Isolation of AF2 (KHEYLRFamide) from Caenorhabditis elegans: Evidence for the presence of more than one FMRFamide related peptide-encoding gene

Numerous FMRF amide-related peptides (FaRPs) have been isolated and sequenced from extracts of free-living and parasitic nematodes. The most abundant FaRP identified in ethanolic/methanolic extracts of the parasitic forms, Ascaris suum and Haemonchus contortus and from the free-living nematode, Panagrellus redivivus, was KHEYLRF amide (AF2). Analysis of the nucleotide sequences of cloned FaRP-precursor genes from C. elegans and, more recently, Caenorhabditis vulgaris identified a series of related FaRPs which did not include AF2. An acid-ethanol extract of Caenorhabditis elegans was screened radioimmunometrically for the presence of FaRPs using a C-terminally directed FaRP antiserum. Approximately 300 pmols of the most abundant immunoreactive peptide was purified to homogeneity and 30 pmols was subjected to Edman degradation analysis and gas-phase sequencing. The unequivocal primary structure of the heptapeptide, Lys-His-Glu-Tyr-Leu-Arg-Phe-NH2 (AF2) was determined following a single gas-phase sequencing run. The molecular mass of the peptide was determined using a time-of-flight mass spectrometer and was found to be 920 (MH(+))(-), which was consistent with the theoretical mass of C-terminally amidated AF2. These results indicate that C. elegans possesses more than one FaRP gene. (C) 1995 Academic Press, Inc.

Isolation of Alternaria alternata from an emollient cream:implications for public health

A specimen of emollient cream, which was observed to be contaminated peripherally with a filamentous fungus was examined for the presence of fungi and the resulting fungal colonies were examined phenotypically and genotypically. Subsequent DNA extraction and PCR amplification of the large internal transcribed spacer region [ITS1-5.8S-ITS2] yielded an amplicon of 512 bp. Sequence analysis identified this as Alternaria alternata at the 100% homology level with all 512/512 bases called. This organism has been previously reported as a cause of opportunistic infections involving skin and immunocompromised patients. This is the first report of an emollient cream as a source of this organism. It highlights the need for proper management of such preparations in order to minimize the potential spread of fungi to susceptible patient populations.

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

Isolation of epithelial stem cells from dermis by a three-dimensional culture system

Skin is a representative self-renewing tissue containing stem cells. Although many attempts have been made to define and isolate skin-derived stem cells, establishment of a simple and reliable isolation procedure remains a goal to be achieved. Here, we report the isolation of cells having stem cell properties from mouse embryonic skin using a simple selection method based on an assumption that stem cells may grow in an anchorage-independent manner. We inoculated single cell suspensions prepared from mouse embryonic dermis into a temperature-sensitive gel and propagated the resulting colonies in a monolayer culture. The cells named dermis-derived epithelial progenitor-1 (DEEP) showed epithelial morphology and grew rapidly to a more than 200 population doubling level over a period of 250 days. When the cells were kept confluent, they spontaneously formed spheroids and continuously grew even in spheroids. Immunostaining revealed that all of the clones were positive for the expression of cytokeratin-8, -18, -19, and E-cadherin and negative for the expression of cytokeratin-1, -5, -6, -14, -20, vimentin, nestin, a ckit. Furthermore, they expressed epithelial stem cell markers such as p63, integrin beta1, and S100A6. On exposure to TGFbeta in culture, some of DEEP-1 cells expressed alpha-smooth muscle actin. When the cells were transplanted into various organs of adult SCID mice, a part of the inoculated cell population acquired neural, hepatic, and renal cell properties. These results indicate that the cells we isolated were of epithelial stem cell origin and that our new approach is useful for isolation of multipotent stem cells from skin tissues.

Stimulation of cholesteryl ester synthesis in human monocyte-derived macrophages by low-density lipoproteins from type 1 (insulin-dependent) diabetic patients:the influence of non-enzymatic glycosylation of low-density lipoproteins

Diabetes mellitus is an independent risk factor in the development of atherosclerosis. In this study we aimed to demonstrate whether there is an abnormal interaction between low-density lipoproteins from diabetic patients and human macrophages. We measured cholesteryl ester synthesis and cholesteryl ester accumulation in human monocyte-derived macrophages (obtained from non-diabetic donors) incubated with low density lipoproteins from Type 1 (insulin-dependent) diabetic patients in good or fair glycaemic control. Low density lipoproteins from the diabetic patients stimulated more cholesteryl ester synthesis than low density lipoproteins from non-diabetic control subjects (7.19 +/- 1.19 vs 6.11 +/- 0.94 nmol/mg cell protein/20 h, mean +/- SEM, p less than 0.05). The stimulation of cholesteryl ester synthesis by low density lipoproteins isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (p less than 0.02). There were no significant differences in the lipid composition of low density lipoproteins between the diabetic and control groups. Non-enzymatic glycosylation of low density lipoproteins was higher in the diabetic group (p less than 0.01) and correlated significantly with cholesteryl ester synthesis (r = 0.58). Similarly, low-density lipoproteins obtained from non-diabetic subjects and glycosylated in vitro stimulated more cholesteryl ester synthesis in macrophages than control low density lipoproteins. The increase in cholesteryl ester synthesis and accumulation by cells exposed to low density lipoproteins from diabetic patients seems to be mediated by an increased uptake of these lipoproteins by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of in vitro non-enzymatic glycosylation of human skin collagen on susceptibility to collagenase digestion

