Crystal Structure and Comparative Functional Analyses of a Mycobacterium Aldo-Keto Reductase

Aldo-keto reductases (AKRs) are a large superfamily of NADPH-dependent enzymes that catalyze the reduction of aldehydes, aldoses, dicarbonyls, steroids, and monosaccharides. While their precise physiological role is generally unknown, AKRs are nevertheless involved in the detoxification of a broad range of toxic metabolites. Mycobacteria contain a number of AKRs, the majority of which are uncharacterised. Here, we report the 1.9 and 1.6 Å resolution structures of the apoenzyme and NADPH-bound forms, respectively, of an AKR (MSMEG_2407) from Mycobacterium smegmatis, a close homologue of the M. tuberculosis enzyme Rv2971, whose function is essential to this bacterium. MSMEG_2407 adopted the triosephosphate isomerase (α/β)₈-barrel fold exhibited by other AKRs. MSMEG_2407 (AKR5H1) bound NADPH via an induced-fit mechanism, in which the NADPH was ligated in an extended fashion. Polar-mediated interactions dominated the interactions with the cofactor, which is atypical of the mode of NADPH binding within the AKR family. Moreover, the nicotinamide ring of NADPH was disordered, and this was attributed to the lack of an “AKR-conserved” bulky residue within the nicotinamide-binding cavity of MSMEG_2407. Enzymatic characterisation of MSMEG_2407 and Rv2971 identified dicarbonyls as a preferred substrate family for hydrolysis, and the frontline antituberculosis drug isoniazid (INH) was shown to inhibit the enzyme activity of both recombinant MSMEG_2407 and Rv2971. However, differences between the affinities of MSMEG_2407 and Rv2971 for dicarbonyls and INH were observed, and this was attributable to amino acid substitutions within the cofactor- and substrate-binding sites. The structures of MSMEG_2407 and the accompanying biochemical characterisation of MSMEG_2407 and Rv2971 provide insight into the structure and function of AKRs from mycobacteria.     

Steroid regulation of progesterone synthesis in a stable porcine granulosa cell line: a role for progestins

The objective of this investigation was to determine the effect of steroid hormones on the synthesis of progesterone in a stable porcine granulosa cell line, JC-410. We also examined the effect of steroid hormones on expression of the genes encoding the steroidogenic enzymes, cytochrome P450-cholesterol side chain cleavage (P450scc) and 3β-hydroxy-5-ene steroid dehydrogenase (3β-HSD). We observed that 48 h exposure of the JC-410 cells to estradiol-17β (estradiol), androstenedione, 5-dihydrotestosterone, levonorgestrel, and 5-cholesten-3β, 25-diol (25-hydroxycholesterol) resulted in stimulation of progesterone synthesis. 25-Hydroxycholesterol augmented progesterone synthesis stimulated by estradiol, 5-dihydrotestosterone, levonorgestrel and 8-bromoadenosine 3′:5′-cyclic monophosphate (8-Br-cAMP). This increase in progesterone synthesis was additive with estradiol, 5-dihydrotestosterone and levonorgestrel, and synergistic with 8-Br-cAMP. Cholera toxin, progesterone, levonorgestrel and androstenedione increased P450scc mRNA levels, whereas estradiol had no effect. Cholera toxin, progesterone and levonorgestrel increased 3β-HSD mRNA levels, but estradiol and androstenedione had no effect. The results were interpreted to mean that estrogens, androgens and progestins regulate progesterone synthesis in the JC-410 cells. The effect of androgens appears to be mediated by stimulation of P450scc gene expression while progestins stimulate both P450scc and 3β-HSD gene expression. Our results support the concept that progesterone is an autocrine regulator of its own synthesis in granulosa cells.     

Human CD4+ T cells express TLR5 and its ligand flagellin enhances the suppressive capacity and expression of FOXP3 in CD4+CD25+ T regulatory cells

Germline encoded pattern recognition receptors, such as TLRs, provide a critical link between the innate and adaptive immune systems. There is also evidence to suggest that pathogen-associated molecular patterns may have the capacity to modulate immune responses via direct effects on CD4+ T cells. Given the key role of both CD4+CD25+ T regulatory (Treg) cells and the TLR5 ligand flagellin in regulating mucosal immune responses, we investigated whether TLR5 may directly influence T cell function. We found that both human CD4+CD25+ Treg and CD4+CD25- T cells express TLR5 at levels comparable to those on monocytes and dendritic cells. Costimulation of effector T cells with anti-CD3 and flagellin resulted in enhanced proliferation and production of IL-2, at levels equivalent to those achieved by costimulation with CD28. In contrast, costimulation with flagellin did not break the hyporesponsiveness of CD4+CD25+ Treg cells, but rather, potently increased their suppressive capacity and enhanced expression of FOXP3. These observations suggest that, in addition to their APC-mediated indirect effects, TLR ligands have the capacity to directly regulate T cell responses and modulate the suppressive activity of Treg cells.     

