Formation of persisters in Streptococcus mutans biofilms induced by antibacterial dental monomer.

Antibacterial monomers can combat oral biofilm acids and caries; however, little is known on whether quaternary ammonium monomers (QAMs) would induce drug persistence in oral bacteria. The objectives of this study were to investigate the interactions of Streptococcus mutans (S. mutans) with dimethylaminohexadecyl methacrylate (DMAHDM), and determine for the first time whether DMAHDM could induce persisters in S. mutans. DMAHDM was synthesized using a modified Menschutkin reaction. Dose-dependent killing curves and time-dependent killing curves of planktonic S. mutans and biofilms were determined to evaluate drug persistence, using chlorhexidine (CHX) as control. The inheritability assay, minimum inhibitory concentration (MIC) and live/dead biofilm assay were determined to investigate persister characteristics. DMAHDM matched the killing potency of the gold standard CHX against S. mutans biofilms. DMAHDM and CHX induced drug persistence in S. mutans biofilms but not in planktonic bacteria. S. mutans biofilm persistence was not inheritable in that the tolerance to DMAHDM or CHX of the surviving persisters in the initial population was not transferred to subsequent generations, as displayed by the inheritability assay. The MIC of S. mutans parental strain and induced persisters remained the same. The induced persisters in S. mutans biofilms could be eliminated via higher doses of 300 µg/mL of DMAHDM and CHX. In conclusion, this study showed for the first time that (1) DMAHDM induced persisters only in biofilms, but not in planktonic bacteria; and (2) both DMAHDM-induced and CHX-induced S. mutans persister biofilms could be completely eradicated by even higher concentrations of DMAHDM and CHX. More studies are needed on the induction of persisters in oral biofilms for the development and use of a new generation of antibacterial dental monomers and resins.

2-Substituted-2-amino-6-boronohexanoic acids as arginase inhibitors.

Substitution at the alpha center of the known human arginase inhibitor 2-amino-6-boronohexanoic acid (ABH) is acceptable in the active site pockets of both human arginase I and arginase II. In particular, substituents with a tertiary amine linked via a two carbon chain show improved inhibitory potency for both enzyme isoforms. This potency improvement can be rationalized by X-ray crystallography, which shows a water-mediated contact between the basic nitrogen and the carboxylic acid side chain of Asp200, which is situated at the mouth of the active site pocket of arginase II (Asp181 in arginase I). We believe that this is the first literature report of compounds with improved arginase inhibitory activity, relative to ABH, and represents a promising starting point for further optimization of in vitro potency and the identification of better tool molecules for in vivo investigations of the potential pathophysiological roles of arginases.

A multifunctional in situ-forming hydrogel for wound healing.

In this study, a multifunctional in situ-forming hydrogel (MISG) was prepared as a wound dressing designed to stop bleeding, inhibit inflammation, relieve pain, and improve healing. A mixture of poloxamers 407 and 188 was used for the matrix of the MISG. Other ingredients include aminocaproic acid (to stop bleeding), povidone iodine (anti-infective), lidocaine (pain relief), and chitosan (to enhance wound healing and regeneration). The incipient gelation temperature of the MISG was modified by varying the poloxamer concentration. Poloxamer cytotoxicity was evaluated in addition to the effect of the MISG on hemostasis in rabbits, pain relief in mice, bacteriostasis in vitro, and wound healing. The optimal MISG matrix consisted of 30% (w/v) poloxamer (407/188, 1 : 1, w/w) solution and was able to change to a gel within 10 minutes at 37 °C. The poloxamer solution had no cytotoxicity in fibroblasts. Compared to sterile gauze alone, the MISG significantly shortened average hemostasis time and decreased bleeding. The hydrogel showed strong bacteriostatic action similar to povidone iodine solution. It markedly increased the pain threshold and accelerated wound healing compared to the gauze. The MISG is a promising formulation for wound healing in emergency situations.

Role of the ventromedial nucleus of the thalamus in motor behaviour--I. Effects of focal injections of drugs.

