Two distinct pathways for inhibiting pds1 ubiquitination in response to DNA damage

The presence of DNA damage activates a conserved cellular response known as the DNA damage checkpoint pathway. This pathway induces a cell cycle arrest that persists until the damage is repaired. Consequently, the failure to arrest in response to DNA damage is associated with genomic instability. In budding yeast, activation of the DNA damage checkpoint pathway leads to a mitotic cell cycle arrest. Following the detection of DNA damage, the checkpoint signal is transduced via the Mec1 kinase, which in turn activates two kinases, Rad53 and Chk1 that act in parallel pathways to bring about the cell cycle arrest. The downstream target of Rad53 is unknown. The target of Chk1 is Pds1, an inhibitor of anaphase initiation whose degradation is a prerequisite for mitotic progression. Pds1 degradation is dependent on its ubiquitination by the anaphase-promoting complex/cyclosome ubiquitin ligase, acting in conjunction with the Cdc20 protein (APC/CCdc20). Previous studies showed that the Rad53 and Chk1 pathways independently lead to Pds1 stabilization but the mechanism for this was unknown. In the present study we show that both the Chk1 and the Rad53 pathways inhibit the APC/CCdc20-dependent ubiquitination of Pds1 but they affect different steps of the process: the Rad53 pathway inhibits the Pds1-Cdc20 interaction whereas Chk1-dependent phosphorylation of Pds1 inhibits the ubiquitination reaction itself. Finally, we show that once the DNA damage is repaired, Pds1 dephosphorylation is involved in the recovery from the checkpoint induced cell cycle arrest.     

Drosophila KAP interacts with the kinesin II motor subunit KLP64D to assemble chordotonal sensory cilia, but not sperm tails

BACKGROUND: Kinesin II-mediated anterograde intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia in various cell types. Kinesin associated protein (KAP) is identified as the non-motor accessory subunit of Kinesin II, but its role in the corresponding motor function is not understood. RESULTS: We show that mutations in the Drosophila KAP (DmKap) gene could eliminate the sensory cilia as well as the sound-evoked potentials of Johnston's organ (JO) neurons. Ultrastructure analysis of these mutants revealed that the ciliary axonemes are absent. Mutations in Klp64D, which codes for a Kinesin II motor subunit in Drosophila, show similar ciliary defects. All these defects are rescued by exclusive expression of DmKAP and KLP64D/KIF3A in the JO neurons of respective mutants. Furthermore, reduced copy number of the DmKap gene was found to enhance the defects of hypomorphic Klp64D alleles. Unexpectedly, however, both the DmKap and the Klp64D mutant adults produce vigorously motile sperm with normal axonemes. CONCLUSIONS: KAP plays an essential role in Kinesin II function, which is required for the axoneme growth and maintenance of the cilia in Drosophila type I sensory neurons. However, the flagellar assembly in Drosophila spermatids does not require Kinesin II and is independent of IFT.     

Pitfalls in fine needle aspiration cytology of extraadrenal paraganglioma. A report of 2 cases

BACKGROUND: Fine needle aspiration (FNA) is commonly used as an initial diagnostic tool in the evaluation of mass lesions. However, extraadrenal paragangliomas (EAPs) are not commonly seen on FNA and, when encountered, can pose a diagnostic challenge since they have a wide variety of morphologic patterns. CASES: Two intraabdominal EAPs were misdiagnosed as an anaplastic adenocarcinoma of the pancreas with giant cell features and papillary adenocarcinoma of the pancreas. The sources of the pitfalls were inaccurate radiologic localization, misinterpretation of vascular-rich tissue fragments as papillary structures, presence of acinarmicroglandular structures, marked cellular pleomorphism (including multinucleation), prominent nucleoli and some cells with "squamoid" cytoplasm. CONCLUSION: The cytologic features of EAP, although suggestive, are not specific. A high index of suspicion and knowledge of clinical information, exact anatomic location and cytologic morphology combined with appropriate ancillary studies are the key to an accurate diagnosis.     

A general model for amyloid fibril assembly based on morphological studies using atomic force microscopy

Based on atomic force microscopy analysis of the morphology of fibrillar species formed during fibrillation of alpha-synuclein, insulin, and the B1 domain of protein G, a previously described model for the assembly of amyloid fibrils of immunoglobulin light-chain variable domains is proposed as a general model for the assembly of protein fibrils. For all of the proteins studied, we observed two or three fibrillar species that vary in diameter. The smallest, protofilaments, have a uniform height, whereas the larger species, protofibrils and fibrils, have morphologies that are indicative of multiple protofilaments intertwining. In all cases, protofilaments intertwine to form protofibrils, and protofibrils intertwine to form fibrils. We propose that the hierarchical assembly model describes a general mechanism of assembly for all amyloid fibrils.     

