Response of lupins (Lupinus angustifolius L.) and peas (Pisum sativum L.) to Fe deficiency induced by low concentrations of Fe in solution or by addition of HCO3 -

Lupins appear to be more sensitive than peas to Fe deficiency. However, when grown in nutrient solutions between pH 5–6, little difference existed between them in their ability to acidify the solution or to release Fe^III reducing compounds. This experiment was aimed at determining whether differences between species which occurred when Fe deficiency was induced by withholding Fe from an acid solution, are maintained when Fe deficiency is induced by addition of HCO₃ ^-. Lupins and peas were grown in nutrient solutions at 0, 2 and 6 μM of Fe^III EDDHA and either with or without HCO₃ ^- (6 mM). Bicarbonate induced symptoms of Fe deficiency (chlorosis) in both lupins and peas, and markedly decreased the growth of shoots. Symptoms appeared sooner and were more severe in lupins than in peas. Growing plants without HCO₃ ^-, but at the lowest Fe level, decreased the growth and Fe concentration of shoots of lupins but did not induce chlorosis. Growing peas in this treatment, decreased Fe concentrations, but to a lesser extent than in lupins, and did not decrease growth. H⁺-ion extrusion and release of Fe^III reducing compounds was greater in lupins than in peas. Bicarbonate also decreased the growth of roots of lupins but increased the growth of roots of peas. Results indicate that when Fe deficiency is induced by HCO₃ ^-, then the response of lupins and peas are similar to their response in acid solution culture. Differences between species therefore could not be explained by their relative abilities to acidify or release Fe^III reducing compounds. Greater control of the distribution of Fe within the shoots, the presence of a pool of Fe within the roots, a lower threshold for Fe uptake, or a higher content of seed-Fe, may therefore be the reason for the lower sensitivity of peas than lupins to Fe deficiency.     

Transgenic proteoid roots of white lupin: a vehicle for characterizing and silencing root genes involved in adaptation to P stress

White lupin (Lupinus albus L.) has become an illuminating model for the study of plant adaptation to phosphorus (P) deficiency. It adapts to -P stress with a highly coordinated modification of root development and biochemistry resulting in short, densely clustered secondary roots called proteoid (or cluster) roots. In order to characterize genes involved in proteoid root formation and function in a homologous system, we have developed an Agrobacterium rhizogenes-based transformation system for white lupin roots that allows rapid analysis of reporter genes as well as RNA interference (RNA(i))-based gene silencing. We used this system to characterize a lupin multidrug and toxin efflux (Lupinus albus MULTIDRUG AND TOXIN EFFLUX, LaMATE) gene previously shown to have enhanced expression under -P stress. Here, we show that LaMATE had high expression in proteoid roots not only under -P, but also under -Fe, -N, -Mn and +Al stress. A portion containing the putative LaMATE promoter was fused to GUS and enhanced green fluorescence protein (EGFP) reporter genes, and a translational LaMATE::EGFP fusion was constructed under control of the LaMATE promoter. The LaMATE promoter directed P-dependent GUS and EGFP expression to proteoid roots. Confocal microscopy in white lupin and Arabidopsis point to the plasma membrane as the likely location of the LaMATE protein. LaMATE displayed homology to FRD3 in Arabidopsis, but did not complement an Arabidopsis ferric reductase defective 3 (FRD3) mutant. RNA(i)-based gene silencing was shown to effectively reduce LaMATE expression in transformed white lupin roots. LaMATE RNAi-silenced plants displayed an about 20% reduction in dry weight.     

