Refined crystal structures of Escherichia coli and chicken liver dihydrofolate reductase containing bound trimethoprim

Refined crystal structures are reported for complexes of Escherichia coli and chicken dihydrofolate reductase containing the antibiotic trimethoprim (TMP). Structural comparison of these two complexes reveals major geometrical differences in TMP binding that may be important in understanding the stereo-chemical basis of this inhibitor's selectivity for bacterial dihydrofolate reductases. For TMP bound to chicken dihydrofolate reductase we observe an altered binding geometry in which the 2,4-diaminopyrimidine occupies a position in closer proximity (by approximately 1 A) to helix alpha B compared to the pyrimidine position for TMP or methotrexate bound to E. coli dihydrofolate reductase. One important consequence of this deeper insertion of the pyrimidine into the active site of chicken dihydrofolate reductase is the loss of a potential hydrogen bond that would otherwise form between the carbonyl oxygen of Val-115 and the inhibitor's 4-amino group. In addition, for TMP bound to E. coli dihydrofolate reductase, the inhibitor's benzyl side chain is positioned low in the active-site pocket pointing down toward the nicotinamide-binding site, whereas, in chicken dihydrofolate reductase, the benzyl group is accommodated in a side channel running upward and away from the cofactor. As a result, the torsion angles about the C5-C7 and C7-C1' bonds for TMP bound to the bacterial reductase (177 degrees, 76 degrees) differ significantly from the corresponding angles for TMP bound to chicken dihydrofolate reductase (-85 degrees, 102 degrees). Finally, when TMP binds to the chicken holoenzyme, the Tyr-31 side chain undergoes a large conformational change (average movement is 5.4 A for all atoms beyond C beta), rotating down into a new position where it hydrogen bonds via an intervening water molecule to the backbone carbonyl oxygen of Trp-24.     

Functional studies of the domains of talin

The protein talin has two domains of approximately 200 and 47 kD, which can be cleaved apart by a variety of proteases. To examine the function of these two structural domains of talin, we have digested purified talin with a calcium-dependent protease and separated the resulting fragments chromatographically. Both fragments were radioiodinated and used to probe Western blots of whole fibroblasts and chicken gizzard extracts. The large talin fragment bound to vinculin and metavinculin. The small fragment did not demonstrate any binding in this assay. The fragments were labeled fluorescently and microinjected into fibroblasts in tissue culture. The large talin fragment incorporated quickly into focal adhesions where it remained stable for at least 14 h. The small fragment associated with focal adhesions of fibroblasts but was also distributed diffusely in the cytoplasm and the nucleus. These experiments suggest that talin has at least two sites that contribute to its localization in focal adhesions. Intact talin microinjected into Madin-Darby bovine kidney epithelial cells localized to the focal adhesions but was excluded from the zonulae adherentes, despite the localization of vinculin to both of these sites. In contrast, the large talin fragment, when microinjected into these epithelial cells, incorporated into both focal adhesions and zonulae adherentes. The difference in localization between the large talin fragment and intact talin seems to be due to the removal of the small domain. This difference in localization suggests that talin binding sites in zonulae adherentes have limited accessibility.     

Responses of the ticksAmblyomma hebraeum andA. variegatum to known or potential components of the aggregation-attachment pheromone. IV. Attachment stimulation of nymphs

Ten known or potential components of the aggregation-attachment pheromone (AAP) of the ticksAmblyomma hebraeum andA. variegatum, as well as a mixture of these components and extracts of prefed males of the two species, were tested as attachment stimulants for nymphs. Unfed nymphs were confined in linen bags on the ears of rabbits that had been treated with the test compounds, mixture or extracts; the numbers attached were recorded after 24 h. InA. hebraeum, attachment was induced by four compounds (2-methyl propanoic acid, methyl salicylate,o-nitrophenol and salicylaldehyde), the mixture and extracts from both species. InA. variegatum, attachment was induced by three compounds (methyl salicylate,o-nitrophenol and salicylaldehyde), the mixture and extracts from both species. Methyl salicylate ando-nitrophenol are primary components of the AAP of bothA. hebraeum andA. variegatum. 2-methyl propanoic acid is a species-specific attachment stimulant forA. hebraeum. Salicylaldehyde, a phenolic compound, is not a naturally occurring AAP component. Nymphs of both species respond to fewer attachment stimulants than the adults and, as shown by their respective host ranges, are less dependent on the AAP in the regulation of attachment than the adults.     

Molecular shape and self-association of vinculin and metavinculin

Vinculin, a 130,000-dalton protein localized to adhesion plaques, and metavinculin, a 150,-000 dalton protein closely related to vinculin, have been studied using rotary shadowing and electron microscopy. Both proteins have globular head regions attached to rod-shaped tail domains. Vinculin and metavinculin also both form complexes consisting of four to six individual molecules. These multimers are formed by head-to-head as well as tail-to-tail interactions. Talin, another protein which has been localized to adhesion plaques and binds to both vinculin and metavinculin, has also been investigated using shadowing techniques. Talin is an elongated, flexible molecule in high ionic strength buffers, as shown here by rotary shadowing and negative stain electron microscopy.     

