Sample storage for soil enzyme activity and bacterial community profiles.

Storage of samples is often an unavoidable step in environmental data collection, since available analytical capacity seldom permits immediate processing of large sample sets needed for representative data. In microbiological soil studies, sample pretreatments may have a strong influence on measurement results, and thus careful consideration is required in the selection of storage conditions. The aim of this study was to investigate the suitability of prolonged (up to 16 weeks) frozen or air-dried storage for divergent soil materials. The samples selected to this study were mineral soil (clay loam) from an agricultural field, humus from a pine forest and compost from a municipal sewage sludge composting field. The measured microbiological parameters included functional profiling with ten different hydrolysing enzyme activities determined by artificial fluorogenic substrates, and structural profiling with bacterial 16S rDNA community fingerprints by amplicon length heterogeneity analysis (LH-PCR). Storage of samples affected the observed fluorescence intensity of the enzyme assay's fluorophor standards dissolved in soil suspension. The impact was highly dependent on the soil matrix and storage method, making it important to use separate standardisation for each combination of matrix type, storage method and time. Freezing proved to be a better storage method than air-drying for all the matrices and enzyme activities studied. The effect of freezing on the enzyme activities was small (<20%) in clay loam and forest humus and moderate (generally 20-30%) in compost. The most dramatic decreases (>50%) in activity were observed in compost after air-drying. The bacterial LH-PCR community fingerprints were unaffected by frozen storage in all matrices. The effect of storage treatments was tested using a new statistical method based on showing similarity rather than difference of results.

Increased levels of acylation-stimulating protein in interleukin-6-deficient (IL-6(-/-)) mice.

IL-6-deficient (IL-6(-/-)) mice develop obesity at 6-7 months of age. To elucidate the mechanisms of this mature-onset obesity, global gene expression profiles of 3-month-old preobese IL-6(-/-) were compared with those of IL-6(+/+) mice using DNA arrays. Genes that were up-regulated in IL-6(-/-) mice included the factors transthyretin and properdin in white adipose tissue and adipsin in muscle. These factors have been shown to influence the formation of acylation-stimulating protein (ASP), a cleavage product of complement C3. ASP stimulates the synthesis of triacylglycerol in adipocytes, and ASP-deficient mice are resistant to diet-induced obesity. In line with the increases in transthyretin, properdin, and adipsin, ASP levels in serum were increased by 31-54% in IL-6(-/-) compared with IL-6(+/+) mice. Furthermore, IL-6 replacement treatment in IL-6(-/-) mice decreased ASP levels significantly by 25-60%. In conclusion, ASP levels are increased in preobese IL-6(-/-) mice. This increase may result in increased triacylglycerol formation and uptake in IL-6(-/-) adipocytes and thereby contribute to the development of obesity in IL-6(-/-) mice.

The influence of lignin content and temperature on the biodegradation of lignocellulose in composting conditions.

The aim of this research was to study the influence of lignin content and composting temperature on the biodegradation of lignin-containing pulp and paper products in a controlled composting test (European standard prEN 14046). Lignin reduced the biodegradation of the samples, and there was a linear correlation between the lignin content and the biodegradation of pulp and paper products at 58 degrees C. The influence of incubation temperature (35, 50 and 58 degrees C) on biodegradation was studied using bleached kraft paper containing 0.2 wt% lignin and mechanical pulp (stone-ground wood) containing 24-27 wt% lignin. Mechanical pulp biodegraded better at lower temperatures, while kraft paper biodegraded well at all three temperatures. Microbial activity was evaluated by measuring CO(2) evolution and the change in ATP content, and fungal biomass by measuring the ergosterol content during the composting experiments. Kraft paper strongly increased microbial activity during the controlled composting test, but the activity returned to the background level at the end of the composting test. The proportion of sample carbon converted to microbial biomass carbon was considerably higher at lower incubation temperatures. Changes in microbial community structure during biodegradation of mechanical pulp and kraft paper at 50 degrees C were studied by the PCR-based technique denaturing gradient gel electrophoresis. Changes in the microbial community were observed during the intensive degradation phase of kraft paper.

