From Africa to Africa.

Chiropractic, an American-born profession, has grown and developed internationally, especially within the last decade. In reality, however, this growth started long ago in other countries. This article explores the development of the profession in South Africa by examining the life of an American-trained, South African-born, chiropractor. During this time, the international chiropractic community was a small group with close ties to America. This was, in part, due to the fact that chiropractic education was only available in the United States and one was not considered "legitimate" unless he or she was American trained. The struggles of the profession are not unique to America; they have also occurred elsewhere. Chiropractic evolution in South Africa is examined as well as the development of registration (licensing) and tertiary education.

Effect of agents used in perforation repair on osteoblastic cells.

The effects of dental materials on osteoblastic responses in bone were measured using biosynthesis of matrix proteins, osteopontin (OPN), and osteocalcin (OCN) as indices. Materials used in perforation repair were standardly mixed and extracted in sterile water for 10 days. Extracts were added to the medium of osteoblastic ROS 17/2.8 cells and cultured for 48 h. Water incubated in parallel served as the vehicle/dilution control. 1,25-Dihydroxyvitamin D3, which increases biosynthesis of OPN and OCN, served as the positive control. After culture, total cellular RNA was isolated from individual monolayers, and Northern blotting was performed to quantitate mRNA levels encoding OCN and OPN. mRNA levels in treated samples were compared with controls, and significant differences were detected for several materials. Changes in matrix biosynthesis were modest (< 2-fold), compared with 1,25-dihydroxyvitamin D3 (6-fold). Materials used in perforation repair may produce small, but measurable effects on osteoblastic responses.

Kinetic mechanism of Clostridium perfringens phospholipase C. Hydrolysis of a thiophosphate analogue of lysophosphatidylcholine.

The hydrolysis of S-[2-(hexadecanoyloxy)ethyl]thiophosphocholine (I), an analogue of lysophosphatidylcholine, by Clostridium perfringens phospholipase C, was followed at pH 7.5, 37 degrees C and I 1.0 (maintained with KCl), in a continuous assay, by monitoring the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) at 412 nm. Simple saturation kinetics are observed with linear mixed-type slope-intercept effects for the hydrolysis of compound (I) with variable [Ca2+] at fixed concentrations of compound (I) and a simple slope effect as [compound (I)] is varied at fixed concentrations of Ca2+. These data are consistent with a simple ordered rapid-equilibrium mechanism in which Ca2+ binds to the enzyme first followed by substrate. The observed kinetic constants at pH 7.5, 37 degrees C and I 1.0 are K1 = 12.0 mM (Ca2+ dissociation), K2 = 36 microM [compound (I) dissociation] and Vmax. = 552 microM.min-1.mg-1. Alkane diammonium salts inhibit the enzyme by a non-competitive mechanism that involves binding to free enzyme, E.Ca2+ and E.Ca2+.S. The use of the simple micellarized substrate under these conditions allows the determination of kinetic and inhibition constants without complications arising from enzyme-micelle interactions.

Inhibition of the Clostridium perfringens phospholipase C hydrolysis of a thiophosphate analog of lysophosphatidylcholine by micelle-bound ammonium and sulfonium cations.

Cetyltrimethylammonium and n-octadecyldimethylsulfonium bromides inhibit the Clostridium perfringens phospholipase C-catalyzed hydrolysis of 1-S-phosphocholine-2-O-hexadecanoyl-1-mercapto-2-ethanol (1) at pH 7.5, 37 degrees C, mu = 0.15 with KCl. Mixed micelles containing 1 and either inhibitor are substrates for the enzyme and the fraction of activity remaining is a monotonic, but non-linear function of the mole fraction of inhibitor. Simple saturation kinetics are observed as the concentration of 1 is increased in mixed micelles containing a constant mole fraction of inhibitor. Inhibition constants for cetyltrimethylammonium and n-octadecyldimethylsulfonium bromides are 0.66 +/- 0.04 and 0.25 +/- 0.02 mM, respectively. The data suggest that the significant inhibition previously observed for soluble alkyldisulfonium salts (K50 for dodecamethylene-bis(dimethylsulfonium) bromide, 27 microM) is dependent upon bifunctional cationic interactions rather than hydrophobic binding.

Morton's neuroma.

Morton's "neuroma" is a perineurofibrosis of an interdigital nerve. The authors describe various factors that may be responsible for the development of this lesion and relate this information to two case histories. In these cases, treatment with manipulation, various physical therapy modalities, and/or foot orthotics, resulted in the successful resolution of symptoms.

The acid instability of myelin. A model for myelin degeneration in multiple sclerosis.

Electron micrographs of samples of bovine spinal cord which have been briefly acidified (10 mM lactate buffer, pH 5.5, 25 degrees C, 15 minutes) prior to being fixed for EM examination, reveal extensive vesicular disruption of the myelin lamellae; micrographs of control samples incubated under identical conditions at pH 7.0, show normal compact lamellae. Culture of thioglycollate-elicited rat peritoneal macrophages in the presence of derivatized, non-ingestible, bovine CNS material results in the secretion of lactic acid and the acidification of the culture medium to levels which are comparable to those which cause lamellae disruption in the tissue slices. Because of the sensitivity of the myelin lamellae to an acidic microenvironment, it is suggested that a local hyperlactemia, with the resulting decrease in interstitial pH, may be a major pathological process in cell-mediated inflammatory demyelination. Antihyperlactemics may therefore provide a new therapeutic approach to minimizing myelin degeneration in multiple sclerosis and in other CNS disorders characterized by inflammatory demyelination.

The stereoselectivity of the Clostridium perfringens phospholipase C: hydrolysis of thiophosphate analogs of phosphatidylcholine.

