The modified obstetric metabolic equivalent (MET): finding a MET that fits in pregnancy.

The Compendium of Physical Activities (CPA) provides the energy expenditure (EE) for hundreds of daily activities reported in metabolic equivalents (MET). It remains to be determined if the metabolic changes of pregnancy alter the use of the CPA MET (METCPA) in this population. The energy cost of rest, activities of daily living (ADL; typing, folding laundry and sweeping) and treadmill walking [2.0, 2.5, 3.0 mph (0% incline), 3.0 mph (3% incline)] were compared with the METCPA from the 2000 and 2011 CPA in 30 pregnant women (10-14 weeks gestation) using indirect calorimetry (IC). The METCPA for each activity was compared against two measured IC values: METabsolute (3.5 ml O2/kg/min) and METratio (EEactivity/EErest). Means for both comparisons were tested by one-sample t-test. Measured MET correlated with the 2011 METCPA: METabsolute v. METCPA R 2 = 0.906, P < 0.0001; METratio v. METCPA R 2 = 0.861, P < 0.0001. Differences between measured MET values and the 2011 METCPA ranged from 16% underestimation to 48% overestimation. Using the absolute definition, the METCPA significantly overestimated the ADL (P < 0.0005); yet, no significant differences were found between walking at 0% grade and METCPA. Conversely, only folding laundry was significantly different with the ratio definition, whereas walking at a level grade was significantly underestimated (P < 0.0001). Similar observations were found using the 2000 CPA. The use of the METCPA to estimate EE in pregnant women can result in significant over- or underestimation, depending on the activity and the definition of the MET that is used.

A comprehensive post-market review of studies on a probiotic product containing Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011.

The probiotic preparation Lacidofil® has been commercially available in Europe, Asia and North America since 1995. This product is a combination of two strains, Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011. The strains have been evaluated for safety, identity and mechanisms of probiotic action in vitro, in animal models and human clinical trials. The strains adhered to human epithelial cells, helped to maintain the barrier function and blocked the adhesion of a number of pathogens, allowing them to be cleared from the intestine. The strains also elicited an anti-inflammatory response by down-regulating IL-1ß, IL-8 and TNF-α. In various stress models, the probiotic combination facilitated better coping and outcomes which may be through the maintenance of barrier function and suppressing inflammation. Overall, pre-clinical studies suggest a potential anti-infectious role for the strains and the combination. Clinical studies, primarily in children, have identified Lacidofil as an effective supplement for various gastrointestinal diseases such as antibiotic-associated diarrhoea and acute gastroenteritis. Recent research has also indicated that Lacidofil may be beneficial for individuals with atopic dermatitis or vaginal dysbacteriosis.

Activation of CPP32 during apoptosis of neurons and astrocytes.

Members of the interleukin-1 beta-converting enzyme (ICE)/CED-3 protease family have been implicated in apoptosis in both vertebrates and invertebrates. Using primary culture methods, we report that neurons and astrocytes require the activity of the ICE/CED-3 family of proteases to undergo apoptosis induced by staurosporine, ceramide, and serum-free media. We show that specific inhibitors of ICE/CED-3 proteases can inhibit apoptosis and that cytosolic fractions from apoptosing neurons, but not healthy cells, induced apoptosis in a cell-free system. Cell extracts from neurons induced to undergo apoptosis contained ICE/ CED-3 protease activity. To determine which member of the ICE/CED-3 family was activated in neurons and astrocytes during apoptosis, we developed a novel affinity-labeling technique that labeled the active site cysteine and identified a 17-kDa subunit of the activated protease. The affinity-labeled 17-kDa protease subunit shares antigenic and molecular mass identity with the processed form of CPP32 on immunoblots, suggesting that CPP32 may be the principal effector in the apoptotic pathway in neurons and astrocytes. In time-course experiments, the activation of CPP32 preceded the detection of PARP cleavage and DNA laddering, suggesting that processing of CPP32 is a very early event in apoptosis of neurons and astrocytes and may be involved in the proteolytic action on specific cellular targets. The affinity-labeling technique developed and used in this report with neural cells allows for the sensitive detection, purification, and identification of ICE/CED-3 proteases that may be activated in other cells types under a variety of conditions, including certain diseased states.

