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A cesarean section (C-section) is common practice in complicated deliveries that otherwise carry risk of severe complications. However, C-sections themselves carry risks, as do all major surgical procedures. In this paper, I examine the causal effects of C-sections on child and maternal outcomes in a population of high-risk deliveries, namely breech births. To capture the causal effect of C-sections, I exploit an information shock to the medical community on the effectiveness of the procedure. Using Swedish administrative data, I find that C-sections improve the health of children at birth and during childhood. I find no significant impacts on maternal morbidity or labor market outcomes.

Objectives Frontline workers have been a bulwark in the fight against COVID-19, while being subject to major unexpected stressors. These include conflicting news, evolving guidelines, perceived inadequate personal protective equipment, overflow of patients with rising death counts, absence of disaster training, and limitations in the implementation of social distancing. This study investigates the incidence and associated factors of depression, suicidal thoughts, and burnout among physicians during the COVID-19 pandemic. Methods In a cross-sectional survey-based study of resident, fellow, and attending physicians from a tertiary university hospital during the height of the COVID-19 pandemic in New York from April 24 to May 15, 2020, demographics and practice specialty, attending vs. resident/fellow status, call frequency, emotional exhaustion, depersonalization, and depression severity were examined. Results Two hundred twenty-five subjects completed the survey (response rate of 16.3%), with rates of 6.2% depression, 6.6% suicidal ideation, and 19.6% burnout. Depression, suicidal ideation, and burnout were all associated with history of prior depression/anxiety and frequency of on call. Suicidal ideation and burnout were also associated with younger age. There was no difference in rates of depression, suicidal ideation, or burnout between attending and resident physicians. Female physicians reported less work-life balance and more burnout. Conclusions These findings highlight the importance of considering physician mental health during times of peak stress, such as natural or man-made disasters. The prominence of premorbid depression/anxiety as a relevant factor underscores the need to further understand physician mental health and provide early screening and treatment.

Significance We analyzed data from saliva-based COVID-19 screening deployed on the University of Colorado Boulder campus. Our dataset is unique in that all SARS-CoV-2−positive individuals reported no symptoms at the time of saliva collection, and therefore were infected but asymptomatic or presymptomatic. We found that 1) the distribution of viral loads observed in our asymptomatic college population was indistinguishable from what has been reported in hospitalized populations; 2) regardless of symptomatic status, approximately 50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the infection; and 3) just 2% of infected individuals carry 90% of the virions circulating within communities, serving as viral “supercarriers” and likely also superspreaders., We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral “supercarriers” and possibly also superspreaders.

Supplemental Material, sj-pdf-1-gos-10.1177_21514593211035230 for Orthopaedic Surgeon Mental Health During the COVID-19 Pandemic by Samer M. Al-Humadi, Ricardo Cáceda, Brian Bronson, Megan Paulus, Houlin Hong and Samantha Muhlrad in Geriatric Orthopaedic Surgery & Rehabilitation

Supplemental Material, sj-pdf-1-gos-10.1177_21514593211035230 for Orthopaedic Surgeon Mental Health During the COVID-19 Pandemic by Samer M. Al-Humadi, Ricardo Cáceda, Brian Bronson, Megan Paulus, Houlin Hong and Samantha Muhlrad in Geriatric Orthopaedic Surgery & Rehabilitation

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). The test has two steps: 1) heat saliva with a stabilization solution, and 2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections.

This article describes a patient whose cutaneous pyogenic granuloma was mistaken for infection after injury from a fractured smartphone screen. Clinicians should suspect pyogenic granuloma in patients with these types of injuries so that patients can avoid unnecessary procedures, antibiotics, and discomfort.

Histones are located in the cell nucleus. They are positively charged small proteins which became extracellular upon apoptosis, necrosis, and infection – induced cell death. The mixture of extracellular Histones was shown to bundle Actin filaments and digested by bacterial proteases, which was inhibited by DNA and Actin. Here we studied the interaction of five major family of Histones, H2A, H2B, H3.1, H1 and H4, with DNA and Actin filaments. We found that all the Histones studied bound to DNA, increased the viscosity of Actin containing solutions and bundled Actin filaments in various degrees. The bundling of Actin filaments by Histones was inhibited by DNA, NaCl and DNase1. DNA and Actin filaments also inhibited the proteolysis of the five Histones by Subtilisin, Fusolisin and Pseudomonas Aeruginosa bacterial proteases. Both the degree of the proteolysis and its inhibition was different with various Histones. The results indicate that all the Histones studied bind strongly to the negatively charged DNA and to the Actin filaments.

PARN loss-of-function mutations cause a severe form of the hereditary disease dyskeratosis congenita (DC). PARN deficiency affects the stability of non-coding RNAs such as human telomerase RNA (hTR), but these effects do not explain the severe disease in patients. We demonstrate that PARN deficiency affects the levels of numerous miRNAs in human cells. PARN regulates miRNA levels by stabilizing either mature or precursor miRNAs by removing oligo(A) tails added by the poly(A) polymerase PAPD5, which if remaining recruit the exonuclease DIS3L or DIS3L2 to degrade the miRNA. PARN knockdown destabilizes multiple miRNAs that repress p53 translation, which leads to an increase in p53 accumulation in a Dicer-dependent manner, thus explaining why PARN-defective patients show p53 accumulation. This work also reveals that DIS3L and DIS3L2 are critical 3' to 5' exonucleases that regulate miRNA stability, with the addition and removal of 3' end extensions controlling miRNA levels in the cell.

Stress granules and P-bodies are conserved assemblies of nontranslating mRNAs in eukaryotic cells that can be related to RNA–protein aggregates found in some neurodegenerative diseases. Herein, we examine how the Hsp70/Hsp40 protein chaperones affected the assembly and disassembly of stress granules and P-bodies in yeast. We observed that Hsp70 and the Ydj1 and Sis1 Hsp40 proteins accumulated in stress granules and defects in these proteins led to decreases in the disassembly and/or clearance of stress granules. We observed that individual Hsp40 proteins have different effects on stress granules with defects in Ydj1 leading to accumulation of stress granules in the vacuole and limited recovery of translation following stress, which suggests that Ydj1 promotes disassembly of stress granules to promote translation. In contrast, defects in Sis1 did not affect recovery of translation, accumulated cytoplasmic stress granules, and showed reductions in the targeting of stress granules to the vacuole. This demonstrates a new principle whereby alternative disassembly machineries lead to different fates of components within stress granules, thereby providing additional avenues for regulation of their assembly, composition, and function. Moreover, a role for Hsp70 and Hsp40 proteins in stress granule disassembly couples the assembly of these stress responsive structures to the proteostatic state of the cell.

