Organization and function of the cytoskeleton in polarized epithelial cells: a component of the protein sorting machinery.

Development and maintenance of cell-surface polarity in epithelial cells requires specialized localization of proteins to functionally and structurally distinct plasma membrane domains. The organization of these domains is dependent upon targeted delivery of transport vesicles between different membrane compartments, and upon protein sorting in the membranes of the Golgi complex and cell surface. Increasing evidence has been gathered in recent years that cytoskeletal components facilitate these processes.

Golgi spectrin: identification of an erythroid beta-spectrin homolog associated with the Golgi complex.

Spectrin is a major component of a membrane-associated cytoskeleton involved in the maintenance of membrane structural integrity and the generation of functionally distinct membrane protein domains. Here, we show that a homolog of erythrocyte beta-spectrin (beta I sigma*) co-localizes with markers of the Golgi complex in a variety of cell types, and that microinjected beta-spectrin codistributes with elements of the Golgi complex. Significantly, we show a dynamic relationship between beta-spectrin and the structural and functional organization of the Golgi complex. Disruption of both Golgi structure and function, either in mitotic cells or following addition of brefeldin A, is accompanied by loss of beta-spectrin from Golgi membranes and dispersal in the cytoplasm. In contrast, perturbation of Golgi structure without a loss of function, by the addition of nocodazole, results in retention of beta-spectrin with the dispersed Golgi elements. These results indicate that the association of beta-spectrin with Golgi membranes is coupled to Golgi organization and function.

Clathrin assembly protein AP-2 induces aggregation of membrane vesicles: a possible role for AP-2 in endosome formation.

We have examined the in vitro behavior of clathrin-coated vesicles that have been stripped of their surface coats such that the majority of the clathrin is removed but substantial amounts of clathrin assembly proteins (AP) remain membrane-associated. Aggregation of these stripped coated vesicles (s-CV) is observed when they are placed under conditions that approximate the pH and ionic strength of the cell interior (pH 7.2, approximately 100 mM salt). This s-CV aggregation reaction is rapid (t1/2 < or = 0.5 min), independent of temperature within a range of 4-37 degrees C, and unaffected by ATP, guanosine-5'-O-(3-thiophosphate), and in particular EGTA, distinguishing it from Ca(2+)-dependent membrane aggregation reactions. The process is driven by the action of membrane-associated AP molecules since partial proteolysis results in a full loss of activity and since aggregation is abolished by pretreatment of the s-CVs with a monoclonal antibody that reacts with the alpha subunit of AP-2. However, vesicle aggregation is not inhibited by PPPi, indicating that the previously characterized polyphosphate-sensitive AP-2 self-association is not responsible for the reaction. The vesicle aggregation reaction can be reconstituted: liposomes of phospholipid composition approximating that found on the cytoplasmic surfaces of the plasma membrane and of coated vesicles (70% L-alpha-phosphatidylethanolamine (type I-A), 15% L-alpha-phosphatidyl-L-serine, and 15% L-alpha-phosphatidylinositol) aggregated after addition of AP-2, but not of AP-1, AP-3 (AP180), or pure clathrin triskelions. Aggregation of liposomes is abolished by limited proteolysis of AP-2 with trypsin. In addition, a highly purified AP-2 alpha preparation devoid of beta causes liposome aggregation, whereas pure beta subunit does not, consistent with results obtained in the s-CV assay which also indicate the involvement of the alpha subunit. Using a fluorescence energy transfer assay we show that AP-2 does not cause fusion of liposomes under physiological solution conditions. However, since the fusion of membranes necessarily requires the close opposition of the two participating bilayers, the AP-2-dependent vesicle aggregation events that we have identified may represent an initial step in the formation and fusion of endosomes that occur subsequent to endocytosis and clathrin uncoating in vivo.

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.

A spectrin membrane skeleton of the Golgi complex.

The existence of a Golgi-localized membrane cytoskeleton has been revealed by the identification of two major components of the spectrin membrane skeleton, spectrin and ankyrin, that associate with the Golgi complex. Golgi spectrin was identified with an antibody specific for the beta-subunit of the erythroid isoform of spectrin (beta1Sigma1). This antibody recognizes a 220 kDa polypeptide that localizes to discrete regions of the Golgi complex and associates with Golgi membranes in a Brefeldin A sensitive manner. Two isoforms of Golgi ankyrin have been identified: a 119 kDa form (AnkG119) which represents a truncated, alternatively spliced isoform of a recently cloned novel ankyrin of the nervous system AnkG, and a larger 195 kDa ankyrin (Ank195) that cross-reacts with antibodies to erythrocyte ankyrin. A Golgi localized membrane skeleton composed of these unique membrane skeleton isoforms could serve a variety of important functions, including the maintenance of Golgi structural organization and the formation of discrete membrane domains within Golgi compartments.

