Interconversion of structural and contractile actin gels by insertion of myosin during assembly.

Extracts of the soluble cytoplasmic proteins of the sea urchin egg form gels of different composition and properties depending on the temperature used to induce actin polymerization. At temperatures that inactivate myosin, a gel composed of actin, fascin, and a 220,000-mol-wt protein is formed. Fascin binds actin into highly organized units with a characteristic banding pattern, and these actin-fascin units are the structural core of the sea urchin microvilli formed after fertilization and of the urchin coelomocyte filopods. Under milder conditions a more complex myosin-containing gel is formed, which contracts to a small fraction of its original volume within an hour after formation. What has been called "structural" gel can be assembled by combining actin, fascin, and the 220,000-mol-wt protein in 50-100 mM KCl; the aim of the experiments reported here was to determine whether myosin could be included during assembly, thereby interconverting structural and contractile gel. This approach is limited by the aggregation of sea urchin myosin at the low salt concentrations utilized in gel assembly. A method has been devised for the sequential combination of these components under controlled KCl and ATP concentrations that allows the formation of a gel containing dispersed myosin at a final concentration of 60-100 mM KCl. These gels are stable at low (approximately 10 micron) ATP concentrations, but contract to a small volume in the presence of higher (approximately 100 micron) ATP. Contraction can be controlled by forming a stable gel at low ATP and then overlaying it with a solution containing sufficient ATP to induce contraction. This system may provide a useful model for the study of the interrelations between cytoplasmic structure and motility.

Induction of either contractile or structural actin-based gels in sea urchin egg cytoplasmic extract.

The gel formed by warming the 100,000 g supernate of isotonic extracts of sea urchin eggs to 40 degrees C is made up of actin and two additional proteins of mol wt of 58,000 and 220,000. Actin and 58,000 form a characteristic structural unit which has now been identified in the microvilli of the urchin egg and in the filopods of urchin coelomocytes. However, egg extract gels did not contract as those from other cell types do, and the aim of these experiments was to determine the reason for this lack of contraction. Although the extracts are dialyzed to a low ionic strength, myosin is present in soluble form and makes up approximately 1% of the protein of the extract. It becomes insoluble in the presence of high ATP concentrations at 0 degrees C, and the precipitate formed under these conditions consists almost entirely of myosin. This procedure provides a simple method of isolating relatively pure myosin without affecting other extract components and functions. Contraction will follow gelation in these extracts if the temperature and time of incubation used to induce actin polymerization are reduced to minimize myosin inactivation. At the optimal ATP and KCl concentration for contraction, the contracted material has an additional 250,000 component and contains very little 58,000. The conditions found to provide maximum gel yields favor the formation of the actin-58,000-220,000 structural gel, while reduced temperature and increase in KCl concentration results in a contractile gel whose composition is similar to those reported from amoeboid cell types. Both the structural protein cores found in the egg microvilli and a gel contraction related to the amoeboid motion which is seen in later urchin embryonic development can thus be induced in vitro in the same extract.

The mitotic apparatus. Identification of the major soluble component of the glycol-isolated mitotic apparatus.

Mitotic apparatuses (MA) isolated from metaphase sea urchin eggs in 12% hexylene glycol at pH 6.4 can be dissolved rapidly in 0.6 M KCl, and more than one-half of the total protein of the MA is soluble under these conditions. In the phase-contrast microscope, the fibrous structure of the MA can be seen to disintegrate in KCl solution, leaving only granular material which, in the electron microscope has been seen to be largely vesicular, with no evidence of microtubules or other fibrous elements. The KCl-soluble material thus must contain the soluble components of the microtubules and consists of one major, homogeneous component with a sedimentation coefficient of 22 Svedbergs, and a much smaller amount of more heterogeneous material sedimenting at 4-5S. A component similar to the 22S component can be identified in extracts of unfertilized eggs, where it forms approximately 8% of the total cell protein. The amount of this protein present in the cell is considerably in excess of that involved in the MA, as can be shown by its presence in the soluble supernate from a mitotic apparatus isolation. This protein must form part of, or be associated with, the fibrous structure of the MA in some fashion that allows its release only upon the dissolution of the mitotic apparatus.

