Mutational analysis of the Vibrio fischeri LuxI polypeptide: critical regions of an autoinducer synthase

Synthesis of the Vibrio fischeri autoinducer, a signal involved in the cell density-dependent activation of bioluminescence, is directed by the luxI gene product. The LuxI protein catalyzes the synthesis of N-acyl-homoserine lactones from S-adenosylmethionine and acylated-acyl carrier protein. We have gained an appreciation of the LuxI regions and amino acid residues involved in autoinducer synthesis by isolating and analyzing mutations generated by random and site-specific mutagenesis of luxI. By random mutagenesis we isolated 13 different single amino acid substitutions in the LuxI polypeptide. Eleven of these substitutions resulted in no detectable autoinducer synthase activity, while the remaining two amino acid substitutions resulted in reduced but detectable activity. The substitutions that resulted in no detectable autoinducer synthase activity mapped to two small regions of LuxI. In Escherichia coli, wild-type luxI showed dominance over all of the mutations. Because autoinducer synthesis has been proposed to involve formation of a covalent bond between an acyl group and an active-site cysteine, we constructed site-directed mutations that altered each of the three cysteine residues in LuxI. All of the cysteine mutants retained substantial activity as an autoinducer synthase in E. coli. Based on the analysis of random mutations we propose a model in which there are two critical regions of LuxI, at least one of which is an intimate part of an active site, and based on the analysis of site-directed mutations we conclude that an active-site cysteine is not essential for autoinducer synthase activity.     

An interactive activation approach to object processing: effects of structural similarity, name frequency, and task in normality and pathology

We present a computational model of the processes involved in retrieving stored semantic and name information from objects, using a simple interactive activation and competition architecture. We simulate evidence showing a cross-over in normal reaction times to make semantic classification and identification responses to objects from categories with either structurally similar or structurally dissimilar exemplars, and that identification times to objects from these two different classes correlate differentially with measures of the structural similarity of objects within the category and the frequency of the object's name. Structural similarity exerts a negative effect on object decision as well as naming, though this effect is larger on naming. Also, on naming, structural similarity interacts with the effects of name frequency, captured in the model by varying the weight on connections from semantic to name units; frequency effects are larger with structurally dissimilar items. In addition, (1) the range of potential errors for objects from these two classes, when responses are elicited before activation reached a stable state, differ--a wider range of errors occur to objects from categories with structurally similar exemplars; and (2) simulated lesions to different locations within the model produce selective impairments to identification but not to semantic classification responses to objects from categories with structurally similar exemplars. We discuss the results in relation to data on visual object processing in both normality and pathology.     

Formation of unilamellar liposomes from total polar lipid extracts of methanogens

Unilamellar liposomes were formed by controlled detergent dialysis of mixed micelles consisting of acetone-insoluble total polar lipids extracted from various methanogens and the detergent n-octyl-beta-D-glucopyranoside. The final liposome populations were studied by dynamic light scattering and electron microscopy. Unilamellar liposomes with mean diameters smaller than 100 nm were obtained with lipid extracts of Methanococcus voltae, Methanosarcina mazei, Methanosaeta concilii, and Methanococcus jannaschii (grown at 50 degrees C), whereas larger (greater than 100-nm) unilamellar liposomes were obtained with lipid extracts of M. jannaschii grown at 65 degrees C. These liposomes were shown to be closed intact vesicles capable of retaining entrapped [14C]sucrose for extended periods of time. With the exception of Methanospirillum hungatei liposomes, all size distributions of the different liposome populations were fairly homogeneous.     

Histological and biomechanical analysis of bone and interface reactions around hydroxyapatite-coated intramedullary implants of different stiffness: a pilot study on the goat

We hypothesized that reduced stem stiffness of orthopaedic implants contributes to a high risk of loosening, since interface stresses and relative motions may exceed a tolerable range. To study this hypothesis, three types of load-bearing implant with different stiffnesses were inserted into the tibia of the goat. Histological analysis was performed of bone repair after insertion of the implant, bone ingrowth, interface disruption and loosening. A finite element model of the configuration provided the quantitative range of interface stresses and relative motions for the present experiment. The implants were made out of stainless steel, hollow titanium and a thin titanium core covered with a polyacetal coating. The stiffness ratios of these implants were approximately 10:4:1, respectively. All implants were coated with a layer of hydroxyapatite (HA) in order to minimize the possible biological effects of the different implant materials. Irrespective of the type of implant, there was a repair phase that lasted 6-12 weeks. The stiff implants functioned well. Large areas of bone bonding to the HA layer were found after the repair phase at 12 weeks postoperatively. After 24 weeks, some signs of loosening were observed. More loosening occurred with the hollow titanium and polyacetal implants, mainly during the repair phase. Three hollow titanium and three polyacetal coated implants survived this period, and were killed after 24 weeks. The integrity of the HA layer at the bone-implant interface of the titanium implants was good. In the polyacetal implants, the repair reaction of the cortical bone was incomplete. Bone ingrowth into HA was largely lacking. In conclusion, we found significant differences in the repair and interface reactions around implants of different stiffness. Stiff implants showed favourable initial interface conditions for bone ingrowth. Intermediate and flexible implants provoked unfavourable interface conditions for initial bone ingrowth. The finite element study showed that the flexible stems produce larger micromotions and higher interface stresses at the bone-prosthesis interface than the stiff stems, indicating an explanation for the histological findings.     

