Two variable active site residues modulate response regulator phosphoryl group stability

Many signal transduction networks control their output by switching regulatory elements on or off. To synchronize biological response with environmental stimulus, switching kinetics must be faster than changes in input. Two-component regulatory systems (used for signal transduction by bacteria, archaea and eukaryotes) switch via phosphorylation or dephosphorylation of the receiver domain in response regulator proteins. Although receiver domains share conserved active site residues and similar three-dimensional structures, rates of self-catalysed dephosphorylation span a >or= 40,000-fold range in response regulators that control diverse biological processes. For example, autodephosphorylation of the chemotaxis response regulator CheY is 640-fold faster than Spo0F, which controls sporulation. Here we demonstrate that substitutions at two variable active site positions decreased CheY autodephosphorylation up to 40-fold and increased the Spo0F rate up to 110-fold. Particular amino acids had qualitatively similar effects in different response regulators. However, mutant proteins matched to other response regulators at the two key variable positions did not always exhibit similar autodephosphorylation kinetics. Therefore, unknown factors also influence absolute rates. Understanding the effects that particular active site amino acid compositions have on autodephosphorylation rate may allow manipulation of phosphoryl group stability for useful purposes, as well as prediction of signal transduction kinetics from amino acid sequence.     

Cytokinin Regulation of Flower and Pod Set in Soybeans (Glycine max(L.) Merr.)

Exogenous application of cytokinin to raceme tissues of soybean(Glycine max(L.) Merr.) has been shown to stimulate flower productionand to prevent flower abortion. The effects of these hormoneapplications have been ascertained for treated tissues, butthe effects of cytokinins on total seed yields in treated plantshave not been evaluated. Our objectives were to examine theeffects of systemic cytokinin applications on soybean yieldsusing an experimental line of soybeans, SD-87001, that has beenshown to be highly sensitive to exogenous cytokinin application.Soybeans were grown hydroponically or in pots in the greenhouse,and 6-benzylaminopurine (BA) was introduced into the xylem streamthrough a cotton wick for 2 weeks during anthesis. After theplants had matured, the number of pods, seeds per pod, and thetotal seed weight per plant were measured. In the greenhouse,application of 3.4 x 10^-7 moles of BA resulted in a 79% increasein seed yield compared with controls. Results of field trialsshowed much greater variability within treatments, with consistent,but non-significant increases in seed number and total yieldsof about 3%. Data suggest that cytokinin levels play a significantrole in determining total yield in soybeans, and that increasingcytokinin concentrations in certain environments may resultin increased total seed production. Copyright 2001 Annals ofBotany Company Glycine max, soybean, flower abortion, cytokinin, 6-benzylaminopurine, hydroponic, seed yield, wicking     

Analysis of the genomic organisation of a small chromosome of Leishmania braziliensis M2903 reveals two genes encoding GTP-binding proteins, one of which belongs to a new G-protein family and is an antigen

Leishmania braziliensis M2903 contains a highly amplified small chromosome. This work is aimed at resolving its structural organization and determining whether this unusual chromosome contains specific genes encoding proteins with important functions in disease pathology or drug resistance. Our results show that the M2903 250-kb small chromosome contains LD1 sequences and has an inverted repeat structure. The LD1 sequences and two cDNAs (cDNA2 and cDNA53) were mapped on a cosmid contig, and the two cDNAs and the corresponding genomic fragments from the small chromosome were sequenced. The gene encoding cDNA2 predicts a putative GTP-binding protein with homology to other GTP-binding proteins only in the G-1 domain region; however, four other conserved motifs can be recognized. Sequence similarity to cDNA53 is located in at least five chromosomes, and its small chromosome copy is a pseudogene. An open reading frame downstream of the cDNA53 pseudogene predicts another GTP-binding protein that belongs to a new G-protein family with an unusual conserved GTP-binding domain and a newly characterized conserved sequence motif. A portion of this GTP-binding protein gene was studied previously in L. aethiopica as a recombinant antigen that reacts with human antibodies.     

Functional response of <Emphasis Type="Italic">Pteromalus cerealellae</Emphasis> (Hymenoptera: Pteromalidae) on the cowpea weevil, <Emphasis Type="Italic">Callosbruchus maculatus</Emphasis> (Coleoptera: Bruchidae), and interaction between parasitism and cowpea varietal susceptibility

Laboratory studies were carried out to determine the functional response of the parasitoid, Pteromalus cerealellae (Boucek) to cowpea weevil, Callosobruchus maculatus (F.) hosts, and the relationship between parasitism and cowpea variety susceptibility to the weevil. The attack response of P. cerealella to weevil larvae was best described by a type III functional response using an egg limitation model. The mean attack rate (a) of the parasitoid was estimated as 1.7 and the upper limit of the response was 24 weevil larvae per individual parasitoid within a 48-h period. The results also showed that cowpea varieties resistant to weevil infestation supported fewer weevil progeny and facilitated a greater suppression of the weevil by the parasitoid. The parasitoid progeny index, a measure that compares the number of the parasitoid progeny to the number of the beetle progeny was higher in cowpea weevil resistant varieties than in susceptible varieties. Additionally, the presence of P. cerealellae resulted in reduced weight losses of cowpea caused by the weevil infestation in both susceptible and resistant varieties.     

Spontaneous cor pulmonale in laboratory beagles

Right heart failure associated with postmortem evidence of pulmonary hypertension (cor pulmonale) was observed in nearly 1% of the young beagles of a large research colony. During the past 18 years, 176 dogs with cor pulmonale were observed. Most cases occurred between September and April of each year. Nearly equal numbers of males and females were involved, and some siblings were affected. Ninety-six percent of known affected dogs died, and 85% of the deaths occurred by 5 weeks of age. Clinically, most dogs were stunted and exhibited ascites, subcutaneous edema, hypothermia, dyspnea, cyanosis, and systolic murmur. Radiography revealed cardiomegaly, and electrocardiography revealed right axis deviation and an enlarged right atrium. Postmortem evidence of cor pulmonale included subcutaneous edema, ascites, hydrothorax, mediastinal and mesenteric edema, splenomegaly, centrolobular hepatic congestion and necrosis, right ventricular hypertrophy, interstitial pneumonia, and medial hypertrophy of pulmonary arteries and arterioles. The specific cause of the disease was not determined.     

Neuronal Hyperactivity Disturbs ATP Microgradients,Impairs Microglial Motility,and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.     

The ATPase subunit 6 gene sequence predicts that RNA editing is conserved between lizard- and human-infecting Leishmania.

Here we investigate the similarities in the kinetoplastid RNA editing process between human- and lizard-infecting Leishmania species. We present the sequence of the maxicircle-encoded ATPase subunit 6 gene from L. (V.) panamensis, L. (L.) mexicana and L. (L.) donovani species of human-infecting Leishmania. These represent the first available sequences of this gene from Leishmania species other than the lizard-infecting L. tarentolae. The gene sequences are highly conserved, both over the edited and unedited parts of the gene, implying that the RNA editing process is likely to be highly conserved between Leishmania species. Indeed, the first editing domain is absolutely conserved in all three Leishmania species studied and L. tarentolae. A phylogeny based on part of the ATPase subunit 6 gene placed the lizard-infecting Leishmania within the monophyletic Leishmania genus, supporting previous data which suggest that lizard- and human-infecting Leishmania species are closely related.