Leaf anatomy is not correlated to CAM function in a C3+CAM hybrid species,Yucca gloriosa

Background and AimsCrassulacean acid metabolism (CAM) is often considered to be a complex trait, requiring orchestration of leaf anatomy and physiology for optimal performance. However, the observation of trait correlations is based largely on comparisons between C₃ and strong CAM species, resulting in a lack of understanding as to how such traits evolve and the level of intraspecific variability for CAM and associated traits.MethodsTo understand intraspecific variation for traits underlying CAM and how these traits might assemble over evolutionary time, we conducted detailed time course physiological screens and measured aspects of leaf anatomy in 24 genotypes of a C₃+CAM hybrid species, Yucca gloriosa (Asparagaceae). Comparisons were made to Y. gloriosa’s progenitor species, Y. filamentosa (C₃) and Y. aloifolia (CAM).Key ResultsBased on gas exchange and measurement of leaf acids, Y. gloriosa appears to use both C₃ and CAM, and varies across genotypes in the degree to which CAM can be upregulated under drought stress. While correlations between leaf anatomy and physiology exist when testing across all three Yucca species, such correlations break down at the species level in Y. gloriosa.ConclusionsThe variation in CAM upregulation in Y. gloriosa is a result of its relatively recent hybrid origin. The lack of trait correlations between anatomy and physiology within Y. gloriosa indicate that the evolution of CAM, at least initially, can proceed through a wide combination of anatomical traits, and more favourable combinations are eventually selected for in strong CAM plants.     

Response of Wolfiporia cocos to iron availability: alterations in growth, expression of cellular proteins, Fe3+-reducing activity and Fe3+-chelators production

Aims:  The main objective of this study was to evaluate the behaviour of the brown-rot fungus Wolfiporia cocos under differential iron availability.
Methods and Results:  W. cocos was grown under three differential iron conditions. Growth, catecholate and hydroxamate production, and mycelial and extracellular Fe³⁺-reducing activities were determined. Iron starvation slowed fungal growth and accelerated pH decline. Some mycelial proteins of low molecular weight were repressed under iron restriction, whereas others of high molecular weight showed positive iron regulation. Mycelial ferrireductase activity decreased as culture aged, while Fe³⁺-reducing activity of low molecular reductants constantly increased. Hydroxamates production suffered only limited iron repression, whereas catecholates production showed to be more iron repressible.
Conclusions:  W. cocos seems to possess more than one type of iron acquisition mechanism; one involving secretion of organic acids and ferrireductases and/or extracellular reductants, and another relying on secretion of catecholates and hydroxamates chelators.
Significance and Impact of the Study:  This paper is the first to report the kinetic study of brown-rot fungus grown under differential iron availability, and the information provided here contributes to address more traditional problems in protecting wood from brown decay, and also makes a contribution in the general area of the physiology of brown-rot fungi.     

Using phase resetting to predict 1:1 and 2:2 locking in two neuron networks in which firing order is not always preserved

Our goal is to understand how nearly synchronous modes arise in heterogenous networks of neurons. In heterogenous networks, instead of exact synchrony, nearly synchronous modes arise, which include both 1:1 and 2:2 phase-locked modes. Existence and stability criteria for 2:2 phase-locked modes in reciprocally coupled two neuron circuits were derived based on the open loop phase resetting curve (PRC) without the assumption of weak coupling. The PRC for each component neuron was generated using the change in synaptic conductance produced by a presynaptic action potential as the perturbation. Separate derivations were required for modes in which the firing order is preserved and for those in which it alternates. Networks composed of two model neurons coupled by reciprocal inhibition were examined to test the predictions. The parameter regimes in which both types of nearly synchronous modes are exhibited were accurately predicted both qualitatively and quantitatively provided that the synaptic time constant is short with respect to the period and that the effect of second order resetting is considered. In contrast, PRC methods based on weak coupling could not predict 2:2 modes and did not predict the 1:1 modes with the level of accuracy achieved by the strong coupling methods. The strong coupling prediction methods provide insight into what manipulations promote near-synchrony in a two neuron network and may also have predictive value for larger networks, which can also manifest changes in firing order. We also identify a novel route by which synchrony is lost in mildly heterogenous networks.     

The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana

The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.     

