Functional complementation of the yeast divalent cation transporter family SMF by NRAMP2, a member of the mammalian natural resistance-associated macrophage protein family.

The mammalian NRAMP gene family has two members, NRAMP1 and NRAMP2 that encode integral membrane proteins. Nramp1 is expressed exclusively in macrophages where it is found in the phagosomal membrane, and NRAMP1 mutations cause susceptibility to infection by abrogating the capacity of macrophages to control intracellular microbial replication. Nramp2 is highly similar to Nramp1, but is expressed in several tissues and cell types. The Nramp protein family is remarkably conserved throughout evolution, and recent data suggest that the mammalian Nramp2 and the yeast homologues Smf1 and Smf2 transport divalent cations. We tested whether structural similarity between the mammalian Nramp and the yeast Smf proteins results in functional complementation in yeast. Wild-type and mutant variants of the Nramp1 and Nramp2 proteins were expressed in a yeast mutant bearing null alleles at the SMF1 and SMF2 loci, and complementation of the phenotypes of this yeast mutant was investigated. Nramp2, but not Nramp1, was found to complement hypersensitivity to EGTA of the smf1/smf2 mutant under oxidative stress conditions (methyl viologen). We also observed that the smf1/smf2 double mutant is hypersensitive to growth at alkaline pH (pH 7.9) and that Nramp2 could complement this phenotype as well. Complementation by Nramp2 was specific and required a functional protein as independent mutations in residues highly conserved in all members of the Nramp family abrogated Nramp2 complementation. Since Mn2+ was the only divalent cation capable of completely suppressing both the EGTA and pH phenotypes, our results suggest that Nramp2 can transport Mn2+ in yeast.

Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome.

The Nramp1 (natural-resistance-associated macrophage protein 1) locus (Bcg, Ity, Lsh) controls the innate resistance or susceptibility of mice to infection with a group of unrelated intracellular parasites which includes Salmonella, Leishmania, and Mycobacterium. Nramp1 is expressed exclusively in professional phagocytes and encodes an integral membrane protein that shares structural characteristics with ion channels and transporters. Its function and mechanism of action remain unknown. The intracellular localization of the Nramp1 protein was analyzed in control 129/sv and mutant Nramp1-/- macrophages by immunofluorescence and confocal microscopy and by biochemical fractionation. In colocalization studies with a specific anti-Nramp1 antiserum and a panel of control antibodies directed against known cellular structures, Nramp1 was found not to be expressed at the plasma membrane but rather localized to the late endocytic compartments (late endosome/lysosome) of resting macrophages in a Lamp1 (lysosomal-associated membrane protein 1)-positive compartment. Double immunofluorescence studies and direct purification of latex bead-containing phagosomes demonstrated that upon phagocytosis, Nramp1 is recruited to the membrane of the phagosome and remains associated with this structure during its maturation to phagolysosome. After phagocytosis, Nramp1 is acquired by the phagosomal membrane with time kinetics similar to Lamp1, but clearly distinct from those of the early endosomal marker Rab5. The targeting of Nramp1 from endocytic vesicles to the phagosomal membrane supports the hypothesis that Nramp1 controls the replication of intracellular parasites by altering the intravacuolar environment of the microbe-containing phagosome.

Na+-induced transcription of nhaA, which encodes an Na+/H+ antiporter in Escherichia coli, is positively regulated by nhaR and affected by hns.

nhaA encodes an Na+/H+ antiporter in Escherichia coli which is essential for adaptation to high salinity and alkaline pH in the presence of Na+. We used Northern (RNA) analysis to measure directly the cellular levels of nhaA mRNA. NhaR belongs to the LysR family of regulatory proteins. Consistent with our previous data with an nhaA'-'lacZ fusion, NhaR was found to be a positive regulator and Na+ was found to be a specific inducer of nhaA transcription. In the nhaA'-'lacZ fusion, maximal induction was observed at alkaline pH. In contrast, in the nhaA+ strain both the level of nhaA expression and the induction ratio were lower at alkaline pH. This difference may be due to the activity of NhaA in the wild-type strain as NhaA efficiently excreted Na+ at alkaline pH and reduced the intracellular concentration of Na+, the signal for induction. We also showed that although the global regulator rpoS was not involved in nhaA regulation, the global regulator hns played a role. Thus, the expression of nhaA'-'lacZ was derepressed in strains bearing hns mutations and transformation with a low-copy-number plasmid carrying hns repressed expression and restored Na+ induction. The derepression in hns strains was nhaR independent. Most interestingly, multicopy nhaR, which in an hns+ background acted only as an Na+-dependent positive regulator, acted as a repressor in an hns strain in the absence of Na+ but was activated in the presence of the ion. Hence, an interplay between nhaR and hns in the regulation of nhaA was suggested.

