A protein factor inhibiting the magnesium-activated adenosine triphosphatase of desensitized-actomyosin

1. The preparation and properties of a myofibrillar protein factor which inhibits the Mg(2+)-activated adenosine triphosphatase of desensitized actomyosin is described. 2. This factor had negligible effect on the Mg(2+)-activated adenosine triphosphatase of natural actomyosin and on the Ca(2+)-activated adenosine triphosphatases of desensitized actomyosin and myosin. 3. The Mg(2+)-activated inosine triphosphatase activity of desensitized actomyosin was not affected by the factor. 4. The inhibitory effect was sensitive to ionic strength. In addition to their ionic effects Mg(2+) and Ca(2+) appeared to have a specific action in reducing the effect of the inhibitor. 5. F-actin reduced the inhibition whereas Bailey-type tropo-myosin had little effect. 6. As far as can be judged from the reported experiments this factor is different from any of the previously described myofibrillar components.     

3-Methylhistidine in actin and other muscle proteins

1. By the use of the extended elution system for basic amino acid analysis, 3-methylhistidine has been detected in hydrolysates of actin isolated from mammalian, fish and bird skeletal muscle. 2. Evidence is presented to indicate that 3-methylhistidine forms part of the primary structure and that in rabbit actin this residue is restricted to one peptide fraction obtained from the tryptic digest. 3. Rabbit skeletal-muscle actin has a 3-methylhistidine:histidine ratio 1:7.6, indicating a minimum molecular weight of 47600. 4. Adult rabbit myosin contains approximately 2 3-methylhistidine residues/mol. These residues are localized in the heavy meromyosin part of the molecule, and are restricted to the major component obtained after succinylation.     

Direct contact is required between serum albumin and hamster spermatozoa for capacitation in vitro

A dialysis unit was used to test whether direct physical contact between serum albumin and hamster spermatozoa is required for capacitation and/or the acrosome reaction. Sperm and bovine serum albumin (BSA) were incubated cither together (direct incubation) or separated by a dialysis membrane (indirect incubation). Sperm viability was supported with “sperm motility factors” (hypotaurine and epinephrine) and polyvinylalcohol (PVA). Spermatozoa became capacitated and underwent acrosome reactions when directly incubated in medium containing BSA (TALP-PVA), but did not undergo acrosome reactions when indirectly incubated with BSA (medium TLP-PVA). When sperm were first incubated for 4 hr indirectly with BSA, followed by 4 hr direct incubation with BSA, capacitation did not occur during indirect incubation. These findings indicate that an “intimate association” is necessary between serum albumin and spermatozoa to support capacitation under in vitro incubation conditions. The data are consistent with the concept of direct transfer of compounds from sperm to albumin and/or vice versa during sperm capacitation.     

Properties and attempted culture of Pasteuria penetrans, a bacterial parasite of root-knot nematode (Meloidogyne javanica)

When they were subjected to a range of physical and chemical treatments, spores of Pasteuria penetrans showed properties similar to those of other endospore-forming bacteria. The spores did not take up some stains, were resistant to desiccation and sonication and showed extrusion of spore contents ('spore popping') on prolonged exposure to 0.1% KMnO₄ in 0.3 n HNO₃. Calcium and dipicolinic acid (DPA) were present at concentrations of 0.28% and 0.96% of the spore dry weight respectively, giving a Ca: DPA molar ratio of 1.2. The infectivity of P. penetrans spores was reduced to a low level after heating at 100°C for 5 min, but spore attachment was not markedly affected by heating at 100°C for 15 min. Evidence for the presence of catalase in P. penetrans spores was equivocal because the low levels of catalase activity observed in spore suspensions may have been due to contamination from catalase-positive nematode tissue. When P. penetrans spores were exposed to a range of substances known to act as germinants for spores of Bacillus spp., germination or loss of refractility was not observed by phase microscopy. In vitro culture of P. penetrans was attempted by inoculating either spores or vegetative mycelial bodies onto a diverse range of simple and complex media and incubating them in aerobic, reduced oxygen, anaerobic and increased CO₂ environments. Signs of spore germination or growth of vegetative stages were never observed.     

Aminophylline enhances contractility of frog skeletal muscle: an effect dependent on extracellular calcium

The effects of aminophylline (10-500 microM) on isometric twitch and tetanic forces were studied in vitro on frog semitendinosus muscle. Two hypotheses were tested: 1) that micromolar concentrations of aminophylline enhanced contractility of isolated skeletal muscle and 2) that the potentiating effect of aminophylline was dependent on the presence of extracellular calcium ions. Muscles were removed, placed in aerated Ringer solution at 20 degrees C, attached to a force transducer, and stimulated directly. Muscles in normal Ringer and aminophylline Ringer were compared throughout the frequency-force relationship from twitches to maximum tetanic force. Aminophylline increased twitch force significantly at concentrations as low as 25 microM. Over a range of stimulation frequencies, but especially at 10 and 20 Hz, aminophylline increased tetanic force. The potentiating effect of aminophylline (100 microM) was reduced or eliminated in calcium-free Ringer containing 10 mM magnesium. We conclude that aminophylline, at therapeutic concentrations, enhances muscle contractility, and the enhancement is dependent on the presence of extracellular calcium. These findings support the concept that aminophylline is effective in improving respiration in humans with airway obstruction by enhancing diaphragmatic contractility.     

Monoclonal antibody production in hollow fiber bioreactors using serum-free medium

Murine hybridoma cells that produce monoclonal antibody directed against human fibronectin have been cultured in VITAFIBER II and VITAFIBER V hollow fiber bioreactors using defined, serum-free WRC 935 medium. During a two-week growth period, following inoculation of the bioreactors, the cells proliferated to an extent where the bioreactor was filled with cultured cells. Using a 5 sq. ft. VITAFIBER V bioreactor, over 15 grams of antibody were produced during the 40 days of the experiment. This antibody was greater than 95% IgG. During the production period, this packed mass of cells produced 579 +/- 15 mg IgG per day. Because the medium is formulated for air equilibration and high cell densities, WRC 935 medium is especially useful for production of gram quantities of monoclonal antibodies using continuous feed hollow fiber bioreactor cell culture systems.