A Small-Molecule Inhibitor of trans-Translation Synergistically Interacts with Cathelicidin Antimicrobial Peptides To Impair Survival of Staphylococcus aureus.

Staphylococcus aureus is a leading cause of infection in the United States, and due to the rapid development of resistance, new antibiotics are constantly needed. trans-Translation is a particularly promising antibiotic target because it is conserved in many bacterial species, is critical for bacterial survival, and is unique among prokaryotes. We have investigated the potential of KKL-40, a small-molecule inhibitor of trans-translation, and find that it inhibits both methicillin-susceptible and methicillin-resistant strains of S. aureus KKL-40 is also effective against Gram-positive pathogens, including a vancomycin-resistant strain of Enterococcus faecalis, Bacillus subtilis, and Streptococcus pyogenes, although its performance with Gram-negative pathogens is mixed. KKL-40 synergistically interacts with the human antimicrobial peptide LL-37, a member of the cathelicidin family, to inhibit S. aureus but not other antibiotics tested, including daptomycin, kanamycin, or erythromycin. KKL-40 is not cytotoxic to HeLa cells at concentrations that are 100-fold higher than the effective MIC. We also find that S. aureus develops minimal resistance to KKL-40 even after multiday passage at sublethal concentrations. Therefore, trans-translation inhibitors could be a particularly promising drug target against S. aureus, not only because of their ability to inhibit bacterial growth but also because of their potential to simultaneously render S. aureus more susceptible to host antimicrobial peptides.

Age related changes in the tunica media of the vertebral artery: implications for the assessment of vessels injured by trauma.

AIMS: To provide an illustrated, detailed semiquantitative analysis of the important degenerative changes along the length of the vertebral artery so that pathologists faced with investigating a fatal arterial injury can identify important pre-existing wall abnormalities. METHODS: Ten transverse annuli were taken along 34 vertebral arteries from 17 subjects and stained sections were prepared using haematoxylin and eosin and the picro-sirius red method. After routine microscopy, the elastic fibres, collagen, and smooth muscle nuclei in the tunica media were quantified using an eyepiece graticule. An estimate of the severity and extent of elastic tissue fragmentation, collagenous scarring, and intimal thickening/atheroma was then undertaken. RESULTS: Smooth muscle counts remained constant along the artery but collagen counts were higher and elastic counts substantially lower within the intracranial segment. Elastic fibre fragmentation was recognised in infancy and was moderately advanced by early adulthood but considerable collagenous scarring developed later in life. Some individuals demonstrated severe fragmentation and scarring before the age of 35 years. The degenerative changes were often focal and spared the intracranial segment almost completely. Atheroma increased with age but was rarely severe and appeared not to worsen appreciably beyond the age of 40 years. An unusual arrangement of the collagenous tissue was described within the upper cervical loops. CONCLUSION: Damaged vertebral arteries need to be sampled extensively to allow a proper histological assessment. The picro-sirius red method was successful in delineating the fine connective tissue structure of the wall and early degenerative changes. An understanding of the age and site specific changes should allow the pathologist to recognise important pre-existing abnormalities more easily.

Exosite interactions determine the affinity of factor X for the extrinsic Xase complex.

The initiation of coagulation results from the activation of factor X by an enzyme complex (Xase) composed of the trypsin-like serine proteinase, factor VIIa, bound to tissue factor (TF) on phospholipid membranes. We have investigated the basis for the protein substrate specificity of Xase using TF reconstituted into vesicles of phosphatidylcholine, phosphatidylserine, or pure phosphatidylcholine. We show that occupation of the active site of VIIa within Xase by a reversible inhibitor or an alternate peptidyl substrate is sufficient to exclude substrate interactions at the active site but does not alter the affinity of Xase for factor X. This is evident as classical competitive inhibition of peptidyl substrate cleavage but as classical noncompetitive inhibition of factor X activation by active site-directed ligands. This implies that the productive recognition of factor X by Xase arises from a multistep reaction requiring an initial interaction at sites on the enzyme complex distinct from the active site (exosites), followed by active site interactions and bond cleavage. Exosite interactions determine protein substrate affinity, whereas the second binding step influences the maximum catalytic rate for the reaction. We also show that competitive inhibition can be achieved by interfering with exosite binding using factor X derivatives that are expected to have limited or abrogated interactions with the active site of VIIa within Xase. Thus, substrate interactions at exosites, sites removed from the active site of VIIa within the enzyme complex, determine affinity and binding specificity in the productive recognition of factor X by the VIIa-TF complex. This may represent a prevalent strategy through which distinctive protein substrate specificities are achieved by the homologous enzymes of coagulation.

