Phenotypic characteristics of coliform and noncoliform bacteria from a public water supply compared with regional and national clinical species.

During the summer and fall of 1984, elevated total coliform counts were observed in the distribution system of a public water supply serving 350,000 people in south central Connecticut. As part of an investigation of possible health risks associated with the presence of bacteria in the water supply, bacterial isolates from the distribution system were compared with bacterial isolates of the same species obtained from a large regional teaching hospital and from a national compendium of clinical isolates. Characteristics analyzed included phenotypic metabolic activity, antimicrobial susceptibilities to clinically utilized antibiotics, temperature tolerance at 44.5 degrees C, and beta-glucuronidase activity in single-test form and on a selective medium. Environmental isolates lacked known plasmid-mediated characteristics, with the exception of one Escherichia coli isolate which showed some antibiotic resistance. Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter agglomerans from all sources were temperature tolerant and yielded positive fecal coliform tests. Only E. coli showed beta-glucuronidase activity (both in a single biochemical test and on a selective medium). No single characteristic analyzed was sufficient to establish an organism as either environmental or clinical in origin.     

Carbohydrates on human Fc gamma receptors. Interdependence of the classical IgG and nonclassical lectin-binding sites on human Fc gamma RIII expressed on neutrophils

To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes. Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots. This binding is specifically inhibitable by alpha-methylmannoside. Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor. Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion. Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions. Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site. Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site.     

Sandhoff disease in Argentina: high frequency of a splice site mutation in the HEXB gene and correlation between enzyme and DNA-based tests for heterozygote detection

The level of -hexosaminidase activity in plasma and leukocytes and the frequency of three known HEXB mutations were studied in an Argentinean deme with high incidence of infantile Sandhoff disease. Two mutations were previously identified in one of two Sandhoff patients from the region, a splice mutation, IVS-2+1 GA, and a 4-bp deletion, CTTT_782–785. These mutations, and a 16kb deletion from the 5' end of the HEXB gene common in non-Argentineans, were screened in 9 Sandhoff patients (all unrelated), 24 obligate heterozygotes, 33 additional individuals belonging to families with affected members, and 64 randomly ascertained individuals from the high risk region. Of 31 independent alleles examined, including those of the two patients previously reported, 30 had the IVS-2 splice mutation and only the originally reported patient had the CTTT deletion. The 16-kb deletion was not observed. Further, among the 57 unaffected members of families with a previous history of Sandhoff disease, and absolute correlation was found between carrier diagnosis by enzyme assay of leukocytes and the DNA-based tests for mutation. One of the 64 controls was classified as a carrier by enzyme assay but did not have one of the three mutations screened. We conclude that a single mutation predominates in this Argentinean population and that the DNA-based test can be an effective supplement or alternative to enzyme-based testing.     

Use of 31P magnetisation transfer magnetic resonance spectroscopy to measure ATP changes after 670 nm transcranial photobiomodulation in older adults

Mitochondrial function declines with age, and many pathological processes in neurodegenerative diseases stem from this dysfunction when mitochondria fail to produce the necessary energy required. Photobiomodulation (PBM), long-wavelength light therapy, has been shown to rescue mitochondrial function in animal models and improve human health, but clinical uptake is limited due to uncertainty around efficacy and the mechanisms responsible. Using ³¹P magnetisation transfer magnetic resonance spectroscopy (MT-MRS) we quantify, for the first time, the effects of 670 nm PBM treatment on healthy ageing human brains. We find a significant increase in the rate of ATP synthase flux in the brain after PBM in a cohort of older adults. Our study provides initial evidence of PBM therapeutic efficacy for improving mitochondrial function and restoring ATP flux with age, but recognises that wider studies are now required to confirm any resultant cognitive benefits.     

Fc gamma receptor III induces actin polymerization in human neutrophils and primes phagocytosis mediated by Fc gamma receptor II.

