Postmortale bildung von 5,7-dihydroxychromon-7-rutinosid in Mentha longifolia

5,7-Dihydroxychromone 7-rutinoside isolated from Mentha longifolia must be considered to be a product of postmortem processes. It is only formed after heating fresh plant material and its production is connected with the degradation of flavonoids, and particularly of eriodictyol 7-rutinoside. Investigations with four other Mentha species confirm these results. Eriodictyol 7-rutinoside and eriodictyol are degraded up to 50%, by horseradish peroxidase but 5,7-dihydroxychromone-7-rutinoside cannot be detected.     

Chemical and morphological diversity in wild populations of Mentha longifolia in Israel

Populations of Mentha longifolia, an endangered species in Israel, were tested for essential oil composition and conservational ability. In 2002-2003, 25 wild populations country-wide were tested, indicating population divergence into two chemotypes. Chemotype A was characterized by high levels of menthone and pulegone, and chemotype B by high levels of piperitenone oxide and piperitone oxide. Chemotype A was more abundant (22 of 25 populations) than chemotype B (11 of 25 populations). However, a chemotype/population interaction was not recorded (P?>?0.05). In spring 2003, seven of the 25 wild populations were resampled, propagated, and cultivated at the Newe Ya'ar campus. Then, in 2004, the propagated plants were tested for essential oil composition. The propagated plants maintained the essential oil composition as well as the chemotype-frequency distribution of the original wild population from which they were obtained. Since a chemotype/population interaction was not recorded, and the cultivated plants displayed the wild population essential oil composition, it can be concluded that i) the chemotype diversity is genetically based, and ii) the M. longifolia populations sampled can be horticulturally conserved.     

In vitro cultures of Drosera aliciae as a source of a cytotoxic naphthoquinone: ramentaceone

A protocol for the in vitro propagation of Drosera aliciae to increase the yield of the naphthoquinone, ramentaceone, was developed. The highest micropropagation coefficient was obtained using half-strength Murashige–Skoog medium supplemented with 0.4 μM 6-benzyladenine (BA). The genetic fidelity and stability of the regenerated plants was confirmed with RAPD markers. The activity of the isolated ramentaceone was determined against four human tumor cell lines: U937, HeLa, MCF-7, HCT-116 with the highest cytotoxic activity towards the leukemic U937 cell line with an IC₅₀ value of 3.2 μM.     

Animal cell cultures as a source of hormones

Current methodology now makes it possible to establish in culture a variety of mammalian cells which perform organ-specific functions during serial propagation for periods of months or years. This report describes the results of experiments with animal and human cells that produce growth hormone, adrenocortical steroid hormones, thyrocalcitonin, and parathyroid hormone. Within the next decade it should be possible to use cell culture methods for manufacturing purposes to produce hormones and other valuable cellular products which are difficult to obtain in other ways. At first it may be necessary to use neoplastic cells for this purpose; but evidence is accumulating to suggest that it may eventually be possible to establish in culture normal, functional animal and human cells.     

Growth and monoterpene production by transformed shoot cultures of Mentha citrata and Mentha piperita in flasks and fermenters

This paper reports studies on the growth and biosynthesis of monoterpenes by transformed shoot cultures of Mentha citrata and Mentha piperita, originally developed 5 years ago and since maintained by regular subculturing. Throughout this time, the M. citrata culture has stably maintained production of an oil closely resembling that of the parent plant in which linalool and linalyl acetate are the predominant components. However, M. piperita, which initially showed a divergence from the parent plant in producing significant amounts of menthofuran in addition to the characteristic oil components menthol and menthone, has now been found to produce pulegone and menthofuran as the major components. The cultures were subjected to different environmental conditions of varying periods of light and temperature in an attempt to restore menthol and menthone production. Increased illumination reduced the yields of pulegone and menthofuran but did not stimulate the production of either menthol or menthone, which remained only at trace levels (below 0.2 g/g fresh weight). Cultures of M. citrata were, however, stimulated by increased illumination, and produced more linalool and linalyl acetate. Shoot cultures of M. citrata and M. piperita were grown in 14–1 fermenters for up to 60 dys during which the biomass increased from approximately 100 g to 2.5 kg and 3.5 kg respectively. Both cultures rapidly consumed sucrose with a concomitant release of glucose, and the uptake of inorganic ions was similar except that M. citrata consumed far less Na+ during the fermentation. The total yields of monoterpenes from the fermentations were 1.16 g (M. piperita) and 0.18 g (M. citrata). *** DIRECT SUPPORT *** AG903062 00005     

Persistently infected cultures as a source of hepatitis A virus

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.     

