Translational coupling to an upstream gene promotes folding of the mycobacterial plasmid pAL5000 replication protein RepB and thereby its origin binding activity

In the mycobacterial plasmid pAL5000 replication region, the replication genes repA and repB are organized in an operon. Earlier, a RepB-dependent origin binding activity was detected in Escherichia coli cells expressing the repA-repB operon. This activity was maximal when expression of the two genes was coupled (A. Basu, M. Chawla-Sarkar, S. Chakrabarti, and S. K. Das Gupta, J. Bacteriol. 184:2204-2214, 2002). In this study we have shown that translational coupling makes a significant difference in the structure and function of RepB. When repB expression was coupled to repA, the polypeptide folded into an active structure (referred to as RepB*), which possessed higher helical content than RepB expressed independently. RepB* could also be distinguished from the less active RepB on the basis of sensitivity to OmpT, an outer membrane protease of E. coli: RepB* was sensitive to the protease, whereas RepB was resistant. Similar conformational differences between RepB* and RepB could be observed when repA was replaced with an unrelated gene, malE (encoding maltose binding protein). These results show that translational coupling of repB to an upstream gene is necessary for better folding and origin binding activity. It is speculated that in coupled systems where translation machinery is passed on from the upstream to the downstream open reading frame, cotranslational folding of the polypeptide expressed from the downstream open reading frame is enhanced due to increased folding competence of translationally primed ribosomes.     

Sequential 2'-O-methylation of archaeal pre-tRNATrp nucleotides is guided by the intron-encoded but trans-acting box C/D ribonucleoprotein of pre-tRNA

Haloferax volcanii pre-tRNA(Trp) processing requires box C/D ribonucleoprotein (RNP)-guided 2'-O-methylation of nucleotides C34 and U39 followed by intron excision. Positioning of the box C/D guide RNA within the intron of this pre-tRNA led to the assumption that nucleotide methylation is guided by the cis-positioned box C/D RNPs. We have now investigated the mechanism of 2'-O-methylation for the H. volcanii pre-tRNA(Trp) in vitro by assembling methylation-competent box C/D RNPs on both the pre-tRNA and the excised intron (both linear and circular forms) using Methanocaldococcus jannaschii box C/D RNP core proteins. With both kinetic studies and single nucleotide substitutions of target and guide nucleotides, we now demonstrate that pre-tRNA methylation is guided in trans by the intron-encoded box C/D RNPs positioned in either another pre-tRNA(Trp) or in the excised intron. Methylation by in vitro assembled RNPs prefers but does not absolutely require Watson-Crick pairing between the guide and target nucleotides. We also demonstrate for the first time that methylation of two nucleotides guided by a single box C/D RNA is sequential, that is, box C'/D' RNP-guided U39 methylation first requires box C/D RNP-guided methylation of C34. Methylation of the two nucleotides of exogenous pre-tRNA(Trp) added to an H. volcanii cell extract also occurs sequentially and is also accomplished in trans using RNPs that pre-exist in the extract. Thus, this trans mechanism is analogous to eukaryal pre-rRNA 2'-O-methylation guided by intron-encoded but trans-acting box C/D small nucleolar RNPs. This trans mechanism could explain the observed accumulation of the excised H. volcanii pre-tRNA(Trp) intron in vivo. A trans mechanism would also eliminate the obligatory refolding of the pre-tRNA that would be required to carry out two cis-methylation reactions before pre-tRNA splicing.     

Morphologic spectrum of papillary carcinoma of the thyroid: role of cytology in identifying the variants

