Diminished exoproteome of Frankia spp. in culture and symbiosis.

Frankia species are the most geographically widespread gram-positive plant symbionts, carrying out N(2) fixation in root nodules of trees and woody shrubs called actinorhizal plants. Taking advantage of the sequencing of three Frankia genomes, proteomics techniques were used to investigate the population of extracellular proteins (the exoproteome) from Frankia, some of which potentially mediate host-microbe interactions. Initial two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatants indicated that cytoplasmic proteins appeared in supernatants as cells aged, likely because older hyphae lyse in this slow-growing filamentous actinomycete. Using liquid chromatography coupled to tandem mass spectrometry to identify peptides, 38 proteins were identified in the culture supernatant of Frankia sp. strain CcI3, but only three had predicted export signal peptides. In symbiotic cells, 42 signal peptide-containing proteins were detected from strain CcI3 in Casuarina cunninghamiana and Casuarina glauca root nodules, while 73 and 53 putative secreted proteins containing signal peptides were identified from Frankia strains in field-collected root nodules of Alnus incana and Elaeagnus angustifolia, respectively. Solute-binding proteins were the most commonly identified secreted proteins in symbiosis, particularly those predicted to bind branched-chain amino acids and peptides. These direct proteomics results complement a previous bioinformatics study that predicted few secreted hydrolytic enzymes in the Frankia proteome and provide direct evidence that the symbiosis succeeds partly, if not largely, because of a benign relationship.

The use of neostigmine to treat postoperative ileus in orthopedic spinal patients.

Ileus is a common complication of spinal surgery, affecting 5% to 12% of all patients. Often this ileus is secondary to acute colonic pseudo-obstruction. This study is a prospective clinical trial of neostigmine in seven spinal patients with ileus after surgery to demonstrate its efficacy. All patients had evidence of the Ogilvie syndrome that was unresponsive to 24 hours of conservative therapy. Patients received 2 mg neostigmine, and abdominal circumference, clinical response, and radiographic colonic measurements were recorded. Patients were followed for recurrence of ileus for their remaining time in the hospital. Six patients had prompt colonic decompression, and no patient had recurrence of colonic distension. Side effects were minimal. These results suggest that postoperative spinal patients with ileus secondary to acute colonic pseudo-obstruction that is unresponsive to conservative therapy benefit from treatment with neostigmine, resulting in safe, rapid decompression of the colon.

Probing the differences between rat liver outer mitochondrial membrane cytochrome b5 and microsomal cytochromes b5.

Two distinct forms of cytochrome b5 exist in the rat hepatocyte. One is associated with the membrane of the endoplasmic reticulum (microsomal, or Mc, cyt b5) while the other is associated with the outer membrane of liver mitochondria (OM cyt b5). Rat OM cyt b5, the only OM cyt b5 identified so far, has a significantly more negative reduction potential and is substantially more stable toward chemical and thermal denaturation than Mc cytochromes b5. In addition, hemin is kinetically trapped in rat OM cyt b5 but not in the Mc proteins. As a result, no transfer of hemin from rat OM cyt b5 to apomyoglobin is observed at pH values as low as 5.2, nor can the thermodyamically favored ratio of hemin orientational isomers be achieved under physiologically relevant conditions. These differences are striking given the similarity of the respective protein folds. A combined theoretical and experimental study has been conducted in order to probe the structural basis behind the remarkably different properties of rat OM and Mc cytochromes b5. Molecular dynamics (MD) simulations starting from the crystal structure of bovine Mc cyt b5 revealed a conformational change that exposes several internal residues to the aqueous environment. The new conformation is equivalent to the "cleft-opened" intermediate observed in a previously reported MD simulation of bovine Mc cyt b5 [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693]. The rat OM protein does not adopt a comparable conformation in MD simulations, thus restricting access of water to the protein interior. Subsequent comparisons of the protein sequences and structures suggested that an extended hydrophobic network encompassing the side chains of Ala-18, Ile-32, Leu-36, and Leu-47 might contribute to the inability of rat OM cyt b5 to adopt the cleft-opened conformation and, hence, stabilize its fold relative to the Mc isoforms. A corresponding network is not present in bovine Mc cyt b5 because positions 18, 32, and 47, are occupied by Ser, Leu, and Arg, respectively. To probe the roles played by Ala-18, Ile-32, and Leu-47 in endowing rat OM cyt b5 with its unusual structural properties, we have replaced them with the corresponding residues in bovine Mc cyt b5. Hence, the I32L (single), A18S/L47R (double), and A18S/L47R/I32L (triple) mutants of rat OM cyt b5 were prepared. The stability of these proteins was found to decrease in the following order: WT rat OM > rat OM I32L > rat OM A18S/L47R > rat OM A18S/L47R/I32L > bovine Mc cyt b5. The decrease in stability of the rat OM protein correlates with the extent to which the hydrophobic cluster involving the side chains of residues 18, 32, 36, and 47 has been disrupted. Complete disruption of the hydrophobic network in the triple mutant is confirmed in a 2.0 A resolution crystal structure of the protein. Disruption of the hydrophobic network also facilitates hemin loss at pH 5.2 for the double and triple mutants, with the less stable triple mutant exhibiting the greater rate of hemin transfer to apomyoglobin. Finally, 1H NMR spectroscopy and side-by-side comparisons of the crystal structures of bovine Mc, rat OM, and rat OM A18S/L47R/I32L cyt b5 allowed us to conclude that the nature of residue 32 plays a key role in controlling the relative stability of hemin orientational isomers A and B in rat OM cyt b5. A similar analysis led to the conclusion that Leu-70 and Ser-71 play a pivotal role in stabilizing isomer A relative to isomer B in Mc cytochromes b5.

