A series of meso-tris (N-methyl-pyridiniumyl)-(4-alkylamidophenyl) porphyrins: synthesis, interaction with DNA and antibacterial activity.

A series of meso-5,10,15-tris(N-methyl-4-pyridiniumyl)-20-(4-alkylamidophen yl) porphyrins were synthesized by derivatizing the amino group on the phenyl ring with the following hydrophobic groups: -C(O)C7F15, -C(O)CH=CH2, C(O)CH3, -C(O)C7H15, and -C(O)C15H31. The cationic tris-pyridiumyl porphyrin core serves as a DNA binding motif and a photosensitizer to photomodify DNA molecules. The changes of the UV-Vis absorption spectra during the titration of these porphyrins with calf thymus DNA revealed a large bathochromic shift (up to 14 nm) and a hypochromicity (up to 55%) of the porphyrins Soret bands, usually considered as proof of porphyrin intercalation into DNA. Association constants (K) calculated according to the McGhee and von Hippel model, were in the range of 10(6)-10(7) M(-1). An increase in hydrophobicity of the substituents at the 20-meso-position produced higher binding affinity. These porphyrins caused photomodification of the supercoiled plasmid DNA when a green laser beam at 532 nm was applied. Those with higher surface activity acted more efficiently as DNA photomodifiers. The porphyrin with a perfluorinated alkyl chain (-COC7F15) at the meso-20-position inhibited the growth of gram-positive bacteria (S. aureus, or S. epidermidis). Other porphyrins exhibited moderate activity against both gram-negative and gram-positive organisms.

Cardiotoxin from cobra venom affects the Ca-Mg-ATPase of cardiac sarcolemmal membrane vesicles.

Highly purified sarcolemmal (SL) membrane vesicles were prepared from bovine cardiac tissue and used to evaluate the effects of cardiotoxin (CTX) on Ca2+ transport systems of the SL membrane. The addition of CTX, at 1.0 microM and 10 microM, stimulated the ATP-dependent transport of 45Ca2+ by the Ca2(+)-Mg2(+)-ATPase to 138% and 193% of control levels, respectively. The increase in Ca2+ accumulation by the SL vesicles in the presence of CTX was not affected by 100 microM ouabain but was reduced to 50% of control uptake levels by the addition of 0.5 mM dicyclohexylcarbodiimide. Additionally, no effect of CTX (1.0 microM to 10 microM) was seen on 45Ca2+ transport by the Na-Ca exchange system. These effects of CTX on the SL membrane do not appear to be due to non-specific membrane disruption since SL vesicles preloaded with 45Ca2+ did not release (efflux) the accumulated Ca2+ more rapidly in the presence of CTX (1.0 microM to 250 microM). The rate of hydrolysis of ATP by the SL Ca2(+)-Mg2(+)-ATPase was observed to increase (29% to 52%) as CTX concentration increased (1.0 microM to 10 microM). The same concentrations of CTX had no effect on ATP hydrolysis by Na(+)-K(+)-ATPase. We conclude that 10 microM CTX stimulates the rate of ATP hydrolysis and Ca2+ transport by the SL Ca2(+)-Mg2(+)-ATPase.

A bioassay for cobra cardiotoxin activity using semi-isolated cockroach heart.

A semi-isolated cockroach heart preparation was used to rapidly determine the activity of cobra cardiotoxin, monitored as a direct response on heart rate. This preparation produced a dose-response curve in the presence of active cardiotoxin and demonstrated that cardiotoxin retained its biological activity after boiling, although cardiotoxin activity was destroyed by heating in the presence of dithiothreitol. Experiments that cross-linked radiolabeled cardiotoxin to solubilized cockroach heart membranes suggested that cardiotoxin bound specifically to a 59,000 mol. wt membrane protein in this tissue.

Calreticulin is the major Ca2+ storage protein in the endoplasmic reticulum of the pea plant (Pisum sativum).