Host defense peptides are effector molecules of the innate immunity that possess antimicrobial and health-promoting properties. Due to their potential therapeutic activities, host defense peptides are being explored as alternatives for antibiotics. The human LL-37 and its shorter, cost-effective, bactericidal core peptide derivates have been suggested for their therapeutic potential. Bacteria evade host defense peptides by proteolytic inactivation. Actin released from necrotized cells and abundant in infected sites was shown to bind and protect LL-37 from microbial proteolytic degradation, and to enable the peptide’s antimicrobial action despite the presence of the proteases. Here, we characterized the interactions of the 10–13 residues long LL-37 core peptides with actin. We show that the LL-37 core peptides associate with actin with a lower affinity than that of LL-37. Their association with actin, which is very ionic strength sensitive, is mainly based on electrostatic interactions. Likewise, the antimicrobial activity against Escherichia coli of the minimal antimicrobial peptide KR-12 but not FK-13 nor LL-37 is also very sensitive to salts. In addition, the antimicrobial activity of the FK-13 core peptide is protected by actin against the tested bacterial proteases in a similar manner to that of LL-37, supporting its potential for therapeutic use.

Purpose To explore the efficacy of injectable collagenase Clostridium histolyticum (CCH) in distal interphalangeal (DIP) joint contractures in Dupuytren disease . Methods A retrospective review was conducted for patients with DIP joint contractures treated with CCH. Baseline contracture and posttreatment residual contracture were recorded. The primary end point was achievement of 0° to 5° of residual contracture 30 days postmanipulation. Recurrence and adverse events are also reported. Results Twenty-one patients were treated with collagenase for DIP joint contractures and had a mean follow-up of 2.6 months. Seventeen of 21 patients reached the primary end point of less than 5° residual flexion contracture . Eleven of 21 patients experienced an adverse event, most commonly bruising and edema. No serious complications such as neurovascular injuries or tendon ruptures were recorded. Two patients had recurrence of disease. Conclusions Injection with CCH is an option for the treatment of DIP joint contractures in Dupuytren disease. Type of study/level of evidence Therapeutic IV.

The objective of this paper is the presentation of the European Road Safety Knowledge System, which was developed within the DaCoTA research project of the 7th Framework Programme of the European Commission. This knowledge system includes a number of components, concerning data and tools, road safety issues and countries. A wealth of data was gathered, on road fatalities, exposure, safety performance indicators, socioeconomic indicators, health and causation indicators, road user attitudes & behaviours, traffic laws and regulations, road safety management etc., for 30 European countries. This data was used in road safety analyses leading to the Basic Fact Sheets, and the Annual Statistical report. Comprehensive syntheses of the literature on important road safety issues and methodologies were carried out, in the form of web-texts. Country analyses were also carried out, including road safety management “profiles”, “country overviews” and country forecasts. These were integrated through a web-based Road Safety Knowledge System. This System can be a very useful support for road safety research in Europe, and ideal to link research and policy making.

The objective of this paper is the analysis of road safety management in European countries and the identification of “good practice”. A road safety management investigation model was created, based on several “good practice” criteria. Road safety management systems have been thoroughly investigated in 14 European countries on 2010, by means of interviews with both governmental representatives and independent experts, who filled in an extensive questionnaire. A reliable and accurate picture (“profile”) was created for each country, allowing country comparisons. Then, statistical methods were used to make rankings of countries, and analyse the relationship between road safety management and road safety performance. The results of the analyses suggest that it is not possible to identify one single “good practice”. Nevertheless, there were several elements that emerged as “good practice” criteria. On the basis of the results, recommendations are proposed at national and European level.

The objective of this research is the assessment of current needs for evidence-based road safety decision making in Europe, through the consultation of a panel of road safety experts. The members of this Experts Panel have extensive knowledge of road safety management processes and needs in their country, being either directly involved in decision making, or working closely with decision makers. Two consultation methods were implemented: semi-directive interviews and written contributions. The synthesis of the results was carried out by means of a predefined matrix, in which the road safety management tasks were separated into their components, and were then cross-tabulated with distinct categories of needs. The results provide valuable information on the current and future needs for evidence-based road safety management in Europe. A number of key issues were brought forward with wide consensus among Experts, such as the need to make the consideration of scientific evidence in road safety decisions compulsory in all countries. The establishment of appropriate procedures was emphasized, including institutional arrangements for road safety management, with the necessary links and interactive procedures for local needs. The results also include useful recommendations, for the setting of targets, the use of cost-benefit analyses, the analysis of combined effects of measures, the collection of data on measures implementation, the collection of exposure and behavioural data, the estimation of injury under-reporting and the standardization of analysis methods.

Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4 A) MTS-1 (1,1-Methanedyl Bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.

Cap hydrolysis is a critical control point in the life of eukaryotic mRNAs and is catalyzed by the evolutionarily conserved Dcp1-Dcp2 complex. In Saccharomyces cerevisiae, decapping is modulated by several factors, including the Lsm family protein Edc3, which directly binds to Dcp2. We show that Edc3 binding to Dcp2 is mediated by a short peptide sequence located C terminal to the catalytic domain of Dcp2. This sequence is required for Edc3 to stimulate decapping activity of Dcp2 in vitro, for Dcp2 to efficiently accumulate in P-bodies, and for efficient degradation of the RPS28B mRNA, whose decay is enhanced by Edc3. In contrast, degradation of YRA1 pre-mRNA, another Edc3-regulated transcript, occurs independently from this region, suggesting that the effect of Edc3 on YRA1 is independent of its interaction with Dcp2. Deletion of the sequence also results in a subtle but significant defect in turnover of the MFA2pG reporter transcript, which is not affected by deletion of EDC3, suggesting that the region affects some other aspect of Dcp2 function in addition to binding Edc3. These results raise a model for Dcp2 recruitment to specific mRNAs where regions outside the catalytic core promote the formation of different complexes involved in mRNA decapping.