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.

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.

Examination of frozen cross sections of cervical spinal intersegments in nine horses with cervical vertebral malformation: lesions associated with spinal cord compression.

Nine horses with clinical and radiographic findings of cervical vertebral malformation that were necropsied and examined using frozen cervical spinal cord cross sections were reviewed. Only cases with actual distortion of the spinal cord due to compression were selected. The goal of the study was to determine the morphologic features responsible for narrowing of the spinal canal and compression of the spinal cord. In individual cases, bony changes are associated with osteochondrosis and osteomyelitis of the dorsal articular facets and osteosclerosis of the dorsal cervical lamina. Soft tissue pathology associated with spinal cord compression included ligamentum flavum hypertrophy, joint capsule swelling and hypertrophy, and synovial cysts. In most cases, a combination of abnormalities was found in horses with spinal cord compression.

Recognition of Pneumocystis carinii in foals with respiratory distress.

Five 3-month-old foals presenting with fever and respiratory disease were found to have pulmonary abscesses with patchy to diffuse alveolar and interstitial pneumonia on post-mortem examination. All affected foals had evidence of Rhodococcus equi infection and had few to abundant Pneumocystis carinii cysts in the sections of affected lung. Of the 5 foals examined radiographically, 3 had a distinct reticulonodular (miliary) pattern which may aid in the ante-mortem diagnosis of P. carinii pneumonia (PCP). Leukocyte counts of foals with PCP were significantly greater than in the control group of foals with uncomplicated bacterial pneumonia. Foals with PCP tended to be more tachypnoeic than the control foals and 4 of the 5 PCP+ foals appeared dyspnoeic before death. The ante-mortem recognition of PCP may be expedited by bronchoalveolar lavage and successful treatment of foals with PCP may require the administration of adequate levels of potentiated sulphonamides.

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.

Lack of residual lung damage in horses in which Rhodococcus equi-induced pneumonia had been diagnosed.

The effect of prior Rhodococcus equi-induced pneumonia on pulmonary health was investigated in 5 horses (< 24 months old) using endoscopy, radiography, hematologic and bronchoalveolar lavage analyses, and pulmonary function testing. Rhodococcus equi-induced pneumonia had been diagnosed in principal horses when they were foals. Diagnosis was based on positive results of transtracheal aspiration and thoracic radiography at the time of initial clinical examination. Results of reevaluation of the respiratory system of these horses (R+) were compared with those of 5 age-matched healthy horses (R-) that lacked clinical or historical evidence of foalhood pneumonia. Significant differences in variables between the 2 groups of horses were not evident. In both groups, most horses had radiographic evidence of an accentuated bronchointerstitial pattern, although results of analysis of bronchoalveolar lavage specimens were normal and mononuclear cells predominated. Variability in results of the pulmonary function tests was observed within the between the 2 groups of horses. Only normalized dynamic lung compliance was slightly lower in the previously infected horses, but this difference was not significant. We concluded that horses previously infected with and successfully treated for R equi-induced pneumonia do not have detectable evidence of residual lung damage.

Effects of intramuscular administration of glycosaminoglycan polysulfates on signs of incipient hip dysplasia in growing pups.

We tested the hypothesis that treatment of growing, susceptible (to hip dysplasia) pups by IM administration of glycosaminoglycan polysulfates would mitigate the signs of incipient hip dysplasia. In 1 experiment, 7 pups, selected at random from 2 litters, were administered glycosaminoglycan polysulfates (2.5 mg/kg of body weight, IM) twice weekly, and 7 control pups from the same litters were given sterile buffered 0.9% saline solution from the age of 6 weeks to 8 months. Hip joints were examined by radiography, with pups in the standard, limbs-extended position. At 8 months of age, all pups in this experiment did not manifest femoral head subluxation radiographically. The Norberg angle, a measure of coxofemoral congruity, improved from a mean +/- SEM value of 102 degrees +/- 1 degrees in controls to 106 degrees +/- 1 degrees in treated pups (P = 0.008). Pups were not subjected to necropsy. In the second experiment, 8 pups were selected at random from 2 litters and were administered 5 mg of glycosaminoglycan polysulfates/kg, IM, twice weekly from 6 weeks to 8 months of age. Similarly, 8 control pups were administered saline solution. At 8 months of age, hip joints were examined by radiography with pups in the standard position; at necropsy, intra-articular tissues were evaluated macroscopically and biochemically. Of 8 treated pups, none had subluxation radiographically, whereas 4 of 8 control dogs had femoral head subluxation. Mean Norberg angle on the radiographs was 109.7 degrees +/- 1.6 degrees for the treated group and was 101.5 degrees +/- 1.6 degrees for controls, representing a mean improvement in coxofemoral congruity of 8.2 degrees in the treated pups.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Joint laxity and its association with hip dysplasia in Labrador retrievers.