A 56kDa protein with high similarity in its N-terminal amino acid sequence to animal calreticulin and 100% homology with the N-terminal amino acids of spinach calreticulin has been identified in seeds of the pea plant (Pisum sativum). A new purification procedure is described by which the calreticulin-like protein was selectively solubilized by incubation with deoxycholate and HgCl2 from microsomes enriched for endoplasmic reticulum. Following Mono Q ion exchange chromatography of the deoxycholate extract by fast protein liquid chromatography, the calreticulin-like protein was obtained in nearly pure form. This purified protein is similar to animal calreticulin in apparent mass, characteristic blue staining with Stains-all dye and calcium-binding ability. In addition, this protein is recognized only by affinity purified antibodies against rabbit calreticulin and is not recognized by anti-calsequestrin antibodies. Our data suggested that calreticulin rather than calsequestrin functions as the Ca(2+)-storage protein in the endoplasmic reticulum of pea plants.

Membrane lateral phase separations and chlortetracycline transport by Bacillus megaterium.

Chlortetracycline, a fluorescent probe of its own active transport, has been used to study lateral phase separations of membrane lipid in Bacillus megaterium cells. Arrhenius plots of initial accumulation rates are triphasic, with transitions or characteristic temperatures of 20 degrees and 9.5 degrees . At the higher temperature, the mobility of the chloretracycline, as measured by fluorescence polarization, is markedly altered. Chlortetracycline transport exhibits saturation kinetics, and fluorescence energy transfer from protein to bound antibiotic can be observed. N-Phenyl-1-naphthylamine, a lipophilic fluorescent probe, responds to changes in the hydrophobic regions of the membrane that are distinct from membrane protein. The fluorescent properties of N-phenylnaphthylamine in partitioning and polarization experiments are altered most significantly at the lower characteristic temperature. No fluorescence energy transfer between N-phenylnaphthylamine and membrane protein or bound tetracycline can be detected. In correlative electron spin resonance experiments on the partitioning of a lipid-soluble spin label, the same characteristic temperatures detected in the fluorescence studies were measured. These data suggest that different probes may respond to either or both of the characteristic temperatures describing the lateral phase separation. Between these characteristic temperatures the chlortetracycline transport system is most intimately associated with relatively immobile lipids that are surrounded by a more mobile lipid phase.

Cardiac sarcolemmal and sarcoplasmic reticulum membrane vesicles exhibit distinctive (Ca-Mg)-ATPase substrate specificities.

The nucleoside 5'-triphosphate (NTP) substrate specificities for Ca-stimulated ATPase and ATP-dependent Ca2+ uptake activities have been examined in cardiac sarcolemma (SL) and sarcoplasmic (SR) membrane vesicles. The results indicate that SL membrane vesicles exhibit a much narrower range of NTP substrate specificities than SR membranes. In SR membrane vesicles, the Ca-stimulated Mg-dependent hydrolysis of ATP and dATP occurred at nearly equivalent rates, whereas the rates of hydrolysis of GTP, ITP, CTP, and UTP ranged from 16-33% of that for ATP. All of the above nucleotides also supported Ca2+ transport into SR vesicles; dATP was somewhat more effective than ATP while GTP, ITP, CTP, and UTP ranged from 28-30% of the activity for ATP. In the presence of oxalate, the initial rate of Ca accumulation with dATP was 4-fold higher than for ATP, whereas the activity for GTP, ITP, CTP, and UTP ranged from 35-45% of that for ATP. For the SL membranes, Ca-activated dATP hydrolysis occurred at 60% of the rate for ATP; GTP, ITP, CTP, and UTP were hydrolyzed by the SL preparations at only 7-9% of the rate for ATP. NTP-dependent Ca2+ uptake in SL membranes was supported only by ATP and dATP, with dATP 60% as effective as ATP. GTP, ITP, CTP, and UTP did not support the transport of Ca2+ by SL vesicles. The results indicate that the SL and SR membranes contain distinctly different ATP-dependent Ca2+ transport systems.

Site-specific photomodification of DNA by porphyrin-oligonucleotide conjugates synthesized via a solid phase H-phosphonate approach.

meso-Tris(4-pyridyl)[[(omega-hydroxyhexamethylene)carbamoyl]phenyl ] porphyrin was converted to its H-phosphonate derivative and conjugated using solid phase synthesis with the 5'-hydroxyl group of deoxyribonucleotides d(TCTTCCCA) and d(T)12. These conjugates were transformed into their (N-methylpyridiniumyl)porphyrin analogs in the reaction with methyl iodide. A 532 nm laser beam was utilized to photoactivate both types of the conjugates in the presence of the target 22-mer and 16-mer oligonucleotides. Photoactivation of porphyrin-oligonucleotide conjugates resulted in site-specific DNA modification characterized by a main reaction site size of approximately 5 bases.