Platelets and whole blood coagulation.

In our early work in developing activated clotting time (ACT) assays, it became apparent that changes occurred in coagulation times as a whole blood sample aged (0-6 h). Subsequent studies showed that the coagulation parameters of plasma obtained from the samples remained stable during this time frame. These changes in whole blood clotting times during storage were eventually traced to the platelets. Several years of work demonstrated that this change was due to the removal of the blood from the vascular lining. This recalled a mechanism that was originally put forth in the 1970s with the discovery of prostacyclin. In this postulated mechanism, platelets are 'time-bombs'. They are kept under control by prostacyclin (PGI2) secreted by the vascular lining. Without this prostacyclin, platelets 'preactivate'. Since that time, additional substances secreted by the vascular endothelium have been identified, such as nitric oxide, that also influence platelet activity. The 'preactivation' of platelets in a blood sample can be followed using an ACT. In the same donor, the preactivation is uniform and reproducible over an extended period (months). There is, however, considerable variability between donors. Some donors' platelets preactivate dramatically, while other donors show hardly any change. Prostacyclin, added to the blood sample when it is collected, prevents this preactivation. The clinical significance of these observations has yet to be clearly established, but these observations raise a number of questions with respect to methods for improving platelet function during bypass and in evaluating the risk of platelet-mediated cardiovascular disease.

Otolaryngologic features of Laurence-Moon-Bardet-Biedl syndrome.

The most common otolaryngologic features associated with LMBBS include SNHL, speech and language disorders, and oral and dental abnormalities. Early otolaryngologic, audiologic, speech pathology, and dental evaluation of these individuals is recommended. This is the first reported case of bifid epiglottis, a rare congenital laryngeal anomaly, found in association with LMBBS. Most patients with bifid epiglottis have additional congenital anomalies, most commonly polysyndactyly. Polysyndactyly is a feature of both LMBBS and bifid epiglottis and may be an early hallmark for the presence of other congenital anomalies.

Regulation of extrinsic pathway factor Xa formation by tissue factor pathway inhibitor.

Tissue factor (TF) pathway inhibitor (TFPI) regulates factor X activation through the sequential inhibition of factor Xa and the VIIa.TF complex. Factor Xa formation was studied in a purified, reconstituted system, at plasma concentrations of factor X and TFPI, saturating concentrations of factor VIIa, and increasing concentrations of TF reconstituted into phosphatidylcholine:phosphatidylserine membranes (TF/PCPS) or PC membranes (TF/PC). The initial rate of factor Xa formation was equivalent in the presence or absence of 2.4 nM TFPI. However, reaction extent was small (<20%) relative to that observed in the absence of TFPI, implying the rapid inhibition of VIIa.TF during factor X activation. Initiation of factor Xa formation using increasing concentrations of TF/PCPS or TF/PC in the presence of TFPI yielded families of progress curves where both initial rate and reaction extent were linearly proportional to the concentration of VIIa.TF. These observations were consistent with a kinetic model in which the rate-limiting step represents the initial inhibition of newly formed factor Xa. Numerical analyses of progress curves yielded a rate constant for inhibition of VIIa.TF by Xa.TFPI (>10(8) M-1.s-1) that was substantially greater than the value (7.34 +/- 0.8 x 10(6) M-1.s-1) directly measured. Thus, VIIa.TF is inhibited at near diffusion-limited rates by Xa.TFPI formed during catalysis which cannot be explained by studies of the isolated reaction. We propose that the predominant inhibitory pathway during factor X activation may involve the initial inhibition of factor Xa either bound to or in the near vicinity of VIIa.TF on the membrane surface. As a result, VIIa.TF inhibition is unexpectedly rapid, and the concentration of active factor Xa that escapes regulation is linearly dependent on the availability of TF.