Diversity of Geotrichum candidum strains isolated from traditional cheesemaking fabrications in France.

The diversity of French fungus-ripened cheeses is due partly to the succession of fungi that colonize the cheese during ripening. Geotrichum candidum appears in the early stages of ripening on soft cheeses such as Camembert and semihard cheeses such as St. Nectaire and Reblochon. Its lipases and proteases promote flavor development, and its aminopeptidases reduce bitterness imparted by low-molecular-weight peptides in cheese. We assessed the genetic diversity of G. candidum strains by using random amplification of polymorphic DNA (RAPD)-PCR correlated with phenotypic tests for carbon assimilation and salt tolerance. Strains were isolated from milk, curd, and cheese collected in seven major cheesemaking regions of France. Sixty-four isolates were characterized. We found high genetic diversity of G. candidum even within the same cheesemaking regions. Strains did not group according to region. All of the strains from the Haute-Savoie were able to assimilate lactate as the sole source of carbon, while lactate assimilation varied among strains from the Auvergne. Strains varied in D-mannitol assimilation, and none used citrate as the sole source of carbon. Yeast-like colony morphology predominated in Reblochon, while all of the strains isolated from St. Nectaire were filamentous. The RAPD-PCR technique readily differentiated Geotrichum fragrans isolated from milk and curd in a St. Nectaire cheesemaking facility. This study reveals an enormous diversity of G. candidum that has been empirically selected through the centuries by the cheesemakers of France.

The role of acute decompression and restoration of spinal alignment in the prevention of post-traumatic syringomyelia: case report and review of recent literature.