Natural resistance to intracellular infections: Nramp1 encodes a membrane phosphoglycoprotein absent in macrophages from susceptible (Nramp1 D169) mouse strains.

The mouse Nramp1 gene (Bcg/Ity/Lsh) controls innate defense to infection with intracellular parasites such as Mycobacterium, Salmonella, and Leishmania. Sequence analysis of Nramp1 predicts a hydrophobic, membrane-associated protein expressed exclusively in monocyte/macrophage lineages. A single G169D substitution within the fourth predicted transmembrane domain of Nramp1 is associated with susceptibility to infection (Bcg) in inbred mouse strains. To initiate the biochemical characterization of the Nramp1 protein and to analyze the molecular basis of susceptibility associated with the Nramp1(D169) allele, oligopeptides derived from Nramp1 and two fusion proteins containing the first 54 and the last 35 residues of Nramp1 were used to raise specific anti-Nramp1 antisera. In addition, a c-Myc epitope (EQKLISEEDL) was introduced in-frame at the C terminus of Nramp1 to follow its expression in a yeast heterologous system. Western analysis of crude membrane fractions from yeast demonstrated that Nramp1 is indeed an integral membrane protein (resistant to urea extraction). In resident Bcg(r) (129sv, Nramp1(G169)) macrophages, one of the anti-Nramp1 antisera identified by immunoprecipitation a protein of 90,000 to 100,000 apparent m.w. that was absent in macrophages from knockout mice bearing a null mutation at Nramp1. The Nramp1 protein could be metabolically labeled with orthophosphate and was sensitive to glycosylase treatment, verifying that Nramp1 is an integral membrane phosphoglycoprotein. Parallel analysis of macrophage extracts from Bcg(s) mouse strains (C57BL6/J and BALB/c, Nramp1(D169)) failed to detect mature Nrampl protein in these cells, suggesting that the G169D mutation at Nramp1 prevents proper maturation or membrane integration of the protein, resulting in its rapid degradation.

Parenting predictors of early conduct problems in urban, high-risk boys.

OBJECTIVE: As part of a larger, prospective study, the authors examined concurrent and prospective relations among parenting and child antisocial behavior in inner-city boys at high risk for delinquent behavior. METHOD: One hundred twenty-six younger brothers (aged 6 to 10 years) of convicted delinquents in New York City and their parents were assessed; 15 months later 112 boys were reassessed. Demographics, parenting, and child diagnosis were examined as they relate to child externalizing behavior problems. Hierarchical multiple regression analyses predicted changes in Externalizing scores from year I parenting. RESULTS: At years I and II, 22% and 27% of boys, respectively, scored above the clinical cutoff for Externalizing. Controlling for earlier Externalizing, each of three domains of parenting still made significant independent contributions to later Externalizing scores, explaining 17% of the variance. Altogether this model explained 51% of the variance in year II Externalizing scores. CONCLUSIONS: Data support a cumulative risk model, whereby each of several adverse parenting factors further compounds the likelihood of child conduct problems.

Amiloride and harmaline are potent inhibitors of NhaB, a Na+/H+ antiporter from Escherichia coli.

The diuretic drug amiloride is a specific inhibitor of sodium transporting proteins in several cell types. Attempts to inhibit this activity in membrane vesicles derived from various bacteria, did not yield clear results. Therefore, we tested the effect of amiloride and its derivatives on the purified Na+/H+ antiporters of E. coli reconstituted in functional form in proteoliposomes. Whereas NhaA is not inhibited by amiloride, both amiloride and harmaline are potent inhibitors of NhaB with K0.5 of 6 and 15 microM, respectively. The pattern of inhibition by amiloride derivatives is different from that reported for mammalian antiporters but similar to that reported for the Na+/H+ antiporter of D. salina [Katz, A., Kleyman, T.R. and Pick, U. (1994) Biochemistry 33, 2389-2393]. Clonidine is a poor inhibitor (K0.5 = 200 microM) while cimetidine had no effect on the antiporter up to concentration of 1 mM. These new potent inhibitors provide us with important tools for the study of the mechanism of action of NhaB.

