Semen impairment and occurrence of SARS-CoV-2 virus in semen after recovery from COVID-19.

STUDY QUESTION: How is the semen quality of sexually active men following recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection? SUMMARY ANSWER: Twenty-five percent of the men with recent SARS-Cov-2 infections and proven healing were oligo-crypto-azoospermic, despite the absence of virus RNA in semen. WHAT IS KNOWN ALREADY: The presence of SARS-CoV-2 in human semen and its role in virus contagion and semen quality after recovery from coronavirus disease 2019 (COVID-19) is still unclear. So far, studies evaluating semen quality and the occurrence of SARS-CoV-2 in semen of infected or proven recovered men are scarce and included a limited number of participants. STUDY DESIGN, SIZE, DURATION: A prospective cross-sectional study on 43 sexually active men who were known to have recovered from SARS-CoV2 was performed. Four biological fluid samples, namely saliva, pre-ejaculation urine, semen, and post-ejaculation urine, were tested for the SARS-CoV-2 genome. Female partners were retested if any specimen was found to be SARS-CoV-2 positive. Routine semen analysis and quantification of semen leukocytes and interleukin-8 (IL-8) levels were performed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Questionnaires including International Index of Erectile Function and Male Sexual Health Questionnaire Short Form were administered to all subjects. The occurrence of virus RNA was evaluated in all the biological fluids collected by RT-PCR. Semen parameters were evaluated according to the World Health Organization manual edition V. Semen IL-8 levels were evaluated by a two-step ELISA method. MAIN RESULTS AND THE ROLE OF CHANCE: After recovery from COVID-19, 25% of the men studied were oligo-crypto-azoospermic. Of the 11 men with semen impairment, 8 were azoospermic and 3 were oligospermic. A total of 33 patients (76.7%) showed pathological levels of IL-8 in semen. Oligo-crypto-azoospermia was significantly related to COVID-19 severity (P < 0.001). Three patients (7%) tested positive for at least one sample (one saliva; one pre-ejaculation urine; one semen and one post-ejaculation urine), so the next day new nasopharyngeal swabs were collected. The results from these three patients and their partners were all negative for SARS-CoV-2. LIMITATIONS, REASONS FOR CAUTION: Although crypto-azoospermia was found in a high percentage of men who had recovered from COVID-19, clearly exceeding the percentage found in the general population, the previous semen quality of these men was unknown nor is it known whether a recovery of testicular function was occurring. The low number of enrolled patients may limit the statistical power of study. WIDER IMPLICATIONS OF THE FINDINGS: SARS-CoV-2 can be detected in saliva, urine, and semen in a small percentage of men who recovered from COVID-19. One-quarter of men who recovered from COVID-19 demonstrated oligo-crypto-azoospermia indicating that an assessment of semen quality should be recommended for men of reproductive age who are affected by COVID-19. STUDY FUNDING/COMPETING INTEREST(S): None. TRIAL REGISTRATION NUMBER: N/A.

Isothermal microcalorimetry minimal inhibitory concentration testing in extensively drug resistant Gram-negative bacilli: a multicentre study.

OBJECTIVES: To evaluate the performance of an isothermal microcalorimetry (IMC) method for determining the MICs among extensively drug-resistant Gram-negative bacilli. METHODS: A collection of 320 clinical isolates (n = 80 of each) of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii from Sweden, Spain, Italy and the Netherlands were tested. The MICs were determined using the IMC device calScreener (Symcel, Stockholm, Sweden) and ISO-broth microdilution as the reference method. Essential agreement, categorical agreement, very major errors (VME), major errors (ME) and minor (mE) errors for each antibiotic were determined. RESULTS: Data from 316 isolates were evaluated. Four errors (two ME, one VME, one mE) among 80 K. pneumoniae, six errors (four ME, one VME, one mE) among 79 E. coli, 15 errors (seven VME, three ME, five mE) among 77 P. aeruginosa and 18 errors (12 VME, two ME, four mE) among 80 A. baumannii were observed. Average essential agreement and categorical agreement of the IMC method were 96.6% (95% confidence interval, 94.2-99) and 97.1% (95% confidence interval, 95.4-98.5) respectively when the MICs were determined at the end of 18 hours. Categorical agreement of the IMC method for prediction of MIC by the end of 8 hours for colistin, meropenem, amikacin, ciprofloxacin and piperacillin/tazobactam were 95%, 91.4%, 94%, 95.2% and 93.7% respectively. CONCLUSIONS: The IMC method could accurately determine the MICs among extensively drug-resistant clinical isolates of E. coli, K. pneumoniae, P. aeruginosa and A. baumannii isolates.

