Assembly, secretion, and vacuolar delivery of a hybrid immunoglobulin in plants

Secretory immunoglobulin (Ig) A is a decameric Ig composed of four alpha-heavy chains, four light chains, a joining (J) chain, and a secretory component (SC). The heavy and light chains form two tetrameric Ig molecules that are joined by the J chain and associate with the SC. Expression of a secretory monoclonal antibody in tobacco (Nicotiana tabacum) has been described: this molecule (secretory IgA/G [SIgA/G]) was modified by having a hybrid heavy chain sequence consisting of IgG gamma-chain domains linked to constant region domains of an IgA alpha-chain. In tobacco, about 70% of the protein assembles to its final, decameric structure. We show here that SIgA/G assembly and secretion are slow, with only approximately 10% of the newly synthesized molecules being secreted after 24 h and the bulk probably remaining in the endoplasmic reticulum. In addition, a proportion of SIgA/G is delivered to the vacuole as at least partially assembled molecules by a process that is blocked by the membrane traffic inhibitor brefeldin A. Neither the SC nor the J chain are responsible for vacuolar delivery, because IgA/G tetramers have the same fate. The parent IgG tetrameric molecule, containing wild-type gamma-heavy chains, is instead secreted rapidly and efficiently. This strongly suggests that intracellular retention and vacuolar delivery of IgA/G is due to the alpha-domains present in the hybrid alpha/gamma-heavy chains and indicates that the plant secretory system may partially deliver to the vacuole recombinant proteins expected to be secreted.     

The impact of aerobic and anaerobic training regimes on blood pressure in normotensive and hypertensive rats: focus on redox changes

Molecular and Cellular Biochemistry - Melatonin is a crucial neurohormone synthesized in the pineal gland that influences the physiology of animals. The molecular mechanism of norepinephrine...     

Annexin A2–dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament–bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2–induced actin bundling is apparently essential for generating active exocytotic sites.     

Highly sensitive method for the determination of melatonin by normal-phase high-performance liquid chromatography with fluorometric detection

In the present study a new chromatographic method was developed to quantify melatonin in rat pineal that can be extended to other tissues. Melatonin was extracted from an acid homogenate with ethyl acetate to avoid amine interference. HPLC was performed with silica normal-phase column and fluorescence detection. This method is sensitive enough for detecting melatonin in a single pineal gland with a detection limit of 3 pg/mg tissue.     

Efficient Haploid Induction in Microspore Suspension Culture of Aesculus hippocastanum and Karyotype Analysis

Suspension culture was more efficient method for haploid production than anther culture. All analysed androgenic regenerants originating from embryogenic microspores in suspension culture of Aesculus hippocastanum L. had a haploid number of chromosomes (n=20), while 50 % of those derived from anther culture were diploids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic Diversity and Pathogenicity of Fusarium oxysporum f.sp. melonis Races from Different Areas of Italy

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis (FOM) is a devastating disease of melon worldwide. Pathogenicity tests performed with F. oxysporum isolates obtained from Italian melon‐growing areas allowed to identify thirty‐four FOM isolates and the presence of all four races. The aims of this work were to examine genetic relatedness among FOM isolates by race determination and to perform phylogenetic analyses of identified FOM races including also other formae speciales of F. oxysporum of cucurbits. Results showed that FOM race 1,2 was the most numerous with a total of eighteen isolates, while six and nine isolates were identified as race 0 and 1, respectively, and just one isolate was assigned to race 2. Phylogenetic analysis was performed by random amplified polymorphic DNA (RAPD) profiling and by translation elongation factor‐1α (TEF‐1α) sequencing. The analysis of RAPD profiles separated FOM races into two distinct clades. Clade 1, which included races 0, 1 and 1,2, was further divided into ‘subclade a’ which grouped almost all race 1,2 isolates, and into ‘subclade b’ which included race 0 and 1 isolates. Clade 2 comprised only race 2 isolates. The phylogenetic analysis based on TEF‐1α separated FOM from the other formae speciales of F. oxysporum. Also with TEF‐1α analysis, FOM races 0, 1 and 1,2 isolates grouped in one single clade clearly separated from FOM race 2 isolates which grouped closer to F. oxysporum f.sp. cucumerinum. RAPD technique was more effective than TEF‐1α in differentiating FOM race 1,2 isolates from those belonging to the closely related races 0 and 1. Both phylogenetic analyses supported the close relationship between the three different FOM races which might imply the derivation from one another and the different origin of FOM race 2.     

