Vibrational spectra and assignments of 5-amino-2-chlorobenzoic acid by ab initio Hartree-Fock and density functional methods.

The Fourier transform Raman and Fourier transform infrared spectra of 5-amino-2-chlorobenzoic acid (5A2CBA) were recorded in the solid phase. Geometry opitimizations were done without any constraint and harmonic-vibrational wavenumber and several thermodynamic parameters were calculated for the minimum energy conformer at ab initio and DFT levels invoking 6-311G(d,p) basis set and the results are compared with the experimental values with the help of three specific scaling procedures, the observed vibrational wavenumbers in FTIR and FT-Raman spectra were analyzed and assigned to different normal modes of the molecule. Most of the modes have wavenumbers in the expected range, the error obtained was in general very low. The appropriate theoretical spectrograms for the FTIR spectra of the title molecule were also constructed.

Inhibition of nitric oxide and caspase-3 mediated apoptosis by a tetrapeptide derivative (PEP1261) in cultured synovial fibroblasts from collagen-induced arthritis.

In this study, the effect of (Boc-Lys (Boc)-Arg-Asp-Ser (tBu)-OtBu), a tetrapeptide derivative (PEP1261) was examined for antiproliferative potency and apoptotic induction. Synovial fibroblasts were isolated from collagen-induced arthritic (CIA) rats and exposed to peptides viz., PEP1261, and parental peptides (KRDS and RGDS). Viability of the cells decreased in the presence of PEP1261 at a lower concentration (0.1 mM) when compared to RGDS and KRDS (1 mM). The treatment of cells with peptides showed induction of apoptosis, resulting in the cleavage of caspase-3 as well as its substrate poly-(ADP-ribose) polymerase (PARP). Pretreatment of cells with caspase-3 inhibitor prevented inhibition of [(3)H] thymidine incorporation, DNA fragmentation, and cleavage of caspase-3 and PARP as confirmed by western blotting as well as annexin-V/PI-staining using flow cytometry. However, caspase-1 and caspase-2 inhibitors did not prevent the peptides from inducing apoptosis indicating that caspase-3 might have a role in the process of apoptosis induced by peptides. Treatment of synovial fibroblasts with nitric oxide donor, S-nitroso-N-acetyl-DL: -penicillamine (SNAP) (500 microM) showed significant elevation of nitric oxide levels and resulted in absence of apoptosis by preventing the inhibition of [(3)H] thymidine incorporation. This was further evidenced by annexin V/propidium iodide (PI) staining and absence of DNA fragmentation, intra cellular caspase-3 activity and PARP cleavage. In contrast, SNAP followed by PEP1261 and parental peptides-induced apoptosis by lowering the levels of nitric oxide. These results suggested that PEP1261 suppressed the proliferation and induced apoptosis in cultured synovial fibroblasts from CIA rats. This study also confirmed that PEP1261 inhibited nitric oxide level in cultured synovial fibroblasts.

Effect of a derivatized tetrapeptide from lactoferrin on nitric oxide mediated matrix metalloproteinase-2 production by synovial fibroblasts in collagen-induced arthritis in rats.

Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteolytic enzymes, which degrade several components of extracellular matrix, in arthritic synovial cells. In cultured synovial fibroblasts, both nitric oxide (NO) and reactive oxygen species (ROS) are potent inducers of MMPs production. PEP1261, a tetrapeptide derivative used in this study, corresponds to residues of 39-42 human lactoferrin. The parent protein lactoferrin is able to inhibit the production of free radicals in rheumatoid joints and it regulates many aspects of inflammation. This study is aimed to examine the effects of PEP1261 on MMP-2 production in the presence of nitric oxide donor in cultured synovial fibroblasts from collagen-induced arthritic rats. PEP1261 affects a significant reduction in nitrite levels as well as in MMP-2 production in SNAP stimulated synovial fibroblasts and this is validated by gelatin zymography and immunoblot analysis. Furthermore, RTPCR analysis has demonstrated that PEP1261 inhibits MMP-2 mRNA expression in SNAP treated synovial fibroblasts. The results of this study suggest that PEP1261 possesses antiarthritic activity by inhibiting nitrite levels as well as MMP-2 expression better than control peptides viz., KRDS and RGDS.

Reaction mechanism of electron transfer from FeII(CN)6(4-) or W(IV)(CN)8(4-) to the cupric ions in human copper, zinc superoxide dismutase.

The electron transfer reactions from FeII(CN)6(4-) and W(IV)(CN)8(4-) to the cupric ions in human copper, zinc superoxide dismutase were followed by the micro-stopped-flow method. The kinetic rate data clearly indicate that FeII(CN)6(4-) or W(IV)(CN)8(4-) first forms an adduct with the enzyme through the interaction with Arg143 of the active cavity and then an electron from FeII(CN)6(4-) or W(IV)(CN)8(4-) of the adduct transfers to the cupric ion in the enzyme. The dissociation constants of the adducts of FeII(CN)6(4-) and W(IV)(CN)8(4-) were 4.0(+/-0.3) x 10(-3) and 2.2(+/-0.3) x 10(-3) M, respectively. In spite of the difference between the standard redox potentials of FeIII(CN)6(3-)/FeII(CN)6(4-) (468 mV) and W(V)(CN)8(3-)/W(IV)(CN)8(4-) (556 mV), the electron transfer rate constant (0.148(+/-0.005) s(-1) of FeII(CN)6(4-) at 25 degrees C is very similar to that of W(IV)(CN)8(4-) (0.072(+/-0.011) s(-1)). The entropy values of the adduct formations and the activation energies of the electron transfer rates were determined by the temperature dependence of the dissociation constants of the adducts and the electron transfer rates. The enthalpy values of the formation of adducts are almost zero, so that the driving forces to form the adducts are mainly derived from the entropy. The activation energy of the electron transfer rate of FeII(CN)6(4-) is very similar to that of W(IV)(CN)8(4-). The formation of the adduct between FeII(CN)6(4-) and the enzyme was inhibited by the presence of various anions (ClO4-, SO4(2-), SCN-, and N3-). The bulky anions SO4(2-) and ClO4- behave as competitive inhibitors for FeII(CN)6(4-); these anions should interact mainly with Arg143, as it has a positive charge at the entrance of the active cavity. The competitive inhibition constants of ClO4-, SO4(2-), and SCN- were 0.010, 0.012, and 0.008 M. The azide ion, which is smaller than SO4(2-) or ClO4-, shows mixed inhibition, because N3- can interact with Arg143 (competitive inhibition) and also directly binds to the cupric ion in h-SOD (noncompetitive inhibition). The competitive and noncompetitive inhibition constants of N3- were 0.004 and 0.016 M, respectively.