A mathematical model and quantitative comparison of the small RNA circuit in the Vibrio harveyi and Vibrio cholerae quorum sensing systems.

Quorum sensing is the process by which bacteria regulate their gene expression based on the local cell-population density. The quorum sensing systems of Vibrio harveyi and Vibrio cholerae are comprised of a phosphorelay cascade coupled to a small RNA (sRNA) circuit. The sRNA circuit contains multiple quorum regulated small RNA (Qrr) that regulate expression of the homologous master transcriptional regulators LuxR (in V. harveyi) and HapR (in V. cholerae). Their quorum sensing systems are topologically similar and homologous thereby making it difficult to understand why repression of HapR is more robust than LuxR to changes in Qrr. In this work we formulate and parameterize a novel mathematical model of the V. harveyi and V. cholerae sRNA circuit. We parameterize the model by fitting it to a variety of empirical data from both species. We show that we can distinguish all of the parameters and that the parameterizations (one for each species) are robust to errors in the data. We then use our model to propose some experiments to identify and explain kinetic differences between the species. We find that V. cholerae Qrr are more abundant and more sensitive to changes in LuxO than V. harveyi Qrr and argue that this is why expression of HapR is more robust than LuxR to changes in Qrr.

5-aminolevulinate synthase: catalysis of the first step of heme biosynthesis.

5-Aminolevulinate synthase is a homodimeric pyridoxal 5'-phosphate-dependent enzyme that catalyzes the first step of the heme biosynthetic pathway in animals, fungi, and the alpha-subclass of the photosynthetic purple bacteria. The reaction cycle involves condensation of glycine with succinyl-coenzyme A to yield 5-aminolevulinate, carbon dioxide, and CoA. Mutations in the human erythroid-specific aminolevulinate synthase gene are associated with the erythropoietic disorder X-linked sideroblastic anemia. Recent kinetic and crystallographic data have facilitated an unprecedented understanding of how this important enzyme produces 5-aminolevulinate, and suggest possible directions for future research that may lead to treatments not only for X-linked sideroblastic anemia, but also other diseases.

Pre-steady-state reaction of 5-aminolevulinate synthase. Evidence for a rate-determining product release.

5-Aminolevulinate synthase (ALAS) is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the alpha-subclass of purple bacteria. The pyridoxal 5'-phosphate cofactor at the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the kinetics of these reactions spectroscopically. Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase demonstrate that reaction with glycine plus succinyl-CoA results in a pre-steady-state burst of quinonoid intermediate formation. Thus, a step following binding of substrates and initial quinonoid intermediate formation is rate-determining. The steady-state spectrum of the enzyme is similar to that formed in the presence of 5-aminolevulinate, suggesting that release of this product limits the overall rate. Reaction of either glycine or 5-aminolevulinate with ALAS is slow (kf = 0.15 s-1) and approximates kcat. The rate constant for reaction with glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme that is accelerated by succinyl-CoA. The slow rate of reaction of 5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme. Reaction of succinyl-CoA with the enzyme-glycine complex to form a quinonoid intermediate is a biphasic process and may be irreversible. Taken together, the data suggest that turnover is limited by release of 5-aminolevulinate or a conformational change associated with 5-aminolevulinate release.

Lysine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates.

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to form CoA, carbon dioxide, and 5-aminolevulinate. This represents the first committed step of heme biosynthesis in animals and some bacteria. Lysine 313 (K313) of mature murine erythroid 5-aminolevulinate synthase forms a Schiff base linkage to the pyridoxal 5'-phosphate cofactor. In the presence of glycine and succinyl-CoA, a quinonoid intermediate absorption is transiently observed in the visible spectrum of purified murine erythroid ALAS. Mutant enzymes with K313 replaced by glycine, histidine, or arginine exhibit no spectral evidence of quinonoid intermediate formation in the presence of glycine and succinyl-CoA. The wild-type 5-aminolevulinate synthase additionally forms a stable quinonoid intermediate in the presence of the product, 5-aminolevulinate. Only conservative mutation of K313 to histidine or arginine produces a variant that forms a quinonoid intermediate with 5-aminolevulinate. The quinonoid intermediate absorption of these mutants is markedly less than that of the wild-type enzyme, however. Whereas the wild-type enzyme catalyzes loss of tritium from [2-3H2]-glycine, mutation of K313 to glycine results in loss of this activity. Titration of the quinonoid intermediate formed upon binding of 5-aminolevulinate to the wild-type enzyme indicated that the quinonoid intermediate forms by transfer of a single proton with a pK of 8.1 +/- 0.1. Conservative mutation of K313 to histidine raises this value to 8.6 +/- 0.1. We propose that K313 acts as a general base catalyst to effect quinonoid intermediate formation during the 5-aminolevulinate synthase catalytic cycle.