Preparation and purification of polymerized actin from sea urchin egg extracts.

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F-actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.

Direct isolation of the hyaline layer protein released from the cortical granules of the sea urchin egg at fertilization.

Treatment of the eggs of the sea urchin with a 1 M solution of glycerol at fertilization allows the recovery from this solution of the protein released from the cortical granules, including that which would normally give rise to the hyaline layer. The calcium-gelable protein previously extracted from whole eggs and from isolated cortical material was found to be present in the glycerol solution, confirming its localization in the cortical granules and its role in the hyaline layer. Quantitative measurements on the eggs of two Hawaiian species, Colobocentrotus atratus and Pseudoboletia indiana, which have the widest variation in the gel protein content, demonstrated that a proportionate amount of this material was released at fertilization in these species, which correlates with the thickness of the hyaline layer in the two cases. In addition, the calcium-insoluble fraction of Sakai can be extracted from these eggs after removal of the hyaline protein by glycerol, showing that this is a different material. A simple method for the separation of the hyaline protein from the calcium-insoluble fraction in solution is provided.

Actin polymerization and interaction with other proteins in temperature-induced gelation of sea urchin egg extracts.

The gel which forms on warming the extracts of the cytoplasmic proteins of sea urchin eggs has been separated into two fractions, one containing F-actin and the other containing two proteins of 58,000 and 22,000 mol wt. When combined in 0.1 M KCl, even at 0 degrees C, these components will form gel material identical to that formed by warming extracts. This gel is a network of laterally aggregated F-actin filaments which are in register and which display a complex cross-banding pattern generated by the presence of the other two proteins. Low concentrations of calcium block the assembly of these proteins to form this complex structure, which may play some cytoskeletal role in the cytoplasm. This association of F-actin with the other proteins to form a gel is very likely the last step fo the process occurring in warmed extracts. At low temperatures, gelation of extracts is limited by the relative absence of F-actin, as demonstrated by the inability to sediment it at 100,000 g and also by the fact that gelation occurs immediately if exogenous F-actin is added to cold extracts. The transformation of the G-actin present in the extract to the F-form is apparently repressed at low temperatures. This is shown directly by the failure of added G-actin to polymerize at low temperatures in the presence of extract. These observations resemble those which have been reported on preparations from amoeboid cells and may be significant in the involvement of actin and these other proteins in cell division and later developmental processes.

A comparative study of the isolation of the cortex and the role of the calcium-insoluble protein in several species of sea urchin egg.

A comparative study was made of the isolation of the cortex in the eggs of several sea urchin species. Since the isolation method developed by Sakai depends on the presence of magnesium in the medium, the protein composition of the cortex was investigated to determine whether the protein component of the egg described by Kane and Hersh which is gelled by divalent ions, is present in these cortices. Isolation of the cortex was found to require the same divalent ions at the same concentrations as protein gelation, and in the eggs of some species much of the gel protein of the cell was found in the isolated cortical material. In the eggs of other species a smaller fraction of this protein was found in the isolated cortex, although it was more concentrated there than in the endoplasm, and in one species this protein appeared to be uniformly distributed throughout the cell. These results indicate that this protein is localized in the cortical region of the eggs of some species of sea urchin, possibly in the cortical granules, but also point up the fact that results from one species cannot be uncritically extrapolated to others.

Some properties of hyalin: the calcium-insoluble protein of the hyaline layer of the sea urchin egg.

The principal protein component of the hyaline layer of sea urchin eggs is the calcium-insoluble protein first described by Kane and Hersh. The protein hyalin is abnormally high in acidic amino acids, almost devoid of basic amino acids, and characteristically rich in valine and proline. Essentially all of the cysteine present is found in the disulfide form; no evidence points to intermolecular disulfide linkages. Hyalin from several species has a minimal subunit weight of about 100,000, though evidence exists for a particle three times this weight in urea or guanidine hydrochloride from one species. Optical rotatory dispersion measurements indicate no alpha-helix content, though the dispersion has unique characteristic features. Addition of small quantities of calcium causes hyalin to gel to a birefringent fibrous form. The fibrous, birefringent form of hyalin is rendered isotropic upon addition of EDTA, but the birefringence is restored with re-addition of divalent cation.