Variable performance of different commercial systems for testing carbapenem susceptibility of KPC carbapenemase-producing Escherichia coli.

OBJECTIVES: The aim was to evaluate different methods for testing carbapenem susceptibility of Escherichia coli producing KPC-type carbapenemase. METHODS: Susceptibility to imipenem, meropenem and ertapenem was assayed using the reference broth microdilution method and several commercial methods (Vitek2, MicroScan, Etest, MIC Test Strip) starting from the same bacterial suspension. Susceptibility to imipenem and meropenem was also tested by Sensititre and disc diffusion (Bio-Rad). Results were interpreted according to EUCAST clinical breakpoints. Essential agreement (EA), category agreement (CA) and error rates were calculated as described by the International Organization for Standardization (ISO) guidelines and also considering the new EUCAST definitions. Genotypic diversity of isolates was evaluated with a RAPD profiling protocol. RESULTS: Of 54 KPC-positive E. coli isolates, 5.6%, 7.4% and 0% were susceptible standard dosing regimen (S), 55.6%, 72.2% and 0% susceptible increased exposure (I), and 38.9%, 20.4% and 100.0% resistant (R) to imipenem, meropenem and ertapenem, respectively, using the reference broth microdilution method. CA lower than 90% were observed with all systems for imipenem and meropenem using both the ISO and the modified EUCAST criteria. With ertapenem, CA >90% was observed with all methods except Vitek2. RAPD profiling revealed a remarkable genotypic diversity of the isolates, supporting that results were not biased by an oligoclonal nature of the collection. CONCLUSIONS: Commercial methods can be unreliable for testing susceptibility to carbapenems of KPC-producing E. coli. Susceptibility should be confirmed by reference broth microdilution.

A simple phenotypic method for screening of MCR-1-mediated colistin resistance.

OBJECTIVES: To evaluate a novel method, the colistin-MAC test, for phenotypic screening of acquired colistin resistance mediated by transferable mcr-1 resistance determinants, based on colistin MIC reduction in the presence of dipicolinic acid (DPA). METHODS: The colistin-MAC test consists in a broth microdilution method, in which colistin MIC is tested in the absence or presence of DPA (900 µg/mL). Overall, 74 colistin-resistant strains of Enterobacteriaceae (65 Escherichia coli and nine other species), including 61 strains carrying mcr-1-like genes and 13 strains negative for mcr genes, were evaluated with the colistin-MAC test. The presence of mcr-1-like and mcr-2-like genes was assessed by real-time PCR and end-point PCR. For 20 strains, whole-genome sequencing data were also available. RESULTS: A ≥8-fold reduction of colistin MIC in the presence of DPA was observed with 59 mcr-1-positive strains, including 53 E. coli of clinical origin, three E. coli transconjugants carrying MCR-1-encoding plasmids, one Enterobacter cloacae complex and two Citrobacter spp. Colistin MICs were unchanged, increased or at most reduced by twofold with the 13 mcr-negative colistin-resistant strains (nine E. coli and four Klebsiella pneumoniae), but also with two mcr-1-like-positive K. pneumoniae strains. CONCLUSIONS: The colistin-MAC test could be a simple phenotypic test for presumptive identification of mcr-1-positive strains among isolates of colistin-resistant E. coli, based on a ≥8-fold reduction of colistin MIC in the presence of DPA. Evaluation of the test with a larger number of strains, species and mcr-type resistance determinants would be of interest.