Buforin is a cationic antimicrobial peptide (AMP) from the stomach of toads. Buforin II is a derivative of this naturally occurring peptide. Buforin IIB is a synthetic analog of buforin II containing a model α-helical sequence (3xRLLR) at the C-terminus. To further increase the antimicrobial activity and decrease toxicity to eukaryotic cells, new derivatives (buforin III analogs) were designed by substituting amino acids in the buforin IIB sequence. In this work, the antimicrobial activity and the actin- and DNA-binding characteristics of buforin IIIB (RVVRQWPIGRVVRRVVRRVVR) and the newly synthetized buforin IIIE (RLLLRQWPIGRLLRRLLRRLLR) were studied. The antimicrobial activity of buforin IIIB (measured against E. coli and E. faecalis ) was significantly greater than that of buforin IIIE, while both peptides were nontoxic to macrophages at the minimal concentrations required to inhibit microbial growth. Actin, which inhibited the antimicrobial activity of the two buforin III analogs, was bundled by both peptides; however, less buforin IIIE than buforin IIIB was needed for bundling. Higher levels of NaCl were needed to unbundle actin bundled by buforin IIIE than actin bundled by buforin IIIB, which indicates that buforin IIIE binds more strongly to actin than buforin IIIB. Actin bundled by either peptide was dissociated with the same concentration of DNA; however, buforin IIIE bound more strongly to DNA than buforin IIIB. These results contribute to the understanding of the antimicrobial mechanism of cationic AMPs in general and histone-derived peptides in particular.

Recent results indicate that nontranslating mRNAs in eukaryotic cells exist in distinct biochemical states that accumulate in P bodies and stress granules, although the nature of interactions between these particles is unknown. We demonstrate in Saccharomyces cerevisiae that RNA granules with similar protein composition and assembly mechanisms as mammalian stress granules form during glucose deprivation. Stress granule assembly is dependent on P-body formation, whereas P-body assembly is independent of stress granule formation. This suggests that stress granules primarily form from mRNPs in preexisting P bodies, which is also supported by the kinetics of P-body and stress granule formation both in yeast and mammalian cells. These observations argue that P bodies are important sites for decisions of mRNA fate and that stress granules, at least in yeast, primarily represent pools of mRNAs stalled in the process of reentry into translation from P bodies.

Summary A critical step in mRNA degradation is the removal of the 5′ cap structure, which is catalyzed by the Dcp1-Dcp2 complex. The crystal structure of an S . pombe Dcp1p-Dcp2n complex combined with small-angle X-ray scattering analysis (SAXS) reveals that Dcp2p exists in open and closed conformations, with the closed complex being, or closely resembling, the catalytically more active form. This suggests that a conformational change between these open and closed complexes might control decapping. A bipartite RNA-binding channel containing the catalytic site and Box B motif is identified with a bound ATP located in the catalytic pocket in the closed complex, suggesting possible interactions that facilitate substrate binding. Dcp1 stimulates the activity of Dcp2 by promoting and/or stabilizing the closed complex. Notably, the interface of Dcp1 and Dcp2 is not fully conserved, explaining why the Dcp1-Dcp2 interaction in higher eukaryotes requires an additional factor.

Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is a key regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs the filaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactions with subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a cryptic cofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, which targets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal alpha-actin and WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but was strongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysis of the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 on recombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analytical ultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealed oligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bound a second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Under polymerizing conditions the cross-linked AC formed mostly short filaments, which according to image reconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actin mutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actin to Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involved in a weak binding of cofilin to G-actin.

There are two A-type cyclins in higher vertebrates, cyclin A1 and A2. Targeted mutagenesis has shown that cyclin A2 is essential for early embryonic development while cyclin A1 is required only for male meiosis. The embryonic lethality of cyclin A2 knockout mice has obviated understanding its role in other aspects of mammalian development, including the germ line. We reported previously that cyclin A2 expression in the male germ line is consistent with a role in both mitotic and meiotic cell cycles. Usingin situhybridization and immunohistochemistry, we now observe high levels of cyclin A2 in granulosa cells and less-abundant but readily detectable expression in ovarian and ovulated oocytes. A decrease in cyclin A2 protein was observed in oocytes from embryonic stages to post-natal and adult ovaries. Interestingly, cyclin A2 protein was nuclear in oocytes from embryonic day 13.5 to 15.5, changing to largely cytoplasmic in oocytes from embryonic day 16.5 to post-natal and adults. Readily detectable expression of the cyclin-dependent kinases Cdk1 and Cdk2, two common partners for the A-type cyclins, was observed in granulosa cells and oocytes at all stages of folliculogenesis. Cdk1 was predominantly cytoplasmic, whereas Cdk2 was both cytoplasmic and nuclear in oocytes. No cyclin A1 expression, at either the mRNA level or the protein level was detected in either embryonic or adult ovaries, consistent with the full fertility observed in female cyclin A1-deficient mice. These results suggest that in the female germ line, cyclin A2 but not cyclin A1 has distinct roles in both mitosis and meiosis.

The decapping of eukaryotic mRNAs is a key step in their degradation. The heteroheptameric Lsm1p–7p complex is a general activator of decapping and also functions in protecting the 3′ ends of deadenylated mRNAs from a 3′-trimming reaction. Lsm1p is the unique member of the Lsm1p–7p complex, distinguishing that complex from the functionally different Lsm2p–8p complex. To understand the function of Lsm1p, we constructed a series of deletion and point mutations of the LSM1 gene and examined their effects on phenotype. These studies revealed the following: (i) Mutations affecting the predicted RNA-binding and inter-subunit interaction residues of Lsm1p led to impairment of mRNA decay, suggesting that the integrity of the Lsm1p–7p complex and the ability of the Lsm1p–7p complex to interact with mRNA are important for mRNA decay function; (ii) mutations affecting the predicted RNA contact residues did not affect the localization of the Lsm1p–7p complex to the P-bodies; (iii) mRNA 3′-end protection could be indicative of the binding of the Lsm1p–7p complex to the mRNA prior to activation of decapping, since all the mutants defective in mRNA 3′ end protection were also blocked in mRNA decay; and (iv) in addition to the Sm domain, the C-terminal domain of Lsm1p is also important for mRNA decay function.

A major mechanism of eukaryotic mRNA degradation initiates with deadenylation followed by decapping and 5' to 3' degradation. We demonstrate that the yeast EDC1 mRNA, which encodes a protein that enhances decapping, has unique properties and is both protected from deadenylation and undergoes deadenylation-independent decapping. The 3' UTR of the EDC1 mRNA is sufficient for both protection from deadenylation and deadenylation-independent decapping and an extended poly(U) tract within the 3' UTR is required. These observations highlight the diverse forms of decapping regulation and identify a feedback loop that can compensate for decreases in activity of the decapping enzyme. Surprisingly, the decapping of the EDC1 mRNA is slowed by the loss of Not2p, Not4p, and Not5p, which interact with the Ccr4p/Pop2p deadenylase complex. This indicates that the Not proteins can affect decapping, which suggests a possible link between the mRNA deadenylation and decapping machinery.

