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Chemicals/CAS: benzo[a]pyrene, 50-32-8; calcium ion, 14127-61-8; retinol, 68-26-8, 82445-97-4

In vitro assays that permit cloning of tumour cells in soft agar have been improved during the last 5 years. Two of them are claimed to be useful as test systems for the screening of new anticancer drugs and even for drug sensitivity testing of individual human tumours in devising individualized cancer chemotherapy regimens. Three assays were investigated for this report: those of Toshio Kuroki (TK), and Hamburger and Salmon (HS), and that in use for bone marrow cell cultures (BM). Cells of various origins were tested for their growth capacity and colony formation in these three assays. Included were cells of 10 established lines classified as malignant or nonmalignant according to the in vivo malignancy test. Cells freshly derived from two tumours and those from five tumours after 2-10 passages in monolayer culture were also used as test cells. The BM assay gave the best results. Up to now, a 100 per cent correlation has been found between the in vivo and in vitro test. Investigations are under way to determine whether this assay can also be used as a transformation assay using cells with a low transformation rate. Chemicals/CAS: agar, 9002-18-0; Agar, 9002-18-0; Culture Media

If cells of the bacterium Escherichia coliC infected with bacteriophage φX174 are lysed artificially in the cold, the phage obtained is relatively heat resistant at temperatures around 65° and unable to adsorb to host cells at 4°. At 37° the phage particles become more thermolabile with respect to inactivation at 65° and, concomitantly, obtain the ability to adsorb to E. coli C in the cold. A minor fraction remaining thermoresistant at 37° does not adsorb to host cells even at this temperature. Optimal conditions for the transition of the resistant into the sensitive form in phosphate buffer are 37°, pH ≤ 7 and a concentration of phosphate ≤= 25 mM. At lower pH and high titers of phage the thermosensitive form changes partly back into the thermoresistant one. © 1967.

In vitro, high-throughput methods have been widely recommended as an approach to screen chemicals for the potential to cause developmental neurotoxicity and prioritize them for additional testing. The choice of cellular models for such an approach will have important ramifications for the accuracy, predictivity and sensitivity of the screening assays. In recent years neuroprogenitor cells from rodents and humans have become more widely available and may offer useful models having advantages over primary neuronal cultures and/or transformed cell lines. To date, these models have been utilized in only a limited number of toxicity studies. This review summarizes the state of the science regarding stem and neuroprogenitor models that could be used for screening assays, provides researchers in this field with examples of how these cells have been utilized to date, and discusses the advantages, limitations and knowledge gaps regarding these models. Data are available from both rodent and human stem and neuroprogenitor cell models that indicate that these models will be a valid and useful tool for developmental neurotoxicity testing. Full potential of these models will only be achieved following advances in neurobiology that elucidate differentiation pathways more clearly, and following further evaluation of larger sets of developmentally neurotoxic and non-toxic chemicals to define the sensitivity and predictivity of assays based on stem or progenitor cell models.

Identifying the qualities of primary care that have the potential to produce optimal health outcomes is only half the story. The MOCHA project has explored how to transfer these to other national contexts, but also which successful components should be transferred. It is important to assess the population criteria of the identified sociodemographic, cultural and social characteristics, and the population perspectives on a care system’s components. The project analysed public experiences and perceptions of the quality of primary care for children from a representative sample of the general public in five EU Member States. The public perception of children’s primary care services, in particular the perceived quality of care and expectations of children and their care is important to understand before MOCHA lessons can be effectively adopted in a country. We found that the socio-cultural characteristics of a country inform the population perceptions and preferences with regard to the care system. In the five countries surveyed there was agreement about aspects of quality of care – such as accessible opening hours, confidential consultations for children and timeliness of consultation for an illness; but there was a difference in opinion about giving priority to items such as making an appointment without a referral, or a child’s right to a confidential consultation. The cultural context of transferability and the means of addressing this such as defining the target audience and the different means of disseminating important messages to the wider community to address contextual factors can act as barriers or facilitators to the introduction of new components of primary care models.

