Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana

Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100 g per 5 × 10⁵ protoplasts), inclusion of carrier DNA (50 g), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3 and 5 to the marker gene yielded the highest levels of GUS expression.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MU methylumbelliferone - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid     

Modulation of drug-induced cytotoxicity by a bispecific monoclonal antibody that recognizes the epidermal growth factor receptor and doxorubicin

A hybrid hybridoma secreting a bispecific hybrid mAb (bsmAb), which recognizes both the epidermal growth factor receptor (EGF-R) and the drug doxorubicin, was produced by somatic hybridization of two hybridomas. The bsmAb obtained was able to retarget doxorubicin cytotoxicity in vitro specifically on EGF-R-positive cells exerting at the same time an antidotal effect on cells that did not overexpress the EGF-R. Distribution studies in mice indicate that the bsmAb selectively delivers the drug to tumour cells and modifies doxorubicin biodistribution with a statistically significant decrease of drug concentration in the intestine, which is the main target of early anthracycline toxicity. In keeping with this finding is the remarkable antidotal activity exerted by bsmAb in mice treated with doxorubiein, which is proved by retardation in loss of body weight and mortality. The effectiveness on tumour growth of the mAb followed by the administration of doxorubicin appears to be equal to that of the drug alone; however, the bsmAb exerts a remarkable antidotal activity.     

Interaction sites on phosphorylase kinase for calmodulin

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the subunit and a peptide from the subunit present in a region deleted in the isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca²⁺ binding to calmodulin in presence of peptides was measured. By this way, the peptides 542–566, 547–571, 660–677 and 597–614 have been found to bind specifically to calmodulin.Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the and subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in as well as to two regions in and . Exogenous calmodulin can bind to two regions in and in . A binding stoichiometry of 0.8mol of calmodulin/ protomer of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23nM calmodulin which is in the affinity range of calmodulin binding peptides from to calmodulin. Therefore, binding of exogenous calmodulin to activates the enzyme. A model for switching endogenous calmodulin between , and and modulation of ATP binding to as well as Mg²⁺/ADP binding to by calmodulin is presented.     

Patterns of cellular proliferation and migration in the developing tectum mesencephali of the frog Rana temporaria and the salamander Pleurodeles waltl

The development of the tectum mesencephali was studied in the frog Rana temporaria and the salamander Pleurodeles waltl by means of nuclear staining and by labeling of cells with bromodeoxyuridine (BrdU). The general spatial and temporal pattern of cell proliferation and cell migration is the same in both species, despite drastic differences in overall tectal morphology. However, the salamander species differs from the frog species by (1) a generally lower cell proliferation rate, (2) a reduction in the activity of the lateral proliferation zone, and (3) a reduction in the formation of superficial cellular layers. Because point (3) affects processes that occur late in ontogeny, our experiments provide evidence that the simple morphology of the tectum of Pleurodeles waltl, compared with the multilayered tectum of Rana, is a consequence of a paedomorphic alteration of the ancestral developmental pattern of the amphibian tectum.     

Wood distillate (pyroligneous acid) boosts nutritional traits of potato tubers

Potato is the fourth most widely consumed staple food in the world. This study investigated the effectiveness of 0.2% wood distillate (WD), a biostimulant derived from the pyrolysis of waste plant biomass, in boosting the nutritional quality of potato tubers. The results showed that application of WD significantly increased the content of soluble sugars (sucrose +56.3%; glucose +44.9%; fructose +62.2%), starch (+35.1%) and total carbohydrates (+16.8%). Antioxidants (total antioxidant power, polyphenols, flavonoids) and most mineral elements (K, Mg, Ca, Na, Fe, Zn) were not affected. A lower content of Cu (−17.8%) and P (−24.5%) was found in WD-treated potato.     

The distribution of coastal fish eDNA sequences in the Anthropocene

Aim

Coastal fishes have a fundamental role in marine ecosystem functioning and contributions to people, but face increasing threats due to climate change, habitat degradation and overexploitation. The extent to which human pressures are impacting coastal fish biodiversity in comparison with geographic and environmental factors at large spatial scale is still under scrutiny. Here, we took advantage of environmental DNA (eDNA) metabarcoding to investigate the relationship between fish biodiversity, including taxonomic and genetic components, and environmental but also socio-economic factors.