Human polymorphonuclear leukocytes (PMN) express two classes of Fc gamma R: Fc gamma RII the 42-kDa receptor with a traditional membrane spanning domain and cytoplasmic tail and Fc gamma RIIIPMN the 50- to 80-kDa receptor with a glycosyl-phatidylinositol membrane anchor expressed on PMN. To explore the capacity of Fc gamma RIIIPMN to generate intracellular signals, we have analyzed the ability of Fab and F(ab')2 anti-Fc gamma R mAb to induce actin filament assembly, a prerequisite for motile behaviors. Multivalent ligation of Fc gamma RIIIPMN, independent of Fc gamma RII, results in an increase in F-actin content that is [Ca2+]i dependent. Multivalent ligation of Fc gamma RII also initiates actin polymerization but uses a [Ca2+]i-independent initial pathway. In addition to providing a mechanism for Fc gamma RIIIPMN triggered effector functions, the increase in F-actin and [Ca2+]i generated by Fc gamma RIIIPMN ligation also serves as a "priming" signal to modify PMN responses to other stimuli. Experiments using erythrocytes specifically coated with anti-Fc gamma RII Fab demonstrate that cross-linking of Fc gamma RIIIPMN with anti-Fc gamma RIII F(ab')2 enhances phagocytosis mediated by Fc gamma RII. Thus, Fc gamma RIIIPMN, a glycosyl-phosphatidylinositol anchored protein, may contribute directly to an intracellular program of actin assembly that may trigger and prime neutrophil effector functions.     

Efficacy of beta-glucuronidase assay for identification of Escherichia coli by the defined-substrate technology.

In 1976, Kilian and Bulow described the association of beta-glucuronidase with the genus Escherichia (97% positive) and suggested that a beta-glucuronidase assay would be a useful identification test. Since that report, papers about the sensitivity and specificity of this enzyme for the identification of Escherichia coli from clinical sources, food, seawater, potable-water supplies, and various environmental sources have appeared. A study was undertaken to determine the efficacy and specificity of the defined-substrate technology beta-glucuronidase (Colilert) assay for the identification of this species from fecal samples. A total of 460 human, 105 cow, and 55 horse E. coli isolates were tested. Results showed 95.5% beta-glucuronidase-positive isolates in 24 h and 99.5% positive after 28 h of incubation. Only one E. coli isolate was negative. There were no significant differences in the percentage of beta-glucuronidase-positive isolates among the human or animal isolates. There were no non-E. coli isolates that were positive. All subjects carried beta-glucuronidase-positive E. coli.     

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the β₂ integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both β₂ integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.     

Antibacterial Flavonoids and Other Compounds from the Aerial Parts of Vernonia guineensis Benth. (Asteraceae)

An extensive phytochemical study of the aerial parts of Vernonia guineensis Benth. (Asteraceae) led to the isolation of a new flavone, vernoguinoflavone and a naturally isolated glycerol ester, eicosanoic acid 2‐hydroxy‐1,3‐propanediyl ester, together with eighteen known secondary metabolites including quercetin, luteolin, vernopicrin, vernomelitensin, β‐amyrin, oleanolic acid, ursolic acid, lupeol, betulinic acid, β‐carotene, a mixture of stigmasterol and β‐sitosterol, β‐sitosterol‐3‐O‐β‐D‐glucoside, 2,3‐dihydroxypropyl heptacosanoate, pentacosanoic acid, docosan‐1‐ol, tritriacontan‐1‐ol, and heptatriacontan‐1‐ol. Eleven compounds are reported herein for the first time from this species. The structures of these compounds were elucidated on the basis of extensive spectroscopic analyses, particularly 1D and 2D NMR, and HR‐ESI‐MS and by comparison of their data with those reported in the literature. The crude extract, fractions and some isolated compounds were evaluated for their antibacterial activity against Gram‐negative bacteria: Escherichia coli (ATCC 25922), Shigella flexineri (NR 518), Salmonella muenchen, Salmonella typhimurium and Salmonella typhi (ATCC 19430). All the tested compounds demonstrated inhibitory activities against the tested enteric bacteria with MIC values ranging from 3.12 to 100 μg/ml. Three flavonoids isolated from the most active fraction demonstrated the best bioactivities against Escherichia coli, Salmonella muenchen and Salmonella typhimurium with MIC values ranging from 3.12 to 25 μg/mL.