Effects of interleukin-8 on granulation tissue maturation

The inflammatory alpha-chemokine, interleukin-8 (IL-8), affects the function and recruitment of various inflammatory cells, fibroblasts, and keratinocytes. Gap junctions are anatomical channels that facilitate the direct passage of small molecules between cells. The hypothesis is that IL-8 enhances gap junctional intercellular communication (GJIC) between fibroblasts in granulation tissue, which increases the rate of granulation tissue maturation. In vitro, human dermal fibroblasts were incubated with IL-8 prior to scrape loading, a technique that quantifies GJIC. Polyvinyl alcohol (PVA) sponges were implanted within subcutaneous pockets in rats and received local injections of either IL-8 or saline and were harvested on day 11. In vitro, IL-8 treated fibroblasts demonstrated an increase in GJIC by scrape loading compared to saline treated controls. In vivo, IL-8 treated PVA sponges demonstrated a decrease in cell density and an increase in vascularization compared to saline controls by H&E staining. Polarized light viewed Sirius red-stained specimens demonstrated greater collagen birefringence intensity, indicating thicker, more-mature collagen fibers. IL-8 increases GJIC in cultured fibroblasts and induces a more rapid maturation of granulation tissue.     

Clonal variation in maximum specific growth rate and susceptibility towards antimicrobials

AIMS: To examine associations between growth rate within bacterial populations and survival patterns following treatment with antimicrobial agents. METHODS AND RESULTS: Time survival data were generated for the inactivation of Escherichia coli populations, grown as batch and continuous cultures, exposed to ciprofloxacin, benzalkonium chloride and tetracycline. Time-survivor plots were biphasic. Surviving cells were collected and immediately re-exposed to agent or were regrown and then re-exposed. Survivors were resistant to immediate challenge with any of the treatment agents. This resistance was lost on regrowth suggesting that survival reflects an expressed phenotype within a subset of the culture (persisters) rather than individual resistant clones or nonspecific quenching of the test agent. The fraction of persisters increased with decreasing growth rate when cultures were prepared in continuous culture. CONCLUSIONS: Clonal growth rates within populations were determined by culture of individual cells within microtitre plate wells. The fraction of clones, in batch cultures, growing maximally at rates below the apparent threshold for susceptibility to the test agents was sufficient to explain the results of continuous culture experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of persisters in populations of bacteria relate to small subset of cells that are growing only slowly or are metabolically quiescent.     

Nuclease activities and DNA fragmentation during programmed cell death of megagametophyte cells of white spruce (Picea glauca) seeds

The haploid megagametophyte of white spruce (Picea glauca) seeds undergoes programmed cell death (PCD) during post-germinative seedling growth. Death of the megagametophyte storage parenchyma cells was preceded by reserve mobilization and vacuolation. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling)-positive nuclei indicated that the first megagametophyte cells to die were those closest to the radicle at the micropylar end of the seed as well as those that comprised the most peripheral and innermost layers at the chalazal end of the seed. The death process was accompanied by nuclear fragmentation and internucleosomal DNA cleavage and the sequential activation of several nucleases. The latter comprised at least two groups: those induced relatively early during post-germinative seedling growth, that had pH optima in the neutral range (33, 31, 17 and 15 kDa), and those induced later that had pH optima in the acidic range (73, 62, 48, 43 and 29 kDa). Activities of all of the nucleases were stimulated by Ca²⁺, Mg²⁺ and Mn²⁺; only the nucleases active at neutral pH were inhibited by Zn²⁺. The temporal pattern of induction of the neutral and acidic nucleases may suggest that the latter function after tonoplast rupture.     

Novel short oligonucleotide conjugates as inhibitors of human telomerase

A series of oligonucleotide conjugates were designed and synthesized as novel inhibitors of human telomerase. These compounds contain a relatively short (6-7-mer) oligonucleotide domain, with an N3'-->P5' phosphoramidate (np) or thio-phosphoramidate (nps) backbone, targeted to the template region of the RNA component of the enzyme and various pendant groups attached to either their 5'- or preferably to the 3'-termini. The most potent compounds in the series inhibited telomerase with low nM IC50 values in biochemical assays whereas the cognate oligonucleotides without the pendant groups were significantly less active having IC50 values 100-1000-fold higher.     

Ovine uterine gland knock-out model: effects of gland ablation on the estrous cycle