Location

Tropical, temperate and polar coastal areas.

Time period

Present day.

Major taxa studied

Marine fishes.

Methods

We analysed fish eDNA in 263 stations (samples) in 68 sites distributed across polar, temperate and tropical regions. We modelled the effect of environmental, geographic and socio-economic factors on α- and β-diversity. We then computed the partial effect of each factor on several fish biodiversity components using taxonomic molecular units (MOTU) and genetic sequences. We also investigated the relationship between fish genetic α- and β-diversity measured from our barcodes, and phylogenetic but also functional diversity.

Results

We show that fish eDNA MOTU and sequence α- and β-diversity have the strongest correlation with environmental factors on coastal ecosystems worldwide. However, our models also reveal a negative correlation between biodiversity and human dependence on marine ecosystems. In areas with high dependence, diversity of all fish, cryptobenthic fish and large fish MOTUs declined steeply. Finally, we show that a sequence diversity index, accounting for genetic distance between pairs of MOTUs, within and between communities, is a reliable proxy of phylogenetic and functional diversity.

Main conclusions

Together, our results demonstrate that short eDNA sequences can be used to assess climate and direct human impacts on marine biodiversity at large scale in the Anthropocene and can further be extended to investigate biodiversity in its phylogenetic and functional dimensions.     

Food intake and mass gain of hand-reared brown bear cubs

Five orphaned European brown bear cubs (Ursus arctos) from 3 litters were hand-reared from the ages of 1–4 months. Body mass initially ranged from 1.7 to 2.8 kg. Growth rates were monitored with reference to diet. Over a period of 3 years, 6 different feed formulas were used. The first 4 formulas were given with bottles until an average age of 133 days. Conversion to mass in the first 10 months ranged from 3.5 to 32.0 g of food per gram of body mass (or 38.1–192.6 kJ of gross energy/gram body mass), and was affected by type of diet. High fat content increased, whereas high carbohydrate content decreased the conversion rates. Formula 3, with 12.0% protein, 23.9% fat, and only 0.2% carbohydrates, simulated values found in bears' milk and produced the best growth rates. Formula 6 (bread, fruits, and meat) was used from ages 7 to 35 months, and over this period, the efficiency of gross energy conversion decreased gradually, by an eventual factor of 3.8. Hand-reared cubs ranged from 1.3 to 2.7 times heavier than 17 wild cubs measured at matching ages. Wild mass is probably limited by maternal hibernation, and by the largely herbivorous nature of the diet. © 1993 Wiley-Liss, Inc.     

A novel and high-throughput approach to assess photosynthetic thermal tolerance of kelp using chlorophyll α fluorometry

Foundation seaweed species are experiencing widespread declines and localized extinctions due to increased instability of sea surface temperature. Characterizing temperature thresholds are useful for predicting patterns of change and identifying species most vulnerable to extremes. Existing methods for characterizing seaweed thermal tolerance produce diverse metrics and are often time-consuming, making comparisons between species and techniques difficult, hindering insight into global patterns of change. Using three kelp species, we adapted a high-throughput method – previously used in terrestrial plant thermal biology – for use on kelps. This method employs temperature-dependent fluorescence (T–F₀) curves under heating or cooling regimes to determine the critical temperature (T_crit) of photosystem II (PSII), i.e., the breakpoint between slow and fast rise fluorescence response to changing temperature, enabling rapid assays of photosynthetic thermal tolerance using a standardized metric. This method enables characterization of T_crit for up to 48 samples per two-hour assay, demonstrating the capacity of T–F₀ curves for high-throughput assays of thermal tolerance. Temperature-dependent fluorescence curves and their derived metric, T_crit, may offer a timely and powerful new method for the field of phycology, enabling characterization and comparison of photosynthetic thermal tolerance of seaweeds across many populations, species, and biomes.