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K A Beck

Publications and source records attributed to K A Beck.

At least 37 records · Page 2Linked to original sources
PubMed1991

Clathrin domains involved in recognition by assembly protein AP-2.

The domains on clathrin responsible for interaction with the plasma membrane-associated assembly protein AP-2 have been studied using a novel cage binding assay. AP-2 bound to pure clathrin cages but not to coat structures already containing AP that had been prepared by coassembly. Binding to preassembled cages also occurred in the presence of elevated Tris-HCl concentrations (greater than or equal to 200 mM) which block AP-2 interactions with free clathrin. AP-2 interactions with assembled cages could also be distinguished from AP-2 binding to clathrin trimers by sodium tripolyphosphate (NaPPPi), which binds to the alpha subunit of AP-2 (Beck, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4442-4447). At concentrations of 1-5 mM, NaPPPi blocked clathrin-triskelion binding; in contrast, interactions with cages persisted in the presence of 25 mM NaPPPi. To begin to identify the region(s) of the clathrin molecule important in recognition by AP-2, clathrin cages were proteolyzed to remove heavy chain terminal domains and portions of the distal leg as well as all of the light chains. AP-2 bound to these "clipped cages"; however, unlike the interaction with native cages, binding of AP-2 to clipped cages was sensitive to the lower concentrations of both Tris-HCl and NaPPPi which disrupt interactions of AP-2 with clathrin trimers. Reconstitution of the clipped cages with clathrin light chains did not restore resistance of AP-2 binding to Tris-HCl. We conclude that one binding site for AP-2 resides on the hub and/or proximal part of the clathrin triskelion whereas a second site is likely to involve the terminal domain and/or distal leg; the second site is manifested only in the assembled lattice structure. We suggest that these two distinct binding interactions may be mediated by the two unique large subunits within the AP-2 complex, acting sequentially during assembly.

Animals↗
PubMed1991

Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2.

Several components of the phosphoinositide cycle have been found to interact specifically and at physiological concentrations with the plasma membrane-associated clathrin assembly (adaptor) protein AP-2. These include phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, which are present at the plasma membrane, as well as other polyphosphoinositols. ATP and other polyphosphate molecules complete with the polyphosphoinositols, however, they are at least 80-fold less potent. Also, the effect of ATP, unlike the polyphosphoinositols, is blocked by physiological concentrations of Mg2+. Photoaffinity labeling of AP-2 by [alpha-32P]8-azidoadenosine 5'-triphosphate and its competition by polyphosphoinositols has been used to identify the alpha subunit of the AP-2 complex as the site of specific interaction with the polyphosphoinositols and to confirm direct ultrafiltration binding experiments. Proteolytic dissection of the labeled AP-2 demonstrated that binding occurred exclusively on the N-terminal portion of the alpha subunit. Interaction of purified AP-2 with sub-microM concentrations of polyphosphoinositols has inhibitory effects on a novel AP-2 self-association described in the accompanying paper (Beck, K. A., and Keen, J. H., J. Biol. Chem. 266, 4437-4441), and at higher concentrations on the binding of AP-2 to dissociated clathrin trimers as well as AP-2-mediated clathrin coat assembly. Review of the literature shows that several physiological stimuli that are known to result in increased coat pit formation in intact cells correlate with increased phosphoinositide turnover. These in vivo correlations and the in vitro observations reported here suggest that coated membrane and phosphoinositide cycles may be interdependent within cells.

Adaptor Proteins, Vesicular Transport↗
PubMed1991

Self-association of the plasma membrane-associated clathrin assembly protein AP-2.

A self-association reaction involving the plasma membrane-associated clathrin assembly protein AP-2 has been detected by incubating AP-2 alone under solution conditions that would favor the assembly of complete coat structures if clathrin were present. Self-association was rapid, unaffected by nonionic detergents, readily reversible, and gave rise to sedimentable aggregates. Only the AP subtype AP-2 exhibited self-association: the structurally or functionally related assembly proteins AP-1 and AP-3 and unrelated proteins neither self-associated nor were incorporated into the AP-2 aggregate. AP-2 interactions responsible for self-association were of high affinity, with an apparent Kd of approximately 10(-8)M. By proteolytic dissection, the self-association domain was localized to the core of the molecule containing the intact 50- and 16-kDa polypeptides in association with the truncated 60-66-kDa moieties of the parent alpha/beta polypeptides. Self-association of the intact AP-2 molecule was pH-dependent, exhibiting an apparent pKa approximately 7.4. While it is unlikely that the large AP-2 aggregates formed in solution are themselves biologically relevant structures, the AP-2 interactions involved in their formation have properties consistent with their occurrence in intact cells and thus may be important in cellular functions of the plasma membrane-localized assembly protein.

