CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28–1 mutant and to a lesser extent in a cdc7–1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.     

AGR3 in Breast Cancer: Prognostic Impact and Suitable Serum-Based Biomarker for Early Cancer Detection

Blood-based early detection of breast cancer has recently gained novel momentum, as liquid biopsy diagnostics is a fast emerging field. In this study, we aimed to identify secreted proteins which are up-regulated both in tumour tissue and serum samples of breast cancer patients compared to normal tissue and sera. Based on two independent tissue cohorts (n = 75 and n = 229) and one serum cohort (n = 80) of human breast cancer and healthy serum samples, we characterised AGR3 as a novel potential biomarker both for breast cancer prognosis and early breast cancer detection from blood. AGR3 expression in breast tumours is significantly associated with oestrogen receptor α (P<0.001) and lower tumour grade (P<0.01). Interestingly, AGR3 protein expression correlates with unfavourable outcome in low (G1) and intermediate (G2) grade breast tumours (multivariate hazard ratio: 2.186, 95% CI: 1.008-4.740, P<0.05) indicating an independent prognostic impact. In sera analysed by ELISA technique, AGR3 protein concentration was significantly (P<0.001) elevated in samples from breast cancer patients (n = 40, mainly low stage tumours) compared to healthy controls (n = 40). To develop a suitable biomarker panel for early breast cancer detection, we measured AGR2 protein in human serum samples in parallel. The combined AGR3/AGR2 biomarker panel achieved a sensitivity of 64.5% and a specificity of 89.5% as shown by receiver operating characteristic (ROC) curve statistics. Thus our data clearly show the potential usability of AGR3 and AGR2 as biomarkers for blood-based early detection of human breast cancer.     

Uric acid is a strong independent predictor of renal dysfunction in patients with rheumatoid arthritis

Introduction  

Recent evidence suggests that uric acid (UA), regardless of crystal deposition, may play a direct pathogenic role in renal disease. We have shown that UA is an independent predictor of hypertension and cardiovascular disease (CVD), and that CVD risk factors associate with renal dysfunction, in patients with rheumatoid arthritis (RA). In this study we investigated whether UA associates with renal dysfunction in patients with RA and whether such an association is independent or mediated through other comorbidities or risk factors for renal impairment.     

Strain-specific genomic regions of Ruminococcus flavefaciens FD-1 as revealed by combinatorial random-phase genome sequencing and suppressive subtractive hybridization

Two closely related strains of the Gram-positive, cellulolytic ruminal bacterium Ruminococcus flavefaciens were compared at the genomic level by suppressive subtractive hybridization. The two strains investigated in this study differ by 1.94% in their respective 16S rDNA genes. Three hundred and eighty-four PCR-amplified products were cloned and then screened for their strain identity by dot blot hybridization. Based on redundancy percentages of the clones sequenced, 9.5% of the genome of the R. flavefaciens FD-1 strain is not present in the JM1 strain. The majority of identities of individual cloned subtracted products (642 bp average length) bore no relation to deposited sequences in GenBank (42% of the subtracted library), whereas of those with putative assigned functions 7% are loosely associated with fibre-degradation, 6% with insertion elements, transposons and phage-like ORFs, 5% with cell membrane associated proteins and 3% with signal transduction. Subtracted sequences were then supplemented with the draft (2 x coverage) genome sequence of R. flavefaciens FD-1 to indicate potential regions of rearrangement within the genome, including a novel insertion sequence.     

The imbalanced supertree of flowering-plant phylogeny

Two contrasting approaches have been used to construct the overall tree of life from molecular data: one involves the analysis of single large datasets, while the other involves joining many independent smaller analyses into a supertree. A recent study uses the latter approach to produce the most complete phylogeny yet of flowering plant families.     

Determination of iduronic acid and glucuronic acid in glycosaminoglycans after stoichiometric reduction and depolymerization using high-performance liquid chromatography and ultraviolet detection

The reduction of uronic acids in glycosaminoglycans (GAGs) prior to depolymerization reactions is one way in which the uronic acid content of polysaccharides can be studied without major losses. The obtained monosaccharides can be recovered from the subsequent depolymerization with a yield better than 95%. Following reduction, depolymerization, and lyophilization, D-glucuronic acid is converted to D-Glc and L-iduronic acid to 1,6-anhydro-idose. Per-O-benzoyl derivatives of these monosaccharides can be separated and detected in nanogram amounts using reversed phase HPLC. A linear detector response was obtained for injections up to 22 nmol (4 micrograms) of Glc and 1,6-anhydro-idose and the detection limit was 5 and 7 pmol, respectively. Reduction, depolymerization, and derivatization with subsequent chromatography of various GAGs can be readily performed in the 1- to 30-micrograms range.     

