Derivation of human embryonic stem cell lines

Human embryonic stem cells (hESC) are undifferentiated cells derived from an early embryo that can grow in vitro indefinitely, while retaining their capability to differentiate into specialized somatic cell types. Over the last decade there has been great interest in derivation and culture of these cells, as they can potentially provide a supply of readily available differentiated cells and tissues of all types to be used for therapeutic purposes in cell transplantation in humans, as well as for other medical uses such as drug discovery. The source of hESC lines is usually excess human embryos from in vitro fertilization treatments, although novel ways of producing hESCs have been suggested recently. The actual methods of hESC derivation have not changed greatly since the first report by Thomson et al. in 1998 . However, the main emphasis over the last several years has been in finding defined conditions for derivation and culture of hESCs, because to enable the clinical use of hESC for cell transplantation, the use of animal derived biological components is no longer acceptable. For basic research, the aim is to replace even human derived materials with completely defined systems. In this paper we describe methods utilized in our laboratory for hESC derivation and describe two studies conducted in an attempt to improve derivation efficiency and to enable research outcomes to be achieved using fewer embryos.     

Wound-induced oxidative responses in mountain birch leaves

AIMS: The aim of the study was to examine oxidative responses in subarctic mountain birch, Betula pubescens subsp. czerepanovii, induced by herbivory and manual wounding. METHODS: Herbivory-induced changes in polyphenoloxidase, peroxidase and catalase activities in birch leaves were determined. A cytochemical dye, 3,3-diaminobenzidine, was used for the in situ and in vivo detection of H2O2 accumulation as a response to herbivory and wounding. To localize peroxidase activity in leaves, 10 mm H2O2 was applied to the dye reagent. KEY RESULTS: Feeding by autumnal moth, Epirrita autumnata, larvae caused an induction in polyphenoloxidase and peroxidase activities within 24 h, and a concomitant decrease in the activity of antioxidative catalases in wounded leaves. Wounding also induced H2O2 accumulation, which may have both direct and indirect defensive properties against herbivores. Wound sites and guard cells showed a high level of peroxidase activity, which may efficiently restrict invasion by micro-organisms. CONCLUSION: Birch oxidases together with their substrates may form an important front line in defence against herbivores and pathogens.     

The Generation of Six Clinical-Grade Human Embryonic Stem Cell Lines

The edict for producing clinically compliant human embryonic stem cells (hESCs) necessitates adherence to global ethical standards for egg procurement and embryo donation, conformity to regulations controlling clinical-grade cell and tissue product development, and compliance with current good tissue and manufacturing practices (cGTPs and cGMPs, respectively). For example, the U.S. FDA Center for Biologics Evaluation and Research recently promulgated regulations regarding human cells and cellular-based products (HCT/Ps) intended for tissue repair or replacement. Issued under Code of Federal Regulations parts 1270 and 1271 (Code of Federal Regulations, 2006a, 2006b), the rules are broadened by requirements for donor selection and cGMPs for HCT/Ps. By adhering to regulations and in anticipation of future standards, we have generated six clinical-grade hESC lines. Here we describe their manufacture, from embryo procurement to line characterization, including sterility and pathogen testing (Figure 1). To our knowledge, the lines represent the first to have been produced in compliance with international regulatory requirements, suitable for therapeutic use.     

Effects of fasting and exogenous melatonin on annual rhythms in the blue fox (Alopex lagopus)

The arctic fox (Alopex lagopus) is a winter-active inhabitant of the high arctic with extreme fluctuations in photoperiod and food availability. The blue fox is a semi-domesticated variant of the wild arctic fox reared for the fur industry. In this study, 48 blue foxes were followed for a year in order to determine the effects of exogenous melatonin and wintertime food deprivation on their reproductive and thyroid axes. Half of the animals were treated with continuous-release melatonin capsules in July 2002, and in November-January, the animals were divided into three groups and either fed continuously or fasted for one or two 22-day periods. Food deprivation decreased the plasma triiodothyronine and thyroxine concentrations probably in order to preserve energy due to a decreased metabolic rate. The same was observed in the plasma testosterone levels of the males but not in the plasma estradiol concentrations of the females. Exogenous melatonin advanced the autumn moult and seasonal changes in the voluntary food intake. It also advanced the onset of the testosterone peak in the males. The plasma estradiol levels of the females were unaffected, but the progesterone levels peaked more steeply in the sham-operated females. Melatonin exerted a strong influence not only on the reproductive axis of the males but also on the seasonal food intake. The species seemed quite resistant to periodic involuntary food deprivation.     

Sequence-selective cleavage of oligoribonucleotides by 3d transition metal complexes of 1,5,9-triazacyclododecane-functionalized 2'-O-methyl oligoribonucleotides

2'-O-Methyl oligoribonucleotides bearing a 3'-[2,6-dioxo-3,7-diaza-10-(1,5,9-triazacyclododec-3-yl)decyl phospate conjugate group have been shown to cleave in slight excess of Zn(2+) ions complementary oligoribonucleotides at the 5'-side of the last base-paired nucleotide. The cleavage obeys first-order kinetics and exhibits turnover. The acceleration compared to the monomeric Zn(2+) 1,5,9-triazacyclododecane chelate is more than 100-fold. In addition, 2'-O-methyl oligoribonucleotides having the 1,5,9-triazacyclododec-3-yl group tethered to the anomeric carbon of an intrachain 2-deoxy-beta-d-erythro-pentofuranosyl group via a 2-oxo-3-azahexyl, 2,6-dioxo-3,7-diazadecyl, or 2,9-dioxo-3,10-diazatridecyl linker have been studied as cleaving agents. These cleave as zinc chelates a tri- and pentaadenyl bulge opposite to the conjugate group approximately 50 times as fast as the monomeric chelate and show turnover. The cleavage rate is rather insensitive to the length of linker. Interestingly, a triuridyl bulge remains virtually intact in striking contrast to a triadenyl bulge. Evidently binding of the zinc chelate to a uracil base prevents its catalytic action. Replacement of Zn(2+) with Cu(2+) or Ni(2+) retards the cleaving activity of all the cleaving agents tested.     