Cofilin/ADF affects strongly the structure of actin filaments and especially the intermolecular contacts of the DNase I binding loop (D-loop) in subdomain 2. In G-actin, the D-loop is cleaved by subtilisin between Met47 and Gly48, while in F-actin this cleavage is inhibited. Here, we report that yeast cofilin, which is resistant to both subtilisin and trypsin, accelerates greatly the rate of subtilisin cleavage of this loop in F-actin at pH 6.8 and at pH 8.0. Similarly, cofilin accelerates strongly the tryptic cleavage in F-actin of loop 60-69 in subdomain 2, at Arg62 and Lys68. The acceleration of the loops' proteolysis cannot be attributed to an increased treadmilling of F-actin for the following reasons: (i) the rate of subtilisin cleavage is independent of pH between pH 6.8 and 8.0, unlike F-actin depolymerization, which is pH-dependent; (ii) at high concentrations of protease the cleavage rate of F-actin in the presence of cofilin is faster than the rate of monomer dissociation from the pointed end of TRC-labeled F-actin, which limits the rate of treadmilling; and (iii) cofilin also accelerates the rate of subtilisin cleavage of F-actin in which the treadmilling is blocked by interprotomer cross-linking of the D-loop to the C terminus on an adjacent protomer. This suggests a substantial flexibility of the D-loop in the cross-linked F-actin. The increased cleavage rates of the D-loop and loop 60-69 reveal extensive exposure of subdomain 2 in F-actin to proteolytic enzymes by cofilin.

The effect of yeast cofilin on lateral contacts between protomers of yeast and skeletal muscle actin filaments was examined in solution. These contacts are presumably stabilized by the interactions of loop 262-274 of one protomer with two other protomers on the opposite strand in F-actin. Cofilin inhibited several-fold the rate of interstrand disulfide cross-linking between Cys265 and Cys374 in yeast S265C mutant F-actin, but enhanced excimer formation between pyrene probes attached to these cysteine residues. The possibility that these effects are due to a translocation of the C terminus of actin by cofilin was ruled out by measurements of fluorescence resonance energy transfer (FRET) from tryptophan residues and eATP to acceptor probes at Cys374. Such measurements did not reveal cofilin-induced changes in FRET efficiency, suggesting that changes in Cys265-Cys374 cross-linking and excimer formation stem from the perturbation of loop 262-274 by cofilin. Changes in lateral interactions in F-actin were indicated also by the cofilin-induced partial release of rhodamine phalloidin. Disulfide cross-linking of S265C yeast F-actin inhibited strongly and reversibly the release of rhodamine phalloidin by cofilin. Overall, this study provides solution evidence for the weakening of lateral interactions in F-actin by cofilin.

Aberrant expression of several key regulators controlling the G1/S phase of the cell cycle has been implicated in human male germ cell tumorigenesis. Given the critical role of cyclin A2 at both the G1/S and G2/M transitions and the essential role for cyclin A1 in male germ cell development, our present study focused on the involvement of the A-type cyclins in the transformation and progression of male germ cell tumors (GCTs). The expression of the A-type cyclins and their catalytic partners Cdk1 and Cdk2 was examined in all types and stages of human male GCTs, including carcinoma in situ(CIS), seminoma and non-seminoma GCTs, along with normal testis samples. Elevated levels of cyclin A2, Cdk1 and Cdk2 were detected in the majority of GCTs and were correlated with the invasiveness of the tumors (p < 0.05). Cyclin A1 expression was virtually undetectable in CIS and seminoma, but was aberrantly expressed in all non-seminomatous GCTs. Cyclin A2 expression was strongly correlated with that of its catalytic partners Cdk1 and Cdk2 in all types of testicular tumors examined (p < 0.05), whereas a strong correlation between cyclin A1 and Cdk1 or Cdk2 was only seen in non-seminomatous GCTs (p < 0.05). Histone kinase activities of cyclin A1/Cdks and cyclin A2/Cdks were found to be elevated in tumors. Our data suggest that aberrant expression of A-type cyclins and their Cdks is a significant factor in male germ cell tumorigenesis. The abundant ectopic expression of cyclin A1 in non-seminomatous GCTs and its absence in CIS and seminomas is likely linked to the tumor transformation and progression and may be relevant to clinical prognosis.

The myosin motor protein generates force in muscle by hydrolyzing Adenosine 5′-triphosphate (ATP) while interacting transiently with actin. Structural evidence suggests the myosin globular head (subfragment 1 or S1) is articulated with semi-rigid catalytic and lever-arm domains joined by a flexible converter domain. According to the prevailing hypothesis for energy transduction, ATP binding and hydrolysis in the catalytic domain drives the relative movement of the lever arm. Actin binding and reversal of the lever-arm movement (power stroke) applies force to actin. These domains interface at the reactive lysine, Lys84, where trinitrophenylation (TNP-Lys84-S1) was observed in this work to block actin activation of myosin ATPase and in vitro sliding of actin over myosin. TNP-Lys84-S1's properties and interactions with actin were examined to determine how trinitrophenylation causes these effects. Weak and strong actin binding, the rate of mantADP release from actomyosin, and actomyosin dissociation by ATP were equivalent in TNP-Lys84-S1 and native S1. Molecular dynamics calculations indicate that lever-arm movement inhibition during ATP hydrolysis and the power stroke is caused by steric clashes between TNP and the converter or lever-arm domains. Together these findings suggest that TNP uncouples actin activation of myosin ATPase and the power stroke from other steps in the contraction cycle by inhibiting the converter and lever-arm domain movements.

Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)

The osteogenic growth peptide (OGP) is a key factor in the mechanism of the systemic osteogenic response to local bone marrow injury. When administered in vivo, OGP stimulates osteogenesis and hematopoiesis. The C-terminal pentapeptide OGP(10-14) is the minimal amino acid sequence that retains the full OGP-like activity. Apparently, it is also the physiologic active form of OGP. Residues Tyr(10), Phe(12), Gly(13), and Gly(14) of OGP are essential for the OGP(10-14) activity. The present study explored the functional role of the peptide bonds, carboxyl and amino terminal groups, and conformational freedom in OGP(10-14). Transformations replacing the peptide bonds with surrogates such as Psi(CH(2)NH), Psi(CONMe), and Psi(CH(2)CH(2)) demonstrated that amide bonds do not contribute significantly to OGP(10-14) bioactivity. End-to-end cyclization yielded the fully bioactive cyclic pentapeptide c(Tyr-Gly-Phe-Gly-Gly). The retroinverso analogue c(Gly-Gly-phe-Gly-tyr), a cyclostereoisomer of c(Tyr-Gly-Phe-Gly-Gly), is at least as potent as the parent cyclic pentapeptide. The unique structure-activity relations revealed in this study suggest that the spatial presentation of the Tyr and Phe side chains has a major role in the productive interaction of OGP(10-14) and its truncated and conformationally constrained analogues with their cognate cellular target.

Resume De maniere generale, dans les pays a faibles et moyens revenus, ou la motorisation est un phenomene recent, l'insecurite routiere devient un probleme de plus en plus aigu. Cet article analyse les raisons specifiques de ce probleme et apporte un certain nombre d'elements de reponse qui concernent tant les apports possibles d'une cooperation bien comprise avec les pays plus avances que la politique des bailleurs de fond internationaux sur cette question.

Background The SPE-8 group gene products transduce the signal for spermatid activation initiated by extracellular zinc in C. elegans. Mutations in the spe-8 group genes result in hermaphrodite-derived spermatids that cannot activate to crawling spermatozoa, although spermatids from mutant males activate through a pathway induced by extracellular TRY-5 protease present in male seminal fluid. Results Here, we identify SPE-8 as a member of a large family of sperm-expressed non-receptor-like protein-tyrosine kinases. A rescuing SPE-8::GFP translational fusion reporter localizes to the plasma membrane in all spermatogenic cells from the primary spermatocyte stage through spermatids. Once spermatids become activated to spermatozoa, the reporter moves from the plasma membrane to the cytoplasm. Mutations in the spe-8 group genes spe-12, spe-19, and spe-27 disrupt localization of the reporter to the plasma membrane, while localization appears near normal in a spe-29 mutant background. Conclusions These results suggest that the SPE-8 group proteins form a functional complex localized at the plasma membrane, and that SPE-8 is correctly positioned only when all members of the SPE-8 group are present, with the possible exception of SPE-29. Further, SPE-8 is released from the membrane when the activation signal is transduced into the spermatid.

Intrastrand cross-linking of actin filaments by ANP, N-(4-azido-2-nitrophenyl) putrescine, between Gln-41 in subdomain 2 and Cys-374 at the C-terminus, was shown to inhibit force generation with myosin in the in vitro motility assays [Kim et al. (1998) Biochemistry 37, 17801-17809]. To clarify the immobilization of which of these two sites inhibits the actomyosin motor, the properties of actins with partially overlapping cross-linked sites were examined. pPDM (N,N'-p-phenylenedimaleimide) and ABP [N-(4-azidobenzoyl) putrescine] were used to obtain actin filaments cross-linked ( approximately 50%) between Cys-374 and Lys-191 (interstrand) and Gln-41 and Lys-113 (intrastrand), respectively. ANP, ABP, and pPDM cross-linked filaments showed similar inhibition of their sliding speeds and force generation with myosin ( approximately 25%) in the in vitro motility assays. In analogy to ANP cross-linking of actin, pPDM and ABP cross-linkings did not change the strong S1 binding to actin and the V(max) and K(m) parameters of actomyosin ATPase. The similar effects of these three cross-linkings reveal the tight coupling between structural elements of the subdomain 2/subdomain 1 interface and show the importance of its dynamic flexibility to force generation with myosin. The possibility that actin cross-linkings inhibit rate-limiting steps in motion and force generation during myosin cross-bridge cycle was tested in stopped-flow experiments. Measurements of the rates of mantADP release from actoS1 and ATP-induced dissociation of actoS1 did not reveal any differences between un-cross-linked and ANP cross-linked actin in these complexes. These findings are discussed in terms of the uncoupling between force generation and other aspects of actomyosin interactions due to a constrained dynamic flexibility of the subdomain 2/subdomain 1 interface in cross-linked actin filaments.

Dynamic properties of F-actin structure prompted suggestions (Squire, J. M., and Morris, E. P. (1998) FASEB J. 12, 761-771) that actin subdomain 2 movements play a role in thin-filament regulation. Using fluorescently labeled yeast actin mutants Q41C, Q41C/C374S, and D51C/C374S and azidonitrophenyl putrescine (ANP) Gln(41)-labeled alpha-actin, we monitored regulation-linked changes in subdomain 2. These actins had fully regulated acto-S1 ATPase activities, and emission spectra of regulated Q41C(AEDANS)/C374S and D51C(AEDANS)/C374S filaments did not reveal any calcium-dependent changes. Fluorescence energy transfer in these F-actins mostly occurred from Trp(340) and Trp(356) to 5-(2((acetyl)amino)ethyl)amino-naphthalene-1-sulfonate (AEDANS)-labeled Cys(41) or Cys(51) of adjacent same strand protomers. Our results show that fluorescence energy transfer between these residues is similar in the mostly blocked (-Ca(2+)) and closed (+Ca(2+)) states. Ca(2+) also had no effect on the excimer band in the pyrene-labeled Q41C-regulated actin, indicating virtually no change in the overlap of pyrenes on Cys(41) and Cys(374). ANP quenching of rhodamine phalloidin fluorescence showed that neither Ca(2+) nor S1 binding to regulated alpha-actin affects the phalloidin-probe distance. Taken together, our results indicate that transitions between the blocked, closed, and open regulatory states involve no significant subdomain 2 movements, and, since the cross-linked alpha-actin remains fully regulated, that subdomain 2 motions are not essential for actin regulation.

