A model for the misfolded bis-His intermediate of cytochrome c: the 1-56 N-fragment

We have characterized the ferric and ferrous forms of the heme-containing (1-56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV-visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His-His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57-104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.     

Biotechnological applications of bioluminescence and chemiluminescence

Recent progress in molecular biology has made available several biotechnological tools that take advantage of the high detectability and rapidity of bioluminescence and chemiluminescence spectroscopy. These developments provide inroads to in vitro and in vivo continuous monitoring of biological processes (e.g. gene expression, protein-protein interaction and disease progression), with clinical, diagnostic and drug discovery applications. Furthermore, combining luminescent enzymes or photoproteins with biospecific recognition elements at the genetic level has led to the development of ultrasensitive and selective bioanalytical tools, such as recombinant whole-cell biosensors, immunoassays and nucleic acid hybridization assays. The high detectability of the luminescence analytical signal makes it appropriate for miniaturized bioanalytical devices (e.g. microarrays, microfluidic devices and high-density-well microtiter plates) for the high-throughput screening of genes and proteins in small sample volumes.     

Microglia proliferation as a response to activation in the freshwater snail Planorbarius corneus: a BrdU incorporation study

Invertebrate microglia constitute a class of cells resident in the ganglionic nervous system which are activated after tissue injury or by the presence of pathogens. The microglia activation response includes graduated morpho-functional and biochemical changes and cell proliferation. In this study we verified in the freshwater snail Planorbarius corneus that an activation caused by a traumatic event may induce microglia division. Cell proliferation was assessed immunocytochemically using BrdU incorporation technique and documented on both ganglionic sections and microglia cultured cells at different experimental conditions and times after activation. In addition, we studied the possibility of increasing microglia proliferation by adding to the cultured medium the Macrophage-Colony Stimulating Factor (M-CSF) that has been shown to stimulate specifically this process in vertebrates.     

Meridianins, a new family of protein kinase inhibitors isolated from the ascidian Aplidium meridianum

Meridianins are brominated 3-(2-aminopyrimidine)-indoles which are purified from Aplidium meridianum, an Ascidian from the South Atlantic (South Georgia Islands). We here show that meridianins inhibit various protein kinases such as cyclin-dependent kinases, glycogen synthase kinase-3, cyclic nucleotide-dependent kinases and casein kinase 1. Meridianins prevent cell proliferation and induce apoptosis, a demonstration of their ability to enter cells and to interfere with the activity of kinases important for cell division and cell death. These results suggest that meridianins constitute a promising scaffold from which more potent and selective protein kinase inhibitors could be designed.     

The K(ATP)+ channel is involved in a low-amplitude permeability transition in plant mitochondria

Pea (Pisum sativum) stem mitochondria, energized by NADH, succinate or malate plus glutamate, underwent a spontaneous low-amplitude permeability transition (PT), which could be monitored by dissipation of the electrical potential (deltapsi) or swelling. The occurrence of the latter effects was dependent on O2 availability, because O2 shortage anticipated the manifestation of both deltapsi dissipation and swelling. Spontaneous deltapsi collapse was also monitored in sucrose-resuspended mitochondria and again O2 deprivation caused an anticipation of the phenomenon. However, in this case deltapsi dissipation was not accompanied by a parallel mitochondrial swelling. The latter effect was, indeed, evident only if mitochondria were resuspended in KCl (as osmoticum), or other cations with a molecular mass up to 100 Da (choline+). PT was also induced by protonophores (carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or free fatty acids) or valinomycin (only in KCl). The FCCP-induced dissipation of deltapsi and swelling were inhibited by ATP and stimulated (anticipated) by cyclosporin A or O2 shortage. The FCCP-induced PT was accompanied by the release of pyridine nucleotides from the matrix and of cytochrome c from the intermembrane space of KCl-resuspended mitochondria. The spontaneous and FCCP-induced low-amplitude PT of plant mitochondria are interpreted as due to the activity of a recently identified K(ATP)+ channel whose open/closed state is dependent on polarization of the inner membrane and on the oxidoreductive state of some sulfhydryl groups.     