Use of the scanning electron microscope in the study of isssolated ribosomes and polysomes

A procedure is described by which the ribosome assemblies isolated from the buds of Scots pine ( Pinus sylvestris L.) as well as the ribosome and polysome fractions purified by sucrose density gradient centrifagation can be prepared for study by scanning electron microscopy. The technique has also been used to illustrate ribosomes extracted froai leaves and roots of Nicotiana tabacum L., from roots of Tuiipa gesneriana hybr. and from commercial wheat germ. The sample under study is spread on a piece of coverslip', frozen and dried at room temperature. The coverslip, with attached material, is taken through the critical point drying procedure, glued on a specimen retainer aod coated with gold-palladium. The coverslip serves as a sufficiently stable specimen support to allow the study of ribosomes at magnifications of several hundred thousand. Use of the method makes it possible to study the structure of polysomes, to check the purity of fractioned samples and to investigate differences in the ribosome assembly of the developing plant tissues.     

Murine cathepsin D deficiency is associated with dysmyelination/myelin disruption and accumulation of cholesteryl esters in the brain

Cathepsin D (CTSD) deficiencies are fatal neurological diseases that in human infants and in sheep are characterized by extreme loss of neurons and myelin. To date, similar morphological evidence for myelin disruption in CTSD knockout mice has not been reported. Here, we show that CTSD deficiency leads to pronounced myelin changes in the murine brain: myelin-related proteolipid protein and myelin basic protein were both markedly reduced at postnatal day 24, and the amount of lipids characteristically high in myelin (e.g. plasmalogen-derived alkenyl chains and glycosphingolipid-derived 20- and 24-carbon acyl chains) were significantly lowered compared with controls. These changes were accompanied by ultrastructural alterations of myelin, including significant thinning of myelin sheaths. Furthermore, in CTSD knockout brains there was a pronounced accumulation of cholesteryl esters and abnormal levels of proteins related to cholesterol transport, with an increased content of apolipoprotein E and a reduced content of ATP-binding cassette transporter A1. These results provide evidence for dysmyelination and altered trafficking of cholesterol in brains of CTSD knockout mice, and warrant further studies on the role of lipid metabolism in the pathogenesis of CTSD deficiencies.     

History of agriculture in Mikkeli Orijärvi,eastern Finland as reflected by palynological and archaeological data

The introduction and development of cultivation in eastern Finland was studied by pollen and charcoal analysis of a palaeomagnetically dated sediment profile from Lake Orijärvi, in the vicinity of permanent prehistoric fields. The earliest changes of possibly anthropogenic origin are visible in the pollen data from 1630 b.c. onwards and indications of human impact become more evident from 500 b.c. onwards. According to finds of cereal pollen and AMS-dating of charred cereal grains from the oldest field layer, the onset of cultivation can be dated to the Merovingian period around a.d. 600. To a significant extent the pollen data reflect only the cultivation of Secale during the first 600 years. The marked intensification of agricultural activities including cultivation in permanent fields only becomes evident in the pollen data from about a.d. 1050 to 1080 onwards and the most intensive land use phase dates to a.d. 1300–1965. Archaeological and palaeoecological material indicate that swidden cultivation and permanent field cultivation were in use simultaneously during the late Iron Age. The combination of these techniques together with animal husbandry and hunting formed a subsistence strategy in the climatic border-zone outside the centres of the agricultural core areas.     

Accuracy and repeatability of Roentgen stereophotogrammetric analysis (RSA) for measuring knee laxity in longitudinal studies

Roentgen stereophotogrammetric analysis (RSA) can be used to assess temporal changes in anterior-posterior (A-P) knee laxity. However, the accuracy and precision of RSA is dependent on many factors and should be independently evaluated for a particular application. The objective of this study was to evaluate the use of RSA for measuring A-P knee laxity. The specific aims were to assess the variation or "noise" inherent to RSA, to determine the reproducibility of RSA for repeated A-P laxity testing, and to assess the accuracy of these measurements. Two experiments were performed. The first experiment utilized three rigid models of the tibiofemoral joint to assess the noise and to compare digitization errors of two independent examiners. No differences were found in the kinematic outputs of the RSA due to examiner, repeated trials, or the model used. In a second experiment, A-P laxity values between the A-P shear load limits of +/-60 N of five cadaver goat knees were measured to assess the error associated with repeated testing. The RSA laxity values were also compared to those obtained from a custom designed linkage system. The mean A-P laxity values with the knee 30 degrees, 60 degrees, and 90 degrees of flexion for the ACL-intact goat knee (+/-95% confidence interval) were 0.8 (+/-0.25), 0.9 (+/-0.29), and 0.4 (+/-0.22) mm, respectively. In the ACL-deficient knee, the A-P laxity values increased by an order of magnitude to 8.8 (+/-1.39), 7.6 (+/-1.32), and 3.1 (+/-1.20)mm, respectively. No significant differences were found between the A-P laxity values measured by RSA and the independent measurement technique. A highly significant linear relationship (r(2)=0.83) was also found between these techniques. This study suggests that the RSA method is an accurate and precise means to measure A-P knee laxity for repeated testing over time.