A single Fc binding domain--alkaline phosphatase gene fusion expresses a protein with both IgG binding ability and alkaline phosphatase enzymatic activity.

A recombinant gene fusion was created and cloned using a previously constructed gene encoding a monodomain IgG Fc binding protein and the gene coding for bacterial alkaline phosphatase. The construct was able to express and secrete a fusion protein that exhibited both IgG binding and alkaline phosphatase enzymatic activities. Greater than 60% of the protein demonstrating both biological activities was detected from periplasmic space preparations. Nanogram concentrations of the Fc binding--alkaline phosphatase fusion protein allowed primary IgG antibody detection without the use of conjugated secondary antibodies. Removal of the domain coding for alkaline phosphatase resulted in decreased resistance of the protein to proteolytic degradation and the loss of IgG Fc binding ability. Using affinity-purified fusion protein, the specificity of binding to IgG, IgM and IgA was examined; binding was strong to IgG and barely detectable against IgM or IgA. Affinity for binding of the fusion protein to IgG (Kd = 6.7 x 10(-8) M) was determined to be equal to or greater than previously reported for protein A.

Ca(2+)-induced folding and aggregation of skeletal muscle sarcoplasmic reticulum calsequestrin. The involvement of the trifluoperazine-binding site.

Calsequestrin is an intermediate affinity, high capacity Ca(2+)-binding protein found in the lumen of the sarcoplasmic reticulum of both skeletal and cardiac muscle cells. Previous sequence analysis suggested that calsequestrin may contain a hydrophobic binding site for the drug trifluoperazine, a site shared by the calmodulin family and shown to play a role in calmodulin/calmodulin receptor interaction. Previous studies showed that, upon Ca2+ binding, calsequestrin undergoes a conformational change, burying the trifluoperazine-binding site, folding into a more compact structure that is trypsin-resistant, and increasing the negative ellipticity of the circular dichroism spectrum. In this study, the structural and functional roles of the trifluoperazine-binding site in the Ca(2+)-induced conformational change of calsequestrin are further studied using the calmodulin antagonists trifluoperazine and melittin. If trifluoperazine or melittin is added to calsequestrin prior to Ca2+ addition, then Ca(2+)-induced folding is inhibited as determined by the changes in circular dichroism spectra and protein sensitivity to trypsin digestion. If, however, Ca2+ is added prior to trifluoperazine or melittin, calsequestrin remains resistant to trypsin digestion, just as if the calmodulin antagonists are not present, suggesting that the conformational change is not affected. Aggregates of calsequestrin that exhibit high Ca2+ binding capacity have previously been shown to occur at high Ca2+ and calsequestrin concentrations. By preventing a prerequisite folding step, trifluoperazine or melittin also prevents the Ca(2+)-induced aggregation of calsequestrin, thus decreasing the maximal Ca2+ binding by calsequestrin. These data suggest that the trifluoperazine-binding site is critically involved in the Ca(2+)-induced intramolecular folding step required for the intermolecular interactions leading to high capacity Ca(2+)-binding by calsequestrin.

Apoptosis induced by Staphylococcus aureus in epithelial cells utilizes a mechanism involving caspases 8 and 3.

In this study, we demonstrate that the mechanism by which Staphylococcus aureus induces apoptosis in bovine mammary epithelial (MAC-T) cells involves caspases 8 and 3, two key components of a proteolytic cascade leading to apoptosis. In addition, internalized S. aureus induces expression of the inflammatory cytokines tumor necrosis factor alpha and interleukin-1beta by MAC-T cells. These data suggest that the internalization of S. aureus could induce specific cellular responses in vivo that may ultimately impact the course of infection.

Site-directed mutagenesis of cys148 in the lac carrier protein of Escherichia coli.

