RiPP enzyme heterocomplex structure-guided discovery of a bacterial borosin α-N-methylated peptide natural product.

Amide peptide backbone methylation is a characteristic post-translational modification found in a family of ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) called borosins. Previously, we bioinformatically identified >1500 putative borosin pathways in bacteria; however, none of the pathways were associated with a known secondary metabolite. Through in-depth characterization of a borosin pathway in Shewanella oneidensis MR-1, we have now identified a bacterially derived borosin natural product named Shewanellamide A. Borosin identification was facilitated by the creation and analysis of a series of precursor variants and crystallographic interrogation of variant precursor and methyltransferase complexes. Along with assaying two proteases from S. oneidensis, probable boundaries for proteolytic maturation of the metabolite were projected and confirmed via comparison of S. oneidensis knockout and overexpression strains. All in all, the S. oneidensis natural product was found to be a 16-mer linear peptide featuring two backbone methylations, establishing Shewanellamide A as one of the few borosin metabolites yet identified, and the first from bacteria.

Suppressive effects of selectin inhibitor SKK-60060 on the leucocyte infiltration during endotoxin induced uveitis.

BACKGROUND: It is well known that selectin is involved in the development of endotoxin induced uveitis (EIU), and has a major role in leucocyte infiltration. Recently, a novel selectin inhibitor (SKK-60060) that can block P and L selectins in vitro has been developed. This study was designed to investigate the anti-inflammatory effects of SKK-60060 on the inflammatory reaction during EIU in rats by studying leucocyte-endothelium interactions. METHODS: EIU was induced in Lewis rats by footpad injection of lipopolysaccharide (LPS). SKK-60060 was administered 15 minutes before LPS injection, and its suppressive effects on inflammatory leucocyte behaviour were evaluated in vivo with acridine orange digital fluorography; the diameters of retinal arteries and veins were also measured. After these studies, aqueous humour was collected to evaluate leucocyte infiltration and protein leakage. RESULTS: After LPS injection, rolling leucocytes were observed in major retinal veins, followed by leucocyte infiltration into the vitreous cavity. Following treatment with SKK-60060, leucocyte rolling was significantly inhibited in the retinal veins (p <0.01), and subsequent leucocyte infiltration into the vitreous cavity was also significantly suppressed (p <0.01). Retinal vasodilation was also substantially suppressed in SKK-60060 treated rats (p <0.01). Similarly, leucocyte infiltration and protein leakage into the aqueous humour were reduced significantly by SKK-60060 (p <0.01). CONCLUSIONS: SKK-60060 treatment significantly inhibited the inflammatory reaction induced by LPS. Its inhibitory effects on P and L-selectin resulted in suppression of leucocyte infiltration and the subsequent inflammatory reaction caused by accumulated leucocytes. The current findings suggest that SKK-60060 may be useful in the management of uveitis.

Effects of a new synthetic selectin blocker in an acute rat thrombotic glomerulonephritis.

In an attempt to explore a novel therapeutic approach, a new synthetic sulfatide derivative (SKK60037) was evaluated in an acute rat model of P-selectin and leukocyte-dependent thrombotic glomerulonephritis (TG). In vitro, SKK60037 inhibits the function of P- and L-selectin more effectively than sialyl Lewis X (sLe(x)), a well-established selectin blocker. TG was induced by the intravenous administration of nephrotoxic globulin (NTG) to rats pretreated with a subclinical dose of lipopolysaccharide. In this model, platelet accumulation was remarkable within 10 minutes after induction of disease, followed by the infiltration of leukocytes, mainly neutrophils and macrophages. Thrombus formation and fibrinogen deposition in the glomeruli were observed within 1 hour, and they proceeded until 6 hours. P-selectin was highly expressed in glomeruli, whereas E-selectin and L-selectin ligands were not detected. We tested the effects of SKK60037 in this model in comparison with sLe(x) and antirat P-selectin monoclonal antibody (ARP2-4). SKK60037 blocked platelet accumulation in glomerular capillaries at 10 minutes after NTG injection. At 6 hours, leukocyte infiltration and thrombosis were significantly suppressed. Protective effects of SKK60037 were similar to those of ARP2-4, whereas sLe(x) showed minimum effect. The superior effects and more favorable characteristics of SKK60037 to sLe(x) suggest the potential of SKK60037 for clinical application.

Protective effects of selectin ligands/inhibitor (SKK-60060) against retinal ischemia-reperfusion injury.