Role of the activation peptide domain in human factor X activation by the extrinsic Xase complex.

The activation of factor X by the extrinsic coagulation system results from the action of an enzyme complex composed of factor VIIa bound to tissue factor on phospholipid membranes in the presence of calcium ions (extrinsic Xase complex). Proteolysis at the Arg52-Ile53 peptide bond in the heavy chain of factor X leads to the formation of the serine protease, factor Xa, and the generation of a heavily glycosylated activation peptide comprising residues 1-52 of the heavy chain. The role of the activation peptide region in mediating substrate recognition and cleavage by the extrinsic Xase complex is unclear. The protease Agkistrodon rhodostoma hydrolase gamma (ARHgamma), from the venom of the Malayan pit viper, was used to selectively cleave human factor X in the activation peptide region. Three cleavage sites were found within this region and gave products designated Xdes1-34, Xdes1-43, and Xdes1-49. The products were purified to yield Xdes 1-49 and a mixture of Xdes 1-34 and Xdes 1-43. Reversed phase high pressure liquid chromatography analysis indicated that the cleaved portion of the activation peptide was likely removed during purification. All cleaved species were inactive and could be completely activated to factor Xa by the extrinsic Xase complex or by a purified activator from Russell's viper venom. Steady state kinetic studies using tissue factor reconstituted into membranes yielded essentially equivalent kinetic constants for the activation of intact factor X and the cleaved derivatives under a wide range of conditions. Since Xdes 1-49 lacks all but three residues of the activation peptide and is devoid of the carbohydrate present in this region, the data suggest that the specific recognition of human factor X by the extrinsic Xase complex is not achieved through specific interactions with residues 1-49 of the activation peptide or with carbohydrate structures attached to these residues.

Otolaryngology manifestation of postpolio syndrome.

Twenty-one patients with postpolio syndrome were surveyed to determine otolaryngologic symptoms. An alteration in voice, dysphagia, and fatigue were the most common symptoms reported. Prevailing etiologic theories are presented, and treatment recommendations are offered.

Eagle's syndrome: a reappraisal.

W.W. Eagle in 1937 described two patients with elongated styloid processes, cervico-facial pain and a history of pharyngeal trauma. Selected case reports are used to illustrate the spectrum of Eagle's Syndrome. An analysis of the prevailing theories of etiology and causation is undertaken correlating anatomy, embryology, and physiology to derive a clearer understanding of Eagle's Syndrome.

Airway manifestations of chondrodysplasia punctata.

Chondrodysplasia punctata is a heterogeneous skeletal dysplasia characterized by small focal calcifications in articular and other cartilages in infancy, referred to as stippled epiphyses, with subsequent epiphysial dysplasia and associated anomalies of the face, eyes and skin. Nasal hypoplasia is commonly seen but secondary respiratory distress is infrequently described. We present two siblings with different degrees of involvement and a review of the different forms of this disorder. When an infant presents with a small nasal airway, the diagnosis of chondrodysplasia punctata should be considered and appropriate evaluations obtained.

Heparinase in the activated clotting time assay: monitoring heparin-independent alterations in coagulation function.

The activated clotting time (ACT) is routinely used to monitor heparin during cardiopulmonary bypass surgery. Activated clotting times may be influenced by a number of factors other than heparin. The presence of heparin in blood samples disguises the occurrence of non-heparin-related changes in coagulation function. During cardiopulmonary bypass, it is difficult to ascertain baseline clotting time fluctuations with ACT alone. Previous attempts to establish accurate baseline data were imprecise and involved extensive sample handling. In this study, we present data obtained using a modified (ACT) assay that incorporates heparinase. The heparinase test cartridge (HTC) instantaneously, specifically, and completely removes heparin in the blood sample at the initiation of the test. In conjunction with standard ACT techniques, the clinician is provided with heparin-independent (baseline) and functional clotting data. The HTC/ACT assay was used in a case study involving 19 patients undergoing cardiopulmonary bypass surgery. The data gathered indicate the usefulness of this assay in monitoring incidents of baseline drift, hemodilution, and hypercoagulation and the efficacy of protamine reversal.