Aspartate-279 in aminolevulinate synthase affects enzyme catalysis through enhancing the function of the pyridoxal 5'-phosphate cofactor.

5-Aminolevulinate synthase (ALAS) catalyzes the first step in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes, which is the condensation of glycine with succinyl-coenzyme A to yield coenzyme A, carbon dioxide, and 5-aminolevulinate. ALAS requires pyridoxal 5'-phosphate as an essential cofactor and functions as a homodimer. D279 in murine erythroid enzyme was found to be conserved in all aminolevulinate synthases and appeared to be homologous to D222 in aspartate aminotransferase, where the side chain of the residue stabilizes the protonated form of the cofactor ring nitrogen, thus enhancing the electron sink function of the cofactor during enzyme catalysis. D279A mutation in ALAS resulted in no detectable enzymatic activity under standard assay conditions, and the conservative D279E mutation reduced the catalytic efficiency for succinyl-CoA 30-fold. The D279A mutation resulted in a 19-fold increase in the dissociation constant for binding of the pyridoxal 5'-phosphate cofactor. UV-visible and CD spectroscopic analyses indicated that the D279A mutant binds the cofactor in a different mode at the active site. In contrast to the wild-type and D279E mutant, the D279A mutant failed to catalyze the formation of a quinonoid intermediate upon binding of 5-aminolevulinate. Importantly, this partial reaction could be rescued in D279A by reconstitution of the mutant with the cofactor analogue N-methyl-PLP. The steady-state kinetic isotope effect when deuteroglycine was substituted for glycine was small for the wild-type enzyme (kH/kD = 1.2 +/- 0.1), but a strong isotope effect was observed with the D279E mutant (kH/kD = 7.7 +/- 0.3). pH titration of the external aldimine formed with ALA indicated the D279E mutation increased the apparent pKa for quinonoid formation from 8.10 to 8.25. The results are consistent with the proposal that D279 plays a crucial role in aminolevulinate synthase catalysis by enhancing the electron sink function of the cofactor.

Role of arginine 439 in substrate binding of 5-aminolevulinate synthase.

5-Aminolevulinate synthase (EC 2.3.1.37) catalyzes the first reaction in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes. Homology sequence modeling between 5-aminolevulinate synthase and some other alpha-family pyridoxal 5'-phosphate-dependent enzymes indicated that the residue corresponding to the Arg-439 of murine erythroid 5-aminolevulinate synthase is a conserved residue in this family of pyridoxal 5'-phosphate-dependent enzymes. Further, this conserved arginine residue in several enzymes, e.g., aspartate aminotransferase, for which the three-dimensional structure is known, has been shown to interact with the substrate carboxyl group. To test whether Arg-439 is involved in substrate binding in murine erythroid 5-aminolevulinate synthase, Arg-439 and Arg-433 of murine erythroid 5-aminolevulinate synthase were each replaced by Lys and Leu using site-directed mutagenesis. The R439K mutant retained 77% of the wild-type activity; its K(m) values for both substrates increased 9-13-fold, while the activity of R433K increased 2-fold and the K(m) values for both substrates remained unchanged. R439L had no measurable activity as determined using a standard 5-aminolevulinate synthase enzyme-coupled activity assay. In contrast, the kinetic parameters for R433L were comparable to those of the wild-type. Dissociation constants (Kd) for glycine increased 5-fold for R439K and at least 30-fold for R439L, while Kd values for glycine for both R433K and R433L mutants were similar to those of the wild-type. However, there was not much difference in methylamine binding among the mutants and the wild-type, excepting of a 10-fold increase in K(d)methylamine for R439L. R439K proved much less thermostable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 8.3 degrees C lower than that of the wild-type enzyme. In addition, in vivo complementation analysis demonstrated that in the active site of murine erythroid 5-aminolevulinate synthase, R439 is contributed from the same subunit as K313 (which is involved in the Schiff base linkage of the pyridoxal 5'-phosphate cofactor) and D279 (which interacts electrostatically with the ring nitrogen of the cofactor), while another subunit provides R149. Taken together, these findings suggest that Arg-439 plays an important role in substrate binding of murine erythroid 5-aminolevulinate synthase.