Complete nucleotide sequence of Tn10

The complete nucleotide sequence of Tn10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis. The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals.     

Community Occupational Therapy for people with dementia and family carers (COTiD-UK) versus treatment as usual (Valuing Active Life in Dementia [VALID]) study: A single-blind,randomised controlled trial

BackgroundWe aimed to estimate the clinical effectiveness of Community Occupational Therapy for people with dementia and family carers–UK version (Community Occupational Therapy in Dementia–UK version [COTiD-UK]) relative to treatment as usual (TAU). We hypothesised that COTiD-UK would improve the ability of people with dementia to perform activities of daily living (ADL), and family carers’ sense of competence, compared with TAU.Methods and findingsThe study design was a multicentre, 2-arm, parallel-group, assessor-masked, individually randomised controlled trial (RCT) with internal pilot. It was conducted in 15 sites across England from September 2014 to January 2018. People with a diagnosis of mild to moderate dementia living in their own home were recruited in pairs with a family carer who provided domestic or personal support for at least 4 hours per week. Pairs were randomised to either receive COTiD-UK, which comprised 10 hours of occupational therapy delivered over 10 weeks in the person with dementia’s home or TAU, which comprised the usual local service provision that may or may not include standard occupational therapy. The primary outcome was the Bristol Activities of Daily Living Scale (BADLS) score at 26 weeks. Secondary outcomes for the person with dementia included the following: the BADLS scores at 52 and 78 weeks, cognition, quality of life, and mood; and for the family carer: sense of competence and mood; plus the number of social contacts and leisure activities for both partners. Participants were analysed by treatment allocated. A total of 468 pairs were recruited: people with dementia ranged from 55 to 97 years with a mean age of 78.6 and family carers ranged from 29 to 94 with a mean of 69.1 years. Of the people with dementia, 74.8% were married and 19.2% lived alone. Of the family carers, 72.6% were spouses, and 22.2% were adult children. On randomisation, 249 pairs were assigned to COTiD-UK (62% people with dementia and 23% carers were male) and 219 to TAU (52% people with dementia and 32% carers were male). At the 26 weeks follow-up, data were available for 364 pairs (77.8%). The BADLS score at 26 weeks did not differ significantly between groups (adjusted mean difference estimate 0.35, 95% CI −0.81 to 1.51; p = 0.55). Secondary outcomes did not differ between the groups. In total, 91% of the activity-based goals set by the pairs taking part in the COTiD-UK intervention were fully or partially achieved by the final COTiD-UK session. Study limitations include the following: Intervention fidelity was moderate but varied across and within sites, and the reliance on primarily proxy data focused on measuring the level of functional or cognitive impairment which may not truly reflect the actual performance and views of the person living with dementia.ConclusionsProviding community occupational therapy as delivered in this study did not improve ADL performance, cognition, quality of life, or mood in people with dementia nor sense of competence or mood in family carers. Future research should consider measuring person-centred outcomes that are more meaningful and closely aligned to participants’ priorities, such as goal achievement or the quantity and quality of activity engagement and participation.Trial RegistrationCurrent Controlled Trials ISRCTN10748953.

Jennifer Wenborn and colleagues investigate the effectiveness of community occupational therapy program in improving activities of daily living, cognition, and mood for people with dementia.     

Protein-DNA contacts and conformational changes in the Tn10 transpososome during assembly and activation for cleavage.

IHF or supercoiling is required early in Tn10 transposition, but at later stages they inhibit the reaction in a classic homeostatic loop. We investigated the mechanism of transpososome assembly and regulation using hydroxyl radical DNA protection and interference. We present a three-dimensional molecular model for the IHF-bent end of Tn10 wrapped around a transposase core. Contacts span some 80 bp at the transposon end, but after assembly of an active complex containing metal ion, most contacts become dispensable. These include transposase contacts beyond the IHF site that chaperone assembly of the complex and are needed for efficient cleavage. Single and double-end breaks do not affect the complex but divalent metal ions promote large conformational changes at bp +1 and the flanking DNA.