Preparation and use of recombinant protein G-gold complexes as markers in double labelling immunocytochemistry

Summary Recombinant protein G (RPG) was conjugated to colloidal gold particles and used for immunocytochemistry. In this report, the preparation of RPG—gold conjugates (RPGG) and the application of these conjugates in spot blot tests and in double immunolabelling are described. The immunolabelling was performed on ultracryosections of pig small intestine using antibodies directed against aminopeptidase N and sucrase—isomaltase. The labelling efficiency of RPGG was compared to that of protein A—gold conjugates (PAG) in different compartments of the enterocyte. Quantification showed that the labelling intensity was dependent on the size of the marker as well as on the kind of protein used for complex formation. The distributions for RPGG and PAG were respectively: for the 12nm particles, 10.3 and 6.2 particles/µm of length of microvillar membrane, 3.5 and 1.0 particles/µm² of Golgi profile and 5.9 and 2.0 particles/µm² of multivesicular body profile; and for the 6nm particles, 49.6 and 15.7 particles/µm of length of microvillar membrane, 24.4 and 5.0 particles/µm² of Golgi profile and 25.4 and 3.4. particles/µm² of multivesicular body profile. Controls showed very little non-specific gold labelling (<0.02 gold particles/µm² of section).     

Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein–DNA interactions

A p53 hot-spot mutation found frequently in human cancer is the replacement of R273 by histidine or cysteine residues resulting in p53 loss of function as a tumor suppressor. These mutants can be reactivated by the incorporation of second-site suppressor mutations. Here, we present high-resolution crystal structures of the p53 core domains of the cancer-related proteins, the rescued proteins and their complexes with DNA. The structures show that inactivation of p53 results from the incapacity of the mutated residues to form stabilizing interactions with the DNA backbone, and that reactivation is achieved through alternative interactions formed by the suppressor mutations. Detailed structural and computational analysis demonstrates that the rescued p53 complexes are not fully restored in terms of DNA structure and its interface with p53. Contrary to our previously studied wild-type (wt) p53-DNA complexes showing non-canonical Hoogsteen A/T base pairs of the DNA helix that lead to local minor-groove narrowing and enhanced electrostatic interactions with p53, the current structures display Watson–Crick base pairs associated with direct or water-mediated hydrogen bonds with p53 at the minor groove. These findings highlight the pivotal role played by R273 residues in supporting the unique geometry of the DNA target and its sequence-specific complex with p53.     

Delayed Contrast Extravasation MRI for Depicting Tumor and Non-Tumoral Tissues in Primary and Metastatic Brain Tumors

The current standard of care for newly diagnosed glioblastoma multiforme (GBM) is resection followed by radiotherapy with concomitant and adjuvant temozolomide. Recent studies suggest that nearly half of the patients with early radiological deterioration post treatment do not suffer from tumor recurrence but from pseudoprogression. Similarly, a significant number of patients with brain metastases suffer from radiation necrosis following radiation treatments. Conventional MRI is currently unable to differentiate tumor progression from treatment-induced effects. The ability to clearly differentiate tumor from non-tumoral tissues is crucial for appropriate patient management. Ten patients with primary brain tumors and 10 patients with brain metastases were scanned by delayed contrast extravasation MRI prior to surgery. Enhancement subtraction maps calculated from high resolution MR images acquired up to 75 min after contrast administration were used for obtaining stereotactic biopsies. Histological assessment was then compared with the pre-surgical calculated maps. In addition, the application of our maps for prediction of progression was studied in a small cohort of 13 newly diagnosed GBM patients undergoing standard chemoradiation and followed up to 19.7 months post therapy. The maps showed two primary enhancement populations: the slow population where contrast clearance from the tissue was slower than contrast accumulation and the fast population where clearance was faster than accumulation. Comparison with histology confirmed the fast population to consist of morphologically active tumor and the slow population to consist of non-tumoral tissues. Our maps demonstrated significant correlation with perfusion-weighted MR data acquired simultaneously, although contradicting examples were shown. Preliminary results suggest that early changes in the fast volumes may serve as a predictor for time to progression. These preliminary results suggest that our high resolution MRI-based delayed enhancement subtraction maps may be applied for clear depiction of tumor and non-tumoral tissues in patients with primary brain tumors and patients with brain metastases.     

