Synthesis, X-ray crystal structure, anti-fungal and anti-cancer activity of [Ag2(NH3)2(salH)2] (salH2=salicylic acid)

[Ag(2)(NH(3))(2)(salH)(2)] (salH(2)=salicylic acid) was synthesised from salicylic acid and Ag(2)O in concentrated aqueous NH(3) and the dimeric Ag(I) complex was characterised using X-ray crystallography. The complex is centrosymmetric with each metal coordinated to a salicylate carboxylate oxygen and to an ammonia nitrogen atom in an almost linear fashion. The two [Ag(NH(3))(salH)] units in the complex are linked by an Ag-Ag bond. Whilst metal-free salH(2) did not prevent the growth of the fungal pathogen Candida albicans [Ag(2)(NH(3))(2)(salH)(2)], [Ag(2)(salH)(2)] and some simple Ag(I) salts greatly inhibited cell reproduction. SalH(2), [Ag(2)(NH(3))(2)(salH)(2)] [Ag(2)(salH)(2)] and AgClO(4) produced a dose-dependent cytotoxic response against the three human derived cancer cell lines, Cal-27, Hep-G2 and A-498, with the Ag(I)-containing reagents being the most effective.     

Physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese

The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).     

Glucagon-like peptide 1 modulates calcium responses to glutamate and membrane depolarization in hippocampal neurons

Glucagon-like peptide 1 (GLP-1) activates receptors coupled to cAMP production and calcium influx in pancreatic cells, resulting in enhanced glucose sensitivity and insulin secretion. Despite evidence that the GLP-1 receptor is present and active in neurons, little is known of the roles of GLP-1 in neuronal physiology. As GLP-1 modulates calcium homeostasis in pancreatic beta cells, and because calcium plays important roles in neuronal plasticity and neurodegenerative processes, we examined the effects of GLP-1 on calcium regulation in cultured rat hippocampal neurons. When neurons were pre-treated with GLP-1, calcium responses to glutamate and membrane depolarization were attenuated. Whole-cell patch clamp analyses showed that glutamate-induced currents and currents through voltage-dependent calcium channels were significantly decreased in neurons pre-treated with GLP-1. Pre-treatment of neurons with GLP-1 significantly decreased their vulnerability to death induced by glutamate. Acute application of GLP-1 resulted in a transient elevation of intracellular calcium levels, consistent with the established effects of GLP-1 on cAMP production and activation of cAMP response element-binding protein. Collectively, our findings suggest that, by modulating calcium responses to glutamate and membrane depolarization, GLP-1 may play important roles in regulating neuronal plasticity and cell survival.     

Nutrient supply to dairy cows from processed white lupines

The objective of this study was to compare the DVE/OEB system (DVE = truly absorbed protein in the small intestine; OEB = degraded protein balance (DPB) in Dutch) and the NRC-2001 model in the prediction of supply of protein to dairy cows from processed white lupines (Lupinus albus L.). Comparisons were made in terms of (1) ruminally synthesized microbial CP, (2) truly absorbed protein in the small intestine, and (3) degraded protein balance. In addition, the systematic investigation of roasting of the white lupines at various temperatures (110, 130, or 150 degrees C) and times (15, 30 or 45 min) on manipulation of digestive behaviour and the potential nutrient supply to dairy cows were also carried out, to obtain information on best processing conditions as intestinal protein sources (to achieve target values for potential high net absorbable protein in the small intestine while holding any N loss in the rumen to a low level). The results showed that the predicted values from the DVE/OEB system and the NRC-2001 model had significant correlations with high R ( > 0.83) values. However, using the DVE/OEB system, the overall average microbial protein supply based on available energy was 11% higher and the truly absorbed protein in the small intestine was 7% higher than that predicted by the NRC-2001 model. The difference was also found in the prediction of the degraded protein balances (DPB), which was 8% higher based on data from the NRC-2001 model. These differences are due to considerably different factors used in calculations in the two models, although both are based on similar principles. This indicates that a further refinement is needed for a modern protein evaluation and prediction system. In addition, this study showed that the roasting at higher temperature and time was effective in shifting protein degradation from rumen to intestines and it increased the DVE or MP values without reaching the negative degraded protein balance. The processing at 15 degrees C for 30 or 45 min might be the best treatments for white lupine due to its higher DVE and MP values and the very low DPB values.     

The importance of the nine-amino acid C-terminal sequence of exendin-4 for binding to the GLP-1 receptor and for biological activity

Exendin-4, a 39-amino acid (AA) peptide, is a long-acting agonist at the glucagon-like peptide-1 (GLP-1) receptor. Consequently, it may be preferable to GLP-1 as a long-term treatment for type 2 diabetes mellitus. Exendin-4 (Ex-4), unlike GLP-1, is not degraded by dipeptidyl peptidase IV (DPP IV), is less susceptible to degradation by neutral endopeptidase, and possesses a nine-AA C-terminal sequence absent from GLP-1. Here we examine the importance of these nine AAs for biological activity of Ex-4, a sequence of truncated Ex-4 analogs, and native GLP-1 and GLP-1 analogs to which all or parts of the C-terminal sequence have been added. We found that removing these AAs from Ex-4 to produce Ex (1-30) reduced the affinity for the GLP-1 receptor (GLP-1R) relative to Ex-4 (IC50: Ex-4, 3.22+/-0.9 nM; Ex (1-30), 32+/-5.8 nM) but made it comparable to that of GLP-1 (IC50: 44.9+/-3.2 nM). The addition of this nine-AA sequence to GLP-1 improved the affinity of both GLP-1 and the DPP IV resistant analog GLP-1 8-glycine for the GLP-1 receptor (IC50: GLP-1 Gly8 [GG], 220+/-23 nM; GLP-1 Gly8 Ex (31-39), 74+/-11 nM). Observations of the cAMP response in an insulinoma cell line show a similar trend for biological activity.     

Divided genomes: negotiating the cell cycle in prokaryotes with multiple chromosomes

Historically, the prokaryotic genome was assumed to consist of a single circular replicon. However, as more microbial genome sequencing projects are completed, it is becoming clear that multipartite genomes comprised of more than one chromosome are not unusual among prokaryotes. Chromosomes are distinguished from plasmids by the presence of essential genes as well as characteristic cell cycle-linked replication kinetics; unlike plasmids, chromosomes initiate replication once per cell cycle. The existence of multipartite prokaryotic genomes raises several questions regarding how multiple chromosomes are replicated and segregated during the cell cycle. These divided genomes also introduce questions regarding chromosome evolution and genome stability. In this review, we discuss these and other issues, with particular emphasis on the cholera pathogen Vibrio cholerae.