Differential role of lipid rafts in the functions of CD4+ and CD8+ human T lymphocytes with aging

Lipid rafts are critical to the assembly of the T-cell receptor (TCR) signaling machinery. It is not known whether lipid raft properties differ in CD4+ and CD8+ T cells and whether there are age-related differences that may account in part for immune senescence. Data presented here showed that time-dependent interleukin-2 (IL-2) production was different between CD4+ and CD8+ T cells. The defect in IL-2 production by CD4+ T cells was not due to lower levels of expression of the TCR or CD28. There was a direct correlation between the activation of p56(Lck) and LAT and their association/recruitment with the lipid raft fractions of CD4+ and CD8+ T cells. p56Lck, LAT and Akt/PKB were weakly phosphorylated in lipid rafts of stimulated CD4+ T cells of elderly as compared to young donors. Lipid rafts undergo changes in their lipid composition (ganglioside M1, cholesterol) in CD4+ and CD8+ T cells of elderly individuals. This study emphasizes the differential role of lipid rafts in CD4+ and CD8+ T-cell activation in aging and suggests that the differential localization of CD28 may explain disparities in response to stimulation in human aging.     

Frequent appearance of novel protein-coding sequences by frameshift translation

Genomic duplication, followed by divergence, contributes to organismal evolution. Several mechanisms, such as exon shuffling and alternative splicing, are responsible for novel gene functions, but they generate homologous domains and do not usually lead to drastic innovation. Major novelties can potentially be introduced by frameshift mutations and this idea can explain the creation of novel proteins. Here, we employ a strategy using simulated protein sequences and identify 470 human and 108 mouse frameshift events that originate new gene segments. No obvious interspecies overlap was observed, suggesting high rates of acquisition of evolutionary events. This inference is supported by a deficiency of TpA dinucleotides in the protein-coding sequences, which decreases the occurrence of translational termination, even on the complementary strand. Increased usage of the TGA codon as the termination signal in newer genes also supports our inference. This suggests that tolerated frameshift changes are a prevalent mechanism for the rapid emergence of new genes and that protein-coding sequences can be derived from existing or ancestral exons rather than from events that result in noncoding sequences becoming exons.     

NMR solution structure of the acylphosphatase from Escherichia coli

The solution structure of Escherichia coli acylphosphatase (E. coli AcP), a small enzyme catalyzing the hydrolysis of acylphosphates, was determined by (1)H and (15)N NMR and restrained modelling calculation. In analogy with the other members of AcP family, E. coli AcP shows an alpha/beta sandwich domain composed of four antiparallel and one parallel beta-strand, assembled in a five-stranded beta-sheet facing two antiparallel alpha-helices. The pairwise RMSD values calculated for the backbone atoms of E. coli and Sulfolobus solfataricus AcP, Bovine common type AcP and Horse muscle AcP are 2.18, 5.31 and 5.12 A, respectively. No significant differences are present in the active site region and the catalytic residue side chains are consistently positioned in the structures.     

Antibacterial essential oils in malodorous cancer patients: clinical observations in 30 patients.

Malodorous necrotic ulcers in cancer patients are of major concern as it leads to social isolation and poor quality of life. Current medications and topical therapies have proven inadequate in their ability to reduce foul smell to acceptable levels. We report the positive experience we have had in using antibacterial essential oils in patients with incurable head and neck cancer and associated malodorous necrotic ulcers. All patients received a standard course of therapy with oral or systemic antibiosis. In addition, we rinsed the ulcers with an antibacterial essential oil mix (mainly based on Eucalyptus oil) twice a day. All patients experienced complete resolution of the foul smell by only the third or fourth day of therapy. As a secondary effect we saw that besides smell reduction the oils had anti-inflammatory effects on neoplastic ulcers. In some patients ulcers started to heal and achieved complete re-epithiliazation. The patients experienced great personal relief upon resolution of their malodorous conditions. Quality of life improved significantly with the resulting reintroduction of social contact with friends and relatives.     