STUDY DESIGN: Case report. INTRODUCTION: Acute post-traumatic syringomyelia formation after spinal cord injury has been considered a rare complication. At this writing, most recent reports have surfaced in neurosurgical journals. As an entity, post-traumatic syringomyelia has not been widely appreciated. It has been confused with conditions such as Hansen's disease or ulnar nerve compression at the cubital tunnel. One study also demonstrated that the occurrence of syrinx is significantly correlated with spinal stenosis after treatment, and that an inadequate reduction of the spine may lead to the formation of syrinx. This reported case describes a patient in whom post-traumatic syringomyelia began to develop 3 weeks after injury, which improved neurologically after adequate decompression. SUMMARY OF BACKGROUND DATA: A 30-year-old man sustained a 20-foot fall at work. He presented with a complete spinal cord injury below T4 secondary to a T4 fracture dislocation. The patient underwent open reduction and internal fixation of T1-T8. After 3 weeks, the patient was noted to have ascending weakness in his bilateral upper extremities and some clawing of both hands. METHODS: A computed tomography myelogram demonstrated inability of contrast to pass through the T4-T5 region from a lumbar puncture. An incomplete reduction was noted. The canal showed significant stenosis. A magnetic resonance image of the patient's C-spine showed increased signal in the substance of the cord extending into the C1-C2 area. The patient returned to the operating room for T3-T5 decompressive laminectomy and posterolateral decompression including the pedicles, disc, and posterior aspect of the body. Intraoperative ultrasound monitoring showed a good flow of cerebrospinal fluid past the injured segment. RESULTS: On postoperative day 1, the clawing posture of the patient's hands was significantly diminished, and the patient noted an immediate improvement in his hand and arm strength. Over the next few days, the patient's strength in the bilateral upper extremities increased to motor Grade 4/5 on manual testing. A magnetic resonance image 4 weeks after decompression showed significant improvement in the cord diameter and signal. CONCLUSIONS: Post-traumatic syringomyelia has not been reported at so early a stage after injury. This disorder is an important clinical entity that must be recognized to prevent potentially fatal or devastating complications. As evidenced by the reported patient and the literature, if this disorder is discovered and treated early, permanent deficit can be avoided. The prevention of post-traumatic syringomyelia requires anatomic realignment and stabilization of the spine without stenosis, even in the case of complete injuries, to maintain the proper dynamics of cerebrospinal fluid flow.

Serodiagnosis of human granulocytic ehrlichiosis by using novel combinations of immunoreactive recombinant proteins.

A panel of seven recombinant antigens, derived from Ehrlichia phagocytophila (the agent of human granulocytic ehrlichiosis), was evaluated by class-specific enzyme-linked immunosorbent assays (ELISAs) for utility in the diagnosis of the infection. Fourteen genomic fragments, obtained by serologic expression screening, contained open reading frames (ORFs) encoding 16 immunodominant antigens. Eleven of these antigens were members of the major surface protein (MSP) multigene family. Alignment of their predicted protein sequences revealed a pattern of conserved sequences, which contained short direct repeats, flanking a variable region. In addition, two genomic clones contained two and three MSP ORFs, respectively, indicating that these genes are clustered in tandem copies. The implications for this pattern of both genomic and protein arrangements in antigenic variations of MSPs and in their utilities in a diagnostic assay are discussed. In addition to two MSP recombinant antigens (rHGE-1 and -3) and a fusion protein of these antigens (rErf-1), five further recombinants were evaluated by ELISA. Two of these antigens (rHGE-14 and -15) were novel, while a third (rHGE-2), with no known function, has been described. The final two recombinant antigens (rHGE-9 and -17) represent overlapping segments of the ankyrin gene (ank). The addition of rHGE-9 ELISA data resulted in the detection of 78% (21 of 27) of acute-phase sera. When serologic data for all recombinants are combined, 96.2% (26 of 27) of convalescent-phase patient serum samples and 85.2% (23 of 27) of acute-phase patient serum samples are detected, indicating the potential of these antigens for use in the development of a rapid serologic assay for the detection of E. phagocytophila infection.

Serological expression cloning and immunological evaluation of MTB48, a novel Mycobacterium tuberculosis antigen.

Improved diagnostics are needed for the detection of Mycobacterium tuberculosis, especially for patients with smear-negative disease. To address this problem, we have screened M. tuberculosis (H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity with M. tuberculosis lysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of the M. tuberculosis H37Rv isolate and showed 99% identity to the Mycobacterium bovis and M. bovis BCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing the esat-6 gene that is deleted in the M. bovis BCG isolate. The mtb48 1,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in the M. tuberculosis and M. bovis isolates tested but not in other mycobacterial species tested, including Mycobacterium leprae and Mycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and the M. tuberculosis 38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity for M. tuberculosis infection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.

The effect of anterior osteophytes and flexural position on thoracic trabecular strain.

