Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Similarity of maize and sorghum genomes as revealed by maize RFLP probes

Summary Densely saturated genetic maps of neutral genetic markers are a prerequisite either for plant breeding programs to improve quantitative traits in crops or for evolutionary studies. cDNA and genomic clones from maize were utilized to initiate the construction of a RFLP linkage map in Sorghum bicolor. To this purpose, an F₂ population was produced from starting parental lines IS 18729 (USA) and IS 24756 (Nigeria) that were differentiated with regard to many morphological and agronomical traits. A total of 159 maize clones were hybridized to the genomic DNA of the two parents in order to detect polymorphism: 154 probes hybridized to sorghum and 58 out of these were polymorphic. In almost all of the cases hybridization patterns were similar between maize and sorghum. The analysis of the segregation of 35 polymorphic clones in an F₂ population of 149 individuals yielded five linkage groups. The three principal ones recall regions of maize chromosomes 1, 3 and 5: in general, colinearity was maintained. A possible inversion, involving a long region of maize chromosome 3, was detected. Simulations were also performed to empirically obtain a value for the lowest number of individuals of the F₂ population needed to obtain the same linkage data.Prof. E. Ottaviano, to whom this paper is dedicated, suddenly died on June 7^th, 1991     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

Methylation and acetylation of 15-hydroxyanandamide modulate its interaction with the endocannabinoid system

The biological activity of endocannabinoids like anandamide (AEA) and 2-arachidonoylglycerol (2-AG) is subjected in vivo to a “metabolic control”, exerted mainly by catabolic enzymes. AEA is inactivated by fatty acid amide hydrolase (FAAH), that is inhibited competitively by hydroxyanandamides (HAEAs) generated from AEA by lipoxygenase activity. Among these derivatives, 15-HAEA has been shown to be an effective (K_i ∼0.6 μM) FAAH inhibitor, that blocks also type-1 cannabinoid receptor (CB1R) but not other components of the “endocannabinoid system (ECS)”, like the AEA transporter (AMT) or CB2R. Here, we extended the study of the effect of 15-HAEA on the AEA synthetase (NAPE-PLD) and the AEA-binding vanilloid receptor (TRPV1), showing that 15-HAEA activates the former (up to ∼140% of controls) and inhibits the latter protein (down to ∼70%). We also show that 15-HAEA halves the synthesis of 2-AG and almost doubles the transport of this compound across the membrane. In addition, we synthesized methyl and acetyl derivatives of 15-HAEA (15-MeOAEA and 15-AcOAEA, respectively), in order to check their ability to modulate FAAH and the other ECS elements. In fact, methylation and acetylation are common biochemical reactions in the cellular environment. We show that 15-MeOAEA, unlike 15-AcOAEA, is still a powerful competitive inhibitor of FAAH (K_i ∼0.7 μM), and that both derivatives have negligible interactions with the other proteins of ECS. Therefore, 15-MeOAEA is a FAAH inhibitor more selective than 15-HAEA. Further molecular dynamics analysis gave clues to the molecular requirements for the interaction of 15-HAEA and 15-MeOAEA with FAAH.     

A kinematic and anthropometric study of the upper cervical spine and the occipital condyles

The center of rotation (COR) of the upper cervical spine (UCS) is an important biomechanical landmark that is used to determine upper neck moment, particularly when evaluating injury risk in the automotive environment. However, neither the location of the UCS CORs nor the occipital condyles (OCs), which are frequently the referenced landmark for UCS CORs, have been measured with respect to known cranial landmarks. This study determines the CORs using pure bending (+/-3.5 N m), 3D digitization, and image analysis. Landmarks digitized included the OCs, external auditory meatus (EAM), infraorbital foramen, zygion, nasion, and the foramen magnum. The centroid of each occipital condylar surface (area 301+/-29.8 mm(2); length 25.4+/-3.2 mm) was located 18.4 mm posterior, 54.4 mm medial, and 31.0 mm inferior of the EAM. The UCS CORs were distinct: On average, OC-C1 CORs (22.5 mm posterior and 22.6 mm inferior to the left EAM) were superior and more posterior of OCs; C1-C2 CORs (7.4 mm posterior and 46.7 mm inferior to the left EAM) were inferior and more anterior of OC; and OC-C2 CORs (17.0 mm posterior and 33.1 mm inferior to the left EAM) were aligned with OC. There was a statistically significant difference between the percentage of UCS rotation in C1-C2 and OC-C1; 45% of the flexion and 71% of the extension occurred in OC-C1. Details of an anatomical variant with two pairs of distinct condylar surfaces are also presented.