Outer membrane protein A deficient Escherichia coli activates neutrophils to produce superoxide and shows increased susceptibility to antibacterial peptides

The outer membrane protein A (OmpA) of Gram-negative bacteria has been ascribed multiple functions including maintenance of structural membrane integrity and porin activity. OmpA has also been implicated in various host defense processes in that it contributes to bacterial serum resistance and activates certain immune cells. Recently, OmpA was shown to be the molecular target for neutrophil elastase (NE), and Escherichia coli mutants lacking OmpA were resistant to the bactericidal effects of NE. In addition to NE, neutrophils use a variety of other antibacterial effector molecules such as oxygen radicals and bactericidal peptides or proteins. The aim of this study was to investigate the role of E. coli OmpA regarding susceptibility to other neutrophil-derived defense systems. We found that OmpA-deficient (OmpA(-)), but not wild-type isogenic, E. coli activated human neutrophils to produce oxygen radicals intracellularly. This activation was found to require an intact neutrophil cytoskeleton but was independent of bacterial phagocytosis. Furthermore, we found that the OmpA(-) strain was more susceptible to membrane-acting bactericidal peptides than the wild-type strain, although the susceptibility to different oxygen radicals was independent of the presence of OmpA. Taken together, these data suggest an important role for OmpA in the context of bacteria vs. host interactions.     

Clinical relevance of matrix metalloproteinase-13 determined with a new highly specific and sensitive ELISA in ascitic fluid of advanced ovarian carcinoma patients

Matrix metalloproteinases (MMPs) are involved in many physiological and pathophysiological processes, including tumor cell invasion and metastasis. For one member of this family, MMP-13 (collagenase-3), a new, highly specific ELISA with a sensitivity of 0.5 ng MMP-13/ml was established. The protein levels of MMP-13 in ascitic fluids of 30 patients with advanced ovarian cancer FIGO stage III (n = 19) and IV (n = 11) were measured with this ELISA. Using a cut-off value of 0.5 ng MMP-13/mg total protein, two patient subpopulations with short (median 16 months) and long (median 36 months) overall survival were identified. Together with other prognostic markers, determination of MMP-13 in ascitic fluid may help to identify patients at risk for early death and help to individualize adjuvant therapy.     

Specific regulation of lipocalin-type prostaglandin D synthase in mouse heart by estrogen receptor beta

Estrogens have important physiological roles in the cardiovascular system. We use DNA microarray technology to study the molecular mechanism of estrogen action in the heart and to identify novel estrogen-regulated genes. In this investigation we identify genes that are regulated by chronic estrogen treatment of mouse heart. We present our detailed characterization of one of these genes, lipocalin-type prostaglandin D synthase (L-PGDS). Northern and Western blot analysis revealed that L-PGDS was induced both by acute and chronic estrogen treatment. Northern blot analysis, using estrogen receptor (ER)-disrupted mice, suggests that L-PGDS is specifically induced by ERbeta in vivo. In further support of ERbeta-selective regulation, we identify a functional estrogen-responsive element in the L-PGDS promoter, the activity of which is up-regulated by ERbeta, but not by ERalpha. We demonstrate that a one-nucleotide change (A to C) in the L-PGDS estrogen-responsive element affects receptor selectivity.     

Hormone replacement therapy in rheumatoid arthritis is associated with lower serum levels of soluble IL-6 receptor and higher insulin-like growth factor 1

Hormone replacement therapy (HRT) modulates the imbalance in bone remodeling, thereby decreasing bone loss. Sex hormones are known to influence rheumatic diseases. The aim of this study was to investigate the effects of HRT on the serum levels of hormones and cytokines regulating bone turnover in 88 postmenopausal women with active rheumatoid arthritis (RA) randomly allocated to receive HRT plus calcium and vitamin D3 or calcium and vitamin D3 alone for 2 years. An increase in estradiol (E2) correlated strongly with improvement of bone mineral density in the hip (P < 0.001) and lumbar spine (P < 0.001). Both baseline levels and changes during the study of IL-6 and erythrocyte sedimentation rate were correlated positively (P < 0.001). HRT for 2 years resulted in an increase of the bone anabolic factor, insulin-like growth factor 1 (IGF-1) (P < 0.05) and a decrease of serum levels of soluble IL-6 receptor (sIL-6R) (P < 0.05), which is known to enhance the biological activity of IL-6, an osteoclast-stimulating and proinflammatory cytokine. Baseline levels of IL-6 and IGF-1 were inversely associated (P < 0.05), and elevation of IGF-1 was connected with decrease in erythrocyte sedimentation rate (P < 0.05) after 2 years. Interestingly, increase in serum levels of E2 was associated with reduction of sIL-6R (P < 0.05) and reduction of sIL-6R was correlated with improved bone mineral density in the lumbar spine (P < 0.05). The latter association was however not significant after adjusting for the effect of E2 (P = 0.075). The influences of IGF-1 and the IL-6/sIL-6R pathways suggest possible mechanisms whereby HRT may exert beneficial effects in RA. However, to confirm this hypothesis future and larger studies are needed.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.     

