Plasmid encoded AcrAB-TolC tripartite multidrug-efflux system in Acidiphilium symbioticum H8.

Acidophilic bacterium, Acidiphilium symbioticum H8, is resistant to high levels of several heavy metals, hydrophobic agents, and organic solvents. The approximately 9.6 kb plasmid pASH8, was purified, digested with HindIII, and sub-cloned in pUC19 at the respective site. Three different fragment size clones were achieved. The clones were completely sequenced and analyzed. The first clone encodes for a single putative open reading frame (ORF), which showed significant homology to several rusticyaninA1 proteins. The second clone encodes for a 43-kDa protein, which has conserved domain homology with several outer envelop TolC proteins. The clone with pASH8 tolC gene can functionally complement an Escherichia coli tolC mutant strain, making it resistant to several toxic hydrophobic agents, earlier for which it was sensitive. The tolC gene was found to be essential for imparting resistance to the clone toward these toxic hydrophobic agents. The third clone encodes for a putative 318-aa AcrA (acriflavine resistance protein A) protein and the clone was resistance to plasmid curing dye acriflavine. The clone also has a truncated ORF, which showed significant homology to cation-efflux pump AcrB. This study is the first to report a multi-drug efflux system to be encoded on a plasmid of any Acidiphilium strain.

Potential therapeutic application of gold nanoparticles in B-chronic lymphocytic leukemia (BCLL): enhancing apoptosis.

B-Chronic Lymphocytic Leukemia (CLL) is an incurable disease predominantly characterized by apoptosis resistance. We have previously described a VEGF signaling pathway that generates apoptosis resistance in CLL B cells. We found induction of significantly more apoptosis in CLL B cells by co-culture with an anti-VEGF antibody. To increase the efficacy of these agents in CLL therapy we have focused on the use of gold nanoparticles (GNP). Gold nanoparticles were chosen based on their biocompatibility, very high surface area, ease of characterization and surface functionalization. We attached VEGF antibody (AbVF) to the gold nanoparticles and determined their ability to kill CLL B cells. Gold nanoparticles and their nanoconjugates were characterized using UV-Visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). All the patient samples studied (N = 7) responded to the gold-AbVF treatment with a dose dependent apoptosis of CLL B cells. The induction of apoptosis with gold-AbVF was significantly higher than the CLL cells exposed to only AbVF or GNP. The gold-AbVF treated cells showed significant down regulation of anti-apoptotic proteins and exhibited PARP cleavage. Gold-AbVF treated and GNP treated cells showed internalization of the nanoparticles in early and late endosomes and in multivesicular bodies. Non-coated gold nanoparticles alone were able to induce some levels of apoptosis in CLL B cells. This paper opens up new opportunities in the treatment of CLL-B using gold nanoparticles and integrates nanoscience with therapy in CLL. In future, potential opportunities exist to harness the optoelectronic properties of gold nanoparticles in the treatment of CLL.

The pharmacotherapy of depressive illness in adolescents: an open-label comparison of fluoxetine with imipramine-treated historical controls.

BACKGROUND: This open-label, 6-week clinical trial investigated the response to fluoxetine in medication-naive adolescents hospitalized for treatment of major depression. METHOD: A total of 52 consecutively admitted patients (mean age = 15.7 years) fulfilling Research Diagnostic Criteria for unipolar, nonpsychotic major depression received fluoxetine monotherapy (mean dose = 33.2 mg/day) in conjunction with psychosocial therapies. Outcome was assessed weekly using the Hamilton Rating Scale for Depression (HAM-D) and the Clinical Global Impressions Scale (CGI). Response in this cohort was compared with that observed in 28 historical controls treated with imipramine (mean dose = 217 mg/day) who were consecutively admitted patients to this same facility and assessed in an identical, standardized, open-label protocol. RESULTS: HAM-D scores decreased by a mean of 13.2 in the fluoxetine group compared with 10.2 in the group receiving imipramine (p<.002). The mean percentage decreases in HAM-D scores in the 2 groups were 54.3% and 41.4%, respectively (p<.003). The percentages of patients classified as responders based on a final CGI score of 2 or less were 48.1% and 17.9%, respectively (p = .009). Medications were generally well tolerated with only 5 patients failing to complete the full 6 weeks of their original treatment. CONCLUSION: In spite of the uncontrolled nature of these data, the findings add to recent evidence suggesting more favorable response to selective serotonin reuptake inhibitors than tricyclics in adolescents with depressive illness.

A family with Alport syndrome and psychosis.

A family is described with a history of both hereditary nephritis (Alport syndrome) and chronic psychosis in multiple family members. Although the disorders do not segregate together in all cases, the finding of this family may provide a clue for the location of a psychosis gene. Alport syndrome has been mapped to the long arm of the X chromosome. Some studies also support a genomic locus on the X chromosome in at least some cases of manic-depressive disorder and schizophrenia.