The atomic structures for several myosin head isoforms in different nucleotide states have been determined in recent years. The comparison of these structures is complicated by the use of myosin subfragment 1 (S1) constructs of different length in different studies. Several atomic structures of the S1 nucleotide complex were obtained using Dictyostelium discoideum S1dC, a genetically truncated form of S1 lacking the light chain binding domain (LCBD) and both light chains. The goal of the present study has been to assess the effects of such a truncation on the solution properties of S1 and in particular, on its active site, actin binding site and the converter region. The nucleotide and actin binding properties, CD spectra and the reactivities of Lys-84 (corresponds to the 'reactive lysine', Lys-83 in rabbit skeletal S1) and Cys-678 (corresponds to the 'SH2-group', Cys-697 in rabbit S1) were compared for the full length (flS1) and the truncated (S1dC) forms of Dictyostelium S1. The two forms showed similar nucleotide binding properties. However, SldC had a lower structural stability and a significantly higher Km value for actin-activated ATPase as compared to flS1. Differences were found also in the near-UV CD spectrum between flS1 and S1dC. SH2 reactivity in SldC appeared to be greatly inhibited compared with that in flS1. The modification of Lys-84 caused a greater increase in the MgATPase activity in S1dC than in flS1. ADP inhibited this activation for both SldC and flS1. Taken together our results identify both truncation-caused differences between S1dC and flS1, as well as isoform-related differences between skeletal and Dictyostelium S1.

The amino acid sequence of osteogenic growth peptide (OGP) consists of 14 residues identical to the C-terminal tail of histone H4. Native and synthetic OGP are mitogenic to osteoblastic and fibroblastic cells and enhance osteogenesis and hematopoiesis in vivo. The C-terminal truncated pentapeptide of OGP, H-Tyr-Gly-Phe-Gly-Gly-OH [OGP(10–14)], is a naturally occurring osteoblastic mitogen, equipotent to OGP. The present study assesses the role of individual amino acid residues and side chains in the OGP(10–14) mitogenic activity which showed a very high correlation between osteoblastic and fibroblastic cell cultures. Truncation of either Tyr10 or its replacement by Ala or d-Ala resulted in substantial, but not complete, loss of activity. Nevertheless, only a small loss of activity was observed following removal of the Tyr10 amino group. No further loss occurred consequent to the monoiodination of desaminoTyr10 on meta-position. However, a marked decrease in proliferative activity followed removal of the Tyr10 phenolic or the Phe12 aromatic group. Loss of activity of a similar magnitude also occurred subsequent to replacing Gly11 with l- or d-Ala. Approximately 50% loss of mitogenic activity occurred subsequent to truncation of Gly14 or blocking the C-terminal group as the methyl ester. All other modifications of the C-terminus and l- or d-Ala substitution of Gly13 resulted in 70–97% decrease in activity. Collectively, these data suggest that the integrity of the pharmacophores presented by Tyr and Phe side chains, as well as the Gly residues at the C-terminus, are important for optimal bioactivity of OGP(10–14).

Cys 10 is located in subdomain 1 of actin, which has an important role in the interaction of actin with myosin- and actin-binding proteins. Cys 10 was modified with fluorescence probes N -(iodoacetyl) N′ -(5-sulfo-1-naphthyl)ethylene diamine (IAEDANS), 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromo bimane (MBB) by the method of Drewes and Faulstich (1991, J. Biol. Chem. 266:5508–5513). The specificity of Cys 10 modification was verified by showing that the 33-kDa subtilisin fragment of actin (residues 48–375), which contains all of the actin thiols but Cys 10 , is not fluorescent. Cys 10 modification exposed a new site on actin to subtilisin cleavage. Edman degradation revealed this site to be between Ala 19 and Gly 20 . The modification slightly increased the rate of ϵATP-ATP exchange and decreased the rates of G-actin ATPase and polymerization. The activation of S1 ATPase by Cys 10 -modified F-actin showed small probe-dependent changes in the values of V max and K M . The sliding speed of actin filaments in the in vitro motility assay remained unchanged upon modification of Cys 10 . These results indicate that although the labeling of Cys 10 perturbs the structure of subdomain 1, the modified actin remains fully functional. The binding of S1 to actin filaments decreases the accessibility of Cys 10 probes to acrylamide and nitromethane quenchers. Because Cys 10 does not participate directly in either actin polymerization or S1 binding, our results indicate that actin-actin and actin-myosin interactions induce dynamic, allosteric changes in actin structure.

Subdomain 2 of actin is a dynamic segment of the molecule. The cross-linking of Gln-41 on subdomain 2 to Cys-374 on an adjacent monomer in F-actin inhibits actomyosin motility and force generation (Kim et al., 1998; Biochemistry 37, 17,801–17,809). To shed light on this effect, additional modifications of the Gln-41 site on actin were carried out. Both intact G-actin and G-actin cleaved by subtilisin between Met-47 and Gly-48 in the DNase 1 binding loop of subdomain 2 were treated with bacterial transglutaminase. According to the results of Edman degradation, transglutaminase introduced an intramolecular zero-length cross-linking between Gln-41 and Lys-50 in both intact and subtilisin cleaved actins. This cross-linking perturbs G-actin structure as shown by the inhibition of subtilisin and tryptic cleavage in subdomain 2, an allosteric inhibition of tryptic cleavage at the C-terminus and decrease of modification rate of Cys-374. The cross-linking increases while the subtilisin cleavage dramatically decreases the thermostability of F-actin. The Mg- and S1-induced polymerizations of both intact and subtilisin cleaved actins were only slightly influenced by the cross-linking. The activation of S1 ATPase by actin and the sliding speeds of actin filaments in the in vitro motility assays were essentially unchanged by the cross-linking. Thus, although intramolecular cross-linking between Gln-41 and Lys-50 perturbs the structure of the actin monomer, it has only a small effect on actin polymerization and its interaction with myosin. These results suggest that the new cross-linking does not alter the intermonomer interface in F-actin and that changes in actomyosin motility reported for the Gln-41–Cys-374 intrastrand cross-linked actin are not due to decreased flexibility of loop 38–52 but to constrains introduced into the F-actin structure and/or to perturbations at the actin's C-terminus.

The osteogenic growth peptide (OGP) is a 14-amino acid stromal cell mitogen that stimulates in vivo osteogenesis and hematopoiesis. In the blood circulation and cell culture conditioned medium immunoreactive OGP (irOGP), identified using antibodies raised against the OGP C-terminal region, presents free and bound forms. The bound form consists entirely of the full length peptide. The present study was designed to investigate the identity of free irOGP under nondenaturing conditions. Fresh human serum and culture medium conditioned with murine osteoblastic MC3T3 E1 cells were fractionated using ultrafiltration (3000 molecular weight cut-off). Hydrophobic chromatography of the ultrafiltrate, immunoscreening of chromatographic fractions with antibodies directed against the OGP C-terminal region and amino acid sequencing of immunoreactive peaks demonstrated the presence of two mitogens, the full length OGP and a C-terminal truncated form, OGP(10–14). The OGP(10–14) derived from both serum and conditioned medium, as well as the synthetic pentapeptide [sOGP(10–14)], shared the in vitro OGP proliferative activity. However, in a competitive binding assay, devised to assess the OGP–OGP binding protein (OGPBP) complex formation, sOGP(10–14) failed to compete out radiolabeled OGP from the complex. It is concluded that OGP(10–14) is a naturally occuring human and murine mitogen. In addition, the data suggests that the OGP(10–14) is generated from OGP by proteolytic cleavage upon dissociation of the OGP–OGPBP complexes.