Aposymbiotic culture of the sepiolid squid Euprymna scolopes: role of the symbiotic bacterium Vibrio fischeri in host animal growth, development, and light organ morphogenesis

The sepiolid squid Euprymna scolopes forms a bioluminescent mutualism with the luminous bacterium Vibrio fischeri, harboring V. fischeri cells in a complex ventral light organ and using the bacterial light in predator avoidance. To characterize the contribution of V. fischeri to the growth and development of E. scolopes and to define the long-term effects of bacterial colonization on light organ morphogenesis, we developed a mariculture system for the culture of E. scolopes from hatching to adulthood, employing artificial seawater, lighting that mimicked that of the natural environment, and provision of prey sized to match the developmental stage of E. scolopes. Animals colonized by V. fischeri and animals cultured in the absence of V. fischeri (aposymbiotic) grew and survived equally well, developed similarly, and reached sexual maturity at a similar age. Development of the light organ accessory tissues (lens, reflectors, and ink sac) was similar in colonized and aposymbiotic animals with no obvious morphometric or histological differences. Colonization by V. fischeri influenced regression of the ciliated epithelial appendages (CEAs), the long-term growth of the light organ epithelial tubules, and the appearance of the cells composing the ciliated ducts, which exhibit characteristics of secretory tissue. In certain cases, aposymbiotic animals retained the CEAs in a partially regressed state and remained competent to initiate symbiosis with V. fischeri into adulthood. In other cases, the CEAs regressed fully in aposymbiotic animals, and these animals were not colonizable. The results demonstrate that V. fischeri is not required for normal growth and development of the animal or for development of the accessory light organ tissues and that morphogenesis of only those tissues coming in contact with the bacteria (CEAs, ciliated ducts, and light organ epithelium) is altered by bacterial colonization of the light organ. Therefore, V. fischeri apparently makes no major metabolic contribution to E. scolopes beyond light production, and post-embryonic development of the light organ is essentially symbiont independent. J. Exp. Zool. 286:280-296, 2000.     

Retrograde tracing and neuropeptide immunohistochemistry of sensory neurones projecting to the cartilaginous distal femoral epiphysis of young rats

Although cartilage is considered to be devoid of innervation, axons occur in the perichondrium and during development in cartilage canals, thereby having a relatively close spatial relationship to chondroblasts and chondrocytes. The present study locates the source of the sensory innervation of the femoral cartilaginous epiphyses of young rats and investigates whether the neuropeptide calcitonin gene-related peptide (CGRP) can influence chondrocytes. Retrograde tracing from the distal femoral epiphysis of young rats with Fast Blue (FB) showed labelled neuronal profiles in the L2-L5 dorsal root ganglia. Sample countings indicated that 50% of the FB-labelled neuronal profiles were located at the L3 level and 25% at the L4 level. The labelled neurones had diameters of 15-40 microm, with a peak at 25-30 microm. Immunohistochemistry showed that about 50% of the FB-labelled profiles contained CGRP. Together with the finding that CGRP influences bone cells to generate the second messenger cAMP, this result suggested the hypothesis that chondrocytes might be similarly influenced by CGRP. However, stimulation of cartilage slices with CGRP in vitro followed by an assay of the cAMP content did not provide support for this hypothesis. We conclude that primary sensory neurones containing CGRP project to the perichondrium and to cartilage canals of growing cartilage, and that exogenous CGRP does not elevate the cAMP content of cartilage slices in vitro.     

Identification of the rrmA Gene Encoding the 23S rRNA m1G745 Methyltransferase in Escherichia coli and Characterization of an m1G745-Deficient Mutant

An Escherichia coli mutant lacking the modified nucleotide m¹G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83–100, 1970). In this study, we localize the position of the m¹G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. The rrmA gene encoding 23S rRNA m¹G745 methyltransferase was mapped to bp 1904000 on the E. coli chromosome and identified to be identical to the previously sequenced gene yebH.     

The expression of the salt-responsive gene salT from rice is regulated by hormonal and developmental cues

The expression pattern of the salT gene was analyzed in different cell types and organs of rice (Oryza sativa L.) in response to saline and hormonal treatments to obtain detailed information on the physiological cues controlling gene expression. Gel blot analysis of RNA and in-situ hybridization performed on seedlings grown for 10 ds in the presence of 1% NaCl revealed that salT was expressed mainly in the younger tissues of the plant. In contrast, 6-week-old plants exhibited maximal salT mRNA accumulation in sheaths of older leaves. In addition, salT was normally expressed in rapidly dividing suspension-cultured cells, but not in quiescent ones. Altogether, these results may indicate that salT expression in each region of the plant is dependent on the metabolic activity of the cells as well as on whether or not they are stressed. The effects of two growth regulators, abscisic acid (ABA) and gibberellic acid, were investigated in combination with the effects of NaCl. Gibberellic acid had a synergistic effect on the induction of the salT gene when combined with 0.5% NaCl, but did not induce salT on its own. At 10 μM, ABA induced salT both in the absence of NaCl and in its presence. Whereas 1 μM ABA acted additively with NaCl to induce gene expression, 5 μM ABA with NaCl was only as effective as NaCl alone. This may indicate that the two stimuli act independently and possibly through antagonistic signal transduction pathways. Received: 26 March 1998 / Accepted: 11 July 1998