Liver-derived IGF-I regulates GH secretion at the pituitary level in mice.

We have reported that liver-specific deletion of IGF-I in mice (LI-IGF-I-/-) results in decreased circulating IGF-I and increased GH levels. In the present study, we determined how elimination of hepatic IGF-I modifies the hypothalamic-pituitary GH axis to enhance GH secretion. The pituitary mRNA levels of GH releasing factor (GHRF) receptor and GH secretagogue (GHS) receptor were increased in LI-IGF-I-/- mice, and in line with this, their GH response to ip injections of GHRF and GHS was increased. Expression of mRNA for pituitary somatostatin receptors, hypothalamic GHRF, somatostatin, and neuropeptide Y was not altered in LI-IGF-I-/- mice, whereas hypothalamic IGF-I expression was increased. Changes in hepatic expression of major urinary protein and the PRL receptor in male LI-IGF-I-/- mice indicated an altered GH release pattern most consistent with enhanced GH trough levels. Liver weight was enhanced in LI-IGF-I-/- mice of both genders. In conclusion, loss of liver-derived IGF-I enhances GH release by increasing expression of pituitary GHRF and GHS receptors. The enhanced GH release in turn affects several liver parameters, in line with the existence of a pituitary-liver axis.

The somatomedin hypothesis revisited in a transgenic model.

Studies of insulin-like growth factor I (IGF-I) gene knockout mice models have clearly shown that IGF-I is necessary for prenatal as well as postnatal body growth in mice. Clinical studies of a patient with an IGF-I gene defect which caused complete absence of IGF-I, verified that it is important for intrauterine and postnatal growth. Recent studies of mice with liver-specific and inducible IGF-I gene knockout indicated that liver-derived IGF-I is not necessary for postnatal body growth, although serum IGF-I levels are decreased by more than 80% in these mice. Therefore, extrahepatic IGF-I is sufficient for maintenance of postnatal body growth in mice. Further investigations are needed to assess whether liver-derived circulating IGF-I is essential for other biological functions.

Liver-derived IGF-I is of importance for normal carbohydrate and lipid metabolism.

IGF-I is important for postnatal body growth and exhibits insulin-like effects on carbohydrate metabolism. The function of liver-derived IGF-I is still not established, although we previously demonstrated that liver-derived IGF-I is not required for postnatal body growth. Mice whose IGF-I gene in the liver was inactivated at 24 days of age were used to investigate the long-term role of liver-derived IGF-I for carbohydrate and lipid metabolism. Serum levels of leptin in these mice were increased by >100% at 3 months of age, whereas the fat mass of the mice was decreased by 25% at 13 months of age. The mice became markedly hyperinsulinemic and yet normoglycemic, indicating an adequately compensated insulin resistance. Furthermore, they had increased serum levels of cholesterol. We conclude that liver-derived IGF-I is of importance for carbohydrate and lipid metabolism.

Retarded liver growth in interleukin-6-deficient and tumor necrosis factor receptor-1-deficient mice.

The liver size in adult mammals is tightly regulated in relation to body weight, but the hormonal control of this is largely unknown. We investigated the roles of interleukin-6 (IL-6) and tumor necrosis factor (TNF) receptor-1 in the regulation of intact liver weight in adult mice. The relative liver wet and dry weights of older adult (5- to 10-month-old) IL-6 knockout (IL-6(-/-)) mice were decreased by 22-28%, and total contents of DNA and protein were decreased compared with those in age-matched wild-type mice. Weights of other visceral organs were unaffected. Older adult (6- to 8-month-old) TNF receptor-1 knockout (TNFR1(-/-)) mice displayed decreased relative liver weight. Treatment with a single injection of IL-6 increased liver wet and dry weights in IL-6(-/-) and wild-type mice, but not TNFR1(-/-) mice. Treatment with TNFalpha enhanced liver weight and DNA synthesis of nonparenchymal liver cells at 24 h in wild-type, but not IL-6(-/-), mice. At 48 h, TNFalpha induced DNA synthesis in nonparenchymal cells and hepatocytes of both wild-type and IL-6(-/-) mice. In conclusion, TNF receptor-1 stimulation and IL-6 production are both necessary for normal liver weight gain in older adult mice. The results of TNFalpha and IL-6 treatment further indicate that the effects of TNF receptor-1 and IL-6 depend on each other for full stimulation of liver growth.

