Transcriptomic Analysis of the Dual Response of Rhodococcus aetherivorans BCP1 to Inorganic Arsenic Oxyanions.

Bacterial strains belonging to the genus Rhodococcus are able to degrade various toxic organic compounds and tolerate high concentrations of metal(loid)s. We have previously shown that Rhodococcus aetherivorans BCP1 is resistant to various levels of the two arsenic inorganic species, arsenite [As(III)] and arsenate [As(V)]. However, while arsenite showed toxic effects at concentrations as low as 5 mM, arsenate at 30 mM boosted the growth rate of BCP1 cells and was toxic only at concentrations of >100 mM. Since such behavior could be linked to peculiar aspects of its metabolism, the transcriptomic analysis of BCP1 cells exposed to 5 mM As(III) and 30 mM As(V) was performed in this work. The aim was to clarify the mechanisms underlying the arsenic stress response of the two growth phenotypes in the presence of the two different oxyanions. The results revealed that As(III) induced higher activity of reactive oxygen species (ROS)-scavenging enzymes than As(V) in relation to the expression of enzymes involved in cellular damage recovery and redox buffers/cofactors (ergothioneine, mycofactocin, and mycothiol). Further, As(III) downregulated pathways related to cell division, while both oxyanions downregulated genes involved in glycolysis. Notably, As(V) induced the expression of enzymes participating in the synthesis of metallophores and rearranged the central and energetic metabolism, also inducing alternative pathways for ATP synthesis and glucose consumption. This study, in providing transcriptomic data on R. aetherivorans exposed to arsenic oxyanions, sheds some light on the plasticity of the rhodococcal response to arsenic stress, which may be important for the improvement of biotechnological applications. IMPORTANCE Members of the genus Rhodococcus show high metabolic versatility and the ability to tolerate/resist numerous stress conditions, including toxic metals. R. aetherivorans BCP1 is able to tolerate high concentrations of the two inorganic arsenic oxyanions, arsenite [As(III)] and arsenate [As(V)]. Despite the fact that BCP1 intracellularly converts As(V) into As(III), this strain responds very differently to the presence of these two oxyanions in terms of cell growth and toxic effects. Indeed, while As(III) is highly toxic, exposure to specific concentrations of As(V) seems to boost cell growth. In this work, we investigated the transcriptomic response, ATP synthesis, glucose consumption, and H2O2 degradation in BCP1 cells exposed to As(III) and As(V), inducing two different growth phenotypes. Our results give an overview of the transcriptional rearrangements associated with the dual response of BCP1 to the two oxyanions and provide novel insights into the energetic metabolism of Rhodococcus under arsenic stress.

Stability and Species Specificity of Renal VEGF-A Splicing Patterns in Kidney Disease.

Vascular endothelial growth factor A (VEGF-A) is essential for maintaining the glomerular filtration barrier. Absolute renal levels of VEGF-A change in patients with diabetic nephropathy and inflammatory kidney diseases, but whether changes in the renal splicing patterns of VEGF-A play a role remains unclear. In this study, we investigated mRNA splicing patterns of pro-angiogenic isoforms of VEGF-A in glomeruli and whole kidney samples from human patients with kidney disease and from mouse models of kidney disease. Kidney biopsies were obtained from patients with acute rejection following kidney transplantation, patients with diabetic nephropathy, and control subjects. In addition, kidney samples were obtained from mice with lupus nephritis, mice with diabetes mellitus, and control mice. The relative expression of each VEGF-A splice variant was measured using RT-PCR followed by quantitative fragment analysis. The pattern of renal VEGF-A splice variants was unchanged in diabetic nephropathy and lupus nephritis and was stable throughout disease progression in acute transplant rejection and diabetic nephropathy; these results suggest renal VEGF-A splicing stability during kidney disease. The splicing patterns were species-specific; in the control human kidney samples, VEGF-A 121 was the dominant isoform, whereas VEGF-A 164 was the dominant isoform measured in the mouse kidney samples.

Multiple aggressive squamous skin cancers in association with nonbullous congenital ichthyosiform erythroderma.