Gene Expression and Synthesis of Phytohemagglutinin in the Embryonic Axes of Developing Phaseolus vulgaris Seeds

Phytohemagglutinin (PHA), the major seed lectin of the common bean (Phaseolus vulgaris), is found largely in the cotyledons, but is also present in the embryonic axis. At mid-maturation, the percentage of total protein synthesis which is directed towards making PHA is 5 to 10 times greater in the cotyledons than in the axes. This lower rate of synthesis in the axes is correlated with a lower abundance of mRNA for PHA, as determined by dot blot hybridization using a cDNA clone for PHA. Manen and Pusztai (Planta 1982 155: 328-334) have claimed on the basis of immunocytochemical evidence that, in the axis, PHA is found in the cytosol although it is present in protein bodies in the cotyledons. In the cotyledons, PHA is synthesized on rough endoplasmic reticulum, and its transport to the protein bodies via the Golgi complex is associated with specific posttranslational processing steps (Vitale and Chrispeels, J Cell Biol 1984 In press). A cytosolic localization of axis PHA would be an indication of a different site of synthesis and transport pathway. The results presented here indicate that the site of synthesis of PHA and the posttranslational modifications of PHA are the same in the axes as in the cotyledons. Since in the cotyledons these modifications take place in the endoplasmic reticulum, the Golgi, and the protein bodies, it appears that the transport pathway and the site of accumulation of PHA in the axes is similar to that in the cotyledons. On the basis of our evidence, we suggest that the subcellular localization of PHA in the axes should be reexamined.     

Pathogenetic mechanisms in hereditary dysfunctions of complex I of the respiratory chain in neurological diseases

This paper covers genetic and biochemical aspects of mitochondrial bioenergetics dysfunction in hereditary neurological disorders associated with complex I defects. Three types of hereditary complex I dysfunction are dealt with: (i) homozygous mutations in the nuclear genes NDUFS1 and NDUFS4 of complex I, associated with mitochondrial encephalopathy; (ii) a recessive hereditary epileptic neurological disorder associated with enhanced proteolytic degradation of complex I; (iii) homoplasmic mutations in the ND5 and ND6 mitochondrial genes of the complex, cohexistent with mutation in the nuclear PINK1 gene in familial Parkinsonism. The genetic and biochemical data examined highlight different mechanisms by which mitochondrial bioenergetics is altered in these hereditary defects of complex I. This knowledge, besides clarifying molecular aspects of the pathogenesis of hereditary diseases, can also provide hints for understanding the involvement of complex I in sporadic neurological disorders and aging, as well as for developing therapeutical strategies.     

Effect of motor abilities on performing the Hvar folk dance cicilion in 11-year-old girls

A battery of 21 motor tests as predictor variables and evaluation of motor performance of cicilion, a Croatian folk dance from the island of Hvar as a criterion variable, were used in a sample of 101 female fifth-grade students aged 11 years. Regression analysis showed the changed group of motor variables to be a good predictor of success in performing the cicilion folk dance with multiple correlation of 0.63. The flexibility variable of astride trunk bending forwards and frequency variable of hand tapping had greatest impact on the criterion. A considerable favorable impact was also recorded for the variables for assessment of rhythmic coordination, balance, agility on ground, and repetitive strength of the trunk.