Temperature-dependent regulation of PLP/DM20 and CNP gene expression in two conditionally-immortalized jimpy oligodendrocyte cell lines.

We conditionally immortalized jimpy primary oligodendrocytes (ODCs) with the temperature-sensitive SV40 large T antigen. Two cell lines (clones JP1.1 and JP1.2) were generated that expressed a number of ODC markers. Both jimpy cell lines expressed DM20 mRNAs at the proliferative temperature of 34 degrees C, but not at the "differentiation" temperature of 39 degrees C. Interestingly, at 39 degrees C neither cell line appeared to differentiate further, and neither survived longer than 7 days, in contrast to other ODC cell lines from normal animals that survive many weeks at 39 degrees C. These findings are not consistent with the notion that a PLP/DM20 gene product is the cause of oligodendrocyte cell death in jimpy, since neither jimpy cell line survived at 39 degrees C, and neither line expressed PLP or DM20 proteins. Analysis of the expression of the CNP (2'3' cyclic nucleotide-3'-phosphodiesterase) gene indicated that in both cell lines only one of the two CNP isoforms was expressed at 34 degrees C. Raising the temperature to 39 degrees C caused a greater reduction in the levels of CNP protein than CNP mRNA. Taken together, the DM20 and CNP data suggest that at least some of the decline in myelin/oligodendrocyte components observed in jimpy brains may not be due simply to fewer mature oligodendrocytes, but also to a down regulation of expression of these genes at several levels including transcriptional and post-transcriptional events. Our results provide two cell models for in vitro investigations into the nature of the jimpy mutation at several cellular and molecular levels.

Bcl-2 expression in neural cells blocks activation of ICE/CED-3 family proteases during apoptosis.

The ICE/CED-3 family of proteases has been implicated in playing a fundamental role in programmed cell death. Bcl-2 protein represses a number of apoptotic death programs, but the biochemical mechanism of its action is not known. We investigated the activation of ICE/CED-3 proteases induced by three apoptotic stimuli (staurosporine, ceramide, and serum withdrawal) in the neuronal cell line GT1-7 and in cells overexpressing Bcl-2. Rapid activation of a 17 kDa subunit of an activated member of the ICE/CED-3 family is demonstrated by affinity-labeling GT1-7 extracts from apoptotic controls cells with a biotinylated ICE/CED-3 inhibitor. This activation corresponds to an increased ICE/CED-3-like protease activity in extracts measured by a fluorogenic substrate assay. In a cell-free system, these extracts induce apoptotic morphological changes in intact nuclei. All three activities are readily inhibited by treatment of control extracts with ICE/CED-3-like protease inhibitors. Overexpressed Bcl-2 inhibits the activation of the 17 kDa protein, the ICE/CED-3-like protease activity in the fluorogenic assay, and the induction of apoptotic morphological changes in HeLa nuclei in the cell-free system, similar to results obtained with ICE/CED-3 protease inhibitors. At the mRNA level, overexpression of Bcl-2 did not alter expression of five members of the ICE/CED-3 family: CPP32, ICE, Mch 2, Nedd 2, and TX. Overexpression of Bcl-2 prevented the apoptosis-induced processing of pro-Nedd 2 to the cleaved form. These data suggest that Bcl-2 participates upstream from the function of ICE/CED-3 proteases and may inhibit apoptosis by preventing the post-translational activation of ICE/CED-3 proteases.

Isoprenyl phenyl ethers from liverworts of the genus Trichocolea: cytotoxic activity, structural corrections, and synthesis.

The main cytotoxic component in New Zealand collections of the liverwort Trichocolea mollissima was identified as methyl 4-[(5-oxogeranyl)oxy]-3-methoxybenzoate, a structure that has not been reported previously. Two double-bond isomers of this geranyl ether were present at lower levels. Reinvestigation of the benzoates from Japanese collections of Trichocolea tomentella led to the identification of four geranyl ethers (including two of the three compounds identified from T. mollissima), which had previously been assigned incorrect geranyl ester structures. One compound, previously reported as a 3,3-dimethylallyl ester, could not be reisolated from T. tomentella, but was found in a New Zealand collection of Trichocolea lanata. It was shown to be a 3,3-dimethylallyl ether by synthesis from methyl vanillate. Several of these compounds were active in cytotoxic and antifungal assays.