Wide intra-genomic G+C heterogeneity in human and chicken is mainly due to strand-symmetric directional mutation pressures: dGTP-oxidation and symmetric cytosine-deamination hypotheses

The intra-strand Parity Rule 2 of DNA (PR2) states that A=T and G=C within each strands. Useful corollaries of PR2 are G/(G+C)=A/(A+T)=0.5, G/(G+A)=C/(C+T)=G+C, G/(G+T)=C/(C+A)=G+C. Here. A, T, G, and C represent relative contents of the four nucleotide residues in a specific strand of DNA, so that A+T+G+C=1. Thus, deviations from the PR2 is a sign of strand-specific (or asymmetric) mutation and/or selection pressures. The present study delineates the symmetric and asymmetric effects of mutations on the intra-genomic heterogeneity of the G+C content in the human genome. The results of this study on the human genome are: (1) When both two- and four-codon amino acids were combined, only slight departures from the PR2 were observed in the total ranges of G+C content of the third-codon position. Thus, the G+C heterogeneity is likely to be caused by symmetric mutagenesis between the two strands. (2) The above result makes the deamination of cytosine due to double-strand breathing of DNA [Mol. Biol. Evol. 17 (2000) 1371] and/or incorporation of the oxidized guanine (8-oxo-guanine) opposite adenine during DNA replication (dGTP-oxidation hypothesis) as the most likely candidates for the major cause of the diversities of the G+C content. (3) Patterns of amino acid-specific PR2-biases detected by plotting PR2 corollaries against the G+C content of third codon position revealed that eight four-codon amino acids can be divided into three types by the second codon letter: (a) C₂-type (Ala, Pro, Ser4, and Thr), (b) G₂-type (Arg4 and Gly), and (c) T₂-type (Leu4 and Val). (4) Most of the asymmetric plot patterns of the above three classes in PR2 biases can be explained by C₂→T₂ deamination of C₂pG₃ of C₂-type to T₂pG₃ (T₂-type) in both human and chicken. This explains the existence of some preferred codons in human and chicken. However, these biases (asymmetric) hardly contribute to the overall G+C content diversity of the third codon position.     

High-density physical maps reveal that the dominant male-sterile gene Ms3 is located in a genomic region of low recombination in wheat and is not amenable to map-based cloning

In wheat it is essential to know whether a gene is located in a high or low recombination region of the genome before initiating a map-based cloning approach. The objective of this study was to explore the potential feasibility of map-based cloning of the dominant male-sterile gene Ms3 of wheat. High-density physical maps of the short arms of the group-5 chromosomes (5AS, 5BS, and 5DS) of Triticum aestivum L. were constructed by mapping 40 DNA markers on a set of 17 homozygous deletion lines. One hundred RFLP loci were mapped: 35 on 5AS, 37 on 5BS, and 28 on 5DS. A consensus physical map was colinearly aligned with a consensus genetic map of the group-5 short arms. Sixteen of the 17 markers in the consensus genetic map encompass a genetic distance of 25 cM and correspond to the distal region (FL 0.56–0.97) of the consensus physical map. Two rice probes, RG463 and RG901, previously identified to be linked to markers CDO344 and CDO749 (group-5 short arm of wheat), respectively, in the genetic map of rice chromosome 12, map between FL 0.56 and 0.63 in the consensus map. Thus at least a part of the group-5 short arm is homoeologous to a region of chromosome 12 of rice. The genetic map of chromosome arm 5AS was constructed using a population of 139 BC₁ plants derived from a cross between the euploid wheat ”Chris” carrying a dominant male-sterile gene Ms3 and a disomic substitution line in which chromosome 5A of T. aestivum cv Chinese Spring was substituted by chromosome 5A from Triticum turgidum ssp. dicoccoides. The map has a genetic length of 53.4 cM with 11 DNA markers. The initial map showed that the gene Ms3 cosegregated with three markers, WG341, BCD1130 and CDO677. High-resolution mapping using an additional 509 BC₁ plants indicated that the marker WG341 was closely linked to Ms3 at a genetic distance of 0.8 cM. The Ms3 was mapped physically in the region spanning 40% of the arm length from the centromere of 5AS. Therefore, map-based cloning of the Ms3 is not feasible, although WG341 can be used as a useful tag for the Ms3 gene for breeding purposes. Received: 12 December 2000 / Accepted: 26 January 2001     