Possible roles of insulin-like growth factor in regulation of physiological and pathophysiological liver growth.

BACKGROUND/AIMS: Almost all circulating insulin-like growth factor-1 (IGF-1) is produced and secreted from the liver. However, the possible role of IGF-1 in local regulation of liver functions including liver growth is unclear. In the present study, we investigated the role of IGF-1 on liver growth in vivo and in hepatic stellate cell function in vitro. RESULTS: Liver-specific knock-out of the IGF-1 gene by use of the cre-loxP system caused enhanced liver growth, possibly reflecting increased growth hormone (GH) secretion due to decreased negative feedback by IGF-1. Studies on cultured rat hepatic stellate cells (HSC) showed that IGF-1 and hepatocyte-conditioned medium (PCcM) time- and dose-dependently increased hepatocyte growth factor (HGF) mRNA and HGF immunoreactivity. IGF-1 and PCcM also enhanced DNA synthesis in the HSC cultures. The PCcM did not contain bioactive IGF-1 and was also able to stimulate proliferation when prepared under serum- and hormone-free conditions. CONCLUSION: In vivo results show that IGF-1 is not essential for normal growth of the intact liver. The in vitro results indicate that both IGF-1 and IGF- 1-independent factor(s) from hepatocytes can stimulate HGF production by HSC. It remains to be investigated whether these effects are of importance for liver regeneration or pathological conditions.

Extraction and purification of DNA in rhizosphere soil samples for PCR-DGGE analysis of bacterial consortia.

Application of DNA fingerprinting methods enables the detection of diverse members of soil bacterial consortia, even including those bacteria not yet cultivated. However, extraction and purification of DNA from soil samples without bias is difficult. We compared five different DNA isolation methods and three purification methods for rhizosphere soil samples. Purified DNA extracts were amplified in PCR using universal bacterial primers and the PCR products were analysed with denaturing gradient gel electrophoresis (DGGE) for the visualisation of DNA bands representing dominant bacterial species. Both the isolation and purification methods affected the apparent bacterial community structure of the samples.

Enteral suspension of nifedipine for neonates. Part 1. Formulation of nifedipine suspension for hospital use.

OBJECTIVE: To formulate an oral suspension of nifedipine for paediatric use and to assess its content uniformity as well as the microbiological and physical stabilities of the hypromellose solution that was used in the formulation. METHOD: Six concentrations (0.5-3.0%) of hypromellose colloids and water as a blank were compounded with nifedipine, both as a powder and as crushed tablets, to a concentration of 1 mg/mL. Four different screening tests were used to find the most homogenous and dose-accurate combination. First, nifedipine suspensions were stored in vials for one month and visual homogenity of the redispersed suspensions was observed. Second, the homogenity of the suspensions was studied by measuring the nifedipine concentration from upper, middle and lower parts of the redispersed suspension. Next, the nifedipine concentration was measured from the suspensions immediately, 1 min and 2 min after shaking to ensure dose accuracy during the administration period. Finally, suspensions were packaged into oral disposable syringes and nifedipine concentrations were determined after one month of storage. Content uniformity of the packaged single-dose syringe suspensions was studied according to a method established by the European Pharmacopoeia. Microbiological stability, density, pH, osmolality, viscosity and surface tension of the hypromellose solution were studied over a 12-month storage period. RESULTS: From the results of the screening tests of hypromellose solution, 1.0% hypromellose was chosen as the vehicle for nifedipine enteral suspensions, made from both crushed tablets and nifedipine powder. Nifedipine suspensions made from hypromellose 1.0% were easiest to redisperse as a homogenous solution, and it also appeared best on visual inspection. The content uniformity of the suspension complied with the test recommended by the European Pharmacopoeia. The 1.0% hypromellose solution was found to be microbiologically stable for 6 months and physically stable for 12 months.