New N-arylsulfonyl-N-alkoxyaminoacetohydroxamic acids as selective inhibitors of gelatinase A (MMP-2)

New N-arylsulfonyl-substituted alkoxyaminoaceto hydroxamic acid derivatives of types 8 and 10 designed as oxa-analogues of known sulfonamide-based MMPi of types 2 and 7 were synthesized and tested for their inhibitory activities on some matrix metalloproteinases. The combination of a biphenylsulfonamide group with oxyamino oxygen in the pharmacophoric central skeleton of sulfonamide-based MMPi obtained in the new sulfonamides 10 seems to be able to give selectivity for MMP-2 over MMP-1. The most potent derivative of this type, 10a, shows similar anti-invasive properties to the analogue reference drug CGS27023A, 2, in an in vitro model of invasion on matrigel, carried out on cellular lines of fibrosarcoma HT1080 (tumoural cells over-expressing MMP-2 and MMP-9).     

Effective genetic vaccination with a widely shared endogenous retroviral tumor antigen requires CD40 stimulation during tumor rejection phase

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8(+), but not CD4(+), T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8(+) T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8(+) T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.     

The domains of glycoprotein D required to block apoptosis induced by herpes simplex virus 1 are largely distinct from those involved in cell-cell fusion and binding to nectin1

Glycoprotein D (gD) interacts with two alternative protein receptors, nectin1 and HveA, to mediate herpes simplex virus (HSV) entry into cells. Fusion of the envelope with the plasma membrane requires, in addition to gD, glycoproteins gB, gH, and gL. Coexpression of the four glycoproteins (gD, gB, gH, and gL) promotes cell-cell fusion. gD delivered in trans is also capable of blocking the apoptosis induced by gD deletion viruses grown either in noncomplementing cells (gD(-/-)) or in complementing cells (gD(-/+)). While ectopic expression of cation-independent mannose-6 phosphate receptor blocks apoptosis induced by both stocks, other requirements differ. Thus, apoptosis induced by gD(-/-) virus is blocked by full-length gD (or two gD fragments reconstituting a full-length molecule), whereas ectopic expression of the gD ectodomain is sufficient to block apoptosis induced by gD(-/+) virus. In this report we took advantage of a set of gD insertion-deletion mutants to map the domains of gD required to block apoptosis by gD(-/-) and gD(-/+) viruses and those involved in cell-cell fusion. The mutations that resulted in failure to block apoptosis were the same for gD(-/-) and gD(-/+) viruses and were located in three sites, one within the immunoglobulin-type core region (residues 125, 126, and 151), one in the upstream connector region (residues 34 and 43), and one in the C-terminal portion of the ectodomain (residue 277). A mutant that carried amino acid substitutions at the three glycosylation sites failed to block apoptosis but behaved like wild-type gD in all other assays. The mutations that inhibited polykaryocyte formation were located in the upstream connector region (residues 34 and 43), at the alpha1 helix (residue 77), in the immunoglobulin core and downstream regions (residue 151 and 187), and at the alpha3 helix (residues 243 and 246). Binding of soluble nectin1-Fc to cells expressing the mutant gDs was generally affected by the same mutations that affected fusion, with one notable exception (Delta277-310), which affected fusion without hampering nectin1 binding. This deletion likely identifies a region of gD involved in fusion activity at a post-nectin1-binding step. We conclude that whereas mutations that affected all functions (e.g., upstream connector region and residue 151) may be detrimental to overall gD structure, the mutations that affect specific activities identify domains of gD involved in the interactions with entry receptors and fusogenic glycoproteins and with cellular proteins required to block apoptosis. The evidence that glycosylation of gD is required for blocking apoptosis supports the conclusion that the interacting protein is the mannose-6 phosphate receptor.