Assembly of Saccharomyces cerevisiae 60S ribosomal subunits: role of factors required for 27S pre-rRNA processing

The precise functions of most of the ～200 assembly factors and 79 ribosomal proteins required to construct yeast ribosomes in vivo remain largely unexplored. To better understand the roles of these proteins and the mechanisms driving ribosome biogenesis, we examined in detail one step in 60S ribosomal subunit assembly-processing of 27SA(3) pre-rRNA. Six of seven assembly factors required for this step (A(3) factors) are mutually interdependent for association with preribosomes. These A(3) factors are required to recruit Rrp17, one of three exonucleases required for this processing step. In the absence of A(3) factors, four ribosomal proteins adjacent to each other, rpL17, rpL26, rpL35, and rpL37, fail to assemble, and preribosomes are turned over by Rat1. We conclude that formation of a neighbourhood in preribosomes containing the A(3) factors establishes and maintains stability of functional preribosomes containing 27S pre-rRNAs. In the absence of these assembly factors, at least one exonuclease can switch from processing to turnover of pre-rRNA.     

The tissue microlocalisation and cellular expression of CD163, VEGF, HLA-DR, iNOS, and MRP 8/14 is correlated to clinical outcome in NSCLC

Background

We have previously investigated the microlocalisation of M1 and M2 macrophages in NSCLC. This study investigated the non-macrophage (NM) expression of proteins associated with M1 and M2 macrophages in NSCLC.

Methods

Using immunohistochemistry, CD68⁺ macrophages and proteins associated with either a cytotoxic M1 phenotype (HLA-DR, iNOS, and MRP 8/14), or a non-cytotoxic M2 phenotype (CD163 and VEGF) were identified. NM expression of the markers was analysed in the islets and stroma of surgically resected tumours from 20 patients with extended survival (ES) (median 92.7 months) and 20 patients with poor survival (PS) (median 7.7 months).

Results

The NM expression of NM-HLA-DR (p<0.001), NM-iNOS (p = 0.02) and NM-MRP 8/14 (p = 0.02) was increased in ES compared to PS patients in the tumour islets. The tumour islet expression of NM-VEGF, was decreased in ES compared to PS patients (p<0.001). There was more NM-CD163 expression (p = 0.04) but less NM-iNOS (p = 0.002) and MRP 8/14 (p = 0.01) expression in the stroma of ES patients compared with PS patients. The 5-year survival for patients with above and below median NM expression of the markers in the islets was 74.9% versus 4.7% (NM-HLA-DR p<0.001), 65.0% versus 14.6% (NM-iNOS p = 0.003), and 54.3% versus 22.2% (NM-MRP 8/14 p = 0.04), as opposed to 34.1% versus 44.4% (NM-CD163 p = 0.41) and 19.4% versus 59.0% (NM-VEGF p = 0.001).

Conclusions

Cell proteins associated with M1 and M2 macrophages are also expressed by other cell types in the tumour islets and stroma of patients with NSCLC. Their tissue and cellular microlocalisation is associated with important differences in clinical outcome.     

A machine learning based method for the prediction of secretory proteins using amino acid composition, their order and similarity-search

Most of the prediction methods for secretory proteins require the presence of a correct N-terminal end of the preprotein for correct classification. As large scale genome sequencing projects sometimes assign the 5'-end of genes incorrectly, many proteins are encoded without the correct N-terminus leading to incorrect prediction. In this study, a systematic attempt has been made to predict secretory proteins irrespective of presence or absence of N-terminal signal peptides (also known as classical and non-classical secreted proteins respectively), using machine-learning techniques; artificial neural network (ANN) and support vector machine (SVM). We trained and tested our methods on a dataset of 3321 secretory and 3654 non-secretory mammalian proteins using five-fold cross-validation technique. First, ANN-based modules have been developed for predicting secretory proteins using 33 physico-chemical properties, amino acid composition and dipeptide composition and achieved accuracies of 73.1%, 76.1% and 77.1%, respectively. Similarly, SVM-based modules using 33 physico-chemical properties, amino acid, and dipeptide composition have been able to achieve accuracies of 77.4%, 79.4% and 79.9%, respectively. In addition, BLAST and PSI-BLAST modules designed for predicting secretory proteins based on similarity search achieved 23.4% and 26.9% accuracy, respectively. Finally, we developed a hybrid-approach by integrating amino acid and dipeptide composition based SVM modules and PSI-BLAST module that increased the accuracy to 83.2%, which is significantly better than individual modules. We also achieved high sensitivity of 60.4% with low value of 5% false positive predictions using hybrid module. A web server SRTpred has been developed based on above study for predicting classical and non-classical secreted proteins from whole sequence of mammalian proteins, which is available from http://www.imtech.res.in/raghava/srtpred/.     

