Structure of apo- and monometalated forms of NDM-1--a highly potent carbapenem-hydrolyzing metallo-β-lactamase

The New Delhi Metallo-β-lactamase (NDM-1) gene makes multiple pathogenic microorganisms resistant to all known β-lactam antibiotics. The rapid emergence of NDM-1 has been linked to mobile plasmids that move between different strains resulting in world-wide dissemination. Biochemical studies revealed that NDM-1 is capable of efficiently hydrolyzing a wide range of β-lactams, including many carbapenems considered as "last resort" antibiotics. The crystal structures of metal-free apo- and monozinc forms of NDM-1 presented here revealed an enlarged and flexible active site of class B1 metallo-β-lactamase. This site is capable of accommodating many β-lactam substrates by having many of the catalytic residues on flexible loops, which explains the observed extended spectrum activity of this zinc dependent β-lactamase. Indeed, five loops contribute "keg" residues in the active site including side chains involved in metal binding. Loop 1 in particular, shows conformational flexibility, apparently related to the acceptance and positioning of substrates for cleavage by a zinc-activated water molecule.     

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19(Arf) and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19(Arf)-p53 signaling, and induces senescence in primary cells.     

Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity

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Host recognition by the specialist endoparasitoidMicroplitis croceipes (Hymenoptera: Braconidae): Role of host- and plant-related volatiles

The specialist parasitoidMicroplitis croceipes Cresson can parasitize only noctuid larvae in the generaHelicoverpa andHeliothis. To be successful in their search for hosts, the ability to distinguish hosts from nonhosts feeding on the same plant is beneficial. In flight tunnel experiments, we found that prior to landing on the odor sourceM. croceipes were able to distinguish volatiles released from frass of host larvae(Helicoverpa zea Boddie) and nonhost larvae (Spodoptera exigua Hübner andSpodoptera frugiperda J. E. Smith) fed on cotton. However, an initial contact experience with frass of cotton-fed host larvae appeared to be critical for this ability. Wasps that had antennated frass of host larvae fed pinto bean diet were equally attracted to frass of host and nonhost larvae fed on pinto bean diet. In short-range walking experiments, wasps located cotton-fed host larvae faster than diet-fed larvae, regardless of their experience. Wasps that had antennated frass of cotton-fed host larvae were less attracted to cotton-fed nonhost larvae, compared to host larvae, and preferred to sting host larvae. Plant-related volatiles in host frass and larvae appear to play a major role in the successful location of host larvae.     

Effects of i.c.v. administration of leptin on copulatory and ingestive behavior in STZ-induced diabetic male rats.

It is well known that circulating leptin concentrations correlate with adiposity in both humans and rodents and decrease after fasting, energy restriction, or weight loss. The goal of the present study was to confirm whether the decreases of copulatory behavior and the increases of ingestive behavior in STZ-induced diabetic male rats could be reversed by i.c.v. administration of leptin. Adult male Wistar-Imamichi rats aged 9 weeks were used for the studies. Males received a single injection of STZ (60 mg/kg, i.p.) and vehicle. During the experiment, individual body weight, and food and water intake were measured. The copulatory and ingestive behaviors in STZ-induced diabetic males were observed at 2 and 4 weeks after STZ. At 6 weeks after STZ, leptin (10 microg/10 microl) or aCSF (artificial cerebrospinal fluid) was injected through a lateral ventricle cannula and the above two behaviors were observed again. The i.c.v. leptin injection to STZ-induced diabetic males resulted in a significant increase of ejaculation frequencies (3.6 +/- 0.26 vs. 2.9 +/- 0.30 times) and a significant decrease in amount of food ingested (36.2 +/- 1.93 vs. 23.2 +/- 3.76 g), compared with the aCSF-injected control (p<0.01). These findings suggest that the copulatory and ingestive behaviors in i.c.v. leptin-injected STZ diabetic males were restored to levels equivalent to those in control males.     

Selective Inhibition of the Mitochondrial Permeability Transition Pore Protects against Neurodegeneration in Experimental Multiple Sclerosis

The mitochondrial permeability transition pore is a recognized drug target for neurodegenerative conditions such as multiple sclerosis and for ischemia-reperfusion injury in the brain and heart. The peptidylprolyl isomerase, cyclophilin D (CypD, PPIF), is a positive regulator of the pore, and genetic down-regulation or knock-out improves outcomes in disease models. Current inhibitors of peptidylprolyl isomerases show no selectivity between the tightly conserved cyclophilin paralogs and exhibit significant off-target effects, immunosuppression, and toxicity. We therefore designed and synthesized a new mitochondrially targeted CypD inhibitor, JW47, using a quinolinium cation tethered to cyclosporine. X-ray analysis was used to validate the design concept, and biological evaluation revealed selective cellular inhibition of CypD and the permeability transition pore with reduced cellular toxicity compared with cyclosporine. In an experimental autoimmune encephalomyelitis disease model of neurodegeneration in multiple sclerosis, JW47 demonstrated significant protection of axons and improved motor assessments with minimal immunosuppression. These findings suggest that selective CypD inhibition may represent a viable therapeutic strategy for MS and identify quinolinium as a mitochondrial targeting group for in vivo use.     

