The devil's in the dosing: severe drug-induced liver injury in a hydroxychloroquine-naive patient with subacute cutaneous lupus erythematosus and porphyria cutanea tarda.

A 29-year-old woman with a 1.5 year history of photosensitive skin lesions on her hands presented with a malar rash, bullous lesions on her hands, and was diagnosed with subacute lupus erythematosus after serologies revealed a positive antinuclear antibody test (1:2560), and antibodies to Ro/SSA and dsDNA. Hydroxychloroquine (400 mg/day) was prescribed and the patient developed severe drug-induced liver injury. Biopsy of her bullous skin lesions was consistent with porphyria cutanea tarda, as were her serological and urinary exams. She was successfully treated with therapeutic phlebotomy. This case identifies porphyria cutanea tarda as an important differential diagnosis for the rheumatologist to consider when evaluating patients with bullous skin lesions. Hydroxychloroquine in lower doses is an effective treatment for porphyria cutanea tarda; at doses used to treat systemic lupus erythematosus and subacute cutaneous lupus, there is a potentially life-threatening complication of hepatotoxicity.

Molecular basis for resistance to silver cations in Salmonella.

Here we report the genetic and proposed molecular basis for silver resistance in pathogenic microorganisms. The silver resistance determinant from a hospital burn ward Salmonella plasmid contains nine open reading frames, arranged in three measured and divergently transcribed RNAs. The resistance determinant encodes a periplasmic silver-specific binding protein (SilE) plus apparently two parallel efflux pumps: one, a P-type ATPase (SilP); the other, a membrane potential-dependent three-polypeptide cation/proton antiporter (SilCBA). The sil determinant is governed by a two-component membrane sensor and transcriptional responder comprising silS and silR, which are co-transcribed. The availability of the sil silver-resistance determinant will be the basis for mechanistic molecular and biochemical studies as well as molecular epidemiology of silver resistance in clinical settings in which silver is used as a biocide.

Colicin-tolerant mutants of Escherichia coli: resistance of membranes to colicin E1.

Colicin E1 blocks proline accumulation by membrane vesicles prepared from wild-type sensitive Escherichia coli. Two classes of mutant cells are unaffected by colicin. Vesicles from colicin-resistant strains are sensitive to colicin E1, whereas vesicles from colicin-tolerant strains are unaffected by colicin El. These results suggest that the colicin E1 receptor is on the cell membrane and that colicin-tolerant strains have altered membranes while colicin-resistant strains have altered cell walls.

Bacterial resistance ATPases: primary pumps for exporting toxic cations and anions.

Bacterial plasmid resistance systems that maintain low intracellular levels of toxic heavy metals by pumping the substrates out as rapidly as they accumulate sometimes work at the biochemical level as efflux ATPases. The two systems responsible for arsenic and cadmium resistance have recently been sequenced. Comparison of the deduced amino acid sequences with those of better characterized ATPases has revealed certain structural and sequence similarities.

gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes.

True gamma or gamma 2 genes, unlike alpha, beta, and gamma 1 (beta gamma) genes of herpes simplex virus 1 (HSV-1), stringently require viral DNA synthesis for their expression. We report that gamma 2 genes resident in cells were induced in trans by infection with HSV-1 but that the induction did not require amplification of either the resident gene or the infecting viral genome. Specifically, to test the hypothesis that expression of these genes is amplification dependent, we constructed two sets of gamma 2-thymidine kinase (TK) chimeric genes. The first (pRB3038) consisted of the promoter-regulatory region and a portion of 5'-transcribed noncoding region of the domain of a gamma 2 gene identified by Hall et al. (J. Virol. 43:594-607) in the HSV-1(F) BamHI fragment D' to the 5'-transcribed noncoding and coding regions of the TK gene. The second (pRB3048) contained, in addition, an origin of HSV-1 DNA replication. Cells transfected with either the first or second construct and selected for the TK+ phenotype were then tested for TK induction after superinfection with HSV-1(F) delta 305, containing a deletion in the coding sequences of the TK gene, and viruses containing, in addition, a ts lesion in the alpha 4 regulatory protein (ts502 delta 305) or in the beta 8 major DNA-binding protein (tsHA1 delta 305). The results were as follows: induction by infection with TK- virus of chimeric TK genes with or without an origin of DNA replication was dependent on functional alpha 4 protein but not on viral DNA synthesis; the resident chimeric gene in cells selected for G418 (neomycin) resistance was regulated in the same fashion; the chimeric gene recombined into the viral DNA was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis; the gamma 2-chimeric genes resident in the host and in viral genomes were transcribed from the donor BamHI fragment D' containing the promoter-regulatory domain of the gamma 2 gene. The significance of the differential regulation of gamma 2 genes in the environments of host and viral genomes by viral trans-acting factors is discussed.