STUDY DESIGN: Compressive and shear trabecular strains were evaluated using six cadaveric thoracic spines that included anterior osteophytes. The treatments were divided into three groups: 1) osteophytes intact and the specimen in the neutral position, 2) osteophytes removed and the specimen in the neutral position, and 3) osteophytes removed and the specimen with 5 degrees of additional flexion. OBJECTIVES: To investigate the influence of osteophytes and flexural position on vertebral trabecular strain during axial compression. SUMMARY OF BACKGROUND DATA: In the thoracic spine, the incidence of anterior wedge fractures increases with the severity of kyphosis. It is unclear whether the role of anterior osteophytes in the thoracic spine is to restrict progressive kyphosis, conduct axial load anteriorly, or both. METHODS: Thoracic motion segments, T10-T12, were axially loaded in compression, and the minimum principal and maximum shear strains were measured using texture correlation. RESULTS: No dramatic changes were found in the spatial distribution of the strains following removal of the anterior osteophytes. Conversely, after removal of the osteophytes and orienting the specimen in 5 degrees of additional flexion, the strain distribution shifted anteriorly and the magnitude increased. CONCLUSIONS: This study demonstrated that osteophytes seem to restrict progressive kyphosis rather than conduct axial load anteriorly.

Segmental instrumentation for thoracic and thoracolumbar fractures: prospective analysis of construct survival and five-year follow-up.

BACKGROUND CONTEXT: Segmental instrumentation systems have replaced nonsegmental systems in all areas of spine surgery. Construct patterns for fracture stabilization have been adapted from deformity experience and from biomechanical studies using nonsegmental systems. Few studies have been completed to validate the use of these implants in trauma or to assess their relative strengths and weaknesses. PURPOSE: To substantiate the safety and efficacy of segmental spinal instrumentation used to treat patients with unstable spinal fractures and to identify successful construct strategies and potential pitfalls. STUDY DESIGN: A prospective, longitudinal single cohort study of patients treated with segmental instrumentation for fractures of the spine. Minimum 2-year follow-up. PATIENT SAMPLE: Seventy-five consecutive patients with unstable fractures of the thoracic, thoracolumbar and lumbar vertebrae, admitted to a level 1 trauma center. All patients sustained high-energy injuries: fifty-five (79%) were injured in motor vehicle accidents, 27 (38%) sustained two or more major additional injuries and 39 (56%) had neurological injuries. OUTCOME MEASURES: Perioperative morbidity and mortality, blood loss, surgical time; postoperative recovery, neurological recovery, complications, thromboembolic and pulmonary disease; long-term outcome measures of fusion, sagittal spinal alignment, construct survival, patient pain and function measures, and return to work and activity. METHODS: A longitudinal, prospective study of surgical outcome after segmental spinal instrumentation. Multifactorial assessment was carried out at prescribed intervals to a mean follow-up of 5 years (range, 2 to 8 years) from the time of surgery. Seventy patients were included in the final analysis. There were 17 thoracic, 36 thoracolumbar and 17 lumbar fractures. RESULTS: At 52 months mean follow-up, 57 of 62 patients (92%) had solid fusion with acceptable spinal alignment. Perioperative complications and mortality were less than expected, based on historical controls matched for injury severity. Rod and hook constructs had 97% good to excellent functional results, with no hardware complications. Six of 11 (55%) patients with short-segment pedicle instrumentation (SSPI) with no anterior column reconstruction had greater than 10 degrees of sagittal collapse during the fracture healing period. Twenty six of 36 neurologically injured patients (72%) experienced (mean) 1.5 Frankel grades recovery after decompression and stabilization. Residual neurological deficit determined return to work: 43 patients (70%) returned to work, 33 without restrictions, 10 with limitations. Five other patients (8%) were fit but unemployed. Fifteen percent experienced some form of hardware failure, but only three (5%) required revision. Hardware complications and fair to poor outcomes occurred after pedicle instrumentation without anterior reconstruction. Patients with anterior reconstruction had 100% construct survival, no sagittal deformity, and less pain. CONCLUSION: Segmental instrumentation allowed immediate mobilization of these severely injured patients, eliminating thromboembolic and pulmonary complications, and reducing overall morbidity and mortality. Segmental instrumentation produced a high rate of fusion with no rod breakage or hook failure. Pedicle screw constructs had a high rate of screw complications associated with anterior column insufficiency, but revision was not always necessary. Eighty percent of these severely injured patients were capable of returning to full-time employment, and 70% did so.

Transitions from alpha to pi helix observed in molecular dynamics simulations of synthetic peptides.