Isolation and chemical study of the glycosaminoglycans from squid cornea

1. Oversulphated chondroitin sulphate (ca 93% of tissue glycosaminoglycans) with average molecular weight 72,500, chondroitin sulphate (5%) and small amounts of lowsulphated chondroitin sulphate were isolated from squid cornea. 2. The sulphation pattern of oversulphated chondroitin sulphate was delta di-4S (52%), delta di-diSD (28%), delta di-6S (9%) and delta di-OSCS (11%) and that of chondroitin sulphate 49, 1, 20 and 30% respectively. 3. All glycosaminoglycans contained neutral monosaccharides, glucose being the predominant neutral monosaccharide in oversulphated chondroitin sulphate and chondroitin sulphate and fucose in low-sulphated chondroitin sulphate. 4. Although L-iduronic acid was not detected, the digestion of oversulphated chondroitin sulphate with chondroitinases ABC and AC gave unexpected results.     

Aspergillus niger and Aspergillus carbonarius in Corinth Raisin and Wine-Producing Vineyards in Greece: Population Composition, Ochratoxin A Production and Chemical Control

Vineyard surveys of Corinth raisin cultivar carried out in the Peloponnese region of Greece during 2002 and of wine‐producing grape cultivars (Cabernet Sauvignon and Grenache Rouge) on the island of Rhodes, Greece, during 2003, demonstrated the occurrence of various Aspergillus spp. in berries of bunches at harvest. Aspergillus niger and A. carbonarius were predominantly isolated from sampled berries. Although the prevailing Aspergillus spp. isolates belonged mainly to A. niger aggregate, isolates of A. carbonarius were by far the most efficient Ochratoxin A (OTA) producers as revealed by the enzyme‐linked immunosorbent assay test. This study provides the first evidence concerning the composition of Aspergillus populations in raisin and wine‐producing vineyards and offers convincing data for their ability to produce various levels of OTA in Corinth raisins and wine‐producing grapes in Greece. Furthermore, it demonstrates that chemical applications with the fungicide Switch, especially under low to intermediate Aspergillus infection of vineyards, could both significantly reduce the occurrence of OTA‐producing Aspergillus spp. and restrict sour rot severity. In contrast, vineyard applications with the fungicides Carbendazim or Chorus were ineffective in controlling the fungus in Corinth raisin cultivar.     

Cytotoxicity and hepatoprotective attributes of methanolic extract of Rumex vesicarius L.

Background

To evaluate the hepatoprotective potential and invitro cytotoxicity studies of whole plant methanol extract of Rumex vesicarius L. Methanol extract at a dose of 100 mg/kg bw and 200 mg/kg bw were assessed for its hepatoprotective potential against CCl₄-induced hepatotoxicity by monitoring activity levels of SGOT (Serum glutamic oxaloacetic transaminase), SGPT (Serum glutamic pyruvic transaminase), ALP (Alkaline phosphatase), TP (Total protein), TB (Total bilirubin) and SOD (Superoxide dismutase), CAT (Catalase), MDA (Malondialdehyde). The cytotoxicity of the same extract on HepG2 cell lines were also assessed using MTT assay method at the concentration of 62.5, 125, 250, 500 μg/ml.

Results

Pretreatment of animals with whole plant methanol extracts of Rumex vesicarius L. significantly reduced the liver damage and the symptoms of liver injury by restoration of architecture of liver. The biochemical parameters in serum also improved in treated groups compared to the control and standard (silymarin) groups. Histopathological investigation further corroborated these biochemical observations. The cytotoxicity results indicated that the plant extract which were inhibitory to the proliferation of HepG2 cell line with IC50 value of 563.33 ± 0.8 μg/ml were not cytotoxic and appears to be safe.

Conclusions

Rumex vesicarius L. whole plant methanol extract exhibit hepatoprotective activity. However the cytotoxicity in HepG2 is inexplicable and warrants further study.