CryoEM and Molecular Dynamics of the Circadian KaiB–KaiC Complex Indicates That KaiB Monomers Interact with KaiC and Block ATP Binding Clefts

The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. The KaiB–KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein–protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.     

DEOXYRIBONUCLEASES IN DEVELOPING AND ADULT RAT CEREBELLUM: TRANSIENT AND LONG DURATION ACTIONS OF METHYLAZOXYMETHANOL

—Acid and alkaline DNase activities were determined in postnatal and adult rat cerebellum. Normal animals were compared with those injected with 10 mg/kg methylazoxymethanol acetate using varying schedules after birth in order to induce transient damage to proliferating cell populations. In all animals studied, both immature and adult, methylazoxymethanol caused an increase in acidic and decrease in alkaline DNase activity. These effects were not specific to the dividing populations and occurred in nonmitotic cells as well. When injections were given to young adults, these effects appeared irreversible and persisted up to 3 months after birth.     

Yersinia enterocolitica produces superantigenic activity.

We have recently observed that antigenic preparations from Yersinia enterocolitica are capable of inducing strong proliferative responses in normal murine spleen cell cultures. As a consequence of this observation, we evaluated whether Yersinia-derived Ag possess superantigenic activity. Stimulatory activity can be found in culture supernatants, as well as membrane and cytoplasmic fractions of Y. enterocolitica. Cell depletion studies indicate that the primary responding cell is a CD4+ T cell, which requires the presence of APC for responsiveness to Y. enterocolitica Ag. Furthermore, these APC must express MHC class II Ag, as evidenced by the fact that either antibody depletion of class II+ APC or addition of anti-class II antibodies (that block class II Ag on the surface of APC) eliminates the proliferative response. Evaluation of TCR usage by BALB/c T cells responsive to Y. enterocolitica revealed that those T cells bearing V beta 3, 6, and 11 and possibly 7 and 9 were expanded after exposure to Y. enterocolitica Ag preparations. By using a panel of T cell hybridomas, we have shown that hybridomas bearing V beta 3, 7, 8.1, 9, and 11 but not 2, 8.2, 8.3, and 13 respond to Yersinia. When cytoplasmic fractions of Y. enterocolitica were subjected to column chromatography, proliferative activity was enriched approximately 27-fold, and the elution characteristics of the active material suggest that it possesses hydrophobic regions and is, therefore, probably membrane associated. These data indicate that Y. enterocolitica produces antigenic material that has properties consistent with those of T cell superantigens.     

Elimimation of external and internal contaminants in rhizomes of Zantedeschia aethiopica with commercial fungicides and antibiotics

Plant Cell, Tissue and Organ Culture (PCTOC) -     

Structural Studies on D2 Dopamine Receptors: Mutation of a Histidine Residue Specifically Affects the Binding of a Subgroup of Substituted Benzamide Drugs

Abstract: A histidine residue (His³⁹⁴) that is likely to be located in the ligand-binding region of the D₂ dopamine receptor has been mutated to a leucine (Leu³⁹⁴), and the properties of the mutant receptor have been determined. For a range of antagonists the mutation has only a minor effect on the affinity of the receptor for the antagonist. The mutation does, however, elicit a structurally specific effect on the affinity with which certain members of the substituted benzamide class of antagonist bind to the receptor. Some of these drugs, e.g., sulpiride, sultopride, and tiapride, bind with reduced affinity to the mutated receptor, whereas others, e.g., clebopride and metoclopramide, bind with increased affinity. However, the Na⁺/H⁺ sensitivity of the binding of sulpiride to the receptor is not reduced by the mutation. These findings have been interpreted in terms of the productive or unfavourable interaction of the His³⁹⁴ residue with these compounds.     

Nucleotide sequence of a repetitive element isolated from Leptospira interrogans serovar hardjo type hardjo-bovis.

A repetitive element from the genome of Leptospira interrogans serovar hardjo type hardjo-bovis ('L. hardjo-bovis') was identified, cloned and sequenced. Similar sequences were shown by hybridization to be encoded by a further eight of 32 other leptospiral serovars tested. An undefined number of repetitive elements were located in the L. hardjo-bovis genome; sequence degeneracy of the elements was observed and no significant open reading frames were identified within the AT-rich (60%) 1467 bp repetitive element. The termini encoded a GC-rich 8 bp repeat motif and two variants showed rearrangements centred on these motifs. The nucleotide sequences of the chromosomal regions flanking the repetitive elements were determined but showed no similarities, with one exception which had a GAAC repeat directly adjacent to both termini. Similar hybridization patterns were shown by Southern transfers of L. hardjo-bovis total genomic digests probed with the repetitive element. Oligonucleotide primer pairs designed from sequences internal to the repetitive element and adjacent chromosomal regions were used in polymerase chain reaction experiments. With one primer pair all L. hardjo-bovis isolates, but no other serovar, gave identical amplified products. Evidence that the repetitive element may have derived from an acquired insertion sequence that is now inactive and chromosomally fixed is discussed.     

Patient-physician sexual involvement: a Canadian survey of obstetrician-gynecologists.

OBJECTIVE: To determine obstetrician-gynecologists'' (ob-gyns'') awareness of and experience with sexual abuse of patients and former patients and their opinions about appropriate consequences. DESIGN: Mailed survey. SETTING: Canada. PARTICIPANTS: All 792 members of the Society of Obstetricians and Gynaecologists of Canada (SOGC); 618 (78%) responded. Approximately half of all ob-gyns in Canada belong to the SOGC. MAIN OUTCOME MEASURES: Knowledge of sexual involvement by an ob-gyn colleague with a patient or former patient (as defined by the respondents and by the College of Physicians and Surgeons of Ontario [CPSO]), self-report of such involvement, attitudes toward physician sexual abuse, desirable length of time a physician should wait before seeing a former patient in a situation that could lead to a sexual encounter, suggested consequences of sexual abuse. RESULTS: Overall, 10% of the respondents indicated that they knew about another ob-gyn who at some time had been sexually involved with a patient. In all, 3% of the male respondents and 1% of the female respondents reported sexual involvement with a patient; the corresponding proportions of those who reported having been accused of sexual abuse by a patient were 4% and 2%. Significantly more of the female ob-gyns than of their male counterparts (37% v. 19%) reported awareness of a colleague''s sexual involvement with a patient that would meet the CPSO''s definition of sexual impropriety, transgression or violation. Most of the respondents felt that the consequence of proven sexual impropriety should be reprimand and fine (chosen by 33%) or rehabilitation without loss of licence (28%). Most of the physicians supported loss of licence for proven sexual transgression (57%) or proven sexual violation (74%), but fewer felt that loss of licence should be permanent for these types of abuse (4% and 24% respectively). The female ob-gyns supported stronger sanctions against sexual transgression and sexual violation than the male ob-gyns. A wide range of opinion was seen regarding the propriety of sexual relationships with former patients. CONCLUSIONS: Ob-gyns have varied opinions about how sexual abuse of patients should be defined and how it should be sanctioned. There is a discrepancy between proposed public policy and the beliefs of physicians to whom the policy is to be applied.