Identification of the veratryl alcohol binding site in lignin peroxidase by site-directed mutagenesis

Site-directed mutagenesis was used to identify the veratryl alcohol binding site of lignin peroxidase. The cDNA encoding isozyme H8 was mutated at Glu146 to both an Ala and a Ser residue. The H8 polypeptide was produced by E. coli as inclusion bodies and refolded to yield active enzyme. The wild type recombinant enzyme and the mutants were purified to homogeneity and characterized by steady state kinetics. The kcat is decreased for both mutants of Glu146. The reactivity of mutants (kcat/Km) toward H2O2 were not affected. In contrast, the kcat/Km of the mutants for veratryl alcohol were decreased by at least half. The oxidation of guaiacol by these mutants were more significantly affected. These results collectively suggest that E146 plays a central role in the binding of veratryl alcohol by lignin peroxidase.     

A statistical analysis of spot variation using the two-dimensional polyacrylamide gel electrophoresis

For the development of valid algorithms for matching protein spots between two-dimensional gels, it is essential that one has an understanding of the relative roles of the many sources of variability affecting the location of spots. We first consider the contribution of observers to the measurement variability of spot location, arriving at a simple model for these effects. Next we present an analysis of the variability in spot locations for a sample of gels containing duplicate gels for each sample. Our data indicate that both differences between duplicates and between samples are considerable, and that the size of the effects depends on the region of the gel being considered. In the discussion we examine several matching strategies that match large groups of gels based on algorithms which match two gels at a time.     

Luteinizing hormone releasing hormone (LHRH) neurons maintained in nasal explants decrease LHRH messenger ribonucleic acid levels after activation of GABA(A) receptors

Inhibition of the LHRH system appears to play an important role in preventing precocious activation of the hypothalamic-pituitary-gonadal axis. Evidence points to gamma-aminobutyric acid (GABA) as the major negative regulator of postnatal LHRH neuronal activity. Changes in LHRH messenger RNA (mRNA) levels after alterations of GABAergic activity have been reported in vivo. However, the extent to which GABA acts directly on LHRH neurons to effect LHRH mRNA levels has been difficult to ascertain. The present work evaluates the effect of GABAergic activity, via GABA(A) receptors, on LHRH neuropeptide gene expression in LHRH neurons maintained in olfactory explants generated from E11.5 mouse embryos. These explants maintain large numbers of primary LHRH neurons that migrate from bilateral olfactory pits in a directed manner. Using in situ hybridization histochemistry and single cell analysis, we report dramatic alterations in LHRH mRNA levels. Inhibition of spontaneous synaptic activity by GABA(A) antagonists, bicuculline (10(-5) M) or picrotoxin (10(-4) M), or of electrical activity by tetrodotoxin (TTX, 10(-6) M) significantly increased LHRH mRNA levels. In contrast, LHRH mRNA levels decreased in explants cultured with the GABA(A) receptor agonist, muscimol (10(-4) M), or KCl (50 mM). The observed responses suggest that LHRH neurons possess functional pathways linking GABA(A) receptors to repression of neuropeptide gene expression and indicate that gene expression in embryonic LHRH neurons, outside the CNS, is highly responsive to alterations in neuronal activity.     

Outbreak of pneumonia in a long-term care facility: antecedent human parainfluenza virus 1 infection may predispose to bacterial pneumonia

