Genetic and biochemical characterization of the chromosomal class A beta-lactamases of Raoultella (formerly Klebsiella) planticola and Raoultella ornithinolytica

Enterobacterial strains of Raoultella spp. display a penicillinase-related beta-lactam resistance pattern suggesting the presence of a chromosomal bla gene. From whole-cell DNA of Raoultella planticola strain ATCC 33531(T) and Raoultella ornithinolytica strain ATCC 31898(T), bla genes were cloned and expressed into Escherichia coli. Each gene encoded an Ambler class A beta-lactamase, named PLA-1 and ORN-1 for R. planticola and R. ornithinolytica, respectively. These beta-lactamases (291 amino acids), with the same pI value of 7.8, had a shared amino acid identity of 94%, 37 to 47% identity with the majority of the chromosome-encoded class A beta-lactamases previously described for Enterobacteriaceae, and 66 to 69% identity with the two beta-lactamases LEN-1 and SHV-1 from Klebsiella pneumoniae. However, the highest identity percentage (69 to 71%) was found with the plasmid-mediated beta-lactamase TEM-1. PLA-1, which displayed very strong hydrolytic activity against penicillins, also displayed significant hydrolytic activity against cefepime and, to a lesser extent, against cefotaxime and aztreonam, but there was no hydrolytic activity against ceftazidime. Such a substrate profile suggests that the Raoultella beta-lactamases PLA-1 and ORN-1 should be classified into the group 2be of the beta-lactamase classification of K. Bush, G. A. Jacoby, and A. A. Medeiros (Antimicrob. Agents Chemother. 39:1211-1233, 1995). The highly homologous regions upstream of the bla(PLA-1A) and bla(ORN-1A) genes comprised a nucleotide sequence identical to the -35 region and another one very close to the -10 region of the bla(LEN-1) gene. From now on, as the bla gene sequences of the most frequent Raoultella and Klebsiella species are available, the bla gene amplification method can be used to differentiate these species from each other, which the biochemical tests currently carried out in the clinical laboratory are unable to do.     

Asbestos in the sputum, crackles in the lungs, and radiologic changes in women exposed to asbestos

In a rural community in South Africa historically exposed to asbestos environmentally and occupationally, 200 women who had worked with asbestos and applied for medical examination to determine compensable asbestos disease were evaluated. Clinical and radiologic evaluation, sputum collection, and microscopic analysis were done. A questionnaire elicited type of exposure, duration, decade of first work exposure, and environmental exposure. Crackles were present in the lungs of 166 women and asbestos fibers and ferruginous bodies were present in 122. Asbestosis was identified in 26 and plural plaques in 62. Auscultation for crackles (rales) is useful in the initial examination of former asbestos workers in rural communities of developing countries.     

Differential effects of X-ALK fusion proteins on proliferation, transformation, and invasion properties of NIH3T3 cells

Majority of anaplastic large-cell lymphomas (ALCLs) are associated with the t(2;5)(p23;q35) translocation, fusing the NPM (nucleophosmin) and ALK (anaplastic lymphoma kinase) genes (NPM-ALK). Recent studies demonstrated that ALK may also be involved in variant translocations, namely, t(1;2)(q25;p23), t(2;3)(p23;q21), t(2;17)(p23;q23) and inv(2)(p23q35), which create the TPM3-ALK, TFG-ALK5, CLTC-ALK, and ATIC-ALK fusion genes, respectively. Although overexpression of NPM-ALK has previously been shown to transform fibroblasts, the transforming potential of variant X-ALK proteins has not been precisely investigated. We stably transfected the cDNAs coding for NPM-ALK, TPM3-ALK, TFG-ALK, CLTC-ALK or ATIC-ALK into nonmalignant NIH3T3 cells. All X-ALK variants are tyrosine phosphorylated and their subcellular distribution was in agreement with that observed in tumors. Moreover, our results show that the in vitro transforming capacity of NIH3T3-transfected cells are in relation to the level of X-ALK fusion proteins excepted for TPM3-ALK for which there is an inverse correlation. The differences between the five X-ALK variants with regard to proliferation rate, colony formation in soft agar, invasion, migration through the endothelial barrier and tumorigenicity seem to be due to differential activation of various signaling pathways such as PI3-kinase/AKT. These findings may have clinical implications in the pathogenesis and prognosis of ALK-positive ALCLs.     

