Rapid detection of intestinal carriage of Klebsiella pneumoniae producing KPC carbapenemase during an outbreak.

Two different approaches are described for rapid detection of intestinal carriage of Klebsiella pneumoniae producing KPC-type carbapenemase (KPC-KP), based on PCR amplification of DNA extracts from rectal swabs (K-PCR), and on direct plating of rectal swabs on to MacConkey agar with a meropenem disc and a meropenem plus 3-aminophenylboronic acid disc (direct KPC screening test, DKST). K-PCR and DKST were tested with a total of 101 samples from 65 patients, during an outbreak. Although less sensitive, DKST could detect high-level carriage, which appears to be common among infected and colonised patients, while being very cheap and easy to perform, and requiring only basic facilities.

Functional assessment of the thyrotropin receptor-beta subunit.

Posttranslational processing of the TSH receptor (TSHR) involves proteolysis of a single chain holoreceptor into TSHR-alpha (or A) and TSHR-beta (or B) subunits, which remain associated via disulfide bonds and which may then form oligomers. As both uncleaved and cleavage-derived forms of this receptor have been reported to bind TSH and transduce signals, reasons for this cleavage into alpha- and beta-subunits have remained enigmatic. Recently we suggested that TSHR cleavage was related to receptor oligomerization and now we have asked if cleavage influenced the binding of G proteins to this receptor. Furthermore, as TSHR-alpha subunits are subject to shedding from the cell surface membrane, we have examined whether the remaining TSHR-beta subunits could mediate signaling themselves, either constitutively and /or ligand-induced. We found that only the cleaved form of the TSHR in transfected Chinese hamster ovary cells was able to bind Gsalpha protein, suggesting that cleavage of the native TSH receptor was associated with receptor activation. We also found that independently expressed TSHR-beta subunits on stable cell lines were unable to mediate either constitutive or TSH-induced signaling, as monitored by their inability to induce cAMP accumulation. These data suggested that receptor cleavage was intimately associated with receptor activation in the wild-type TSH receptor and that the residual TSHR-beta subunits left on the thyroid cell membrane, after TSHR cleavage and subsequent TSHR-alpha shedding, were essentially silent and did not participate in signal transduction.

Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

cAMP signaling selectively influences Ras effectors pathways.

Thyrotropin (TSH) stimulates survival and growth of thyroid cells via a seven transmembrane G protein-coupled receptor. TSH elevates the intracellular cyclic AMP (cAMP) levels activating protein kinase A (PKA). Recent evidence indicates that p21 Ras is required for TSH-induced mitogenesis, but the molecular mechanism(s) is not known. Here we report that Ras p21 activity is necessary for the Go- G1 transition in TSH induced cycle and that the downstream effector of Ras upon TSH signaling is p85-p110 PI3K. We show that PI3K inhibitors block TSH-induced DNA synthesis, cAMP-PKA stimulate the formation of the complex PI3K-p21 Ras and reduce the complex Ras-Raf1 in thyroid and other cells types. Moreover, PKA phosphorylates immunoprecipitated p85 and PKA phosphorylation of cell extracts significantly stimulates the formation of the complex PI3K-Ras. We suggest that PKA phosphorylates p85 and stabilizes the complex p110-p85, enhancing the interaction PI3K and p21 Ras. Simultaneously, cAMP inhibits Raf-1-ERK signaling by decreasing Raf1 availability to Ras. Under these circumstances PI3K signaling is favored. These results indicate that PI3K is an important mediator of Ras effects in cAMP-induced proliferation and illustrates how cAMP can selectively influence Ras effector pathways.

Mutations of thyrotropin receptor isolated from thyroid autonomous functioning adenomas confer TSH-independent growth to thyroid cells.

TSH receptor mutants in the VI transmembrane segment, found in thyroid autonomously functioning adeonomas, have been expressed in differentiated thyroid cells. All mutant receptors constitutively stimulated adenylyl cyclase. The biological activity, measured as cAMP production relative to the wild type receptor, was specific for each mutant in transient and stable transfection assays. Cells expressing these mutants proliferated in the absence of TSH. The rate of growth in the absence of TSH paralleled basal cAMP production for each mutant receptor. Low TSH concentrations stimulated the growth of mutant receptor-expressing cells, and not of the cells expressing the wild type receptor. Also, the entry in the cell cycle and the plating efficiency were markedly stimulated by the expression of the mutant receptors. These data provide a molecular link between the occurrence of TSH receptor mutations and thyroid autonomously functioning adenomas.