OBJECTIVES: To determine the causes of an outbreak of lobar pneumonia. DESIGN: Matched (1:2) case-control study. SETTING: A 70-bed chronic care facility for older people. PARTICIPANTS: Residents of the facility. RESULTS: Ten residents developed pneumonia over a 10-day period. Two residents died. One case-patient had Streptococcus pneumoniae bacteremia; another had polymerase chain reaction evidence of S. pneumoniae infection. No other etiologic agent was identified. Only four of 10 case-patients had received routine diagnostic cultures of blood or sputum before the administration of antibiotics. Symptoms of upper respiratory illness (URI) among residents before the pneumonia outbreak corresponded with elevation of antibodies to human parainfluenza virus 1 (HPIV1). In a matched case-control study, six of 10 case-patients, compared with five of 20 controls, had symptoms of URI during the preceding month (matched odds ratio (MOR) = 4.5, 95% CI = 0.8-33). Nine case-patients had serum available, and five of these had both serologic evidence of recent HPIV1 infection and recent URI, compared with two of 18 controls (MOR = 9.0, 95% CI = 1.2-208). Only three residents had documentation of pneumococcal vaccination. CONCLUSIONS: Noninfluenza viral infections may play a role in the pathogenesis of some bacterial pneumonias. S. pneumoniae was the cause of at least two pneumonias; lack of preantibiotic cultures may have interfered with isolation of S. pneumoniae in others. Recent HPIV1 infection was epidemiologically linked to subsequently developing pneumonia. Spread of HPIV1 in the facility may have contributed to increased susceptibility to S. pneumoniae and, potentially, to other bacterial pathogens.     

Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma

PURPOSE: Standard therapies of head and neck squamous cell carcinoma (HNSCC) often cause profound morbidity and have not significantly improved survival over the last 30 years. Preclinical studies showed that adenoviral vector delivery of the wild-type p53 gene reduced tumor growth in mouse xenograft models. Our purpose was to ascertain the safety and therapeutic potential of adenoviral (Ad)-p53 in advanced HNSCC. PATIENTS AND METHODS: Patients with incurable recurrent local or regionally metastatic HNSCC received multiple intratumoral injections of Ad-p53, either with or without tumor resection. Patients were monitored for adverse events and antiadenoviral antibodies, tumors were monitored for response and p53 expression, and body fluids were analyzed for Ad-p53. RESULTS: Tumors of 33 patients were injected with doses of up to 1 x 10(11) plaque-forming units (pfu). No dose-limiting toxicity or serious adverse events were noted. p53 expression was detected in tumor biopsies despite antibody responses after Ad-p53 injections. Clinical efficacy could be evaluated in 17 patients with nonresectable tumors: two patients showed objective tumor regressions of greater than 50%, six patients showed stable disease for up to 3.5 months, and nine patients showed progressive disease. One resectable patient was considered a complete pathologic response. Ad-p53 was detected in blood and urine in a dose-dependent fashion, and in sputum. CONCLUSION: Patients were safely injected intratumorally with Ad-p53. Objective antitumor activity was detected in several patients. The infectious Ad-p53 in body fluids was asymptomatic, and suggests that systemic or regional treatment may be tolerable. These results suggest the further investigation of Ad-p53 as a therapeutic agent for patients with HNSCC.     

Novel sulfated oligosaccharides containing 3-O-sulfated glucuronic acid from king crab cartilage chondroitin sulfate K. Unexpected degradation by chondroitinase ABC

We prepared a series of oligosaccharides from king crab cartilage chondroitin sulfate K after exhaustive digestion with testicular hyaluronidase, and determined the structures of four tetrasaccharides and a pentasaccharide by fast atom bombardment mass spectrometry, high performance liquid chromatography analysis of chondroitinase AC-II digests, and 500-MHz 1H NMR spectroscopy. The tetrasaccharides shared the common core structure GlcAbeta1-3GalNAcbeta1-4GlcAbeta1-3GalNAc with various sulfation profiles. One structure was GlcAbeta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S), whereas three of them have the following hitherto unreported structures including a novel glucuronate 3-O-sulfate: GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcAbeta1-3GalNAc(4S), GlcAbeta1-3GalNAc(4S)beta1-4GlcA(3S)beta1-3GalNAc(4S), and GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcA(3S)beta1-3GalNAc(4S), where 3S or 4S represents 3-O- or 4-O-sulfate, respectively. The structure of the pentasaccharide was determined as GlcA(3S)beta1-3GalNAc(4S)beta1-4GlcA(3S)beta1- 3GalNAc(4S)beta1-4GlcA. Chondroitinase ABC digestion of the tetrasaccharides with GlcA(3S) at the internal position destroyed the disaccharide unit containing GlcA(3S) derived from the reducing side and resulted in only the disaccharide unit from the non-reducing side. In contrast, these tetrasaccharides remained totally resistant to chondroitinase AC-II. The results indicated that it is necessary to reevaluate the disaccharide composition of chondroitin sulfate poly- or oligosaccharides purified from various biological sources, since they were usually determined after chondroitinase ABC digestion. It is probable that the structures containing GlcA(3S) would not have been detected.     