Long-term outcomes after upper limb arterial injuries.

OBJECTIVE: To assess long-term outcomes in multisystem trauma victims who have arterial injuries to upper limbs. DESIGN: A retrospective case series. SETTING: Tertiary care regional trauma centre in a university hospital. PATIENTS: All consecutive severely injured patients (Injury Severity Score greater than 15) with an upper limb arterial injury treated between January 1986 and January 1995. Demographic data and the nature and management of the arterial and associated injuries were determined from the trauma registry and the hospital records. OUTCOME MEASURES: Death rate, discharge disposition, residual disabilities and functional outcomes as measured by the Glasgow Outcome Scale. RESULTS: Twenty-five (0.6%) of 4538 trauma patients assessed during the study period suffered upper extremity arterial injuries. Nineteen of them were victims of blunt trauma. The death rate was 24%. There were 10 primary and no secondary amputations. An autogenous vein interposition graft was placed in 10 patients. Concomitant fractures or nerve injuries in the upper limb were present in 80% and 86% of the patients, respectively. Long-term follow-up data (mean 2 years) were obtained in 16 of the 19 who survived to hospital discharge. The residual disability rate was high. It included upper limb joint contractures, pain and persistent neural deficits (69%). Associated injuries in other body areas also contributed to overall disability. Only 21% of the patients recovered completely or had only minor disabilities. CONCLUSIONS: Associated injuries, rather than the vascular injury, cause long-term disability in the multisystem trauma victim who has upper extremity involvement. Persistent neural deficits, joint contractures and pain are the principal reasons for long-term impairment of function.

Lower limb compartment syndrome: course after delayed fasciotomy.

OBJECTIVE: To determine the end result of patients who underwent delayed fasciotomy, i.e., more than 35 hours for an established lower limb compartment syndrome. DESIGN: A retrospective review of patients undergoing delayed treatment for a closed injury of the lower extremity, where fasciotomy should ideally have been performed earlier. MATERIALS AND METHODS: Nine fasciotomies in five patients were identified where there was a delay of more than 35 hours after the injury. The average ischemic time was 56 hours (range 35-96 hours). RESULTS: One patient died of multiorgan failure and septicemia. The remaining four patients required lower limb amputation, because of local infection and septicemia. The one late amputation was performed 6 months after the injury, because the patient was left with a functionless insensate foot. Where recognition of an established compartment syndrome is delayed for more than 8 to 10 hours, we propose that the traditional inevitable fasciotomy be reassessed.

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.

A continuous spectrophotometric assay for 5-aminolevulinate synthase that utilizes substrate cycling.

A continuous spectrophotometric assay for determining 5-aminolevulinic acid synthase activity is described. The assay is based upon coupling the production of coenzyme A by 5-aminolevulinic acid synthase to the reduction of NAD+ by alpha-ketoglutarate dehydrogenase and monitoring the increase in absorbance at 340 nm. Reduction of NAD+ is stoichoimetric with formation of 5-aminolevulinic acid. Kinetic parameters for glycine and succinyl-CoA are similar to those reported for other assays which measure the formation of 5-aminolevulinic acid. Regeneration of succinyl-CoA in the alpha-ketoglutarate dehydrogenase reaction facilitates determination of initial rates at subsaturating concentrations of this substrate. This assay will permit the rapid accumulation of kinetic data and aid in mechanistic analyses of both 5-aminolevulinic acid synthase and its recombinant mutants.

The management of long bone fractures in the head-injured polytrauma patient.

