Isolation and cultivation of spirochetes and other spiral-shaped bacteria associated with the cecal mucosa of rats and mice.

A method for the culture of spiral-shaped bacteria associated with the intestinal mucosa of rodents is described. The appearance in culture of a spiral organism from rat ceca and a spirochete from mouse ceca is illustrated; these organisms are morphologically similar to the major inhabitants of the cecal mucosa in each animal species.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: analysis of the initial rates

A study was conducted on the kinetics of enzymatic hydrolysis of pure insoluble cellulose using unpurified culture filtrate Trichoderma reesei, with the emphasis on the initial reaction period. The initial hydrolysis rate and extent of enzyme (soluble protein)adsorption, either apparent or initial, were evaluated under various experimental conditions. It has been found that the various mass-transfer steps do not control the overall hydrolysis rate and that the hydrolysis rate is mainly controlled by the surface reaction step promoted by the adsorbed enzyme. It has also been found that the initial hydrolysis rate strongly depends on the initial extent of soluble protein adsorption and the effectiveness of the adsorbed soluble protein to promote the hydrolysis. The initial extent of soluble protein adsorption, in turn, is related to the initial cellulose concentration, enzyme concentration, and specific surface area of cellulose, whereas the effectiveness of the initially adsorbed soluble protein to promote the derived to interrelate these parameters without resorting to the Michaelis-Menten kinetics. The present result appear to imply that the role of enzyme-substrate complex formation should not be ignored in deriving a mechanistic kinetic model for enzymatic hydrolysis of cellulose.     

Alamethicin-induced changes in lipid bilayer morphology

We have found that alamethicin, in the absence of an electric field, modifies both the hydrophilic surface and hydrophobic core of lipid bilayers. As shown by freeze-fracture and X-ray diffraction experiments with multiwalled vesicles, alamethicin increases the fluid space between bilayers by as much as 50 nm, and at the same time perturbs the hydrocarbon regions of the bilayers. For suspensions of gel-state lipid treated with alamethicin, uniformly spaced rows of particles cover the fracture faces and corresponding linear arrays of stain-collecting depressions cover the hydrophilic surfaces. In the liquid-crystalline state, alamethicin induces an irregular granular texture on the fracture faces.     

Annular and non-annular binding sites on the (Ca2+ + Mg2+)-ATPase

Quenching of the fluorescence of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

Preparation of new ω-aldehydroalkyl 1-thio-d-glycopyranosides,and their coupling to bovine serum albumin by reductive alkylation

Synthesis of two ω-aldehydoalkyl 1-thioglycosides of d-glucopyranose and of d-galactopyranose is described. 3-Oxopropyl and 2-oxoethyl 1-thioglycosides were prepared by treating a tetra-O-acetyl-1-thioaldose with either acrolein or 2-bromoacetaldehyde, followed by O-deacetylation under mild conditions. These ω-aldehydoalkyl 1-thioglycosides were successfully attached to bovine serum albumin (BSA) by reductive alkylation as described previously. With the 3-oxopropyl 1-thioglycosides, much higher levels of sugar attachment (e.g., ～80 mol of sugar per mol of BSA) were attained than hitherto possible with any sugar derivative tested.     

The synthesis of 2,1′-anhydro-,2,1′:3,6-dianhydro-, and 2,1′:3,6: 3′,6′-trianhydro-sucrose

1′-O-Mesyl-6,6′-di-O-tritylsucrose and the corresponding 1′-O-tosyl derivative were prepared from 6,6′-di-O-tritylsucrose by selective sulphonylation. Both sulphonates underwent intramolecular cyclisation reactions, to give 2,1′-anhydrosucrose in high yields rather than the isomeric 1′,4′-anhydride. Sequential benzoylation, detritylation, and mesylation of the 2,1′-anhydride afforded 2,1′-anhydro-6,6′-di-O-mesylsucrose tetrabenzoate which, in the presence of base, gave 2,1′:3,6:3′,6′-trianhydrosucrose that was not identical with the product previously claimed to have this structure. Several derivatives of 2,1′-anhydrosucrose were prepared possessing different functional groups at either the 6,6′- or 4,6′-positions. Dimolar mesitylene-sulphonylation of 3,3′,4′6′-tetra-O-acetylsucrose gave the 6,1′-disulphonate, which, in the presence of alkali, gave 2,1′:3,6-dianhydrosucrose, which was transformed into the 2,1′:3,6:3′,6′-trianhydride by sequential bromination at C-6′ (carbon tetrabromide-triphenylphosphine) and base-catalysed cyclisation. Treatment of 3,3′,4′,6′-tetra-O-benzoylsucrose with sulphuryl chloride furnished the 4,6,1′-trichloro derivative, which, on alkaline hydrolysis, was converted into 2,1′:3,6-dianhydro-4-chloro-4-deoxy-galacto-sucrose.