Changes in the migration and adhesive activity of polymorphonuclear leukocytes as 1 of the characteristics of Gram-negative bacteria isolated from patients with suppurative lung destruction

The influence of Gram-negative bacteria on the migratory and adhesive activity of polymorphonuclear leukocytes (PMNL) in the peripheral blood of clinically normal donors has been studied by the specially developed method with the use of Boyden chambers. Pseudomonas and enterobacteria have been found to produce complex and various effects on the above-mentioned properties of PMNL. When incubated in fresh serum, Gram-negative bacteria are capable of enhancing the migratory activity of PMNL, this property being least pronounced in P. aeruginosa. The incubation of live bacteria from the authors' collection in the patients' sera or in sera obtained from normal donors and inactivated by heating induces no hemotaxis of PMNL, and P. aeruginosa strains even suppress it under such conditions. The isolated Gram-negative bacteria under study increase the number of highly adhesive PMNL in the population used in this investigation, but P. aeruginosa cultures do not produce such effect.     

Frequency of Erwinia carotovora in the Alyth Burn in eastern Scotland and the sources of the bacterium

Contamination of the Alyth Burn by Erwinia carotovora was monitored monthly over 2 years at nine sites spread over a distance of ca 20 km. The bacterium was detected only once in the upper reaches of the river where it flows in uninhabited moorland but frequency of detection and contamination level tended to increase progressively as the river flowed through the middle reaches mostly in grassland to the lower reaches in arable land where the bacterium was almost always present. Erwinia populations rose from < 10² cells/1 before May to frequently > 10³ but < 10⁴ cells/l thereafter at sites in the arable land zone. A similar pattern was found in the grassland zone except that erwinia numbers were lower. Erwinia numbers at one site in the arable land zone were positively and negatively correlated with the river water temperature and flow rate respectively when there was a 1 month lag between the environmental data and the population recorded. More than 80% of isolates tested were E. carotovora subsp. carotovora.
Water from field drains in arable fields, especially those recently planted with potatoes, was frequently contaminated by E.c. carotovora , with numbers and a temporal pattern similar to those of the Alyth Burn. Drainage water from non-arable fields was rarely contaminated. Infected and rotting potatoes deposited in rivers temporarily contaminated the water. Survival of E. carotovora in dilute phosphate buffer was greater at pH 5˙7 than at pH 7˙7 and they survived for at least 10 d in river water.     

Partial-digest DNA sequencing.

A technique called partial-digest sequencing that permits DNA of 4-6 kb in length to be sequenced without subcloning is described. The method exploits the specific cuts introduced by partial digestion with restriction endonucleases that have 4-base recognition sites to produce ordered ladders of PCR-amplified fragments. The staggered ends contain PCR primers and can thus be individually sequenced using conventional methods to yield overlapping sequences covering the entire region. This method should have significant impact on both large and small DNA sequencing projects and find many applications in general manipulations in which ordered sets of deletions need to be produced.     

Parental spleen cells accelerate the development of intestinal brush border structure and function in neonatal mice

The influence of parental spleen cells on the postnatal development of brush border microvillus membrane structure and the ability to transport lysine and alanine has been studied in the mouse jejunum during the second week of postnatal life. Control tissue taken from 7-11 day old mice has an unchanging crypt-villus structure and a low enterocyte migration rate of about 1 micron hr-1. Microvillus elongation in crypt enterocytes takes 6 days to complete under these conditions. Lysine and alanine transport begin 2 days after structural differentiation has ceased. Parental spleen cells injected into 1-2-day-old F1 mice cause crypt cell hyperplasia, villus shortening and a 3-6-fold increase in enterocyte migration rate after a period of 8 days. These effects are associated with large reductions in the time needed to complete microvillus membrane development and first express absorptive function. Lysine and alanine transport begin approximately 6 hr after structural differentiation has ceased under these conditions. Adaptive changes in the development of enterocyte structure and function, induced by injection of parental spleen cells, bear some resemblance to other changes found to occur normally at weaning and in adult animals subjected to controlled changes in diet and environmental temperature. The possibility that common principles govern enterocyte adaptation and that some of these still apply in an intestine undergoing an immune reaction is discussed.     

Efficacy of phototherapy in non-haemolytic hyperbilirubinaemia.

