The use of biochemical markers, serotype and fimbriation in the detection of Escherichia coli clones

Biochemical reactions, O and K serotypes and presence of P-fimbriae were analysed in 116 Escherichia coli strains isolated in blood cultures from patients with bacteraemia and in 99 faecal strains isolated from healthy individuals. By using biochemical typing, the strains could be grouped into six main clusters with similarity index less than 0.8 (Gower, 1971) and altogether 16 subclusters with similarity index 0.82-0.89. The most discriminating tests between the clusters were fermentation of D-tagatose, saccharose, salicin and sorbose. No single biochemical property could differentiate bacteraemic isolates from faecal strains, although strains isolated from blood were significantly more often found in certain subclusters, whereas other subclusters contained mainly control strains. Bacteraemic strains possessed P-fimbriae more often, especially strains isolated from patients with E. coli in the urine concomitantly with bacteraemia. Equally, no single reaction could separate P-fimbriated from non-P-fimbriated strains. D-Tagatose was fermented more often by the P-fimbriated strains; on the other hand, melibiose and lactose fermentation tests were less often positive. Certain O serotypes (O1, O4, O6, O7, O18 and O25) were more common among bacteraemic isolates than controls. K serotypes such as K1, K5 and K52 were also more frequent among blood isolates. We conclude that a combination of biochemical tests, fimbriation and serotyping might be used to identify potentially pathogenic clusters of E. coli.     

Cell matrix adhesion-related proteins VLA-1 and VLA-2: regulation of expression on T cells

The mitogens phytohemagglutinin (PHA) and concanavalin A inhibited the appearance of the very late activation antigen (VLA)-1, but did not inhibit VLA-2 expression on cultured activated T cells. In contrast to diminished VLA-1 expression, mitogen treatment caused increased cell surface expression of other activation antigens such as T10, HLA-DR, interleukin 2 (IL 2) receptor, and 4F2, and greater cell proliferation. Conversely, when T cells were not repetitively restimulated with mitogen, these less proliferative "postactivated" T cells had elevated VLA-1 expression. The diminished expression of VLA-1 caused by PHA was reversible since subsequent removal of mitogen was associated with increased VLA-1, paralleled by a decrease in interleukin 2 receptor levels. In addition to preventing or delaying the initial appearance of VLA-1, PHA stimulation also was somewhat effective in causing the disappearance of VLA-1 already present, especially on recently established cultures. However, cultures that had either never seen PHA, not seen PHA for several weeks, or been stimulated regularly with PHA, but were several months old, did not lose VLA-1 in response to PHA stimulation, suggesting that a state of insensitivity to PHA effects could be attained. Unlike PHA-stimulated T cells, T cells repetitively restimulated with alloantigen or the monoclonal antibody T3 did not show a marked absence of VLA-1 but rather showed an increased level of VLA-2 relative to VLA-1. Taken together, results of stimulation by either mitogen, alloantigen, or anti-T3 monoclonal antibody support the conclusion that T cell stimulation in general can cause a decreased VLA-1:VLA-2 ratio, whether by decreased VLA-1 or increased VLA-2. These shifts in VLA-1:VLA-2 ratios are probably not simply the result of shifts in the relative proportions of different subpopulations, because similar growth-related changes in this ratio were observed on the T cell line ANITA, which is a homogeneous population of cells. Because both VLA-1 and VLA-2 are differentially regulated on cultured, long term activated T cells depending on stage of activation and growth conditions, and are members of a family of at least five heterodimers that includes cell matrix adhesion molecules, we suggest that these studies will provide clues to novel aspects of T cell growth regulation, perhaps relating to T cell-matrix adhesion.     

Characterization of polymers of adenosine diphosphate ribose generated in vitro and in vivo

Methods have been developed and applied to determine the size and branching frequency of polymers of ADP-ribose synthesized in nucleotide-permeable cultured mouse cells and in intact cultured cells. Polymers were purified by affinity chromatography with a boronate resin and were fractionated according to size molecular sieve high-performance liquid chromatography. Fractions were enzymatically digested to nucleotides, which were separated by strong anion exchange high-performance liquid chromatography. From these data, average polymer size and branching frequency were calculated. A wide range of polymer sizes was observed. Polymers as large as 190 residues with at least five points of branching per molecule were generated in vitro. Polymers of up to 67 residues containing up to two points of branching per molecule were isolated from intact cells following treatment with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Cells treated with hyperthermia prior to DNA damage contained polymers of an average maximum size of 244 residues containing up to six points of branching per molecule. The detection of large polymers of ADP-ribose in intact cells suggests that alterations in chromatin organization effected by poly(ADP-ribosylation) may extend beyond the covalently modified proteins and very likely involve noncovalent interactions of poly(ADP-ribose) with other components of chromatin.     

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbillus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially reinactivated, suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.Deceased on 2 Jan. 1987     

Cloning of a human S-phase cell cycle gene: use of transient expression for screening.