MH1, a second-site revertant of an Escherichia coli mutant lacking Na+/H+ antiporters (delta nhaA delta nhaB), regains Na+ resistance and a capacity to excrete Na+ in a delta microH(+)-independent fashion.

The Escherichia coli mutant delta nhaA delta nhaB (EP432), which lacks the two specific Na+/H+ antiporter genes, is incapable of efficiently excreting Na+. Accordingly at low K+ (6 mM) medium, its intracellular Na+ concentration is only slightly lower (1.5-2x) than the extracellular concentration (50 mM), explaining the high sensitivity to Na+ (> or = 30 mM) of the mutant. This Na+ sensitivity is shown to be a powerful selection for spontaneous second-site suppressor mutations that allow growth on high Na+ (< or = 0.6 M) with a rate similar to that of the wild type. One such mutation, MH1, maps at 25.7 min on the E. coli chromosome. It confers Na+ but not Li+ resistance upon delta nhaA delta nhaB cells and exposes a Na(+)-excreting capacity, maintaining a Na+ gradient of about 8-10 (at 50 mM extracellular Na+), which is similar to that of the wild type. Although lower, Na+ excretion capacity is also observed in the delta nhaA delta nhaB mutant when grown in medium containing higher K+ (70 mM). This capacity is accompanied with a shift in the sensitivity of the mutant to higher Na+ concentrations (> or = 300 mM). Whereas Na+ excretion by a wild type carrying delta unc is uncoupler sensitive, that of MH1 delta unc is dependent on respiration in an uncoupler-insensitive fashion. It is concluded that under some conditions (high K+ in the medium or in MH1-like mutants), a primary pump driven by respiration is responsible for Na+ extrusion when the Na+/H+ antiporters are not active.

Kinetic properties of NhaB, a Na+/H+ antiporter from Escherichia coli.

NhaB, a Na+/H+ antiporter from Escherichia coli, was overproduced, purified, and reconstituted in a functional state, demonstrating that a single polypeptide, the product of the nhaB gene, can catalyze full activity. NhaB is a minor protein that accounts for less than 0.1% of the total membrane protein. The use of proteoliposomes made possible the determination of important kinetic and pharmacological properties in the absence of passive and mediated leaks. The activity of NhaB was found to have some pH dependence; the apparent Km for Na+ changes by 10-fold from 1.55 mM at pH 8.5 to 16.66 mM at pH 7.2, while the Vmax remains constant. It was demonstrated that NhaB is electrogenic and translocates more H+ than Na+ per cycle; the rate of sodium efflux from proteoliposomes was accelerated by a membrane potential, negative inside, and NhaB activity generated a membrane potential as monitored by two techniques. The stoichiometry of NhaB was estimated by a thermodynamic method in which the magnitude of delta psi generated by NhaB was measured at various Na+ gradients. A kinetic method, in which the electrophoretic movement of 86Rb+ (in the presence of valinomycin) was monitored in parallel with measurements of NhaB-mediated 22Na+ uptake, allowed us to determine a stoichiometry of 3H+/2Na+. The significance of the existence of two antiporters with different stoichiometries, NhaA and NhaB, active in the same cell, is discussed.

Psychosocial rehabilitation of cranial trauma and stroke patients.

This paper presents the results of a longitudinal psychosocial study of 22 cranial trauma patients and 14 stroke patients from the time preceding injury (using retrospective data), through a 4-5 month intensive rehabilitation programme, to a follow-up 1 year after completion of the programme. Although the two groups of patients differed on several demographic and medical characteristics, essentially similar patterns for psychosocial decline following injury and improvement following rehabilitation could be observed. For both groups, the proportion in marital or cohabitational relationships returned to pre-injury levels, and for both groups the proportion requiring assistance in their living situation declined following rehabilitation, as did use of the health services. Virtually all patients in both groups had been in employment or undergoing education at the time of the injury, and although this percentage declined in practice to a small minority of both groups post-injury, there was a significant increase in the proportions working or in education following the rehabilitation programme. Similarly, the pattern of leisure-time activities in both groups declined post-injury and was restored following rehabilitation. Since both groups entered the programme at over 2.5 years post-injury, these generally encouraging results seem less likely to reflect spontaneous recovery than a beneficial effect of the programme itself.

Cloning and characterization of a putative Ca2+/H+ antiporter gene from Escherichia coli upon functional complementation of Na+/H+ antiporter-deficient strains by the overexpressed gene.

