Synthesis, crystal structures and, antibacterial and antiproliferative activities in vitro of palladium(II) complexes of triphenylphosphine and thioamides

Palladium(II) complexes with triphenylphosphine (PPh₃) and thioamides of the general formulae, [Pd(L)₂(PPh₃)₂]Cl₂ and [Pd(L)₂(PPh₃)₂] have been prepared and characterized by elemental analysis, IR and NMR (¹H, ¹³C and ³¹P) methods, and two of them (trans-[Pd(PPh₃)₂(Dmtu)₂]Cl₂·(H₂O)(CH₃OH)_0.5 (1) and trans-[Pd(PPh₃)₂(Mpy)₂] (2)) by X-ray crystallography; where L = thiourea (Tu), methylthiourea (Metu), N,N′-dimethylthiourea (Dmtu), tetramethylthiourea (Tmtu), 2-mercaptopyridine (Mpy), 2-mercaptopyrimidine (Mpm) and thionicotinamide (Tna). The spectral data of the complexes are consistent with the sulfur coordination of thioamides to palladium(II). The crystal structures of the complexes show that (1) has ionic character consisting of [Pd(PPh₃)₂(Dmtu)₂]⁺² cations and uncoordinated Cl⁻ ions, while (2) is a neutral complex with Mpy behaving as anionic thiolate ligand. The coordination environment around palladium in (2) is nearly regular square-planar, while in (1) the trans angles show significant distortions from 180°. The complexes were screened for antibacterial effects, brine shrimps lethality bioassay and antitumor activity. These complexes showed significant activities in most of the cases against the tested bacteria as compared to that of a standard drug. Their antitumor activity against prostate cancer cells (PC3) is comparable with doxorubicin, together with no cytotoxic effects in brine shrimps lethality bioassay study.     

MMP25 (MT6-MMP) is highly expressed in human colon cancer, promotes tumor growth, and exhibits unique biochemical properties

MMP25 (MT6-MMP) is one of the two glycosylphosphatidylinositol-anchored matrix metalloproteinases (MMPs) that have been suggested to play a role in pericellular proteolysis. However, its role in cancer is unknown, and its biochemical properties are not well established. Here we found a marked increase in MT6-MMP expression within in situ dysplasia and invasive cancer in 61 samples of human colon cancer. Expression of MT6-MMP in HCT-116 human colon cancer cells promoted tumori-genesis in nude mice. Histologically, the MT6-MMP-expressing tumors demonstrated an infiltrative leading edge in contrast to a rounded leading edge in vector control tumors. Biochemical and biosynthesis analyses revealed that MT6-MMP displayed on the cell surface exists as a major form of 120 kDa that likely represents enzyme homodimers linked by disulfide bonds. Upon reduction, a single 57-kDa active MT6-MMP was detected. Interestingly, neither membrane-anchored nor phosphatidylinositol-specific phospholipase C-released MT6-MMPs were found to be associated with tissue inhibitor of metalloproteinases (TIMPs) and did not activate pro-gelatinases (pro-MMP-2 and pro-MMP-9) even in the presence of exogenous TIMP-2 or TIMP-1. A catalytic domain of MT6-MMP was inhibited preferentially by TIMP-1 (K(i) = 0.2 nm) over TIMP-2 (K(i) = 2.0 nm), because of a slower association rate. These results show that MT6-MMP may play a role in colon cancer and exhibit unique biochemical and structural properties that may regulate proteolytic function at the cell surface.     

Investigation of the dimerization of proteins from the epidermal growth factor receptor family by single wavelength fluorescence cross-correlation spectroscopy

Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS), introduced to study biomolecular interactions, has recently been reported to monitor enzyme activity by using a newly developed fluorescent protein variant together with cyan fluorescent protein. Here, for the first time to our knowledge, SW-FCCS is applied to detect interactions between membrane receptors in vivo by using the widely used enhanced green fluorescent protein and monomeric red fluorescent protein. The biological system studied here is the epidermal growth factor/ErbB receptor family, which plays pivotal roles in the development of organisms ranging from worms to humans. It is widely thought that a ligand binds to the monomeric form of the receptor and induces its dimeric form for activation. By using SW-FCCS and F?rster resonance energy transfer, we show that the epidermal growth factor receptor and ErbB2 have preformed homo- and heterodimeric structures on the cell surface and quantitation of dimer fractions is performed by SW-FCCS. These receptors are major targets of anti-cancer drug development, and the receptors' homo- and heterodimeric structures are relevant for such developments.     