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.     

Aspergillus myositis in a patient with a myelodysplastic syndrome

We present the case of an elderly man who, while being treated with corticosteroids for a myelodysplastic syndrome, developed myositis of the calf due to Aspergillus fumigatus. Despite therapy with amphotericin B the myositis failed to resolve and he died. At autopsy, a localized necrotizing myositis of the right calf was found with no evidence of disseminated Aspergillus infection. Myositis in the setting of disseminated candidiasis or cryptococcosis has been previously reported. This case is unique in that it is the first reported case of localized fungal myositis and of myositis caused by Aspergillus.     

Mesodermal metamerism in the teleost, Oryzias latipes (the medaka)

Previous studies of the metameric pattern in mesodermal tissues of chick, mouse, turtle, and amphibian embryos have indicated that segmental characteristics exist along the entire length of the embryo. This paper describes this phenomenon in a fish embryo, for some differences in the cranial segmental plan exist between the anamniote and the amniote embryos hitherto studied. Embryos of the cyprinodont, Oryzias latipes, were fixed at various times, the examined by means of stereo scanning electron microscopy. As in other vertebrate embryos, the first indication of mesodermal metamerism in this fish embryo is the occurrence of somitomeres, which are orderly, tandemly arranged units of uncondensed mesenchymal cells in the paraxial mesoderm. As many as ten somitomeres can be observed caudal to the last formed somite to the elongating tail region. In addition, 7 somitomeres are present rostral to the first definitive somite, which is segment number eight. As in other vertebrate embryos examined, somitomeres in Oryzias embryos are circular, bilaminar arrays of paraxial mesoderm that form before any indications of segmentation can be seen with the light microscope. In the trunk region these mesodermal units condense to give rise to definitive somites, but in the head they eventually disperse. Despite a fundamentally different mode of gastrulation and a relatively small number of cells in the newly formed somitomeres, cranial segmentation in Oryzias embryos was found to be more similar in number to the metameric pattern of the embryos of the bird, reptile, and mammal than to the situation found in the two amphibians studied thus far.     

Embryo transfer in the White-tailed deer: A reproductive model for endangered deer species of the world

A model for artificial propagation of wild, seasonal breeding deer was developed using the White-tailed deer, Odocoileus , virginianus . Preseason estrus induction following removal of vaginal pessaries containing medroxyprogesterone acetate (MPA) was successful in four of seven animals. Synchronization of estrus was achieved in seven of nine attempts when does were treated with prostaglandin F(2) alpha (PGF(2)alpha) 7 to 14 d following observed estrus. Superovulation was achieved in two of five does treated with 1000 IU of pregnant mare's serum gonadotropin (PMSG) at the time of vaginal pessary MPA withdrawal. Superovulation was achieved in two of three does treated with 1000 IU of PMSG at the time of estrus induction with PGF(2)alpha. Surgical embryo recoveries attempted on six does resulted in a 68% embryo recovery rate based on numbers of corpora lutea observed. Surgical embryo transfers from two donors to three recipient does resulted in one pregnancy maintained to full term.     

Effects of sepiapterin and 6-acetyldihydrohomopterin on the guanosine triphosphate cyclohydrolase I of mouse, rat and the fruit-fly Drosophila.

The regulation of GTP cyclohydrolase I would lead to the regulation of tetrahydrobiopterin, an important cofactor for synthesis of neurotransmitters. In an attempt to extend a previous finding [Bellahsene, Dhondt, & Farriaux (1984) Biochem. J. 217, 59-65] that GTP cyclohydrolase I of rat liver is inhibited by subnanomolar concentrations of reduced biopterin and sepiapterin, we found that this could not be verified with the enzyme from mouse liver, fruit-fly (Drosophila) heads or, indeed, from rat liver. It was shown, however, that 12 microM-sepiapterin inhibited mouse liver GTP cyclohydrolase I. Another compound, namely 6-acetyldihydrohomopterin, was also employed in the present study to explore its effect on enzymes that lead to its synthesis in Drosophila and for effects on mammalian systems; at 2-5 microM this compound was shown to stimulate one form of mouse liver GTP cyclohydrolase I and then to inhibit at higher concentrations (40 microM). Neither sepiapterin nor 6-acetyldihydrohomopterin caused any effect on the Drosophila head enzyme. On the other hand, the sigmoid GTP concentration curve for the Drosophila enzyme may indicate a regulatory characteristic of this enzyme. Another report, on the lower level of GTP cyclohydrolase I in mutant mouse liver [McDonald, Cotton, Jennings, Ledley, Woo & Bode (1988) J. Neurochem. 50, 655-657], was confirmed and extended. Instead of having 10% activity, we find that the hph-1 mouse mutant has less than 2% activity in the liver. These studies demonstrate that micromolar levels of reduced pterins may have regulatory effects on GTP cyclohydrolase I and that a mouse mutant is available that has low enough activity to be considered as a model for human atypical phenylketonuria.