Serological similarity of flagellar and mitotic microtubules.

An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins.

Metolazone and spironolactone in cirrhosis and the nephrotic syndrome.

Eighteen patients with hepatic cirrhosis or nephrotic syndrome and having edema and/or ascites were treated during successive periods with metolazone 5 to 40 mg/day, spironolactone 100 mg/day, and with both diuretics concurrently. Metolazone alone produced a marked diuresis, natriuresis, and weight loss in 8 patients. Spironolactone alone had little effect, but the addition of metolazone renewed diuresis and natriuresis and resulted in additional substantial weight losses in all patients responsive to metolazone alone. Concurrent spironolactone and metolazone also induced moderate diuretic effects in some patients who failed to respond to either drug alone. The drugs were well tolerated; the administration of spironolactone with metolazone prevented decreases in serum potassium, which had occurred during treatment with metolazone alone.

A controlled trial of chlorofluorocarbon-free triamcinolone acetonide inhalation aerosol in the treatment of adult patients with persistent asthma. Azmacort HFA Study Group.

STUDY OBJECTIVE: To compare the dose response, efficacy, and safety of inhaled triamcinolone acetonide (TAA) with a hydrofluoroalkane (HFA) propellant (75 microg/puff), TAA with a chlorofluorocarbon propellant (dichlorodifluoromethane [P-12]; 75 microg/puff), and placebo in adult patients with persistent asthma. DESIGN: Multicenter, randomized, double-blind, placebo-controlled, parallel-group study of 514 adult patients with persistent asthma. INTERVENTIONS AND MEASUREMENTS: Patients received 8 weeks of treatment with 150, 300, or 600 microg/d of TAA HFA, the same doses of TAA P-12, or placebo following a 5- to 21-day baseline period. Efficacy was assessed by spirometry, and by daily recordings of albuterol use, peak expiratory flow (PEF), asthma symptom ratings, and nighttime awakenings throughout the study. RESULTS: Linear trend analysis showed that both formulations of TAA at all doses produced statistically significant improvements compared with placebo in spirometry, asthma symptom scores, albuterol use, and PEF. Significant improvement was seen as early as 24 h for morning PEF and as early as 1 week for FEV(1) (TAA HFA, 600 microg/d; TAA P-12, 300 and 600 microg/d) and albuterol use (all doses of both formulations). The P-12 and HFA formulations had comparable efficacy. A dose response showing greater improvement with higher doses was evident for the majority of parameters for both formulations. The incidences of adverse events were similar across all treatment groups with no dose-related trends. CONCLUSION: HFA and P-12 formulations of TAA inhalation aerosol were therapeutically equivalent and showed comparable safety and dose-related efficacy in the treatment of patients with persistent asthma.

A 250K-molecular-weight actin-binding protein from actin-based gels formed in sea urchin egg cytoplasmic extract.