The glycosidic precursor of (Z)-5-ethylidene-2(5H)-furanone in Halocarpus biformis juvenile foliage.

A new glycosidic lactone, (5R,6R)-5-(1-hydroxyethyl)-2(5H)-furanone beta-D-glucopyranoside, has been identified as the principal precursor of (Z)-5-ethylidene-2(5H)-furanone in juvenile foliage of the New Zealand tree Halocarpus biformis. Three related lactone glycosides were isolated in smaller amounts, together with the known phenolic glycosides pyroside, arbutin and picein. The principal lactone glycoside underwent facile elimination of glucose, in neutral or basic conditions, to yield (Z)-5-ethylidene-2(5H)-furanone and its E-isomer. This lactone glycoside was also detected in foliage of H. bidwillii and H. kirkii.

Cytotoxicity and antimicrobial activity of plants from New Zealand's subantarctic islands.

A total of 86 species of bryophytes, lichens and vascular plants were collected from New Zealand's subantarctic Campbell and Auckland Islands, including 18 subantarctic endemic species. Extracts of these species have been screened for antimicrobial activity and toxicity towards a P388 cell line. Of the main taxonomic groups collected, the proportion of extracts with some biological activity was highest for liverworts (25/29), intermediate for flowering plants (17/26) and lowest for mosses and horn worts (4/22). Extracts of six species showed repeatable, high biological activity warranting further investigation. The cytotoxicity of Ranunculus pinguis was due to the presence of ranunculin.

Cloning and characterization of a G protein alpha-subunit-encoding gene from the basidiomycete, Coprinus congregatus.

Complementary DNA (cDNA) clones encoding two G protein alpha-subunit proteins (CGP alpha 1 and CGP alpha 2) were isolated from a Coprinus congregatus (Cc) hyphal tip cell (HTC) library using PCR-generated biotinylated G protein probes. Sequence analysis of the Cc cgp alpha 1 gene indicates that the gene contains an open reading frame (ORF) that translates into a putative 353-amino-acid (aa) product. The predicted CGP alpha 1 protein exhibits similarity to all known G protein alpha-subunits (it has all of the consensus regions for a GTP-binding protein), especially the mammalian retinal G protein, transducin. The CGP alpha 1 aa sequence is 50% identical overall to the transducin subfamily, cgp alpha 1 shares the same aa size grouping as transducin alpha-subunits and, unlike many other G proteins, both CGP alpha 1 and transducin seem to possess a cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive site. Preliminary reverse transcription PCR (RT-PCR) analysis of cgp alpha 1 and cgp alpha 2 mRNA expression revealed that, unlike cgp alpha 2 which seems to be constitutively expressed, cgp alpha 1 is expressed only in HTC that are competent in responding to light. Thus, the cgp alpha 1 product, CGP alpha 1, is a likely candidate for regulating the blue light-induced signal transduction photomorphogenesis system found in Cc.

Sesquiterpene/quinol from a New Zealand liverwort, Riccardia crassa.

A new sesquiterpene/quinol, with mild cytotoxic and antibacterial activity, has been isolated from a New Zealand collection of the liverwort Riccardia crassa. The structure of this compound, riccardiphenol C [3], was established by nmr spectroscopy. Closely related compounds previously isolated from a Japanese collection of R. crassa were not detected in this collection.

Conditionally immortalized oligodendrocyte cell lines migrate to different brain regions and elaborate 'myelin-like' membranes after transplantation into neonatal shiverer mouse brains.

Five immortalized oligodendrocyte cell lines, representing a spectrum of different stages of oligodendrocyte maturation, were transplanted into neonatal shiverer mouse brains and examined for their ability to survive, multiply, and migrate in vivo. Each of the cell lines migrated to different regions of the brain with remarkable consistency when injected into the mouse forebrain, suggesting that the cells might be responding to different environmental cues present in the neonatal mouse brain. These results are consistent with the fact that cells at different stages in the oligodendrocyte lineage probably possess different sets of surface molecules and receptors. Significant differences were also observed in the survival and proliferation of the lines examined, even when the lines tested had quite similar in vitro phenotypes. Interestingly, the cell line with the most mature in vitro phenotype, N20.1, appeared to elaborate membranous processes when transplanted into the brain, reminiscent of oligodendrocytes ensheathing axonal segments. The experiments suggest that these immortalized cells could be useful models to study the cellular and molecular mechanisms involved in the development, maturation and possibly formation of myelin by oligodendrocytes in the mammalian brain.