Influence of zinc on the status of hepatic trace elements and biokinetics of 65Zn in ethanol treated rats

Whole body counting studies of 65Zn indicated that the Tb1 (the faster component) was significantly decreased while the slower component (Tb2) was increased significantly following ethanol treatment. Interestingly, following zinc treatment to ethanol treated rats, slower component (Tb2) of 65Zn came back to within normal limits while the faster component (Tb1) got significantly elevated in comparison to ethanol treatment. Percent uptake values of 65Zn were found to be increased in liver, intestine, muscle, brain and kidney, and decreased in bone under alcoholic conditions. Interestingly, the uptake values of 65Zn in all the organs except muscle were reverted back to within normal limits upon zinc supplementation to these ethanol intoxicated animals. A significant decrease in zinc contents was noticed in ethanol treated rats, which, however, were raised to normal levels upon zinc supplementation: Copper levels, on the other hand, were significantly enhanced in both ethanol fed and combined ethanol + zinc treated rats. Calcium levels were significantly decreased in both ethanol and zinc treated rats, which however were further reduced upon zinc supplementation to ethanol fed rats. However, no significant change was observed in the concentrations of sodium and potassium in any of the treatment groups. In conclusion, zinc appears to play a protective role by normalizing the turnover of 65Zn in whole body as well as in its uptake in different organs under alcoholic conditions.     

Role of biliary brush cytology in primary sclerosing cholangitis

OBJECTIVE: To assess the role of brush cytology in the routine evaluation of patients with primary sclerosing cholangitis (PSC). STUDY DESIGN: From January 1995 to June 2000, 64 brush cytology specimens were obtained from 21 patients who had at least one cytologic sample obtained during endoscopic retrograde cholangiography. All patients had a diagnosis of primary sclerosing cholangitis. Cases were classified as benign, atypical or malignant according to major cytologic criteria (nuclear contour and chromatin irregularities) and minor cytologic criteria (polarity, cellularity, nuclear enlargement, mitosis, increased nuclear/cytoplasmic ratio) used by us to diagnose biliary brush cytology. Follow-up was available in all cases. RESULTS: Diagnoses were benign (13), atypical (5) and malignant (3) on cytology. Follow-up of the 13 benign cases showed bile duct stones (2), gallbladder adenocarcinoma at cholecystectomy (1), ascending cholangitis (1) and clinically/cytologically by benign follow-up (9). Five of 13 benign cases had subsequent liver transplantation for liver failure, with explants showing changes of primary sclerosing cholangitis. Of the 3 malignant cases, 1 had carcinoma in situ on biopsy, with the explanted liver showing high grade dysplasia; the second patient had cholangiocarcinoma on explant; and the third had hepatocellular carcinoma on liver five needle aspiration. The 5 patients with atypical cytology were reclassified on review as reactive (3) and atypical not otherwise specified (2). Follow-up showed benign disease in 3 of 3 atypical cases reclassified as reactive; 2 of 2 reclassified as atypical not otherwise specified showed low grade dysplasia in the explant. CONCLUSION: The overall incidence of malignancy was low (3 of 21) in patients with PSC. Bile duct brushing is a sensitive method of detecting neoplasia in the setting of PSC when well-defined cytologic criteria are applied.     

Biokinetics of 65Zn in liver and whole body and its bio-distribution in nickel treated protein deficient rats

This study was designed to determine the effect of nickel treatment on biological half-lives of 65Zn in whole body and liver as well as on distribution of 65Zn in different organs of protein deficient rats. Nickel sulfate at a dose level of 800mg/l in drinking water was administrated to normal control as well as to protein deficient rats for 8 weeks. A significant increase was found in fast and slow components of biological half lives of 65Zn in whole body and only fast component in liver of protein deficient rats. Interestingly, slow component in whole body and fast component in liver of nickel treated protein deficient rats were not different from normal controls though they were significantly elevated in protein deficient rats. On the other hand, slow component of 65Zn was also not altered in nickel treated protein deficient rats, which however, was significantly decreased in nickel treated rats. Protein deficiency led to a marked elevation in per cent uptake of 65Zn in brain and caused significant depression in liver, kidney and intestine. However, uptake of 65Zn in brain showed a significant depression in nickel treated rats, whereas the uptake was elevated in brain in nickel treated protein deficient rats. In conclusion, protein deficient conditions seem to be playing a dominant role in context with the distribution of 65Zn in different organs when nickel is administered to protein deficient rats. However nickel alone is seen to cause adverse effect on the distribution of 65Zn.     

Retinoids assist the cellular folding of the autosomal dominant retinitis pigmentosa opsin mutant P23H

The clinically common mutant opsin P23H, associated with autosomal dominant retinitis pigmentosa, yields low levels of rhodopsin when retinal is added following induction of the protein in stably transfected HEK-293 cells. We previously showed that P23H rhodopsin levels could be increased by providing a 7-membered ring, locked analog of 11-cis-retinal during expression of P23H opsin in vivo. Here we demonstrate that the mutant opsin is effectively rescued by 9- or 11-cis-retinal, the native chromophore. When retinal was added during expression, P23H rhodopsin levels were 5-fold (9-cis) and 6-fold (11-cis) higher than when retinal was added after opsin was expressed and cells were harvested. Levels of P23H opsin were increased approximately 3.5-fold with both compounds, but wild-type protein levels were only slightly increased. Addition of retinal during induction promoted the Golgi-specific glycosylation of P23H opsin and transport of the protein to the cell surface. P23H rhodopsins containing 9- or 11-cis-retinal had blue-shifted absorption maxima and altered photo-bleaching properties compared with the corresponding wild-type proteins. Significantly, P23H rhodopsins were more thermally unstable than the wild-type proteins and more rapidly bleached by hydroxylamine in the dark. We suggest that P23H opsin is similarly unstable and that retinal binds and stabilizes the protein early in its biogenesis to promote its cellular folding and trafficking. The implications of this study for treating retinitis pigmentosa and other protein conformational disorders are discussed.