Characterization and pro-inflammatory responses of spore and hyphae samples from various mold species.

Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory-related diseases. We characterized X-ray-inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, ß-glucan and protease content/activity. The pro-inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS-2B) and THP-1 monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin-degrading protease that was either of the metallo- or serine type, while one remains unclassified. Mycotoxin levels were generally low. Detectable levels of ß-glucans were found mainly in hyphae particle preparations. PMA-differentiated THP-1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm2 . Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum. Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP-9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro-inflammatory potential.

Proteases in Plasma and Kidney of db/db Mice as Markers of Diabetes-Induced Nephropathy.

Db/db mice are overweight, dyslipidemic and develop diabetic complications, relevant for similar complications in human type 2 diabetes. We have used db/db and db/+ control mice to investigate alterations in proteinase expression and activity in circulation and kidneys by SDS-PAGE zymography, electron microscopy, immunohistochemistry, Western blotting, and in situ zymography. Plasma from db/db mice contained larger amounts of serine proteinases compared to db/+ mice. Kidneys from the db/db mice had a significantly larger glomerular surface area and somewhat thicker glomerular basement membranes compared to the db/+ mice. Furthermore, kidney extracts from db/+ mice contained metalloproteinases with M(r) of approximately 92000, compatible with MMP-9, not observed in db/db mice. These results indicate that higher levels of serine proteinases in plasma may serve as potential markers for kidney changes in db/db mice, whereas a decrease in MMP-9 in the kidney may be related to the glomerular changes.

Medium- and short-chain dehydrogenase/reductase gene and protein families : Structure-function relationships in short-chain alcohol dehydrogenases.

The structure-function relationships of alcohol dehydrogenases from the large family of short-chain dehydrogenase/reductase (SDR) enzymes are described. It seems that while mammals evolved with a medium-chain alcohol dehydrogenase family (MDR), fruit flies utilized an ancestral SDR enzyme. They have modified its function into an efficient alcohol dehydrogenase to aid them in colonizing the emerging ecological niches that appeared around 65 million years ago. To the scientific community, Drosophila has now served as a model organism for quite some time, and Drosophila alcohol dehydrogenase is one of the best-studied members of the SDR family. The availability of a number of high-resolution structures, accurate and thorough kinetic work, and careful theoretical calculations have enabled an understanding of the structure-function relationships of this metal-free alcohol dehydrogenase. In addition, these studies have given rise to various hypotheses about the mechanism of action of this enzyme and contribute to the detailed knowledge of the large superfamily of SDR enzymes.

Cerebral hemodynamic response to unpleasant odors in the preterm newborn measured by near-infrared spectroscopy.

Newborn infants in intensive care units are exposed to several unfamiliar smells, mostly related to the nosocomial environment. How the preterm baby perceives these olfactory stimulations remains unclear. Near-infrared spectroscopy can be performed noninvasively above the olfactory cortex to monitor changes of cerebral blood flow as an indicator of cortical activation. The aim of this study was to explore by near-infrared spectroscopy how odorous substances routinely used in the neonatal intensive care unit influence bilateral cortical hemodynamics in the olfactory region of the brains of preterm infants. Specifically, a detergent (Neomidil) and an adhesive remover (Remove) have been tested. Twenty preterm neonates of gestational age 30-37 wk (mean 33.7 +/- 2.3 SD) and postconceptional age 32-37.3 wk (mean 35.5 +/- 2.75 SD) were monitored by near-infrared spectroscopy. Two optode pairs were placed above the anterior orbitofrontal gyri, which is involved in olfactory processing, on each side of the skull. Fifteen babies were exposed to the smell of a disinfectant and five babies to that of a detergent, both applied to small cotton pads. Changes of oxygenated Hb and deoxygenated Hb were recorded before, during, and after a 10-s stimulus. In 17 out of 20 babies, there was a decrease in oxygenated Hb and total Hb after the exposure to the substances. The decrease was significantly greater in the right side than in the left side. This change was different from that observed in our previous study after exposure to colostrum and the pleasant smell of vanilla, which elicited an increase in blood oxygenation in the same region. The biologic significance of this finding is unknown. We conclude that cortical hemodynamic modifications occur in the preterm newborn after exposure to preparations commonly used in the neonatal intensive care unit. A lateralization seems to occur in processing unpleasant olfactory cues.

Macrophages secrete matrix metalloproteinase 9 covalently linked to the core protein of chondroitin sulphate proteoglycans.

