Peptide nucleic acids (PNAs) can bind to homopurine/homopyrimidine sequences of double-stranded DNA targets in a sequence-specific manner and form [PNA]2/DNA triplexes with single-stranded DNA D-loop structures at the PNA binding sites. These D-loop structures have been found to have a capacity to initiate transcription in vitro. If this strategy can be used to induce transcription of endogenous genes, it may provide a novel approach for gene therapy of many human diseases. Human [beta] globin disorders such as sickle cell anemia and beta-thalassemia are very common genetic diseases that are caused by mutations in the beta-globin gene. When gamma-globin genes are highly expressed in sickle cell patients, the presence of high levels of fetal hemoglobin (HbF, alpha2gamma2) can compensate for the defective beta-globin gene product and such patients have much improved symptoms or are free of disease. However, the gamma-globin genes are developmentally regulated and normally expressed at very low levels (>1%) in adult blood cells. We have investigated the possibility of inducing gamma-globin gene expression with PNAs. Using PNAs designed to bind to the 5' flanking region of the gamma-globin gene, induction of expression of a reporter gene construct was demonstrated both in vitro and in vivo. More importantly, PNA-mediated induction of endogenous gamma-globin gene expression was also demonstrated in K562 human erythroleukemia cells. This result suggests that induction of gamma-globin gene expression with PNAs might provide a new approach for the treatment of sickle cell disease. PNA-induced gene expression strategy also may have implications in gene therapy of other diseases such as genetic diseases, cancer and infectious diseases.

DNA damage can lead to either DNA repair with cell survival or to apoptotic cell death. Although the biochemical processes underlying DNA repair and apoptosis have been extensively studied, the mechanisms by which cells determine whether the damage will be repaired or the apoptotic pathway will be activated is largely unknown. We have studied the role of nucleotide excision repair (NER) in cisplatin DNA damage-induced apoptotic cell death using both normal human fibroblasts and NERdefective xeroderma pigmentosum (XP) XPA and XPG cells. It also showed that a functional XPC protein was required for the association of the ATM protein to genomic DNA. These results suggest that the NER process may prevent the cisplatin treatment-induced apoptosis by activating the ATM protein, and that the presence of the XPC protein is essential for recruiting the ATM protein to the DNA template.Many anticancer drugs are targeted to the genomic DNA of cancer cells to generate DNA damage and block DNA replication and/or gene transcription, resulting in cell cycle arrest and apoptotic cell death (apoptosis). However, cancer cells can avoid this DNA damage-induced cell death through several mechanisms. DNA repair is one of the most important mechanisms that prevent DNA damage-induced cell death (1). Although many studies have been done regarding DNA repair and apoptosis (2-7), the molecular mechanism that determines whether the damage will be repaired or that the damaged cells will undergo apoptosis is largely unknown. The lack of such knowledge has significantly limited our understandings of cancer cell drug resistance and hindered our abilities in the design and development of new drugs for effective cancer treatment.Nucleotide excision repair (NER) 2 is the major DNA repair pathway utilized in the repair of bulky DNA damage generated by most environmental insults and therapeutic drugs (1,8,9). The NER process is initiated by DNA damage recognition and the binding of the XPC-HR23B complex to damaged DNA (10 -14), which further recruits other NER components including XPA, TFIIH, XPG, and XPF/ERCC1 to the damaged site (12,13,15,16). The XPG protein makes a 3Ј incision, which is followed by a 5Ј incision made by the XPF/ERCC1, resulting in a singlestranded gap of 27-32 nucleotides (17). The DNA polymerases (pol ⑀ or pol ␦) fill the gap and the DNA ligase seals the gap to complete the DNA repair process. Interestingly, defects in most of the NER proteins, including XPA, XPB, XPD, XPF and XPG, lead to elevated sensitivities of the cells to many DNA damaging reagents. However, defects of XPC and XPE proteins do not cause increased sensitivity of the cells to DNA damaging treatment (18). Therefore, studying the DNA damage-mediated signaling process in these NER-defective cells will provide important insights into the mechanism of DNA repair in preventing DNA damage-induced apoptosis.DNA damage also promotes cell cycle checkpoint regulation. Both ATM and ATR proteins play important roles in DNA damage-induced cell cycle checkpoint regulation...

Puer tea is a unique Chinese fermented tea with natural flora manufactured in Yunnan Province of China. Very complex changes take place to form special quality and flavor characteristics in Puer tea due to the coordination of microbial metabolic action and natural oxidation. This paper investigates the isolation and identification of fungi responsible for the fermentation and the development of main volatile compounds of Puer tea during the fermentation process by means of GC/MS. Aldehydes and ketones in parched green tea (raw material) with low boiling‐points decreased significantly, while the amount of terpene alcohols – such as linalool and linalool oxides, methoxybenzene and derivatives, and indole – increased remarkably from the parched green tea to the Puer tea product after the manufacturing process. Degradation caused by heat and microbial growth at the piling stage likely played a key role in the generation of these compounds that contributed to the aromatic characteristics of Puer tea. It is hypothesized that the fungus Aspergillus niger plays a decisive role in the development of the volatile compounds.

The intestinal epithelial barrier plays a key protective role in the gut lumen. Bovine lactoferrin (bLF) has been reported to improve the intestinal epithelial barrier function, but its impact on tight junction (TJ) proteins has been rarely described. Human intestinal epithelial crypt cells (HIECs) were more similar to those in the human small intestine, compared with the well-established Caco-2 cells. Accordingly, both HIECs and Caco-2 cells were investigated in this study to determine the effects of bioactive protein bLF on their growth promotion and intestinal barrier function. The results showed that bLF promoted cell growth and arrested cell-cycle progression at the G2/M-phase. Moreover, bLF decreased paracellular permeability and increased alkaline phosphatase activity and transepithelial electrical resistance, strengthening barrier function. Immunofluorescence, western blot and quantitative real-time polymerase chain reaction revealed that bLF significantly increased the expression of three tight junction proteins—claudin-1, occludin, and ZO-1—at both the mRNA and protein levels, and consequently strengthened the barrier function of the two cell models. bLF in general showed higher activity in Caco-2 cells, however, HIECs also exhibited desired responses to barrier function. Therefore, bLF may be incorporated into functional foods for treatment of inflammatory bowel diseases which are caused by loss of barrier integrity.

We have shown previously that cells lacking myosin II are impaired in multicellular motility. We now extend these results by determining whether myosin contractile function is necessary for normal multicellular motility and shape control. Myosin from mutants lacking the essential (mlcE(-)) myosin light chain retains the ability to form bipolar filaments that bind actin, but shows no measurable in vitro or in vivo contractile function. The contractile function is necessary for cell shape control since mlcE(-) cells, like myosin heavy-chain null mutants (mhcA(-)), were defective in their ability to control their three-dimensional shape. When mixed with wild-type cells in chimeric aggregation streams, the mlcE(-) cells were able to move normally, unlike mhcA(-) cells which accumulated at the edges of the stream and became distorted by their interactions with wild-type cells. When mhcA(-) cells were mixed with mlcE(-) streams, the mhcA(-) cells were excluded. The normal behavior of the mlcE(-) cells in this assay suggests that myosin II, in the absence of motor function, is sufficient to allow movement in this constrained, multicellular environment. We hypothesize that myosin II is a major contributor to cortical integrity even in the absence of contractile function.