Molecular dynamics simulations were carried out for three 13-residue peptides of the form AcNH-A-A-E-X-A-E-A-H-A-A-E-K-A-CONH(2) with X = A, F, and W. All three peptides exhibited unexpected dynamical behavior, undergoing a transition from an alpha-helical to a pi-helical structure in the course of 5-ns trajectories in aqueous solution. Analysis of peptide length, accessible surface, interaction energies, hydrogen bonding, and dihedral angles was consistent with alpha --> pi transitions at 2800, 500, and 800 ps for X = A, F and W, respectively. The transitions occurred sequentially and cooperatively, propagating from the C- to the N-terminus for X = A and W and from the center toward both termini for X = F. The time scale of the overall transition ranged from 300 to 500 ps. For all three peptides the backbone structural transition was accompanied by a concerted rearrangement of the charged side chains, including a 3 A increase in the distance between carboxylate groups of Glu-3 and Glu-6. During the transition the peptide backbone hydrogen-bonding patterns were disrupted at the interface between the alpha-helical and nascent pi-helical regions, with peptide groups forming water-bridged hydrogen bonds. The peptide structures exhibited significant fluidity, with individual residues sampling alpha-, pi-, and 3(10)-helical conformations, as well as a "coil" state, without any intramolecular hydrogen bonds. The studied peptides have been designed to form alpha-helices when incorporated in novel hemoprotein model compounds, peptide-sandwiched mesohemes, which consist of two identical peptides covalently attached to an Fe(III) mesoporphyrin [Liu, D., Williamson, D. A., Kennedy, M. L., Williams, T. D., Morton, M. M., and Benson, D. R. (1999) J. Am. Chem. Soc. 121, 11798-11812]. The possibility of adopting pi-helical structures by the constituent peptides may influence the properties of the hemoprotein models.

Hemin is kinetically trapped in cytochrome b(5) from rat outer mitochondrial membrane.

Cytochrome b(5) from the outer mitochondrial membrane of rat liver (OM cyt b(5)) is substantially more stable to thermal and chemical denaturation than cytochrome b(5) from the endoplasmic reticulum of bovine liver (microsomal, or Mc cyt b(5)). In contrast, the corresponding apoproteins have similar stability, suggesting stronger interactions between hemin and the polypeptide in OM cyt b(5). Whereas complete transfer of hemin from bovine Mc cyt b(5) to apomyoglobin at pH 5.2 takes less than 1 h, hemin transfer from OM cyt b(5) is unmeasurably slow. Coupled with the previously reported 1:1 ratio of hemin orientational isomers in OM cyt b(5), this finding suggests that the cofactor is kinetically trapped under physiologically relevant conditions. This conclusion is confirmed by (1)H NMR studies which show that the hemin isomeric ratio changes when the protein is incubated for several hours at 68 degrees C. Interestingly, the orientational isomer favored in OM cyt b(5) is the form less favored in all other known cytochromes b(5).

Guest dechlorination and covalent capture following photoexcitation of inclusion complexes in water.

Photoexcitation of complexes between cyclophane 1 and 1- or 2-chloronaphthalene in aqueous solution leads to rapid dechlorination of the guest, a reaction driven by electron transfer from host to excited guest. The main photoproducts contain a naphthyl group covalently attached to the host framework. The results may lead to new approaches for remediating water contaminated with chlorinated aromatic compounds.

Serological expression cloning of novel immunoreactive antigens of Babesia microti.

Increased recognition of the prevalence of human babesiosis in the United States, together with rising concern about the potential for transmission of this infection by blood transfusion, has provided motivation to develop definitive serologic and molecular tests for the causative agent, Babesia microti. To develop more sensitive and specific assays for B. microti, we screened a genomic expression library with patient serum pools. This screening resulted in the identification of three classes of novel genes and an additional two novel, unrelated genes, which together encode a total of 17 unique B. microti antigens. The first class (BMN1-2 family) of genes encodes seven closely related antigens with a degenerate six-amino-acid repeat that shows limited homology to Plasmodium sp. merozoite and sporozoite surface antigens. A second class (BMN1-8 family) of genes encodes six related antigens, and the third class (BMN1-17 family) of genes encodes two related antigens. The two remaining genes code for novel and unrelated sequences. Among the three classes of antigens and remaining novel sequences, five were chosen to code for the most immunodominant antigens (BMN1-2, -9, -15, and -17 and MN-10). Western blot analysis with the resulting recombinant proteins indicated that these antigens were targets of humoral immune responses during B. microti infection in humans.