Matrix metalloproteinases (MMPs) secreted from the leukemic macrophage cell-line THP-1 have been investigated. Under serum-free conditions, this cell-line synthesizes and secretes proMMP-9, which was detected in the culture medium as a monomer of 92 kDa, and in dimeric forms, including a homodimer of approximately 225 kDa. In addition, a new heterodimer complex is described, in which proMMP-9 is covalently linked to the core protein of chondroitin sulphate proteoglycan (CSPG) through one or more disulphide bridges. After SDS-PAGE electrophoresis, at least two forms of this complex were detected, a large form in the stacking gel and a smaller form with an estimated size of 300 kDa. When the CS chains were removed by chondroitin ABC lyase treatment, heterodimers of proMMP-9/CSPG core protein of approximately 145, 127 and 109 kDa were found, based on zymography and Western blots. Since as much as 10-15 % of the total proMMP-9 secreted from THP-1 cells was covalently linked to CSPG, this association may have important implications for transport, targetting and regulation of the enzyme activity.

Activation of olfactory cortex in newborn infants after odor stimulation: a functional near-infrared spectroscopy study.

In mammals, perception of smells during the first hours of life is an essential prerequisite for adaptation of the newborn to the new extrauterine world. Functional magnetic resonance studies have shown that olfactory impression is processed in the lateral and anterior orbito-frontal gyri of the frontal lobe. Near-infrared spectroscopy (NIRS) can detect changes in oxygenated [Hb O2], and deoxygenated [Hb H] Hb during cortical activation. The aim of this study was to assess by NIRS olfactory cortex activity in newborn infants receiving olfactory stimuli. Twelve males and 11 females were studied when awake at 6 h to 8 d after birth. NIRS monitoring was carried out using two optodes placed above the left anterior orbito-frontal gyri. Each newborn was exposed for 30 s to two different smell stimuli-mother's colostrum and vanilla-and to a negative control, distilled water. Changes in Hb concentration were measured over the orbitofrontal region. During exposure to vanilla, [Hb O2] increased significantly over the left orbito-frontal area in all babies. The magnitude of the [Hb O2] increase over the illuminated region during colostrum exposure was inversely related to postnatal age. We conclude that monitoring Hb changes by NIRS can be valuable in assessing olfactory responsiveness in infants.

Vaccination with FimH adhesin protects cynomolgus monkeys from colonization and infection by uropathogenic Escherichia coli.

Escherichia coli FimH adhesin mediates binding to the bladder mucosa. In mice, a FimH vaccine protects against bacterial challenge. In this study, 4 monkeys were inoculated with 100 microgram of FimCH adhesin-chaperone complex mixed with MF59 adjuvant, and 4 monkeys were given adjuvant only intramuscularly. After 2 doses (day 0 and week 4), a booster at 48 weeks elicited a strong IgG antibody response to FimH in the vaccinated monkeys. All 8 monkeys were challenged with 1 mL of 108 E. coli cystitis isolate NU14. Three of the 4 vaccinated monkeys were protected from bacteruria and pyuria; all control monkeys were infected. These findings suggest that a vaccine based on the FimH adhesin of E. coli type 1 pili may have utility in preventing cystitis in humans.

The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies.

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.

S100A4 involvement in metastasis: deregulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in osteosarcoma cells transfected with an anti-S100A4 ribozyme.

The biological function of the metastasis-associated gene S100A4 is not fully understood, although there is evidence indicating interactions between the gene product and the cytoskeleton. We have examined whether an association could exist between S100A4 and the regulation of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). For these studies, three clones of a highly metastatic human osteosarcoma cell line (OHS) transfected with a hammerhead ribozyme directed against the S100A4 gene transcript were used. The clones demonstrated different expression levels of S100A4 and also different metastatic capacity. In the clone with the most prominent down-regulation of S100A4, the mRNA levels of MMP2, membrane type (MT) 1-MMP, and TIMP-1 were significantly reduced in exponentially growing cultures. Western blots, gelatin zymography, and ELISA showed similar expression patterns of MMPs and TIMPs at the protein level. In the clones with an intermediate expression of S100A4, reduced expression of MT1-MMP and TIMP-1 was detected, whereas the expression of MMP-2 was at the same level as in the control cells. In contrast to the other factors, TIMP-2 was up-regulated in all of the clones independent of the extent of ribozyme-induced down-regulation of S100A4. The transwell chamber assay demonstrated that the capacity of the ribozyme-transfected cells to cross uncoated filters was reduced, relative to control cells, according to the reduction in the S100A4 expression level. The clone with the lowest reduction in S100A4 did not demonstrate different motility compared with control cells, whereas transfectants with only 5% S100A4 mRNA showed a 50% reduction in motility. Interestingly, this trend was even more striking when the capacity to cross Matrigel-coated filters was analyzed, as all the clones demonstrated between 40 and 75% reduced invasion. It is concluded that S100A4 may exert its effect on metastasis formation not only by stimulating the motility of tumor cells but also by affecting their invasive properties through influencing the expression of MMPs and their endogenous inhibitors.

