Treatment of HeLa cells with bacterial water extracts inhibits Shigella flexneri invasion

Abstract Pathogenesis mediated by Shigella flexneri requires invasion of the gastrointestinal epithelium. It has been previously shown that HeLa cells challenged with S. flexneri show alterations in their phosphotyrosine-containing protein profile. In this report, we demonstrated that bacterial water extracts (WE) abrogated the invasion of HeLa cells by S. flexneri in a dose-dependent manner. A proteinaceous component of S. flexneri was shown to be responsible for this inhibitory activity. Proteins encoded on the 140-MDa plasmid were not responsible for the observed inhibition. WE from other Gram-negative bacteria also inhibited Shigella invasion of HeLa cells. HeLa cells pretreated with WE showed changes in the profile and the intensity of phosphotyrosine-containing protein bands. These data were consistent with a surface protein component in WE which initiated aberrant host cell signaling at the membrane which may account for the inhibition of bacterial entry.     

Fetal ultrasonography: Normal biometric ranges in the baboon (Papio hamadryas)

Normal biometric ranges for fetal growth in a captive breeding baboon (Papio hamadryas) colony are described. Measurements include crown-rump length, biparietal diameter, binocular distance, head circumference, abdominal circumference, femur length and amniotic fluid index. The pattern of fetal growth is compared with other baboon subspecies and man. The uses and limitations of such data for breeding colony management and optimum utilisation of experimentally derived data are discussed.     

Molecular Definition of Pericentric Inversion Breakpoints Occurring during the Evolution of Humans and Chimpanzees

High-resolution G-banding analysis has demonstrated remarkable morphological conservation of the chromosomes of the Hominidae family members (humans, chimpanzees, gorillas, and orangutans), with the most notable differences between the genomes appearing as changes in heterochromatin distribution and pericentric inversions. Pericentric inversions may have been important for the establishment of reproductive isolation and speciation of the hominoids as they diverged from a common ancestor. Here the previously published primate karyotype comparisons, coupled with the resources of the Human Genome Project, have been used to identify pericentric inversion breakpoints seen when comparing the human karyotype to that of chimpanzee. Yeast artificial chromosome (YAC) clones were used to detect, by fluorescencein situhybridization, five evolutionary pericentric inversion breakpoints present on the chimpanzee chromosome equivalents of human chromosomes 4, 9, and 12. In addition, two YACs from human 12p that detect a breakpoint in chimpanzee detect a similar rearrangement in gorilla.     

Application of morphometric techniques to postnatal rat testes in organ culture: insights into testis growth

The extent of Sertoli cell proliferation during fetal and neonatal development determines the final adult testis size and potential for sperm output. To gain further knowledge of the factors that regulate Sertoli cell proliferation, the present study used a new approach to analyse changes in morphology and proliferation in the postnatal testis by combining organ culture with morphometric analysis. Fragments of rat testes from days 0 to 10 postpartum were cultured in contact with DMEM for 6 h or 72 h and fixed. The effects of ovine follicle-stimulating hormone (FSH) and activin were studied in an additional 72-h organ culture experiment using day 9 testes. Bromodeoxyuridine (BrdU) was added for the last 6 h of culture to mark proliferating cells. Two-microm sections of the fragments were analysed for morphological changes of the seminiferous cords, and the proportion of BrdU-labelled Sertoli and germ cells was determined. Assessment of 6-h samples revealed growth characteristics consistent with those observed in vivo during days 1-10 of postnatal development. From day 2 onwards, the volume fraction of seminiferous cords began to increase, while significant growth in cross-sectional area of the cords occurred only after day 6. In these culture conditions, germ cell proliferation and testicular architecture was consistent with that expected for the age of the tissue at time of explant. The proportion of dividing Sertoli cells declined from 15-20% at days 0-4 postpartum to below % at day 10 postpartum in the 6-h culture, and it was low or abolished in the 3-day culture at all time points. Activin and FSH together, but not singly, stimulated Sertoli cell proliferation in the 72-h culture. This paper presents a new approach to analysis of in vitro testis development. The combination of fragment culture and stereological analysis permits rigorous and detailed assessment of developmental changes in the postnatal testis.     

