The Yin and Yang of centromeric cohesion of sister chromatids: mitotic kinases meet protein phosphatase 2A

Accurate chromosome segregation during meiosis and mitosis is essential for the maintenance of genomic stability. Defects in the regulation of chromosome segregation during division predispose cells to undergo mitotic catastrophe or neoplastic transformation. Cohesin, a molecular glue holding sister chromatids together, is removed from chromosomes in a stepwise fashion during mitosis and meiosis. Cohesin at centromeres but not on chromosome arm remains intact until anaphase onset during early mitosis and the initiation of anaphase II during meiosis. Several recent studies indicate that the activity of protein phosphatase 2A is essential for maintaining the integrity of centromeric cohesin. Shugoshin, a guardian for sister chromatid segregation, may cooperate with and/or mediate PP2A function by suppressing the phosphorylation status of centromeric proteins including cohesin

Studies on translational mechanisms of RNA viruses

Ph.DDOCTOR OF PHILOSOPH

Role and Relevance of PHT1 in Brain Disposition and Pharmacokinetic of L-Histidine

PHT1 (SLC15A4) is responsible for translocating L-histidine (L-His), di/tripeptides and peptide-like drugs across biological membranes. Previous studies have indicated that PHT1 is located in brain parenchyma, however, its role and significance in brain, along with its impact on the biodistribution of substrates is unknown. In the present study, adult gender-matched Pht1-competent (wildtype) and Pht1-deficient (null) mice were used to investigate the effect of PHT1 on L-His brain disposition via in vitro slice and in vivo pharmacokinetic approaches. Initial phenotyping of the two genotypes and expression measurements of select transporters/enzymes were also performed. No significant differences were observed between genotypes in serum chemistry, body weight, viability and fertility. Polymerase chain reaction (PCR) analyses indicated that Pept2 had a compensatory up-regulation in Pht1 null mice (about 2-fold) as compared to wildtype animals, which was consistent in different brain regions and confirmed by immunoblots. The uptake of L-His was reduced in brain slices by 50% during PHT1 ablation. The L-amino acid transporters accounted for 30% of the uptake, and passive (other) pathways for 20% of the uptake. During the in vivo revealed that, when sampled 5 min after dosing, L-His values were 28–48% lower in Pht1 null mice as compared to wildtype animals, in brain parenchyma but not cerebrospinal fluid. Concentration-time profiles of the in vivo samples were then analyzed using nonlinear mixed effects modeling with NONMEM v7.3. In addition to active PHT1- mediated uptake into brain parenchyma, influx and efflux rate constants of L-His between plasma, brain parenchyma and CSF were modeled as first-order processes. Diffusion between brain parenchyma and CSF, CSF bulk flow and tissue volumes were obtained from the literature. The disposition kinetics of L-His in plasma, CSF and brain parenchyma was best described by a four-compartment model. We observed that the plasma and CSF PK profiles of L-His were comparable in WT and KO mice. However, a more rapid uptake of L-His occurred in the brain parenchyma of WT mice due to active transport by PHT1, which was modeled with a Michaelis- Menten term (Km = 39.9 μM and Vmax = 0.140 nmol/min). Our model quantitatively described the transport kinetics of PHT1-mediated uptake of L-His in brain, for the first time, under in vivo conditions. The results suggest that PHT1 may play an important role in histidine transport in brain, and resultant effects on histidine/histamine homeostasis and neuropeptide regulation. The findings also provide a valuable tool in predicting the disposition of L-His in brain and the potential of PHT1 as a drug target to treat serious CNS diseases.pharmacokinetic (PK) studies, plasma concentration-time profiles of L-His were comparable between the two genotypes after intravenous administration. Still, biodistribution studiesPHDPharmaceutical SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138485/1/xiaoxing_1.pd

Identification of Hepta- and Octo-Uridine stretches as sole signals for programmed +1 and −1 ribosomal frameshifting during translation of SARS-CoV ORF 3a variants

Programmed frameshifting is one of the translational recoding mechanisms that read the genetic code in alternative ways. This process is generally programmed by signals at defined locations in a specific mRNA. In this study, we report the identification of hepta- and octo-uridine stretches as sole signals for programmed +1 and −1 ribosomal frameshifting during translation of severe acute respiratory syndrome coronavirus (SARS-CoV) ORF 3a variants. SARS-CoV ORF 3a encodes a minor structural protein of 274 amino acids. Over the course of cloning and expression of the gene, a mixed population of clones with six, seven, eight and nine T stretches located 14 nt downstream of the initiation codon was found. In vitro and in vivo expression of clones with six, seven and eight Ts, respectively, showed the detection of the full-length 3a protein. Mutagenesis studies led to the identification of the hepta- and octo-uridine stretches as slippery sequences for efficient frameshifting. Interestingly, no stimulatory elements were found in the sequences upstream or downstream of the slippage site. When the hepta- and octo-uridine stretches were used to replace the original slippery sequence of the SARS-CoV ORF 1a and 1b, efficient frameshift events were observed. Furthermore, the efficiencies of frameshifting mediated by the hepta- and octo-uridine stretches were not affected by mutations introduced into a downstream stem–loop structure that totally abolish the frameshift event mediated by the original slippery sequence of ORF 1a and 1b. Taken together, this study identifies the hepta- and octo-uridine stretches that function as sole elements for efficient +1 and −1 ribosomal frameshift events