Adaptor Proteins, Vesicular Transport↗
PubMed1991

Duchenne's cardiomyopathy in a canine model: electrocardiographic and echocardiographic studies.

Thirteen dogs affected with X-linked Duchenne's muscular dystrophy and 11 female carrier dogs were studied by electrocardiography (ECG) and echocardiography. Twelve of the affected dogs were studied as immature animals and followed at 1 to 6 month intervals until they were 7 to 46 months of age. Compared with control dogs, affected dogs had significantly increased (p less than 0.02) Q/R ratios in ECG leads II, III, aVF, CV6LL (V2) and CV6LU (V4). Carrier dogs had significantly increased (p less than 0.02) Q/R ratios in leads V2 and V4. The Q/R ratio increased in three of six dogs followed up from age 6 months to greater than 2 years. The PR intervals were significantly shorter (p less than 0.02) in affected dogs. Ventricular arrhythmias were identified in four of six mature affected dogs. Two-dimensional echocardiography revealed distinctive hyperechoic lesions in 12 of the 13 affected dogs and in 6 of the 11 carrier dogs. Hyperechoic lesions corresponded to calcified myocardium and surrounding dense connective tissue. This study establishes the dog affected with Duchenne's muscular dystrophy as an animal model of Duchenne's cardiomyopathy and demonstrates that the heart in carrier dogs is affected by the dystrophic process.

Animals↗
PubMed1991

Evaluation of praziquantel for treatment of experimentally induced paragonimiasis in dogs and cats.

Praziquantel was used successfully for treatment of a small number of dogs and 1 cat infected with Paragonimus kellicotti. To further evaluate the usefulness of this drug in treating such infections, 7 cats and 7 dogs were inoculated orally with metacercariae (12 and 20 to 22, respectively) obtained from crayfish, then were treated after the infections became patent; 2 cats and 2 dogs served as noninfected controls. Beginning 1 week before infection, and continuing weekly thereafter, physical, hematologic, and fecal examinations were performed on each animal; thoracic radiography was performed every other week. By postinoculation week 6, all dogs given metacercariae had patent infection diagnosed on the basis of positive results of fecal examination. By postinoculation week 7, 5 cats had confirmed patent infection, but 2 cats given metacercariae never had patent infection or had signs of infection. Clinical signs of infection were minor and included increased respiratory tract noise, slight inducible cough, or mild dyspnea. Transient eosinophilia was detected in dogs around postinoculation week 3. Pretreatment radiography revealed cavitated lesions in cats only; pleural lines and patchy infiltrates in cats and dogs; or pneumothorax in dogs only. The treatment regimen consisted of 23 mg of praziquantel/kg of body weight given every 8 hours for 3 days; 1 infected cat and dog were not treated. By 11 days after treatment, eggs had disappeared from the feces of infected animals, and marked resolution of lung lesions was evident radiographically.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Identification of the clathrin-binding domain of assembly protein AP-2.

The clathrin binding domain of the assembly protein AP-2 has been identified by proteolytically cleaving AP-2 into 2 discrete moieties, termed light and heavy mero-AP (LM-AP and HM-AP), and testing their ability to bind to clathrin assembled into cage structures or to clathrin trimers immobilized on Sepharose. The smaller product (LM-AP), which contains 20-40-kD fragments of the parent 100-kD polypeptides and which comprises two small appendages in the native AP-2 molecule, did not significantly interact with clathrin under either condition. In contrast, the HM-AP complex, which forms the larger central mass of the native AP-2 structure and contains uncleaved 50-kD and 16-kD polypeptides as well as 60-66-kD fragments of the parent 100-kD polypeptides, retained binding activity for both dissociated and assembled clathrin.

Adaptor Proteins, Vesicular Transport↗
PubMed1988

Experimental respiratory decompression sickness in sheep.