Ovine endometrial gland development is a postnatal event that can be inhibited epigenetically by chronic exposure of ewe lambs to a synthetic progestin from birth to puberty. As adults, these neonatally progestin-treated ewes lack endometrial glands and display a uterine gland knockout (UGKO) phenotype that is useful as a model for study of endometrial function. Here, objectives were to determine: 1) length of progestin exposure necessary from birth to produce the UGKO phenotype in ewes; 2) if UGKO ewes display normal estrous cycles; and 3) if UGKO ewes could establish and/or maintain pregnancy. Ewe lambs (n = 22) received a Norgestomet (Nor) implant at birth and every two weeks thereafter for 8 (Group I), 16 (Group II), or 32 (Groups III and IV) weeks. Control ewe lambs (n = 13) received no Nor treatment (Groups V and VI). Ewes in Groups I, II, III, and VI were hemihysterectomized (Hhx) at 16 weeks of age. After puberty, the remaining uterine horn in Hhx ewes was removed on either Day 9 or 15 of the estrous cycle (Day 0 = estrus). Histological analyses of uteri indicated that progestin exposure for 8, 16, or 32 weeks prevented endometrial adenogenesis and produced the UGKO phenotype in adult ewes. Three endometrial phenotypes were consistently observed in Nor-treated ewes: 1) no glands, 2) slight glandular invaginations into the stroma, and 3) limited numbers of cyst- or gland-like structures in the stroma. Overall patterns of uterine progesterone, estrogen, and oxytocin receptor expression were not different in uteri from adult cyclic control and UGKO ewes. However, receptor expression was variegated in the ruffled luminal epithelium of uteri from UGKO ewes. Intact UGKO ewes displayed altered estrous cycles with interestrous intervals of 17 to 43 days, and they responded to exogenous prostaglandin F(2 approximately ) (PGF) with luteolysis and behavioral estrus. During the estrous cycle, plasma concentrations of progesterone in intact control and UGKO ewes were not different during metestrus and diestrus, but levels did not decline in many UGKO ewes during late diestrus. Peak peripheral plasma concentrations of PGF metabolite, in response to an oxytocin challenge on Day 15, were threefold lower in UGKO compared to control ewes. Intact UGKO ewes bred repeatedly to intact rams did not display evidence of pregnancy based on results of ultrasound. Collectively, results indicate that 1) transient, progestin-induced disruption of ovine uterine development from birth alters both structural and functional integrity of the adult endometrium; 2) normal adult endometrial integrity, including uterine glands, is required to insure a luteolytic pattern of PGF production; and 3) the UGKO phenotype, characterized by the absence of endometrial glands and a compact, disorganized endometrial stroma, limits or inhibits the capacity of uterine tissues to support the establishment and/or maintenance of pregnancy.     

Targeted insertion of a lacZ reporter gene into the mouse Cer1 locus reveals complex and dynamic expression during embryogenesis

The mouse Cer1 (mCer1, Cer-l, Cerr1) gene encodes one member of a family of cytokines structurally and functionally related to the Xenopus head-inducing factor, Cerberus (xCer). We generated a mouse line in which the Cer1 gene was inactivated by replacing the first coding exon with a lacZ reporter gene. Mice homozygous for this allele (Cer1(lacZ)) showed no apparent perturbation of embryogenesis or later development. However, the lacZ reporter revealed a number of hitherto uncharacterised sites of Cer1 expression in late fetal and adult tissues. Preliminary analysis suggests that Cer1 is not essential for their morphogenesis, differentiation, or homeostasis.     

Testing the robustness of the new Haseman-Elston quantitative-trait loci-mapping procedure

Variance components (VC) techniques have emerged as among the more powerful methods for detection of quantitative-trait loci (QTL) in linkage analysis. Allison et al. found that, with particularly marked leptokurtosis in the phenotypic distribution and moderate-to-high residual sibling correlation, maximum likelihood (ML) VC methods may produce a severe excess of type I errors. The new Haseman-Elston (NHE) method is a least-squares-based VC method for mapping of QTL in sib pairs (Elston et al.). Using simulation, we investigate the robustness of the NHE to marked nonnormality, by means of the same distributions and worst-case conditions identified by Allison et al. for the ML approach (i.e., 100 pairs; high residual sibling correlation). Results showed that, when marked nonnormality is present, the NHE can be used without severe type I error-rate inflation, even at very small alpha levels.     

Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site

The impact of a specific region of the envelope protein E of tick-borne encephalitis (TBE) virus on the biology of this virus was investigated by a site-directed mutagenesis approach. The four amino acid residues that were analyzed in detail (E308 to E311) are located on the upper-lateral surface of domain III according to the X-ray structure of the TBE virus protein E and are part of an area that is considered to be a potential receptor binding determinant of flaviviruses. Mutants containing single amino acid substitutions, as well as combinations of mutations, were constructed and analyzed for their virulence in mice, growth properties in cultured cells, and genetic stability. The most significant attenuation in mice was achieved by mutagenesis of threonine 310. Combining this mutation with deletion mutations in the 3'-noncoding region yielded mutants that were highly attenuated. The biological effects of mutation Thr 310 to Lys, however, could be reversed to a large degree by a mutation at a neighboring position (Lys 311 to Glu) that arose spontaneously during infection of a mouse. Mutagenesis of the other positions provided evidence for the functional importance of residue 308 (Asp) and its charge interaction with residue 311 (Lys), whereas residue 309 could be altered or even deleted without any notable consequences. Deletion of residue 309 was accompanied by a spontaneous second-site mutation (Phe to Tyr) at position 332, which in the three-dimensional structure of protein E is spatially close to residue 309. The information obtained in this study is relevant for the development of specific attenuated flavivirus strains that may serve as future live vaccines.