Molecular evolution of an arsenate detoxification pathway by DNA shuffling.

Functional evolution of an arsenic resistance operon has been accomplished by DNA shuffling, involving multiple rounds of in vitro recombination and mutation of a pool of related sequences, followed by selection for increased resistance in vivo. Homologous recombination is achieved by random fragmentation of the PCR templates and reassembly by primerless PCR. Plasmid-determined arsenate resistance from plasmid pl258 encoded by genes arsR, arsB, and arsC was evolved in Escherichia coli. Three rounds of shuffling and selection resulted in cells that grew in up to 0.5 M arsenate, a 40-fold increase in resistance. Whereas the native plasmid remained episomal, the evolved operon reproducibly integrated into the bacterial chromosome. In the absence of shuffling, no increase in resistance was observed after four selection cycles, and the control plasmid remained episomal. The integrated ars operon had 13 mutations. Ten mutations were located in arsB, encoding the arsenite membrane pump, resulting in a fourfold to sixfold increase in arsenite resistance. While arsC, the arsenate reductase gene, contained no mutations, its expression level was increased, and the rate of arsenate reduction was increased 12-fold. These results show that DNA shuffling can improve the function of pathways by complex and unexpected mutational mechanisms that may be activated by point mutation. These mechanisms may be difficult to explain and are likely to be overlooked by rational design.

Nephrotic syndrome associated with chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Chronic graft-versus-host disease (cGVHD) is the most common late complication of allogeneic hematopoietic cell transplantation (HCT) causing significant morbidity and mortality. The kidneys are not considered a target organ for cGVHD in humans, although animal models show renal damage. Renal involvement in patients with cGVHD, presenting as nephrotic syndrome (NS), has rarely been reported in patients who received allogeneic transplantation. Herein we describe, by far, the largest series of nine patients with NS associated with cGVHD, including two patients who received a reduced-intensity regimen. Pathological features of membranous nephropathy were the most common finding on renal biopsy. The clinical course of the NS was temporally associated with the classical features of cGVHD in all but one of the nine cases. The clinicopathologic features of NS in our series as well as reports in the literature demonstrate an immunopathologic process typical of antibody-mediated damage consistent with cGVHD. Treatment directed against antibody-mediated damage, such as anti-B-cell antibody may play an important role in ameliorating NS associated with cGVHD.

Resistance to arsenic compounds in microorganisms.

Arsenic ions, frequently present as environmental pollutants, are very toxic for most microorganisms. Some microbial strains possess genetic determinants that confer resistance. In bacteria, these determinants are often found on plasmids, which has facilitated their study at the molecular level. Bacterial plasmids conferring arsenic resistance encode specific efflux pumps able to extrude arsenic from the cell cytoplasm thus lowering the intracellular concentration of the toxic ions. In Gram-negative bacteria, the efflux pump consists of a two-component ATPase complex. ArsA is the ATPase subunit and is associated with an integral membrane subunit, ArsB. Arsenate is enzymatically reduced to arsenite (the substrate of ArsB and the activator of ArsA) by the small cytoplasmic ArsC polypeptide. In Gram-positive bacteria, comparable arsB and arsC genes (and proteins) are found, but arsA is missing. In addition to the wide spread plasmid arsenic resistance determinant, a few bacteria confer resistance to arsenite with a separate determinant for enzymatic oxidation of more-toxic arsenite to less-toxic arsenate. In contrast to the detailed information on the mechanisms of arsenic resistance in bacteria, little work has been reported on this subject in algae and fungi.