The polytrauma patient who sustains a significant head injury (head and neck Abbreviated Injury Scale of 3 or greater) will require prolonged and technically demanding operative intervention for musculoskeletal and associated soft tissue trauma. The presence of a head injury may delay the immediate surgical intervention for long bone injuries, which has proven to have major advantages for patient care and well-being. This retrospective review of the Sunnybrook Health Science Centre experience between January 1, 1986, and June 1, 1988, identified 153 polytrauma patients with a significant head injury. Forty-five died from complications unrelated to their long bone injuries or treatment thereof, not surviving long enough to reach the operating room for stabilization of their long bone fractures. The 108 survivors sustained 188 long bone injuries, 63 of which were open fractures. Twenty patients were treated nonoperatively. The 88 patients treated operatively had 12 complications: one peroneal nerve palsy; five cases of sepsis (three in open fractures and all resolving with removal of fixation devices); three malunions; and three cases of delayed union. Sixty-nine patients (78%) were available for long-term follow-up, 64 (93%) making a full recovery. Seven required additional surgery to achieve this goal and another patient awaits an ankle arthrodesis. Examining the head and neck AIS and Injury Severity Score of this group showed that 50 (46%) of these patients were expected to die and 22 (44%) made a full recovery.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct muscle neurotization recovers gastrocnemius muscle function.

This case report describes a devastating injury in which a distal motor nerve had been avulsed from the muscle belly and was not available for reconstruction. This otherwise irreparable posterior tibial nerve injury was successfully treated by direct neurotization of the muscle belly. The patient did have one small, intact, muscular branch to the medial gastrocnemius muscle; however, electrodiagnostic and clinical examination and recovery pattern suggested the neurotization procedure was responsible for the functional recovery. In the rare situation where no distal nerve is available, and tendon transfers or arthrodesis are inappropriate, direct muscle neurotization can be considered as a salvage technique.

Psychological factors leading to amputations in adults.

Psychological factors may lead to a small number of amputations in adults. They may be classified as being due to: 1. chronic pain syndrome; 2. artefactualists; 3. self-mutilation; 4. attempted murder. An understanding of these potential factors will make the amputee clinic team aware of this problem, and help them deal with the rehabilitation of the patient.

Liquefaction and calcification of a chronic compartment syndrome of the lower limb.

We present an uncommon late sequela of a compartment syndrome of the leg that presented as liquefaction and calcification. Our experience with this clinical situation, along with the available literature review, suggests an approach to this diagnostic and therapeutic problem. We recommend that repeated needle aspiration be performed to lessen the risk of secondary infection, chronic sinus formation, and amputation, which may occur after debridement and drainage of the lesion.

In vitro and in vivo characterization of herpes simplex virus clinical isolates recovered from patients infected with human immunodeficiency virus.

A total of 100 herpes simplex viruses isolated from lesions not responding to acyclovir (ACV) therapy were recovered from 51 patients infected with human immunodeficiency virus. In vitro analysis of these isolates included testing their susceptibility to ACV and determining their thymidine kinase (TK) phenotypes. Of the 100 isolates evaluated, 23 were ACV sensitive and 77 were ACV resistant. Seventy-four of these ACV-resistant isolates were of the TK-deficient or low-TK-producer phenotype and three were of the TK-altered phenotype. The TKs isolates that represented each of the different autoradiographic phenotypes were further characterized by enzyme kinetics. The ability of selected isolates to cause disease in vivo was evaluated by using several mouse virulence models. Cutaneous virulence in normal and immunocompromised mice was evaluated, and neurovirulence in normal mice was determined. Latent infections were assayed by the cocultivation of trigeminal ganglia recovered from mice that had survived acute infection. These reactivated viruses were evaluated in vitro and compared with the original infecting isolate. The mechanisms of resistance and pathogenicity of these herpes simplex virus isolates recovered from patients positive for human immunodeficiency virus are similar to those reported for isolates recovered from normal and immunocompromised patients without AIDS.

Microdialysis studies of brain norepinephrine, serotonin, and dopamine release during ingestive behavior. Theoretical and clinical implications.