Comparison of responses to T-kinin and bradykinin in the mesenteric vascular bed of the cat

Responses to T-kinin and bradykinin were compared in the mesenteric vascular bed of the cat. Under constant-flow conditions, injection of T-kinin and bradykinin into the perfusion circuit induced similar dose-related decreases in perfusion pressure. Responses to T-kinin and bradykinin were inhibited by the kinin B2 receptor antagonist Hoe-140, but were not altered by the B1 receptor antagonist des-Arg9-[Leu8]-BK, the histamine H1 antagonist pyrilamine, the histamine H2 receptor antagonist cimetidine, or the H3 receptor antagonist thioperamide. Vasodilator responses to T-kinin and bradykinin were attenuated by the nitric oxide synthase inhibitor, N omega Nitro-L-arginine methyl ester (L-NAME), but were not altered by the cyclooxygenase inhibitor, sodium meclofenamate, or the K+ ATP channel antagonist, U37883A. These data suggest that vasodilator responses to T-kinin and bradykinin are mediated by kinin B2 receptor stimulated release of nitric oxide from the endothelium, but that the activation of kinin B1 receptors, the release of vasodilator prostaglandins, or the opening of K+ ATP channels are not involved in the response to T-kinin in the mesenteric vascular bed of the cat.     

A comparison of orocaecal transit times assessed by the breath hydrogen test and the sulphasalazine/sulphapyridine method in healthy beagle dogs

Orocaecal transit time (OCTT) was assessed in six healthy beagles by means of the breath hydrogen test (BH2T) and the sulphasalazine/sulphapyridine method (SLZ) after the administration of a test meal of canned food mixed with sulphasalazine. Orocaecal transit time was defined as the time taken from the oral administration of the test meal to the time when the first portion of the meal reached the colon. In five of the dogs the OCTTs assessed by the BH2T were shorter than those measured by the SLZ method by 30, 15, 45, 30 and 45 minutes. However, the median OCTT assessed by the BH2T (135 minutes, range 120 to 195 minutes) was not significantly different from that measured by the SLZ (180 minutes, range 150 to 210 minutes) and was highly correlated with it (r = 0.94, P = 0.016). The sixth dog maintained baseline hydrogen and plasma sulphapyridine readings throughout the monitoring period and the OCTT could not be measured.     

Mutational activation of the Cpx signal transduction pathway of Escherichia coli suppresses the toxicity conferred by certain envelope-associated stresses

Anticipation and suspicion are critical aspects to any discussion of intrauterine adhesions. Curettage between the second and fourth week postpartum is more likely to cause adhesions than is any other endometrial trauma. Infertility, recurrent abortion, or menstrual aberrations after any uterine trauma should cause the physician to suspect the presence of intrauterine adhesions. Hysterosalpingography and hysteroscopy are the ideal methods to make the diagnosis of IUA, and the latter is the safest, least traumatic, and most precise method of treating adhesions. The addition of an intrauterine splint and high-dose estrogen therapy completes the therapeutic approach. Before attempting conception the cavity should be inspected to verify its normality.     

Immunoglobulin G responses against human papillomavirus type 16 virus-like particles in a prospective nonintervention cohort study of women with cervical intraepithelial neoplasia

BACKGROUND: Infection with cancer-linked human papillomavirus (HPV) types such as HPV type 16 (HPV16) is the most important risk factor in the development of cervical cancer. It has been shown that immunoglobulin G (IgG) antibody responses against HPV16 virus-like particles (VLPs) are specifically associated with genital HPV16 infection. PURPOSE: The aim of this study was to determine the temporal relationships between the presence of HPV16 VLP-specific IgGs, HPV16 infection patterns, and the course of premalignant cervical disease. METHODS: Plasma samples from 133 women who had been diagnosed originally with mild to moderate cervical dyskaryosis and enrolled in a prospective non-intervention cohort study conducted in Amsterdam, The Netherlands, from 1991 through 1996 were analyzed for the presence of HPV16 VLP-specific IgGs by use of an enzyme-linked immunosorbent assay. A detailed analysis was performed on 43 women with different HPV16 infection patterns during a follow-up period of 10-34 months. Progression or regression of cervical intraepithelial neoplasia (CIN) lesions was monitored by cytologic and colposcopic testing at intervals of 3-4 months. HPV typing in cervical smears was performed by use of a polymerase chain reaction-based assay. Statistical analysis of the serologic data was performed by use of the Mann-Whitney U test or 2 x 2 table analyses. RESULTS: The presence of HPV16 VLP-specific IgGs in the plasma of the patients was found to be associated with the presence of HPV16 DNA in the cervical smear. Significantly higher proportions of patients with persistent HPV16 infections (i.e., who were polymerase chain reaction positive in three to 11 consecutive tests) than of patients with cleared HPV16 infections were found to be positive for the presence of HPV16 VLP-specific IgGs (18 [69.2%] of 26 versus nine [28.1%] of 32, respectively; P = .003). HPV16 VLP-specific IgGs were consistently detected in all women (n = 11) who were persistently HPV16 DNA positive during follow-up and whose disease ultimately progressed to CIN III (histologically diagnosed severe dysplasia or carcinoma in situ). CONCLUSION: HPV16 VLP-specific IgG responses are present in the plasma of a majority of patients with persistent HPV16 infections and histologically confirmed high-grade lesions but only in a smaller subset of patients with cleared HPV16 infections and either normal cervical histology or low-grade CIN lesions. IMPLICATIONS: These results suggest that HPV16 VLP-specific antibodies are not responsible for the clearance of virally induced CIN lesions but that they might, in patients with persistent HPV16 infections, be indicative of an increased cervical cancer risk.