Nonbullous congenital ichthyosiform erythroderma (NBCIE) is one of the autosomal recessive inherited non-syndromic ichthyoses and is currently diagnosed on clinical grounds alone. Skin cancer is not a recognized complication of NBCIE. We report here two NBCIE patients who have developed multiple aggressive nonmelanoma skin cancers, predominantly cutaneous squamous cell carcinoma. NBCIE may be a risk factor for skin cancer development.

p53-specific CD8+ T-cell responses in individuals with cutaneous squamous cell carcinoma.

BACKGROUND: Systemic immunosuppression is a significant risk factor for cutaneous squamous cell carcinoma (SCC). p53 is mutated and overexpressed in up to 90% of cutaneous SCC lesions. Despite considerable evidence that the immune response is important in the control of cutaneous SCC, there are no studies documenting potential tumour-associated antigens. OBJECTIVES: We tested the hypothesis that individuals with cutaneous SCC have functional circulating CD8+ T cells specific for p53. METHODS: Interferon-gamma immunosorbent assays were used to screen peripheral blood mononuclear cells for reactivity to six p53-derived HLA-A*0201-restricted epitopes from HLA-A*0201-positive patients and controls. RESULTS: We observed significantly elevated frequencies of p53-specific CD8+ T cells in seven of 26 individuals with cutaneous SCC and in one of 10 controls. The degree of lymphocytic infiltrate significantly correlated with the frequency of CD8+ T cells specific for p53 epitopes, but not with control epitopes. CONCLUSIONS: Overall, these data suggest that p53 may represent a target for CD8+ T cells in a proportion of individuals with cutaneous SCC.

Spontaneous clinical improvement in HIV-associated follicular syndrome.

Since 1991 infrequent reports have described a distinctive triad of nodulocystic acne, striking follicular spines and an eruption resembling pityriasis rubra pilaris (PRP) in HIV-positive patients. It has been suggested that this may represent a subtype of PRP, or alternatively that it should be viewed as a unique HIV-associated follicular occlusion triad. Clinical manifestations may be severe, and in several cases have been ultimately fatal, with death occurring due to complications of cutaneous sepsis. We describe a case demonstrating severe conglobate acne, follicular keratotic spines and histologically confirmed PRP in association with HIV infection. Clinical features and treatment modalities of previously reported cases are reviewed. Despite refusing all topical and systemic treatment our patient showed spontaneous remission of skin signs after 2 years.

Multiple scrotal epidermolytic acanthomas; secondary to trauma?

Epidermolytic hyperkeratosis (EH) is an abnormality of epidermal maturation, most commonly due to mutations in keratins 1 and 10, which may be a congenital or an acquired defect. The term epidermolytic acanthoma was applied to a solitary discrete epidermal proliferation characterized by EH. Subsequently there have been several reports of disseminated epidermolytic acanthomas. We report a rare case of multiple epidermolytic acanthomas localized to the scrotum. With the aetiology of epidermolytic acanthoma unknown, trauma has been postulated as a possible cause. Our patient repetitively scratched his scrotum for 5 years and we believe that this action triggered his multiple scrotal epidermolytic acanthomas.

Glutathione is a target in tellurite toxicity and is protected by tellurite resistance determinants in Escherichia coli.

Tellurite (TeO3(2-)) is highly toxic to most microorganisms. The mechanisms of toxicity or resistance are poorly understood. It has been shown that tellurite rapidly depletes the reduced thiol content within wild-type Escherichia coli. We have shown that the presence of plasmid-borne tellurite-resistance determinants protects against general thiol oxidation by tellurite. In the present study we observe that the tellurite-dependent depletion of cellular thiols in mutants of the glutathione and thioredoxin thiol:redox system was less than in wild-type cells. To identify the type of low-molecular-weight thiol compounds affected by tellurite exposure, the thiol-containing molecules were analyzed by reverse phase HPLC as their monobromobimane derivatives. Results indicated that reduced glutathione is a major initial target of tellurite reactivity within the cell. Other thiol species are also targeted by tellurite, including reduced coenzyme A. The presence of the tellurite resistance determinants kilA and ter protect against the loss of reduced glutathione by as much as 60% over a 2 h exposure. This protection of glutathione oxidation is likely key to the resistance mechanism of these determinants. Additionally, the thiol oxidation response curves were compared between selenite and tellurite. The loss of thiol compounds within the cell recovered from selenite but not to tellurite.

A controlled study of ultraviolet A sunbed treatment of psoriasis.