A newly developed selSep;71(3)28 to block P- and L-selectins in vitro. We examined its inhibition of leukocyte-endothelial interactions in vivo against retinal ischemia-reperfusion injury and protective effects on ischemia-induced retinal damage. Retinal ischemia was induced by temporary ligation of the optic sheath for 60 min in anesthetized pigmented rats. SKK-60060 was administered 5 min before reperfusion and 4, 12, 24 and 48 hr thereafter, and leukocyte dynamics in the retinal microcirculation were evaluated using acridine orange digital fluorography. After 7 days of reperfusion, ischemia-induced retinal damage was also assessed histologically.SKK-60060 treatment suppressed leukocyte rolling during the reperfusion period; their numbers in the SKK-60060-treated rats were reduced by 67.0% (P < 0. 01) and 53.2% (P < 0.01) at 12 and 24 hr, respectively. The subsequent leukocyte accumulation was also inhibited in SKK-60060-treated rats; accumulated leukocytes in the SKK-60060-treated rats were reduced by 72.8% (P < 0.01) and 53.4% (P < 0.01) at 12 and 24 hr, respectively. Retinal venous vasodilation in SKK-60060-treated rats were significantly suppressed at each time point (P < 0.05). Histological examination demonstrated protective effects of SKK-60060 on ischemia-induced retinal damage, which were more substantial in the inner retina (P < 0.01).SKK-60060 significantly inhibits the leukocyte rolling along the major retinal veins and their accumulation during the reperfusion period. These results suggest therapeutic potential of SKK-60060 for ischemia-reperfusion injury.

Glucose intolerance caused by a defect in the entero-insular axis: a study in gastric inhibitory polypeptide receptor knockout mice.

Mice with a targeted mutation of the gastric inhibitory polypeptide (GIP) receptor gene (GIPR) were generated to determine the role of GIP as a mediator of signals from the gut to pancreatic beta cells. GIPR-/- mice have higher blood glucose levels with impaired initial insulin response after oral glucose load. Although blood glucose levels after meal ingestion are not increased by high-fat diet in GIPR+/+ mice because of compensatory higher insulin secretion, they are significantly increased in GIPR-/- mice because of the lack of such enhancement. Accordingly, early insulin secretion mediated by GIP determines glucose tolerance after oral glucose load in vivo, and because GIP plays an important role in the compensatory enhancement of insulin secretion produced by a high insulin demand, a defect in this entero-insular axis may contribute to the pathogenesis of diabetes.

Synthetic studies on selectin ligands/inhibitors. Synthesis and biological evaluation of sulfated and phosphorylated beta-D-galacto- and lactopyranosides containing fatty-alkyl residues of different carbon chain lengths.

To investigate the biological selectin-ligand interactions, fourteen sulfated and eight phosphorylated beta-D-galacto- and lactopyranosides containing branched fatty-alkyl residues in place of the ceramide have been synthesized. Regioselective sulfation of the parent glycolipids through the dibutylstannylene acetal with a certain amount of sulfur trioxide-trimethylamine complex produced the target sulfated glycolipids, while stepwise phosphorylation by treatment of the properly protected diol with dibenzyloxy(diisopropylamino)phosphine gave the phosphorylated glycolipids. The synthetic glycolipids showed an interesting mode of inhibition of the binding of HL-60 cells to immobilized P-, L- and E-selectins during in vitro experiments. In addition, using computer modeling techniques, we examined the molecular basis for the ligand-selectin complex formation. These glycolipids may be useful as therapeutic agents against selectin-dependent inflammation.

Structure-activity relationships of cyclic enediynes related to dynemicin A. I. Synthesis and antitumor activity of 9-acetoxy enediynes equipped with aryl carbamate moieties.

A series of the 9-acetoxy enediyne compounds, 6a-k which were simplified from natural dynemicin A, and designed to be equipped with various aryl carbamate moieties, was synthesized and evaluated for DNA-cleaving ability, in vitro cytotoxicity, and in vivo antitumor activity. As a result of this study of the structure-activity relationships (SAR) with regard to the Rt substituent, both compounds 6a and 6f with the phenyl carbamate and 4-chlorophenyl carbamate moiety, respectively, were found to exhibit significant activity (T/C > 200%) against murine P388 leukemia in mice, in spite of having IC50 values in the micromolar range. In particular, compound 6f showed the most potent activity with a maximum T/C of 256% at a daily dosage of 4.0 mg/kg for four days. Furthermore, both compounds 6a and 6f were effective against Meth A sarcoma in mice and inhibited 71 and 77% of the tumor growth at 2.0 and 3.0 mg/kg dosages, respectively. In contrast to 6f, compound 6i possessing the 2-nitrophenyl carbamate moiety showed only a slight in vivo activity, while it had about one order of magnitude higher in vitro cytotoxicity than 6f. For the stereochemistry-activity relationships at the C9 position, the (9R*)-isomers of 6c, 6g, and 6j were found to show higher in vitro and in vivo potencies than the corresponding (9S*)-isomers.