DNA libraries from alkaliphilic Bacillus firmus OF4 had been screened earlier (Ivey, D.M., Guffanti, A.A., Bossewitch, J. S., Padan, E., and Krulwich, T. A. (1991) J. Biol. Chem. 266, 23483-23489) for clones that would functionally complement a strain of Escherichia coli (NM81) with a deletion in one of its Na+/H+ antiporter genes. During those studies, an alkaliphile antiporter gene was hypothesized to have been incorporated into the chromosome of strain NM81, producing Na(+)-resistant NM8191. After introduction of a deletion in the second known E. coli Na+/H+ antiporter gene, libraries were prepared from NM8191 and screened for complementation of Na+/H+ antiporter-deficient mutants of E. coli. Instead of retrieving an alkaliphile gene, an unexpected E. coli gene was identified on the basis of its ability to restore Na+ resistance and membrane Na+/H+ antiporter activity to such mutant strains. The active open reading frame in the clone maps at 27 min on the E. coli chromosome and is identical in sequence to a wild type counterpart. It would be predicted to encode an extremely hydrophobic protein with multiple membrane-spanning regions and a molecular weight of 39,200. A region in one of the predicted hydrophilic loops in the gene product structure possesses striking sequence similarity to calsequestrin. The Ca2+/H+ antiporter activity of membranes from an E. coli transformant with a clone possessing only this open reading frame was indeed found to have enhanced pH-independent Ca2+/H+ antiporter activity. The Ca2+/H+ and Na+/H+ antiporter activities conferred by the clone were both inhibited by Mg2+. The gene was designated chaA and is proposed to be the structural gene for a Ca2+/H+ antiporter whose overexpression leads to resistance to growth inhibition by both calcium and sodium.

Physiological role of nhaB, a specific Na+/H+ antiporter in Escherichia coli.

The nhaB gene which codes for Na+/H+ antiporter activity in Escherichia coli was recently cloned (Pinner, E., Padan, E., and Schuldiner, S. (1992) J. Biol. Chem. 267, 11064-11068). In order to elucidate the role of nhaB in Na+ and H+ ions physiology and its interaction with nhaA, we generated mutants in which the chromosomal gene has been inactivated by insertion/deletion. A mutant devoid of both nhaA and nhaB is extremely sensitive to Na+ and Li+ at all pH values, and membranes prepared from this strain show no Na+/H+ antiporter activity. As opposed with the delta nhaA mutant which contains NhaB, the pH independent Na+/H+ antiporter (Padan, E., Maisler, N., Taglicht, D., Karpel, R., and Schuldiner, S. (1989) J. Biol. Chem. 264, 20297-20302), the delta nhaB mutant, containing NhaA, shows Na+/H+ antiporter activity highly dependent on pH. nhaB, in the absence of nhaA, confers a certain tolerance to Na+ which decreases with increasing pH. In the absence of NhaB, NhaA alone confers complete halotolerance under all conditions tested. However, when grown on agar in minimal medium on substrates which are symported with Na+ (proline, serine, and glutamate) at pH 6 and at low Na+ concentrations (< 10 mM), delta nhaB grows slower than the wild type and its Na+ dependent transport of glutamate and proline is markedly inhibited. Since both of these defects of the delta nhaB strain are alleviated upon transformation of the mutant with multicopy plasmid bearing nhaA, we conclude that nhaB is crucial when the level of NhaA activity is growth limiting, when nhaA is not sufficiently induced, and/or when NhaA is not activated.

Cloning, sequencing, and expression of the nhaB gene, encoding a Na+/H+ antiporter in Escherichia coli.

In Escherichia coli, expulsion of sodium ions is driven by proton flux via at least two distinct Na+/H+ antiporters, NhaA and NhaB. When the nhaA gene is deleted from the chromosome, the cell becomes sensitive to high salinity and alkaline pH (Padan, E., Maisler, N., Taglicht, D., Karpel, R., and Schuldiner, S. (1989) J. Biol. Chem. 264, 20297-20302). In the current work we cloned the nhaB gene by complementation of the delta nhaA strain. The gene codes for a membrane protein 504 amino acids long. Hydropathic analysis of the sequence indicates the presence of 12 putative transmembrane helices. NhaB has been specifically labeled with [35S]methionine; it is a membrane protein and displays an apparent M(r) of 47,000, slightly lower than that predicted from its amino acid sequence. Membranes from cells containing multiple dose of nhaB display enhanced Na+/H+ antiporter activity, as measured by the ability of Na+ to collapse a preformed pH gradient or by direct measurement of 22Na+ fluxes. In contrast to NhaA, whose activity increases with pH, NhaB is practically insensitive to pH. Limited homologies with Na+ transporters have been identified.