A study was done to determine whether radiographic-distraction measurement of coxofemoral joint (hip) laxity at 4 and 8 months of age can serve as a predictor of hip dysplasia in older Labrador Retrievers. The method of Smith, Biery, and Gregor was used for radiologic examination of hips and for evaluation of radiographs. Mean (+/- SEM) distraction laxity (ie, distraction index) for 10 adult disease-free dogs was 0.29 +/- 0.05, whereas a group of 8 dogs with dysplastic hips had mean distraction index of 0.60 +/- 0.10 (P < 0.05). Mean distraction index at 4 months of age for 11 pups of 4 litters from matings between dogs with normal hips was 0.39 +/- 0.07, and was 0.54 +/- 0.04 for 31 pups of 7 litters from matings between dogs with hip dysplasia. The distraction index and, thus, joint laxity at that age was significantly (P = 0.0351) different for the 2 groups. The distraction index at 4 months correlated positively with the distraction index at a later age at necropsy (r = 0.43; P = 0.0289). Distraction index < 0.4 at 4 months of age predicted normal hips in 88% of cases and distraction index > or = 0.4 predicted hip dysplasia in 57% of the dogs. Logistic regression modeling indicated that the odds of a hip being normal decreased with increasing distraction index, and thus, with increasing joint laxity. The logistic regression models provided a reasonable mathematical description of the data. Based on the logistic model of the data, distraction indexes between 0.4 and 0.7 at either 4 or 8 months of age were not associated strongly enough with evidence of disease to be clinically reliable in predicting, on an individual basis, the outcome for dysplastic hip conformation when dogs were older. Index > 0.7 was associated with high probability for developing dysplastic joints and distraction index < 0.4 predicted normal hips with high probability.

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)

Caudocranial horizontal beam radiographic projection for evaluation of femoral fracture and osteotomy repair in dogs and cats.

A new radiographic projection of the femur was evaluated for use in the assessment of fracture or osteotomy repair in small animals. The view is obtained by directing the x-ray beam horizontally through the hind limb, from caudad to craniad, with the animal positioned in lateral recumbency, the hip flexed, and the stifle extended. Views obtained, using the new projection, were compared with the standard ventrodorsal views of the pelvis, with hind limbs extended. Osteotomy lines in the femoral shaft were significantly (P less than 0.01) more visible on the horizontal beam view. Significant difference was not evident in visibility of fracture lines between the 2 radiographic projections. The horizontal beam view was easily obtained, and equivalent to the standard ventrodorsal view for radiographic evaluation of femoral fracture and osteotomy repair.

Use of fenbendazole for treatment of Crenosoma vulpis infection in a dog.

An 8-month-old Labrador Retriever was examined because of a 1-month history of productive coughing unresponsive to ampicillin treatment. Larvae of Crenosoma vulpis were found in fecal samples examined by zinc sulfate centrifugation and Baermann technique. Physical examination abnormalities or larvae in fecal samples were not detected 6 weeks after treatment with prednisone (1 mg/kg, PO, q 24 h, for 7 days, then 0.5 mg/kg, PO, q 48 h, for 8 days) and fenbendazole granules (50 mg/kg, PO, q 24 h, for 3 days). This report suggests that fenbendazole may be effective for treating Crenosoma vulpis infection in dogs.

Thoracotomy in adult dairy cattle: 14 cases (1979-1991).

Fourteen cows were subjected to thoracotomy as an aid in the treatment of either septic pericarditis (n = 7) or unilateral pleuritis (fibrous or purulent; n = 7). Thoracic lesions were primary in 4 cows, secondary to traumatic reticuloperitonitis in 9 cows, and secondary to extension of a liver abscess in 1 cow. Thoracotomy was performed on 9 cows under local anesthesia. Surgery was performed on 5 cows under general anesthesia; 2 died during anesthesia, and 2 others were euthanatized. Of the 10 cows allowed to recover from surgery, 4 had pericarditis and 6 had pleuritis. Four cows with pleuritis had thoracic abscesses. All but 1 cow with pericarditis died or were euthanatized, and 5 of the 6 cows with pleuritis were discharged from the hospital. A year after surgery, 1 cow was culled because of infertility, and the other 5 cows were returned to production.

Automobile-induced obstruction of the intrathoracic caudal vena cava in a dog.

A two-year-old labrador retriever was examined after a year's history of persistent ascites and exercise intolerance that began shortly after the dog was struck by an automobile. Contrast venography showed tortuosity of the intrathoracic caudal vena cava with partial obstruction caused by kinking of the vessel. Surgical resection of a fibrous connective band that was found to be tethering the intrathoracic vena cava ventrally, creating a partial obstruction, was performed. A hypothesis of the aetiology of this phenomenon is presented.