Proteins involved in electron transfer to Fe(III) and Mn(IV) oxides by Geobacter sulfurreducens and Geobacter uraniireducens.

Whole-genome microarray analysis of Geobacter sulfurreducens grown on insoluble Fe(III) oxide or Mn(IV) oxide versus soluble Fe(III) citrate revealed significantly different expression patterns. The most upregulated genes, omcS and omcT, encode cell-surface c-type cytochromes, OmcS being required for Fe(III) and Mn(IV) oxide reduction. Other electron transport genes upregulated on both metal oxides included genes encoding putative menaquinol : ferricytochrome c oxidoreductase complexes Cbc4 and Cbc5, periplasmic c-type cytochromes Dhc2 and PccF, outer membrane c-type cytochromes OmcC, OmcG and OmcV, multicopper oxidase OmpB, the structural components of electrically conductive pili, PilA-N and PilA-C, and enzymes that detoxify reactive oxygen/nitrogen species. Genes upregulated on Fe(III) oxide encode putative menaquinol : ferricytochrome c oxidoreductase complexes Cbc3 and Cbc6, periplasmic c-type cytochromes, including PccG and PccJ, and outer membrane c-type cytochromes, including OmcA, OmcE, OmcH, OmcL, OmcN, OmcO and OmcP. Electron transport genes upregulated on Mn(IV) oxide encode periplasmic c-type cytochromes PccR, PgcA, PpcA and PpcD, outer membrane c-type cytochromes OmaB/OmaC, OmcB and OmcZ, multicopper oxidase OmpC and menaquinone-reducing enzymes. Genetic studies indicated that MacA, OmcB, OmcF, OmcG, OmcH, OmcI, OmcJ, OmcM, OmcV and PccH, the putative Cbc5 complex subunit CbcC and the putative Cbc3 complex subunit CbcV are important for reduction of Fe(III) oxide but not essential for Mn(IV) oxide reduction. Gene expression patterns for Geobacter uraniireducens were similar. These results demonstrate that the physiology of Fe(III)-reducing bacteria differs significantly during growth on different insoluble and soluble electron acceptors and emphasize the importance of c-type cytochromes for extracellular electron transfer in G. sulfurreducens.

Characterization of metabolism in the Fe(III)-reducing organism Geobacter sulfurreducens by constraint-based modeling.

Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.

Outer membrane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens.

The potential role of outer membrane proteins in electron transfer to insoluble Fe(III) oxides by Geobacter sulfurreducens was investigated because this organism is closely related to the Fe(III) oxide-reducing organisms that are predominant in many Fe(III)-reducing environments. Two of the most abundant proteins that were easily sheared from the outer surfaces of intact cells were c-type cytochromes. One, designated OmcS, has a molecular mass of ca. 50 kDa and is predicted to be an outer membrane hexaheme c-type cytochrome. Transcripts for omcS could be detected during growth on Fe(III) oxide, but not on soluble Fe(III) citrate. The omcS mRNA consisted primarily of a monocistronic transcript, and to a lesser extent, a longer transcript that also contained the downstream gene omcT, which is predicted to encode a second hexaheme outer membrane cytochrome with 62.6% amino acid sequence identity to OmcS. The other abundant c-type cytochrome sheared from the outer surface of G. sulfurreducens, designated OmcE, has a molecular mass of ca. 30 kDa and is predicted to be an outer membrane tetraheme c-type cytochrome. When either omcS or omcE was deleted, G. sulfurreducens could no longer reduce Fe(III) oxide but could still reduce soluble electron acceptors, including Fe(III) citrate. The mutants could reduce Fe(III) in Fe(III) oxide medium only if the Fe(III) chelator, nitrilotriacetic acid, or the electron shuttle, anthraquinone 2,6-disulfonate, was added. Expressing omcS or omcE in trans restored the capacity for Fe(III) oxide reduction. OmcT was not detected among the sheared proteins, and genetic studies indicated that G. sulfurreducens could not reduce Fe(III) oxide when omcT was expressed but OmcS was absent. In contrast, Fe(III) oxide was reduced when omcS was expressed in the absence of OmcT. These results suggest that OmcS and OmcE are involved in electron transfer to Fe(III) oxides in G. sulfurreducens. They also emphasize the importance of evaluating mechanisms for Fe(III) reduction with environmentally relevant Fe(III) oxide, rather than the more commonly utilized Fe(III) citrate, because additional electron transfer components are required for Fe(III) oxide reduction that are not required for Fe(III) citrate reduction.