Clinical experience of phototherapy for non-haemolytic hyperbilirubinaemia in 3999 infants in Kandang Kerbau Hospital, Singapore, is documented. Phototherapy was most effective in extremely preterm infants with very low birth weight (gestation less than or equal to 32 weeks, birth weight less than or equal to 1500 g) and least effective in full term infants with very low birth weight (gestation greater than or equal to 37 weeks, birth weight less than or equal to 1500 g) and large preterm infants (gestation less than 37 weeks, birth weight greater than 2270 g). Overall, phototherapy was effective in almost all the infants, with a failure rate of only 2.00/1000 infants. No characteristic features common to all the failures could be detected. The bilirubin rebound was usually mild; repeat phototherapy was required in only 30 infants (7.50/1000), with the response to the second exposure comparable to that to the first. No infant required a third exposure. All the infants tolerated phototherapy well, none developing any illness that could be attributed to the treatment. This clinical experience shows that phototherapy for the treatment of nonhaemolytic hyperbilirubinaemia is effective and safe.     

The major herpes simplex virus DNA-binding protein holds single-stranded DNA in an extended configuration

Properties of the major DNA-binding protein found in herpes simplex virus-infected cells were investigated by using a filter binding assay and electron microscopy. Filter binding indicated that the stoichiometry of binding of the protein with single-stranded DNA is approximately 40 nucleotides per protein molecule at saturation. Strong clustering of the protein in DNA-protein complexes, indicative of cooperative binding, was seen with the electron microscope. Measurements of single-stranded fd DNA molecules saturated with protein and spread for electron microscopy by using both the aqueous and formamide spreading techniques indicated that the DNA is held in an extended configuration with a base spacing of approximately 0.13 nm per base.     

Mutation of residues 423 (Met/Ile), 444 (Thr/Met), and 506 (Asn/Ser) confer cholesteryl esterase activity on rat lung carboxylesterase. Ser-506 is required for activation by cAMP-dependent protein kinase.

Site-directed mutagenesis is used to identify amino acid residues that dictate reported differences in substrate specificity between rat hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) and rat lung carboxylesterase (LCE), proteins differing by only 4 residues in their primary sequences. Beginning with LCE, the substitution Met(423) --> Ile(423) alone or in combination with other mutations increased activity with p-nitrophenylcaprylate (PNPC) relative to more hydrophilic p-nitrophenylacetate (PNPA), typical of hncCEH. The substitution Thr(444) --> Met(444) was necessary but not sufficient for expression of cholesteryl esterase activity in COS-7 cells. The substitution Asn(506) --> Ser(506), creating a potential phosphorylation site, uniformly increased activity with both PNPA and PNPC, was necessary but not sufficient for expression of cholesteryl esterase activity and conferred susceptibility to activation by cAMP-dependent protein kinase, a property of hncCEH. The 3 mutations in combination were necessary and sufficient for expression of cholesteryl esterase activity by the mutated LCE. The substitution Gln(186) --> Arg(186) selectively reduced esterase activity with PNPA and PNPC but was not required for cholesteryl esterase activity. Homology modeling from x-ray structures of acetylcholinesterases is used to propose three-dimensional models for hncCEH and LCE that provide insight into the effects of these mutations on substrate specificity.     

Interaction of gentamicin and spermine with bilayer membranes containing negatively charged phospholipids

We measured the electrophoretic mobility of multilamellar phospholipid vesicles, the 31P NMR spectra of both sonicated and multilamellar vesicles, and the conductance of planar bilayer membranes to study the binding of spermine and gentamicin to membranes. Spermine and gentamicin do not bind significantly to the zwitterionic lipid phosphatidylcholine. We measured the concentrations of gentamicin and spermine that reverse the charge on vesicles formed from a mixture of phosphatidylcholine and either phosphatidylserine or phosphatidylinositol. From these measurements, we determined that the intrinsic association constants of the cations with these negative lipids are all about 10 M-1. This value is orders of magnitude lower than the apparent binding constants reported in the literature by other groups because the negative electrostatic surface potential of the membranes and the resultant accumulation of these cations in the aqueous diffuse double layer adjacent to the membranes have not been explicitly considered in previous studies. Our main conclusion is that the Gouy-Chapman-Stern theory of the aqueous diffuse double layer can describe surprisingly well the interaction of gentamicin and spermine with bilayer membranes formed in a 0.1 M NaCl solution if the negative phospholipids constitute less than 50% of the membrane. Thus, the theory should be useful for describing the interactions of these cations with the bilayer component of biological membranes, which typically contain less than 50% negative lipids. For example, our results support the suggestion of Sastrasinh et al. [Sastrasinh, M., Krauss, T. C., Weinberg, J. M., & Humes, H. D. (1982) J. Pharmacol. Exp. Ther. 222, 350-358] that phosphatidylinositol is the major binding site for gentamicin in renal brush border membranes.     

Neutral glycosphingolipids of murine lymphoma EL-4

Neutral glycosphingolipids of murine T-lymphoma EL-4 were studied. The major glycolipid components were identified as GlcCer, LacCer, GgOse3Cer and GgOse4Cer. It has been shown for the first time that not only gangliosides but also neutral glycolipids are shed from the cell surface into the outer medium.