Methanolic extract of Cuminum cyminum inhibits ovariectomy-induced bone loss in rats

Several animal and clinical studies have shown that phytoestrogens, plant-derived estrogenic compounds, can be useful in treating postmenopausal osteoporosis. Phytoestrogens and phytoestrogen-containing plants are currently under active investigation for their role in estrogen-related disorders. The present study deals with anti-osteoporotic evaluation of phytoestrogen-rich plant Cuminum cyminum, commonly known as cumin. Adult Sprague-Dawley rats were bilaterally ovariectomized (OVX) and randomly assigned to 3 groups (10 rats/group). Additional 10 animals were sham operated. OVX and sham control groups were orally administered with vehicle while the other two OVX groups were administered 0.15 mg/kg estradiol and 1 g/kg of methanolic extract of Cuminum cyminum fruits (MCC) in two divided doses for 10 weeks. At the end of the study blood, bones and uteri of the animals were collected. Serum was evaluated for calcium, phosphorus, alkaline phosphatase and tartarate resistant acid phosphatase. Bone density, ash density, mineral content and mechanical strength of bones were evaluated. Scanning electron microscopic (SEM) analysis of bones (tibia) was performed. Results were analyzed using ANOVA and Tukeys multiple comparison test. MCC (1 g/kg, p.o.) significantly reduced urinary calcium excretion and significantly increased calcium content and mechanical strength of bones in comparison to OVX control. It showed greater bone and ash densities and improved microarchitecture of bones in SEM analysis. Unlike estradiol it did not affect body weight gain and weight of atrophic uterus in OVX animals. MCC prevented ovariectomy-induced bone loss in rats with no anabolic effect on atrophic uterus. The osteoprotective effect was comparable with estradiol.     

Interactions among Ytm1, Erb1, and Nop7 required for assembly of the Nop7-subcomplex in yeast preribosomes

In Saccharomyces cerevisiae, more than 180 assembly factors associate with preribosomes to enable folding of pre-rRNA, recruitment of ribosomal proteins, and processing of pre-rRNAs to produce mature ribosomes. To examine the molecular architecture of preribosomes and to connect this structure to functions of each assembly factor, assembly subcomplexes have been purified from preribosomal particles. The Nop7-subcomplex contains three assembly factors: Nop7, Erb1, and Ytm1, each of which is necessary for conversion of 27SA(3) pre-rRNA to 27SB(S) pre-rRNA. However, interactions among these three proteins and mechanisms of their recruitment and function in pre-rRNPs are poorly understood. Here we show that Ytm1, Erb1, and Nop7 assemble into preribosomes in an interdependent manner. We identified which domains within Ytm1, Erb1, and Nop7 are necessary for their interaction with each other and are sufficient for recruitment of each protein into preribosomes. Dominant negative effects on growth and ribosome biogenesis caused by overexpressing truncated Ytm1, Erb1, or Nop7 constructs, and recessive phenotypes of the truncated proteins revealed not only interaction domains but also other domains potentially important for each protein to function in ribosome biogenesis. Our data suggest a model for the architecture of the Nop7-subcomplex and provide potential functions of domains of each protein.     

VirulentPred: a SVM based prediction method for virulent proteins in bacterial pathogens

Background  

Prediction of bacterial virulent protein sequences has implications for identification and characterization of novel virulence-associated factors, finding novel drug/vaccine targets against proteins indispensable to pathogenicity, and understanding the complex virulence mechanism in pathogens.     

Phosphonate-linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates: synthesis, DNA-binding affinity and cytotoxicity

Pyrrolobenzodiazepine-diethylphosphonate conjugates have been designed and synthesized that link through two different types of spacers that are simple alkane chain and also a piperazine moiety side-armed with the alkane chains. These pyrrolobenzodiazepine conjugates have exhibited remarkable DNA-binding affinity and improved solubility in water, a representative compound 7d showing promising in vitro cytotoxicity.     

Remarkable DNA binding affinity and potential anticancer activity of pyrrolo[2,1-c][1,4]benzodiazepine-naphthalimide conjugates linked through piperazine side-armed alkane spacers

A series of pyrrolobenzodiazepine-naphthalimide conjugates tethered through a piperazine ring system have been designed, synthesized, and evaluated for their anticancer activity. These new conjugates exhibit very high DNA binding affinity and cytotoxic activity against a number of cell lines.     

Apolipoprotein E levels in cerebrospinal fluid and the effects of ABCA1 polymorphisms

Background

Animal studies suggest that brain apolipoprotein E (apoE) levels influence amyloid-β (Aβ) deposition and thus risk for Alzheimer's disease (AD). We have previously demonstrated that deletion of the ATP-binding cassette A1 transporter (ABCA1) in mice causes dramatic reductions in brain and cerebrospinal fluid (CSF) apoE levels and lipidation. To examine whether polymorphisms in ABCA1 affect CSF apoE levels in humans, we measured apoE in CSF taken from 168 subjects who were 43 to 91 years old and were either cognitively normal or who had mild AD. We then genotyped the subjects for ten previously identified ABCA1 single nucleotide polymorphisms (SNPs).

Results

In all subjects, the mean CSF apoE level was 9.09 μg/ml with a standard deviation of 2.70 μg/ml. Levels of apoE in CSF samples taken from the same individual two weeks apart were strongly correlated (r² = 0.93, p < 0.01). In contrast, CSF apoE levels in different individuals varied widely (coefficient of variation = 46%). CSF apoE levels did not vary according to AD status, APOE genotype, gender or race. Average apoE levels increased with age by ~0.5 μg/ml per 10 years (r² = 0.05, p = 0.003). We found no significant associations between CSF apoE levels and the ten ABCA1 SNPs we genotyped. Moreover, in a separate sample of 1225 AD cases and 1431 controls, we found no association between the ABCA1 SNP rs2230806 and AD as has been previously reported.

Conclusion

We found that CSF apoE levels vary widely between individuals, but are stable within individuals over a two-week interval. AD status, APOE genotype, gender and race do not affect CSF apoE levels, but average CSF apoE levels increase with age. Given the lack of association between CSF apoE levels and genotypes for the ABCA1 SNPs we examined, either these SNPs do not affect ABCA1 function or if they do, they do not have strong effects in the CNS. Finally, we find no evidence for an association between the ABCA1 SNP rs2230806 and AD in a large sample set.