Addition of growth hormone secretion signal to basic fibroblast growth factor results in cell transformation and secretion of aberrant forms of the protein.

Basic fibroblast growth factor (bFGF) is a potent mitogen for a wide variety of cell types. Unlike most growth factors, the primary translation product for bFGF appears to lack a secretory signal peptide. To explore the normal mode of bFGF release, as well as to investigate the growth factor's oncogenic potential, expression vectors were created for a bFGF cDNA and for a chimeric molecule in which the bFGF coding sequence was linked to the human growth hormone signal peptide sequence. Transfection of NIH3T3 cells with the bFGF cDNA vectors caused the synthesis of high levels of biologically active, cell-associated bFGF, but no evidence of transformation was detected. In contrast, the chimeric bFGF-signal peptide expression vector induced foci of transformation at a very high frequency. The transformed cells grew in soft agar and were tumorigenic in nude mice. The majority of the immunoreactive bFGF species made by the transformed cells was found in the conditioned medium and appeared to be posttranslationally modified, indicating that the chimeric bFGF-signal peptide molecule was processed through the secretory pathway. The secreted bFGF exhibited little mitogenic activity, suggesting that interaction of bFGF with its receptor likely occurs while the fusion protein is being processed along the secretory pathway.

Clinical trials: are they a good buy?

PURPOSE: Concern that clinical trials may be too costly has been used to justify traditionally restrictive insurer policies regarding clinical trials. Additionally, fear of insurer reimbursement denial can be a significant barrier to clinical trial participation. In this study, we reviewed the empirical data on costs of clinical trials versus standard care and summarized the current status of policy initiatives related to clinical trial insurance reimbursement. METHODS: Electronic and print data sources were searched for studies on the costs of oncology clinical trials. Information on policy initiatives for clinical trial reimbursement was obtained from the American Society of Clinical Oncology, the American Society of Hematology, and the Coalition of National Cancer Cooperative Groups and from searches of World Wide Web sites. RESULTS: Five pilot studies provided information for 377 patients on phase II/III clinical trials matched with controls on standard care. Cost estimates ranged from 10% lower to 23% higher costs/charges for clinical trials in comparison to standard medical care. Medicare, 14 states, and several private insurers now cover the costs of patient care in "qualifying" clinical trials. CONCLUSION: Findings from small pilot studies suggest that phase II and III clinical trials result in at most modest increases in cost over standard treatment costs. Also, an increasing number of policy makers have decided to support clinical trial reimbursement initiatives. It is hoped that economic data from large observational studies will facilitate widespread and permanent decisions that support reimbursement for phase I, II, and III clinical trial participation.

Bone marrow transplants from HLA-identical siblings in advanced Hodgkin's disease.

PURPOSE: To determine the outcome of HLA-identical sibling bone marrow transplants in advanced Hodgkin's disease. PATIENTS AND METHODS: We reviewed the data on 100 consecutive patients with Hodgkin's disease who received HLA-identical sibling bone marrow transplants between April 1, 1982 and August 12, 1992, reported to the International Bone Marrow Transplant Registry (IBMTR). The median interval from diagnosis to transplant was 2.5 years (range, < 1 to 14). All had advanced disease. Eighty-nine of 100 patients were not in remission at the time of transplant. Fifty had pretransplant Karnofsky scores less than 90% and 27 had active infection in the week before transplant. Patients received a variety of conditioning regimens; 45 received total-body radiation. RESULTS: The 100-day probability of acute graft-versus-host disease (GVHD) was 35% (95% confidence interval [CI], 26% to 46%); the 3-year probability of chronic GVHD was 45% (95% CI, 31% to 59%). The 3-year probability of relapse was 65% (95% CI, 50% to 78%). The 3-year probability of survival was 21% (95% CI, 14% to 30%). The 3-year disease-free survival rate was 15% (95% CI, 9% to 24%). CONCLUSION: HLA-identical sibling bone marrow transplants have a limited role in advanced Hodgkin's disease.