The lac y gene of Escherichia coli which encodes the lac carrier protein has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys148 is converted to a glycine residue. Cells bearing the mutated lac y gene exhibit initial rates of lactose transport that are about 4-fold lower than cells bearing the wild type gene on a recombinant plasmid. Furthermore, transport activity is less sensitive to inactivation by N-ethylmaleimide, and strikingly, galactosyl 1-thio-beta-D-galactopyranoside affords no protection against inactivation. The findings suggest that although cys148 is essential for substrate protection against sulfhydryl inactivation, it is not obligatory for lactose: proton symport and that another sulfhydryl group elsewhere within the lac carrier protein may be required for full activity.

Site-directed mutagenesis of cysteine-148 in the lac permease of Escherichia coli: effect on transport, binding, and sulfhydryl inactivation.

By subjecting the lac y gene of Escherichia coli to oligonucleotide-directed, site-specific mutagenesis, Cys148 in the lac permease has been replaced with a Gly residue [Trumble, W. R., Viitanen, P. V., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1984) Biochem. Biophys. Res. Commun. 119, 860]. Recombinant plasmids bearing wild-type or mutated lac y were constructed and used to transform E. coli T184. Steady-state levels of lactose accumulation, the apparent Km for lactose under energized conditions, and the KD for p-nitrophenyl alpha-D-galactopyranoside are comparable in right-side-out vesicles containing wild-type or mutant permease. In contrast, the Vmax for lactose transport in vesicles containing mutant permease is significantly decreased. Although antibody binding studies reveal that vesicles from the mutant contain almost as much permease as wild-type vesicles, surprisingly only about one-fourth of the altered molecules bind p-nitrophenyl alpha-D-galactopyranoside with high affinity. Mutant permease is less sensitive to inactivation by N-ethylmaleimide, although the alkylating agent is still capable of completely inhibiting transport activity. Importantly, beta-galactosyl 1-thio-beta-D-galactopyranoside affords complete protection of wild-type permease against N-ethylmaleimide but has no protective effect whatsoever in the mutant. The rate of inactivation of wild-type and mutant permeases by N-ethylmaleimide is increased at alkaline pH and by the presence of a proton electrochemical gradient (interior negative and alkaline), and these phenomena are exaggerated in vesicles containing mutant permease. Finally, p-(chloromercuri)benzenesulfonate, which completely displaces bound p-nitrophenyl alpha-D-galactopyranoside from wild-type permease, does not affect binding in the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells.

Staphylococcus aureus expresses several surface proteins that promote adherence to host cell extracellular matrix proteins, including fibronectin (Fn). Since this organism has recently been shown to be internalized by nonprofessional phagocytes, a process that typically requires high-affinity binding to host cell receptors, we investigated the role of its Fn binding proteins (FnBPs) and other surface proteins in internalization by the bovine mammary gland epithelial cell line (MAC-T). Efficient internalization of S. aureus 8325-4 required expression of FnBPs; an isogenic mutant (DU5883), not expressing FnBPs, was reduced by more than 95% in its ability to invade MAC-T cells. Moreover, D3, a synthetic peptide derived from the ligand binding domain of FnBP, inhibited the internalization of the 8325-4 strain in a dose-dependent fashion and the efficiency of staphylococcal internalization was partially correlated with Fn binding ability. Interestingly, Fn also inhibited the internalization and adherence of S. aureus 8325-4 in a dose-dependent manner. In contrast to internalization, adherence of DU5883 to MAC-T was reduced by only approximately 40%, suggesting that surface binding proteins, other than FnBPs, can mediate bacterial adherence to cells. Adherence via these proteins, however, does not necessarily result in internalization of the staphylococci. An inhibitor of protein tyrosine kinase, genistein, reduced MAC-T internalization of S. aureus by 95%, indicating a requirement for a host signal transduction system in this process. Taken together, these results indicate that S. aureus invades nonprofessional phagocytes by a mechanism requiring interaction between FnBP and the host cell, leading to signal transduction and subsequent rearrangement of the host cell cytoskeleton.

Crystal structure of calsequestrin from rabbit skeletal muscle sarcoplasmic reticulum.