Activation of cAMP-PKA signaling in vivo inhibits smooth muscle cell proliferation induced by vascular injury.

Injury of the arterial wall induces the formation of the neointima. This structure is generated by the growth of mitogenically activated smooth muscle cells of the arterial wall. The molecular mechanism underlying the formation of the neointima involves deregulated cell growth, primarily triggered by the injury of the arterial wall. The activated gene products transmitting the injury-induced mitogenic stimuli have been identified and inhibited by several means: transdominant negative expression vectors, antisense oligodeoxynucleotides, adenovirus-mediated gene transfer, antibodies and inactivating drugs. Results of our study show that local administration of 3',5'-cyclic AMP and phosphodiesterase-inhibitor drugs (aminophylline and amrinone) to rats markedly inhibits neointima formation after balloon injury in vivo and in smooth muscle cells in vitro. The growth inhibitory effect of aminophylline was completely reversed by the inhibition of cAMP-dependent protein kinase A (PKA). These findings indicate an alternative approach to the treatment of diseases associated with injury-induced cell growth of the arterial wall, as stimulation of cAMP signaling is pharmacologically feasible in the clinical setting.

Somatic mutations in the VI transmembrane segment of the thyrotropin receptor constitutively activate cAMP signalling in thyroid hyperfunctioning adenomas.

We have discovered two somatic mutations in the VI transmembrane domain of the thyrotropin receptor gene in thyroid hyperfunctioning adenomas. The mutated amino acid residues are phenylalanine 631 (to cysteine) and threonine 632 (to isoleucine). Cloning and expression of the mutated versions of the receptor in COS cells increased significantly the basal and the TSH-induced cAMP levels compared to the wild type receptor. Moreover, the expression of a reporter gene under the control of the cAMP-inducible promoter, was likewise constitutively activated in cells expressing the 631 and 632 TSH receptor mutants relative to the wild type. These data indicate that the VI transmembrane segment in the TSH receptor and presumably in the other G-protein coupled receptors is a critical domain for the activation of G-protein signalling and that the mutations described here may be the cause of the thyroid hyperfunctioning adenoma.

Novel mutations of thyrotropin receptor gene in thyroid hyperfunctioning adenomas. Rapid identification by fine needle aspiration biopsy.

Hyperfunctioning thyroid adenomas are clonal neoplasms with the intrinsic capacity of growing and differentiate independently of thyroid-stimulating hormone (TSH). We analysed the mRNA encoding thyrotropin receptor of 11 adenomas obtained by fine needle aspiration biopsy (FNAB) and found 7 mutants all located in three aminoacids clustered in the sixth transmembrane domain of the receptor. These mutations were somatic and specifically present in the tumour tissue. DNA sequence revealed that 80 to 90% of the mutations can be rapidly screened and identified by restriction enzyme analysis of the amplified cDNA obtained from the FNABs. The mutation Thr->Ile was introduced in the wild type receptor and expressed in mouse fibroblasts. These cells constitutively activate the transcription of a reporter gene under the control of cyclic AMP responsive element.

Expression of thyrotropin-receptor mRNA in healthy and Graves' disease retro-orbital tissue.

It is not clear whether the ophthalmopathy present in Graves' disease is related to autoimmune reactions to common thyroid/orbit components or is due to specific ocular antigens unrelated to the thyroid. In Graves' disease, serum antibodies to the thyrotropin receptor (TSH-R) can stimulate thyroid function, but no link between the TSH-R and ocular tissues has been found. We have shown, by polymerase chain reaction amplification of specific cDNA, that mature TSH-R mRNA is expressed in the retro-orbital tissue of both healthy subjects and patients with Graves' disease. Of other tissues and cells tested (fibroblasts, lymphocytes, muscle, and thyroid), only thyroid tissue expressed the TSH-R mRNA.