OBJECTIVE: To evaluate the efficacy of fine needle aspiration cytology (FNAC) in the diagnosis of morphologic variants of papillary carcinoma of the thyroid (PCT) and to determine the reasons for misdiagnosis in discrepant cases on cytology. STUDY DESIGN: Fine needle aspiration smears from 158 histologically proven cases of PCT were blindly reviewed and an attempt made to subclassify them into different variants on the basis of various architectural and morphologic features. Cytohistologic correlation was performed to assess the efficacy of cytology in correctly identifying these variants. RESULTS: In cases with satisfactory aspirates, the diagnosis of papillary carcinoma was correctly made in 112 of 139 (80.5%) histologically proven cases of PCT. Subclassification was correct in 87 of 96 (90.6%) cases of classic papillary carcinoma and in 25 of 43 (58.1%) of the other variants of PCT with adequate aspirates. Cytohistologic agreement was 100% in columnar cell variant (CCV) and high grade variant (HGV). Although there was overlap in the morphologic features of tall cell variant (TCV) and Hürthle cell variant, cytology correctly identified 60% and 76.4% of these cases, respectively. The accuracy of cytology was limited in diagnosing follicular variant as only 50% of these cases could be correctly typed on cytology. Nodular fascitis-like stroma and diffuse sclerosis variants could not be diagnosed on cytology. CONCLUSION: Though FNAC is of limited value in typing the variants of PCT due to overlapping morphologic features, it can provide clues to the diagnosis in certain aggressive variants such as TCV, CCV and HGV. Early diagnosis in these cases can assist clinicians with management.     

Platelet storage under in vitro condition is associated with calcium-dependent apoptosis-like lesions and novel reorganization in platelet cytoskeleton

Platelets are cleared from circulation after a life span of 8-10 days. The molecular mechanisms underlying platelet senescence remain poorly characterized. Here we report that, progressive functional impairment in the platelets incubated in vitro in a plasma-free isotonic medium for up to 24 h at 37 degrees C is associated with release of cytochrome c from platelet mitochondria and cleavage of procaspase-9, but without evidence of caspase-3 activation. Concomitantly, there was proteolysis of survival proteins like focal adhesion kinase, Src, gelsolin, and specific cytoskeleton-associated peptides, in a manner regulated by extracellular calcium and calpain activity. Cytoskeleton played a critical role as evidenced from the association of these proteins and their degradation products, as well as procaspase-3 and the actin regulatory small GTPase, CDC42Hs, with the cytoskeleton of the stored platelets. The cytoskeletal enrichment with specific proteins was not associated with increase in the content of F-actin and was cytochalasin-resistant, thus signifying a novel mechanism of interaction of the translocating proteins with the pre-existing cytoskeleton. There was progressive exposure of phosphatidylserine on the outer leaflet of platelet membrane and specific electron microscopic changes suggestive of apoptotic lesions. Based on these observations we discuss the caspase-independent but calpain-mediated signaling events in the stored platelets resembling the features of apoptosis in the nucleated cells.     

Amblyomma americanum salivary gland homolog of nSec1 is essential for saliva protein secretion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins assemble in tight core complexes which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and distributed in distinct subcellular compartments. All vesicle transport mechanisms known to date have an essential requirement for a member of the Sec1 protein family, including the nSec1 in regulated exocytosis. A homolog of nSec1 was cloned and sequenced from the salivary glands of partially fed female ticks. Double-stranded RNA was used to specifically reduce the amount of nSec1 mRNA and protein in female adult tick salivary glands. This reduction was accompanied by a decrease in anticoagulant protein release by the glands and by abnormalities in feeding by dsRNA treated ticks. We report the efficacy of double-stranded RNA-mediated interference in "knocking down" nSec1 both in vivo and in vitro in tick salivary glands and the applicability of this technique for studying the mechanism of exocytosis in tick salivary glands.     

Regulation of HAX-1 anti-apoptotic protein by Omi/HtrA2 protease during cell death