BACKGROUND: Ultraviolet (UV) A sunbeds are widely used by patients with psoriasis in an attempt to treat their skin disease. However, there is little evidence that UVA therapy improves psoriasis, and the long-term risks of sunbed exposure are not known. OBJECTIVES: To perform a randomized, placebo-controlled study of UVA sunbed therapy for psoriasis. METHODS: A sunbed and canopy unit was modified to allow UVA exposure on one side of the body (front and back), and 'placebo' visible light exposure on the other side of the body. We treated 38 patients with psoriasis, giving 12 exposures over a period of 4 weeks. Assessment was made using a modified Psoriasis Area and Severity Index (PASI) score, individual plaque assessment and patient questionnaire. RESULTS: In 17 patients (47%) the PASI score showed a greater reduction on the UVA side compared with placebo, in 11 patients (31%) no difference was recorded between the two sides, and in eight (22%) the improvement was greater on the placebo-treated side. Overall, the median pretreatment half-body modified PASI score was 4.4 units, reducing to 3.9 units on the UVA-treated side and 4.2 units on the placebo-treated side (P = 0. 044 for difference in response). Breakdown of the plaque score into the individual components of erythema, scale and thickness revealed significant improvement only with the score for erythema. Although the degree of improvement was small, 64% of patients felt that the response was sufficiently good that they would use a sunbed again to treat their psoriasis. CONCLUSIONS: Our results show that a short course of sunbed treatment does improve psoriasis in some patients, but that the degree of improvement is small.

Escherichia coli TehB requires S-adenosylmethionine as a cofactor to mediate tellurite resistance.

The Escherichia coli chromosomal determinant for tellurite resistance consists of two genes (tehA and tehB) which, when expressed on a multicopy plasmid, confer resistance to K(2)TeO(3) at 128 microg/ml, compared to the MIC of 2 microg/ml for the wild type. TehB is a cytoplasmic protein which possesses three conserved motifs (I, II, and III) found in S-adenosyl-L-methionine (SAM)-dependent non-nucleic acid methyltransferases. Replacement of the conserved aspartate residue in motif I by asparagine or alanine, or of the conserved phenylalanine in motif II by tyrosine or alanine, decreased resistance to background levels. Our results are consistent with motifs I and II in TehB being involved in SAM binding. Additionally, conformational changes in TehB are observed upon binding of both tellurite and SAM. The hydrodynamic radius of TehB measured by dynamic light scattering showed a approximately 20% decrease upon binding of both tellurite and SAM. These data suggest that TehB utilizes a methyltransferase activity in the detoxification of tellurite.

The role of cysteine residues in tellurite resistance mediated by the TehAB determinant.

TehATehB is a tellurite (TeO(2-)(3)) resistance determinant found on the Escherichia coli chromosome. Normally silent, it specifies a minimal inhibitory concentration (MIC) of 2 microg K(2)TeO(3)/ml unless upregulated or present on a multicopy plasmid which results in an MIC of 128 microg/ml. Both TehA and TehB have three cysteine residues. Oligonucleotide site-directed mutagenesis was carried out to systematically replace all six cysteine residues by alaninies. The results showed that cysteine residues in both TehA and TehB play a role in tellurite resistance: A single cysteine change had no effect, however increasing combinations of two or three cysteine substitutions demonstrated strong phenotypic effects with minimal inhibitory concentrations ranging from 16-64 microg K(2)TeO(3)/ml. A cysteine-free mutant in which all six cysteine residues were replaced by alanines maintained a MIC of 16 microg/ml. Further investigations on the role of cysteines in resistance were studied using thiol reactive reagents on the soluble subunit TehB. These studies confirmed that TehB is a dimer and undergoes a conformational change with tellurite and S-adenosyl-l-methionine binding. Studies using native and SDS denaturing PAGE under reducing and oxidizing conditions suggested that a cysteine in TehB is involved in binding tellurite.

Arterial flow induces changes in saphenous vein endothelium proteins transduced by cation channels.