The effect of glycosylation on susceptibility of skin collagen to collagenase digestion was studied in a skin sample obtained at autopsy from the interscapular region of a 24 year old white male who had died of an acute illness and who had no history of diabetes. Homogeneous suspensions of insoluble collagen were prepared, and were incubated in 50 mmol l-1 dextrose at pH 7.35 and 37 degrees C for 7 days. Non-enzymatic glycosylation measured by the weak acid hydrolysis/thiobarbituric acid method increased from 13.1 +/- 1.0 (n = 5) to 45.2 +/- 5.5 (n = 8) nmol fructose per 10 mg collagen (P less than 0.001). Digestion of collagen using clostridial collagenase was monitored by measuring (a) hydroxyproline content and (b) absorption at 206 nm of the supernatant after centrifugation to remove substrate. The rate of digestion was similar in glycosylated and control collagen. We conclude that the ketoamine link formed in non-enzymatic glycosylation does not increase the resistance of collagen to enzymatic digestion. The possibility remains that subsequent rearrangement of this link could be important in this respect.

Non-enzymatic glycosylation of skin collagen in patients with type 1 (insulin-dependent) diabetes mellitus and limited joint mobility

Forearm skin biopsies were obtained from diabetic subjects with and without limited joint mobility, and from non-diabetic control subjects. Collagen purified from these samples was assayed for non-enzymatic glycosylation. The level in all diabetic patients was significantly greater than that in control subjects (p less than 0.001), but those diabetic patients with limited joint mobility had a level of collagen glycosylation similar to that in those with normal joints (15.3 +/- 1.3 and 16.5 +/- 1.3 nmol fructose/10 mg protein, respectively; mean +/- SEM). Glycosylation of collagen in the diabetic patients correlated with glycosylated haemoglobin measured at the time of skin biopsy (r = 0.60). These results do not support the hypothesis that non-enzymatic glycosylation of collagen, as reflected by the ketoamine link, plays an important role in the development of limited joint mobility in diabetes.

Molecularly Imprinted Polymers for the isolation of bioactive naphthoquinones from plant extracts

Molecularly Imprinted Polymers (MIPs) targeting shikonin, a potent antioxidant and wound healing agent, have been prepared using methacrylic acid (MAA) and 2-diethylaminoethyl methacrylate (DEAEMA) as functional monomers. An investigation of solution association between shikonin and both acidic and basic functional monomers by UV-Vis titrations, suggested stronger affinity towards the basic functionality. Strong inhibition of the co-polymerisation reaction of such basic monomers was observed, but was overcome by reduction of the amount of template used during polymer synthesis. Polymer morphology was severely impacted by the template’s radical scavenging behaviour as demonstrated by solid state NMR spectroscopy measurements. HPLC evaluation of the final materials in polar conditions revealed limited imprinting effects and selectivity, with the MAA polymers exhibiting marginally better performance. During application of the polymers as MI-SPE sorbents in non-polar solvents it was found that the DEAEMA based polymer was more selective towards shikonin compared to the MAA counterpart, while shikonin recoveries of up to 72% were achieved from hexane solutions of a commercial sample of shikonin, hexane extract of Alkanna tinctoria roots and a commercial pharmaceutical ointment.

Isolation of promoter for cytosolic phospholipase A2 (cPLA2)

Cytosolic phospholipase A2 (cPLA2) releases arachidonic acid from membrane phospholipids and is believed to be the rate-limiting enzyme in the arachidonic acid pathway. We report herein the isolation of a 3 kb fragment of rodent genomic DNA containing part of the first intron, the first exon and 5'-flanking sequence. The start site of transcription was mapped by 5'-rapid amplification of cDNA ends and corroborated by ribonuclease protection assay. The gene has a TATAless promoter with no classical Sp1 binding sites or initiator element. A microsatellite series of CA repeats was noted in the 5'-flanking region of both the rodent and human promoters. Deletion constructs have been analysed for luciferase activity and confirmed promoter activity.

Isolation of Aeromonas hydrophila in piglets

The production of Alentejano breed pig started a recovery two decades ago due to increasing demand for gourmet products. These pigs are raised in rotational semi-extensive or extensive outdoor production systems in the “Montado” (green and cork oak forest), grazing and feeding acorns and other associated food resources. Bacteria of the genus Aeromonas are considered as emerging pathogens of importance for man and animals, but its involvement in swine is not well documented. In the context of a study made at the University of Évora to assess the specific diseases of Alentejano swine, diseased piglets from two farms were submitted for pathological and bacteriological examinations. Pathological examinations revealed changes characteristic of septicemia, and Aeromonas hydrophila was isolated in pure culture from multiple organs of piglets from both farms. Antibiotic sensitivity tests showed that the isolates from one of the farms were susceptible to gentamicin, oxitetracycline, neomycin, enrofloxacin, colistin sulfate, trimethoprim, ceftiofur, and amoxicillin plus clavulanic acid. In contrast, the A. hydrophila isolated in the other farm was resistant to all drugs tested but enrofloxacin. This is the first report in the world showing the relationship between septicemia and A. hydrophila infection in piglets. The importance of this finding is further reinforced by the fact that these bacteria can be highly resistant to antimicrobial agents.