Temporal patterns of the cerebral inflammatory response in the rat lithium-pilocarpine model of temporal lobe epilepsy

To better understand the role of inflammatory responses in temporal lobe epilepsy, we characterized Interleukin1-beta (IL1-beta), Nuclear Factor-kappaB (NF-kappaB), and Cyclooxygenase-2 (COX-2) expression together with neurodegeneration in the rat lithium-pilocarpine model. The immunohistochemical expression of IL1-beta, NF-kappaB, and COX-2 started by 12 h post-injection, persisted for 24 h (status epilepticus period), and returned to basal levels by 3 and 6 days (latent period). The regional distribution of IL1-beta, NF-kappaB, and COX-2 occurred mainly in structures prone to develop neuronal damage. Using double-staining protocols, we detected IL1-beta expression in glial cells, COX-2 expression in neurons, and NF-kappaB in both cell types. The presence of Fluoro-Jade-B-positive degenerating neurons was associated with IL1-beta, NF-kappaB, and COX-2 proteins expression during status epilepticus but not during the latent period while neurons were still degenerating. These data suggest that seizure-related IL1-beta, NF-kappaB, and COX-2 expression may contribute to the pathophysiology of epilepsy by inducing neuronal death and astrocytic activation.     

Novel long-circulating liposomes containing peptide library-lipid conjugates: synthesis and in vivo behavior

Rapid uptake of intravenously injected liposomes by the mononuclear phagocyte system has limited their use as drug delivery vehicles. Recently, various long-circulating liposomes have been prepared by incorporating glycolipids or other amphiphilic molecules into the lipid bilayer of conventional liposomes. The purpose of the present study was to design a new class of biodegradable membrane modifiers that would increase the half-life of liposomes in vivo. Using solid-phase peptide synthesis, synthesized were 30-residue random libraries consisting of a random sequence of glycine, beta-alanine and gamma-aminobutyric acid. The libraries were coupled to stearic acid (SA) or phosphatidylethanolamine (PE). The resulting amphiphilic conjugates were mixed with egg phosphatidylcholine (PC) and cholesterol (Chol) in a 6:47:47 ratio, and unilamellar liposomes were prepared. For comparison, plain PC/Chol (50:50) liposomes, as well as liposomes containing polyethylene glycol (PEG)-SA/PC/Chol (6:47:47) and PEG-PE/PC/Chol (6:47:47) were also prepared. Calcein was entrapped in the liposomes, which were given intravenously to rats at a dose of 9.2 mumol lipid/kg, and the amount of intact liposomes present in serum was followed with time. While the conventional liposomes had a short elimination half-life (28 min), the liposomes modified with library-PE had a much longer half-life (170 min), while library-SA provided no improvement of the liposome pharmacokinetics. PEG-PE greatly improved the half-life of the liposomes (400 min) while PEG-SA only provided a marginal improvement. All liposome preparations were cleared in a biphasic fashion. In conclusion, a novel biodegradable lipopeptide conjugate was designed that endows liposomes with a prolonged circulation time in vivo. The pharmacokinetic profile of these modified liposomes was drastically improved over that of conventional liposomes. Since the library is prepared by solid-phase synthesis, length and/or composition could easily be modified in order to modulate the clearance profile of the liposomes. Tailoring of the pharmacokinetic profile of the liposomes depending on their intended application may allow for a greater flexibility of use than PEG-PE.     

Relationship between intratumoral expression of genes coding for xenobiotic-metabolizing enzymes and benefit from adjuvant tamoxifen in estrogen receptor alpha-positive postmenopausal breast carcinoma

Introduction  

Little is known of the function and clinical significance of intratumoral dysregulation of xenobiotic-metabolizing enzyme expression in breast cancer. One molecular mechanism proposed to explain tamoxifen resistance is altered tamoxifen metabolism and bioavailability.     

