The IRREGULAR TRICHOME BRANCH loci regulate trichome elongation in Arabidopsis

The proper control of cell expansion is vital to plant development. It is responsible for shaping individual cells and, together with cell division, it plays a lead role in shaping plant organs. Much of the underlying mechanism by which plant cells expand anisotropically is not understood. We are taking a genetic approach to cell expansion by isolating mutants that affect the branching pattern of Arabidopsis trichomes. Here we report the identification of four new loci that control trichome morphogenesis. These loci were named the IRREGULAR TRICHOME BRANCH (ITB) loci because of the deleterious effects on branch position and length in the mutants. Our analysis of branch expansion in itb mutants shows that the ITB genes act as positive regulators of branch elongation, and that the branch position defects are caused by altered expansion of the trichome stalk. The itb mutations display synergistic effects in double mutant combinations with certain branch number mutations, suggesting that the ITB genes also play key roles in branch initiation. These results demonstrate that the ITB genes are key regulators of anisotropic cell expansion in trichomes.     

Targeting of proConA to the plant vacuole depends on its nine amino-acid C-terminal propeptide

Concanavalin A (ConA) is a well characterized and extensively used lectin accumulated in the protein bodies of jack bean cotyledons. ConA is synthesized as an inactive precursor proConA. The maturation of inactive proConA into biologically active ConA is a complex process including the removal of an internal glycopeptide and a C-terminal propeptide (CTPP), followed by a head-to-tail ligation of the two largest polypeptides. The cDNA encoding proConA was cloned and expressed in tobacco BY-2 cells. ProConA was slowly transported to the vacuole where its maturation into ConA was similar to that in jack bean cotyledons, apart from an incomplete final ligation. To investigate the role of the nine amino acid CTPP, a truncated form lacking the propeptide (proConADelta9) was expressed in BY-2 cells. In contrast to proConA, proConADelta9 was rapidly chased out of the endoplasmic reticulum (ER) and secreted into the culture medium. The CTPP was then fused to the C-terminal end of a secreted form of green fluorescent protein (secGFP). When expressed in tobacco BY-2 cells and leaf protoplasts, the chimaeric protein was located in the vacuole whereas secGFP was located in the culture medium and in the vacuole. Altogether, our results show we have isolated a new C-terminal vacuolar sorting determinant.     

Overexpression of torsinA in PC12 cells protects against toxicity

Childhood-onset dystonia is an autosomal dominant movement disorder associated with a three base pair (GAG) deletion mutation in the DYT1 gene. This gene encodes a novel ATP-binding protein called torsinA, which in the central nervous system is expressed exclusively in neurons. Neither the function of torsinA nor its role in the pathophysiology of DYT1 dystonia is known. In order to better understand the cellular functions of torsinA, we established PC12 cell lines overexpressing wild-type or mutant torsinA and subjected them to various conditions deleterious to cell survival. Treatment of control PC12 cells with an inhibitor of proteasomal activity, an oxidizing agent, or trophic withdrawal, resulted in cell death, whereas PC12 cells that overexpressed torsinA were significantly protected against each of these treatments. Overexpression of mutant torsinA failed to protect cells against trophic withdrawal. These results suggest that torsinA may play a protective role in neurons against a variety of cellular insults.     

Sex differences in salivary cortisol in response to acute stressors among healthy participants, in recreational or pathological gamblers, and in those with posttraumatic stress disorder

Sex differences in incidence and severity of some stress-related, neuropsychiatric disorders are often reported to favor men, suggesting that women may be more vulnerable to aberrant hypothalamic-pituitary-adrenal (HPA) axis responses to stress. In this review, we discuss several investigations that we, and others, have conducted assessing salivary cortisol as a measure of HPA function. We have examined basal cortisol among healthy men and women and also following acute exposure to stressors. Among healthy participants, men had higher basal cortisol levels than did women. In response to acute stressors, such as carbon dioxide or noise, respectively, cortisol levels were comparable between men and women or higher among women. We have also examined cortisol levels among those with problem eating, gambling, or posttraumatic stress disorder (PTSD). Women with restrained eating habits have higher basal cortisol levels than do women without restrained eating habits. Pathological gamblers have more aberrant stress response to gambling stimuli than do recreational gamblers, and these effects are more prominent among men than women. Men who have motor vehicle accident related PTSD, demonstrate more aberrant cortisol function, than do their female counterparts. Although these sex differences in cortisol seem to vary with type of stress exposure and/or pathophysiological status of the individual, other hormones may influence cortisol response. To address this, cortisol levels among boys and girls with different stress-related experiences, will be the subject of future investigation.     