Schizophrenia is not associated with the ERBB3 gene in a Han Chinese population sample: Results from case-control and family-based studies

ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3), encoding a receptor of neuregulin-1 (NRG1), has been considered a functional candidate gene for schizophrenia susceptibility. In order to investigate a relationship between ERBB3 gene and schizophrenia in the Chinese population, case-control and family-based studies were carried out in 470 cases matched by controls, and in 532 family trios. Our results failed to show any evidence of significant association between the ERBB3 rs2292238 polymorphism and schizophrenia.     

Minimal mutation of the cytoplasmic tail inhibits the ability of E-cadherin to activate Rac but not phosphatidylinositol 3-kinase: direct evidence of a role for cadherin-activated Rac signaling in adhesion and contact formation

Classic cadherins are adhesion-activated cell signaling receptors. In particular, homophilic cadherin ligation can directly activate Rho family GTPases and phosphatidylinositol 3-kinase (PI3-kinase), signaling molecules with the capacity to support the morphogenetic effects of these adhesion molecules during development and disease. However, the molecular basis for cadherin signaling has not been elucidated, nor is its precise contribution to cadherin function yet understood. One attractive hypothesis is that cadherin-activated signaling participates in stabilizing adhesive contacts (Yap, A. S., and Kovacs, E. M. (2003) J. Cell Biol. 160, 11-16). We now report that minimal mutation of the cadherin cytoplasmic tail to uncouple binding of p120-ctn ablated the ability of E-cadherin to activate Rac. This was accompanied by profound defects in the capacity of cells to establish stable adhesive contacts, defects that were rescued by sustained Rac signaling. These data provide direct evidence for a role of cadherin-activated Rac signaling in contact formation and adhesive stabilization. In contrast, cadherin-activated PI3-kinase signaling was not affected by loss of p120-ctn binding. The molecular requirements for E-cadherin to activate Rac signaling thus appear distinct from those that stimulate PI3-kinase, and we postulate that p120-ctn may play a central role in the E-cadherin-Rac signaling pathway.     

On the Use of Chiral Compounds for Probing the DNA Handedness: Z to B Conversion in Poly(dGm5dC) Upon Binding of Fe(phen)3 2+ and Ru(phen)3 2+

Abstract

In order to examine whether chiral metal complexes can be used to discriminate between right- and left-handed DNA conformational states we have studied the enantioselective interactions of Fe(phen)₃ ²⁺ and Ru(phen)₃ ²⁺ (phen = 1,10-phenanthroline)with poly(dGm⁵dC) under B- and Z-form conditions. With the inversion-labile Fe(phen)₃ ²⁺, enantioselectivity leads to shifts in the diastereomeric binding equilibria. This effect, known as the “Pfeiffer effect” (1–4), is monitored as a slowly emerging circular dichroism of the solution, corresponding to a net excess of the favoured enantiomer. With Ru(phen)₃ ²⁺, which is stable to intramolecular inversion, the difference in DNA-binding strengths of the enantiomers results in an excess of the less favoured enantiomer in the bulk solution. This excess is detected in the dialysate of the DNA/metal complex solution. With both complexes we find that the Δ-enantiomer is favoured when the polynucleotide adopts the B-form, as previously shown, but also when it initially adopts the Z-form conformational state.

This observation, together with evidence from UV-circular dichroism and binding data, indicates that the binding of these metal complexes induces a Z- to B-form transition in Z- form poly(dGm⁵dC). Consequently, neither of the studied chiral DNA-binders can easily be used to discriminate the DNA handedness.     