An assortment of drugs was injected into one or both ventromedial nuclei of the thalamus, to see how these influenced stereotypy, locomotion and posture in spontaneously behaving and actively rotating rats. Unilateral intrathalamic muscimol promoted weak ipsiversive circling, while bilateral treatment gave catalepsy. Similar injections of 4-amino-hex-5-enoic acid, which inhibits gamma-aminobutyrate metabolism, raised gamma-aminobutyrate levels in the ventromedial nuclei more than three-fold yet had none of these behavioural effects. The indirectly acting gamma-aminobutyrate agonists flurazepam and cis-1,3-aminocyclohexane carboxylic acid had little effect on posture and locomotion and, like muscimol and 4-amino-hex-5-enoic acid, elicited only very weak stereotypies. Procaine behaved like the gamma-aminobutyrate antagonist bicuculline, provoking vigorous locomotor hyperactivity and teeth chattering if given uni- or bilaterally. Pretreatment of one ventromedial nucleus with muscimol or 4-amino-hex-5-enoic acid, and to a lesser extent flurazepam or cis- 1,3-aminocyclohexane carboxylic acid, gave rise to pronounced ipsilateral asymmetries when combined with a large systemic dose of apomorphine. Contraversive rotations were initiated by unilateral stereotaxic injection of muscimol into the substantia nigra pars reticulata, or with apomorphine from the supersensitive striatum in unilaterally 6-hydroxydopamine lesioned rats. Drug treatments in the ipsilateral ventromedial nucleus showed a similar rank order of potency at inhibiting these circling behaviours, seemingly by reducing apomorphine-induced posture and muscimol-induced hypermotility. The suppression of circling by muscimol in these tests was highlighted by introducing the compound into the ventromedial nucleus at the height of circling activity. Both types of circling stimulus lost the capacity to increase locomotion, but still caused head turning and stereotypy in rats made cataleptic with bilateral ventromedial muscimol. Treating one ventromedial thalamus with muscimol greatly intensified any pre-existing posture directed towards that side, and vice versa. These data suggest that the ventromedial nucleus is not involved with the expression of stereotyped behaviours, but can profoundly influence posture and locomotion, especially in the presence of some other motor stimulus. The recovery of circus movements in rats with impaired ventromedial nucleus function implies this nucleus is not essential for the execution of circling in these models.

A study of two methods for estimating plasma fibrinogen and the effect of epsilon aminocaproic acid and protamine.

Fibrinogen concentrations were determined in normal plasma and in plasma from patients with high and low levels. There was a good correlation between the results of a rapid semi-quantitative fibrinogen titre technique and those of a quantitative assay of coagulable fibrinogen. In normal subjects fibrinogen levels were not significantly influenced by taking blood into epsilon aminocaproic acid (EACA) or by the addition of protamine to plasma. In patients with the defibrination syndrome in whom increased plasma fibrinolysis was not detected, fibrinogen levels were not affected by taking blood into EACA but considerably increased levels were observed after the addition of protamine to plasma. In patients undergoing thrombolytic therapy the fibrinogen levels measured were increased both in blood taken into EACA and in plasma containing protamine. It is suggested that EACA acted by preventing lysis in vitro whilst protamine counteracted abnormal fibrin polymerization. The pattern of results may be of diagnostic importance.

Extended experimental and preliminary surgical findings with autologous fibrin tissue adhesive made from patients own blood.

A surgical tissue adhesive can be made from the patient's own blood. We have been refining the procedures for manufacturing autologous fibrin tissue adhesive to facilitate its use in the operating room and to increase its bonding strength. Fibrin tissue adhesive efficacy depends on fibrinogen concentration. We found that fibrinogen precipitation using the ammonium sulfate method produced the highest concentration. Bonding power was compared with that of the commercial glue 10 minutes and 30 minutes after glueing two pieces of 1 X 1 cm2 human dura together. Bonding strength of the autologous product was close to that of the commercial product. Comparisons of fibrinolysis inhibition time of autologous fibrin tissue adhesive and commercial glue in experiments on rats over a period of one hour to six days after subcutaneous injection are described.

Esterases in human saliva.

A sensitive method for quantitative determination of esterase activity has been applied to human saliva and used for substrate and inhibitor studies. By means of gel isoelectrofocusing the whole saliva esterases were divided into two main groups, each resolved into several bands. Comparison of isoelectrophoretic zymograms of whole, submandibular and parotid saliva indicates that one of the groups originates from the submandibular fluid. With the aid of gel filtration and ion-exchange chromatography the saliva esterases were partially separated and purified. The classification of the main saliva esterases as carboxylesterases (EC 3.1.1.1) is discussed in terms of their substrate and inhibitor properties.