Manganese(II) complexes of di-2-pyridinylmethylene-1,2-diimine di-Schiff base ligands: Structures and reactivity

Manganese(II) complexes [Mn(L)X₂] were prepared and characterized, where L is a neutral di-Schiff base ligand incorporating pyridylimine donor arms, including (1R,2R)-N,N′-bis(2-pyridylmethylidene)-1,2-diphenylethylenediimine (L¹), (1R,2R)-N,N′-bis(6-methyl-2-pyridylmethylidene)-1,2-cyclohexyldiimine (L²), or (1R,2R)-, (1S,2S)- or racemic N,N′-bis(2-pyridylmethylidene)-1,2-cyclohexyldiimine (L³), and X⁻ =  or Cl⁻. Product complexes were structurally characterized, specifically including [Mn(R,R-L¹)(NCCH₃)₃](ClO₄)₂, [Mn(R,R-L²)(OH₂)₂](ClO₄)₂ and racemic [Mn(L³)Cl₂]. The first of these complexes features a heptacoordinate ligand field in a distorted pentagonal bipyramid, and the latter two are hexacoordinate, but retain equatorially monovacant pentagonal bipyramidal structures. Complexes [Mn(L³)X₂] (X⁻ = Cl⁻, ) were reacted with the primary phosphine FcCH₂PH₂ (Fc = -C₅H₄FeC₅H₅), H₂O and ethyldiazoacetate (EDA). The first two substrates prompted reactivity at a single ligand imine bond, resulting in hydrophosphination and hydrolysis, respectively. Complexes of the derivative ligands were also structurally characterized. Evidence for EDA activation was obtained by electrospray ionization mass spectrometry, but catalytic carbene transfer was not obtained.     

Toxoplasma gondii: Flat-mounting of retina as a new tool for the observation of ocular infection in mice

Ocular toxoplasmosis is the principal cause of posterior uveitis and a leading cause of blindness. Animal models are required to improve our understanding of the pathogenesis of this disease. The method currently used for the detection of retinal cysts in animals involves the observation, under a microscope, of all the sections from infected eyes. However, this method is time-consuming and lacks sensitivity. We have developed a rapid, sensitive method for observing retinal cysts in mice infected with Toxoplasma gondii. This method involves combining the flat-mounting of retina - a compromise between macroscopic observation and global analysis of this tissue - and the use of an avirulent recombinant strain of T. gondii expressing the Escherichia coli β-galactosidase gene, visually detectable at the submacroscopic level. Single cyst unilateral infection was found in six out of 17 mice killed within 28 days of infection, whereas a bilateral infection was found in only one mouse. There was no correlation between brain cysts number and ocular infection.     

Preservation of female germ cells from ovaries of cat species

This review provides an overview on recent knowledge on female germ cell population within cat ovaries; on isolation, culture and cryopreservation of feline preantral follicles and on ovarian tissue preservation.     

Overexpression of VMAT-2 and DT-diaphorase protects substantia nigra-derived cells against aminochrome neurotoxicity

We tested the hypothesis that both VMAT-2 and DT-diaphorase are an important cellular defense against aminochrome-dependent neurotoxicity during dopamine oxidation. A cell line with VMAT-2 and DT-diaphorase over-expressed was created. The transfection of RCSN-3 cells with a bicistronic plasmid coding for VMAT-2 fused with GFP-IRES-DT-diaphorase cDNA induced a significant increase in protein expression of VMAT-2 (7-fold; P<0.001) and DT-diaphorase (9-fold; P<0.001), accompanied by a 4- and 5.5-fold significant increase in transport and enzyme activity, respectively. Studies with synaptic vesicles from rat substantia nigra revealed that VMAT-2 uptake of 3H-aminochrome 6.3 ± 0.4nmol/min/mg was similar to dopamine uptake 6.2 ± 0.3nmol/min/mg that which were dependent on ATP. Interestingly, aminochrome uptake was inhibited by 2μM lobeline but not reserpine (1 and 10μM). Incubation of cells overexpressing VMAT-2 and DT-diaphorase with 20μM aminochrome resulted in (i) a significant decrease in cell death (6-fold, P<0.001); (ii) normal ultra structure determined by transmission electron microscopy contrasting with a significant increase of autophagosome and a dramatic remodeling of the mitochondrial inner membrane in wild type cells; (iii) normal level of ATP (256 ± 11μM) contrasting with a significant decrease in wild type cells (121±11μM, P<0.001); and (iv) a significant decrease in DNA laddering (21 ± 8pixels, P<0.001) cells in comparison with wild type cells treated with 20μM aminochrome (269 ± 9). These results support our hypothesis that VMAT-2 and DT-diaphorase are an important defense system against aminochrome formed during dopamine oxidation.     

About the structural role of disulfide bridges in serum albumins: evidence from protein simulated unfolding

The role of the 17 disulfide (S-S) bridges in preserving the native conformation of human serum albumin (HSA) is investigated by performing classical molecular dynamics (MD) simulations on protein structures with intact and, respectively, reduced S-S bridges. The thermal unfolding simulations predict a clear destabilization of the protein secondary structure upon reduction of the S-S bridges as well as a significant distortion of the tertiary structure that is revealed by the changes in the protein native contacts fraction. The effect of the S-S bridges reduction on the protein compactness was tested by calculating Gibbs free energy profiles with respect to the protein gyration radius. The theoretical results obtained using the OPLS-AA and the AMBER ff03 force fields are in agreement with the available experimental data. Beyond the validation of the simulation method, the results here reported provide new insights into the mechanism of the protein reductive/oxidative unfolding/folding processes. It is predicted that in the native conformation of the protein, the thiol (-SH) groups belonging to the same reduced S-S bridge are located in potential wells that maintain them in contact. The -SH pairs can be dispatched by specific conformational transitions of the peptide chain located in the neighborhood of the cysteine residues.