Structure-activity relationships of cyclic enediynes related to dynemicin A. II. Synthesis and antitumor activity of 9- and 12-substituted enediynes equipped with aryl carbamate moieties.

Novel enediyne compounds 4-8, simple analogues of dynemicin A (1) equipped with the phenyl or 4-chlorophenyl carbamate moiety, were synthesized and evaluated for DNA-cleaving ability, in vitro cytotoxicity, and in vivo antitumor activity. As a result of the SAR study, it was revealed that the size and character of the substituents (R1 and R2) at the C9 position critically influenced both the stability and antitumor activity of the enediyne compounds. We found that the 9-deoxy compound 6a, a stable and less bulky enediyne having a hydrogen as the R1 and R2 substituents, showed a significant in vivo activity with a T/C of 215% at a daily dosage of 2.0 mg/kg for 4 days. The incorporation of an oxygen-containing functional group as the R3 substituent on a benzene ring resulted in considerable abolishing of both the in vitro and in vivo potencies. In a series of 9-acyloxy compounds, incorporation of the basic aromatic moiety such as 8e was effective for the in vitro activity, but it was ineffective for the in vivo activity. Furthermore, for the stereochemistry-activity relationships at the C9 position, the (9R*)-isomers of 8c, 8e, and 8f were found to show higher both in vitro and in vivo than the corresponding (9S*)-isomers. For the mechanistic studies, compound 6a underwent Bergman cycloaromatization via a diradical pathway under acidic conditions, whereas it scarcely showed DNA-cleaving activity due to the chemical stability of the aryl carbamate moiety under neutral conditions.

Synthesis and antitumor activity of water-soluble enediyne compounds related to dynemicin A.

The enediyne compounds 9-14, simple dynemicin A (1) analogues equipped with aryl carbamate moieties with various aliphatic amino or hydroxy groups at the C9 position, were synthesized and evaluated for DNA-cleaving ability, in vitro cytotoxicity, and in vivo antitumor activity. We found that the water-soluble compounds, in which the tert-amines such as the 2-(dimethylamino)ethyl (10b, 14b), 2-(pyrrolidino)ethyl (10c), or 1-azabicyclo[3.3.0]oct-5-ylmethyl (10d, 12d, 14d) group were attached, showed not only the enhanced in vivo antitumor activity but also the decreased toxicity compared to the corresponding 9-acetoxy enediyne compounds 6-8. In particular, compound 10c showed the most enhanced in vivo antitumor activity (T/C = 222% at a daily dose of 1.25 mg/kg for 4 days) at about half of the dose of 6. These results suggest that both the enhanced antitumor activity and the reduced toxicity might be due to the improved bioavailability or disposition of compounds 6-8 by their water-solubilization.

Synthesis and biological evaluation of novel cyclic enediyne compounds related to dynemicin A as antitumor agents.

Novel cyclic enediyne compounds, which are simple functional analogs of dynemicin A (1) having the bicyclo-[7.3.1]tridec-4-ene-2,6-diyne system, were synthesized and evaluated for the DNA-cleaving ability, in vitro cytotoxicity and in vivo antitumor activity. All of the sulfones 19-24, which were equipped with a 2-(arylsulfonyl)-ethoxycarbonyl group or the 2-(methylsulfonyl)ethoxycarbonyl group as a triggering device, showed both potent DNA-cleaving activity and cytotoxicity against various tumor cell lines. However, these compounds were entirely inactive or only slightly active against murine P388 leukemia in mice. On the other hand, the enediyne 2a having a phenyl carbamate moiety as a stable N-protecting group showed effective antitumor activity both in vitro and in vivo. In particular, it exhibited significant antitumor activity against Lewis lung carcinoma in mice. These results show that the character of the carbamate moiety of the cyclic enediynes strikingly affects their biological activities, that is, the sulfonylethyl carbamate moiety is an effective triggering device for both DNA-cleaving activity and cytotoxicity, and the phenyl carbamate moiety is significant for antitumor activity in vivo. As part of a mechanistic study, the reactivities of 2a and 21 were examined under a weakly basic condition (pH 9.3); both compounds failed to give the Bergman cycloaromatization product.

A simple single-tube procedure of PCR assay for the detection of hepatitis C virus RNA.

