Effect of R factors and other plasmids on ultraviolet susceptibility and host cell reactivation property of Escherichia coli

Some R factors, like some colicin factors, confer partial protection against the bactericidal effect of ultraviolet (UV) irradiation. Of 31 plasmids (17 R, 3 col, and 11 R-col factors) tested in Escherichia coli K-12, 15 protected, 11 had little or no effect, and 5 caused increased UV susceptibility. The effect of representative plasmids was qualitatively the same in K-12 of wild-type UV sensitivity, lambda-lysogenic or non-lysogenic, and in UV-sensitive mutants of classes uvrA, uvrB, uvrC, and recA (except that a sensitizing factor did not increase the sensitivity of two recA hosts). It is inferred that the UV-protecting effect of some plasmids does not result from their specifying enzymes similar to those deficient in such mutants. UV killing of multiply auxotrophic K-12, of wild-type sensitivity or recA or uvrC, was reduced by deprivation of required amino acids for 2 hr before irradiation, and further reduced if "starvation" was continued for 2 hr after irradiation. The plasmids tested in these conditions produced qualitatively the same effects as in nonstarved cells-except that in K-12 of wild-type UV sensitivity the effect of protecting plasmids was reversed (i.e. they caused decreased survival) when the cells were starved after irradiation. Two UV-protecting R factors reduced the ability of HCR(+) K-12 to support growth of irradiated phage T1.     

Combined use of111In-labeled pentetreotide and three-step immunoscintigraphy with antichromogranin a monoclonal antibody in the diagnosis of pituitary adenomas

For various pituitary adenomas, it has been demonstrated that somatostatin receptor can be present. Pilot studies have shown that radio-indium labeled pentetreotide allows very good scintigraphic localization of somatostatin receptor-bearing cell masses. Recently, the presence of CgA in pituitary adenomas has also been demonstrated. MAb A11, raised against CgA, has been successfully used with a three-step ISG for the diagnosis of neuroendocrine tumors. Therefore the combined use of three-step ISG with MAb A11 and radiolabeled somatostatin can be useful in the diagnosis of pituitary adenomas. Twelve patients, 5 secreting (group A) and 7 nonsecreting (group B) pituitary adenomas, were enrolled in the study. All patients underwent three-step ISG, and, 2 wk later, scintigraphy with¹¹¹In-labeled pentetreotide (Octreoscan). Three-step ISG consisted of iv injection of 1 mg of biotinylated MAb A11 (first step), followed by 10 mg of avidin (second step) and [^99mTc]PnAO-biotin (third step). Tomographic imaging were acquired for three-step ISG and Octreoscan at 2 and 4 h after radiotracer injection, respectively. The results are the following: 2 patients of group A (secreting tumors) had a positive three-step ISG, whereas all the patients but one of the same group had a positive pentetreotide study; all the patients of group B (nonsecreting tumors) had a positive three-step ISG and 4 had a positive pentetreotide scintigraphy. These data suggest the utility of the combined use of these techniques for a better diagnosis of pituitary adenomas.     

R-factor-mediated suppression of the galactose-sensitive phenotype of Escherichia coli K-12 galE mutants

Plasmids S-a and Rts1 suppress the galactose-sensitive phenotype of galE mutants of Escherichia coli K-12, giving rise to both galactose-fermenting and nonfermenting strains. Fermenting strains produce normal inducible UDP-galactose epimerase. Plasmids extracted from either a fermenting or a nonfermenting strain are indistinguishable when examined by either measurements of length of relaxed circular molecules by electron microscopy or electrophoretic pattern of restriction endonuclease digestion products. The phenomenon could be explained by reversible recombination between a plasmid-borne epimerase gene and homologous chromosomal sequences.     

A high throughput assay for inhibitors of HIV-1 protease. Screening of microbial metabolites

A novel method for discovery of HIV-1 protease inhibitors in complex biological samples has been developed. The assay is based on two specific reagents: a recombinant protein constituted by a portion of the HIV-1 Gag polyprotein comprising the p17-p24 cleavage site, fused to E. coli beta-galactosidase, and a monoclonal antibody which binds the fusion protein in the Gag region. Binding occurs only if the fusion protein has not been cleaved by the HIV-1 protease. The assay has been adapted for the screening of large numbers of samples in standard 96-well microtiter plates. Using this method about 12000 microbial fermentation broths have been tested and several HIV-1 protease inhibitory activities have been detected. One of these has been studied in detail.     

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS-CoV-2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS-CoV-2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS-CoV-2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state-of-the-art nucleic acid sequencing technologies, we can follow in detail how SARS-CoV-2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS-CoV-2 variants across the globe should be of key interest in our fight against the pandemic.     