Normal pharmacologically-induced, but decreased regenerative liver growth in interleukin-6-deficient (IL-6(-/-)) mice.

BACKGROUND/AIMS: In the absence of liver damage, rapid liver growth can be induced pharmacologically by so-called primary liver growth promoters. The importance of the acute-phase cytokines interleukin-6 and tumor necrosis factor-alpha for the actions of these compounds is not clear. This study aimed to investigate the importance of IL-6 and TNF-receptor-1 in pharmacologically-induced liver growth. METHODS: IL-6 knockout (IL-6(-/-)), TNF-receptor-1 knockout (TNFR1(-/-)) and wild-type mice were treated with the peroxisome proliferator nafenopin and the anti-androgen cyproterone acetate (CPA) in one single injection or for 6 days with daily injections, and examined at 24 or 48 h after treatment. In a control experiment, IL-6(-/-) mice were subjected to two-thirds partial hepatectomy. RESULTS: Nafenopin treatment increased relative liver weight and DNA synthesis similarly in IL-6(-/-), TNFR1(-/-) and wild-type mice. CPA increased liver weight similarly in all groups, but did not increase DNA synthesis. Expression of peroxisome proliferator activated receptor-alpha mRNA was increased in both IL-6(-/-) and wild-type mice by nafenopin treatment, but not by CPA treatment. After hepatectomy DNA synthesis was suppressed in IL-6(-/-) mice compared to wild-type mice. CONCLUSIONS: Liver growth induced by nafenopin and CPA was not dependent on the presence of IL-6 or TNF receptor-1, whereas liver regeneration was decreased in IL-6(-/-) mice.

Characterization of hepatocyte-derived mitogenic activity on hepatic stellate cells.

BACKGROUND/AIMS: Hepatic stellate cells (HSC) are located in close proximity to hepatocytes in Disse's space. Hepatocyte derived factors have earlier been implicated in the paracrine regulation of HSC proliferation. The aim of the present study was to further characterize this mitogenic activity of the parenchymal cell conditioned medium (PCcM). METHODS: Primary rat HSC were cultured for 4 days. DNA synthesis was measured by 3H-thymidine incorporation. TGFbeta1 immunoreactivity was quantified by ELISA. PCcM was obtained from hepatocytes cultured in medium without serum or hormones for two days. RESULTS: Incubation of 4-day-old HSC on plastic surface with PCcM for 2 days increased DNA synthesis, while no effect was seen in HSC cultured on Matrigel. Heat-, acid-, and protease-treatment of PCcM abolished its stimulatory effect. Size fractionations with spin columns indicated that the stimulatory effect was contained in the fractions of a molecular size between 30 and 100 kD. The addition of LY 294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, dose-dependently inhibited the PCcM induced increase in DNA synthesis to about 9% of the control values. The specific MAP kinase (MAPK) inhibitor, PD 98059 only suppressed the PCcM induced DNA synthesis to 35% of control cultures at the highest dose (10 microM). DNA content in the cultures was not affected by either blocker. HSC seemed to produce immunoreactive TGFbeta1. However, addition of latency-associated peptide (LAP), a potent TGFbeta1 blocker, stimulated DNA synthesis to a much less extent than PCcM. CONCLUSIONS: The factor(s) that stimulate DNA synthesis in HSC from hepatocytes are most likely protein(s) with a molecular size between 30-100 kD. These factor(s) rely more on PI3-K than on MAPK for their mitogenic effect and are probably not acting via TGFbeta1 inhibition.

Insulin-like growth factor signaling pathways in rat hepatic stellate cells: importance for deoxyribonucleic acid synthesis and hepatocyte growth factor production.