Hydrated conidia of Metarhizium anisopliae release a family of metalloproteases

Metarhizium anisopliae spores release isoforms of metalloprotease during hydration over a 4-day incubation period. The isoforms were identified and characterized by using one-dimensional native PAGE (1-DE nPAGE) and one-dimensional SDS non-dissociating (1-DE nSDS-PAGE) zymography. The ability of these isozymes to degrade gelatin varied as revealed by 2-D spot densitometry. 1-DE nPAGE zymography revealed five isoforms of gelatinase from Tween wash of conidia. Where as, one to three activities with different intensities appeared on gel from washing of conidia to incubation in water till day 4. The relative migrations of these activities on 1-DE nPAGE zymograms appeared as fast, medium and slow on gel. The 2-D spot densitometry of zymograms indicated isoforms have different proteolytic activity as quantified by pixel intensities. SDS-PAGE zymography indicated the release of two isozymes of Mr 103 and 12 kDa during Tween treatment of conidia. However, during the first washing step with water and incubation of spores at day 2 and 3, respectively, only 12 kDa protein was evident. Majority of these proteases were inhibited by EDTA, but stimulated by CaCl(2), and MgCl(2). The presence of isozymes in conidia and their release during hydration must have functional significance for fungi and in this case it should provide advantages to M. anisopliae in its saprobic or pathogenic modalities. To our knowledge this is the first report describing release of metalloprotease isozymes from conidia.     

Streptococcus pneumoniae hyaluronate lyase contains two non-cooperative independent folding/unfolding structural domains: characterization of functional domain and inhibitors of enzyme

Hyaluronate lyase contributes directly to bacterial invasion by degrading hyaluronan, the major component of host extracellular matrix of connective tissues. Streptococcus pneumoniae hyaluronate lyase (SpnHL) is built from two structural domains that interact through interface residues, in addition to being connected by a peptide linker. For the first time we demonstrate that the N- and C-terminal domains of SpnHL fold/unfold independent of each other suggesting the absence of any significant cooperative interactions between them. The C-terminal domain of SpnHL is less stable than the N-terminal domain against thermal and guanidine hydrochloride denaturation. The intact N-terminal domain was purified after limited proteolysis of SpnHL under conditions where only the C-terminal domain was unfolded. Isolated N-terminal domain of SpnHL had similar thermal stability as when present in the native enzyme and was found to be enzymatically active demonstrating that it is capable of carrying out enzymatic reaction on its own. Functional studies demonstrated that guanidine hydrochloride, guanidine isothiocyanate, l-arginine methyl ester, and l-arginine inhibit the enzymatic activity of SpnHL at very low concentrations. This provides a lead for new chemical entities that can be exploited for designing effective inhibitors of SpnHL.     

In vitro propagation of Caralluma tuberculata and evaluation of antioxidant potential

Present study describes rapid in vitro propagation of Caralluma tuberculata, a traditional medicinal plant, and antioxidant potential of calli and plants extracts. The highest callus induction rate (93.3%) with maximum weight of calli 5.2 g was achieved from shoot tip explants on MS medium supplemented with 9.04 μM 2,4-D and 4.44 μM BA. The maximum shoot induction rate (71.1%) with mean number of shoots 3.66 ± 1.53 and 4.6 cm average shoot length was observed on 13.32 μM BA, 4.52 μM 2,4-D and 2.89 μM GA3 appended in MS medium. The developed shoots were best rooted in the presence of 5.07 μM IAA with 3.0 ± 0.15 roots per plantlet. The plants were successfully acclimatized under in vivo conditions. The plants and calli extracts exhibited good antioxidant activities, however, plant extract activities were more pronounced. The phenolic compounds in plant and calli extracts were 0.16% and 0.057%, respectively. While the flavonoids were 0.092% in plant and 0.039% in calli extract. Total Phenolics, flavonoids; DPPH radical scavenging activity and reducing power potential distributed among different fractions depending upon polarity of the solvent. The highest DPPH scavenging activity and reducing power was exhibited by water fractions; 4.95 mg/mL and 0.729 OD at 10 mg/mL, respectively. The micropropagation protocol can be successfully used for large-scale multiplication and conservation of germplasm of this threatened plant. Furthermore, antioxidant value describes importance of this valuable plant as food and medicine.