Increased expression of CD80, CD86 and CD70 on T cells from HIV-infected individuals upon activation in vitro: regulation by CD4+ T cells

T cells express CD28 and CD27 which transduce co-stimulatory signals after interaction with their ligands on antigen-presenting cells (APC). These ligands, CD80, CD86 and CD70, are also expressed to some extent on activated T cells. Here, we show that in human immunodeficiency virus (HIV)-infected individuals, CD28 and CD27 expression is decreased on CD8+ T cells. On the other hand, T cell stimulation in vitro induced high CD80, CD86 and CD70 expression on T cells from HIV-infected individuals. It appeared that an inverted CD4:CD8 T cell ratio could explain this enhanced expression of co-stimulatory ligands. Indeed, high expression levels of CD80, CD86 and CD70 were found on activated CD8+ T cells from HIV- individuals cultured in the absence of CD4+ T cells. Addition of CD4+ T cells prevented this up-regulation. However, in HIV-infected individuals, addition of excess autologous or healthy control CD4+ T cells did not completely counteract up-regulation of co-stimulatory ligand expression on CD8+ T cells. Thus, to some extent, CD8+ T cells in HIV-infected individuals appeared to be refractory to CD4+ T cell-mediated regulation of ligand expression in vitro. Activated T cells from HIV-infected individuals and activated CD8+ T cells from healthy controls were able to act as accessory cells in CD3-induced T cell proliferation, which was dependent on cell-cell contact. Thus, we showed that T cells from HIV-infected individuals express enhanced levels of co-stimulatory ligands upon activation, which provides them with accessory cell properties. Enhanced stimulatory potential of these nonprofessional APC may contribute to persistently high levels of immune activation in HIV infection related to disease progression.     

Posterior fast atrioventricular node pathways: implications for radiofrequency catheter ablation of atrioventricular node reentrant tachycardia

OBJECTIVES: This study sought to present evidence that fast atrioventricular (AV) node pathways with posterior exit sites may participate in typical AV node reentry. BACKGROUND: Catheter ablation of the slow AV node pathway in the posteroseptal right atrium is the preferred therapeutic approach in patients with AV node reentrant tachycardia. Despite the success achieved with this approach, electrophysiologic changes consistent with fast pathway ablation are occasionally observed. One potential explanation is the presence of an aberrant posterior fast pathway. METHODS: The location of fast and slow AV node pathways was determined by atrial activation mapping along the tricuspid valve annulus during tachycardia and was further confirmed by the effect of radiofrequency catheter ablation. RESULTS: Seven patients with AV node reentrant tachycardia had evidence of a posterior fast pathway near the coronary sinus os. Abolition of anterograde and retrograde fast pathway conduction followed radiofrequency ablation in the posteroseptal region in six patients. Consistent with fast pathway ablation, the AH interval increased from 70 +/- 24 to 195 +/- 35 ms (mean +/- SD), and tachycardia was no longer inducible. Selective slow pathway ablation was performed in one other patient with a posterior fast pathway. CONCLUSIONS: Functionally fast AV node pathways may be located in the posteroseptal right atrium, where slow pathway modification is performed. These data delineate the limitation of an anatomically guided slow pathway ablative approach and emphasize the importance of detailed mapping and localization of the retrograde fast pathway exit site before ablation. Failure to recognize the presence of posterior fast AV node pathways may account for sporadic examples of AV block, complicating posteroseptal ablation in patients with AV node reentry.