Respiratory decompression sickness (RDCS, "the chokes") is a potentially lethal consequence of ambient pressure reduction. Lack of a clearly suitable animal model has impeded understanding of this condition. RDCS, unaccompanied by central nervous system signs, occurred in 17 of 18 unanesthetized sheep exposed to compressed air at 230 kPa (2.27 ATA) for 22 h, returned to normal pressure for approximately 40 min, and taken to simulated altitude (0.75 ATA, 570 Torr). Respiratory signs, including tachypnea, sporadic apnea, and labored breathing, were accompanied by precordial Doppler ultrasound evidence of marked venous bubble loading. Pulmonary arterial pressures exceeded 30 Torr in five catheterized sheep that died or became moribund. Hypoxemia (arterial Po2 less than 40 Torr), neutropenia, and thrombocytopenia were observed. Peribronchovascular edema was the most prominent necropsy finding. Chest radiography indicated interstitial edema in most affected sheep. High body weight and catheterization predisposed the sheep to severe RDCS. It appears that this protocol reliably provides a useful animal model for studies of RDCS and obstructive pulmonary hypertension, that the precipitating event is massive pulmonary embolization by bubbles, and that venous bubbles, detected by Doppler ultrasound, can signal impending RDCS.

Altitude↗
PubMed1987

The clathrin coat assembly polypeptide complex. Autophosphorylation and assembly activities.

A 50-kDa polypeptide that is rapidly phosphorylated on addition of [gamma-32P]ATP to isolated clathrin-coated vesicles is shown here to be identical to the 50-kDa component (AP50) of the clathrin assembly protein (AP), a complex that promotes the assembly of clathrin coat structures under physiological conditions of pH and ionic strength. Phosphorylation of the AP50 occurred readily at 0 degrees C, almost exclusively on a threonyl residue(s). This reaction is attributable to autophosphorylation, since the AP50 was able to covalently incorporate 32P from [gamma-32P]ATP after separation by either one- or two-dimensional sodium dodecyl sulfate gel electrophoresis. Kinetic studies in solution were consistent with an intramolecular phosphorylation event; in addition, a concentration-dependent increase in AP50 phosphorylation was observed that may reflect intermolecular AP-AP activation of autophosphorylation. The phosphorylated AP50 was resistant to several inorganic phosphatases tested but was a substrate for protein phosphatases 1 and 2A, suggesting that a physiological phosphorylation-dephosphorylation cycle may exist. The phosphorylation state of the AP50 did not affect the ability of the AP to promote in vitro clathrin coat assembly. These and other data suggest that unique structural domains of the assembly protein are responsible for assembly (the 100-kDa components) and autophosphorylation (the AP50) and that the latter may be active as a protein kinase in the intact cell.

Adaptor Protein Complex 2↗
PubMed1987

Proteases occurring in the cell membrane: a possible cell receptor for the Bowman-Birk type of protease inhibitors.

The legume-derived Bowman-Birk trypsin and chymotrypsin protease inhibitors (BBI) are effective anticarcinogens in vivo and in vitro. The chymotrypsin-inhibitory domain has been shown to be responsible for this anticarcinogenic action. In this study we identify hydrolytic enzymes by their ability to hydrolyze the relatively specific chymotrypsin substrate succinyl-Ala-Ala-Pro-Phe-aminomethyl coumarin. Results presented in this study show: there is an approximately 2-fold increase in the activity of these enzyme(s) between normal and transformed C3H/10T1/2 cells; there are five such enzymes associated with transformed cells (separated by diethylaminoethyl-cellulose chromatography); only two of these enzymes are inhibited by BBI; the BBI-inhibitable enzymes are membrane associated; the BBI-inhibitable enzymes are similar to each other but different from pancreatic chymotrypsin. BBI has thus distinguished a subpopulation of enzymes capable of hydrolyzing succinyl-Ala-Ala-Pro-Phe-aminomethyl coumarin which may mediate the transformation of C3H/10T1/2 cells.

Cell Line↗
PubMed1982

Indirect imaging of the canine optic nerve, using metrizamide (optic thecography).

