Diethyl phthalate, a chemotactic factor secreted by Helicobacter pylori.

The structure of a small-molecule, non-peptide chemotactic factor has been determined from activity purified to apparent homogeneity from Helicobacter pylori supernatants. H. pylori was grown in brucella broth media until one liter of solution had 0.9 absorbance units. The culture was centrifuged, and the bacteria re-suspended in physiological saline and incubated at 37 degrees C for 4 h. A monocyte migration bioassay revealed the presence of a single active chemotactic factor in the supernatant from this incubation. The chemotactic factor was concentrated by solid phase chromatography and purified by reverse phase high pressure liquid chromatography. The factor was shown to be indistinguishable from diethyl phthalate (DEP) on the basis of multiple criteria including nuclear magnetic resonance spectroscopy, electron impact mass spectroscopy, UV visible absorption spectrometry, GC and high pressure liquid chromatography retention times, and chemotactic activity toward monocytes. Control experiments with incubated culture media without detectable bacteria did not yield detectable DEP, suggesting it is bacterially derived. It is not known if the bacteria produce diethyl phthalate de novo or if it is a metabolic product of a precursor molecule present in culture media. DEP produced by H. pylori in addition to DEP present in man-made products may contribute to the high levels of DEP metabolites observed in human urine. DEP represents a new class of chemotactic factor.     

Menstruation and control of diabetes.

Seventy per cent of the patients aged 45 years or under and suffering from diabetic ketoacidosis who were seen in one diabetic clinic over five years were women. The association of menstruation with ketoacidosis was assessed over two and a half years, and it was found that menstruation was associated with ketoacidosis more often than would be expected by chance (P less than 0-01). Two hundred women were interviewed and 76 observed that menstruation changed their diabetic control. Fifty-three found that control deteriorated and hyperglycaemia occurred, while 23 found that control improved and hypoglycaemia was a common problem. Menstruation appears to be an important factor in influencing control of diabetes. The mechanism of the changes observed has not yet been determined, but it seems to be a subject worthy of further investigation.     

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies

Steady-state kinetic parameters have been obtained for the pure 8-hydroxy-5-deazaflavin-reducing hydrogenase. With H2 and 8-hydroxy-5-deazariboflavin (F0) as substrates, Km (H2) = 12 microM, Km (F0) = 26 microM, and Kcat = 225 s-1. In the back-direction, F0H2 is reoxidized (anaerobically) at 225 s-1. Initial velocity patterns, product inhibition patterns, dead-end inhibition by carbon monoxide, and transhydrogenation to Procion Red HE-3B suggest a two-site hybrid ping-pong mechanism. A kinetic derivation for the rate equation is provided in the Appendix. Studies with D2 and with D2O reveal that no steps involving D transfer are substantially rate determining. Further, D2 yields F0H2 with no deuterium at C5 while in D2O a 5-monodeuterio F0H2 product is formed, indicating complete exchange of hydrogens from H2 with solvent before final transfer of a hydride ion out from reduced enzyme to C5 of F0.     

Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization

The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000. At the optimum temperature (50 degrees C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine). Resolution and reconstitution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme.     

Multiple copies of phosphorylated filaggrin in epidermal profilaggrin demonstrated by analysis of tryptic peptides

The precursor of mouse (c57/B16) epidermal filaggrin (profilaggrin) is a very large (ca. 500 000 daltons), highly phosphorylated protein containing multiple copies of filaggrin (26 000 daltons). The conversion of profilaggrin to filaggrin late in epidermal cell differentiation involves dephosphorylation and proteolysis to yield the unphosphorylated filaggrin, which polymerizes with keratin filaments into macrofibrils. In order to gain insight in the nature of these processes, we compared tryptic digests of profilaggrin with those of filaggrin by reverse-phase liquid chromatography. Approximately 80% of the profilaggrin mass consists of multiple copies of filaggrin. Twenty peptides purified in good yield from both profilaggrin and filaggrin accounted for most of the filaggrin sequence. A detailed analysis of the yield of several peptides provided an estimate of the size and frequency of the repeat unit within profilaggrin. These data indicate that the repeating substructure of profilaggrin contains about 265 amino acids and that about 50 residues are removed per filaggrin domain as the precursor is processed to filaggrin. Assuming a molecular weight of 500 000 (as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis), this indicates there are 16 repeats. Analysis of phosphopeptides isolated from profilaggrin showed that 66% of the phosphate was located on peptides that are unphosphorylated in filaggrin. Analysis of peptide recoveries confirmed the repeat size and showed that every copy of filaggrin was phosphorylated in profilaggrin.     