Thiophosphate containing analogs of phosphatidylcholine have been synthesized with varying degrees of structural complexity. These analogs have been used in a continuous spectrophotometric assay for phospholipase C from Clostridium perfringens in order to examine the requirement for substrate ester functionalities and the stereoselectivity of the enzyme. The substrate analogs with ester groups in the nonpolar portion of the molecule were acceptable substrates for phospholipase C, while those analogs without ester functionalities were not hydrolyzed. Substrate analogs with chiral centers were resolved using the stereospecificity of phospholipase A2 from Crotalus atrox venom. These resolved substrates were used to study the biphasic hydrolytic time courses observed when rac-dioctanoylphosphatidylthiocholine was used as substrate. The "naturally occurring" enantiomer with R absolute configuration was rapidly hydrolyzed in the presence of phospholipase C while the "nonnaturally occurring" enantiomer with S configuration was slowly hydrolyzed only after a long induction or "lag" period. The selectivity for the R enantiomer over the S enantiomer can be lessened by altering the composition of the substrate micelles resulting in accelerated rates of hydrolysis of the S enantiomer.

Synthesis of a thiophosphate analog of dioctanoylphosphatidylcholine: a phospholipase C substrate.

Dioctanoylthiophosphatidylcholine, a racemic thiophosphate analog of L-alpha-dioctanoylphosphatidylcholine, has been synthesized and isolated by flash chromatography. In contrast with the didecanoylthiophosphatidylcholine synthesized previously, the analog is easily dispersed on sonication in aqueous media and is rapidly hydrolyzed to produce a free thiol group in the presence of the extracellular phospholipase C from either Bacillus cereus or Clostridium perfringens. When 5,5'-dithiobis (2-nitro-benzoic acid) was included as a thiol reactive chromogenic agent, the resultant measurement of product release, as an increase in absorbance at 412 nm, showed a linear relationship with added enzyme.

A continuous spectrophotometric assay for the Bacillus cereus phospholipase C using a thiophosphate substrate analog: evaluation of assay conditions and chromogenic agents.

A thiophosphate analog of dioctanoylphosphatidylcholine has been used as the substrate in a continuous spectrophotometric assay for the Bacillus cereus phospholipase C. The reaction has been monitored at 412 nm using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and at 324 nm using 4,4'-dithiopuridine (DTP) as the respective thiol-reactive chromogenic agents. An optimum pH 6.0 was determined for the phospholipase C-catalyzed reaction which was independent of the chromogen utilized. Although the reaction rates observed when DTP was used were increased over those seen with DTNB, the rates were insensitive to changes in the concentration of the chromogen normally used for the assay. The initial velocities were shown to be linearly dependent upon the amount of enzyme added over at least a 20-fold enzyme concentration range. The dependency of the initial velocity on the concentration of substrate showed a discontinuity at [S] = 40 microM when either DTP or DTNB was used. This was consistent with a value of 56 microM estimated for the substrate critical micelle concentration by an independent measurement. While the substrate data measured using DTP could not be fit to existing equations based on Michaelis-Menten kinetics, the data obtained using DTNB as the chromogen conformed with the model proposed by Wells for enzymes acting upon micelle-forming substrates (M. A. Wells (1974, Biochemistry 13, 2248-2257). This allowed for the estimation of monomer and micelle Michaelis-Menten parameters for the B. cereus phospholipase C-catalyzed reaction with a thiophosphate analog substrate.

Bacillus cereus phospholipase C: carboxylic acid ester specificity and stereoselectivity.

Thiophosphate analogs of phosphatidylcholine have been synthesized with varying structural complexity. These analogs have been used in a continuous spectrophotometric assay for phospholipase C (Bacillus cereus) to estimate the minimal structural requirements associated with the non-polar portion of the substrate phospholipid. The analogs were of three types containing zero, one or two carboxylic acid ester functionalities. The analogs with one or two ester groups acted as substrates for phospholipase C, while those without an ester functionality were not hydrolyzed. The rac-phosphatidylcholine analog with two ester functionalities gave biphasic time-course results, and was subsequently resolved into enantiomers by selective hydrolysis with a sterospecific phospholipase A2 (Crotalus atrox). The enantiomer with R absolute configuration was rapidly hydrolyzed by the phospholipase C while the enantiomer with the S configuration was slowly hydrolyzed after a long induction period. The results suggest that the B. cereus phospholipase C is specific for an ester functionality and is stereoselective for the R absolute configuration at glycerol C-2.

Synthesis of choline and ethanolamine phospholipids with thiophosphoester bonds as substrates for phospholipase C.

Spectrophotometric assays of esterases are sensitive, rapid, and quite specific when thioester substrates are used. Glycerophospholipids with thiophosphoester bonds may be useful as substrates for phospholipase C (EC 3.1.4.3). These have been made from mercaptoglycerol and mercaptoethanol. The thiols were oxidized to disulfides, acylated, and reduced with dithiothreitol. Phosphocholine derivatives were made by the classical methods for oxyphosphoesters. The phosphatidyl choline analogue was converted to the phosphatidyl ethanolamine analogue by transphosphatidylation with cabbage phospholipase D and ethanolamine. Structures were proved with enzymic hydrolysis, infrared spectra, TLC behavior, and elemental analyses. The synthesized compounds were rac-1-S-phosphocholine-2,3-O-didecanoyl-1-mercapto-2,3-propanediol, 1-S-phosphoethanolamine-2,3-O-didecanoyl-1-mercapto-2,3-propanediol, and 1-S-phosphocholine-2-O-hexadecanoyl-1-mercapto-2-ethanol.