A rapid and sensitive 125I-fibrin solid-phase fibrinolytic assay for plasmin.

125I-fibrinogen, adsorbed to polystyrene tubes at low ionic strength and treated with thrombin, serves as a substrate for a rapid, convenient, and sensitive test tube assay for plasmin and activators and inhibitors of this enzyme. 125I-labeled digestion products released from the 125I-fibrin-polystyrene matrix are readily separated and quantitated and behave, on gel permeation, in the same manner as plasmin-generated degradation products from an unlabeled conventional fibrin clot. The 125I-fibrin, in probable non-cross-linked form, is firmly bound to the polystyrene and is resistant to nonspecific release, with control (no enzyme) values equivalent to 15.2 ng +/- 1.2 (SD) fibrin (1% of the total bound 125I-fibrin). This fact permits consistent detection of lysis of 30-50 ng 125I-fibrin, which exceeds published sensitivities (1000-5000 ng) using 125I- or fluorochrome-labeled fibrin clots as substrate. The sensitivity for plasmin (0.2 mug/ml) is tenfold greater than that of the fibrin-plate method (2.0-2.5 mug/ml), while sensitivities for streptokinase and urokinase activation of plasmin are 0.02 U/ml and 0.04 CTA U/ml, respectively (sensitivity of fibrin-plate method, 0.5 U/ml for both). The method provides a reasonable analogue of the solid-phase nature of fibrin under physiologic conditions, and the ease of preparation of large batches of tubes makes the method suitable for large-scale screening of factors modulating the plasminogen-plasmin system.

A new concept in the control of acute gingival hemorrhage.

The topical application of epsilon-aminocaproic acid may be life-saving therapy for control of acute gingival hemorrhage in patients who are debilitated because of systemic diseases. When low doses were used, there have been no reports of adverse reactions.

Solubilization of Escherichia coli nitrate reductase by a membrane-bound protease.

Nitrate reductase extracted from the membrane of Escherichia coli by alkaline heat treatment was purified to homogeneity and used to prepare specific antibody. Nitrate reductase, precipitated by this antibody from Triton extracts of the membrane, contained a third subunit not present in the purified enzyme used to prepare the antibody. Nitrate reductase precipitated by antibody from alkaline heat extracts was composed of peptide fragments of various sizes. These fragments were produced by a membrane-bound protease which was activated by alkaline pH and heat. It is the action of this protease that releases the enzyme from the membrane, as shown by the observations that protease inhibitors decreased the amount of solubilization of the enzyme, and the enzyme remaining in the membrane after heating showed much less proteolytic cleavage than that which was released.

Effects of synthetic peptides and protease inhibitors on the interaction of a human ovarian cancer cell line (NIH:OVCAR-3) with a reconstituted basement membrane (Matrigel).

We have investigated the adhesive properties and invasiveness of cells of the human ovarian carcinoma line, NIH:OVCAR-3, in vitro. OVCAR-3 cells exhibited a similar rate of adhesion to all substrates tested including laminin, fibronectin, and collagens I and IV. The synthetic peptide YIGSR-NH2, which corresponds to an attachment site in laminin, inhibited the adhesion of the cells to laminin, but not to fibronectin. In contrast, a GRGDS-NH2 peptide blocked adhesion to fibronectin but not to laminin. OVCAR-3 cells invaded and formed branched colonies on Matrigel. Colony formation was retarded by both YIGSR-NH2 and GRGDS-NH2 peptides. Serine protease inhibitors and human recombinant TIMP, the tissue inhibitor of metalloproteases, inhibited ovarian tumor cell invasion while a synthetic collagenase IV inhibitor (SC-44463) had no effect. These studies suggest that metalloproteases other than collagenase IV may be important for the invasive activity of ovarian cancer cells. It is possible that synthetic peptides with antiadhesive cellular activity and certain antiproteases could be used to control the progressive colonization and invasion of peritoneal surfaces by malignant ovarian cancer cells.