Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli.

We investigated the functional roles of putative active site residues in Escherichia coli CheA by generating nine site-directed mutants, purifying the mutant proteins, and quantifying the effects of those mutations on autokinase activity and binding affinity for ATP. We designed these mutations to alter key positions in sequence motifs conserved in the protein histidine kinase family, including the N box (H376 and N380), the G1 box (D420 and G422), the F box (F455 and F459), the G2 box (G470, G472, and G474), and the "GT block" (T499), a motif identified by comparison of CheA to members of the GHL family of ATPases. Four of the mutant CheA proteins exhibited no detectable autokinase activity (Kin(-)). Of these, three (N380D, D420N, and G422A) exhibited moderate decreases in their affinities for ATP in the presence or absence of Mg(2+). The other Kin(-) mutant (G470A/G472A/G474A) exhibited wild-type affinity for ATP in the absence of Mg(2+), but reduced affinity (relative to that of wild-type CheA) in the presence of Mg(2+). The other five mutants (Kin(+)) autophosphorylated at rates slower than that exhibited by wild-type CheA. Of these, three mutants (H376Q, D420E, and F455Y/F459Y) exhibited severely reduced k(cat) values, but preserved K(M)(ATP) and K(d)(ATP) values close to those of wild-type CheA. Two mutants (T499S and T499A) exhibited only small effects on k(cat) and K(M)(ATP). Overall, these results suggest that conserved residues in the N box, G1 box, G2 box, and F box contribute to the ATP binding site and autokinase active site in CheA, while the GT block makes little, if any, contribution. We discuss the effects of specific mutations in relation to the three-dimensional structure of CheA and to binding interactions that contribute to the stability of the complex between CheA and Mg(2+)-bound ATP in both the ground state and the transition state for the CheA autophosphorylation reaction.

[General aspects of sexual behavior among in-coming university students in Guatemala City]. / Aspectos generales de la conducta sexual del estudiante pre-universitario en la Ciudad de Guatemala.

PIP: 880 randomly selected new students and 270 graduating students at the University of San Carlos in Guatemala City were surveyed between July 1990 and June 1991 during routine physical examinations on various aspects of their sexual behavior. Questionnaires were completed anonymously and confidentially. 61% of the new students and 70% of the graduating students were male. 82% of new students were aged 18-23 years and 68% of graduating students were 23-28 years old. Among new students, 37.5% of men and 2.8% of women reported having their first sexual contact by age 15, while 19.4% of the men and 78.9% of the women had never had sexual relations. 33.4% of the men reported their first sexual contact was with a prostitute. 2.8% of new students reported having been victims of sexual abuse in the past. Among new students, 6% of men and 2% of women reported at least one homosexual experience, while 24% of graduating men and 10% of graduating women did so. Among new and graduating students, respectively 18% and 9% of men and 1% and 0% of women reported having more than one regular sexual partner at the time of the survey. Among new students, 13.3% of men and 3.4% of women reported always using condoms, while 15.3% of men and 1.1% of women reported using them nearly always. 60% of graduating men and 37% of graduating women reported using condoms always or nearly always. 19% of new male students and 2.4% of new female students reported a history of sexually transmitted diseases, compared to 33% of graduating men and 7% of graduating women. 22.8% of new male students and 56% of graduating male students reported having had contact with prostitutes.^ieng

Alkaline phosphatase and peptidase levels in invertebrate cartilage.

Cartilage is encountered in the skeletons of many advanced invertebrates, yet it never calcifies or is replaced by bone. In an attempt to account for the absence of bone in invertebrates, we tested a hypothesis proposing that absence or inadequate quantities of several enzymes associated with vertebrate osteogenesis may underlie the failure of the invertebrates to evolve bone. The enzymes examined were alkaline phosphatase, alanyl beta-naphthylamidase, and neutral protease. Their activities were measured in the gill cartilage of the Atlantic horseshoe crab, Limulus polyphemus, and the odontophore cartilage of the marine whelk, Busycon canaliculatum. Animals were collected from the Cape Cod area. Samples of cartilage of Limulus perichondrium, various non-skeletal tissues, and neonatal rat calvaria, the latter as a reference standard, were homogenized in 0.1 M phosphate buffer (pH 7.1) and analyzed for protein content and the above-mentioned enzyme activities. Alkaline phosphatase specific activity was readily detected in most tissues except the invertebrate cartilage specimens in which it was present only at near-trace levels. Naphthylamidase and protease activities were present in all tissues. In a single experiment, higher phosphatase values were recorded for Limulus cartilage retaining perichondrium, but in a subsequent trial assaying cartilage retaining perichondrium, denuded cartilage, and isolated perichondrium separately, it was demonstrated that phosphatase activity resided primarily within the perichondrium. Exposure of thick cryostat sections to p-nitrophenyl phosphate confirmed the suspicion that alkaline phosphatase activity was present principally in the perichondrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Early detection of extravasation of radiographic contrast medium. Work in progress.

Microwave radiometry is a passive and noninvasive technique that allows quick detection of subcutaneous temperature changes. The feasibility of this technique for differentiating normal intravenous infusions of radiographic contrast medium from extravasations of contrast medium was tested in anesthetized dogs. Room-temperature and heated ionic and nonionic contrast media were administered at flow rates ranging from 0.2 to 9.9 mL/sec by means of a power injector. On the basis of these experiments, an algorithm to adjust for extravasation detection thresholds as a function of injection flow rates was developed. With this algorithm, results showed a false-positive rate of 0% at all infusion rates and false-negative rates of 2%, 2%, and 4% at pump speeds of 0.2, 1.0, and 9.9 mL/sec, respectively. The times of these extravasation "alarms" corresponded to maximum extravasated volumes, respectively, of 4, 6.5, and 8 mL. Microwave radiometry has clinical potential for early detection of extravasation of contrast medium administered with power injectors.