Satellite cereal yellow dwarf virus-RPV (satRPV) RNA requires a douXble hammerhead for self-cleavage and an alternative structure for replication.

The 110 nt hammerhead ribozyme in the satellite RNA of cereal yellow dwarf virus-RPV (satRPV RNA) folds into an alternative conformation that inhibits self-cleavage. This alternative structure comprises a pseudoknot with base-pairing between loop (L1) and a single-stranded bulge (L2a), which are located in hammerhead stems I and II, respectively. Mutations that disrupt this base-pairing, or otherwise cause the ribozyme to more closely resemble a canonical hammerhead, greatly increase self-cleavage. In a more natural multimeric sequence context containing the full-length satRPV RNA and two copies of the hammerhead, wild-type RNA cleaves much more efficiently than in the 110 nt context. Mutations in the upstream hammerhead, including a knock-out in the catalytic core, affect cleavage at the downstream cleavage site, indicating that multimers of satRPV RNA cleave via a double hammerhead. The double hammerhead includes base-pairing between two copies of the L1 sequence which extends stem I. Disruption of L1-L1 base-pairing slows cleavage of the multimer. L1-L2a base-pairing is required for efficient replication of satRPV RNA in oat protoplasts. Mutations that affect self-cleavage of the multimer do not correlate with replication efficiency, indicating that the ability to self-cleave is not a primary determinant of replication. We present a replication model in which multimeric satRPV RNA folds into alternative conformations that cannot form in the monomer. One potential metastable intermediate conformation involves L1-L2a base-pairing that may facilitate formation of the double hammerhead. However, we conclude that L1-L2a also performs some other essential function in the satRPV RNA replication cycle, because the L1-L2a base-pairing is more important than efficient self-cleavage for replication.

Clinical protocol. Purging of autologous stem cell sources with bcl-x(s) adenovirus for women undergoing high-dose chemotherapy for stage IV breast carcinoma.

High-dose chemotherapy (HDCT) and autologous bone marrow transplantation (BMT) is frequently used to treat patients with metastatic cancer including breast cancer and neuroblastoma. However, the bone marrow of such patients is often contaminated with tumor cells. Recently, we have found that a recombinant adenovirus vector that contains a bcl-x, minigene (a dominant negative inhibitor of the bcl-2 family), called the bcl-x(s) adenovirus, is lethal to cancer cells derived from epithelial tissues, but not to normal human hematopoietic cells. To determine the mechanism, by which this virus spares normal hematopoietic cells, we isolated normal mouse hematopoietic stem cells and infected them with an adenovirus that contains a beta-galactosidase minigene. Such cells do not express beta-galactosidase, indicating that hematopoietic stem cells do not express transgene encoded by adenovirus vectors based upon the RSV-AD5 vector system. When breast cancer cells mixed with hematopoietic cells were infected with the bcl-x(s) adenovirus, cancer cells were selectively killed by the suicide adenoviruses. Hematopoietic cells exposed to the suicide vectors were able to reconstitute the bone marrow of mice exposed to lethal doses of y-irradiation. These studies suggest that adenovirus suicide vectors may provide a simple and effective method to selectively eliminate cancer cells derived from epithelial tissue that contaminate bone marrow to be used for autologous BMT. We therefore propose to initiate a phase I clinical trial to test the safety of this virus in women with breast cancer undergoing high does chemotherapy and autologous BMT.

Rapid eye movement sleep disturbance in posttraumatic stress disorder.