A trinitrophenylated hexapeptide, with sequence imitating that near the reactive lysyl residue (Lys84 or RLR) in skeletal muscle myosin, has an induced circular dichroism (CD) signal in the absorption band of the trinitrophenyl group (TNP) characteristic to the TNP in a structured environment. Nuclear Overhauser Effect (NOE) and coupling constant data obtained with 1H NMR confirm that the TNP-hexapeptide (TNP-6p) exists as an ensemble of closely related 3-dimensional structures. A simulated annealing procedure constrained by the NOE distances produced a solution set of 47 structures for the TNP-6p with potential energies less than or approximately equal to the root-mean-squared energy fluctuation expected for this peptide. The CD signals induced in the three lowest-energy electronic transitions of the TNP absorption bands were computed for each structure in the solution set using the matrix method implemented for TNP as the signal donor group. The computed CD signals distinguish two subsets of structures ...

Bertrand et al. [Bertrand, R., Derancourt, J.Kassab, R. (1995) Biochemistry 34, 9500-9507] reported that 6-[fluoresceine-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS) selectively modifies Lys553, which is part of the strong actin-binding site of myosin subfragment 1 (S1). We found that the reaction of FHS with Lys533 is accompanied by a decrease in the fluorescence intensity of the reagent. The rate of the FHS reaction increased with increasing pH implying that the unprotonated form of the epsilon-amino group of Lys553 reacts with FHS. Addition of 0.4 M KCl reduced the rate of reaction significantly, which indicates ionic strength-dependent changes in the structure of S1. Limited trypsinolysis of S1 before the FHS reaction also decreased the rate of the reaction showing that the structural integrity of S1 is needed for the reactivity of Lys553. ATP, ADP, ADP.BeF(x), ADP.AlF(4), ADP.V(i) and pyrophosphate significantly decreased the rate of Lys553 labelling, suggesting nucleotide-induced conformational changes in the environment of Lys553. The fluorescence emission spectrum of the Lys553-bound FH moiety and the quenching of its fluorescence by nitromethane was not influenced by nucleotides, implying that the chemical reactivity but not the accessibility of Lys553 was decreased by the nucleotide-induced conformational change. In the presence of ATP when the M(**)ADP.P(i) state of the ATPase cycle is predominantly populated, the reaction rate decreased more than in the case of the S1.ADP.AlF(4)(-) and S1.ADP.V(i) complexes, which are believed to mimic the M(**)ADP.P(i) state. This indicates that the conformation of the S1-ADP.AlF(4)(-) and S1.ADP.V(i) complexes in the vicinity of Lys553 does not resemble the structure of the M(**)ADP.P(i) state. The rate of Lys553 labelling decreased strongly in the presence of actin. The nitromethane quenching of the Lys553-bound FHS was not influenced by actin, which indicates that the reduced reaction rate is not due to steric hindrance caused by the bulky protein but by actin induced conformational changes in the vicinity of Lys553.

Actin filaments partially cross-linked with ANP (N-(4-azido-2-nitrophenyl)-putrescine between Gln-41 and Cys-374 on adjacent monomers in the long-pitch helix were depolymerized and fractionated into pools of longitudinal cross-linked dimers (s(o)20,w = 5.55 +/- 0.22 S), trimers (s(o)20,w = 6.93 +/- 0.12 S), and higher-order oligomers. Competition binding experiments of myosin subfragment (S1) to cross-linked dimers in the presence of pyrenyl G-actin revealed about 2 orders of magnitude stronger binding of the first than that of the second S1 molecule to actin dimer. Under similar conditions the unpolymerized cross-linked actin species activated the MgATPase of S1 only severalfold compared to 70-fold activation by F-actin. The cross-linked dimers, trimers, and oligomers were polymerized into filaments by MgCl2 faster than un-cross-linked actin. In electron micrographs these filaments appeared sometimes shorter and had greater tendency to bend than un-cross-linked actin filaments. Small amounts of cross-linked actin dimers nucleated S1-induced polymerization of actin, but the polymerization by S1 was inhibited for pure populations of cross-linked dimers, trimers, and oligomers. The cross-linked dimers did not decrease the kinetic difference between the polymerization of actin by S1 isozymes S1(A1) and S1(A2). According to electron microscopy evidence, cross-linked actin oligomers polymerized by S1 yielded much shorter arrowhead structures than the un-cross-linked actin. These results indicate the importance of lateral actin-actin interaction for the activation of myosin ATPase and the polymerization of actin by S1.

A new heterobifunctional photo-cross-linking reagent, N-(4-azido-2-nitrophenyl)-putrescine (ANP), was synthesized and covalently bound to Gln-41 of rabbit skeletal muscle actin by a bacterial transglutaminase-mediated reaction. Up to 1.0 mol of the reagent was incorporated per mole of G-actin; at least 90% of it was bound to Gln-41 while a minor fraction (about 8%) was attached to Gln-59. The labeled G-actin was polymerized, and the resulting F-actin was intermolecularly cross-linked by irradiation with UV light. The labeled and cross-linked peptides were isolated from either a complete or limited tryptic digest of cross-linked actin. In the limited digest the tryptic cleavage was restricted to arginine by succinylation of the lysyl residues. N-terminal sequencing and mass spectrometry indicated that the cross-linked peptides contained residues 40-50 (or 40-62 in the arginine limited digest) and residues 373-375, and that the actual cross-linking took place between Gln-41 and Cys-374. This latter finding was also supported by the inhibition of Cys-374 labeling with a fluorescent probe in the cross-linked actin. The dynamic length of ANP, between 11.1 and 12.5 A, constrains to that range the distance between the gamma-carboxyl group of Gln-41 in one monomer and the sulfur atom of Cys-374 in an adjacent monomer. This is consistent with the distances between these two residues on adjacent monomers of the same strand in the long-pitch helix in the structural models of F-actin [Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 347, 44-49 and Lorenz, M., Popp, D., and Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836]. The effect of cross-linking on the function of actin is described in the companion papers.

