Production and characterization of recombinant P1 adhesin essential for adhesion,gliding, and antigenic variation in the human pathogenic bacterium,Mycoplasma pneumoniae

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6?×?His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.     

Evaluation of the Duty Ratio of the Bacterial Flagellar Motor by Dynamic Load Control

The bacterial flagellar motor is one of the most complex and sophisticated nanomachineries in nature. A duty ratio D is a fraction of time that the stator and the rotor interact and is a fundamental property to characterize the motor but remains to be determined. It is known that the stator units of the motor bind to and dissociate from the motor dynamically to control the motor torque depending on the load on the motor. At low load, at which the kinetics such as proton translocation speed limits the rotation rate, the dependency of the rotation rate on the number of stator units N implies D: the dependency becomes larger for smaller D. Contradicting observations supporting both the small and large D have been reported. A dilemma is that it is difficult to explore a broad range of N at low load because the stator units easily dissociate, and N is limited to one or two at vanishing load. Here, we develop an electrorotation method to dynamically control the load on the flagellar motor of Salmonella with a calibrated magnitude of the torque. By instantly reducing the load for keeping N high, we observed that the speed at low load depends on N, implying a small duty ratio. We recovered the torque-speed curves of individual motors and evaluated the duty ratio to be 0.14 ± 0.04 from the correlation between the torque at high load and the rotation rate at low load.     

Identification of asymptomatic Entamoeba histolytica infection by a serological screening test: A cross-sectional study of an HIV-negative men who have sex with men cohort in Japan

BackgroundAmebiasis, caused by Entamoeba histolytica, is spreading in developing countries and in many developed countries as a sexually transmitted infection. Here, we evaluated the efficacy of serological screening to identify asymptomatic E. histolytica infection as a potential epidemiological control measure to limit its spread.Methodology/Principal findingsThis cross-sectional study was carried out between January and March 2021 in an HIV-negative men who have sex with men (MSM) cohort at the National Center for Global Health and Medicine. Serological screening was performed using a commercially available ELISA kit. For seropositive individuals, we performed stool polymerase chain reaction (PCR) to determine current E. histolytica infection. We performed E. histolytica serological screening of 312 participants. None had a history of E. histolytica infection prior to the study. The overall E. histolytica seropositivity was 6.7% (21/312), which was similar to that found by the rapid plasma reagin test (17/312). We identified current infection in 8 of 20 seropositive participants (40.0%) by stool PCR.Conclusions/SignificanceOur serological screening approach constitutes a potentially practical epidemiological strategy. Active epidemiological surveys, in combination with an effective screening strategy for asymptomatically infected individuals, should be applied to help reduce sexually transmitted E. histolytica infections.     

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.     

Disturbed activation of endoplasmic reticulum stress transducers by familial Alzheimer's disease-linked presenilin-1 mutations

Recent studies have shown independently that presenilin-1 (PS1) null mutants and familial Alzheimer's disease (FAD)-linked mutants should both down-regulate signaling of the unfolded protein response (UPR). However, it is difficult to accept that both mutants possess the same effects on the UPR. Furthermore, contrary to these observations, neither loss of PS1 and PS2 function nor expression of FAD-linked PS1 mutants were reported to have a discernable impact on the UPR. Therefore, re-examination and detailed analyses are needed to clarify the relationship between PS1 function and UPR signaling. Here, we report that PS1/PS2 null and dominant negative PS1 mutants, which are mutated at aspartate residue 257 or 385, did not affect signaling of the UPR. In contrast, FAD-linked PS1 mutants were confirmed to disturb UPR signaling by inhibiting activation of both Ire1alpha and ATF6, both of which are endoplasmic reticulum (ER) stress transducers in the UPR. Furthermore, PS1 mutants also disturbed activation of PERK (PKR-like ER kinase), which plays a crucial role in inhibiting translation during ER stress. Taken together, these observations suggested that PS1 mutations could affect signaling pathways controlled by each of the respective ER-stress transducers, possibly through a gain-of-function.     

Scratch cytologic findings on surgically resected solitary fibrous tumors of the pleura

OBJECTIVE: To ascertain the cytologic characteristics of solitary fibrous tumors of the pleura (SFTPs) on smear preparations. STUDY DESIGN: Fine needle aspiration cytology (FNAC) was initially attempted preoperatively in five cases, but the specimens were inappropriate for interpretation because only a few tumor cells were obtained. Therefore, scratch smears made at the time of operation were used. Papanicolaou and immunocytochemical staining was performed in all 10 cases, 2 of which were malignant. RESULTS: As expected, cellular tumors yielded more cells. The cytologic appearance was variable, showing spindle/bipolar, dendritic/stellate and intermediate cells. Atypical cells reminiscent of sarcoma were also present in cellular, benign tumors. Highly atypical epithelioid cells were obtained in two malignant cases. Immunocytochemically, the tumor cells were positive for CD34 and vimentin and negative for cytokeratin, regardless of histologic differences and cell shape. CONCLUSION: It seems difficult to diagnose SFTPs with certainty by FNAC, partly because the cell morphology of SFTPs resembles a wide variety of heterogeneous groups of spindle cell tumors and partly because only a few tumor cells were available in the FNAC specimens in the present study. However, a cytologic diagnosis of SFTP is possible if cytologic preparations yield CD34-positive cells with spindle/bipolar or dendritic/stellate morphology.     

Different functional aspects of the group II subfamily (Types IIA and V) and type X secretory phospholipase A(2)s in regulating arachidonic acid release and prostaglandin generation. Implications of cyclooxygenase-2 induction and phospholipid scramblase-mediated cellular membrane perturbation.

We have recently reported that members of the heparin-binding group II subfamily of secretory PLA(2)s (sPLA(2)s) (types IIA and V), when transfected into 293 cells, released [(3)H]arachidonic acid (AA) preferentially in response to interleukin-1 (IL-1) and acted as "signaling" PLA(2)s that were functionally coupled with prostaglandin biosynthesis. Here we show that these group II subfamily sPLA(2)s and the type X sPLA(2) behave in a different manner, the former being more efficiently coupled with the prostaglandin-biosynthetic pathway than the latter, in 293 transfectants. Type X sPLA(2), which bound only minimally to cell surface proteoglycans, augmented the release of both [(3)H]AA and [(3)H]oleic acid in the presence of serum but not IL-1. Both types IIA and V sPLA(2), the AA released by which was efficiently converted to prostaglandin E(2), markedly augmented IL-1-induced expression of cyclooxygenase (COX)-2 in a heparin-sensitive fashion, whereas type X sPLA(2) lacked the ability to augment COX-2 expression, thereby exhibiting the poor prostaglandin E(2)-biosynthetic response unless either of the COX isozymes was forcibly introduced into type X sPLA(2)-expressing cells. Implication of phospholipid scramblase, an enzyme responsible for the perturbation of plasma membrane asymmetry, revealed that the scramblase-transfected cells became more sensitive to types IIA and V, but not X, sPLA(2), releasing both [(3)H]AA and [(3)H]oleic acid in an IL-1-independent manner. Thus, although phospholipid scramblase-mediated alteration in plasma membrane asymmetry actually led to the increased cellular susceptibility to the group II subfamily of sPLA(2)s, several lines of evidence suggest that it does not entirely mimic their actions on cells after IL-1 signaling. Interestingly, coexpression of type IIA or V, but not X, sPLA(2) and phospholipid scramblase resulted in a marked reduction in cell growth, revealing an unexplored antiproliferative aspect of particular classes of sPLA(2).