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A Hirschman

Publications and source records attributed to A Hirschman.

At least 19 recordsLinked to original sources
PubMed2001

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.

Adenosine Triphosphate↗
PubMed1992

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)

Alkaline Phosphatase↗
PubMed1991

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.

Adrenergic beta-Agonists↗
PubMed1989

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.

Alkaline Phosphatase↗
PubMed1983

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.

Alkaline Phosphatase↗
PubMed1979

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.

Animals↗
PubMed1977

Naphthylamidase (arylaminopeptidase) activities in rat cartilages and bone.

Extracts of epiphyseal articular and costal cartilages and of metaphyseal bone showed different profiles of activity against 21 aminoacyl beta-naphthylamides. Except for the activity against arginyl and lysyl beta-naphthylamides, most of the activities required the presence of Triton X-100 for extraction, suggesting that in situ, the enzymes are bound to or within membranous structures. Histochemical studies demonstrated that most, if not all, activity was intracellular. The activity is enhanced by Co2+. TAME (2 X 10(-4) mol/l) inhibits activity against leucyl beta-naphthylamide and some others, but has little, if any, inhibition of activity against alanyl beta-naphthylamide. Gel filtration of the extract showed a high molecular weight active fraction eluting with proteoglycan, and an active fraction with an approximate molecular weight of about 230,000. Two active fractions are obtained by gradient elution from DEAE-cellulose.

Amino Acids↗
PubMed1976

In vitro accumulation of mineral components by invertebrate cartilage.

In vitro mineralization of the gill cartilage of Limulus (horse-shoe crab) has been reported previously (Eilberg et al. 1975). The present study demonstrates that cranial cartilage of Loligo (squid) and odontophore cartilage of Busycon (marine snail) also mineralize in vitro in hydroxyapatite-metastable media, but not in carbonate-metastable media. In all three of these cartilages, light phase-contrast microscopy revealed that the mineral phase occured in the form of spherical or ovoid granules ranging between 2 and 9 mum in diameter. During mineralization, the granules appeared successively in the perichondrium, in the matrix, and finally, within chondrocytes. Mineralization occurred more rapidly in Eusycon odontophore cartilage which has a significantly higher content of phosphatidyl serine than in Limulus gill or Loligo head cartilages. In all tissues the mineralization process is related to temperature, taking place most actively at 37 degrees C and only weakly at 50 degrees C.

Animals↗
PubMed1976

Woodpeckers and head injury.

The woodpecker is an experiment in Nature, a model for the investigation of mechanisms of basic importance for head injury and its prevention. A preliminary anatomical study of the woodpecker's head suggests that it may be fruitful to explore impact protective systems which are radically different from those in common use.

Animals↗
PubMed1969

Soluble sulfatase in growing bone of rats.

Soluble sulfatase has been found in epiphyseal, articular, and rib cartilages and in metaphysis and bone marrow of the rat. The greatest activity in young rats is in the metaphysis. Young rats had higher levels of activity in epiphyseal and articular cartilage and in the metaphysis than did the older rats.

Aging↗