Sequence of the mglB gene from Escherichia coli K12: comparison of wild-type and mutant galactose chemoreceptors

Summary The mglB gene of Escherichia coli codes for a galactose-binding protein (GBP) that serves both as the galactose chemoreceptor and as the recognition component of the -methylgalactoside transport system. The mglB551 mutation eliminates the chemotactic function of GBP without altering its transport or substrate-binding properties. To investigate the interaction between GBP and Trg, the chemotactic signal transducer for galactose, we sequenced the mglB genes from wild-type and mglB551 mutant strains. The mutation causes the replacement of Gly₇₄ of GBP by Asp. This residue is located in alpha-Helix III at the tip of the P domain in the GBP tertiary structure farthest removed from the substrate-binding cleft between the P and Q domains. We conclude that Helix III must be part of, or at least adjacent to, the recognition site for Trg. Our sequence also included part of the mglA gene, which is immediately distal to mglB. The amino acid sequence deduced for the beginning of the MglA protein showed homology with a family of polypeptides that contain an ATP-binding site and are components of binding-protein-dependent transport systems.     

Expression and regulation of a Bacteroides fragilis sucrose utilization system cloned in Escherichia coli

A Bacteroides fragilis strain isolated from human feces was the source of chromosomal DNA in the construction of plasmid pBS100. The cloned 6-kilobase insert of plasmid pBS100 conferred a sucrose positivity phenotype on transformed cells of Escherichia coli JA221. E. coli JA221(pBS100) cells were able to utilize sucrose as the sole source of carbon because of the presence of sucrase enzyme and sucrose uptake activities. Sucrase activity was inducible in B. fragilis but constitutive in E. coli JA221(pBS100) cells. In sucrose-minimal medium, both B. fragilis and E. coli JA221(pBS100) produced intracellular and extracellular sucrase activities throughout the growth cycle. Osmotic shock experiments performed on E. coli JA221(pBS100) indicated that up to 55% of the sucrase activity was localized in the periplasmic space, 30% was in the cytoplasm, and the remaining 15% was in the cell-free extracellular supernatant fluid. B. fragilis and E. coli JA221(pBS100) actively transported sucrose. Sucrose uptake was induced by sucrose in B. fragilis, whereas the uptake activity in E. coli JA221(pBS100) was constitutive. E. coli JA221(pBS100) appeared to transport sucrose by a phosphotransferase-independent system. B. fragilis transported sucrose only under strictly anaerobic conditions. No uptake activity was detected under aerobic conditions with or without addition of catalase.     

Family Aggregation and Risk Factors in Phobic Disorders over Three-Generations in a Nation-Wide Study

Objective

This nation-wide register-based study investigated how often phobic disorders (PHO) and co-morbid disorders occur in affected families compared to control families. Furthermore, the study addressed the impact of sex, year of birth, and degree of urbanization in terms of risk factors.

Method

A total of N = 746 child and adolescent psychiatric participants born between 1969 and 1986 and registered in the Danish Psychiatric Central Research Register (DPCRR) with a diagnosis of a mental disorder before the age of 18, and developed PHO at some point during their life-time until a maximum age of 40 years were included. In addition, N = 2229 controls without any diagnosis of mental disorders before age 18 and that were matched for age, sex, and residential region were included. Diagnoses of mental disorders were also obtained from the first- degree relatives as a part of the Danish Three Generation Study (3GS). A family load component was obtained by using various mixed regression models.

Results

PHO occurred significantly more often in case than in control families, in particular, in mothers and siblings. Substance use disorders (SUD), Depressive disorders (DEP), anxiety disorders (ANX) and personality disorders (PERS) in the family were significantly associated with specific phobia in the case-probands. After controlling for various mental disorders comorbid to PHO it was found that some of the family transmission could be caused by various other mental disorders in family members rather than the PHO itself. Female sex and more recent year of birth were further risk factors while region of residence was not related to the manifestation of PHO. Case-relatives did not develop PHO earlier than control relatives. After adjusting for various additional explanatory variables, the family load explained only 0.0013% of the variance in the manifestation of PHO in the case-probands

Discussion

These findings, based on a very large and representative dataset, provide evidence for the family aggregation and further risk factors in PHO. In contrast to anxiety disorders and other major mental disorders the family load of PHO in this nation-wide study was rather low.     

No association between Helicobacter pylori genotypes and antibiotic resistance phenotypes within families

Background. Triple therapy combining a proton pump inhibitor with two antibiotics, e.g. clarythromycin (CLR), metronidazole (MTZ) or amoxicillin (AMX), represents the standard in Helicobacter pylori eradication regimens. Resistance to antimicrobial agents, particularly MTZ (up to 56% in Western countries) and CLR (up to 15% in southern Europe), is frequently observed and may be associated with treatment failure [ 1 ]. Recently, several studies indicated that individual H. pylori colonies from a single anatomic site may not always yield identical genotypes, or the identical patterns of susceptibility to antibiotics [ 2 - 5 ]. Representative for every single patient we analyzed 27 H. pylori antrum isolates for susceptibility to antimicrobial agents in order to test whether identical H. pylori genotypes exhibit a similar pattern of susceptibility to antibiotics. Methods. PCR, RELP, PFGE, antibiotic susceptibility testing. Results. H. pylori genotype and antibiotic susceptibility pattern in families do not segregrate. Conclusion. Molecular typing of H. pylori from family members does not predict antibiotic susceptibility pattern.     