A field method of determining NH 4 + and NO 3 - uptake kinetics in intact roots: Effects of CO2 enrichment on trees and crop species

Models describing plant and ecosystem N cycles require an accurate assessment of root physiological uptake capacity for NH ₄ ⁺ and NO ₃ ^- under field conditions. Traditionally, rates of ion uptake in field-grown plants are determined by using excised root segments incubated for a short period in an assay solution containing N either as a radioactive or stable isotope tracer (e.g., ³⁶ClO₃ as a NH ₄ ⁺ analogue, ¹⁴CH₃NH₃ as an NO ₃ ^- analogue or ¹⁵NH ₄ ⁺ and ¹⁵NO ₃ ^- ). Although reliable, this method has several drawbacks. For example, in addition to radioactive safety issues, purchase and analysis of radioactive and stable isotopes is relatively expensive and can be a major limitation. More importantly, because excision effectively interrupts exchange of compounds between root and shoot (e.g., carbohydrate supply to root and N transport to shoot), the assay must be conducted quickly to avoid such complications. Here we present a novel field method for simultaneous measurements of NH ₄ ⁺ and NO ₃ ^- uptake kinetics in intact root systems. The application of this method is demonstrated using two tree species; red maple (Acer rubrum) and sugar maple (Acer saccharum) and two crop species soybean (Glycine max) and sorghum (Sorghum bicolor). Plants were grown in open-top chambers at either ambient or elevated levels of atmospheric CO₂ at two separate US national sites involved in CO₂ research. Absolute values of net uptake rates and the kinetic parameters determined by our method were found to be in agreement with the literature reports. Roots of the crop species exhibited a greater uptake capacity for both N forms relative to tree species. Elevated CO₂ did not significantly affect kinetics of N uptake in species tested except in red maple where it increased root uptake capacity, V, for NH ₄ ⁺ . The application, reliability, advantages and disadvantages of the method are discussed in detail. This revised version was published online in June 2006 with corrections to the Cover Date.     

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins. J. Cell. Biochem. 68:139–150, 1998. © 1998 Wiley-Liss, Inc.     

The chemokine receptor CCR5 is not a necessary inflammatory mediator in kainic acid-induced hippocampal injury: evidence for a compensatory effect by increased CCR2 and CCR3

Chemokines and their receptors have been strongly implicated in the inflammatory process. However, their roles in excitotoxic brain injury are largely unknown. In this study we used C-C chemokine receptor 5 (CCR5) knockout (KO) mice to investigate the role of CCR5 in neurodegeneration induced by intranasal administration of the excitotoxin kainic acid (KA). Although KA treatment resulted in an increased CCR5 mRNA level in the hippocampi of wild-type mice, a CCR5 deficiency in KO mice did not affect either the clinical and pathological changes in vivo or the neuronal susceptibilities to KA insult in vitro. KA treatment stimulated mRNA expression of the monocyte chemoattractant protein-2 (MCP-2) in both the wild-type and KO mice. KA treatment did not affect mRNA levels for the macrophage inflammatory protein-1alpha (MIP-1alpha) or the regulated upon activation normal T cells expressed and secreted protein (RANTES) in either wild-type or CCR5 KO mice. CCR2 mRNA expression was undetectable in the hippocampi of wild-type mice regardless of KA treatment. In contrast, CCR5 KO mice showed CCR2 mRNA expression that was remarkably increased after KA treatment. KA treatment did not affect CCR3 mRNA expression in the wild-type mice, whereas KO mice showed both a higher basal level of CCR3 mRNA expression as well as a strong upregulation following KA treatment. These results indicate that CCR5 is not a necessary inflammatory mediator in KA induced neurodegeneration. The roles of CCR5 in excitotoxic injury in CCR5 deficient mice are compensated by increased CCR2 and CCR3 expression, which share the common MCP-2 ligand with CCR5.     