Cloning, sequencing and expression of the nhaA and nhaR genes from Salmonella enteritidis.

Na+/H+ antiporter activity is wide-spread and plays essential physiological roles. We found that several Enterobacteriaceae share conserved sequences with nhaA, the gene coding for an E. coli antiporter. A delta nhaA strain, which is sensitive to Na+ and Li+, was used to clone by complementation a DNA fragment from Salmonella enteritidis which confers resistance to the ions. The cloned fragment increased Na+/H+ antiport activity in membranes isolated from strains carrying the respective hybrid plasmid. DNA sequence analysis of the insert revealed two open reading frames. Both encode putative polypeptides which are closely homologous to the nhaA and nhaR gene products from Escherichia coli. The antiporter activity displays properties very similar to that of the E. coli NhaA, namely, it is activated by alkaline pH and recognizes Li+ with high affinity.

Psychosocial outcome following individualized neuropsychological rehabilitation of brain damage.

At the center for Rehabilitation of Brain Damage, University of Copenhagen, 46 consecutively admitted brain-damaged patients with varying pathologies and who were on average 2.9 years post-injury were treated in a daily four-month rehabilitation program in groups of about 10, followed by a six-month period of contact varying according to individual needs. An evaluation of psychosocial outcome is presented. The results, based on comparisons between pre-, post-treatment and follow-up questionnaire data, show continuing functional improvements in the areas of family life and living conditions. Dependence on health services declined. Over 70% of the patients returned to either work, further education or voluntary work activities. For the whole group, leisure activities returned to the pre-injury level. Follow-up at about two years revealed continuing improvements in all areas, suggesting social readaptation to a degree above expectations as judged from the existing literature.

HIV-relevant sexual behavior among a healthy inner-city heterosexual adolescent population in an endemic area of HIV.

The AIDS crisis has devastated segments of the population including the gay community and those who use intravenous drugs. HIV has spread to other groups including prostitutes and those with other sexually transmitted diseases. We have been studying adolescents in a major Northeast city where there is a major HIV/AIDS epidemic. Despite high levels of AIDS related knowledge, these adolescents reported high levels of sex risk behaviors. In addition, our data suggests that even moderate alcohol or marijuana use predicts high risk sexual behaviors. These data indicate the urgent need to develop prevention strategies for the spread of HIV among inner-city youth based upon relevant predictors of risk behaviors. The coupling of HIV in inner-city populations with a high frequency of risk behaviors in adolescents demands an immediate public health response.

Effects of trazodone on aggressive behavior in seven patients with organic mental disorders.

Seven patients with organic mental disorders and physically aggressive behavior were treated with trazodone. Three ceased demonstrating aggressive behavior within 4-6 weeks after starting trazodone treatment. Three had no discernible reduction in aggressiveness. One was unable to complete the trial.

Cognitive function during hypoglycaemia in type I diabetes mellitus.

Neuropsychological testing was carried out in 16 insulin dependent (type I) diabetic men during four periods when mean blood glucose concentrations were (A) 6.3 (SEM 0.13) mmol/l (113.5 (SEM 2.3) mg/100 ml), (B) 2.9 (0.05) mmol/l (52.3 (0.9) mg/100 ml), and (C) 1.8 (0.03) mmol/l (32.4 (0.05) mg/100 ml), all measured during intravenous insulin infusion, and (D) 6.1 (0.13) mmol/l (109.9 (2.3) mg/100 ml), measured after intravenous glucose. The total neuropsychological test score decreased between periods A and B, A and C, and B and C, whereas improvement occurred between periods C and D (all p less than 0.02). These results were not due to changes in individual subjects alone but were consistent for the whole group. During hypoglycaemia there were changes in the patients' estimates of elapsed time, which were underestimated at period C as compared with the estimates at periods A, B, and D (all p less than 0.05). None of the 16 patients noticed symptoms of hypoglycaemia at period A or B, 12 reported symptoms at C, and one at D. Patients with type I diabetes may show a deterioration in neuropsychological skills during periods of asymptomatic subnormal or hypoglycaemic blood glucose concentrations.