A simple and sensitive assay for determining plasma tipranavir concentration in the clinical setting by new HPLC method

A simple method for the quantification of tipranavir, the first non-peptidic HIV protease inhibitor, was developed and validated. Quinoxaline, as internal standard, was added to 50 microl of plasma before a liquid-liquid extraction by 600 microl of protein precipitation solution. The extracts were diluted before being injected in the chromatographic system. Chromatographic separation was made on a C18 column using potassium phosphate buffer (pH 3.2) and acetonitrile with gradient. Detection was performed by an UV detector at 260 nm. Relative error at three control quality concentrations ranged from -1.81 to 1.72%. Intra-day (CV%) and inter-day (CV%) precision ranged from 0.94 to 2.55% and from 3.07 to 4.24%, respectively. LOQ and LOD were 0.090 microg/ml and 0.035 microg/ml, respectively. Mean recovery was 87.1%+/-2.4%. Calibration curve was linear up to 180 microg/ml. Concentration range when optimized (0.703-180 microg/ml) proved to be adequate to measure tipranavir concentration in HIV-1-positive patients, therefore this method could be suitable for therapeutic drug monitoring of this drug.     

NMR and CD studies on the conformation of a synthetic peptide containing epitopes of the human immunodeficiency virus 1 transmembrane protein gp41

CD and nmr characterizations are reported for the 23-mer peptide CMC3, corresponding to residues 577–599 of gp41, the transmembrane glycoprotein of the human immunodeficiency virus 1. Concentration, temperature, and pH dependencies of CD and nmr spectra are indicative of self-association with a consequent stabilization of secondary structural elements in water. The addition to the water solution of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. The amphipathic character of the helix and the presence of three hydrophobic 4/3 heptad repeats suggest that the peptide could be structured in a symmetric association of helices, such as in a coiled-coil structure. This behavior is discussed in terms of a possible role of this segment in the gp41 envelope oligomerization. © 1996 John Wiley & Sons, Inc.     

HIV neutralizing IgA in exposed seronegative subjects recognise an epitope within the gp41 coiled-coil pocket

Human immunodeficiency virus (HIV)-specific IgA can be detected in cervical secretions, saliva, and sera of HIV-infected and HIV-uninfected individuals with a known exposure to the virus. IgA from HIV-uninfected exposed seronegative individuals (ESN) neutralize in vitro primary strains of HIV-1. We analyzed the epitopes of HIV recognized by serum HIV-specific IgA of ESN individuals to identify the antigenic correlates of HIV neutralization in exposed-uninfected subjects, and to verify whether different epitopes would be recognized by HIV-specific IgA of ESN and of HIV-infected patients. Results confirmed that HIV-neutralizing IgA are detected in sera of ESN and showed that neutralization of primary HIV strains is mediated by the recognition of different epitopes in HIV-infected patients and ESN. Thus, whereas IgA of HIV+ individuals recognize epitopes expressed both within gp120 and gp41, IgA of ESN exclusively bind to gp41-expressed epitopes. Epitope mapping revealed that the epitope recognized by serum IgA of ESN on gp41 is restricted to aa 581-584 (LQAR) and corresponds to coiled coil pocket in the alpha helic region. In contrast, the epitope seen by IgA of HIV-infected patients on gp41 is identified by two regions; the first is contained within the cystein loop (aa 589-618), the second correspond to C terminal region in the extra membrane region of gp 41 (aa 642-673). Thus, we have identified and characterized the epitopes that mediate neutralization of HIV in individuals in whom infection does not occur despite multiple exposures to the virus. These results have important implications for the development of a new therapy against HIV infection.     

Polymorphonuclear cell transmigration induced by Pseudomonas aeruginosa requires the eicosanoid hepoxilin A3

Lung inflammation resulting from bacterial infection of the respiratory mucosal surface in diseases such as cystic fibrosis and pneumonia contributes significantly to the pathology. A major consequence of the inflammatory response is the recruitment and accumulation of polymorphonuclear cells (PMNs) at the infection site. It is currently unclear what bacterial factors trigger this response and exactly how PMNs are directed across the epithelial barrier to the airway lumen. An in vitro model consisting of human PMNs and alveolar epithelial cells (A549) grown on inverted Transwell filters was used to determine whether bacteria are capable of inducing PMN migration across these epithelial barriers. A variety of lung pathogenic bacteria, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa are indeed capable of inducing PMN migration across A549 monolayers. This phenomenon is not mediated by LPS, but requires live bacteria infecting the apical surface. Bacterial interaction with the apical surface of A549 monolayers results in activation of epithelial responses, including the phosphorylation of ERK1/2 and secretion of the PMN chemokine IL-8. However, secretion of IL-8 in response to bacterial infection is neither necessary nor sufficient to mediate PMN transepithelial migration. Instead, PMN transepithelial migration is mediated by the eicosanoid hepoxilin A3, which is a PMN chemoattractant secreted by A549 cells in response to bacterial infection in a protein kinase C-dependent manner. These data suggest that bacterial-induced hepoxilin A3 secretion may represent a previously unrecognized inflammatory mechanism occurring within the lung epithelium during bacterial infections.     

New HPLC–MS method for the simultaneous quantification of the antileukemia drugs imatinib,dasatinib, and nilotinib in human plasma

A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple protein precipitation extraction procedure was applied on 250 μl of plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 20 min of analytical run, at flow rate of 1 ml/min. Mean intra-day and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was 1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from 10,000 to 62.5 ng/ml. The limit of quantification was set at 78.1 ng/ml for imatinib and at 62.5 ng/ml for dasatinib and nilotinib. This novel developed methodology allows a specific, sensitive and reliable simultaneous determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in plasma of patients affected by chronic myeloid leukemia.