Ligands of the peripheral benzodiazepine receptor have therapeutic effects in pneumopathies in vivo

In this study, we documented the effects of different peripheral benzodiazepine receptor (PBR) ligands: PK 11195, Ro5-4864 and the newly described SSR 180575 on the development of pulmonary inflammation in vivo. To this aim, we used MRL/lpr mice that develop pathological signs similar to the human lupus erythematosus (LE) signs. We found that a chronic treatment (at 3 mg/kg per i.p. for 30 days) with PBR ligands had a significant beneficial therapeutic action and decreased the inflammatory pulmonary responses and alveolitis onset. When analyzing PBR expression in inflamed tissues, we observed that in addition to the infiltrated leukocytes, PBR was expressed in the bronchial epithelium, and especially we evidenced for the first time that PBR in expressed in Clara cells. Interestingly, we observed that PBR expression in those cells was reduced when MRL/lpr mice developed the pathology and restored upon PBR ligand treatment. These original findings support a role of PBR in pulmonary inflammatory process and suggest new therapeutic applications in auto immune disorders for specific potent PBR ligands.     

Frozen section examination of the endocervical margin of cervical conization specimens

OBJECTIVE: We conducted this retrospective study to determine accuracy of frozen section examination of endocervical margin during cold knife conization. METHODS: Between June 1993 and June 2001, 310 consecutive patients underwent cervical conization for squamous intraepithelial lesion or stage IA1 cervical cancer. Before 1997, the surgical specimens of 149 patients were processed following a standard pathological procedure (historical group). After 1997, a frozen section of the upper endocervical margin was processed during surgery for 161 patients. If the upper endocervical margin was involved with intraepithelial neoplasia, the surgeon performed a second resection if possible. Results of the frozen section examination were compared with the final diagnoses to determine sensitivity, specificity, and positive and negative predictive values. The usefulness of this procedure was evaluated by comparison of positive margin status rate with the one of the historical control group. RESULTS: For the diagnosis of intraepithelial neoplasia involving the endocervical margin, the sensitivity, specificity, and positive and negative predictive values of frozen section were 91%, 100%, 100%, and 98%, respectively. Eleven patients had definitive positive endocervical margin in the frozen section group (three false negatives, six patients without additional resection, and two patients with intraepithelial neoplasia involving the upper margin of the additional resection) and 17 patients in the historical group (P =.16). CONCLUSION: Frozen section examination of the endocervical margin of cervical specimen obtained during cold knife conization is highly accurate. Its clinical relevance has to be demonstrated in a multicenter study.     

TP53 polymorphism at exon 4 in caucasian women from eastern France: lack of correlation with HPV status and grade of cervical precancerous lesions

OBJECTIVE: To investigate the codon 72 TP53 polymorphism in women from eastern France with normal or abnormal cervical cytology.STUDY DESIGN: We analyzed the TP53 allele distribution by denaturant gradient gel electrophoresis assay and the human papillomaviruses (HPV) infection in 138 cervical smears: 50 normal, 20 atypical squamous cells of undetermined significance, 40 low grade squamous intraepithelial lesions, 28 high grade squamous intraepithelial lesions.RESULTS: The viral DNA prevalence increased with cytological abnormalities. The rates of arginine (Arg) and proline (Pro) homozygosity and Arg/Pro heterozygosity were 49, 0.72, and 51%, respectively. No association was found between HPV status and TP53 polymorphism. No differences were observed in the frequency of the TP53 genotypes according to cytology.CONCLUSION: The TP53 Arg/Arg genotype does not appear to represent a risk factor in the progression of HPV associated cervical lesions. We were not able to confirm that the TP53 genotype increases the susceptibility to be infected by HPV or to develop HGSIL, and a fortiori invasive carcinoma of the cervix.     

Ventilatory responses to hypercapnia and hypoxia in heterozygous c-ret newborn mice

The c-ret proto-oncogene encodes a tyrosine-kinase receptor involved in survival and differentiation of neural crest cell lineages. Previous studies have shown that homozygous c-ret-/- mice die soon after birth and have impaired ventilatory responses to hypercapnia. Heterozygous c-ret +/- mice develop normally, but their respiratory phenotype has not been described in detail. We used whole-body flow plethysmography to compare baseline breathing and ventilatory and arousal responses to chemical stimuli in unrestrained heterozygous c-ret +/- newborn mice and their wild-type c-ret +/+ littermates at 10-12 h of postnatal age. The hyperpnoeic and arousal responses to hypoxia and hypercapnia were not significantly different in these two groups. However, the number and total duration of apnoeas and periodic breathing episodes were significantly higher in c-ret +/- than in c-ret +/+ pups during hypoxia and post-hypoxic normoxia. These results are further evidence that respiratory control at birth is heavily dependent on genes involved in the neural determination of neural crest cells.