolution by multiple isomorphous replacement. The structure, comprising residues 2-136 of the polypeptide chain and 14 water molecules, refined to an R value of 0.214 (Rfree = 0.288). The molecule has a novel fold with two domains. The N-terminal domain is a beta sandwich with two five-stranded sheets. The C-terminal domain is formed from two alpha helices arranged in an antiparallel coiled-coil. A series of alanine residues from each helix form the central contacting residues in the helical domain and can be described as an 'alanine zipper'. There is an extensive hydrophobic contact region between the two domains providing a stable interface. The individual domains of the crystal structure closely resemble the structures determined in solution by NMR spectroscopy. CONCLUSIONS: Sequence alignments of a number of epsilon subunits from diverse sources suggest that the C-terminal domain, which is absent in some species, is not essential for function. In the crystal the N-terminal domains of two epsilon subunits make a close hydrophobic interaction across a crystallographic twofold axis. This region has previously been proposed as the contact surface between the epsilon and gamma subunits in the complete F1-ATPase complex. In the crystal structure we observe what is apparently a stable interface between the two domains of the epsilon subunit, consistent with the fact that the crystal and solution structures are quite similar despite close crystal packing. This suggests that a gross conformational change in the epsilon subunit, to transmit the effect of proton translocation to the catalytic domain, is unlikely, but cannot be ruled out.

Nature of the 5' residue in the M2 domain affects function of the human alpha 1 beta 1 GABAA receptor.

The effects on the functional properties of the alpha 1 beta 1 GABAA receptor when the 5' (alpha 1 Val260; beta 1 Ile255) hydrophobic amino acids in the second transmembrane (M2) region were changed to threonine were examined. In response to a saturating concentration of GABA, the current evoked in mutant receptors showed a decreased rate of desensitization and at equilibrium was a greater fraction of the peak current than in wild-type receptors. The half-saturation concentration of the peak current response to GABA in mutant receptors was comparable to that in wild-type receptors, but the Hill coefficient was reduced to less than one. It was concluded that the 5' amino acids in the M2 region have a role in the conformational changes that occur within the alpha 1 beta 1 GABAA receptor in response to GABA.