Methylphenidate and desipramine in hospitalized children: I. Separate and combined effects on cognitive function.

OBJECTIVE: The separate and combined effects of methylphenidate and desipramine on cognitive function were investigated in 16 psychiatrically hospitalized children with primary, secondary, and mixed features of attention-deficit hyperactivity disorder and mood disorder. METHOD: A double-blind, placebo controlled, crossover design was used to investigate drug effects on vigilance, short-term memory, visual problem solving, and higher-order learning. RESULTS: Methylphenidate alone improved vigilance, both drugs positively affected short-term memory and visual problem solving, and combined drugs affected learning of higher-order relationships. CONCLUSIONS: Separate and combined drug effects are related to the specific cognitive domain assessed and have implications for neurotransmitter models of action.

The effects of methylphenidate and lithium on attention and activity level.

Seven psychiatrically hospitalized prepubertal children were treated with methylphenidate, placebo, lithium carbonate alone and in combination with methylphenidate. Children met DSM-III-R criteria for both disruptive behavior disorder and bipolar or major depressive disorder by structured interview and consensus diagnosis. Parents, children, teachers, staff, and raters were blind to medication condition. Children were rated weekly on measures of attention and activity to determine whether medications alone or in combination produced a differential effect on these variables. The nosological and practical implications of the results are discussed.

Side effects of methylphenidate and desipramine alone and in combination in children.

OBJECTIVE: To investigate side effects of methylphenidate and desipramine alone and in combination in hospitalized children with symptoms of attention-deficit hyperactivity disorder and depression. METHOD: A double-blind placebo controlled crossover design was used to investigate each medication alone and in combination. Side effect ratings and EKGs were done weekly. Pulse and blood pressure were monitored daily. RESULTS: Nausea, dry mouth, and tremor were present in at least twice as many children on combined methylphenidate and desipramine compared with any other condition. Nausea/vomiting, headaches, other aches, refusal of food, and feeling "tired" were significantly more frequent during the combined methylphenidate plus desipramine condition when compared with either methylphenidate alone or with baseline. Significantly higher ventricular heart rate was found on combined methylphenidate plus desipramine compared with desipramine alone, methylphenidate alone, and baseline. Prolonged PR interval and significantly higher heart rate occurred during desipramine alone compared with baseline. CONCLUSIONS: During the several-month duration of the study, there were more frequent side effects during combined methylphenidate plus desipramine treatment than with either medication alone. Clinically, side effects present during combined medication appeared to be similar to and no more serious than those associated with desipramine alone.

Complete remission of 'treatment resistant' severe melancholia in adolescents with phenelzine: two case reports.

We describe two cases of adolescents hospitalized with protracted courses of delusional melancholia who responded dramatically to treatment with phenelzine following multiple failed clinical trials with other agents and, in one case, ECT. The two cases are of interest in light of evidence linking delusional subtype of major depression in adolescents to bipolar disease, and the role of monoamine oxidase inhibitors in the management of adults with bipolar depression, as well as unipolar depressions unresponsive to other pharmacological treatments.

No effect of adjunctive fluoxetine on eating behavior or weight phobia during the inpatient treatment of anorexia nervosa: an historical case-control study.

A six-week open label clinical trial investigated the response to fluoxetine in adolescents hospitalized for treatment of anorexia nervosa. Patients were drawn from consecutive admissions to a specialty treatment service and received fluxoetine as an add-on to their multidisciplinary treatment regimen beginning three weeks to one month postintake. Global clinical severity ratings of eating behavior and weight phobia were obtained at baseline and then at biweekly intervals. These ratings were compared to those obtained from matched historical case-controls who received an identical course of inpatient therapy but without adjunctive pharmacotherapy. Analyses failed to show any beneficial or detrimental effect of fluoxetine on these clinical measures. We conclude that fluoxetine has no robust additive or synergistic therapeutic benefit when measured against the effects of intensive, multidisciplinary inpatient therapy.

Risperidone in children and adolescents with pervasive developmental disorder: pilot trial and follow-up.