Drosophila lebanonensis alcohol dehydrogenase: pH dependence of the kinetic coefficients.

The alcohol dehydrogenase (ADH) from Drosophila lebanonensis shows 82% positional identity to the alcohol dehydrogenases from Drosophila melanogaster. These insect ADHs belong to the short-chain dehydrogenase/reductase family which lack metal ions in their active site. In this family, it appears that the function of zinc in medium chain dehydrogenases has been replaced by three amino acids, Ser138, Tyr151 and Lys155. The present work on D. lebanonensis ADH has been performed in order to obtain information about reaction mechanism, and possible differences in topology and electrostatic properties in the vicinity of the catalytic residues in ADHs from various species of Drosophila. Thus the pH dependence of various kinetic coefficients has been studied. Both in the oxidation of alcohols and in the reduction of aldehydes, the reaction mechanism of D. lebanonensis ADH in the pH 6-10 region was consistent with a compulsory ordered pathway, with the coenzymes as the outer substrates. Over the entire pH region, the rate limiting step for the oxidation of secondary alcohols such as propan-2-ol was the release of the coenzyme product from the enzyme-NADH complex. In the oxidation of ethanol at least two steps were rate limiting, the hydride transfer step and the dissociation of NADH from the binary enzyme-NADH product complex. In the reduction of acetaldehyde, the rate limiting step was the dissociation of NAD+ from the binary enzyme-NAD+ product complex. The pH dependences of the kon velocity curves for the two coenzymes were the opposite of each other, i.e. kon increased for NAD+ and decreased for NADH with increasing pH. The two curves appeared complex and the kon velocity for the two coenzymes seemed to be regulated by several groups in the free enzyme. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD+ complex, with a pKa value of 7.1. The kon velocity for acetaldehyde was pH independent and showed that in the enzyme-NADH complex, the pKa value of the catalytic residue must be above 10. The koff velocity of NAD+ appeared to be partly regulated by the catalytic residue, and protonation resulted in an increased dissociation rate. The koff velocity for NADH and the hydride transfer step was pH independent. In D. lebanonensis ADH, the pKa value of the catalytic residue was 0.5 pH units lower than in the ADHS alleloenzyme from D. melanogaster. Thus it can be concluded that while most of the topology of the active site is mainly conserved in these two distantly related enzymes, the microenvironment and electrostatic properties around the catalytic residues differ.

Unique salience of maternal breast odors for newborn infants.

Human infants are particularly responsive to olfactory cues emanating from their mother's nipple/areola region. Beginning within minutes after birth, maternal breast odors elicit preferential head orientation by neonates and help guide them to the nipple. Such odors also influence babies' general motor activity and arousal, which may contribute further to successful nipple localization and sucking. The role of maternal olfactory signals in the mediation of early breast-feeding is functionally analogous to that of nipple-search pheromone as described in nonhuman mammals. To some extent, the chemical profile of breast secretions overlaps with that of amniotic fluid. Therefore, early postnatal attraction to odors associated with the nipple/areola may reflect prenatal exposure and familiarization. Although newborns are generally attracted to breast odors produced by lactating women, breast-fed infants rapidly learn their mother's characteristic olfactory signature while sucking at her breasts and can subsequently recognize her by that unique scent alone. Early odor-based recognition may be an important factor in the development of the infant-mother bond.

Interleukin-1 alpha and basic fibroblast growth factor induction of matrix metalloproteinases and their inhibitors in osteosarcoma cells is modulated by the metastasis associated protein CAPL.