The actin-based gel formed at 35 degrees C in the cytoplasmic extract from eggs of a sea urchin, Tripneustes gratilla, contains several high-molecular-weight proteins. Among them, the 250K-molecular-weight protein was isolated and characterized. This protein migrated slightly more slowly than filamin from chicken gizzard upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It reacted only very weakly with antibodies against chicken gizzard filamin or against a high-molecular-weight actin-binding protein from Physarum plasmodia. It did not react with antibodies against chicken erythrocyte alpha-spectrin nor against the 220K protein from the same egg. A chemical crosslinking experiment revealed the presence of dimers in the purified 250K protein preparation. A rotary shadowed specimen of such a preparation showed wavy single-stranded molecules 120-170 nm long, having five to six globular domains, which may represent dimers. The appearance was different from that of spectrin or actin-binding protein from macrophage or chicken gizzard filamin. This protein increased the viscosity of F-actin solution. It bound to F-actin preferably at low KCl concentrations such as 20 mM. The binding ability was not influenced by pH between 6.0 and 7.5, although it was somewhat reduced above pH 8.0. The binding was insensitive to low Ca ion concentrations. Electron microscopy using the negative staining technique supported the idea that this protein crosslinks actin filaments. In addition, a second protein from egg gels, with a reported molecular weight of about 220K (Kane, R.E., J. Cell Biol. 66, 305-315 (1975)), comigrated with human erythrocyte alpha-spectrin on an SDS-gel and reacted with antibodies against chicken erythrocyte alpha-spectrin. This suggests that this protein is a sea urchin egg spectrin. The role of these proteins in the cytoskeleton formation in the sea urchin egg is discussed.

Long-term diuretic therapy with metolazone of renal failure and the nephrotic syndrome.

The effects of the new diuretic metolazone were studied in ten patients with chronic renal insufficiency and ten with nephrotic syndrome. Patients were maintained on metolazone for up to 44 months. Beneficial effects of treatment included loss of edema and improved control of blood pressure. The natriuretic effect of metolazone facilitated the use of sodium bicarbonate to treat acidosis in several patients. Concurrent administration of metolazone and furosemide produced a dramatic diuresis in one patient resistant to either diuretic alone. Adverse effects of metolazone therapy were those characteristic of other effective diuretics, Including serum electrolyte losses and hyperuricemia. Initial treatment produced small increases in serum creatinine among patients with renal insufficiency, suggesting that GFR was decreased secondary to diuresis-induced volume depletion. The study demonstrates that metolazone is both safe and effective over long periods of time.

Cost savings and economic considerations using home intravenous antibiotic therapy for cystic fibrosis patients.

Our Cystic Fibrosis (CF) Center made an effort to utilize home intravenous antibiotic therapy (HIVAT) as an alternative to continued hospitalization during a 1-year study. After thorough individual clinical and financial evaluation, 27 of 41 CF patients admitted for treatment, including antibiotic therapy, were selected for HIVAT to complete a 14- to 21-day treatment course (mean 15.1 days). The 27 patients (6-28 years old, mean 16 years) incurred a total of $698,587 in hospital charges and physician fees during 96 admissions. The average charge for 974 inpatient days was $717/day ($7,280 per admission). After an average of 10.2 days of inpatient care, the 27 patients underwent 79 courses of HIVAT for an additional 8 days; 21 additional HIVAT courses in six of these patients were initiated on an outpatient basis between frequent readmissions. The 811 days of HIVAT resulted in $85,027 total charges by two home care companies. The charges per day of HIVAT by one company were almost twice that of the other. The average daily cost of HIVAT was $108/day. If the HIVAT patients had remained hospitalized to complete the course of intravenous antibiotic therapy, the projected inpatient costs would have been $589,271. Therefore, the 811 days of HIVAT over a 1-year period resulted in total estimated direct cost savings of $501,770. The average savings per course of HIVAT was $5,017, or $618/day.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the CA 19-9 antigen and Lewis blood group with pulmonary disease severity in cystic fibrosis.