Dopamine receptor antagonists, particularly haloperidol, have been the most effective medications in currently available double-blind placebo-controlled studies for treating the disruptive behaviors often associated with pervasive developmental disorder (PDD). The rationale for trying risperidone in this population includes its dopamine-blocking activity; its seemingly lower incidence of tardive dyskinesia when compared to standard neuroleptics; the possibility that risperidone may ameliorate the social withdrawal of PDD, as it does the negative symptoms in schizophrenia; and substantial effects on serotonergic neurotransmission, which has been shown to be dysregulated in some patients with PDD. This study was an open-label pilot trial of risperidone in 6 subjects (aged 7-14 years, mean = 10.7) who met DSM-III-R criteria for a PDD diagnosis. The mean optimal dose was 2.7 mg daily (range 1-6). Mean duration of risperidone administration was 5.2 months (range 1-8). Despite the small sample size, risperidone treatment appeared to be associated with significant improvements in ratings of angry affect (p = 0.04) and lability of affect (p = 0.03) and with a trend (p = 0.10) toward a reduction of mean hyperactivity scores. Clinical Global Improvement scale ratings were statistically significant (p < 0.001). Increased sociability was reported in 3 subjects by their parents and family following the study. Three patients continued on risperidone for over 2 years, and none showed any loss of its apparent therapeutic effects. Weight gain was observed in 5 of 6 patients, with a median increase of 5.4 kg (12 lbs) in 7 weeks. Other side effects included transient sedation, increased salivation, and stereotypies. One child showed a worsening of pre-existing tic and phobic symptoms after 5 months of successful monotherapy. No loss of therapeutic effect was noted in the 3 subjects who remained on risperidone for over 2 years, but 1 patient developed hepatotoxicity and another developed withdrawal dyskinesia, similar to her prior experience with haloperidol. Overall, 5 of the 6 patients derived significant clinical benefits from risperidone. Pharmacologic alternatives for treating behavioral symptoms in PDD are need, and risperidone may be a promising possibility.

Childhood and adolescent depression: a review.

This paper reviews the recent literature on various aspects of depressive disorders in children and adolescents, including phenomenology, comorbidity, epidemiology, etiology, natural history, and treatment. Depression is an important psychiatric disorder in children and adolescents that increases in frequency with age, often coexists with anxiety disorders and behavior disorders, and is associated with long-term morbidity and risk of suicide. Both pharmacological and psychosocial interventions are currently under investigation to determine their efficacy in the treatment of depression in youth. Given the significant duration, relapse rate, morbidity, and impaired psychosocial functioning associated with depressive disorders in childhood, further exploration of the natural history and treatment of this condition is warranted.

Bioleaching of nickel and cobalt from lateritic chromite overburden using the culture filtrate of Aspergillus niger.

Extraction of metals (Ni, Co) from chromite overburden of Sukinda mines of Orissa, India, with the culture filtrate of Aspergillus niger was studied. Results showed that the amounts of metals leached varied directly with reaction temperature and period of fermentation. The culture filtrate was analyzed for citric and oxalic acids, and contained only oxalic acid-the concentration of which increased with time. Although this acid played the major role in leaching of metals, other unidentified metabolites present in the culture filtrate influenced the dissolution of the metals significantly. Maximum recovery of metals from raw and roasted ore samples was achieved at 80 °C with the 21-day culture filtrate containing the highest amount of oxalic acid. Under identical experimental conditions, much higher amounts of the metals were leached from roasted ore. Microstructures of the ore particles were studied by scanning electron microscopy and transmission electron microscopy; the bonding behaviors of metal compounds were identified by Fourier transform infrared spectroscopy which showed that the metals were leached after chelation with oxalic acid.

Mechanism of cadmium binding on the cell wall of an acidophilic bacterium.

Under certain conditions bacteria can act as a good biosorbent for different toxic heavy metals. However, no study on this aspect has been reported in case of acidophilic, heterotrophic, Gram-negative Acidiphilium strains, which are mostly resistant to several heavy metals. FTIR, SEM, TEM along with sorption experiments using bacterial cells of Acidiphilium symbioticum H8 were conducted to establish the mechanism of Cd(2+) ion sorption. The anionic functional groups present in the cell envelop were the components primarily responsible for the metal-binding capability of the bacterium. Sorption experiments further confirmed that 248.62 mg of cadmium was adsorbed per gram biomass at pH 6.0. The process can better be explained by Langmuir-Freundlich dual isotherm model. Blocking of the functional groups by chemical modification suggested that the binding of cadmium on the biomass occurs through electrostatic reaction and complex formation. Accumulation of cadmium on the cell envelop was supported by fine structure study.

Morphological changes in an acidophilic bacterium induced by heavy metals.

The Acidiphilium strains inhabit acidic mine regions where they are subjected to occasional environmental stresses such as high and low temperatures, exposure to various heavy metals, etc. Change in morphology is one of the strategies that bacteria adopt to cope with environmental stresses; however, no study on this aspect has been reported in the case of Acidiphilium sp. This work is an attempt using the acidophilic heterotrophic bacterium Acidiphilium symbioticum H8. It was observed that the maximum alterations in size occurred when the bacterium was exposed to sub-inhibitory concentrations of Cu and Cd. Loosely packed coccobacillus-type normal cells formed characteristic chains of coccoidal lenticular shape with constrictions at the junctions between them in the presence of Cd; Cu induced transformation of cells to become round shaped; Ni caused the cells to aggregate, but Zn showed no effect. Respective metal depositions on the cell surface were confirmed by scanning electron microscopy equipped with energy dispersive X-ray analysis. Cell bound Ca2+ ions were replaced by these metal ions and measured by inductively coupled plasma mass spectrometry from the culture filtrate. Cell shape changed only after the addition of sub-inhibitory concentrations of the metals, but in growth inhibitory concentrations it was similar to the normal cells.