Loss of proline-rich tyrosine kinase 2 function induces spreading and motility of epithelial prostate cells

Although prostate carcinoma is an aggressive cancer preferentially metastasizing to the bones, many prostate tumors remain localized and confined to the prostate indefinitely. Prediction of the behavior of anatomically localized and moderately differentiated prostate tumors remains difficult because of lack of prognostic markers. Cell motility is an important step in the progression of epithelial tumor toward invasive metastatic carcinomas and changes in the expression and function of adhesion molecules contribute to the acquisition of a more malignant phenotype. Proline-rich tyrosine kinase 2 (Pyk2) is implicated in regulating the organization of actin cytoskeleton, a process critical for cell migration, mitosis, and tumor metastasis. In this report, we investigated whether Pyk2 played a role in the acquisition of an aggressive phenotype in prostate cell. Data reported here demonstrate that loss of Pyk2 kinase function results in induction of cell motility and migration in EPN cells, a line of non-transformed epithelial cells derived from human normal prostate tissue. Changes in motility and migration of prostate cells were associated with changes in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. Ablation of Pyk2 kinase activity caused a dramatic decrease of the expression of E-cadherin and IRS1 and an increase of the expression of alpha5-integrin. In addition, a massive reorganization of actin cytoskeleton was observed. Our data indicate that Pyk2 plays a central role in the mechanism that regulate cell-cell and cell-substrate interaction and lack of its kinase activity induces prostate cells to acquire a malignant, migrating phenotype.     

Immunohistochemical evaluation of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus

Control of the G1/S-phase transition as well as angiogenic switch are two of the most studied mechanisms in cancer. The current study examined the correlation between the immunohistochemical expression of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus (BE). Overall, p53 showed a much higher expression in BE patients (up to 50%) than in controls (1-10%) (P < 0.005). Also p21 showed a downregulation in BE when compared to normal esophagus (70% of cells vs. 65%), but the difference did not show any statistical significance (P = 0.45). pRb2/p130 was detected in 80% of cells in normal controls, but showed positive in only 20% of cells in BE biopsies. Additionally, Rb2/p130 expression was inversely correlated to that of VEGF, EZH2, and PCNA (P < 0.0001, P = 0.0032, P < 0.001, respectively). p27 stained more intensely and in a widespread manner (70%) cells in normal esophageal tissues but about only 30% in BE samples (P < 0.001). Lastly, in accordance with other reports, we also found p16 expressed by immunohistochemistry at high levels in normal controls and at low levels in BE (P < 0.001). In conclusion, p16, p21, p27, and p53 staining confirmed previously published data. Interestingly, pRb2/p130 expression was found significantly decreased in metaplastic epithelium compared to normal controls and showed significant inverse correlation with the expression of other markers, such as VEGF, EZH2, and PCNA. These data, taken together, indicate that these molecular events occurring in Barrett's metaplasia (BM) may represent one of the many steps taking place during esophageal malignant progression such as impairment of cell-cycle control, altered differentiation, and unbalanced angiogenesis.     

Selective Cytochrome c Displacement by Phosphate and Ca2+ in Brain Mitochondria

In brain mitochondria, phosphate- and Ca²⁺-dependent cytocrome c (cyt c) release reveals pools that interact differently with the inner membrane. Detachment of the phosphate-dependent pool did not influence the pool released by Ca²⁺. Cyt c pools were also detected in a system of cyt c reconstituted in cardiolipin (CL) liposomes. Gradual binding of cyt c (1 nmol) to CL/2–[12-(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]dodecanoyl-1-hexadecan oyl-sn-glycero-3-phosphocholine (NBDC₁₂-HPC) liposomes (10 nmol) produced NBD fluorescence quenching up to 0.4 nmol of added protein. Additional bound cyt c did not produce quenching, suggesting that cyt c-CL interactions originate distinct cyt c pools. Cyt c was removed from CL/NBDC₁₂-HPC liposomes by either phosphate or Ca²⁺, but only Ca²⁺ produced fluorescence dequenching and leakage of encapsulated 8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridinium bromide. In mitochondria, complex IV activity and mitochondrial membrane potential (Δψ_m) were not affected by the release of the phosphate-dependent cyt c pool. Conversely, removal of cyt c by Ca²⁺ caused inhibition of complex IV activity and impairment of Δψ_m. In a reconstituted system of mitochondria, nuclei and supernatant, cyt c detached from the inner membrane was released outside mitochondria and triggered events leading to DNA fragmentation. These events were prevented by enriching mitochondria with exogenous CL or by sequestering released cyt c with anti-cyt c antibody.