A transient rise in intracellular Ca2+ is a precursor reaction to the zona pellucida-induced acrosome reaction in mouse sperm and is blocked by the induced acrosome reaction inhibitor 3-quinuclidinyl benzilate.

The acrosome reaction induced by the zona pellucida in mouse sperm has been shown to proceed in two stages experimentally distinguishable by the fluorescent probe chlortetracycline. Entry into the first stage of sperm bound to isolated, structurally intact zonae pellucidae is blocked by the compound 3-quinuclidinyl benzilate. In this study, we show, utilizing the fluorescent Ca2+ indicator fluo-3, that the first stage of the zona-induced acrosome reaction is characterized by an increase in intracellular Ca2+, followed by a decrease as the acrosome reaction proceeds. This calcium transient is completely suppressed by 3-quinuclidinyl benzilate. We conclude that the Ca2+ transient is induced by the zona pellucida and is required for the zona-induced acrosome reaction. Blockage of this sperm intracellular Ca2+ transient provides a mechanism for the inhibitory action of 3-quinuclidinyl benzilate on the zona-induced acrosome reaction in mouse sperm.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Mitochondrial DNA depletion and fatal infantile hepatic failure due to mutations in the mitochondrial polymerase γ (POLG) gene: a combined morphological/enzyme histochemical and immunocytochemical/biochemical and molecular genetic study

Combined morphological, immunocytochemical, biochemical and molecular genetic studies were performed on skeletal muscle, heart muscle and liver tissue of a 16‐months boy with fatal liver failure. The pathological characterization of the tissues revealed a severe depletion of mtDNA (mitochondrial DNA) that was most pronounced in liver, followed by a less severe, but still significant depletion in skeletal muscle and the heart. The primary cause of the disease was linked to compound heterozygous mutations in the polymerase γ (POLG) gene (DNA polymerase γ; A467T, K1191N). We present evidence, that compound heterozygous POLG mutations lead to tissue selective impairment of mtDNA replication and thus to a mosaic defect pattern even in the severely affected liver. A variable defect pattern was found in liver, muscle and heart tissue as revealed by biochemical, cytochemical, immunocytochemical and in situ hybridization analysis. Functionally, a severe deficiency of cytochrome‐c‐oxidase (cox) activity was seen in the liver. Although mtDNA depletion was detected in heart and skeletal muscle, there was no cox deficiency in these tissues. Depletion of mtDNA and microdissection of cox‐positive or negative areas correlated with the histological pattern in the liver. Interestingly, the mosaic pattern detected for cox‐activity and mtDNA copy number fully aligned with the immunohistologically revealed defect pattern using Pol γ, mtSSB‐ and mtTFA‐antibodies, thus substantiating the hypothesis that nuclear encoded proteins located within mitochondria become unstable and are degraded when they are not actively bound to mtDNA. Their disappearance could also aggravate the mtDNA depletion and contribute to the non‐homogenous defect pattern.     

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein,a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

Mycobacterium leprae recA harbors an in‐frame insertion sequence that encodes an intein homing endonuclease (PI‐MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG‐type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI‐MleI is distinctive from other members of the family of LAGLIDADG‐type HEases for its modular structure with functionally separable domains for DNA‐binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI‐MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active‐site residues essential for DNA target site recognition and double‐stranded DNA cleavage, we performed site‐directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild‐type PI‐MleI and its variants disclosed that the two amino acid residues, Asp¹²² (in Block C) and Asp¹⁹³ (in functional Block E), are crucial to the double‐stranded DNA endonuclease activity, whereas Asp²¹⁸ (in pseudo‐Block E) is not. However, despite the reduced catalytic activity, the PI‐MleI variants, like the wild‐type PI‐MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA‐binding affinities, but abolished the double‐stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double‐stranded DNA cleavage activity, compared with the wild‐type PI‐MleI. These results provide compelling evidence that Asp¹²² and Asp¹⁹³ in DOD motif I and II, respectively, are bona fide active‐site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.