Omi/HtrA2 is a nuclear-encoded mitochondrial serine protease that has a pro-apoptotic function in mammalian cells. Upon induction of apoptosis, Omi translocates to the cytoplasm and participates in caspase-dependent apoptosis by binding and degrading inhibitor of apoptosis proteins. Omi can also initiate caspase-independent apoptosis in a process that relies entirely on its ability to function as an active protease. To investigate the mechanism of Omi-induced apoptosis, we set out to isolate novel substrates that are cleaved by this protease. We identified HS1-associated protein X-1 (HAX-1), a mitochondrial anti-apoptotic protein, as a specific Omi interactor that is cleaved by Omi both in vitro and in vivo. HAX-1 degradation follows Omi activation in cells treated with various apoptotic stimuli. Using a specific inhibitor of Omi, HAX-1 degradation is prevented and cell death is reduced. Cleavage of HAX-1 was not observed in a cell line derived from motor neuron degeneration 2 mice that carry a mutated form of Omi that affects its proteolytic activity. Degradation of HAX-1 is an early event in the apoptotic process and occurs while Omi is still confined in the mitochondria. Our results suggest that Omi has a unique pro-apoptotic function in mitochondria that involves removal of the HAX-1 anti-apoptotic protein. This function is distinct from its ability to activate caspase-dependent apoptosis in the cytoplasm by degrading inhibitor of apoptosis proteins.     

Inducible peroxidases mediate nitration of anopheles midgut cells undergoing apoptosis in response to Plasmodium invasion

Plasmodium berghei invasion of Anopheles stephensi midgut cells causes severe damage, induces expression of nitric-oxide synthase, and leads to apoptosis. The present study indicates that invasion results in tyrosine nitration, catalyzed as a two-step reaction in which nitric-oxide synthase induction is followed by increased peroxidase activity. Ookinete invasion induced localized expression of peroxidase enzymes, which catalyzed protein nitration in vitro in the presence of nitrite and H(2)O(2). Histochemical stainings revealed that when a parasite migrates laterally and invades more than one cell, the pattern of induced peroxidase activity is similar to that observed for tyrosine nitration. In Anopheles gambiae, ookinete invasion elicited similar responses; it induced expression of 5 of the 16 peroxidase genes predicted by the genome sequence and decreased mRNA levels of one of them. One of these inducible peroxidases has a C-terminal oxidase domain homologous to the catalytic moiety of phagocyte NADPH oxidase and could provide high local levels of superoxide anion (O(2)), that when dismutated would generate the local increase in H(2)O(2) required for nitration. Chemically induced apoptosis of midgut cells also activated expression of four ookinete-induced peroxidase genes, suggesting their involvement in general apoptotic responses. The two-step nitration reaction provides a mechanism to precisely localize and circumscribe the toxic products generated by defense reactions involving nitration. The present study furthers our understanding of the biochemistry of midgut defense reactions to parasite invasion and how these may influence the efficiency of malaria transmission by anopheline mosquitoes.     

Behavioral and developmental effects of neem extracts on Clavigralla scutellaris (Hemiptera: Heteroptera: Coreidae) and its egg parasitoid, Gryon fulviventre (Hymenoptera: Scelionidae)

Extracts of neem, Azadirachta indica A. Juss, negatively affected feeding and development of Clavigralla scutellaris (Westwood), a coreid pest of pigeonpea, Cajanus cajan (L.) Millspaugh. Labial dabbing, pod wall penetration, and seed damage by fifth instars were significantly reduced on beans, Phaseolus vulgaris (L.), that had been dipped in aqueous, methanolic, or hexane extracts of neem seed kernel. When fourth instars were dipped directly into aqueous extract, developmental abnormalities of the wings occurred at all levels tested and fecundity dropped to zero at concentrations above 0.3125%. The LC50 value was 3.14% (220 ppm azadirachtin) at 8 d. The scelionid wasp Gryon fulviventre (Crawford) is an important natural enemy of Clavigralla spp.; egg mortality from this parasitoid ranged from 37 to 85% during the fall cropping season. Feeding by newly emerged wasps was dramatically reduced when honey was mixed with aqueous neem suspension, but 6-d survivorship of adults did not differ significantly from that of the control. Wasp oviposition behavior was altered slightly when coreid eggs were treated with neem: the period of antennation was significantly extended, but time for drilling, oviposition, and marking was unaffected. Neem-dipped eggs were accepted for oviposition and progeny emerged successfully from these treated eggs. Exposure of already parasitized eggs to neem did not interfere with progeny emergence, longevity, or sex ratio. Thus, neem extract and egg parasitoids seem to be compatible and promising control strategies for C. scutellaris. Our results suggest that use of neem against pod-sucking bugs will not interfere with natural control provided by G. fulviventre.