The subjective sleep disturbance in posttraumatic stress disorder (PTSD), including the repetitive, stereotypical anxiety dream, suggests dysfunctional rapid eye movement (REM) sleep mechanisms. The polysomnograms of a group of physically healthy combat veterans with current PTSD were compared with those of an age-appropriate normal control group. Tonic and phasic REM sleep measures in the PTSD subjects were elevated on the second night of recorded sleep. Increased phasic REM sleep activity persisted in the PTSD group on the subsequent night. During the study, an anxiety dream occurred in a PTSD subject in REM sleep. The results are consistent with the view that a dysregulation of the REM sleep control system, particularly phasic event generation, may be involved in the pathogenesis of PTSD. The finding of a specific disturbance of sleep unique to PTSD may have significant implications for the design of effective treatments for PTSD.

Rapid eye movement sleep changes during the adaptation night in combat veterans with posttraumatic stress disorder.

BACKGROUND: Hyperarousal in posttraumatic stress disorder (PTSD) is manifested during sleep as well as waking. Elevated rapid eye movement sleep (REMS) phasic activity, likely signifying central nervous system alerting, has been identified in PTSD. The authors reasoned that PTSD compared to control subjects would show particularly increased REMS phasic activity on the first night of polysomnography, with adaptation to a novel environment. METHODS: First-night polysomnograms of 17 veterans with PTSD were compared with those of 11 control subjects. Sleep was also studied in subsets of both groups over two nights. RESULTS: On the first night, the PTSD subjects had a higher density of rapid eye movements in the first REMS period. This measure was increased on the first compared to the second night, but there was no interaction effect between night and group. CONCLUSIONS: REMS changes are again demonstrated in veterans with PTSD. Introduction to a novel environment activated a REMS phasic process, but not differentially in PTSD compared to control subjects.

Bacterial resistances to toxic metal ions--a review.

Bacterial plasmids encode resistance systems for toxic metal ions, including Ag+, AsO2-, AsO4(3-), Cd2+, Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, TeO3(2-), Tl+ and Zn2+. The function of most resistance systems is based on the energy-dependent efflux of toxic ions. Some of the efflux systems are ATPases and others are chemiosmotic cation/proton antiporters. The Cd(2+)-resistance ATPase of Gram-positive bacteria (CadA) is membrane cation pump homologous with other bacterial, animal and plant P-type ATPases. CadA has been labeled with 32P from [alpha-32P] ATP and drives ATP-dependent Cd2+ (and Zn2+) uptake by inside-out membrane vesicles (equivalent to efflux from whole cells). Recently, isolated genes defective in the human hereditary diseases of copper metabolism, namely Menkes syndrome and Wilson's disease, encode P-type ATPases that are more similar to bacterial CadA than to other ATPases from eukaryotes. The arsenic resistance efflux system transports arsenite [As(III)], alternatively using either a double-polypeptide (ArsA and ArsB) ATPase or a single-polypeptide (ArsB) functioning as a chemiosmotic transporter. The third gene in the arsenic resistance system, arsC, encodes an enzyme that converts intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. The triple-polypeptide Czc (Cd2+, Zn2+ and Co2+) chemiosmotic efflux pump consists of inner membrane (CzcA), outer membrane (CzcC) and membrane-spanning (CzcB) proteins that together transport cations from the cytoplasm across the periplasmic space to the outside of the cell.

Promoters and transcription of the plasmid-mediated citrate-utilization system in Escherichia coli.

The nucleotide sequence of an 878-bp BamHI-BglII restriction endonuclease fragment from citrate utilization transposon Tn3411 was determined, and was compared with that from plasmid pMS185 [Sasatsu et al., J. Bacteriol. 164 (1985) 983-993]. A long open reading frame for a 379-amino acid (aa) polypeptide (citB) was found 5' to the citA gene (431-aa membrane protein) in Tn3411 as well as in pMS185. Promoter regions were identified by RNA polymerase filter-binding assays, S1 nuclease mapping and cit-lac fusion experiments. The results indicated that two genes (citA and citB) have separate promoters, and the location of the promoter for the citB gene in the Tn3411 nucleotide sequence was different from that in pMS185. The regulation of transcription of the two genes (citA and citB) was characterized by the use of cit-lacZ fusions. The level of the citB promoter activity was about five-fold higher than that of the citA gene promoter, and transcription from both was not induced by citrate. Synthesis of the mRNA for the citB gene (especially with the wild-type Cit+ determinant) was suppressed by citrate, accompanying growth suppression of Escherichia coli. The citB gene expressed in E. coli minicells produced a membrane-associated 37.5-kDa polypeptide.

