G-protein beta subunit of Cochliobolus heterostrophus involved in virulence, asexual and sexual reproductive ability, and morphogenesis

Previous work established that mutations in mitogen-activated protein (MAP) kinase (CHK1) and heterotrimeric G-protein alpha (Galpha) subunit (CGA1) genes affect the development of several stages of the life cycle of the maize pathogen Cochliobolus heterostrophus. The effects of mutating a third signal transduction pathway gene, CGB1, encoding the Gbeta subunit, are reported here. CGB1 is the sole Gbeta subunit-encoding gene in the genome of this organism. cgb1 mutants are nearly wild type in vegetative growth rate; however, Cgb1 is required for appressorium formation, female fertility, conidiation, regulation of hyphal pigmentation, and wild-type virulence on maize. Young hyphae of cgb1 mutants grow in a straight path, in contrast to those of the wild type, which grow in a wavy pattern. Some of the phenotypes conferred by mutations in CGA1 are found in cgb1 mutants, suggesting that Cgb1 functions in a heterotrimeric G protein; however, there are also differences. In contrast to the deletion of CGA1, the loss of CGB1 is not lethal for ascospores, evidence that there is a Gbeta subunit-independent signaling role for Cga1 in mating. Furthermore, not all of the phenotypes conferred by mutations in the MAP kinase CHK1 gene are found in cgb1 mutants, implying that the Gbeta heterodimer is not the only conduit for signals to the MAP kinase CHK1 module. The additional phenotypes of cgb1 mutants, including severe loss of virulence on maize and of the ability to produce conidia, are consistent with CGB1 being unique in the genome. Fluorescent DNA staining showed that there is often nuclear degradation in mature hyphae of cgb1 mutants, while comparable wild-type cells have intact nuclei. These data may be genetic evidence for a novel cell death-related function of the Gbeta subunit in filamentous fungi.     

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.     

Down-regulation of human<Emphasis Type="Italic">NDR</Emphasis> gene in megakaryocytic differentiation of erythroleukemia K562 cells

To study the control of hematopoietic cell differentiation, a human negative differentiation regulator (NDR) gene was identified by the comparative analysis of differentially expressed genes in hemato-lymphoid tissues.NDR is expressed preferentially in the adult bone marrow, fetal liver and testis. Immunocytochemistry with anti-NDR antiserum showed the presence of NDR in human erythroleukemia K562 cell line and CD34⁺ cells sorted from the umbilical cord blood. When fused to the green fluorescent protein (GFP), NDR was directed to the nucleus of mouse 3T3 and K562 cells. Fusion protein with a deletion from residues 7 to 87 was detected in the cytoplasm. NDR appeared not to affect the proliferation of K562 cells when overly expressed. However, its expression was down-regulated during megakaryocytic differentiation of K562 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Down-regulation of NDR correlated well with up-regulation of megakaryocytic markers, CD41 and CD61. Overexpression of the nuclear NDR-GFP in K562 cells inhibited the expression of CD41 and CD61 in megakaryocytic differentiation. Treatment of K562 cells with GF-109203X (GFX), an antagonist of the protein kinase C (PKC), blocked NDR down-regulation, up-regulated expression of CD41/CD61 and TPA-induced megakaryocytic differentiation. These results suggest a novel function of nuclear NDR protein in regulating hematopoietic cell development.     

Unique actinomycin D binding to self-complementary d(CXYGGCCY'X'G) sequences: duplex disruption and binding to a nominally base-paired hairpin

