Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well established, but comparatively little is understood about its 3-D structure. In this analysis, orientation and distance constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. A molecular mechanics-based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models. Seven different structures that satisfy the constraints equally well were generated and compared by superposition to estimate helix positions and orientations. Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pre-tRNA that is consistent with the crosslinking results and the available phylogenetic comparisons.     

Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack

We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.     

Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack

We take advantage of a cell-free system that reconstitutes essentially a single round of transport of the VSV-encoded G protein between Golgi cisternae to identify discrete stages in the maturation of carrier vesicles. Using GTP gamma S and N-ethylmaleimide (NEM) as selective inhibitors to accumulate coated and uncoated vesicles, respectively, we find these to be successive and obligatory transport intermediates. We find that the coated and uncoated vesicles that accumulate when transport is blocked have already transferred from donor to acceptor stacks but not yet fused. Similar coated and uncoated vesicles accumulate in appropriately treated whole cells. Our studies imply that a coated bud (pit)-coated vesicle-uncoated vesicle system analogous to that responsible for receptor-mediated endocytosis carries biosynthetic protein transport across the Golgi stack. However, "Golgi"-coated buds do not contain clathrin and seem to act as bulk carriers, whereas endocytic clathrin-coated pits carry a highly selective cargo.     

Thyroid function during intermittent exposure to hypobaric hypoxia

Circulatory levels of triiodothyronine (T₃) and thyroxine (T₄) and their kinetics were studied in rabbits exposed to intermittent hypobaric hypoxia (5200 m, 395 mm Hg,PO₂ 83 mm Hg) 6 h daily for 5 weeks in a decompression chamber maintained at room temperature of 22°–24° C. Kinetics of T₃ and T₄ were studied on days 21 and 28 of hypoxic exposure. The T₃ and T₄ values were found to be significantly lower on day 8 of exposure to hypoxia compared to the pre-exposure values. The decreased levels were maintained throughout the entire period of hypoxic stress. The metabolic clearance rate, production rate, distribution space and extrathyroidal T₃ and T₄ pools were significantly decreased in animals under hypoxic stress compared to the control animals. The decline in thyroid hormone levels and their production in rabbits under hypoxic stress indicate an adaptive phenomenon under conditions of low oxygen availability.     

Isolation and Characterization of Phosphoenolpyruvate Phosphatase from Germinating Mung Beans (Vigna radiata)

A phosphoenolpyruvate (PEP) phosphatase was purified to homogeneity from germinating mung beans (Vigna radiata). It was found to be a tetrameric protein (molecular mass 240,000 daltons) made up of apparently identical subunits (subunit molecular mass 60,000 daltons). It was free from bound nucleotides. It did not show pyruvate kinase activity. The enzyme showed high specificity for PEP. Pyrophosphate and some esters (nucleoside di- and triphosphates) were hydrolyzed slowly and phosphoric acid monoesters were not hydrolyzed. The enzyme showed maximum activity at pH 8.5. At this pH, the K_m of PEP was 0.14 millimolar and the V_max was equal to 1.05 micromoles pyruvate formed per minute per milligram enzyme protein. Dialysis of the enzyme against 10 millimolar triethanolamine buffer (pH 6.5), led to loss of the catalytic activity, which was restored on addition of Mg²⁺ ions (K_m = 0.12 millimolar). Other divalent metal ions inhibited the Mg²⁺ -activated enzyme. PEP-phosphatase was inhibited by ATP and several other metabolites.     

Pitfalls in the diagnosis of Wegener''s granulomatosis on fine needle aspiration cytology

OBJECTIVE: To review the clinical and pathological findings in six suspected cases of Wegener's granulomatosis (WG) and highlight the diagnostic difficulties faced by the cytopathologist. METHODS: Retrospective review of records of the Cytopathology Department to identify patients who underwent image-guided transthoracic pulmonary fine needle aspiration cytology (FNAC) for pulmonary lesions of suspected WG and those who were subsequently confirmed to have WG. Detailed evaluation of cytomorphological features was carried out. RESULTS: A total of six cases were identified in whom the initial procedure to obtain a pathological diagnosis was transthoracic FNAC. In one case, atypical squamous cells on cytology initially suggested a diagnosis of squamous cell carcinoma while in another a diagnosis of WG was made on cytology; however, a subsequent lung biopsy revealed silicosis. CONCLUSION: Acute inflammation and necrosis are the most consistent cytopathological findings in WG. In selected cases FNAC can provide supportive pathological evidence to establish a diagnosis of WG.     

The role of GRASP55 in Golgi fragmentation and entry of cells into mitosis

GRASP55 is a Golgi-associated protein, but its function at the Golgi remains unclear. Addition of full-length GRASP55, GRASP55-specific peptides, or an anti-GRASP55 antibody inhibited Golgi fragmentation by mitotic extracts in vitro, and entry of cells into mitosis. Phospho-peptide mapping of full-length GRASP55 revealed that threonine 225 and 249 were mitotically phosphorylated. Wild-type peptides containing T225 and T249 inhibited Golgi fragmentation and entry of cells into mitosis. Mutant peptides containing T225E and T249E, in contrast, did not affect Golgi fragmentation and entry into mitosis. These findings reveal a role of GRASP55 in events leading to Golgi fragmentation and the subsequent entry of cell into mitosis. Surprisingly, however, under our experimental conditions, >85% knockdown of GRASP55 did not affect the overall organization of Golgi organization in terms of cisternal stacking and lateral connections between stacks. Based on our findings we suggest that phosphorylation of GRASP55 at T225/T249 releases a bound component, which is phosphorylated and necessary for Golgi fragmentation. Thus, GRASP55 has no role in the organization of Golgi membranes per se, but it controls their fragmentation by regulating the release of a partner, which requires a G2-specific phosphorylation at T225/T249.     

Targeting optimal introns for phylogenetic analyses in non-model taxa: experimental results in Asian pitvipers

Nuclear introns are increasingly used as phylogenetic markers. Here, we present a multidisciplinary approach towards optimal locus selection and amplification using Asian pitvipers as an example of a non‐model taxon, and raise the profile of length variant heterozygotes (LVHs) in intron loci. Taxon‐specific primers were identified using a bioinformatic approach, and also designed from existing exon primed, intron crossing (EPIC) primer amplifications. Eleven further universal EPIC primer pairs were assayed using a range of PCR optimization strategies. Taxon‐specific primers yielded the most consistent amplifications, but assaying a large number of universal EPIC primers yielded another appropriate locus for phylogenetic purposes. Modified Taq DNA polymerases such as JumpStart?Taq either significantly improved the specificity and yield of EPIC PCR amplifications (of low copy number nuclear targets), or resulted in amplifications that were not significantly worse than those derived from a generic Taq DNA polymerase. Finally, LVHs were detected in all loci that were sequenced suggesting that they are relatively common in introns. This study provides an efficient and cost effective template for the successful identification of intron markers for molecular systematics which is universally applicable to other non‐model taxon groups. © The Willi Hennig Society 2005.