Skin explant cultures as a source of keratinocytes for cultivation

Cultivated human keratinocytes can be used successfully in the treatment of burn patients, but efforts to heal burns and other wounds can be hampered by the very small skin biopsies available for cultivation of transplantable keratinocyte sheets. A small biopsy (and correspondingly small number of enzymatically isolated keratinocytes for use in classical cultivation techniques) can lead to a low yield of multilayer sheets for clinical application or unacceptably long cultivation times. One way of addressing this is to make use of skin remnants remaining after enzymatic digestion and culture cells migrating out of these skin explants. Sufficient numbers of explant-derived keratinocytes can be obtained to facilitate additional routine cultivation of these cells. Biopsy remnants can be used to initiate explant cultures repeatedly (we were able to re-use pieces of skin 10 times and still obtain useful numbers of keratinocytes) and this “passaging” yields substantially more cells for classical cultivation than would be available from conventional methodology alone, and in a comparable timeframe. Another advantage of this method is that it does not require additional biopsies to be procured from already-compromised patients and overcomes problems associated with contamination of skin samples with resistant hospital-acquired bacterial infections common during prolonged hospitalization.     

Persistently infected cultures as a source of hepatitis A virus.

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.     

Influence of carbon source on shoot multiplication and adventitious bud regeneration in in vitro beech cultures

Experiments were performed to determine the effects ofcarbon source and concentration on shootmultiplication in shoot cultures of Fagussylvatica (one clone) and F. orientalis (twoclones) and on the induction of adventitious shootbuds from leaf and internode explants of F.orientalis. In general, glucose was the best carbonsource for both axillary branching and adventitiousshoot regeneration. Shoot-tip explants grown on 3–4%glucose medium produced more shoots than those onsucrose or fructose. For maximum shoot length, glucosemedium was best for two of the three clones, and 4%sucrose for the other. The number of shoots was theparameter most influenced by glucose concentration inthe adventitious shoot regeneration experiments, thenumber increasing with sugar concentration. The lowesthyperhydricity rate occurred in the presence ofsucrose in both species. Shoot growth and quality wasnegatively affected by fructose supplied media. Theuse of filter-sterilized rather than autoclavedfructose neither stimulated shoot growth nor reducedthe incidence of hyperhydricity in all three clones.The response of shoot cultures to differentcarbohydrate treatments appears to some extent to begenotype dependent.     

Production and composition of extracellular polysaccharide from cell suspension cultures of Mentha

A suspension culture of Mentha was established from callus which formed on the tips of young shoots of a Mentha hybrid (M. arvenis × M. spicata). Changes in growth parameters during a culture cycle were recorded. The general appearance of cells during division and growth, including the changes in cell form, was also represented.Suspension-cultured cells of Mentha hybrid released a large amount of extracellular polysaccharides (ECP) mainly at the logarithmic phase of the growth cycle. The ECP contained galacturonic acid as major components and arabinose, galactose, glucose, xylose, rhamnose and mannose as minor components. The ratio of the uronic acid content to total sugar content in the ECP was below 40% at day 7, but increased up to 90% at day 21. The relative contents of xylose and glucose in the ECP decreased during the culture period, while the arabinose content increased and those of rhamnose, mannose and galactose remained constant.The IR spectrum suggested that the ECP were low-methoxylated pectic polysaccharides. The presence of lignin and related compounds in the ECP was not detected. The protein content of the ECP was about 10% and the main amino acids were alanine, proline, hydroxyproline, valine, asparticacid and serine, in that order.     

Lippia dulcis shoot cultures as a source of the sweet sesquiterpene hernandulcin

The axenic shoot culture of Lippia dulcis Trev., Verbenaceae, was established on hormone-free Murashige-Skoog solid medium containing 3% sucrose. Shoots were cultured in various liquid or solid media. Woody Plant liquid medium was best for shoot multiplication, but the production of hernandulcin was relatively low. The highest hernandulcin content (2.9% dry wt) was obtained after 28 days of culture on Murashige-Skoog solid medium containing 2% sucrose. The addition of chitosan to the culture media enhanced the growth of shoots as well as the production of hernandulcin, especially with the liquid medium.Abbreviations MS(2%) Murashige-Skoog medium containing 2 % sucrose - MS(3%) Murashige-Skoog medium containing 3 % sucrose - 1/2MS half strength Murashige-Skoog medium containing 2% sucrose - B5 Gamborg B5 medium containing 2% sucrose - WP Woody Plant medium containing 2% sucrose     

Assessment of variability among morphological and molecular characters in wild populations of mint [Mentha longifolia (L.) L.] germplasm

Mentha longifolia is an important medicinal and aromatic perennial herb that exhibits wide distribution range from sub-tropical to temperate regions. In the present study, agro-morphological traits and genetic differences in 19 different populations of M. longifolia were studied to evaluate the level and extent of its diversity. Analysis of variance (ANOVA) showed that the different phenotypic characters show considerable differences among various populations and was significant at p < 0.05. Molecular diversity analysis performed by using arbitrary amplified eleven ISSR primers generated a total of 121 amplicons that range within the size of 200–2500 base pairs (bp). Each primer on average generated 11 amplicons with percentage polymorphism being 100. The analysis of molecular variance (AMOVA) showed more (64%) among population genetic diversity and less (36%) within the populations. Greater genetic differentiation (Gst = 0.6852) among these populations occurs due to low gene flow (Nm = 0.2297) and greater habitat variability. Geographic and genetic distances were positively correlated according to Mantel’s test. In order to remove any kind of biases, we used R software to perform cluster and redundancy analysis to analyse the extent of relatedness among studied populations. In terms of morphological and molecular aspects, the populations were grouped into four and five clusters respectively based on hierarchical clustering method. The results demonstrated that M. longifolia displays a great degree of morphological and genetic variation and can be utilized in breeding, genetic improvement, and gene bank conservation programmes in future.     