Functional domain structure of human heterochromatin protein HP1(Hsalpha): involvement of internal DNA-binding and C-terminal self-association domains in the formation of discrete dots in interphase nuclei

Human heterochromatin protein HP1(Hsalpha) possesses two evolutionarily conserved regions in the N- and C-terminal halves, so-called chromo and chromo-shadow domains, and DNA-binding domain in the internal non-conserved region. Here, to examine its in vivo properties, we expressed HP1(Hsalpha) as a fusion product with green fluorescent protein in human cells. HP1(Hsalpha) was observed to form discrete dots in interphase nuclei and to localize in the centromeric region of metaphase chromosomes by fluorescence microscopy. Interestingly, this dot-forming activity was also found in the N-terminal half retaining the chromo and DNA-binding domains and in the C-terminal chromo-shadow domain. However, the chromo domain alone stained nuclei homogeneously. To correlate this dot-forming activity with self-associating activity in vitro, the chromo and chromo-shadow domain peptides were independently expressed in Escherichia coli, affinity purified, and chemically cross-linked with glutaraldehyde. In a SDS-polyacrylamide gel, the former mainly produced a dimer, while the latter produced a ladder of bands up to a tetramer. When passed through a gel filtration column in a native state, these peptides were exclusively separated as a dimer and a tetramer, respectively. These results suggested that the internal DNA-binding and C-terminal chromo-shadow domains are both involved in heterochromatin formation in vivo.     

Reversal of some viral IL-6 electrostatic properties compared to IL-6 contributes to a loss of alpha receptor component recruitment

Human interleukin-6 (hIL-6) is a pleiotropic mediator of activation and proliferation across a large number of different cell types. Human herpesvirus-8 (HHV-8) has been associated with classical and AIDS-related Kaposi's sarcoma (KS). HHV-8 encodes viral IL-6 (vIL-6), a functional homolog of human interleukin-6, that promotes the growth of KS and of some lymphoma cells. Signaling induced by human IL-6 requires recruitment of the glycoprotein gp130, which acts as the signal transducing chain, and of IL-6Ralpha, which is necessary for cognate recognition and high affinity receptor complex formation. In contrast, the formation of a functional complex between vIL-6 and gp130 does not require the presence of IL-6Ralpha. The physico-chemical properties of vIL-6 have been analyzed and compared to those of hIL-6 and of the receptor chains, gp130 and IL-6Ralpha. Interaction sites on vIL-6 involve more hydrophobic residues than those of hIL-6. The electrostatic fields induced by vIL-6 and IL-6Ralpha are repulsive and prevent interaction between vIL-6 and IL-6Ralpha, whereas the electrostatic field induced by hIL-6 steers the complex formation with IL-6Ralpha. Subsequently, electrostatic binding free energy in the vIL-6/IL-6Ralpha complex is destabilizing, whereas it is stabilizing in the complex comprising hIL-6. These properties result from charge reversals between viral and human IL-6, an unusual phenomenon of amino acid substitutions within a homologous protein family. This suggests a selection pressure for vIL-6 to by-pass the IL-6Ralpha control of host defense against virus infection. This selection pressure has yielded the reversal of electrostatic properties of vIL-6 when compared to hIL-6.     

A Generalization of the Transmission/Disequilibrium Test for Uncertain-Haplotype Transmission

A new transmission/disequilibrium-test statistic is proposed for situations in which transmission is uncertain. Such situations arise when transmission of a multilocus marker haplotype is considered, since haplotype phase is often unknown in a substantial number of instances. Even for single-locus markers, transmission is uncertain if one or both parents are missing. In both these situations, uncertainty may be reduced by the typing of further siblings, whose disease status may be unaffected or unknown. The proposed test is a score test based on a partial score function that omits the terms most influenced by hidden population stratification.     

Effects of plant leachates from four boreal understorey species on soil N mineralization, and white spruce (Picea glauca) germination and seedling growth

BACKGROUND AND AIMS: Natural regeneration of white spruce (Picea glauca) after disturbance has been reported to be very poor. Here a study was made to determine whether C compounds released from understorey species growing together with white spruce could be involved in this regeneration failure, either by (1) changing soil nutrient dynamics, (2) inhibiting germination, and/or (3) delaying seedling growth. METHODS: Foliage leachates were obtained from two shrubs (Ledum palustre and Empetrum hermaphroditum) and one bryophyte (Sphagnum sp.) with high phenolic compound concentrations that have been reported to depress growth of conifers in boreal forests, and, as a comparison, one bryophyte (Hylocomium splendens) with negligible phenolic compounds. Mineral soil from a white spruce forest was amended with plant leachates to examine the effect of each species on net N mineralization. Additionally, white spruce seeds and seedlings were watered with plant leachates to determine their effects on germination and growth. KEY RESULTS: Leachates from the shrubs L. palustre and E. hermaphroditum contained high phenolic compound concentrations and dissolved organic carbon (DOC), while no detectable levels of C compounds were released from the bryophytes Sphagnum sp. or H. splendens. A decrease in net N mineralization was determined in soils amended with L. palustre or E. hermaphroditum leachates, and this effect was inversely proportional to the phenolic concentrations, DOC and leachate C/N ratio. The total percentage of white spruce germination and the growth of white spruce seedlings were similar among treatments. CONCLUSIONS: These results suggest that the shrubs L. palustre and E. hermaphroditum could negatively affect the performance of white spruce due to a decrease in soil N availability, but not by direct effects on plant physiology.