Characterization of active site structure in CYP121. A cytochrome P450 essential for viability of Mycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.     

Examining the Impact of a Public Health Message on Fish Consumption in Bermuda

Background

In 2003 mean cord blood mercury concentrations in pregnant Bermudian women exceeded levels associated with adverse health outcomes in children. The principal mercury source was local fish species. Public health messages were developed suggesting pregnant women reduce consumption of fish species with higher mercury concentrations (e.g. swordfish), substituting species containing lower mercury concentrations, and elevated omega-3 fatty acids (e.g. anchovies). Recent evidence indicates mercury concentrations in Bermuda’s pregnant women have fallen five- fold.

Objectives

Assess whether changes in women’s fish eating patterns during pregnancy are consistent with the public health messaging. Determine who is making changes to their diet during pregnancy and why.

Methods

Mixed methods study with a cross-sectional survey of 121 pregnant women, including 13 opened-ended interviews. Health system, social vulnerability, public health messaging, and socio-demographic variables were characterized and related to changes in fish consumption during pregnancy. Qualitative data were coded according to nutritional advice messages, comprehension of communication strategies, and sources of information.

Results

95% of women surveyed encountered recommendations about fish consumption during pregnancy. 75% reported modifying fish eating behaviors because of recommendations. Principal sources of information about fish consumption in pregnancy were health care providers and the Internet. 71% of women reported reducing consumption of large fish species with greater mercury levels, but 60% reported reduced consumption of smaller, low mercury fish. No participant mentioned hearing about the benefits of fish consumption. More frequent exposure to public health messages during pregnancy was associated with lower reported consumption. Bermudian born women were less likely to reduce consumption of large fish species during pregnancy.

Conclusions

In Bermuda, public health messages advocating reduced consumption of larger, higher mercury-containing fish species appear effective, but masked the nutritional value message of small fish species, with low mercury concentration. Adjustment is needed to better balance the risk communication.     

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.     

Inactivation of the Glucose-Dependent Insulinotropic Polypeptide Receptor Improves Outcomes following Experimental Myocardial Infarction

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Phylogeographic structure,demographic history and morph composition in a colour polymorphic lizard

In polymorphic species, population divergence in morph composition and frequency has the potential to promote speciation. We assessed the relationship between geographic variation in male throat colour polymorphism and phylogeographic structure in the tawny dragon lizard, Ctenophorus decresii. We identified four genetically distinct lineages, corresponding to two polymorphic lineages in the Northern Flinders Ranges and Southern Flinders Ranges/Olary Ranges regions respectively, and a monomorphic lineage in the Mt Lofty Ranges/Kangaroo Island region. The degree of divergence between these three lineages was consistent with isolation to multiple refugia during Pleistocene glacial cycles, whereas a fourth, deeply divergent (at the interspecific level) and monomorphic lineage was restricted to western New South Wales. The same four morphs occurred in both polymorphic lineages, although populations exhibited considerable variation in the frequency of morphs. By contrast, male throat coloration in the monomorphic lineages differed from each other and from the polymorphic lineages. Our results suggest that colour polymorphism has evolved once in the C. decresii species complex, with subsequent loss of polymorphism in the Mt Lofty Ranges/Kangaroo Island lineage. However, an equally parsimonious scenario, that polymorphism arose independently twice within C. decresii, could not be ruled out. We also detected evidence of a narrow contact zone with limited genotypic admixture between the polymorphic Olary Ranges and monomorphic Mt Lofty Ranges regions, yet no individuals of intermediate colour phenotype. Such genetic divergence and evidence for barriers to gene flow between lineages suggest incipient speciation between populations that differ in morph composition.     

Alpha-synuclein and tau: teammates in neurodegeneration?

The accumulation of α-synuclein aggregates is the hallmark of Parkinson’s disease, and more generally of synucleinopathies. The accumulation of tau aggregates however is classically found in the brains of patients with dementia, and this type of neuropathological feature specifically defines the tauopathies. Nevertheless, in numerous cases α-synuclein positive inclusions are also described in tauopathies and vice versa, suggesting a co-existence or crosstalk of these proteinopathies. Interestingly, α-synuclein and tau share striking common characteristics suggesting that they may work in concord. Tau and α-synuclein are both partially unfolded proteins that can form toxic oligomers and abnormal intracellular aggregates under pathological conditions. Furthermore, mutations in either are responsible for severe dominant familial neurodegeneration. Moreover, tau and α-synuclein appear to promote the fibrillization and solubility of each other in vitro and in vivo. This suggests that interactions between tau and α-synuclein form a deleterious feed-forward loop essential for the development and spreading of neurodegeneration. Here, we review the recent literature with respect to elucidating the possible links between α-synuclein and tau.     

Public opinion toward the first,second, and third generations of plant biotechnology

Summary This paper considers public attitudes toward genetically modified plants in the fields or those soon to be planted. Analyzing a regional public opinion survey of 680 respondents in Arkansas, Texas, Louisiana, New Mexico, and Oklahoma carried out in the Spring—Summer of 2004, we look at the importance of public attitudes toward the three generations of agricultural biotechnology in light of the changing regulatory environment. Specifically, we ask questions concerning the first generation of plants with agronomic qualities, comparing our findings with previous studies, then look at perceptions of the second generation of crops with product quality characteristics, and the third generation, which expresses industrial products and pharmaceutical drugs. We look at perceived benefits, the likelihood, that these plants might accidentally enter the food supply, the likelihood that these plants might be eaten by the respondent, as well as how worried and angry the respondent would be as a result. Findings suggest that the public is still largely unaware of food biotechnology and genetically modified food products in their life. When compared with the first and second generation agricultural biotechnology products, survey respondents indicated that third generation products are not only likely to provide greater benefits, but are also potentially the source of more worry and anger if accidentally eaten.     

Gastroenteritis of Infants in Two Canadian Communities

Enteroviruses were isolated from 11 of 1391 rectal swabs collected from 1103 infants aged less than 2 years who were hospitalized in Toronto, Ontario, and New Westminster, British Columbia, between April 1966 and March 1969. Viruses were recovered from 23 of 1231 rectal swabs obtained from 1076 age-matched control patients without gastroenteritis. The dominance of coxsackievirus and echovirus strains in Toronto patients with and without gastroenteritis contrasted sharply to their extremely low incidence in New Westminster where the Sabin attenuated strains of poliovirus composed the majority of isolates from both categories of patient. Although enteropathogenic bacteria were identified in 36 cases of gastroenteritis and 11 control subjects, no patient excreted a bacterial pathogen and a virus simultaneously.