Calsequestrin, the major Ca2+ storage protein of muscle, coordinately binds and releases 40-50 Ca2+ ions per molecule for each contraction-relaxation cycle by an uncertain mechanism. We have determined the structure of rabbit skeletal muscle calsequestrin. Three very negative thioredoxin-like domains surround a hydrophilic center. Each monomer makes two extensive dimerization contacts, both of which involve the approach of many negative groups. This structure suggests a mechanism by which calsequestrin may achieve high capacity Ca2+ binding. The suggested mechanism involves Ca2+-induced collapse of the three domains and polymerization of calsequestrin monomers arising from three factors: N-terminal arm exchange, helix-helix contacts and Ca2+ cross bridges. This proposed structure-based mechanism accounts for the observed coupling of high capacity Ca2+ binding with protein precipitation.

Intracellular Staphylococcus aureus escapes the endosome and induces apoptosis in epithelial cells.

We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a Staphylococcus aureus mastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections. S. aureus cells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of S. aureus, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalization S. aureus escapes the endosome and induces apoptosis in nonprofessional phagocytes.

Ryanodine receptor expression in embryonic avian cardiac muscle.

The cardiac ryanodine receptor serves as a sarcoplasmic reticulum calcium release channel and contributes to the rise in cytosolic calcium necessary for contraction of the heart. We have investigated the presence and oligomeric form of the ryanodine receptor during embryonic development of the avian heart. A single isoform of the ryanodine receptor was identified both in adult and in embryonic cardiac tissue from Days 4 to 20 of development, using anti-receptor monoclonal antibodies in conjunction with [3H]ryanodine binding. The results of sucrose density gradient sedimentation analysis and [3H]ryanodine binding indicated that the cardiac ryanodine receptor is present in a tetrameric form in both the adult and embryos at Day 6 of development. The observation of specific [3H]ryanodine binding in hearts from Days 4 and 5 of embryonic development also indicates the presence of a tetrameric receptor protein. Although the heart begins to beat at approximately 33-38 hr (Day 1.5) of embryonic development, we were unable to detect the cardiac ryanodine receptor, using biochemical or immunological techniques, prior to Embryonic Day 4. Ryanodine was found to alter the chronotropic state of intact hearts as early as Hamburger and Hamilton stages 15-19 (Embryonic Day 3) suggesting that receptor protein may be present in a limited subset of cells involved in pacemaker activity in the very early embryo. After Embryonic Day 4, ryanodine also exerted a negative inotropic effect on embryonic hearts. Our results suggest that a single isoform of the ryanodine receptor is present from Day 4 to Day 20 of embryonic development in avian cardiac muscle and that the ryanodine receptor assumes a tetrameric structure capable of forming a functional calcium release channel that participates excitation-contraction coupling as early as Embryonic Day 4. In addition, a ryanodine receptor-related function may serve as a determinant of chronotropic effects in the very early embryonic heart.

Staphylococcus aureus Agr and Sar global regulators influence internalization and induction of apoptosis.

Staphylococcus aureus was recently shown to be internalized by and to induce apoptosis in a bovine mammary epithelial cell line, suggesting that these processes could be involved in staphylococcal pathogenesis or persistence. To examine the role of virulence factor regulators during internalization, mutant agr and sar strains of S. aureus were analyzed for their abilities to enter and induce apoptosis in epithelial cells. Like a previously characterized bovine mastitis isolate, the standard laboratory strain, RN6390 (wild type), entered the epithelial cells and subsequently induced apoptosis. In contrast, the mutant strains RN6911 (agr), ALC136 (sar), and ALC135 (agr sar) were internalized by the cultured cells at levels reproducibly greater than that for RN6390 but failed to induce apoptosis. The internalization of S. aureus was affected by growth phase, suggesting a role for agr-regulated surface proteins in this process. Furthermore, the ability to induce apoptosis required metabolically active intracellular bacteria. These data indicate that the ability of S. aureus to enter mammalian cells and induce apoptosis is dependent on factors regulated by Agr and Sar. Since transcriptional control by these global regulators is mediated by quorum-sensing and environmental factors, staphylococci may have the potential to induce several alternative effects on cells from an intracellular environment. A model for the function of the agr locus in the context of internalization, intracellular persistence, and dissemination is proposed.