Subunit phosphorylation and activation of skeletal muscle phosphorylase kinase by the cAMP-dependent protein kinase. Divalent metal ion, ATP, and protein concentration dependence

This report provides a characterization of the effects of varying the concentrations of Mg2+, ATP, phosphorylase kinase, and the cAMP-dependent protein kinase on the activation and phosphorylation of phosphorylase kinase. The results show the following. (a) The Km for MgATP2- for the cAMP-dependent protein kinase-catalyzed phosphorylation is decreased by increasing Mg2+, probably as a consequence of decreasing the free ATP:MgATP2- ratio and increasing free Mg2+. (b) Whereas beta subunit phosphorylation of phosphorylase kinase plays a prominent role in determining its activity, alpha subunit phosphorylation can also modulate activity. (c) The phosphorylation of the alpha subunit, which occurs following the initial cAMP-dependent phosphorylation of the beta subunit, is catalyzed by the cAMP-dependent protein kinase and is not a consequence of EGTA-insensitive (or EGTA-sensitive) autophosphorylation occurring as a result of the enhanced phosphorylase kinase activity. (d) The relationship between subunit phosphorylation and phosphorylase kinase activation is complex and particularly dependent upon concentrations of cAMP-dependent protein kinase and phosphorylase kinase in the activation reaction. The data suggest the possibilities that the pathway of phospho-intermediates involved in the activation process probably varies with the activation conditions, that the efficacy of a specific site to be covalently modified is dependent upon the phosphorylation status of other sites, and that the effect of phosphorylation in regulating activity may also be dependent on the phosphorylation status of other sites. It is clear from the data that the activation process for phosphorylase kinase can be very complex, and it is possible that this complexity might have significant physiological ramifications.     

Wound closure of the myelomeningocoele defect

Our experience with 74 neonates with myelomeningocoele is reported. Management in the first phase of the study period consisted of primary closure in 37 patients by wide undermining and skin advancement, marked by a high wound-complication rate. Latissimus dorsi muscle closure, either "reverse" or advanced, was performed in a transitional phase in 5 patients, characterized by increased operative time and blood loss. In the last portion of the study period, 32 patients were managed by immediate dural closure and skin grafts either simultaneously or on a delayed basis at 48 to 72 hours with a low incidence of graft loss, CSF leak, or sepsis. Back ulceration and follow-up in either the primary closure or the skin-grafted group has been infrequent.     

Potentials for progress in laser medicine

Lasers could come to occupy a highly important position in the armament of medicine. They are the brightest known sources of light, man-made or natural, and emit light having such properties as coherence and monochromaticity. Furthermore, lasers have the ability to deliver very brief pulses of light which can cause unique alterations in biological materials. The major obstacle to the increased use of lasers in medicine and surgery is not the availability of laser devices, but the dearth of basic information about laser-tissue interactions. We have recently demonstrated that, even in turbid tissue such as the dermis, it is possible simultaneously to induce microscopically selective thermal damage, localized to millions of selectively absorbing targets, while sparing surrounding tissues. These "targets" may be as small as organelles or as large as blood vessels. Such localized thermal damage is truly unique to pulsed laser exposures. The scope and medical utility of these lesions has yet to be fully understood. Thus, there is much research to be done in describing and characterizing laser-induced injury. There is, however, ample evidence that several laser therapies could be improved by using selectively absorbed, short pulses that lead to the spatial confinement of thermal injury. Treatment of port wine stains, pigmented lesions, atheromatous arterial plaques, and the fragmentation of kidney and gall stones are examples. It should also be possible to use a variety of systems to deliver exogenous laser targets on or within individual types of cells or organelles. Such chromophores may lead to new forms of cancer therapy, for example.     

The branch point effect. Ultrasensitivity and subsensitivity to metabolic control

The interdependence of the activities of branch point enzymes which compete for a common substrate can yield ultrasensitivity or subsensitivity to control, even if the competing enzymes follow Michaelis-Menten kinetics. The nature of this "branch point effect" for a particular system depends on the kinetic parameters of the competing enzymes, the rate of substrate production leading into the branch point and the type of regulatory mechanism involved. With physiologically reasonable parameter values, the branch point effect can give ultrasensitivity equivalent to an allosteric enzyme with a Hill coefficient of 8 or higher. An experimental example of this ultrasensitivity was provided by the branch point between isocitrate lyase (of the glyoxylate bypass) and isocitrate dehydrogenase in Escherichia coli. The glyoxylate bypass is very active during growth on acetate but its flux decreases by a factor of approximately 150 upon addition of glucose. This inhibition is brought about by two relatively modest events: a 4-fold increase in the maximum velocity of isocitrate dehydrogenase and a factor of 5.5 decrease in the rate of isocitrate production. The mechanism which underlies this sensitivity amplification is discussed.