Tonsillar abscess.

This article presents seven cases of patients with tonsillar abscess formation and discusses the pathophysiology of intratonsillar abscess formation.

Heparin monitoring in the coronary care unit after percutaneous transluminal coronary angioplasty.

The rate of acute restenosis in patients after percutaneous transluminal coronary angioplasty (PTCA) is related to thrombotic complications triggered by the PTCA. This risk is reduced by anticoagulating the patients with heparin after the procedure. The anticoagulation state of patients receiving heparin therapy is routinely monitored with the activated partial thromboplastin time (APTT) test. In an effort to provide more timely results regarding the status of patients who are receiving heparin after PTCA, a study was conducted to see whether low-range activated clotting time measurement (LR ACT) performed at the bedside could provide information comparable to that from APTT values determined in the laboratory. The study showed that the LR ACT values were comparable to laboratory-generated APTT values (R2 = 0.68). The LR ACT data generated were superior to the APTT data in terms of timeliness and the wider range of heparin levels covered. Having these values available allowed the CCU staff to react rapidly to changes in the patient's coagulation status.

Vocal rehabilitation of tracheoesophageal speech failures.

Twenty-three tracheoesophageal speech failures were prospectively evaluated by clinical parameters and transnasal air insufflation at 3 L per minute. The results of testing allow an accurate indication of the etiology of the speech failure. Pharyngoesophageal spasm accounted for 79% of the failures; hypopharyngeal strictures for 26%. One patient was found to have both pharyngoesophageal spasm and a hypopharyngeal stricture. A modified air insufflation test result greater than 20 mm Hg reliably identified all tracheoesophageal speech failures prior to tracheoesophageal puncture. Clinical parameters were not helpful in identifying speech failures. Successful treatment of the specific etiology of the failure resulted in a reduction of the measured intraesophageal pressures. Ninety-one percent of the tracheoesophageal speech failures were successfully rehabilitated and achieved fluent tracheoesophageal speech. Successful rehabilitation was associated with long-term tracheoesophageal speech use.

Surgical treatment of pharyngoesophageal spasm.

The clinical course of 17 laryngectomees with pharyngoesophageal spasm who underwent pharyngeal myotomy was studied to determine clinical response and complications. The influence of speech therapy on the development of volitional control of pharyngoesophageal spasm, tracheoesophageal speech fluency, and the efficacy and complications of surgical treatment for pharyngoesophageal spasm were assessed. Volitional control of tracheoesophageal speech was never achieved. Ninety-four percent of the patients (16/17) were successfully rehabilitated following surgical therapy. The complications following pharyngeal myotomy were acceptable.

Supraglottitis in young adults.

Supraglottitis is a disorder with the potential for significant morbidity and mortality in adults. Of the 25 patients we reviewed, 18 were black, representing the largest series of black adults with this condition reported in the medical literature. The mean age for black men was 14 years younger than previously described adults with supraglottitis. The index of suspicion for supraglottitis should be high in young adults with odynophagia out of proportion to findings on oropharyngeal examination. Indirect laryngoscopy will confirm the diagnosis and allow prompt initiation of treatment. Early recognition and treatment will avoid complications.

An objective method for prediction of tracheoesophageal speech production.

Current methods of esophageal air insufflation testing to predict postoperative tracheoesophageal speech success lack procedural objectivity and rely on subjective interpretation of the voice production results. A range of intraesophageal pressure measurements was obtained prior to tracheoesophageal (TE) puncture in each of 27 laryngectomized patients in an attempt to predict TE speech outcome. Postoperatively, three levels of speech production were identified. Fluent speakers, nonfluent speakers, and nonspeakers demonstrated low, intermediate, and high intraesophageal pressures, respectively. Patients with intermediate and high preoperative pressures did not achieve fluent speech without myotomy. This technique offers a reliable, objective preoperative indication of expected TE speech fluency.