It has been shown recently that insulin-like growth factor 1 (IGF-1) increases both DNA synthesis and hepatocyte growth factor (HGF) production in cultured hepatic stellate cells. In this study, we used selective blockers to investigate crucial signaling pathways for these effects of IGF-1 in cultured rat hepatic stellate cells. Both LY 294002 [a phosphatidylinositol 3-kinase (PI3-K) inhibitor], and rapamycin [a blocker of activation of the serine/threonine p70 S6 kinase (p70S6K), a molecule downstream from PI3-K] completely reversed the IGF-1-induced stimulation of DNA synthesis. Mitogen-activated protein kinase (MAPK) inhibition by PD 98059 had a less pronounced suppressory effect, although the used PD 98059 dose was fully effective in inhibiting MAPK phosphorylation. Both LY 294002 and PD 98059 lowered the IGF-1-induced increase of HGF in the medium by about 40%, but LY 294002 was 10 times more potent than PD 98059. Inhibition of p70S6K activation by rapamycin blocked IGF-1-induced DNA synthesis but not the increase in HGF. In conclusion, PI3-K (and, to some extent, MAPK) signaling pathways seem to be important for IGF-1-stimulated DNA synthesis and HGF production. DNA synthesis also seems to be dependent on rapamycin-sensitive activation of the PI3-K effector p70S6K.

The functional deformation of maxillary complete dentures in patients with flabby alveolar ridges. Part I: Before surgery.

Using strain-guaged duplicate maxillary dentures, the deformation patterns and magnitude of functional strain were studied on five complete denture wearing subjects who had varying amounts of soft mobile tissue over their maxillary edentulous alveolar ridge so called flabby ridges. The thickness of mobile tissue--expressed as depth--was measured by a sharpened periodontal probe with aid of a plastic base plate as a locator. The functional test included maximum biting, and the chewing of three test food samples. The results did not point on any direct relationship between the thickness of the soft tissue and the midline deformation pattern and/or magnitude of functional strain. It seems, however, as if the main influence of mobile tissue is on the strain behaviour in the lateral part of the denture.

The functional deformation of maxillary complete dentures in patients with flabby alveolar ridges. Part II: After surgery.

Using strain-gauged maxillary complete dentures, the effects of the surgical removal of soft mobile tissue over the maxillary edentulous alveolar ridges--flabby ridges--on the deformation pattern and magnitude of functional strain were studied in five subjects. The results showed that a 20-90% reduction in denture deformation had taken place after surgery. Furthermore, a pre-operative shear-straining pattern of the buccal flanges and the lateral palatal slopes was not evident post-operatively. The results of this study point on the beneficial value of early surgical removal of flabby maxillary alveolar ridges.

Blood group antigen staining pattern during experimental carcinogenesis in rat palate.

During 4-nitroquinoline-1-oxide-induced carcinogenesis in the rat palate, animals were sacrificed at various intervals and stained for blood group antigens B and H (Type 2 chain) by an immunofluorescent method. In rats without signs of epithelial dysplasia, the staining pattern was identical with that in the normal control rats. In rats with definite or questionable (borderline cases) dysplasias, marked changes in blood group antigen staining pattern were seen. Thus, changes in cell-surface carbohydrates during malignant development in the rat palate seem to follow closely the histomorphological changes. As there is good evidence that carcinomas would eventually develop in all rats if they were not sacrificed, it seems that the blood group antigen staining pattern does not predict malignant development in the absence of histological suspicion.

Bone tissue formation within a sintered microporous glass-fiber network implanted in extraction sockets in the rat.

Implants were prepared by sintering glass-fibers into a three-dimensional network with meshes smaller than 20 micron. Oblong 5 x 2 x 0.15 mm sheets of the test material were implanted in maxillary incisor alveoli in eight white rats. The alveoli were closed with a suture in the overlying mucosa. After 3 months the animals were sacrificed and sections from the site of implantation prepared. In three animals only root remnants were found but in the remaining five the implants were wholly or partially embedded in bone tissue. Sections studied with transmitted light, microradiography, and scanning electron microscopy showed that mineralized tissue had also formed within the pores of the network. The intimate contact between the individual fibers and the bone within the network was corroborated through microchemical analysis (EDX).