Geobacter sulfurreducens can grow with oxygen as a terminal electron acceptor.

Geobacter sulfurreducens, previously classified as a strict anaerobe, tolerated exposure to atmospheric oxygen for at least 24 h and grew with oxygen as the sole electron acceptor at concentrations of 10% or less in the headspace. These results help explain how Geobacter species may survive in oxic subsurface environments, being poised to rapidly take advantage of the development of anoxic conditions.

Eating disorders and aggressiveness among adolescents.

OBJECTIVE: To explore the links between propensity to aggression and eating disorders in a sample of school-aged adolescents in a northeastern area of Italy. METHOD: In a mixed male-female sample of 1000 adolescents (10% of the district's population aged 15-19 years) we administered the Eating Attitudes Test, the Bulimic Investigatory Test of Edinburgh and the Body Attitudes Test, as measures of abnormal eating attitudes and behaviours, and the Aggression Questionnaire, as a measure of the propensity to aggression. RESULTS: Females scored significantly higher than males at all eating disorders inventories (P < 0.0001). Males scored higher than females at the Aggression Questionnaire. In both genders, there was a positive correlation between scores at any of the eating disorders inventories and those at the Aggression Questionnaire (P < 0.0001). CONCLUSION: Overtly expressed aggression might have a negative impact on the course of eating disorders and on the compliance with treatment, also enhancing the risk of suicide.

OmcB, a c-type polyheme cytochrome, involved in Fe(III) reduction in Geobacter sulfurreducens.

Microorganisms in the family Geobacteraceae are the predominant Fe(III)-reducing microorganisms in a variety of subsurface environments in which Fe(III) reduction is an important process, but little is known about the mechanisms for electron transport to Fe(III) in these organisms. The Geobacter sulfurreducens genome was found to contain a 10-kb chromosomal duplication consisting of two tandem three-gene clusters. The last genes of the two clusters, designated omcB and omcC, encode putative outer membrane polyheme c-type cytochromes which are 79% identical. The role of the omcB and omcC genes in Fe(III) reduction in G. sulfurreducens was investigated. OmcB and OmcC were both expressed during growth with acetate as the electron donor and either fumarate or Fe(III) as the electron acceptor. OmcB was ca. twofold more abundant under both conditions. Disrupting omcB or omcC by gene replacement had no impact on growth with fumarate. However, the OmcB-deficient mutant was greatly impaired in its ability to reduce Fe(III) both in cell suspensions and under growth conditions. In contrast, the ability of the OmcC-deficient mutant to reduce Fe(III) was similar to that of the wild type. When omcB was reintroduced into the OmcB-deficient mutant, the capacity for Fe(III) reduction was restored in proportion to the level of OmcB production. These results indicate that OmcB, but not OmcC, has a major role in electron transport to Fe(III) and suggest that electron transport to the outer membrane is an important feature in Fe(III) reduction in this organism.

Deaths by unintentional illicit drug overdose in Italy, 1984-2000.