The altered carbohydrate structure of sputum from patients with cystic fibrosis (CF) has been thought to be due to the inflammatory airway response. Carcinoembryonic antigen (CEA) and CA 19-9 detect sialosylated carbohydrates in mucus. The epitope of CA 19-9 is part of the Lewis A (Le(a)) blood group antigen. Serum concentrations of CEA and CA 19-9 were determined by radioimmunoassay in 41 CF patients, aged 6-34 years; 16 were asymptomatic Outpatients, and 25 had been admitted for pulmonary exacerbations. There was no difference in CEA between groups. The CA 19-9 serum concentration was elevated in 90% of patients who had at least one of the two Lewis antigens. The CA 19-9 concentration of Inpatients with exacerbations was 2.7 times that of stable Outpatients (263 +/- 44 versus 99 +/- 13 U/mL; P less than 0.02). CA 19-9 correlated significantly with age (r = 0.35, P less than 0.05), Brasfield score (r = 0.39, P less than 0.015), pulmonary function tests, cough severity (r = 0.50, P less than 0.001) and NIH clinical score (r = 0.57, P less than 0.001). CA 19-9 concentration of Inpatients decreased by 44% from admission to discharge (302 +/- 45 to 169 +/- 39, P less than 0.02). Fourteen of 25 (56%) of the Inpatients were Le(a) positive versus only 3/15 (20%) of Outpatients who had milder lung disease (P less than 0.002). Of the Inpatients, 25% with more advanced lung disease were Le(a+b+), a rare blood group in the normal population, and one not observed in the Outpatients with milder disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of low, medium, and high carbohydrate formulas for nighttime enteral feedings in cystic fibrosis patients.

This study examined whether the increase in CO2 production (VCO2) and ventilatory demands by carbohydrate loading with different formulas during nighttime enteral feedings could be detrimental in young adult cystic fibrosis patients with moderate to advanced lung disease. Ten patients age 17 to 24 (mean 21.4 years) received 1000 kcal/M2 of a low (Pulmocare), medium (Ensure Plus), and high (Vivonex HN) carbohydrate formula in random order. Eight patients had severe, and two moderate obstructive pulmonary disease; nine used nighttime oxygen therapy. Basal energy expenditure (BEE) without feedings averaged 120% of that predicted by the Harris-Benedict equation. The metabolic expenditure by indirect calorimetry during nighttime feedings was 25 to 36% greater than the BEE. Oxygen consumption (VO2) increased 21 to 27% during nighttime feedings with no difference between formulas. VCO2 increased 29% for Pulmocare, 46% with Ensure Plus, and 53% with Vivonex HN. The increase in VCO2 with Pulmocare was significantly less than Ensure Plus (p less than 0.05) and Vivonex HN (p less than 0.005). The respiratory quotient (RQ) (VCO2-/VO2) for Pulmocare (0.88) was the same as the BEE, but increased with Ensure Plus (1.00), and Vivonex HN (1.08). The 41% increase in minute ventilation with Vivonex HN was greater than the 25 to 28% increase observed for Pulmocare and Ensure Plus (p less than 0.05). Transcutaneous oxygen saturation fell no more than 2% with all formulas. PCO2 changed +/- 5 torr during enteral feedings with similar changes in any patient with all formulas.(ABSTRACT TRUNCATED AT 250 WORDS)

Asymptomatic uveitis in young people with inflammatory bowel disease.

PURPOSE: To ascertain the prevalence of uveitis in a population of pediatric patients with inflammatory bowel disease without ocular symptoms. METHODS: We prospectively evaluated all young people who came to the pediatric gastroenterology clinic with endoscopically proven inflammatory bowel disease between March 1994 and June 1995. All the patients were examined for evidence of ocular manifestations of inflammatory bowel disease. The examination consisted of slit-lamp examination, tonometry, and indirect ophthalmoscopy. None of the patients had visual or ocular symptoms. Eighteen patients had Crohn's disease and 14 had ulcerative colitis. RESULTS: Of the 32 patients evaluated, four (12.5%) had evidence of asymptomatic ocular inflammation, defined as anterior chamber cell and flare. All patients with ocular inflammation were male. Three of these four male patients had Crohn's disease; the other had ulcerative colitis. Five patients had posterior subcapsular cataract, one had esotropia and amblyopia, and one had unilateral high myopia. CONCLUSIONS: The prevalence of asymptomatic uveitis in our population of young people with inflammatory bowel disease was 12.5%. These findings suggest the need for a screening ophthalmologic examination to rule out occult eye disease in young people with inflammatory bowel disease.

Initial results of a program in liver transplantation.

St. Louis University established a liver transplant program in early 1988. The authors report on the program's first 10 months in operation, emphasizing the careful planning and cooperation the medical center must undertake to ensure the program's success.