Shoot tips of wheat as an alternative source for regenerable embryogenic callus cultures

Shoot tips of Triticum aestivum L. cvs. Turbo and Nandu, both summer wheat varieties, were excised from 4 and 10 day-old seedlings, and used for induction of embryogenic callus. A modified L3 medium, supplemented with 10 M 2,4-dichlorophenoxyacetic acid (2,4-d) for culture initiation, and 5 M 2,4-d for subculturing, was optimal; 90% of 4 day-old Turbo seedlings formed embryogenic callus. Optimal plant regeneration was achieved from callus incubated on a modified MS medium without 2,4-d, but supplemented with 2.22 M 6-benzylaminopurine and 0.27 M naphthaleneacetic acid. Plantlets formed via embryogenesis from all embryogenic Turbo calli initiated from 4 day-old explants, with a mean number of 8 regenerants per explant. Regeneration occured via embryogenesis only. Results obtained using Nandu were within the same range.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Correction to: Lactococcus lactis as a safe and inexpensive source of bioactive silver composites

Applied Microbiology and Biotechnology - The authors would like to inform readers of a mistake in the acknowledgement section of the original publication of this article.     

Corroding iron as a hydrogen source for sulphate reduction in growing cultures of sulphate-reducing bacteria

Summary In anaerobic corrosion experiments, hydrogenase-positiveDesulfovibrio strains, grown with limiting lactate concentrations in the presence of steel wool, formed more sulphide than expected or observed with lactate alone. The additional sulphide obviously originated from sulphate reduction with cathodically formed hydrogen from the steel surface. The hydrogenasenegativeD. sapovorans did not produce additional sulphide. The observations agree with the theory of von Wolzogen Kühr and van der Vlugt (1934) that explains anaerobic corrosion as a cathodic depolarization of iron surfaces by hydrogen-consuming sulphate-reducing bacteria. The influence of the iron surface area, the salt concentration and the pH-value on the utilization of cathodically formed hydrogen was investigated. The significance of an additional organic electron donor for the corrosion of iron in aqueous environments is discussed.     

Fish cell cultures as in vitro models of pro-inflammatory responses elicited by immunostimulants

We have tested the elicitation of innate defence-related responses in two stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss) after they were exposed to different concentrations of lipopolysaccharide (LPS), levamisole, or polyinosinic polycytidylic acid (poly-I:C). For comparison, cultures of rainbow trout head kidney macrophages were also included in the study, and the effect of the immunostimulants on the phagocytic activity, the intracellular and extracellular reactive oxygen species and nitric oxide production were assayed. Although the responses varied depending upon the concentration of the immunostimulants and the particular cell line, our results demonstrate that those activities were enhanced in the TSS and TPS-2 cell lines after exposure to any of the immunostimulants. These results indicate that the stromal cells of the main lympho-haemopoietic organs of O. mykiss develop innate defence responses, which are enhanced by well-known immunostimulants. In addition, such enhancement of the defence responses in the TSS and TPS-2 cell lines could be also elicited when they were exposed to conditioned supernatants from levamisole- or poly I:C-stimulated HK macrophage cultures, thus demonstrating that the haemopoietic stromal cells respond to macrophage-derived factors. Moreover, we demonstrate that the stromal cell lines constitutively expressed the Toll-like receptors TLR3, TLR5 and TLR9 genes. The results are discussed considering the role of the lympho-haemopoietic stromal cells in the innate immune responses, and the possibility of using histiotypic cell cultures of non-leucocyte cells of the haemopoietic organs to develop in vitro methods to select new immunostimulant candidates for aquaculture.     

Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium

We describe here a new in vitro protocol for structuring cardiac cell cultures to mimic important aspects of the in vivo ventricular myocardial phenotype by controlling the location and mechanical environment of cultured cells. Microlithography is used to engineer microstructured silicon metal wafers. Those are used to fabricate either microgrooved silicone membranes or silicone molds for microfluidic application of extracellular matrix proteins onto elastic membranes (involving flow control at micrometer resolution). The physically or microfluidically structured membranes serve as a cell culture growth substrate that supports cell alignment and allows the application of stretch. The latter is achieved with a stretching device that can deliver isotropic or anisotropic stretch. Neonatal ventricular cardiomyocytes, grown on these micropatterned membranes, develop an in vivo-like morphology with regular sarcomeric patterns. The entire process from fabrication of the micropatterned silicon metal wafers to casting of silicone molds, microfluidic patterning and cell isolation and seeding takes approximately 7 days.