This minireview deals with the possible roles of monoamines in feeding and feeding disorders. The introduction sketches the results of earlier studies with local drug injections and selective neurotoxins which provided pharmacological evidence that monoamines can influence food intake and body weight. A table summarizing this evidence is used to list monoamine changes that could underlie anorexia or hyperphagia. It is apparent that abnormalities in the monoamines, along with their cotransmitters, could cause many forms of feeding disorder. It is proposed as a working hypothesis that several varieties of hyperphagia leading to obesity have a common element. This common factor is a change in excitability of a lateral hypothalamic reinforcement system as manifested in self-stimulation at a stimulation-bound feeding site. Understanding this feeding reward-aversion system helps us understand hyperphagia and anorexia. The neurochemistry of reward and aversion involves the monoamines. This paper focuses on dopamine and serotonin. The data support the hypothesis that dopamine systems projecting to the nucleus accumbens and other forebrain areas from the mid-brain ventral tegmental area (VTA) are important for approach and positive reinforcement in ingestive behavior and self-stimulation. Serotonin is hypothesized to facilitate satiety and inhibition of feeding reward in the hypothalamus. The next section abstracts our recent experiments that measured pharmacological and physiological release of the monoamines in the hypothalamus and nucleus accumbens during ingestive behavior and self-stimulation. In vivo microdialysis in freely moving rats suggested the following: (1) Norepinephrine was released in the paraventricular nucleus during the active, feeding period of the circadian cycle. (2) The serotonin metabolite 5-HIAA also increased in the PVN at the same time if there was food to eat. (3) Amphetamine infused into the lateral hypothalamus (LH) by reverse dialysis increased synaptic dopamine, norepinephrine, and serotonin. (4) The anorectic drug d-fenfluramine increased synaptic serotonin in the LH and also increased the dopamine metabolite DOPAC, suggesting that serotonin and dopamine in the LH might contribute to fenfluramine-induced satiety. Local d-fenfluramine injection into the LH or local infusion by reverse dialysis again increased serotonin and decreased 5-HIAA and interfered with local dopamine metabolism as reflected in decreased DOPAC and HVA. (5) Tryptophan, a serotonin precursor, given systemically at an anorectic dose, increased extracellular serotonin in the LH, but this effect was only detectable in food-deprived rats. This was seemingly pH independent (between 5.8 and 8). The passage other cations through CFo is strictly suppressed (even at pH 8 and with 300 mM NaCl in the medium).(ABSTRACT TRUNCATED AT 400 WORDS)

Method for relating the centre of pressure locus during dynamic stance to the anatomical structure of the foot.

This study describes a new methodology for relating the centre of pressure locus to the anatomical structure of the foot. This technique involved locating three radio-opaque markers placed over anatomically located marks (the posterior border of the calcaneus and the first and fifth metatarsal heads) and relating these to the coordinates of the corresponding centre of pressure. In this manner, dynamic functional data are related to a standing roentgenogram of the complete foot.

Traumatic partial foot amputations in adults. A long-term review.

A retrospective study of 260 industrial amputees was undertaken to determine the long-term functional results of partial foot amputations following trauma. Follow-up ranged from 1 to 68 years with a mean of 16 years. Of 113 partial foot amputees (118 amputations) who had retained their original amputation, the functional end-results were 43% good, 38% fair and 19% poor. Lisfranc and Chopart amputations were better than those at transmetatarsal or digital levels. Of 260 initial amputations 49 (19%) were revised to a Syme's or a below-knee amputation.

The chlordiazepoxide/pentylenetetrazol discrimination: characterization of drug interactions and homeostatic responses to drug challenges.

Rats were trained to discriminate chlordiazepoxide (CDP) from pentylenetetrazol (PTZ) in a two-lever food motivated discrimination task. Training drug doses were adjusted until subjects emitted approximately 50% of their responses on each of the two drug-appropriate levers during saline injection tests. Tests that followed injection of CDP/PTZ combinations illustrated a reciprocal antagonism between the two drugs. Saline-injection tests that followed large dose injections of CDP revealed a period of predominantly PTZ-appropriate responding that persisted after the initial period of predominantly CDP-appropriate responding. These data are interpreted to suggest that, unlike some other drugs that have been shown to antagonize the behavioral and CNS effects of benzodiazepines, the interoceptive stimulus generated by PTZ occupies a position opposite to that of CDP along some single affective continuum. In addition, these data suggest that drug/drug (DD) discriminations are capable of characterizing the interactions between training drugs. Finally, the data suggest that the CDP/PTZ discrimination is a sensitive detector of bidirectional shifts in interoceptive stimulus state along the CDP/PTZ continuum.