Clenbuterol, a beta 2-receptor agonist, reduces net bone loss in denervated hindlimbs.

Clenbuterol treatment for several weeks prevented up to one-third of the reduction in mineralization of femurs and tibias caused by sectioning of the sciatic nerve in young rats. The normalizing effect of clenbuterol on bone mineral content was directly proportional to similar alterations in muscle mass, which in turn could be abolished by ablation of the triceps surae or hindlimb unweighting and reduced by hypophysectomy. In contrast to the effects of inactivity, ovariectomy caused small reductions (2-4%) in bone density that were not affected by clenbuterol and were not accompanied by changes in ash weight. Together, our results suggest that the ability of beta 2-agonists to retard the loss in net muscle mass and enhance contractile tension can oppose net bone loss caused by denervation. Increases in contractile tension caused by beta 2-agonists may enhance the utility of exercise or electrical stimulation as countermeasures for the effects of scoliosis, prolonged bed rest, spinal cord injury, or weightlessness in space on bone mass.

Information preserving image compression for archiving NMR images.

This paper presents a result on information preserving compression of NMR images for the archiving purpose. Both Lynch-Davisson coding and linear predictive coding have been studied. For NMR images of 256 x 256 x 12 resolution, the Lynch-Davisson coding with a block size of 64 as applied to prediction error sequences in the Gray code bit planes of each image gave an average compression ratio of 2.3:1 for 14 testing images. The predictive coding with a third order linear predictor and the Huffman encoding of the prediction error gave an average compression ratio of 3.1:1 for 54 images under test, while the maximum compression ratio achieved was 3.8:1. This result is one step further toward the improvement, albeit small, of the information preserving image compression for medical applications.

Neutral protein-degrading enzymes in experimental fracture callus: a preliminary report.

The process of endochondral fracture healing is biochemically similar to growth plate calcification. Recent studies have identified potentially important roles for proteoglycan-degrading enzymes in the growth plate. The purpose of the study described herein was to identify, in healing fractures, neutral enzyme activities capable of degrading proteoglycans and other matrix proteins. Two sets of 60 male Sprague-Dawley rats underwent the production of closed femoral fractures. Calluses were retrieved at timed intervals, and cell and matrix vesicle fractions were prepared for electron microscopy, neutral peptidase, and alkaline phosphatase assays. In another group of 10 animals, fractions were prepared from 14-day calluses and examined for proteoglycanase activity. In the cell fractions, alkaline phosphatase, alanyl-beta-naphthylamidase, aminopeptidase, and endopeptidase activities showed somewhat parallel distributions peaking at approximately 14-17 days. In the matrix vesicle fractions, similar relative distributions were observed for alkaline phosphatase and endopeptidase. However, here the peak activities occurred up to 3 days later than they did in the cell fractions. Significant proteoglycanase activity was confirmed in both cell and matrix vesicle fractions. These findings are consistent with the hypotheses that (a) neutral peptidases, by virtue of their temporal expression in parallel with alkaline phosphatase, may be involved in preparing fracture callus matrix for calcification; and (b) matrix vesicles may convey certain of these enzymes to sites of both matrix degradation and calcification, since the same activities found in cells are found in matrix vesicles a few days later. The possibility that some of these enzymes are involved in growth factor activation remains to be investigated.

Neutral peptidase activities in matrix vesicles from bovine fetal alveolar bone and dog osteosarcoma.

Extracellular matrix vesicles from bovine fetal alveolar bone and from a dog osteosarcoma were isolated by differential centrifugation and then fractionated on a discontinuous sucrose density gradient. The fractions were examined by electron microscopy and were analyzed for protein, alkaline phosphatase, aminotripeptidase, and four different beta-naphthylamidase activities. The low-density peak of enzyme activities was shown by electron microscopy to be much more homogeneous than the crude matrix vesicle fraction. Two major peaks of protein and enzyme activities were present, one in the high and one in the low density layers. There was good correlation between the activities of alkaline phosphatase and the various peptidases in the fractions from the sucrose density gradient. These results indicate a coexistence of peptidase and alkaline phosphatase in matrix vesicles. On the other hand, there was generally no correlation between the peptidase and alkaline phosphatase activities in vesicular specimens from bovine liver obtained in the same way. Most of the peptidase activity and about half of the alkaline phosphatase activity were solubilized from bone matrix vesicles by detergents. The extracted alkaline phosphatase and alanyl beta-naphthylamidase activities were separated from each other on a DEAE-cellulose column.

"Exit, Voice, and Loyalty:" further reflections and a survey of recent contributions.

Three years after publication of Exit, Voice, and Loyalty: Responses to Decline in firms, Organizations, and States, Professor Hirschman was called upon to extend his consideration of uses of economic modes of reasoning to dissect political phenomena. The exit-voice framework is extended in connection with the search for optimal ways of organizing public services--from the State itself to day care, to the British National Health Service. These little-known further reflections are reprinted as a service to the increasing number of students of exit-voice themes in health services.

Woodpecker drilling behavior. An endorsement of the rotational theory of impact brain injury.

High-speed cinematograph films of a drilling woodpecker were examined by direct visual inspection and by a microdensitometer and computer-imaging technique. These showed (1) that the drilling trajectory is essentially linear, with very little, if any, rotation of the head; (2) that there is minimal movement after impact; (3) that the impact velocity is of the order of 600 to 700 cm/s; and (4) that the impact deceleration is of the order of 1,000 g. Dynamic and morphologic findings in the woodpecker may be highly relevant to the prevention of concussion and brain injury in man. Taken in the context of modern packaging technology and other animal and mathematical modelling research, they suggest that brain injury preventive systems could be greatly improved over those now in common use.