Antigenic differences between Anabaena azollae fresh from the Azolla fern leaf cavity and free-living cyanobacteria

Fluorescent antibody staining indicated differences in surface antigenicity in Anabaena azollae cells fresh from the leaf cavities of the fern, Azolla caroliniana, and algae which were isolated and subcultured from this fern. Such results suggest that either changes in antigenicity occur in this phycobiont during culturing or that isolation selects for an antigenically different mutant strain capable of in vitro growth.Non-Standard Abbreviations FA fluorescent antibody staining - PBS phosphate buffered saline - W microwatt - Anti-F antiserum prepared against fresh cells - Anti-N antiserum prepared against Newton's culture - FTTC fluorescein isothiocyanate To whom offprint requests should be sent     

High resolution vertical profiles of pH in recent sediments

High resolution (0.1 cm sampling interval) profiles of pH were obtained from some recent estuarine (Long Island Sound, Chesapeake Bay) and freshwater (Lake Erie) sediments and from laboratory microcosms containing homogenized Lake Erie sediment (both with and without tubificid oligochaetes) by incrementally precessing a micro-pH electrode downward through the sediment. These profiles revealed that hydrogen ion undergoes chemical reactions on a scale smaller than can be resolved using classic 1 cm sampling intervals, and that the vertical distribution of hydrogen ion is affected by bioturbation. In all sediments examined, a local pH minimum occurred immediately below the oxidized zone. In estuarine sediments, a second deeper pH minimum was observed. The presence of tubificids prevented profound pH changes from developing in microcosm sediments treated with a layer of activated sewage sludge and resulted in more modest alterations of pH profile in microcosm sediments lacking such a layer. The technique used in this study is by no means limited to pH. In principal, any chemical species that can be directly determined by electrodes (e.g. O₂, S^-2) may be studied. Microelectrode techniques could be especially useful in the study of chemical gradients around animal burrows and in time series studies of whole core diagenesis.     

Tumor promoters and epidermal growth factor stimulate anchorage-independent growth of adenovirus-tranformed rat embryo cells.

Previous studies indicated that the potent tumor promoter 12--0--tetradecanoyl-phorbol-13-acetate (TPA) enhances transformation of rat embryo cells (2 degrees RE) by a mutant of human Ad5 (H5ts125). This study examines the effect of TPA, its structural analogs and epidermal growth factor (EGF) on anchorage-independent growth of a cloned population of H5ts125-transformed 2 degrees RE cells (clone E11). Both TPA and EGF (approximately 10(-8) M) induced a 3--5 fold increase in agar cloning efficiency of E11 cells. In addition, macroscopic colonies appeared earlier and were larger and more diffuse. The TPA analogs phorbol--12,13--didecanoate (PDD) and ingenol--3,20--dibenzoate also enhanced growth in agar of E11 cells, whereas phorbol, 4 alpha PDD and 4--0--meTPA, which are inactive as tumor promoters, failed to enhance agar growth. In contrast to the results obtained with E11 cells, TPA, PDD or ingenol--3,20--bidenzoate failed to induce growth in agar of normal 2 degrees RE cells. Dexamethasone (10(-5)--10(-6) M), trans retinoic acid (10(-5)--10(-6) M) and the protease inhibitors leupeptin, antipain and elastatinol did not inhibit the ability of TPA to enhance the growth of E11 cells in agar. The TPA-enhanced anchorage independence was a stable property, since subclones of E11 cells isolated from TPA-agar plates had a higher agar cloning efficiency than the parental E11 cells when retested in the absence of TPA. This effect of TPA does not appear to reflect simple selection of a subpopulation of cells. When the parental E11 cells were first cloned in monolayer culture in the absence of TPA, all ten randomly picked clones showed enhanced growth in agar in the presence of TPA. In addition, prior growth of E11 cells in monolayer culture in the presence of TPA did not enhance their subsequent growth in agar. This system therefore provides an example in which TPA appears to enhance the acquisition of a stable cell property, and thus may be a useful model for studying mechanisms of tumor promotion and progression.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase: localization of substrate binding sites.

Butanedione in the presence of borate buffer reversibly inhibits Rhodospirillum rubrum chromatophore transhydrogenase complex and the separated membrane-bound and soluble factor components of the complex. NADP⁺ completely protected against inactivation of the membrane-bound component, whereas NAD⁺ was without effect. Soluble factor was maximally protected only partially by either NAD⁺ or NADP⁺, but a mixture of the substrates afforded complete protection. NADP⁺-dependent association of soluble factor with factor-depleted membranes was markedly decreased after incubation of membranes with butanedione in the absence, but not in the presence, of NADP⁺. Soluble factor was bound to agarose-NAD and was eluted by NAD⁺, but not by NADP⁺. These results demonstrate the presence of at least three nicotinamide adenine dinucleotide binding sites on R. rubrum transhydrogenase complex, including separate NADP and NAD binding sites on soluble factor and a NADP binding site on the membrane-bound component.     