A 2.08 Å resolution structure of HLB5, a novel cellulase from the anaerobic gut bacterium Parabacteroides johnsonii DSM 18315

Cellulases play a significant role in the degradation of complex carbohydrates. In the human gut, anaerobic bacteria are essential to the well‐being of the host by producing these essential enzymes that convert plant polymers into simple sugars that can then be further metabolized by the host. Here, we report the 2.08 Å resolution structure of HLB5, a chemically verified cellulase that was identified previously from an anaerobic gut bacterium and that has no structural cellulase homologues in PDB nor possesses any conserved region typical for glycosidases. We anticipate that the information presented here will facilitate the identification of additional cellulases for which no homologues have been identified to date and enhance our understanding how these novel cellulases bind and hydrolyze their substrates.     

Serum proteins and paraproteins in women with silicone implants and connective tissue disease: a case-control study

Prior studies have suggested abnormalities of serum proteins, including paraproteins, in women with silicone implants but did not control for the presence of connective-tissue disease (CTD). This retrospective case-control study, performed in tertiary-care academic centers, assessed possible alterations of serum proteins, including paraproteins, in such a population. Seventy-four women with silicone implants who subsequently developed CTD, and 74 age-matched and CTD-matched women without silicone implants, were assessed in the primary study; other groups were used for additional comparisons. Routine serum protein determinations and high-sensitivity protein electrophoresis and immunofixation electrophoresis were performed for detection of paraproteins. Women with silicone implants, either with or without CTD, had significantly lower serum total protein and alpha1-globulin, alpha2-globulin, beta-globulin, gamma-globulin, and IgG levels compared with those without silicone implants. There was no significant difference, however, in the frequency of paraproteinemia between women with silicone implants and CTD (9.5%) and age-matched and CTD-matched women without silicone implants (5.4%) (odds ratio, 1.82; 95% confidence interval, 0.51-6.45). Paraprotein isotypes were similar in the two groups, and the clinical characteristics of the 13 women with paraproteinemia were comparable with an independent population of 10 women with silicone breast implants, CTD, and previously diagnosed monoclonal gammopathies. In summary, this first comprehensive study of serum proteins in women with silicone implants and CTD found no substantially increased risk of monoclonal gammopathy. Women with silicone implants, however, had unexpectedly low serum globulin and immunoglobulin levels, with or without the subsequent development of CTD. The causes and clinical implications of these findings require further investigation.     

Class B scavenger receptor types I and II and CD36 mediate bacterial recognition and proinflammatory signaling induced by Escherichia coli, lipopolysaccharide, and cytosolic chaperonin 60

Class B scavenger receptors (SR-B) are lipoprotein receptors that also mediate pathogen recognition, phagocytosis, and clearance as well as pathogen-induced signaling. In this study we report that three members of the SR-B family, namely, CLA-1, CLA-2, and CD36, mediate recognition of bacteria not only through interaction with cell wall LPS but also with cytosolic chaperonin 60. HeLa cells stably transfected with any of these SR-Bs demonstrated markedly (3- to 5-fold) increased binding and endocytosis of Escherichia coli, LPS, and chaperonin 60 (GroEL) as revealed by both FACS analysis and confocal microscopy imaging. Increased pathogen (E. coli, LPS, and GroEL) binding to SR-Bs was also associated with the dose-dependent stimulation of cytokine secretion in the order of CD36 > CLA-2 > CLA-1 in HEK293 cells. Pathogen-induced IL-6-secretion was reduced in macrophages from CD36- and SR-BI/II-null mice by 40-50 and 30-40%, respectively. Intravenous GroEL administration increased plasma IL-6 and CXCL1 levels in mice. The cytokine responses were 40-60% lower in CD36(-/-) relative to wild-type mice, whereas increased cytokine responses were found in SR-BI/II(-/-) mice. While investigating the discrepancy of in vitro versus in vivo data in SR-BI/II deficiency, SR-BI/II(-/-) mice were found to respond to GroEL administration without increases in either plasma corticosterone or aldosterone as normally seen in wild-type mice. SR-BI/II(-/-) mice with mineralocorticoid replacement demonstrated an ～40-50% reduction in CXCL1 and IL-6 responses. These results demonstrate that, by recognizing and mediating inflammatory signaling of both bacterial cell wall LPS and cytosolic GroEL, all three SR-B family members play important roles in innate immunity and host defense.