Nucleotide sequence analysis of cryptic plasmid pAM5 from Acidiphilium multivorum.

Plasmid pAM5 of Acidiphilium multivorum JCM-8867 has been completely sequenced by initial cloning of HindIII-PstI fragments followed by primer walking. It has a size of 5161bp and single site for several restriction enzymes as revealed by DNA sequencing. Sequence analysis predicts five putative open reading frames. ORF1 and ORF3 show significant identity with various plasmid encoded mobilization (Mob) and replication initiation (Rep) proteins, respectively. The putative Mob protein has several characteristics of the MOB(Q) family having the motifs with conserved amino acid residues. Upstream of the Mob ORF, there exists a 34bp oriT region having a nic consensus sequence. The constructed plasmid pSK1 bearing pAM5 mob region can be mobilized to Escherichia coli in presence of conjugative plasmid pRK2013. The replication module comprises of several DnaA like boxes, several perfect direct and inverted repeats, a potential prokaryotic promoter and putative rep gene. The rep module is very similar to several theta replicating iteron family plasmids, suggesting pAM5 replication to follow the same course. Any phenotypic character determinant (e.g., metal resistance, antibiotic resistance etc.) gene is absent in pAM5, suggesting this plasmid to be cryptic in nature. However, a pAM5 derivative plasmid named pSK2, containing the putative pAM5 rep region, can replicate and be stably maintained in Acidiphilium, Acidocella, and E. coli strains; it can also carry foreign DNA fragments. Thus, pSK2 could serve as a cloning shuttle vector between these bacteria. It was observed that pAM5 Rep is essential for pSK2 to replicate in acidophiles. In its natural host, A. multivorum JCM-8867, pAM5 maintains a copy number of 50-60, and its derivative pSK2 maintains a comparatively, higher copy number in E. coli than in acidophiles.

High-yielding plasmid extraction method from acidophilic heterotrophic bacteria of the genus Acidiphilium.

Plasmid yield from Acidiphilium strains always had been poor following various standard methods. We adopted some simple modifications in the alkaline lysis procedure to get a better yield of plasmid from these bacteria. An approximately 10- to 20-fold increase in the plasmid yield was achieved when harvested Acidiphilium cells were preincubated 16-20 h at pH 6 in nitrogen-free medium. Another independent approach showed that freezing (-18 to -20 degrees C) of the harvested cells initially and at two subsequent steps in the alkaline lysis procedure of plasmid DNA extraction improved the yield further by 1.5- to 3-fold. The combination of these changes yielded at least 15- to 30-fold more plasmid from various Acidiphilium strains as compared with standard methods.

Integration of metal-resistant determinants from the plasmid of an Acidocella strain into the chromosome of Escherichia coli DH5alpha.

Acidophilic bacteria of mine origin are ideal systems for studying microbial metal resistance because of their ability to grow in the presence of high concentrations of metal salts. We have previously shown that the metal-resistant transformants obtained after transformation of Escherichia coli DH5alpha with plasmid DNA preparation from Acidocella sp. strain GS19h did not contain any plasmid suggesting chromosomal integration of the plasmid(s) (Appl Environ Microbiol 1997; 63: 4523-4527). The present study provides evidence in support of this suggestion. The pulsed field gel electrophoresis (PFGE) pattern of genomic DNA of the plasmidless metal-resistant transformants differed markedly from that of the untransformed DH5alpha strain. Moreover, when the recombinant plasmids constructed by cloning plasmid DNA fragments of the Acidocella strain GS19h in the vector pBluescript II KS+ were used to transform E. coli DH5alpha strain, no plasmid DNA was detected in some of the zinc- and ampicillin-resistant (ZnrAmpr) clones. The PFGE pattern of genomic DNA of such a transformed clone also differed markedly from that of the parent strain, suggesting chromosomal integration of the recombinant plasmid(s) containing both ampicillin- and zinc-resistance determinants. This observation was further supported by hybridization of chromosomal DNA of the plasmidless ZnrAmpr E. coli DH5alpha clone with the probes made from the plasmid DNA of strain GS19h and the vector DNA. Thus, this study corroborates our previous finding and documents the phenomenon of integration of metal-resistant determinants from the Acidocella GS19h plasmid(s) into the chromosome of E. coli DH5alpha.