Distribution of radiolabeled l-glutamate and d-aspartate from blood into peripheral tissues in naive rats: Significance for brain neuroprotection

Excess l-glutamate (glutamate) levels in brain interstitial and cerebrospinal fluids (ISF and CSF, respectively) are the hallmark of several neurodegenerative conditions such as stroke, traumatic brain injury or amyotrophic lateral sclerosis. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As in previous studies, we have established the role of blood glutamate levels in brain neuroprotection, we have now investigated the contribution of the peripheral organs to the homeostasis of glutamate in blood. We have administered naive rats with intravenous injections of either l-[1-¹⁴C] Glutamic acid (l-[1-¹⁴C] Glu), l-[G-³H] Glutamic acid (l-[G-³H] Glu) or d-[2,3-³H] Aspartic acid (d-[2,3-³H] Asp), a non-metabolized analog of glutamate, and have followed their distribution into peripheral organs. We have observed that the decay of the radioactivity associated with l-[1-¹⁴C] Glu and l-[G-³H] Glu was faster than that associated with glutamate non-metabolized analog, d-[2,3-³H] Asp. l-[1-¹⁴C] Glu was subjected in blood to a rapid decarboxylation with the loss of ¹⁴CO₂. The three major sequestrating organs, serving as depots for the eliminated glutamate and/or its metabolites were skeletal muscle, liver and gut, contributing together 92% or 87% of total l-[U-¹⁴C] Glu or d-[2,3-³H] Asp radioactivity capture. l-[U-¹⁴C] Glu and d-[2,3-³H] Asp showed a different organ sequestration pattern. We conclude that glutamate is rapidly eliminated from the blood into peripheral tissues, mainly in non-metabolized form. The liver plays a central role in glutamate metabolism and serves as an origin for glutamate metabolites that redistribute into skeletal muscle and gut. The findings of this study suggest now that pharmacological manipulations that reduce the liver glutamate release rate or cause a boosting of the skeletal muscle glutamate pumping rate are likely to cause brain neuroprotection.     

Identification and Molecular Characterization of Homeologous Δ9-Stearoyl Acyl Carrier Protein Desaturase 3 Genes from the Allotetraploid Peanut (Arachis hypogaea)

The stearoyl–acyl carrier protein (ACP) desaturase (SAD) is a nuclear-encoded, plastid-localized soluble desaturase that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays a key role in the determination of the properties of the majority of cellular glycerolipids. Sad genes from a variety of plant species have been cloned and characterized. However, in peanut (Arachis hypogaea), an important edible and oilseed crop, these genes have not yet been characterized. By searching peanut expressed sequence tag (EST) and parallel sequencing (454) libraries, we have identified three members of the ahSad gene family. Among them, only one gene, ahSad3, was exclusively expressed during seed development and in a manner fully corresponding to oil accumulation. Both ahSad3 homeologous genes (ahSad3A and ahSad3B) were recovered from the allotetraploid peanut, and their mRNA expression levels were characterized. The open reading frames for ahSad3A and ahSad3B are 98% identical and consist of 1,158 bp, encoding a 386-full-amino-acid protein, with one intron in the coding sequence. Comparisons of the sequences of these two homeologous genes revealed seven single-nucleotide polymorphisms and one triplet insertion in the coding region. Southern blot analysis indicated that there are only two copies of the ahSad3 gene in the peanut genome. Homeolog-specific gene expression analysis showed that both ahSad3 homeologs are expressed in developing seeds, but gene expression is significantly biased toward the B genome. Our results point to ahSad3 as a possible target gene for manipulation of fatty acid saturation in A. hypogaea.     

