Ultrastructure ofDigitalis lanata tissue cultures

Summary The anatomy of twoDigitalis lanata tissue culture strains, S-1 and S-2, has been studied. The cardenolide accumulating cell aggregates consisted of highly vacuolated cells with very lobed nuclei in the periphery and a central part of meristematic cells. Sieve tube elements and companion cells and tracheids were observed in some of the cultures. The effect of gibberellic acid (GA₃) and SAN 9789 on the ultrastructure of the cultures was most apparent as regards the plastids and the amounts of mitochondria and ER. The content of starch seemed to be highest in the cardenolide accumulating strain S-2 grown in darkness (S-2 D) with or without GA3, but considerable amounts were also found in S-1 grown in darkness (S-1 D) with or without GA₃, which did not accumulate cardenolides. The amount of plastoglobuli was increased in S-1 by SAN treatment. It was also higher in S-2 D and S-2 D+GA₃ than in S-1 D and S-1 D+GA₃; i.e., it was high in tissues with blocked carotene synthesis. Many large plastoglobuli were also observed in apparently degenerating cells. The amount of ER seemed relatively high in cardenolide producing cultures. The amount of mitochondria was highly variable, but no correlation with cardenolide accumulation could be found.Abbreviations D dictyosome - ER endoplasmic reticulum - M mitochondrion - N nucleus - P plastid - PG plastoglobule - S starch grain - SC sieve cell - V vacuole - W cell wall - GA₃ gibberellic acid - S-1D strain S-1 cultured in darkness - S-1 L strain S-1 cultured in light - S-2 D strain S-2 cultured in darkness - S-2L8 strain S-2 previously cultured in darkness followed by 8 days in light in the present study - SAN SAN 9789 (Norflurazon) 4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-tolyl)-3(2H)-pyridazinone     

Glycation of PDGF results in decreased biological activity

Advanced glycation end products (AGEs) are formed by the non-enzymatic glycation of proteins by reducing carbohydrates or α-oxo-aldehydes such as glyoxal and methylglyoxal and further rearrangements, eliminations and oxidations. AGE-modifications alter peptide structure, function and stability and accumulate under several pathophysiological conditions such as diabetes and are considered a biomarker of ageing. PDGF is a major regulator of wound healing, which is impaired in hyperglycaemia and ageing. We analyzed whether glycated PDGF has impaired activity in cell culture models and occurs in human subjects. PDGF was AGE-modified by the α-oxo-aldehydes glyoxal and methylglyoxal, which was shown by Western-blotting using α-carboxymethyllysine (CML) or α-arginine-pyrimidine (Arg-Pyr) antibodies. In mouse AKR-2B fibroblasts, this AGE-modified PDGF exhibited reduced signalling to AKT and ERK resulting in decreased cell proliferation. In the human osteosarcoma cell line 143B, PDGF signalling towards the AKT-kinase was decreased when using modified PDGF-AA, -AB, and -BB whereas the constitutive active ERK was not affected. Secreted proteins from collagen-activated platelets from diabetic subjects contained more CML-modified proteins compared to healthy controls. PDGF protein as a platelet protein coprecipitated in immunoprecipitation experiments with α-CML-antiserum. In summary, our data suggest that AGE-modification of PDGF contributes to reduced wound healing in diabetic patients.     

Infection-Induced Retrotransposon-Derived Noncoding RNAs Enhance Herpesviral Gene Expression via the NF-κB Pathway

Short interspersed nuclear elements (SINEs) are highly abundant, RNA polymerase III-transcribed noncoding retrotransposons that are silenced in somatic cells but activated during certain stresses including viral infection. How these induced SINE RNAs impact the host-pathogen interaction is unknown. Here we reveal that during murine gammaherpesvirus 68 (MHV68) infection, rapidly induced SINE RNAs activate the antiviral NF-κB signaling pathway through both mitochondrial antiviral-signaling protein (MAVS)-dependent and independent mechanisms. However, SINE RNA-based signaling is hijacked by the virus to enhance viral gene expression and replication. B2 RNA expression stimulates IKKβ-dependent phosphorylation of the major viral lytic cycle transactivator protein RTA, thereby enhancing its activity and increasing progeny virion production. Collectively, these findings suggest that SINE RNAs participate in the innate pathogen response mechanism, but that herpesviruses have evolved to co-opt retrotransposon activation for viral benefit.     

