Letter to the Editor: 1H, 15N and 13C NMR Resonance Assignments of Staphostatin A, a Specific Staphylococcus Aureus Cysteine Proteinase Inhibitor

The increasing antibiotic resistance of an important human pathogen Staphylococcus aureus calls for the development of new therapeutic strategies. Staphylococcal cysteine proteases have been suggested as targets for such therapies. The recent discovery of staphostatins, specific protein inhibitors of these enzymes, gives prospects for the design and production of synthetic, low molecular weight analogs which might become drugs. We have decided to structurally characterize staphostatin A, a representative inhibitor of staphylococcal cysteine proteases, and to assess its binding mode to the target protease with the view of clarifying the specificity determinants. Here we report the (1)H, (15)N and (13)C NMR resonance assignments of staphostatin A.     

Lethality in LPS-induced endotoxemia in C3H/HeCr mice is associated with prevalence of proinflammatory cytokines: lack of protective action of lactoferrin

C3H/HeCr mice are more susceptible to infection compared with other strains. Lactoferrin (LF), a protein involved in innate defense, was shown to protect mice against lethal endotoxemia. In this investigation we attempt to explain the cause of increased susceptibility of C3H/HeCr mice to LPS and lack of protective LF action in these mice. We found that C3H/HeCr mice produced up to 5-fold more serum TNFalpha and 66% higher IFNgamma levels in response to i.v. LPS injection than the control, CBA strain. 24 h pretreatment of C3H/HeCr mice with LF did not cause inhibition of the LPS-induced TNFalpha serum levels, whereas in CBA mice LF significantly decreased TNFalpha level. IL-6 serum levels, in turn, were lowered in C3H/HeCr mice but elevated in CBA mice. That differential regulation of cytokine production by LF in C3H/HeCr mice paralleled a decreased survival after lethal LPS injection - 10% vs. 60% in control, PBS treated mice. In addition, determination of colony forming units (CFU) in livers and spleens after administration of 10(8) Escherichia coli revealed that pretreatment of CBA mice with LF caused a marked reduction of CFU in these organs, whereas in C3H/HeCr mice the changes were insignificant. These results indicate that the altered TNFalpha/IL-6 ratio in C3H/HeCr mice, as compared to control CBA mice, as well as the increased IFNgamma level, may be responsible for the increased susceptibility to endotoxemia in that substrain. We also suggest that an association exists between the LF protective effect against endotoxic sequelae and the insult-induced systemic immune response.     

Organization of repetitive DNAs and the genomic regions carrying ribosomal RNA,<Emphasis Type="Italic"> cob</Emphasis>, and<Emphasis Type="Italic"> atp9</Emphasis> genes in the cucurbit mitochondrial genomes

Plants in the genus Cucumis (cucumber and melon) have the largest mitochondrial genomes known among all plants, due in part to the accumulation of repetitive DNAs of varying complexities. Recombination among these repetitive DNAs should produce highly rearranged mitochondrial genomes relative to the smaller mitochondrial genomes of related plants. We cloned and sequenced mitochondrial genomic regions near the rRNA, atp9 and cob genes from cucumber, melon, squash and watermelon (all members of the Cucurbitaceae family), and compared to the previously sequenced mitochondrial genomes of Arabidopsis thaliana and sugar beet to study the distribution and arrangement of coding and repetitive DNAs. Cucumber and melon had regions of concentrated repetitive DNAs spread throughout the sequenced regions; few repetitive DNAs were revealed in the mitochondrial genomes of A. thaliana, sugar beet, squash and watermelon. Recombination among these repetitive DNAs most likely produced unique arrangements of the rrn18 and rrn5 genes in the genus Cucumis. Cucumber mitochondrial DNA had more pockets of dispersed direct and inverted repeats than melon and the other plants, and we did not reveal repetitive sequences significantly contributing to mitochondrial genome expansion in both cucumber and melon.Disclaimer. Names are necessary to report factually on available data; however, the U.S. Department of Agriculture (USDA) neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.Communicated by R. Hagemann     

Relationships between glutamine, glutamate, and GABA in nerve endings under Pb-toxicity conditions

Glutamine (Gln), glutamate (Glu) and gamma-amino butyric acid (GABA) are essential amino acids for brain metabolism and function. Astrocytic-derived glutamine is the precursor of the two most important neurotransmitters: glutamate, an excitatory neurotransmitter, and GABA, an inhibitory neurotransmitter. In addition to their roles in neurotransmission these neurotransmitters act as alternative metabolic substrates that enable metabolic coupling between astrocytes and neurons. The relationships between Gln, Glu and GABA were studied under lead (Pb) toxicity conditions using synaptosomal fractions obtained from adult rat brains to investigate the cause of Pb neurotoxicity-induced seizures. We have found that diminished transport of [(14)C]GABA occurs after Pb treatment. Both uptake and depolarization-evoked release decrease by 40% and 30%, respectively, relative to controls. Lower expression of glutamate decarboxylase (GAD), the GABA synthesizing enzyme, is also observed. In contrast to impaired synaptosomal GABA function, the GABA transporter GAT-1 protein is overexpressed (possibly as a compensative mechanism). Furthermore, similar decreases in synaptosomal uptake of radioactive glutamine and glutamate are observed. However, the K(+)-evoked release of Glu increases by 20% over control values and the quantity of neuronal EAAC1 transporter for glutamate reaches remarkably higher levels after Pb treatment. In addition, Pb induces decreased activity of phosphate-activated glutaminase (PAG), which plays a role in glutamate metabolism. Most noteworthy is that the overexpression and reversed action of the EAAC1 transporter may be the cause of the elevated extracellular glutamate levels. In addition to the impairment of synaptosomal processes of glutamatergic and GABAergic transport, the results indicate perturbed relationships between Gln, Glu and GABA that may be the cause of altered neuronal-astrocytic interactions under conditions of Pb neurotoxicity.     