Global maximization of Jatropha oil production under semi‐arid conditions by balancing vegetative growth with reproductive capacity

Jatropha curcas L. is a drought tolerant crop that is globally cultivated under semi‐arid conditions as a biodiesel feedstock. Despite its great potential, however, many projects failed to reach commercially viable seed and oil yields. The aim of the study was to provide globally applicable solutions for maximization of Jatropha oil production under semi‐arid conditions. Under extremely low irrigation (10% of potential evapotranspiration; ETp), fruit production was very low and a surprisingly significant portion of the fruits delayed their maturity up to six months post‐bloom. Increasing irrigation to mid‐level (60% ETp) significantly elevated fruit production and speeded up the ripening rate, whereas further increasing irrigation to a higher level (90% ETp) decreased seed and oil yields, probably due to the increased investment in vegetative growth. Nevertheless, maximal seed and oil yields at 60% ETp remained far below targeted yields. Coupling irrigation at 60% ETp, with induction of vegetative arrest, by soil application of a commercial gibberellin synthesis inhibitor, brought forward the second bloom period by two months, reduced vegetative growth, promoted floral production and significantly enhanced reproductive capacity by more than doubling oil production. The results show that under semi‐arid conditions, commercially viable seed and oil yields of Jatropha can be achieved by carefully balancing vegetative growth with reproductive capacity through the combined application of optimal irrigation regimes and induced vegetative arrest.     

Plant-growth regulators derived from the sweetener stevioside

A series of isosteviol derivatives such as aromatic esters and amino acid amides were synthesized. Their effects on seed germination and root elongation of the crop plants, Capsicum annuum, Lens culinaris medicus, Lycopersicon esculentum, Trifollium spp. and Triticum vulgare, were studied. The derivatives could be arranged into four groups: (1) seed germination inhibitors (2) root elongation inhibitors (3) root elongation inducers (4) general inhibitors.     

Ontogenetic development of diffusional restriction to protein at the pial surface of the rat brain: an electron microscopical study

Blood–brain, blood–CSF and ventricular CSF–brain barriers to protein, are present very early in brain development. In order to determine whether the outer pial surface of the brain also restricts free penetration of macromolecules, the dorso-lateral part of the sensorimotor cortex from rats at embryonic day 12 (E12), 14, 16, and 18, the day of birth (P0), and adult rat, was studied by electron microscopical techniques. Potassium ferrocyanide, Ruthenium Red and immunogold labelling of endogenous albumin were used to investigate junctional structures and the sites of restriction to albumin diffusion. At E12, large fenestrated sinusoids were present in the pia-arachnoid and the brain surface was formed by an incomplete layer of neuroepithelial and presumptive radial glial end feet, but capillaries in the pia-arachnoid showed no fenestrations at E14 or later. From E14, we observed the progressive appearance of distinct junctional structures between the glial end feet which, to our knowledge, have not been described before. Analysis of albumin distribution from E16 to P0 suggests that the junctions may contribute to restriction of diffusion between the subarachnoid space and the brain extracellular fluid. The restriction to the penetration of protein at both the pial and the ependymal surfaces may ensure the isolation of the neural environment during a critical phase in development of the nervous system. The changes in the structure of the junctions between E12 and P0 suggests a transitional series of embryonic junctional types, which eventually give way to the mature junctions of the adult. Parallels between the embryonic glial junctions and junctions described in adult invertebrate brain, suggest some interesting parallels in junctional development in phylogeny and ontogeny.     

Is Male Cohabitation Costly for Females of the Spider Stegodyphus lineatus (Eresidae)?

Reproductive interests of females and males can vary over a number of issues, including the number of matings and the occurrence and duration of mate guarding and cohabitation. The mating system of the spider Stegodyphus lineatus (Eresidae) is characterized by an exceptional sexual conflict where males induce females to remate by committing infanticide. Females experience high costs because of this male strategy, while males benefit. During the mating season, males tend to stay with females for several days. We examined whether this male strategy of cohabitation is also disadvantageous for females of S. lineatus. A field experiment revealed that male presence in a female's nest negatively affected her body condition, whereas cohabiting males gained weight because they fed on prey caught in webs of females. As a response, females did not renew their webs when males were present. Thus, females with a cohabiting male experienced the combined cost of prey loss and loss of time available for foraging. These costs are expected to increase with every additional cohabiting male. Mated females behaved more aggressively toward males than did virgin females. This behavior may be interpreted as an adaptive response to reduce mating costs.