2-[(2-Nitrophenyl)amino]ethyl triphosphate (NPhAETP) is the smallest ATP analogue that serves as a substrate for the actin-activated ATPase of myosin subfragment 1 (S1) and supports the development of active tension in skinned fibers. 2-(Phenylamino)ethyl triphosphate (PhAETP), in which the nitro group on the phenyl ring of NPhAETP is substituted by a H atom, is also a substrate of the actin-activated ATPase but does not support active tension [Wang, D., Pate, E., Cooke, R., and Yount, R. (1993) J. Muscle Res. Cell Motil. 14, 484-497]. We compared the S1-catalyzed hydrolysis of these analogues, their ability to support the formation of stable complexes with S1 and phosphate analogues, and their effect on S1 conformation. The analogues were hydrolyzed by S1 under various conditions both in the presence and in the absence of actin. In some cases, the effects of the two analogues are similar to each other and to those of ATP; they protect S1 from heat denaturation at 40 degreesC and inhibit the formation of the N-terminal 29 kDa fragment during the tryptic digestion of S1 and the modification of Lys-83 with trinitrobenzene sulfonate. However, in other cases, the effect of the two analogues is different; the effect of NPhAETP resembles that of ATP. NPhAETP and ATP decrease while PhAETP increases the rate of reaction of the SH1 thiol (Cys-707) with coumarin maleimide. The diphosphate forms of the two analogues induce a much smaller change in the near-UV CD spectrum of S1 than ADP. NPhAEDP forms stable complexes with S1 in the presence of beryllium fluoride (BeFx), aluminum fluoride (AlF4-), or vanadate (Vi) phosphate analogues, while the S1.PhAEDP complex is stable in the presence BeFx but much less stable with AlF4- and Vi. These results indicate that the S1.PhAEDP.Pi state is poorly populated during the PhAETP hydrolysis. The models of the atomic structure of S1 complexed by the two analogues show that PhAETP, unlike NPhAETP or ATP, does not form a H bond with Tyr-134 in S1, which is the probable structural reason of the lack of tension development, with PhAETP as the substrate.

The circular dichroism (CD) spectrum was measured from vanadate (Vi) cyclic esters of chiral vicinal diols, hydroxycarboxylates, and cyclodextrines as a function of Vi concentration ([Vi]) and at the lowest energy transitions of the vanadium. At low [Vi] and in the presence of excess vicinal diols, hydroxycarboxylates, or cyclodextrines the CD signal intensity scales linearly with [Vi] indicating the predominance of a monomeric cyclic ester. At higher [Vi], the signal intensity in the presence of the vicinal diols and hydroxycarboxylates become nonlinear in [Vi], indicating formation of a dimeric cyclic ester. Vanadium-51 NMR (51V-NMR) indicates the coordination geometry of several of these model Vi centers in solution and identifies the CD signals characteristic to Vi trigonal bipyramidal (tbp) and octahedral (Oh) coordination geometries from monomeric and dimeric species. The CD spectra from monomeric and dimeric forms of the tbp-coordinated model compounds have two apparent transitions with amplitudes of opposite sign at wavelengths≥240 nm. Spectra from the monomeric and dimeric Oh coordinated species are distinct from the tbp-type spectra over the same wavelength domain because of the presence of two additional transitions with opposite sign amplitudes. These model spectra were compared to the vanadate CD spectra from Vi bound to rabbit myosin subfragment 1 (S1) in solution, in the presence of divalent metal cations (MeVi-S1) or trapped with MeADP (MeADPVi-S1). Polymeric MeVi binds to the active site of S1 and the vanadate centers in MnVi-S1 or CoVi-S1 produce a CD signal resembling that from the tbp model. The trapped ATPase transition state analog MeADPVi produces a different CD signal resembling that from the Oh model.

G-actin was covalently cross-linked with S1 in a bacterial transglutaminase-catalyzed reaction. The cross-linking sites were identified with the help of fluorescent probes and limited proteolysis as the Gln-41 on the DNase I binding loop of subdomain 2 in G-actin and a lysine-rich loop (residues 636–642) on the S1 heavy chain. The same lysine-rich loop was cross-linked to another region of G-actin in a former study (Combeau, C., D. Didry, and M-F. Carlier. 1992. J. Biol. Chem. 267:14038–14046). This indicates the existence of more than one G-actin-S1 complex. In contrast to G-actin, no cross-linking was induced between F-actin and S1 by the transglutaminase reaction. This shows that in F-actin the inner part of the DNase I binding loop, where Gln-41 is located, is not accessible for S1. The cross-linked G-actin-S1 polymerized upon addition of 2mM MgCl2 as indicated by electron microscopy and sedimentation experiments. The filaments obtained from the polymerization of cross-linked actin and S1 were much shorter than the control actin filaments. The ATPase activity of the cross-linked S1 was not activated by actin, whereas the K+(EDTA)-activated ATPase activity of S1 was unaffected by the cross-linking. The cross-linking between G-actin and S1 was not influenced by the exchange of the tightly bound calcium to magnesium; however, it was inhibited by the exchange of the actin-bound ATP to ADP. This finding supports the view that the structure of the DNase binding loop in ADP-G-actin is somewhere between the structures of ATP-G-actin and F-actin.

The osteogenic growth peptide (OGP) was characterized recently in regenerating bone marrow (BM) and normal serum. In vitro, the OGP regulates stromal-cell proliferation and differentiated functions. In vivo, an increase in serum OGP accompanies the osteogenic phase of postablation BM regeneration. The present results in normal mice show that OGP induces a balanced increase in WBC counts and overall BM cellularity. In mice receiving myeloablative irradiation and syngeneic or semiallogeneic BM transplants, OGP stimulates hematopoietic reconstruction and doubles the survival rate; these effects are dependent on initiating the OGP administration before irradiation. Chimerism measurements in semiallogeneic graft recipients suggest no preferential effect of OGP on residual host cells. The data implicate OGP in the acceleration of hematopoiesis secondary to expansion of the stromal microenvironment and/or enhancement of stroma-derived signals to stem cells. The low-dose effectiveness of OGP is explained by the demonstration of an autocrine positive feedback loop that together with the OGP-binding protein sustains high serum levels of the peptide. A potential OGP-based treatment in combination with chemoradiotherapy is attractive because of the OGP-induced balanced multi-lineage enhancement of hematopoiesis and possible replacement of expensive recombinant cytokines by a readily synthesized peptide.