Limb lymph node response to bone fracture

In previous clinical studies, dilation of afferent lymphatics and enlargement of inguinal lymph nodes (LN) were observed in lymphoscintigrams from patients with persistent posttraumatic edema of lower extremities after fractures and trauma of soft tissues. In this study, changes in rat popliteal and iliac lymph nodes draining lymph from the site of tibial fracture and adjacent soft tissue injury were investigated. The observed parameters were lymph node weight, cell number, phenotype frequency, cell cytokine expression, and reactivity to mitogens. The key observations included: a) increase in the weight and total cell number of the lymph nodes; b) increased autotransformation rate and responsiveness of lymph node cells to mitogen; c) decreased frequency of ED1 macrophages and activated OX8 cytotoxic cells in flow cytometry analysis; d) high expression of OX6 class II-positive, OX7 (stem cells), OX62 (migrating dendritic cells), ED1 (macrophages), and OX12 (B cells) on immunohistochemical sections of LNs with some few HIS48 (granulocytes); e) high expression of NOS3 and TGF beta by lymph node lymphocytes and endothelial cells. In summary, local lymph nodes reacted to internal wounds, such as bone fracture and injury to adjacent tissues, through mobilization of cells from the blood circulation, along with activation of cellular subsets. The molecular mechanism that provides the signal for this reaction remains unknown. The absence of major changes in the frequency of lymph node cell subpopulations indicates that lymph nodes are constitutively prepared for influx of antigens from damaged tissues and react only with increase in cell number and cell activation. The nature of the reaction, including lack of immunization against autoantigens, remains unclear. Further elucidation will require studies on the mechanism of cross-tolerance to self-antigens during wound healing.     

Determination of new derivatives of genistein in culture media by liquid chromatography

Methods for determination of genistein and its four new analogues in culture media have been developed to support studies on their potential anticancer activities. The investigated compounds were extracted from the media using liquid-liquid extraction with appropriate solvent. After evaporation of organic solvents each of the dry extracts was reconstituted in appropriate mobile phase. Reversed-phase HPLC was applied to quantitative determining of tested compounds. The methods are specific, sensitive and technically simple. They were used to evaluate concentration level of investigated compounds in experiments with human promyelocytic leukemia cells (HL-60 cell line).     

Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family

Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties of Staphylococcus epidermidis staphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinant S. epidermidis staphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases from S. aureus cleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue, S. aureus staphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by non-target cysteine proteases. Conversely, S. aureus staphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases.     

Alteration of the disulfide-coupled folding pathway of BPTI by circular permutation

The kinetics of disulfide-coupled folding and unfolding of four circularly permuted forms of bovine pancreatic trypsin inhibitor (BPTI) were studied and compared with previously published results for both wild-type BPTI and a cyclized form. Each of the permuted proteins was found to be less stable than either the wild-type or circular proteins, by 3-8 kcal/mole. These stability differences were used to estimate effective concentrations of the chain termini in the native proteins, which were 1 mM for the wild-type protein and 2.5 to 4000 M for the permuted forms. The circular permutations increased the rates of unfolding and caused a variety of effects on the kinetics of refolding. For two of the proteins, the rates of a direct disulfide-formation pathway were dramatically increased, making this process as fast or faster than the competing disulfide rearrangement mechanism that predominates in the folding of the wild-type protein. These two permutations break the covalent connectivity among the beta-strands of the native protein, and removal of these constraints appears to facilitate direct formation and reduction of nearby disulfides that are buried in the folded structure. The effects on folding kinetics and mechanism do not appear to be correlated with relative contact order, a measure of overall topological complexity. These observations are consistent with the results of other recent experimental and computational studies suggesting that circular permutation may generally influence folding mechanisms by favoring or disfavoring specific interactions that promote alternative pathways, rather than through effects on the overall topology of the native protein.     

The acrAB locus is involved in modulating intracellular acetyl coenzyme A levels in a strain of Escherichia coli CM2555 expressing the chloramphenicol acetyltransferase (cat) gene

Recently, an Escherichia coli CM2555 strain was described as sensitive to chloramphenicol when expressing the chloramphenicol resistance gene (cat) from a multicopy plasmid. This sensitivity was linked to dysfunction of the acrA gene, which encodes a component of the AcrAB-TolC multidrug efflux pump. Preliminary data indicate that the sensitivity phenotype might be due to a decline in intracellular acetyl coenzyme A concentration accompanying the reaction catalyzed by chloramphenicol acetyltransferase, the cat-encoded resistance protein. Here, we demonstrate that the acrA dysfunction is the factor impairing the intracellular acetyl coenzyme A levels in the cat-expressing CM2555 strain. This effect might be alleviated by the interplay of proteins constituting two homologous efflux systems: AcrAB-TolC and AcrEF-TolC. However, our results show also that this is a genetic background-specific phenomenon, as the decrease in acetyl coenzyme A level is not evident in a cat-bearing acrAB derivative of the commonly used strain C600.