Actinomycin D (ACTD) has been shown to bind weakly to the sequence -GGCC-, despite the presence of a GpC site. It was subsequently found, however, that d(CATGGCCATG) binds relatively well to ACTD but exhibits unusually slow association kinetics, contrary to the strong-binding -XGCY- sites. In an effort to elucidate the nature of such binding and to delineate the origin of its interesting kinetic behavior, studies have now been extended to include oligomers with the general sequence motifs of d(CXYGGCCY′X′G)₂. It was found that analogous binding characteristics are observed for these self-duplex decamers and comparative studies with progressively base-truncated oligomers from the 5′-end led to the finding that d(GGCCY′X′G) oligomers bind ACTD considerably stronger than their parent decamers and exhibit 1:1 drug/strand binding stoichiometry. Melting profiles monitored at the drug spectral region indicated additional drug binding prior to the onset of eventual complex disruptions with near identical melting temperatures for all the oligomers studied. These results are consistent with the notion that the related oligomers share a common strong binding mode of a hairpin-type, with the 3′-terminus G folding back to base-pair with the C base of GGC. A binding scheme is proposed in which the oligomers d(CXYGGCCY′X′G) exist predominantly in the duplex form and bind ACTD initially at the central GGCC weak site but subsequently disrupt to accommodate the stronger hairpin binding and thus the slow association kinetics. Such a mechanism is supported by the observation of distinct biphasic fluorescence kinetic traces in the binding of 7-amino-ACTD to these duplexes.     

Troglitazone administration limits infarct size by reduced phosphorylation of canine myocardial connexin43 proteins

Troglitazone, an antidiabetic thiazolidinedione, has been shown to have a scavenging effect on reactive oxygen species, which can modulate expression of connexin43. The study purpose was to evaluate whether troglitazone provides cardioprotection and to assess whether the cardioprotection is associated with an attenuated expression of connexin43 at the border of infarction in a canine model of acute myocardial infarction. Vehicle or troglitazone (1, 5, and 50 mg/kg; n = 14 for each group) was given intravenously 15 min before the coronary artery occlusion. Among the survivors, infarct size was significantly larger in the control than in the supplemented groups. There was a significantly lower infarct size in the high-dose group compared with that in the low-dose group (15 +/- 7% vs. 23 +/- 10% of the risk region in the low-dose group, P = 0.04). Reperfusion caused a significant elevation in superoxide anions as measured by lucigenin-derived chemiluminescence, which was significantly inhibited in animals treated with troglitazone. Connexin43 underwent dephosphorylation in response to ischemia-reperfusion measured by Western blot in control hearts at the border zone; these changes were significantly enhanced by troglitazone administration. Confocal microscopy confirmed the changes of junctional complexes. The magnitude of infarct size positively correlated with the magnitude of phosphorylated connexin43 expression assessed by Western blot analysis (r = 0.73, P < 0.0001). This result demonstrated that the cardioprotective effect of troglitazone as an antioxidant may be associated with reduced phosphorylation of myocardial connexin43 protein.     

An LKB1-interacting protein negatively regulates TNFα-induced NF-κB activation

The Peutz-Jeghers syndrome (PJS) is a hereditary disorder that predisposes an individual to benign and malignant tumors in multiple organ systems. Recently, the locus responsible for PJS was mapped genetically to the LKB1 gene, with a subsequent investigation proving that it is responsible for most cases of PJS. LKB1 encodes a nuclear serine/threonine protein kinase, and potential tumor-suppressing activity has been attributed to LKB1 kinase. However, how LKB1 exerts its tumor-suppressing function remains to be determined. In this report, we describe the identification of a putative human LKB1-interacting protein, FLIP1, using the yeast two-hybrid system. Two regions of the LKB1 sequence have been determined to be crucial for the interaction with FLIP1. FLIP1 encodes a protein of 429 amino acids with a predicted molecular weight of 47 kd. In contrast to LKB1, which is mainly nuclear, FLIP1 is a cytoplasmic protein, and its expression is ubiquitous in all human tissues examined to date. Interestingly, deletion of the 195 N- terminal amino acids allows FLIP1 to enter the nucleus, suggesting the presence of a regulatory mechanism through its N-terminus for nuclear entry. In addition, we found that ectopic expression of FLIP1 selectively blocks cytokine-induced NF-kappaB activation. The involvement of FLIP1 in the regulation of NF-kappaB activity may shed new light on the role of LKB1 in tumor suppression.     