Mercuric reductase structural genes from plasmid R100 and transposon Tn501: functional domains of the enzyme.

The nucleotide sequence for the 2240 bp of plasmid R100 following the merC gene of the mercuric resistance operon has been determined and compared with the homologous sequence of transposon Tn501. The sequences following merC and preceding the next structural gene merA are unrelated between R100 and Tn501 and differ in length, with 72 bp in Tn501 and 509 bp in R100. The R100 sequence has a potential open reading frame (ORF) for a 140 amino acid polypeptide with a reasonable translational start signal preceding it. The merA genes contain 1686 (Tn501) and 1695 (R100) bp respectively. When optimally aligned, the merA sequences differ in 18% of their positions. These differences were clustered in specific regions. In addition, there was one nucleotide triplet in the Tn501 sequence which has no counterpart in the R100 sequence and one dodecyl-nucleotide sequence in the R100 sequence without counterpart in Tn501. Thus the predicted merA polypeptide of Tn501 contains 561 amino acids and the R100 counterpart contains 564 amino acids. Comparison of the R100 mercuric reductase sequences with that for human glutathione reductase [Krauth-Siegel et al.: Eur. J. Biochem. 121 (1982) 259-267], for which there is a 2 A resolution electron density map [Thieme et al.: J. Mol. Biol. 152 (1981) 763-782] shows a strong homology, with 26% identical amino acids and many conservative substitutions. This homology allows the conclusion that the active site of these enzymes and the contact positions for flavin adenine dinucleotide (FAD) and NADPH are highly conserved, while the amino- and carboxyl-terminal sequences differ.

Acute lymphoblastic leukemia with eosinophilia.

Acute lymphoblastic leukemia with eosinophilia is a rare but distinctive clinical entity. The eosinophilia in these patients can present before, concomitantly, or after the diagnosis of leukemia. Patients with this syndrome often suffer from the cardiovascular complications of severe eosinophilia, suffering excess morbidity and mortality as a result of their eosinophilia. Treatment of the eosinophilia in this syndrome consists of administration of induction chemotherapy, followed by prednisone and hydroxyurea if required for persistent eosinophilia. Eosinophilia often resolves with remission of leukemia, only to return at the time of relapse in a high percentage of cases. Patients with this syndrome characteristically have cytogenetic abnormalities involving the long arms of chromosomes 5 and 14. These cytogenetic abnormalities are not commonly seen in acute lymphoblastic leukemia and suggest that this syndrome may have a distinct pathophysiology and etiology. The affected region on chromosome 5 contains genes that control hematopoiesis, including eosinophilopoiesis. Further investigations into these cytogenetic abnormalities may provide insight into the etiology of the leukemia and eosinophilia characteristic of this syndrome.

Adenocarcinoma of the colon simulating primary urinary bladder neoplasia. A report of nine cases.

Nine cases of adenocarcinoma of the colon, secondarily involving the urinary bladder mucosa and histologically mimicking primary bladder neoplasia, are reported. Five patients presented with bladder involvement at the time of diagnosis of colon cancer; four developed vesical lesions 9 to 66 months after resection of their colonic primary. The majority (89%) had genitourinary symptoms at presentation; gastrointestinal manifestations were present in only 60% of those with synchronous colonic involvement. The initial clinical impression, largely based on cystoscopic and radiographic studies, was a bladder primary in four cases and colon cancer in five. Of the former, three (75%) were known to have a history of colon cancer. Histologically, all were enteric-type adenocarcinomas and all had features mimicking a villous adenoma of the bladder. Distinguishing a primary bladder adenocarcinoma from spread of a colonic carcinoma to the bladder may not be possible on histopathologic grounds alone. Consideration should be given to the possibility of an extravesical primary even when symptomatology, cystoscopy, radiographic studies, and histopathology suggest a primary bladder neoplasm.