A polymorphic multigene family encoding an immunodominant protein from Babesia microti.

Human babesiosis in the United States is caused predominantly by Babesia microti, a tick-transmitted blood parasite. Improved testing methods for the detection of infection with this parasite are needed, since asymptomatic B. microti infection represents a potential threat to the blood supply in areas where B. microti is endemic. We performed immunoscreening of an expression library of genomic DNA from a human isolate of B. microti (strain MN1). Among 17 unique immunoreactive clones, we identified 9 which represent a related family of genes with little sequence homology to other known sequences but with an architecture resembling that of several surface proteins of Plasmodium. Within this family, a tandem array of a degenerate six-amino-acid repeat (SEAGGP, SEAGWP, SGTGWP, SGTVGP) was found in various lengths between relatively well conserved segments at the N and C termini. In order to examine within-clone variation, we developed a PCR protocol for direct recovery of a specific bmn1-6 homologue directly from 30 human blood isolates, 4 corresponding hamster isolates, and 5 geographically corresponding Peromyscus leucopus (white-footed mouse) isolates. Isolates from the hamsters had the same sequences as those found in the corresponding human blood, suggesting that genetic variation of bmn1-6 does not occur during passage. However, clones from different patients were often substantially different from each other with regard to the number and location of the degenerate repeats within the bmn1-6 homologue. Moreover, we found that strains that were closely related geographically were also closely related at the sequence level; nine patients, all from Nantucket Island, Mass., harbored clones that were indistinguishable from each other but that were distinct from those found in other northeastern or upper midwestern strains. We conclude that considerable genetic and antigenic diversity exists among isolates of B. microti from the United States and that geographic clustering of subtypes may exist. The nature of the bmn1-6 gene family suggests a mechanism of antigenic variation in B. microti that may occur by recombination, differential expression, or a combination of both mechanisms.

Multiepitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in patients with treated or untreated Chagas' disease.

A tetrapeptide and a recombinant protein, each representing 4 immunodominant epitopes of Trypanosoma cruzi, were tested by use of ELISA for the detection of serum antibodies. Sera from individuals with Chagas' disease, including persons untreated and successfully or unsuccessfully treated, were tested. These assays detected antibody in 100% of the parasitemias. The antibody reactivity decreased based on the success of treatment. Higher sensitivity was observed for tetrapeptide/recombinant protein assays than for lysate-based ELISA, and specificity was improved, particularly with Leishmania sera. The results indicate that multiepitope antigens provide a more sensitive and specific alternative to lysate for detection of anti-T. cruzi antibodies, as required for developing blood screening assays.

Natural diversity of Frankia strains in actinorhizal root nodules from promiscuous hosts in the family Myricaceae.

Actinorhizal plants invade nitrogen-poor soils because of their ability to form root nodule symbioses with N(2)-fixing actinomycetes known as Frankia. Frankia strains are difficult to isolate, so the diversity of strains inhabiting nodules in nature is not known. To address this problem, we have used the variability in bacterial 16S rRNA gene sequences amplified from root nodules as a means to estimate molecular diversity. Nodules were collected from 96 sites primarily in northeastern North America; each site contained one of three species of the family Myricaceae. Plants in this family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. We found that strain evenness varies greatly between the plant species so that estimating total strain richness of Frankia within myricaceous nodules with the sample size used was problematical. Nevertheless, Myrica pensylvanica, the common bayberry, was found to have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families. Myrica gale, sweet gale, yielded a few dominant sequences, indicating either symbiont specialization or niche selection of particular ecotypes. Strains in Comptonia peregrina nodules had an intermediate level of diversity and were all from a single major group of Frankia.

Urgent surgical stabilization of spinal fractures in polytrauma patients.