AIM: To determine whether there has been an increase in deaths by overdose in Italy, as elsewhere in the Western world, over the past fifteen years. METHOD: This study's conclusions are based on analysis of official data on overdose deaths attributed to illicit drug addiction and abuse (ICD-9 codes 304 and 305) from 1984 to 2000, drawn from two archives of drug abuse information: the Direzione Centrale per i Servizi Antidroga (DADE) of the Italian Ministry of the Interior (1984-2000), and the Health Statistics held at the Italian Central Statistics Institute (ISTAT) (1984-1997). Mortality rates have been calculated for both genders in the following age groups: 15-24, 25-34, and 35-44 years. RESULTS: Official data indicate that there has been a steady increase in the number of deaths by overdose in Italy over recent 15 years. This trend has affected both genders, but is more evident among males. Over the whole period females had consistently lower overdose rates than males. In both genders the age group 35-44 was subject to the highest mortality rate increase over the study period, however, the highest overdose rates for both males and females were observed in the 25-34 age group. Consistently higher rates were witnessed in the northern regions of Italy with an overall increase across all latitudes. However, the greatest increase over the study period occurred in the South. In 5190 cases evaluated by the Italian Forensic Toxicology Group, 95.9% of deaths were attributed to heroin, but in about half of these, mixtures of three or more substances (heroin, benzodiazepines, cannabinoids, cocaine, methadone) were found in the deceased at doses that were likely to have contributed to death. CONCLUSIONS: The increase observed in the rates of death by overdose is likely to be a reflection of increased use of illicit drugs in the general population. Reporting practice by forensic pathologists might explain the extent of attribution of cause of death to heroin. Drop-out from addiction treatment is a commonly observed antecedent of fatal opioid overdose, therefore, caution is required when establishing treatment protocol for patients. Interventions that aim specifically at improving patient compliance with medical and psychiatric therapies should be favoured.

Social desirability and eating disorders. A community study of an Italian school-aged sample.

OBJECTIVE: To explore the links between social desirability and eating disorders in a sample of adolescents in a north-east area of Italy. METHOD: A mixed male-female sample of 1000 school-aged adolescents, corresponding to 10% of the young population aged 15-19 years living in the district, were investigated with self-reported questionnaires, including the Eating Attitudes Test (EAT), the Bulimic Investigatory Test of Edinburgh (BITE), the Body Attitudes Questionnaire (BAT), and an Italian version of the Marlowe-Crowne Social Desirability Scale (MC-SDS). RESULTS: Females scored higher than males at all eating disorder inventories. In both genders there was a negative relationship (in all cases P < 0.01) between scores at the eating disorder inventories and those at the MC-SDS. When analysing eating disorder "caseness", as measured by cut-off, "cases" reported significantly lower scores than "non-cases" at the MC-SDS in both genders. CONCLUSION: Personality traits measured by the MC-SDS, such as defensiveness, self-esteem, and dependence from approval, might contribute to the development of abnormal eating patterns at risk of eating disorders.

Development of a genetic system for Geobacter sulfurreducens.

Members of the genus Geobacter are the dominant metal-reducing microorganisms in a variety of anaerobic subsurface environments and have been shown to be involved in the bioremediation of both organic and metal contaminants. To facilitate the study of the physiology of these organisms, a genetic system was developed for Geobacter sulfurreducens. The antibiotic sensitivity of this organism was characterized, and optimal conditions for plating it at high efficiency were established. A protocol for the introduction of foreign DNA into G. sulfurreducens by electroporation was also developed. Two classes of broad-host-range vectors, IncQ and pBBR1, were found to be capable of replication in G. sulfurreducens. In particular, the IncQ plasmid pCD342 was found to be a suitable expression vector for this organism. When the information and novel methods described above were utilized, the nifD gene of G. sulfurreducens was disrupted by the single-step gene replacement method. Insertional mutagenesis of this key gene in the nitrogen fixation pathway impaired the ability of G. sulfurreducens to grow in medium lacking a source of fixed nitrogen. Expression of the nifD gene in trans complemented this phenotype. This paper constitutes the first report of genetic manipulation of a member of the Geobacter genus.

Season and suicide: recent findings from Italy.

