A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus influenzae: a survey of 24 non-typeable H. influenzae strains

In view of the importance of 5-N-acetyl neuraminic acid in bacterial pathogenesis, a sensitive, reproducible and reliable method for the determination of 5-N-acetyl neuraminic acid levels in lipopolysaccharide (LPS) is described and applied to 24 different non-typeable Haemophilus influenzae (NTHi) strains. The method involves analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of terminal 5-N-acetyl neuraminic acid residues released by neuraminidase treatment of O-deacylated LPS. The procedure is relatively fast and the instrumental effort is moderate. The results of the procedure were compared with data obtained by 1H NMR and electrospray ionisation-mass spectrometry (ESI-MS). The analysis of LPS from 24 NTHi strains showed that 5-N-acetyl neuraminic acid was found to be a common constituent of LPS in NTHi. Only one strain (NTHi 432) did not show any sialylation. Molar ratios (LPS/5-N-acetyl neuraminic acid) ranged between 5/1 and 500/1. Several strains in which no 5-N-acetyl neuraminic acid could be determined by other methods including 1H NMR and ESI-MS were shown to contain 5-N-acetyl neuraminic acid by this HPAEC-PAD procedure. The method was applied to determine levels of terminal 5-N-acetyl neuraminic acid in LPS from NTHi strains grown under different conditions and mutant strains containing inactive LPS biosynthetic genes.     

Similar Susceptibility to Excess Irradiance in Sun and Shade Acclimated Saplings of Norway Spruce [Picea Abies (L.) Karst.] and Stone Pine (Pinus Cembra L.)

Photosynthetica - We compared the responses of sun and shade acclimated saplings of Picea abies and Pinus cembra to excess photosynthetic photon flux density (PPFD) equivalently exceeding the level...     

Foetidin,a sesquiterpenoid coumarin from Ferula assa-foetida

A new sesquiterpenoid coumarin, foetidin, has been isolated from the roots of Ferula assa-foetida.     

Further characterization of 4-bromomisonidazole as a potential detector of hypoxic cells

[14C]Bromomisonidazole was prepared by direct bromination of [ring-2] [14C]misonidazole in dioxane. The uptake and binding of the two labeled sensitizers were compared in vitro in 1-mm EMT-6 spheroids which contain a necrotic core. Using liquid scintillation counting it was shown that spheroids incubated with 50 microM [14C]bromomisonidazole concentrated drug above levels in the medium by 1 1/2 hr and achieved maximum concentration by 10 hr with no further increase at 23 hr. Spheroids incubated with 50 microM [14C]misonidazole may concentrate the sensitizer more slowly but ultimately reached the same fivefold increase over levels in the medium by 23 hr as was observed for bromomisonidazole. Autoradiographs prepared from spheroids after incubation with [14C]misonidazole or [14C]bromomisonidazole showed silver grains preferentially located over viable hypoxic cells in the inner half of the spheroid rim adjacent to the necrotic center, with lower grain density over nonviable necrotic areas and many fewer grains over oxic cells at the periphery of the spheroid. The results indicate that both severely and moderately hypoxic cells may preferentially bind [14C]bromomisondiazole. The data support the potential of radiolabeled bromomisonidazole for in vivo imaging pending additional studies of the metabolism of this agent.     

A splice site variant in the SUV39H2 gene in Greyhounds with nasal parakeratosis

Hereditary nasal parakeratosis (HNPK), described in the Labrador Retriever breed, is a monogenic autosomal recessive disorder that causes crusts and fissures on the nasal planum of otherwise healthy dogs. Our group previously showed that this genodermatosis may be caused by a missense variant located in the SUV39H2 gene encoding a histone 3 lysine 9 methyltransferase, a chromatin modifying enzyme with a potential role in keratinocyte differentiation. In the present study, we investigated a litter of Greyhounds in which six out of eight puppies were affected with parakeratotic lesions restricted to the nasal planum. Clinically and histologically, the lesions were comparable to HNPK in Labrador Retrievers. Whole genome sequencing of one affected Greyhound revealed a 4‐bp deletion at the 5′‐end of intron 4 of the SUV39H2 gene that was absent in 188 control dog and three wolf genomes. The variant was predicted to disrupt the 5′‐splice site with subsequent loss of SUV39H2 function. The six affected puppies were homozygous for the variant, whereas the two non‐affected littermates were heterozygous. Genotyping of a larger cohort of Greyhounds revealed that the variant is segregating in the breed and that this breed might benefit from genetic testing to avoid carrier × carrier matings.     

Physicochemical characteristics of triacyl lipid A partial structure OM-174 in relation to biological activity.

The triacylated lipid A partial structure OM-174 was characterized in detail using a variety of physical and biological techniques. OM-174 aggregates adopt the micellar HI structure. The temperature (Tc) of the gel to liquid-crystalline phase transition of the hydrocarbon chains is 0 degrees C, from which high fluidity of the acyl chains at 37 degrees C can be deduced. The molecular area of a single OM-174 molecule at a surface pressure of 30 mN x m-1 is 0.78 +/- 0.04 nm2. Conformational analyses, using IR spectroscopy, of the behavior of the various functional groups of OM-174 as compared with hexa-acyl lipid A suggest altered hydration of the phosphate charges and unusually strong hydration of the ester groups, which is probably related to the high accessibility of these groups to water in the micellar aggregate structure. OM-174 was shown to intercalate into a phospholipid membrane corresponding to the macrophage membrane within seconds in the presence, and within minutes to hours in the absence, of LPS-binding protein. In the Limulus amebocyte lysate assay, the triacyl lipid A is more than 105-fold less active than hexa-acyl lipid A, but only 10-fold less active in inducing IL-6 in human mononuclear cells, and equally active in inducing NO production in murine macrophages. These findings are used to explain the mechanism of the lipid A-induced cell activation.     

Quantitative analysis of cell density and pattern of chondrocytes in transitory hyaline cartilage anlagen of human tarsal bones.]

Changes in the distribution patterns of chondrocytes in the embryonic transitory hyaline cartilage of the tarsus were studied using 56 sagittal section of tarsi from 28 human embryos, with a vertex-foot length from 7.0 to 44.0 cm. The os naviculare and os cuboideum were not sliced in all sections. A total of 93,931 cells were transferred via a mirror to paper, and the resulting patterns were analyzed. Determinations included the numerical surface and volume density of the chondrocytes and the cell distribution patterns using the dispersion index. In the case of the calcaneus, talus, os naviculare and os cuboideum, the number of sliced cells found over a constant area decreases at a continuous linear rate in the course of the embryonic period. In all tarsal primordia the distribution pattern developed from regular to random. Particular conditions prevail immediately below the perichondrium and near the ossification centers which can be found in the calcaneus from the 6th month onwards.     

Modulation of the nuclear antigen p105 as a function of cell-cycle progression

The characterization of the proliferation-associated nuclear antigen designated p105 in quiescent and proliferating lymphocytes is described. Through the use of novel flow cytometric and cell-sorting strategies the intracellular content of p105 was assessed in situ on a per cell basis. These analyses demonstrated the presence of multiple cellular subpopulations within the cell cycle differing significantly in p105 content. The data revealed that the flow cytometric quantitation of p105 levels may effectively discriminate cycling from noncycling cells. Immunogold electron microscopy revealed that the modulation of this interchromatin-associated antigen was correlated with a significant degree of nuclear restructuring. In conjunction with cell sorting, immunogold electron microscopy and immunoblot controls demonstrated that the cell-cycle-related modulation in p105 cannot be accounted for by increased cellular mass or antigen sequestration. The significance of these controls and of the potential role of p105 in cellular proliferation is discussed.