STUDY DESIGN: A prospective, longitudinal study of multiply injured patients treated with segmental instrumentation for spinal fractures with a minimum 2-year follow-up. OBJECTIVES: To determine whether urgent stabilization of spinal fractures in severely injured patients increases the risk of surgery compared with early treatment and historical results. SUMMARY AND BACKGROUND DATA: Opinion in clinical studies is divided about whether operative treatment offers an advantage over nonoperative treatment in isolated spine fractures. Concomitant trauma is rarely discussed relative to decision making or surgical timing. Urgent stabilization of long-bone fractures improves survival and outcome in polytrauma patients. To date, urgent treatment of spine fractures in polytrauma patients has not been considered in the literature. METHODS: Seventy-five consecutive patients treated with segmental instrumentation for spinal trauma were observed prospectively to assess perioperative and longterm outcome. Twenty-seven patients with severe polytrauma (injury severity score, > 26) were separately analyzed. Perioperative and postoperative results were analyzed relative to timing of surgery, injury severity score, and surgical approach. Urgent treatment was defined as that provided within 24 hours of the spinal injury, and early treatment was defined as that provided between 24 and 72 hours after injury. RESULTS: Twenty-five patients (93%) sustained two or more major injuries in addition to the spine fracture, and 17 of 27 (63%) had neurologic injury. The mean injury severity score approached or exceeded the LD50 (50% expected mortality) in each group--36.0 for the early-treatment group and 42.0 for the urgent group--but only one patient in each group died. There were no deep venous thromboses, pulmonary emboli, neurologic injuries, decubiti, deep wound infections, or episodes of sepsis in either group. Blood loss for anterior procedures was significantly higher in the urgent group, but estimated blood loss for posterior procedures was similar for both groups. At 49 months' mean follow-up, no revisions were necessitated by the urgent spinal treatment. CONCLUSIONS: Urgent spinal stabilization is safe and appropriate in polytrauma patients when progressive neurologic deficit, thoracoabdominal trauma, or fracture instability increase the risks of delayed treatment.

A multi-epitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in radioimmunoprecipitation-confirmed and consensus-positive sera.

Peptide epitopes of Trypanosoma cruzi have been identified through expression cloning. A tripeptide (2/D/E) containing three epitopes (TcD, TcE, PEP-2) was used in ELISA to detect antibodies to T. cruzi in 239 of 240 consensus-positive sera and 41 of 42 sera confirmed positive by radioimmunoprecipitation assay. The 1 discrepant consensus-positive serum was used to expression-clone a novel gene that contained a repeat sequence. A peptide corresponding to this sequence, TcLo1.2, was specific for T. cruzi. This antigen detected the discrepant consensus-positive serum and enhanced reactivity of low-positive sera in the tripeptide assay. A branched synthetic peptide, 2/D/E/Lo1.2, or a linear recombinant, r2/D/E/Lo1.2, realized all of the diagnostic features of the four epitopes, including the ability to boost reactivity of low-reactive sera. These studies show that peptides and recombinants containing multiple repeat epitopes are powerful tools for developing assays for T. cruzi antibody detection and have direct application in blood screening.

A cloned antigen (recombinant K39) of Leishmania chagasi diagnostic for visceral leishmaniasis in human immunodeficiency virus type 1 patients and a prognostic indicator for monitoring patients undergoing drug therapy.

Serologic assays using crude antigens for the diagnosis of visceral leishmaniasis in human immunodeficiency virus type 1 (HIV)-seropositive patients have been shown to lack sensitivity and specificity, particularly in AIDS patients. Antibodies to a cloned antigen, recombinant (r) K39, of Leishmania chagasi are specific for members of the Leishmania donovani complex and have been shown to indicate active disease in immunocompetent persons. This study demonstrated that antibodies to rK39 were also detectable in HIV-seropositive patients coinfected with Leishmania infantum. Furthermore, the rK39 ELISA was more sensitive than an IFA for detecting L. infantum infections in patients with AIDS. In addition, antibody titers to rK39 in HIV-negative patients infected with L. infantum or L. chagasi declined during treatment with meglumine antimoniate or liposomal amphotericin B. In contrast, most patients who clinically relapsed showed increased antibody titers to rK39. These data demonstrate the diagnostic and prognostic utility of rK39 in detecting active visceral leishmaniasis.