OBJECTIVES: expression of leukocyte adhesins and proteins controlling thrombosis is likely to be an important determinant of graft patency early following vein bypass. We have previously demonstrated rapid increase in endothelial ICAM-1 and nitric oxide synthase (eNOS) concentrations in human saphenous vein exposed to arterial flow. The aim of this study was to investigate whether ion-channel-blocking drugs could alter these flow-induced changes. METHODS: human saphenous vein segments, freshly excised from patients, were placed in a validated in vitro circuit using flow conditions shown to simulate arterial or venous circulations for 90 min, in the presence or absence of ion-channel blockers. The concentrations of ICAM-1, VCAM-1, eNOS and tissue factor (TF) were assessed by quantitative immunohistochemistry in vein exposed to flow and compared with that in freshly excised vein from the same patient. The endothelial protein concentration was calculated as the mean area of staining as percentage of that for the control protein CD31, using computer-aided image analysis. RESULTS: after arterial flow conditions the area ratio of ICAM-1 increased from 21.4+/-1.4 to 44.6+/-2.0%, of eNOS increased from 50.0+/-5.6 to 70.1+/-5.0%, of VCAM-1 decreased from 16.6+/-3.4 to 3.6+/-1.0%, whereas TF staining area ratio was unchanged. Inclusion of the non-selective K(+)channel blocker, tetraethylammonium, in the arterial perfusion solution abolished all these arterial flow-induced changes. Inclusion of the K(+)ATP channel blocker, glibenclamide, selectively abolished the arterial flow-induced changes in ICAM-1 and VCAM-1. Inclusion of the calcium channel blocker, nifedipine, abolished the arterial flow-induced changes in eNOS and VCAM-1 but increased the TF staining area ratio from 3.0+/-0.4 to 8.5+/-0.7%, p=0.01. Inclusion of the stretch-activated cation-channel blocker, gadolinium, enhanced the arterial flow-induced increase in eNOS, but prevented the arterial flow-induced increase in ICAM-1. CONCLUSIONS: perfusion of veins under arterial flow conditions with gadolinium was associated with low endothelial concentrations of ICAM-1, VCAM-1 and TF, but high levels of eNOS. Such a concentration of endothelial proteins may be advantageous in newly implanted vein grafts. In contrast, nifedipine could have adverse effects by promoting increase in TF concentration.

Evidence that type I, II, and III inositol 1,4,5-trisphosphate receptors can occur as integral plasma membrane proteins.

A number of previous reports have suggested that inositol 1,4, 5-trisphosphate receptors (IP(3)Rs) are present in the plasma membranes of cells. We confirm this directly in the present study by demonstrating that a significant proportion of the IP(3)Rs found in A431 cells, Jurkat cells, and rat parotid acini can be biotinylated by the extracellular application of sulfo-N-hydroxysuccinimide-biotin to intact cells. This labeling cannot be accounted for by the reaction of sulfo-N-hydroxysuccinimide-biotin with intracellular IP(3)Rs since calnexin and the SERCA2 ATPase, both integral membrane proteins of the endoplasmic reticulum, are not labeled under the same experimental conditions. Individual IP(3)R subtypes were detected using subtype-specific antibodies. A431 cells expressed only the type-3 IP(3)R, and 23% of this protein was in the biotinylated (plasma membrane) fraction. Jurkat cells and rat parotid cells expressed all three IP(3)R subtypes. Contrary to earlier results suggesting that only the type-3 IP(3)R might localize to the plasma membrane, we found that significant amounts (5-14%) of all three subtypes could be identified in the biotinylated fractions of Jurkat and rat parotid cells. Our results suggest a role for IP(3)Rs in plasma membrane as well as intracellular membrane function.

A retrospective study of outcome of Mohs' micrographic surgery for cutaneous squamous cell carcinoma using formalin fixed sections.

The surgical management of recurrent or large squamous cell carcinoma (SCC) can be challenging as tumours often extend beyond visible margins. Micrographic surgery is a potentially effective method of ensuring complete clearance of tumour. A retrospective study of all cases of SCC treated by micrographic surgery in this department between 1986 and 1996 has been done. Sixty-one patients were treated using a formalin-fixed paraffin-embedded tissue technique with a median follow-up of 4 years. In two cases there was local recurrence and in three others metastasis to local lymph nodes. The overall cure rate was 92% (56 of 61), which compares favourably with published series using chemosurgery and frozen tissue techniques. The results show that this technique of micrographic surgery is a satisfactory and cost-effective alternative to conventional frozen section techniques in the treatment of SCC. The formalin-fixed tissue method has the advantage of providing high-quality permanent histological sections using existing conventional pathology services.