Effect of protease digestion on the secondary structure of sarcoplasmic reticulum Ca2(+)-ATPase as seen by FT-i.r. spectroscopy

1. Upon controlled protein cleavage the catalytic activity of the Ca2(+)-ATPase from sarcoplasmic reticulum is drastically reduced concomitantly with small but significant changes in secondary structure as seen by Fourier transformed infrared (FT-i.r.) spectroscopy, although no loss of protein bound to the membrane is found. 2. FT-i.r. band fitting procedures show a reduction in the beta-sheet and turns content of the protein which is accompanied by an increase in alpha-helix and/or random structure. 3. These changes in the secondary structure of the protein appear to be well correlated to the tryptic digestion pattern and also to changes in the ATP hydrolysis rate of the Ca2(+)-ATPase. 4. It is concluded that these small changes reflect the disruption of key domains of the protein, which lie outside of the membrane matrix, leading to loss of enzymatic activity. 5. FT-i.r. spectroscopy appears to be a very useful technique to study changes in secondary structure of proteins.     

The marine toxin okadaic acid is a potent neurotoxin for cultured cerebellar neurons.

The tumor promoter okadaic acid (OKA), is a marine toxin of algal origin, identified as a potent inhibitor of protein phosphatases 1 and 2A, and possibly enhancing calcium influx through voltage dependent calcium channels (VSSC). We now report that OKA at concentrations as low as 0.5 nM produced neurotoxicity, characterized first by the desintegration of the neurites and swelling of cell bodies, and later by cellular death. Non-neuronal cells viability and morphology were unaffected up to at least 5 nM OKA. Neurons sensitivity to the toxin changed with age in culture. Maximum neurotoxicity was observed in neurons at 9 DIC, when the OKA concentration producing half of the maximum reduction in neuronal survival (EC50) was approximately 0.65 nM. At 5 DIC or 19 DIC (EC50 approximately 2.5 nM and approximately 4.5 nM respectively), neurons appeared to be less sensitive to OKA. Neurotoxicity by OKA was not reduced by VSCC antagonists such as nifedipine and verapamil, nor by antagonists of excitatory aminoacid (EAA) receptors including APV, MK801 or CNQX. VSCC antagonists and EAA receptors antagonists fully protected from neurotoxicity induced by depolarization with KCl. These results suggest that OKA mechanism of neurotoxicity may not directly involve VSCC, endogenous EAA release and EAA receptors, but may depend upon other neurochemical events.     

The binding of cortisol to adrenal mitochondria

Binding of tritiated cortisol to adrenal zona glomerulosa mitochondria was studied and compared with that of corticosterone. Cortisol was shown to bind specifically to the inner membrane of zona glomerulosa mitochondria. Corticosterone and cortisol had similar apparent association constants (Ka) and concentrations of binding sites. The methodology was validated by obtaining similar Ka from both binding plots and kinetic data. Cortisol binding was inhibited by pretreatment with sodium dithionite, and displaced by deoxycorticosterone, corticosterone, 18-hydroxy-corticosterone, 11 beta-hydroxy-18-ethynyl-progesterone and metyrapone, but not by cholesterol. These results suggest that cortisol and corticosterone bind to the same cytochrome P-450.     

Novel,X-ray supported kinetic imaging of hidden-lifestyle arthropods

Dear Editor,There are several arthropods,which live and develop covertly in plant tissues.The plant tissues surrounding them provide them with shelter during their vulnerable developing stage or ensure overwintering as well as they can supply them with essential food for their ontogenetic development(McNaughton,1983).     

Iron deficiency in peach trees: effects on leaf chlorophyll and nutrient concentrations in flowers and leaves

The effects of iron deficiency on the leaf chlorophyll concentrations and on the macro- (N, P, K, Ca and Mg) and micro-nutrient (Fe, Mn, Zn and Cu) composition of flowers (at full bloom) and leaves (60 and 120 days after full bloom) of field-grown peach (Prunus persica L. Batsch) trees were investigated. Flowers and leaves were taken and analysed from fifty individual trees. Our data indicate that large decreases in leaf chlorophyll concentration were found at the beginning of the season in control trees, possibly associated to a dilution effect by leaf growth, that were later followed by leaf chlorophyll concentration increases. Leaf Fe chlorosis apparently results from two different processes, the dilution of leaf Chl caused by growth and the subsequent inability to produce and/or stabilize new Chl molecules in the thylakoid membrane. Iron chlorosis did not change the seasonal change patterns of any of the nutrients studied. In Fe-deficient trees the K concentration and the K/Ca ratio were high not only in leaves but also in flowers, indicating that this is a characteristic of Fe-deficient plant tissue in the whole fruit tree growing season. Flower Fe concentrations were well correlated with the degree of chlorosis developed later in the season by the trees, suggesting that flower analysis could be used for the prognosis of Fe deficiency in peach.     