Chemical studies on the inactivation of Escherichia coli RTEM beta-lactamase by clavulanic acid.

Incubation of clavulanic acid with the beta-lactamase from Escherichia coli RTEM leads to enzyme-catalyzed depletion of clavulanic acid, to transient inhibition, and to irreversible inactivation of the enzyme. Both the transiently inhibited and the irreversibly inactivated species show a marked increase in the absorbance at 281 nm that is proportional to the decrease in enzyme activity. Hydroxylamine treatment of irreversibly inactivated enzyme restores about one-third of the catalytic activity, with a concomitant decrease in absorbance at 281 nm. Polyacrylamide isoelectric focusing of the irreversibly inactivated enzyme shows three bands of approximately equal intensity, different from native enzyme. Upon hydroxylamine treatment, one of the three bands disappears and now focuses identically with native enzyme. It is evident that the irreversible inactivation of enzyme by an excess of clavulanic acid generates three products, one of which can be reactivated by hydroxylamine.     

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction

Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.     

The Rab11-family interacting proteins reveal selective interaction of mammalian recycling endosomes with the Toxoplasma parasitophorous vacuole in a Rab11- and Arf6-dependent manner

After mammalian cell invasion, the parasite Toxoplasma multiplies in a self-made membrane-bound compartment, the parasitophorous vacuole (PV). We previously showed that Toxoplasma interacts with many host cell organelles, especially from recycling pathways, and sequestrates Rab11A and Rab11B vesicles into the PV. Here, we examine the specificity of host Rab11 vesicle interaction with the PV by focusing on the recruitment of subpopulations of Rab11 vesicles characterized by different effectors, for example, Rab11-family interacting roteins (FIPs) or Arf6. Our quantitative microscopic analysis illustrates the presence of intra-PV vesicles with FIPs from class I (FIP1C, FIP2, FIP5) and class II (FIP3, FIP4) but to various degrees. The intra-PV delivery of vesicles with class I, but not class II, FIPs is dependent on Rab11 binding. Cell depletion of Rab11A results in a significant decrease in intra-PV FIP5, but not FIP3 vesicles. Class II FIPs also bind to Arf6, and we observe vesicles associated with FIP3-Rab11A or FIP3-Arf6 complexes concomitantly within the PV. Abolishing FIP3 binding to both Rab11 and Arf6 reduces the number of intra-PV FIP3 vesicles. These data point to a selective process of mammalian Rab11 vesicle recognition and scavenging mediated by Toxoplasma, suggesting that specific parasite PV proteins may be involved in these processes.     

Effect of clinician information sessions on diagnostic testing for Chagas disease

BackgroundChagas disease is a potentially life-threatening neglected disease of poverty that is endemic in continental Latin America. Caused by Trypanosoma cruzi (T. cruzi), it is one of six parasitic diseases in the United States targeted by the Centers for Disease Control as a public health problem in need of action. An estimated 300,000 people are infected with T. cruzi in the United States (US). Although its morbidity, mortality and economic burden are high, awareness of Chagas disease is lacking among many healthcare providers in the US. The purpose of this analysis is to determine if the number of diagnostic tests performed at a community health center serving an at-risk population for Chagas disease increased after information sessions. A secondary aim was to determine if there was a difference by provider type, i.e., nurse practitioner vs. physician, or by specialty in the number of patients screened.Methodology/Principal findingsWe conducted a retrospective data analysis of the number of Chagas serology tests performed at a community health center before and after information sessions for clinicians. A time series analysis was conducted focusing on the Adult and Family Medicine Departments at East Boston Neighborhood Health Center (EBNHC). Across all departments there were 1,957 T. cruzi tests performed before the sessions vs. 2,623 after the sessions. Interrupted time series analysis across departments indicated that testing volume was stable over time prior to the sessions (pre-period slope = +4.1 per month; p = 0.12), followed by an immediate shift after the session (+51.6; p = 0.03), while testing volume remained stable over time after the session (post-period slope = -6.0 per month; p = 0.11).Conclusion/SignificanceIn this study, Chagas testing increased after information sessions. Clinicians who began testing their patients for Chagas disease after learning of the importance of this intervention added an extra, potentially time-consuming task to their already busy workdays without external incentives or recognition.