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 ± 8 mg (mean ± S.E.) glucoamylase (GAM) L-1 in batch culture and 373 ± 9 mg GAM L-1 in maltodextrin- limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (q(p)) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (q(p), 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 ± 12 to 496 ± 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 ± 0.4 to 16.4 ± 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar glucoamylase (GAM) production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of GAM gene copies in the later strain was reduced, which could explain its reduced GAM production. Shake-flask cultures of morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains. The physiological changes in these morphological mutants which contribute to this decreased level of GAM production were shown

Chemicals/CAS: 2 amino 2 methylpropionic acid, 62-57-7; carbon, 7440-44-0; thymidine, 50-89-5; tritium, 10028-17-8; Aminoisobutyric Acids; Carbon Isotopes; Lectins; Thymidine, 50-89-5; Tritium, 10028-17-8

To understand cartilage degenerative diseases and improve repair procedures, we investigate the influence of glycosaminoglycans (GAGs) on cartilage matrix biochemistry and functionality. Bovine articular chondrocytes were cultured in alginate beads with(out) para-nitrophenyl-beta-d-xyloside (PNPX) to inhibit GAG incorporation into newly formed proteoglycans. As expected, GAG deposition in alginate beads decreased with increasing PNPX concentration. Next to GAGs, collagen deposition and cross-linking also decreased. In the presence of PNPX, GAGs and collagen were deposited further away from the chondrocyte than in the control and increased amounts were found in the culture medium. These changes resulted in decreased functional properties of the construct. We conclude that in our culture system, intact proteoglycans play a role in deposition of collagen and thus the formation of a functional matrix. The effect of less proteoglycans on the collagen network could explain why cartilage repair is ineffective in osteoarthritis and help us with development of new therapies. © 2008 Elsevier Inc. All rights reserved.

In The Netherlands, several pilot projects are carried out on the use of spat collectors as an additional supply of seed for bottom culture of mussels (Mytilus edulis). The method proves to be successful in yielding substantial amounts of seed. One of the conditions for successful application of collector seed on bottom plots is a good yield of the seed on bottom plots. Mussel seed of different origin (from wild littoral and sublittoral beds or from collectors) was offered to predators (crabs Carcinus maenas and starfish Asterias rubens) and seed survival was monitored. In addition, the effect of density and size of collector seed on predation was studied. Circular cages containing predators and seed were placed in a basin with running seawater, or suspended from a jetty in a harbour. Two size classes of predators and three size classes of seed were used. Survival was monitored. Consumption of mussel seed by starfish was much lower than by crabs. Maximum observed consumption rates were 23 seed/day/crab and 1 seed/day/starfish. Consumption rates increased significantly with decreasing seed size. Seed larger than 20 mm were consumed at a significantly lower rate. Seed density did not affect survival. Collector seed was not consumed at higher rates than wild littoral or sublittoral seed. In conclusion, collector seed can be a promising additional source of seed for bottom culture of mussels in The Netherlands.

The production of active dried starter cultures can be influenced at several levels in the production process. In this paper the following process factors are discussed: osmotic stress during growth and cell density prior to drying. Contradicting results are reported in the literature on the influence of osmotic stress during growth on the residual activity after drying. The combined approach in which two process factors were studied at a time resulted in an explanation for the discrepancy in earlier work. The cell density prior to drying had an important influence on the glucose fermenting activity after drying. Residual activities ranging from 0.10 to 0.83 were achieved using initial cell densities between 0.025 and 0.23 g of cell/g of sample, respectively. The drying tolerance of cells grown with osmotic stress of 1 M NaC1 was low (residual activity = 0.06) and was not related to the cell density prior to drying. The influence of osmotic stress during growth on the drying tolerance of Lactobacillus plantarum was dependent on the cell density prior to drying.

Previously tropic effects of extracts from whole chick embryos and from innervated muscles on cultured muscle cells were described. The present study demonstrated similar effects of extracts from 10-days denervated chick muscles. Extracts from innervated as well as from denervated muscles exsanguinated in vivo with saline prior to dissection showed only marginal trophic activity, suggesting a major contribution of serum components to the trophic effects of tissue extracts. Indeed, serum of adult chicks appeared to have a trophic action similar to that of chick embryo extract.

