Comparative Hydrolysis of Sphingomyelin and 2-N-(Hexadecanoyl)-Amino-4-Nitrophenyl-Phosphorylcholine by Normal Human Brain Homogenate at Acid and Neutral pH

Hydrolysis of sphingomyelin and 2-N-(hexade-canoyl)-amino-4-nitrophenyl-phosphorylcholine (HDA-PC), a synthetic analogue of sphingomyelin, by acid and Mg-dependent neutral sphingomyelinases was tested with a homogenate of normal human brain cortex. Results demonstrated quite different substrate specificities for these enzymes. Acid sphingomyelinase, which is neither activated by MgCl₂ nor inhibited by EDTA, hydrolyzed both substrates (the hydrolysis ratio of HDA-PC to sphingomyelin is ?2). In contrast, Mg-dependent neutral sphingomyelinase, which is inhibited by EDTA and reactivated by MgCl₂, hydrolyzed only sphingomyelin (the hydrolysis ratio of HDA-PC to sphingomyelin is ?0-0.05). This synthetic substrate seems to be useful for selective determination of acid sphingomyelinase and for avoiding interference of Mg-dependent neutral sphingomyelinase.     

Taxonomic Implications of Spore Fine Structure in Clostridium bifermentans

Thirty-five strains of Clostridium bifermentans were, in most part, culturally homogeneous by conventional taxonomic criteria but were heterogeneous with respect to spore fine structure. Fourteen of the strains produced spores with appendages, distributed among four distinct ultrastructural types. No consistent correlation existed between spore type and other variable properties of these strains. It is proposed, therefore, that these spore appendage-type strains be considered as "varieties" of C. bifermentans and that they should not be designated as new species.     

The isolation and properties of phenylalanine hydroxylase from human liver

Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The K(m) values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.     

Microbiological and Pharmacological Behavior of 7-Chlorolincomycin

Replacement of the 7-(R) hydroxyl group of lincomycin by a 7-chloro-substituent produced a compound with greater in vitro activity than the parent. Laboratory studies of this compound showed it to be highly active against all of the following strains of gram-positive organisms examined, including penicillinase- and nonpenicillinase-producing staphylococci, Diplococcus pneumoniae, Streptococcus viridans and Streptococcus pyogenes. The enterococci, as well as all the gram-negative organisms tested, with the exception of some strains of Haemophilus, were uniformly insensitive to this agent. The activity of 7-chlorolincomycin was not affected by serum or inoculum size. Resistance developed in a slow stepwise pattern. Peak levels of approximately 2 mug/ml were achieved in the serum of volunteers after ingestion of 150 mg either in the fasting state or after a meal. No untoward effects were noted. The antibiotic appears to be of potential value in the treatment of infections due to gram-positive organisms, with the exception of enterococcus.     

Minicultures of Mammalian Cells in a New Plastic Plate

A new disposable micro tissue culture plate was developed and tested for use in virological procedures. Miniature mammalian cell cultures (minicultures) were grown in these plates. Each plate contained 96 circular cultures in flat wells (7 mm in diameter). Replicate titrations of a number of viruses were performed in various tissues. Excellent reproducibility was demonstrated. Mean infectivity titers determined by miniculture methods were generally within 0.6 log₁₀/ml of macro tube titrations. Standard tissue culture assay techniques such as hemadsorption, interference titration, and microneutralization were easily carried out with this method and were very reproducible. Development of this noncytotoxic disposable micro tissue culture plate now permits the routine performance of rapid, reliable, and reproducible tissue culture tests at a very significant reduction in cost and labor.     

Sensitivity of Pseudomonas aeruginosa to Normal Serum and to Polymyxin

Strains of Pseudomonas aeruginosa isolated from clinical material were very variable in their sensitivity to the bactericidal action of normal serum mediated by the complement system. Fifty per cent killing end points ranged from 0.015 ml to greater than 0.4 ml. Most of the strains with relatively greater sensitivity to serum were isolated from patients with cystic fibrosis. Immunization of rabbits resulted in antisera with enhanced levels of bactericidal antibody, except with one strain which was resistant to the bactericidal action of normal serum and antiserum. When P. aeruginosa was cultivated at 41 C instead of at 37 C, it was significantly more sensitive to serum and to several antibiotics, thereby implicating fever as a host defense mechanism in Pseudomonas infections. In contrast to their heterogeneity to serum bactericidal activity, the strains were relatively homogeneous in their sensitivity to polymyxin, with no apparent association between their sensitivity to the two antimicrobial agents.     

Hydrolysis of RNA to 5′-nucleotide by seed sprouts,particularly malt sprouts

Conditions for the efficient conversion of commercial RNA to nucleoside 5′-monophosphate by means of a phosphodiesterase in malt sprouts have been determined. A comparison of the enzyme content of the rootlets, stems, and kernels of various plant seedlings, including barley, rye, oat, wheat, rice, and beans shows maximum amounts in the rootlets, and minimum quantities in the ungerminated kernels. Of all the seedlings tested, (mung bean, soy bean, oat, wheat, rice, barley) barley gave the highest conversion of RNA to 5′-nucleotides. Commercial malt sprouts prepared from 6 different malted barleys including 2-rowed and 6-rowed samples all showed about the same amount of phosphodiesterase content. Besides phosphodiesterase, other enzymes capable of hydrolyzing RNA and 5′-nucleotides were found in sprouts. These included 3′-phosphodiesterases, 5′-nucleotidases, and nucleosidases. By carefully pretreating both extracts and the solid sprouts at elevated temperatures for a limited time and by the addition of minimum amounts of Zn⁺², the action of these undesirable enzymes was either effectively destroyed or minimized so that the production of 5′-nucleotides was maximized. It was found that suspensions of appropriately washed and treated barley malt rootlets are substantially more effective than aqueous extracts for converting RNA to 5′-nucleotides.