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.^1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.^10-15 Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.^16-21The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.^22-30This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. ³¹Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. ^32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. ³⁷Download video file.^{(62M, mov)}     

Effects of hypothermia and re-warming on the inflammatory response in a murine multiple hit model of trauma

INTRODUCTION: Although, hypothermia is a frequent event after trauma, it is unclear whether its beneficial or detrimental effects are more important. This study aims to quantify the effects of hypothermia and re-warming on the inflammatory response after fracture/hemorrhage and subsequent fracture stabilization with resuscitation. MATERIALS AND METHODS: Eighty-one male C57Bl/6 mice (aged 8-10 weeks, weighing 22.0+/-3.0 g) underwent femoral fracture and hemorrhage followed by resuscitation and splint fixation of the fracture. Animals were sacrificed 3h after induction of hemorrhage and fracture. Besides a sham group (n=6), four experimental groups were created: A: normothermia (n=12), B: hypothermia after trauma (n=21), C: re-warming after resuscitation and before stabilization (n=21), and D: hypothermia before trauma (n=21). Groups B-D were further subdivided into three subgroups according to the degree of hypothermia (subgroup 1: 35-33 degrees C, subgroup 2: 32.9-30.0 degrees C, and subgroup 3: 29.9-27.0 degrees C). Plasma cytokine (TNF-alpha, IL-6, and IL-10) and chemokine (MCP-1) concentrations were determined by ELISA, pulmonary permeability changes were quantified, and histological analysis of lung and liver tissues was performed. RESULTS: Normothermia resulted in a significantly increased early mortality rate. A significantly increased pro-inflammatory and decreased anti-inflammatory responses were also observed in normothermia as compared to hypothermia. The extent of these changes was most pronounced in the severe hypothermic group. Re-warming after mild hypothermia resulted in a pro-inflammatory response comparable to normothermia. CONCLUSION: Hypothermia has a beneficial effect on early survival after trauma, which appears to be independent of the level of hypothermia and re-warming. Re-warming, however, enhanced the pro-inflammatory response. Further studies with a longer posttraumatic observation period are required to investigate the long term effects of the hypothermia and re-warming-induced changes on the pro- and anti-inflammatory responses.     

Characterization of neutralizing epitopes within the major capsid protein of human papillomavirus type 33

Background

Understanding how an organism replicates and assembles a multi-segmented genome with fidelity previously measured at 100% presents a model system for exploring questions involving genome assortment and RNA/protein interactions in general. The virus family Reoviridae, containing nine genera and more than 200 members, are unique in that they possess a segmented double-stranded (ds) RNA genome. Using reovirus as a model member of this family, we have developed the only functional reverse genetics system for a member of this family with ten or more genome segments. Using this system, we have previously identified the flanking 5' sequences required by an engineered s2 ssRNA for efficient incorporation into the genome of reovirus. The minimum 5' sequence retains 96 nucleotides and contains a predicted sequence/structure element. Within these 96 nucleotides, we have identified three nucleotides A-U-U at positions 79–81 that are essential for the incorporation of in vitro generated ssRNAs into new reovirus progeny viral particles. The work presented here builds on these findings and presents the results of an analysis of the required 3' flanking sequences of the s2 ssRNA.

Results

The minimum 3' sequence we localized retains 98 nucleotides of the wild type s2 ssRNA. These sequences do not interact with the 5' sequences and modifications of the 5' sequences does not result in a change in the sequences required at the 3' end of the engineered s2 ssRNA. Within the 3' sequence we discovered three regions that when mutated prevent the ssRNA from being replicated to dsRNA and subsequently incorporated into progeny virions. Using a series of substitutions we were able to obtain additional information about the sequences in these regions. We demonstrate that the individual nucleotides from, 98 to 84, 68 to 59, and 28 to 1, are required in addition to the total length of 98 nucleotides to direct an engineered reovirus ssRNA to be replicated to dsRNA and incorporated into a progeny virion. Extensive analysis using a number of RNA structure-predication software programs revealed three possible structures predicted to occur in all 10 reovirus ssRNAs but not predicted to contain conserved individual nucleotides that we could probe further by using individual nucleotide substitutions. The presence of a conserved structure would permit all ten ssRNAs to be identified and selected as a set, while unique nucleotides within the structure would direct the set to contain 10 unique members.

Conclusion

This study completes the characterization and mapping of the 5' and 3' sequences required for an engineered reovirus s2 ssRNA to be incorporated into an infectious progeny virus and establishes a firm foundation for additional investigations into the assortment and encapsidation mechanism of all 10 ssRNAs into the dsRNA genome of reovirus. As researchers build on this work and apply this system to additional reovirus genes and additional dsRNA viruses, a complete model for genome assortment and replication for these viruses will emerge.     

Modulation of juxtamembrane cleavage ("shedding") of angiotensin-converting enzyme by stalk glycosylation: evidence for an alternative shedding protease.

The role of juxtamembrane stalk glycosylation in modulating stalk cleavage and shedding of membrane proteins remains unresolved, despite reports that proteins expressed in glycosylation-deficient cells undergo accelerated proteolysis. We have constructed stalk glycosylation mutants of angiotensin-converting enzyme (ACE), a type I ectoprotein that is vigorously shed when expressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation did not significantly inhibit release. Introduction of an N-linked glycan directly adjacent to the native stalk cleavage site resulted in a 13-residue, proximal displacement of the cleavage site, from the Arg-626/Ser-627 to the Phe-640/Leu-641 bond. Substitution of the wild-type stalk with a Ser-/Thr-rich sequence known to be heavily O-glycosylated produced a mutant (ACE-JGL) in which this chimeric stalk was partially O-glycosylated; incomplete glycosylation may have been due to membrane proximity. Relative to levels of cell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL were enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr bond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristics of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3, 4-dichloroisocoumarin. These data indicate that (1) stalk glycosylation modulates but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-glycosylated stalk directs cleavage, at least in part, by an alternative shedding protease, which may resemble an activity recently described in TNF-alpha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].