Seasonal asymmetry in yearly suicide occurrence is a long-observed phenomenon in psychiatric, suicidological, and sociological research, and the effects of seasonal factors on suicidal behavior have been the focus of a number of earlier studies. Taking into account limitations of data and methods, these studies have generally favored interpretations based on psychosocial factors. Recent studies have challenged the widely held notion that seasonal effects on suicide are a unitary phenomenon, not influenced by age, gender, or circumstances of the act. In particular, the seasonal occurrence of suicides has been found to differ significantly between the young and the elderly, and differences have also been found between male and female cycles of occurrence. Suicides using violent methods have been shown to follow clearer seasonal patterns than suicides by less violent methods (such as drug or gas poisoning), possibly reflecting the greater impulsive component involved in the choice of a violent lethal means. In this paper, findings from Italy are used to illustrate the clinical implications of studies into the topic of season and suicide, with the aim of developing more effective preventative strategies.

Isoform-specific effects of charged residues at borders of the M1-M2 loop of the Na,K-ATPase alpha subunit.

The Na,K-ATPase is specifically inhibited by the cardiac glycoside, ouabain. Via a largely undefined mechanism, the ouabain affinity of the Na,K-ATPase can be manipulated by mutating the residues at the borders of the first extracellular (M1-M2) loop of the alpha subunit [Price, E. M., Rice, D. A., and Lingrel, J. B. (1990) J. Biol. Chem. 265, 6638-6641]. To address this issue, we compared the effects of two combinations of charged residues at the M1-M2 loop border, R113, D124 and D113,R124 (numbered according to the rat alpha1 subunit), on the ouabain sensitivity of the alpha1 and alpha2 isoforms. We report that ouabain sensitivity is dependent not only upon the identity of the residues at the M1-M2 loop border but also upon the context into which they are introduced. Furthermore, at low concentrations of ATP, the identity of the residues at the M1-M2 loop border affects the regulation of ATP hydrolysis by potassium in an isoform-specific manner. Analysis of chimeric alpha subunits reveals that the effects of potassium are determined primarily by the interaction of the N-terminus and M1-M2 loop with the C-terminal third of the alpha subunit. M1-M2 loop border residues may, therefore, influence ouabain sensitivity indirectly by altering the stability or structure of the intermediate of the Na,K-ATPase catalytic cycle which is competent to bind ouabain.

Extramedullary involvement in patients with acute promyelocytic leukemia: a report of seven cases.

BACKGROUND: Extramedullary involvement is only occasionally observed in patients with acute promyelocytic leukemia (APL) but has been said to occur more frequently after treatment with all- trans retinoic acid (ATRA) than after treatment with cytotoxic drugs. In the literature, 37 well-documented cases have been reported. METHODS: The authors report 7 patients with extramedullary APL documented by cytologic, phenotypic, and molecular analyses among 120 adult APL patients referred to two different institutions during a period of 9 years. RESULTS: In this APL series, extramedullary disease (EMD) occurred in 7 of 120 cases (5.8%). The extramedullary sites were the skin in five patients, the central nervous system in one, and the lymph nodes in one. Molecular analysis of the PML/RARalpha rearrangement was performed on four samples of skin and one of CSF; all patients exhibited the same molecular pattern in the bone marrow (BM) and EMD sites. Of 120 patients, 61 were treated with ATRA plus chemotherapy and 59 with chemotherapy alone. Relapses were observed in 38 patients, 6 of whom had EMD; 1 patient had developed EMD at the onset of APL. Of the relapsed EMD cases, 2 of 61 patients had received ATRA plus chemotherapy and 4 of 59 had received chemotherapy alone. CONCLUSIONS: There is some controversy as to whether treatment of APL with ATRA may predispose patients to the development of extramedullary relapse. The data from this study do not contain evidence that EMD may occur more frequently in APL patients treated with ATRA.

Intracellular localization of Na,K-ATPase alpha2 subunit mutants.