BACKGROUND: Several recent investigations have shown that the expression of the CAPL protein seems to be of importance in the metastatic potential in some types of cancer. However, the mechanisms behind this and other biological functions of CAPL are still largely unknown. The aim of the present work was to investigate whether CAPL could affect the expression of candidate proteolytic facilitators of the metastatic process, i.e. matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). MATERIALS AND METHODS: A highly metastatic osteosarcoma cell-line with a high expression of CAPL was transfected with either a vector containing a ribozyme against this transcript, or with the vector alone as a control. The expression of MMPs and TIMPs was investigated with ELISA and gelatin zymography. RESULTS: The cell-line with a low CAPL expression (III-14) responded to bFGF treatment by an increased synthesis of MMP-1 and MMP-9 and to Il-1 alpha treatment by an increased synthesis of MMP-9. In contrast, the cell-line with a high CAPL expression (pH beta-1) did not respond with an altered expression of these MMPs. Neither of these two cell-lines responded with an altered expression of MMP-2. bFGF treatment resulted in an increased expression of TIMP-1 in both cell-lines, while Il-1 alpha treatment resulted in a decreased production of TIMP-1 in pH beta-1 cells, and III-14 cells were unaffected. CONCLUSIONS: The CAPL protein expressed in cell-cultures appear to block the MMP induction by bFGF and Il-1 alpha. However, the induction of TIMP-1 by bFGF must proceed through a pathway different from the MMP induced pathway, i.e. a pathway unaffected by CAPL. In addition, CAPL appeared to act in synergy with Il-1 alpha to reduce the synthesis of TIMP-1.

[Results of a primary health care study on alcohol: 17 per cent of men, 8 per cent of women were probable heavy consumers]. / Resultat av primärvårdsstudie om alkohol: 17 procent av männen, 8 procent av kvinnorna troliga högkonsumenter.

A questionnaire study designed to elicit an impression of the proportion of heavy drinkers among primary care patients, carried out at five primary facilities in Västerbotten County, showed 12 per cent of the patients (17% of the men, 8% of the women) to be probable heavy alcohol consumers. This emphasises the strategically advantageous position of primary care facilities with a view to early intervention. Age was found to be a significant determinant of alcohol consumption, but not place of residence or the reason for consulting.

The role of liver alcohol dehydrogenase isoenzymes in the oxidation of glycolethers in male and female rats.

The glycolethers 2-methoxyethanol (2-ME), 2-ethoxyethanol (2-EE), and 2-butoxyethanol (2-BE) are used as solvents and have teratogenic, spermatotoxic, and hematotoxic effects. These glycolethers are oxidized to their corresponding alkoxyacetic acids, probably by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). This metabolic conversion of the glycolethers is a prerequisite for development of toxicity, as the toxic effects have been shown to be due to the alkoxyacetic acid metabolites. Three isoenzymes of ADH have been detected in rat tissues. The liver contains two of these isoenzymes, ADH-2 and ADH-3. It has also been shown that the activity level of ADH is strongly sex dependent, with higher activity in females than in males. In the present study, we have investigated whether one or both of the ADH isoenzymes in male and female rat livers were able to oxidize 2-ME, 2-EE, and 2-BE and whether one or both of the ADH isoenzymes in male rat liver were able to oxidize 2-pentyloxyethanol and 2-hexyloxyethanol. Our results indicated that only the ADH-3 isoenzyme effectively oxidized the glycolethers in rat liver. Both ADH-2 and ADH-3 were able to oxidize medium chain aliphatic alcohols with a chain length corresponding to the glycolethers. The activity of ADH is higher in female than in male rat liver. However, it was the same ADH isoenzyme (ADH-3) that oxidized the different glycolethers tested in both male and female rat livers, and the substrate specificity was 2-BE > 2-EE > 2-ME.

Soothing effect of amniotic fluid smell in newborn infants.

Newborn young of several mammalian species are attracted to the odor of amniotic fluid (AF); these chemical cues also appear to calm neonates and help them adapt to their novel postnatal environment. AF odor likewise elicits positive (head orientation) responses by human infants. The present study systematically examined whether the odors of AF and mother's breasts influence the crying of the newborn infant, when separated from its mother. The total crying time from 31-90 min postnatal was registered on tapes in 47 healthy fullterm newborns, allocated to one of three conditions; exposure to either AF or breast odor or no exposure (controls). Babies exposed to AF smell cried significantly less (median 29 s) than babies in the two other groups (breast odor--301 s, controls--135 s). The data are consistent with the hypothesis that the fetus may become familiar with chemical cues present in the intrauterine environment. Our data provide new evidence of the human baby's fine olfactory discrimination capacity, and add to the growing body of evidence indicating that naturally occurring odors play an important role in the mediation of infants' early behavior.