Between the optic nerve and its sheath is a small, CSF-filled space that communicates with the subarachnoid space of the brain. A technique for the radiographic opacification of the optic nerve subarachnoid space following the intrathecal injection of metrizamide was investigated in 8 dogs. The technique enabled indirect visualization of the optic nerves from the optic chiasm to the eyeball and of structures within the subarachnoid basal cisterns of the brain, including the optic chiasm, hypophysis, and blood vessels of the cerebral arterial circle. Displacement and obstruction of the optic nerve subarachnoid space were demonstrated after surgically creating optic nerve lesions to simulate orbital tumors and trauma. The technique was found to be safe, effective, and advantageous over other techniques currently available for the visualization of these structures.

Animals↗
PubMedDate not supplied

The use of a silicone T-tube to treat tracheal stenosis in a llama.

A female llama was presented at 4 days of age with severe dyspnea resulting from bilateral choanal atresia. A tracheostomy was performed before surgical treatment of the airway obstruction. Although the choanal atresia was successfully corrected, tracheal stenosis secondary to mucosal necrosis, malacia of the cartilage rings, and proliferation of intraluminal granulation tissue at and distal to the tracheal stoma developed. The affected segment of the trachea was resected and an end-to-end anastomosis was performed, but the lumen again became obstructed by granulation tissue. A silicone "T-tube" was placed in the trachea to provide a patent airway and intraluminal support. The llama has done well for 12 months with this prosthesis, and the complications that are often seen with long-term use of traditional tracheostomy tubes have not developed.

Animals↗
PubMedDate not supplied

Effect of sagittal plane positioning errors on measurement of the angle of inclination in dogs.

Angles of inclination were calculated from ventrodorsal (VD) and caudocranial horizontal beam (CaCrHB) radiographs of 17 anesthetized dogs, and from radiographs of left femurs of the same dogs positioned 0 degree, 10 degrees, 15 degrees, and 20 degrees from the cassette in the sagittal plane. Angles of inclination also were measured directly from radiographs of the bones rotated to correct for anteversion. Calculated angles of inclination from the bones at 10 degrees, 15 degrees, and 20 degrees from the cassette were significantly different from the 0 degree values obtained by calculation and direct measurement. Inclination angles from live dogs were consistently larger than those from 0 degree bones. Differences between angles of inclination calculated from VD and CaCrHB radiographs of live dogs were not significant.

Animals↗
PubMedDate not supplied

The effect of long-term bone plate application for fixation of radial fractures in dogs.

This study was divided into two phases. In the in vitro phase, a stainless steel bone plate was applied to the cranial surface of the radius in 14 canine limbs. The effect of the presence of a bone plate on bone density analysis using radiographic photodensitometry (RP) was evaluated by comparing the density measurement of the unplated limb to the density measurement of the plated limb. The optical density of the plated bones was 12% greater than that of the unplated bones. This information was used as a correction factor for the in vivo study. In the in vivo phase, 23 dogs with radial and ulnar fractures were examined for complications associated with the long-term application of a stainless steel plate applied to the cranial surface of the radius. In 14 dogs, RP analysis was used to compare the plated limb with the normal, contralateral limb. No significant differences in radial cortical bone density existed between the plated limb and the contralateral limb after taking into account the effect a bone plate had on photodensitometry readings. There was no significant correlation between the change in radial cortical density and the duration of bone plate application, suggesting that a steady state between bone loss and bone production occurs after long-term plate fixation of the fractured canine radius. The majority (87%) of the dogs with a plate applied to the radius greater than 1 year had normal limb usage when standing, walking, or running.

Absorptiometry, Photon↗
PubMedDate not supplied

Fibrodysplasia ossificans progressiva in the cat. A case report.

Clinical, radiographic, electromyographic, and pathologic findings in a cat with fibrodysplasia ossificans progressiva are described. The features of five previously reported cases of this feline disorder are also presented. This disorder affects young adult to middle-aged cats of both sexes. Characteristic clinical features include progressive stiffness of gait, with enlargement of proximal limb musculature. Radiography reveals multiple mineralized densities within the affected musculature. The clinical course is rapid, with development of severe disability within 2 weeks to several months. Electromyographic and pathologic findings suggest that this is a disorder of connective tissue, affecting primarily the epimysium, tendons, and fasciae, and results in marked proliferation of fibrovascular connective tissue, with associated chondroid and osseous metaplasia.

Animals↗