The Na,K-ATPase is an essential plasma membrane transporter of mammalian cells composed of two subunits, alpha and beta, of which there are several isoforms. We investigated the effect of a substitution, S364P, on the subcellular localization and enzymatic activity of the wild-type alpha2 and alpha2L111R,N122D (alpha2RD) subunits. The substitutions, L111R and N122D, lower the affinity of the alpha2 subunit for the inhibitor ouabain roughly one thousand-fold (E. A. Jewell and J. B. Lingrel, J. Biol. Chem. 266, 16925-16930, 1991) and were introduced into the alpha2 subunit to distinguish its enzymatic activity from that of the endogenous alpha1 subunit of COS-7 cells. The S364P substitution is located in the ATP binding site, only five residues from the aspartyl residue which is phosphorylated during the catalytic cycle of the Na,K-ATPase. This substitution dramatically decreases the amount of enzymatic activity associated with expression of the alpha2RD subunit. Despite the fact that S364P substitution does not block association of the alpha2RD subunit with the endogenous beta1 subunit, it prevents the alpha2 and alpha2RD subunits from accumulating in the plasma membrane and results in their localization in the endoplasmic reticulum.

The alpha2L111R,N122D isoform of the Na,K-ATPase expressed in HeLa cells does not undergo an adipocyte-like increase in activity in response to insulin.

In the rat adipocyte, insulin increases potassium uptake by a preferential activation of the alpha2 isoform of the Na,K-ATPase. The question under consideration here is whether expression of the alpha2 isoform is sufficient to replicate its differential activation by insulin. Accordingly, we compared the effect of insulin on the activity of the ouabain resistant rat alpha1 and alpha2RD (alpha2L111R,N122D) isoforms in HeLa cells. In HeLa cells, in contrast to the rat adipocyte, insulin produces an increase of equal magnitude in the rate of 86Rb+/K+ uptake by the ouabain resistant rat alpha1 and rat alpha2RD subunits. We conclude that the mechanism of insulin activation of the alpha2RD isoform in HeLa cells differs from that of the wild type alpha2 isoform in the rat adipocyte.

Ubiquitination of Na,K-ATPase alpha1 and alpha2 subunits.

Na,K-ATPase, an essential transporter of mammalian cells, is an oligomeric transmembrane protein composed of two subunits, alpha and beta, of which there are several isoforms. In this study, we demonstrate that the alpha1 and alpha2 isoforms of the Na,K-ATPase alpha subunit are modified by the covalent attachment of ubiquitin polymers in COS-7 cells. We propose that polyubiquitination of the Na,K-ATPase alpha subunit may play a role in regulating its degradation.

[Diagnosis of mycoplasma pneumonia in children: which is the role of thoracic radiography?]. / La diagnosi di polmonite da mycoplasma nel bambino: quale il ruolo del radiogramma del torace?

Despite its frequency, pneumonia is often surprisingly difficult to diagnose in children and young adults. In particular, the etiologic agent of pneumonia is difficult to recognize in an early stage, which obviously implies an empirical or delayed treatment. Chest radiography is one of the most common procedures required when pneumonia is suspected. This retrospective study was carried out to investigate the capabilities of chest radiography to identify the specific patterns of mycoplasma pneumonia in children. The chest radiographs of 76 children and adolescents (aged 4.2 to 16.4 years) with a radiographic diagnosis of pneumonia were reviewed. All patients were tested twice for serum antimycoplasma antibodies. Thirty-eight subjects (50%) with markedly increased antimycoplasma antibody levels were diagnosed as having mycoplasma infection. In the remaining 38 patients, viral (22 patients, 29%), bacterial (13 patients, 17%) and mixed (3 patients, 4%) infections were diagnosed on the grounds of clinical and laboratory data. Parahilar peribronchial infiltrates were found to be associated with both viral and mycoplasma infections, whereas segmental or lobar consolidation was associated with bacterial, viral and mycoplasma infections. Reticulonodular infiltrates were a specific pattern of mycoplasma pneumonia. The authors conclude that, in the presence of a reticulonodular infiltrate in a lobe, mycoplasma pneumonia can be confidently diagnosed.