A less complicated source of neurons suitable for this type of studies is the parasympathetic ciliary ganglion. In the pigeon and in the chick this ganglion is known to contain only two classes of neurons, both of which are cholinoceptive and cholinergic and that innervate the muscle fibres of the choroid plexus of the eye, the sphincter iridis and the ciliary body (Marwitt et al., 1971). The iris and the ciliary muscle in birds are both striated (Ovio, 1927), whereas in the mammal they are smooth. Neurons of the ciliary ganglion of the chick embryo can be cultivated in tissue culture (Hooisma et al., 1975) either in explants of whole ganglia or after dissociation, as individual cells. Branching neurites grow radially from the ganglia. A corona of neurites with a diameter of 500-1000 μm surrounding the ganglia is formed during 2 days in culture. Mixed cultures of ciliary neurons and skeletal-muscle cells have been used for the study of the process of neuron-to-target recognition.

Bedrijven en overheidsinstanties hebben soms moeite met het identificeren van chronische veiligheidsissues. Op verzoek van DCMR bracht TNO de veiligheidscultuur in kaarr bij veertien BRZO-plichtige bedrijven in de Rijnmondregio. Conclusie: er is ruimte voor verbetering in bijna alle ond erzochte dimensies. Vakmedia

Aspergillus niger B1, a recombinant strain carrying 20 extra copies of the native glucoamylase gene, was grown in glucose-limited chemostat cultures supplemented with various organic nitrogen sources (dilution rate 0.12 ± 0.01 h-1, pH 5.4). In cultures supplemented with L-alanine, L-methionine, casamino acids, or peptone, specific glucoamylase (GAM) production rapidly decreased to less than 20% of the initial level. Reducing the pH of the culture to 4.0 resulted in stable GAM production for up to 400 h. Morphological mutants (a light brown and a dark brown mutant) appeared in each fermentation and generally displaced B1. Light brown mutants had higher selection coefficients relative to B1 than dark brown mutants and became the dominant strain in all fermentations except those maintained at pH 4.0. Several mutants isolated from these cultures had reduced ability to produce GAM in batch culture, although few had lost copies of the glaA gene. Some mutants had methylated DNA. © 2000 Academic Press.

In intervertebral disc herniation with nucleus pulposus (NP) extrusion, the elicited inflammatory response is considered a key pain mechanism. However, inflammatory cytokines are reported in extruded herniated tissue, even before monocyte infiltration, suggesting that the tissue itself initiates the inflammation. Since herniated tissue swells, we investigated whether this simple mechanobiological stimulus alone could provoke an inflammatory response that could cause pain. Furthermore, we investigated whether sustained-release cyclooxygenase-2 (COX2) inhibitor would be beneficial in such conditions. Healthy bovine NP explants were allowed to swell freely or confined. The swelling explants were treated with Celecoxib, applied either as a bolus or in sustained-release. Swelling explants produced elevated levels of interleukin-6 (IL-6) and prostaglandin E2 (PGE2) for 28 days, while confined explants did not. Both a high concentration bolus and 10 times lower concentration in sustained release completely inhibited PGE2 production, but did not affect IL-6 production. Swelling of NP tissue, without the inflammatory system response, can trigger cytokine production and Celecoxib, even in bolus form, may be useful for pain control in extruded disc herniation.

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D) on embryo-like structures (ELS) and plant development from barley microspores was determined. Microspores cultured on filters enabled simple modification of growth regulator concentrations. Regeneration frequencies obtained with 2,4- D as growth regulator were similar to the results achieved with the generally applied cytokinin 6-benzylaminopurine. If 2,4-D was applied after a regular mannitol pretreatment, maximal plant regeneration was achieved if 10-6 mol/L 2,4-D was present continuously or for 7 days. Alternatively, maximal plant formation was induced if 10-4 or 10-5 mol/L 2,4-D was present for 1 h or if present resp. 3 days or 1 day. Induction of plant regeneration by a l h treatment with 10-4 mol/L 2,4-D is a more generally observed phenomenon for single cells or small cell clusters of both dicotyledonous and monocotyledonous species. Without mannitol pretreatment, it was possible to induce plant production after 2,4-D treatment only in anther cultures. Without mannitol pretreatment no embryogenic type of microspores could be recognized at the moment of microspore isolation, and plating efficiency never reached 1%. In anther culture without mannitol pretreatment, a higher molarity and/or longer presence of 2,4-D was required and resulted only in about 1 green plant per anther. After application of a mannitol pretreatment, plant production increased at least 10 times. To our knowledge this is the first report on microspore-derived barley plants via androgenesis without any pretreatment. The combination of 2,4-D and anther pretreatment with mannitol as trigger for microspore differentiation is discussed.