Murine oligodendroglial cells express nerve growth factor.

The studies reported here present evidence for the expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) by an oligodendroglial cell line and of NGF by oligodendrocytes in mouse primary culture. An immortalized oligodendroglial cell line (N19) expressing markers for immature oligodendrocytes stimulated PC12 cells to elaborate processes. Polymerase chain reaction analysis with degenerate primers indicated that the N19 cells expressed the mRNAs for the neurotrophic factors NGF and BDNF. Northern blot analysis confirmed that the N19 cells expressed the 1.3-kb NGF mRNA and the 1.4- and 4-kb BDNF mRNAs. In situ hybridization histochemistry identified the presence of NGF mRNAs in 9-day primary oligodendroglial cultures. Combined immunocytochemistry and in situ hybridization histochemistry colocalized NGF mRNA within primary cultured cells that immunostained for the oligodendrocyte marker galactocerebroside (GC). Double-immunofluorescence analysis also colocalized NGF protein within GC+ cells and within A2B5+ cells, a marker for oligodendrocyte progenitors. These results show that oligodendroglia and their precursor cells can express the neurotrophic factor NGF. They suggest that cells in the oligodendrocyte lineage may play an active role in neurite extension through fiber tracts in addition to myelination.

Antitumour lignans and cytotoxic resin acids from a New Zealand gymnosperm, Libocedrus plumosa.

The antitumour activity of a foliage extract of a New Zealand gymnosperm, Libocedrus plumosa, against P388 leukaemia cells was due to ß-peltatin 3. Deoxypodophyllotoxin 1 and ß-peltatin A-methyl ether 2, P388-active lignans previously reported in L. bidwillii, were found at lower levels. High levels of trans-communic acid 4 and sandaracopimaric acid 5 were found in the L. plumosa extract. These diterpene resin acids showed cytotoxic activity against BSC-1 cells. A rapid "chemical screening" method is described, which further characterises bioactive extracts. Chemical screening and mass spectroscopy results for the two Libocedrus species and for Cupressus macrocarpa suggested that the P388 activity of the latter species was also due to 1 and 2. Extracts of all three of these species inhibited tubulin polymerisation.

Generation and analysis of normal and shiverer temperature-sensitive immortalized cell lines exhibiting phenotypic characteristics of oligodendrocytes at several stages of differentiation.

Normal glial cells immortalized at specific developmental stages would be useful tools with which to study glial cell differentiation in vitro. Similarly, immortalized glial cell lines derived from known neurological mutants with identified developmental, molecular genetic defects would also be useful for the in vitro examination of the effects of the mutation on glial cell function. In this report we describe the immortalization of 19 separate oligodendroglial cell lines, 10 derived from normal mice and 9 derived from the neurological mutant shiverer, which is missing a large segment of the myelin basic protein gene. Enriched oligodendrocyte cultures prepared at 7 days in vitro, a time when the majority of the cells were oligodendrocyte precursors undergoing transition into mature oligodendrocytes, were immortalized with pZIPSVtsA58, which carries a temperature-sensitive immortalizing oncogene. All of the immortalized cell lines grew rapidly at the permissive temperature of 34 degrees C and exhibited a dramatic decrease in growth rate at the nonpermissive temperature of 39 degrees C. The cell lines were characterized by immunocytochemistry and Northern blot analysis for a number of glial cell markers, and the phenotype of all the lines were consistent with their being in the oligodendroglial cell lineage. The phenotypes of the cell lines varied significantly with representatives of oligodendrocyte precursors, and immature and mature oligodendrocytes being present within the population of immortalized lines. All the cell lines appeared to be clonal based upon Southern blot analysis and all have been passaged at least 40 times with retention of stable phenotype.

A rapid method for cosmid cloning.

We present a method for genomic library construction using cosmid vectors. With a combination of backfilling with Klenow enzyme and a cosmid vector with two cos sites, a DNA bank in excess of 500,000 clones can be made from 10 micrograms of genomic DNA. The method is more rapid than conventional protocols because size fractionation of target DNA is not necessary. A further advantage is that libraries can be made from relatively small amounts of genomic DNA.

Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement.

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.