We developed a simple single tube procedure using the PCR (polymerase chain reaction) for the detection of hepatitis C virus RNA. The entire reaction from RNA extraction to PCR occurred in one tube; this was made possible by the use of more than 1,000 micrograms/ml of proteinase K for RNA extraction, instead of the acid-guanidinium thiocyanate-phenol-chloroform method. All necessary reagents were added to the tube, and PCR products were not removed from the tube until the end of PCR, although opening of the tube during the procedure could not be avoided. Therefore, cross-contamination which might theoretically take place during transfer of products between tubes never occurred. This method detected about 1 chimpanzee infectious dose of HCV in H strain plasma.

Quantitation and typing of serum hepatitis C virus RNA in patients with chronic hepatitis C treated with interferon-beta.

We quantified serum hepatitis C virus RNA titers and determined hepatitis C virus subtypes in chronic hepatitis C patients treated with interferon-beta to investigate relationships among serum ALT response, serum hepatitis C virus titer and hepatitis C virus subtype. Of 146 chronic hepatitis C patients who received interferon-beta therapy, 24 patients with sustained serum ALT normalization (complete responders) and 26 patients without serum ALT normalization (nonresponders) were randomly selected. Detection, typing and quantitation of hepatitis C virus were performed by means of the "single-tube" polymerase chain reaction method. Of the 24 complete responders, 21 (87.5%) became negative for hepatitis C virus RNA, whereas 21 (80.8%) of the 26 nonresponders remained positive. Hepatitis C virus infections with types I, II, III, IV, II + III and III + IV occurred in 0 (0%), 22 (51.2%), 10 (23.3%), 1 (2.3%), 7 (16.5%) and 3 (7.9%) patients, respectively. The mean pretreatment hepatitis C virus RNA titer of complete responders (0.4 +/- 2.0 x 10(4) CID50/ml) was significantly lower than that of nonresponders (3.8 +/- 4.5 x 10(4) CID50/ml) (p < 0.01). Regardless of HCV subtype, patients with more than 10(4) CID50/ml of HCV did not show serum ALT normalization, whereas complete serum ALT response was seen in most cases with less than 10(2) CID50/ml HCV. These results show that mixed infections with different hepatitis C virus subtypes appear to be more common than previously reported and that the pretreatment serum level of hepatitis C virus RNA is a more important predictor of outcome of interferon therapy than is virus genotype.

Function of the lipopolysaccharide-binding protein of Periplaneta americana as an opsonin.

Previously, we reported the purification of an LPS-binding protein from the hemolymph of the American cockroach that was specific for E. coli LPS. In this study we found that this protein participated in the clearance of E. coli cells injected into the abdominal cavity of the cockroach, and that hemocytes ingested E. coli cells treated with this LPS-binding protein in vitro. These findings suggest that this LPS-binding protein acts as an opsonin.

Molecular cloning of cDNA for lipopolysaccharide-binding protein from the hemolymph of the American cockroach, Periplaneta americana. Similarity of the protein with animal lectins and its acute phase expression.

A previous paper described the purification of a calcium-dependent lipopolysaccharide-binding protein from the hemolymph of Periplaneta americana (Jomori, T., Kubo, T., and Natori, S. (1990) Eur. J. Biochem. 190, 201-206). This paper describes the molecular cloning and characterization of cDNA for the LPS-binding protein. This protein was found to have a carbohydrate-recognition domain at its carboxyl terminus containing amino acid sequences that are conserved in various mammalian C-type lectins. It was also shown to contain an N-linked carbohydrate chain, and the amino acid residue carrying this chain was assigned as Asn at position 56 (23rd amino acid residue from the amino terminus). Northern blot analysis revealed the presence of multiple mRNAs that hybridized with this cDNA and transient increases in their content after injection of Escherichia coli into adult Periplaneta, suggesting that the LPS-binding protein plays a role in the acute phase response of this insect.

Purification and characterization of lipopolysaccharide-binding protein from hemolymph of American cockroach Periplaneta americana.

A protein having affinity to lipopolysaccharide of Escherichia coli K12 was purified to homogeneity from the hemolymph of Periplaneta americana. This protein, with an average molecular mass of 450 kDa. was a homooligomer of a 28-kDa subunit protein. Comparative studies using lipopolysaccharide molecules of E. coli and Salmonella minnesota suggested that this protein recognizes and binds to a specific carbohydrate structure of E. coli lipopolysaccharide. Ca2+ was required for this protein to bind to lipopolysaccharide, but other divalent cations could not replace Ca2+.