Mutation patterns and structural correlates in human immunodeficiency virus type 1 protease following different protease inhibitor treatments

Although many human immunodeficiency virus type 1 (HIV-1)-infected persons are treated with multiple protease inhibitors in combination or in succession, mutation patterns of protease isolates from these persons have not been characterized. We collected and analyzed 2,244 subtype B HIV-1 isolates from 1,919 persons with different protease inhibitor experiences: 1,004 isolates from untreated persons, 637 isolates from persons who received one protease inhibitor, and 603 isolates from persons receiving two or more protease inhibitors. The median number of protease mutations per isolate increased from 4 in untreated persons to 12 in persons who had received four or more protease inhibitors. Mutations at 45 of the 99 amino acid positions in the protease-including 22 not previously associated with drug resistance-were significantly associated with protease inhibitor treatment. Mutations at 17 of the remaining 99 positions were polymorphic but not associated with drug treatment. Pairs and clusters of correlated (covarying) mutations were significantly more likely to occur in treated than in untreated persons: 115 versus 23 pairs and 30 versus 2 clusters, respectively. Of the 115 statistically significant pairs of covarying residues in the treated isolates, 59 were within 8 A of each other-many more than would be expected by chance. In summary, nearly one-half of HIV-1 protease positions are under selective drug pressure, including many residues not previously associated with drug resistance. Structural factors appear to be responsible for the high frequency of covariation among many of the protease residues. The presence of mutational clusters provides insight into the complex mutational patterns required for HIV-1 protease inhibitor resistance.     

KCl cotransport is an important modulator of human cervical cancer growth and invasion

Cervical cancer is a major world health problem for women, but the pathophysiology of this disease has received scant attention. Here we show that the growth and invasion of cervical cancer cells are strongly linked the expression and activity of the KCl cotransporter (KCC), an important regulator of the ionic and cellular osmotic homeostasis. Functional assays of KCl cotransport activation by osmotic swelling, staurosporine, and N-ethylmaleimide indicate that removal of the N-terminal 117 amino acids from KCC1 produces a dominant-negative loss-of-function phenotype for KCl cotransport in human cervical cancer cells. The capability for regulatory volume decrease is much attenuated in the loss-of-function KCC mutant cervical cancer cells. The loss-of-function KCC mutant cervical cancer cells exhibit inhibited cell growth accompanied by decreased activity of the cell cycle gene products retinoblastoma and cdc2 kinase. Reduced cellular invasiveness is in parallel by reduced expression of alpha v beta 3 and alpha 6 beta 4 integrins, accompanied by decreased activity of matrix metalloproteinase 2 and 9. Inhibition of tumor growth in SCID mice confirms the crucial role of KCC in promoting cervical cancer growth and invasion. Thus, blockade of KCl cotransport may be a useful therapeutic adjunctive strategy to retard or prevent cervical cancer invasion.     

Presence of an unusual GM2 derivative,taurine-conjugated GM2, in Tay-Sachs brain

Tay-Sachs disease (TSD) is a classical glycosphingolipid (GSL) storage disease. Although the genetic and biochemical bases for a massive cerebral accumulation of ganglioside GM2 in TSD have been well established, the mechanism for the neural dysfunction in TSD remains elusive. Upon analysis of GSLs from a variant B TS brain, we have detected a novel GSL that has not been previously revealed. We have isolated this GSL in pure form. Using NMR spectroscopy, mass spectrometry, and chemical synthesis, the structure of this unusual GSL was established to be a taurine-conjugated GM2 (tauro-GM2) in which the carboxyl group of N-acetylneuraminic acid was amidated by taurine. Using a rabbit anti-tauro-GM2 serum, we also detected the presence of tauro-GM2 in three other small brain samples from one variant B and two variant O TSD patients. On the other hand, tauro-GM2 was not found in three normal human brain samples. The presence of tauro-GM2 in TS brains, but not in normal brains, indicates the possible association of this unusual GM2 derivative with the pathogenesis of TSD. Our findings point to taurine conjugation as a heretofore unelucidated mechanism for TS brain to cope with water-insoluble GM2.