Seasonal variations in basal metabolic rate, lower critical temperature and responses to temporary starvation in the arctic fox (Alopex lagopus) from Svalbard

Metabolic rates of four resting, post-absorptive male adult summer- and winter-adapted captive arctic foxes (Alopex lagopus) were recorded. Basal metabolic rates (BMR) varied seasonally with a 36% increase from winter to summer, while body mass was reduced by 17% in the same period. The lower critical temperature (T _1c) of the winter-adapted arctic fox was estimated to −7°C, whereas T _lc during summer was 5°C. The similarity of these values, which are much higher than hitherto assumed (e.g. Scholander et al. 1950b), is mainly due to a significantly (P<0.05) lower BMR in winter than in summer. Body core (stomach) temperature was stable, even at ambient temperatures as low as −45°C, but showed a significant (P<0.05) seasonal variation, being lower in winter (39.3±0.33°C) than in summer (39.8±0.16°C). The thermal conductivity of arctic fox fur was the same during both seasons, whereas the thermal conductance in winter was lower than in summer. This was reflected in an increase in fur thickness of 140% from summer to winter, and in a reduced metabolic response to ambient temperatures below T _lc in winter. Another four arctic foxes were exposed to three periods of forced starvation, each lasting 8 days during winter, when body mass is in decline. No significant reduction in mass specific BMR was observed during the exposure to starvation, and respiratory quotient was unchanged at 0.73±0.02 during the first 5 days, but dropped significantly (P<0.05) to 0.69±0.03 at day 7. Locomotor activity and body core (intraperitoneal) temperature was unaltered throughout the starvation period, but body mass was reduced by 18.5±2.1% during these periods. Upon re-feeding, locomotor activity was significantly (P<0.05) reduced for about 6 days. Energy intake was almost doubled, but stabilised at normal levels after 11 days. Body mass increased, but not to the level before the starvation episodes. Instead, body mass increased until it reached the reduced body mass of ad libitum fed control animals. This indicates that body mass in the arctic fox is regulated according to a seasonally changing set point.     

The stability curve of hen egg white lysozyme

The physiological stability curve--a plot of the free energy of unfolding versus temperature--is calculated for hen egg white lysozyme from a combination of extrapolated unfolding thermodynamic data from reversible conditions and isothermal titrations with guanidine hydrochloride. The shape of the curve suggests the existence of only one folded conformation.     

Clinically failed eggs as a source of normal human embryo stem cells

The promise of human embryo stem cells (hESCs) for regenerative medicine is offset by the ethical and practical challenges involved in sourcing eggs and embryos for this objective. In this study we sought to isolate an hESC line from clinically failed eggs, the usage of which would not conflict with donor interests to conceive. A total of 8 blastocysts were allocated for hESC derivation from a pool of 579 eggs whose fertilization had been clinically assessed to have occurred abnormally (i.e., three pronuclei) or failed (i.e., no pronuclei) following in vitro insemination or intracytoplasmic sperm injection (ICSI). The latter were subjected to a recovery intervention consisting of either reinsemination by ICSI or parthenogenetic stimulation. One hESC line (RCM1) was obtained from a failed-to-fertilize inseminated egg recovered by parthenogenetic activation. Standard in vitro and in vivo characterization revealed this line to possess all of the properties attributed to a normal euploid hESC line. Whole-genome single-nucleotide polymorphism analysis further revealed that the line was biparental, indicating that sperm penetration had occurred, although parthenogenetic stimulation was required for activation. Our results demonstrate the viability of an alternative strategy to generate normal hESC lines from clinically failed eggs, thereby further minimizing the potential to conflict with donor reproductive interest to conceive.     

From 3D to 2D: a review of the molecular imprinting of proteins

Molecular imprinting is a generic technology that allows for the introduction of sites of specific molecular affinity into otherwise homogeneous polymeric matrices. Commonly this technique has been shown to be effective when targeting small molecules of molecular weight <1500, while extending the technique to larger molecules such as proteins has proven difficult. A number of key inherent problems in protein imprinting have been identified, including permanent entrapment, poor mass transfer, denaturation, and heterogeneity in binding pocket affinity, which have been addressed using a variety of approaches. This review focuses on protein imprinting in its various forms, ranging from conventional bulk techniques to novel thin film and monolayer surface imprinting approaches.