Impact of Ramadan intermittent fasting on cognitive function in trained cyclists: a pilot study

This study assessed selected measures of cognitive function in trained cyclists who observed daylight fasting during Ramadan. Eleven cyclists volunteered to participate (age: 21.6±4.8 years, VO₂max: 57.7±5.6 ml kg⁻¹·min⁻¹) and were followed for 2 months. Cognitive function (Cambridge Neuropsychological Test Automated Battery (CANTAB), Reaction Time index (RTI) and Rapid Visual Information Processing (RVP) tests) and sleep architecture (ambulatory EEG) were assessed: before Ramadan (BR), in the 1st week (RA1) and 4th week of Ramadan (RA4), and 2 weeks post-Ramadan (PR). Both cognitive tests were performed twice per day: before and after Ramadan at 8-10 a.m. and 4-6 p.m., and during Ramadan at 4-6 p.m. and 0-2 a.m., respectively. Training load (TL) by the rating of perceived exertion (RPE) method and wellness (Hooper index) were measured daily. If the TL increased over the study period, this variable was stable during Ramadan. The perceived fatigue and delayed onset muscle soreness (DOMS) increased at RA4. Sleep patterns and architecture showed clear disturbances, with significant increases in the number of awakenings and light sleep durations during Ramadan (RA1 and RA4), together with decreased durations of deep and REM sleep stages at PR. RTI (simple and multiple reaction index) reaction and movement times did not vary over the study period. The RVP test showed reduced false alarms during Ramadan, suggesting reduced impulsivity. Overall accuracy significantly increased at RA1, RA4 and PR compared to baseline. At RA4, the accuracy was higher at 0-2 a.m. compared to 4-6 p.m. Despite the observed disturbances in sleep architecture, Ramadan fasting did not negatively impact the cognitive performance of trained cyclists from the Middle East.     

Tension on JAM-A activates RhoA via GEF-H1 and p115 RhoGEF

Junctional adhesion molecule A (JAM-A) is a broadly expressed adhesion molecule that regulates cell–cell contacts and facilitates leukocyte transendothelial migration. The latter occurs through interactions with the integrin LFA-1. Although we understand much about JAM-A, little is known regarding the protein’s role in mechanotransduction or as a modulator of RhoA signaling. We found that tension imposed on JAM-A activates RhoA, which leads to increased cell stiffness. Activation of RhoA in this system depends on PI3K-mediated activation of GEF-H1 and p115 RhoGEF. These two GEFs are further regulated by FAK/ERK and Src family kinases, respectively. Finally, we show that phosphorylation of JAM-A at Ser-284 is required for RhoA activation in response to tension. These data demonstrate a direct role of JAM-A in mechanosignaling and control of RhoA and implicate Src family kinases in the regulation of p115 RhoGEF.     

A new protein of adhesion plaques and ruffling membranes

A protein with a molecular weight on SDS polyacrylamide gels of 215,000 (referred to here as 215K) was purified from chicken gizzard smooth muscle. Antibodies against this protein localized it in fibroblasts to adhesion plaques (focal contacts), to regions underlying cell surface fibronectin, and to ruffling membranes. In the first two distributions it was similar to vinculin in cellular location, and this was confirmed by double-label immunofluorescence microscopy, but the concentration of 215K in membrane ruffles distinguished it from vinculin. There was no cross-reaction of the antibody against 215K with vinculin, and immunoprecipitation and antibody staining of SDS gels of whole cells revealed a single cross-reactive component with a molecular weight of 215,000. Immunoprecipitation from cultures labeled with [32P]phosphate revealed 215K to be a phosphoprotein. Transformation of rat or chicken fibroblasts by Rous sarcoma virus resulted in a reorganization of 215K, in some cases into complex intracellular structures. The localization of 215K where microfilament bundles terminate as well as in close relation to cell surface fibronectin and in membrane ruffles suggests that the protein has some function in the organization of actin filaments at or close to regions of actin-membrane attachment.     

Theileria annulata: cross-reactions between a cell culture schizont antigen and antigens of East African species in the indirect fluorescent antibody test

A schizont antigen for the indirect fluorescent antibody test was prepared from an in vitro culture suspension of lymphoid cells infected with Theileria annulata macroschizonts. Two cattle recovered from T. annulata infection showed marked rises in antibody titer to this schizont antigen, with peak titers of 1:40,960 and 1:2560. Using sera from these recovered cattle, T. annulata cell culture schizont antigen was shown to cross-react markedly with Theileria parva and Theileria lawrencei cell culture schizonts and with Theileria mutans piroplasms in the indirect fluorescent antibody test. In contrast, using high-titer antisera to T. parva, T. lawrencei, and T. mutans, serological cross-reactions with T. annulata schizonts were only detected with T. parva and T. lawrencei antisera, and in both instances these were minimal. The value of the indirect fluorescent antibody test in the differential diagnosis of Theileria species pathogenic for cattle is discussed.