A cross-platform public domain PC image-analysis program for the comet assay

The single-cell gel electrophoresis, also known as the comet assay, has gained wide-spread popularity as a simple and reliable method to measure genotoxic and cytotoxic effects of physical and chemical agents as well as kinetics of DNA repair. Cells are generally stained with fluorescent dyes. The analysis of comets--damaged cells which form a typical comet-shaped pattern--is greatly facilitated by the use of a computer image-analysis program. Although several image-analysis programs are available commercially, they are expensive and their source codes are not provided. For Macintosh computers a cost-free public domain macro is available on the Internet. No ready for use, cost-free program exists for the PC platform. We have, therefore, developed such a public domain program under the GNU license for PC computers. The program is called CASP and can be run on a variety of hardware and software platforms. Its practical merit was tested on human lymphocytes exposed to gamma-rays and found to yield reproducible results. The binaries for Windows 95 and Linux, together with the source code can be obtained from: http://www.casp.of.pl.     

Pre-existing glomerular immune complexes induce polymorphonuclear cell recruitment through an Fc receptor-dependent respiratory burst: potential role in the perpetuation of immune nephritis

In immune complex (IC) diseases, FcR are essential molecules facilitating polymorphonuclear cell (PMN) recruitment and effector functions at the IC site. Although FcR-dependent initial tethering and FcR/integrin-dependent PMN accumulation were postulated, their underlying mechanisms remain unclear. We here addressed potential mechanisms involved in PMN recruitment in acute IC glomerulonephritis (nephrotoxic nephritis). Since some renal cells may be recruited from bone marrow (BM) lineages, reconstitution studies with BM chimeras and PMN transfer between wild-type (WT) and FcR-deficient mice (gamma(-/-)) were performed. Severe glomerular damage was induced in WT and W gamma chimeras (BM from WT to irradiated gamma(-/-)), while it was absent in gamma(-/-) and gamma W chimeras (gamma(-/-) BM to WT). Moreover, WT PMN transfer, but not gamma(-/-) PMN, reconstituted the disease in gamma(-/-), indicating that FcR on resident cells is not a prerequisite for PMN recruitment in this disease. Surprisingly, transferred WT PMN were recruited coincidentally with NF-kappa B activation and TNF-alpha overexpression even in glomeruli with preformed IC (nephrotoxic Ab administered 3 days previously), suggesting that PMN can initially be recruited via its own FcR without previous chemoattractant release. Furthermore, H(2)O(2) inhibition by catalase attenuated the acute WT PMN recruitment and the induction of NF-kappa B and TNF-alpha much more than integrin (CD18) blockade, indicating a role for the respiratory burst before integrin-dependent accumulation. In coculture experiments with IC-stimulated PMN and glomeruli, PMN caused acute glomerular TNF-alpha expression predominantly via FcR-mediated H(2)O(2) production. In conclusion, glomerular IC, even preformed, can cause PMN recruitment and injury through PMN FcR-mediated respiratory burst during initial PMN tethering to IC.     

Galactose induces the expression of penicillin acylase under control of the lac promoter in recombinant Escherichia coli

The expression of penicillin acylase (PA), cloned in the pPA102 plasmid under control of the wild-type lac promoter and using galactose as inducer in Escherichia coli JM101, JM103 and JM105 transformant cells, was analyzed. The E. coli JM101/pPA102 cultures attained the highest specific activity of PA. For large scale PA production based on E. coli JM101/pPA102 a culture media with galactose instead of isopropyl-thio-galactopyranoside as inducer would be as successful and less expensive.     

Modulation of nuclear pore topology by transport modifiers

The nuclear pore complex (NPC) represents the only pathway for macromolecular communication between the nuclear and cytoplasmic compartments of the cell. Nucleocytoplasmic transport requires the interaction of transport receptors with phenylalanine-glycine (FG)-repeats that line the transport pathway through the NPC. Here we examine the effects of transport receptors and amphipathic alcohols on NPC topology using scanning force microscopy. We show that transport receptors that irreversibly bind FG-repeats increase NPC vertical aspect, whereas transport receptors that weakly interact with FG-repeats increase NPC diameter. Interestingly, small polar alcohols likewise increase NPC diameter. These opposing effects agree with the inhibition or enhancement of nuclear transport, respectively, previously ascribed to these agents.     

Local and long-range structural effects caused by the removal of the N-terminal polypeptide fragment from immunoglobulin L chain lambda

The role of the N-terminal polypeptide fragment of the immunoglobulin l-chain in V domain packing stability, and the flexibility of the whole chain was approached by molecular dynamics simulation. The observations were supported by experimental analysis. The N-terminal polypeptide fragment appeared to be the low-stability packing element in the V domain. At moderately elevated temperature it may be replaced at its packing locus by Congo red and then removed by proteolysis. After removal of Congo red by adsorption to (diethylamino)ethyl (DEAE) cellulose, the stability of complete L chain and of L chain devoid of the N-terminal polypeptide fragment were compared. The results indicated that the N-terminal polypeptide fragment plays an essential role in the stability of the V domain. Its removal makes the domain accessible for ANS and Congo red dye binding without heating. The decreased domain stability was registered in particular as increased root mean square (RMS) fluctuation and higher susceptibility to proteolytic attack. The long-range effect was most clearly manifested at 340 K as independent V and C domain fluctuation in the l-chain devoid of the N-terminal polypeptide fragment. This is likely due to the lack of direct connections between the N- and C-termini of the V domain polypeptide. In a complete V domain the connection involves residues 8-12 and 106-110 in particular. Partial or complete disruption of this connection increases the freedom of V domain rotation, while its increased cohesion strengthens the coupling of the V and C domains, making the whole L chain less flexible.