The architecture of the Gram-positive bacterial cell wall.

The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics1,2. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor1,3. In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength4-6. Here we applied atomic force microscopy7-12 to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface13,14, providing information complementary to traditional structural biology approaches.

Histopathological prevalence of subchondral insufficiency fracture of the femoral head.

OBJECTIVE: Subchondral insufficiency fracture of the femoral head (SIF) is a recently recognised cause of acute onset arthritis mostly in older women, which previously had been commonly considered either as osteonecrosis or osteoarthritis. The purpose of this study is to report the histopathological incidence of SIF using surgically removed femoral heads. METHODS: We reviewed 7718 consecutive femoral heads from 7286 patients removed over a 4-year period (2001-2004). There were 4211 women and 3507 men. The age range was from 13 to 96, average age was 60. There were 7349 hips with a preoperative clinical diagnosis of osteoarthritis and 369 with a preoperative clinical diagnosis of osteonecrosis. RESULTS: SIF was diagnosed histologically as the primary process in 501 of 7718 resected femoral heads (6.5%). The age range in these patients was from 20 to 93, with an average age of 68, in which 79% (394 hips) were over 60. They were 305 hips in women and 196 in men. The affected side was the right one in 253 hips and left in 248. The prevalence of SIF in cases with a preoperative diagnosis of osteoarthritis was 6.3% (460 of 7349), and with osteonecrosis was 11.1% (41 of 369). In all cases, callus and granulation tissue were observed histologically along the fracture line. CONCLUSIONS: In this large series of total hip replacements SIF as the cause of disease was diagnosed histologically in 6.5% of the surgically removed femoral heads.

High failure rate of a modern, proximally roughened, cemented stem for total hip arthroplasty.

The role of surface finish on the survivorship of cemented femoral stems continues to be debated. A total of 34 proximally roughened cemented stems were implanted in 33 consecutive patients undergoing total hip arthroplasty by a single surgeon. An alarmingly high failure rate was observed, prompting a retrospective chart review, analysis of radiographs, and evaluation of retrieved stems and pathological specimens. Nineteen patients were available with more than two years follow-up. Of these 19 patients, nine stems had failed (47%) due to severe osteolysis and stem loosening. Failures were significantly more common in the male gender (p<0.005), and young (p=0.05), tall (p<0.002), and heavy patients (p<0.004). All failed revised hips showed severe metallosis, with both gross and microscopic evidence of metallic shedding from the stems. Our findings suggest that this proximally roughened stem is susceptible to early failure. Failure is characterized by stem debonding, subsidence within the cement mantle, shedding of metallic and cement particles due to fretting, and rapidly progressive osteolysis. These findings have been observed with other rough surface finish cemented stems.

The Escherichia coli AmtB protein as a model system for understanding ammonium transport by Amt and Rh proteins.

The Escherichia coli ammonium transport protein (AmtB) has become the model system of choice for analysis of the process of ammonium uptake by the ubiquitous Amt family of inner membrane proteins. Over the past 6 years we have developed a range of genetic and biochemical tools in this system. These have allowed structure/function analysis to develop rapidly, offering insight initially into the membrane topology of the protein and most recently leading to the solution of high-resolution 3D structures. Genetic analysis has revealed a novel regulatory mechanism that is apparently conserved in prokaryotic Amt proteins and genetic approaches are also now being used to dissect structure/function relationships in Amt proteins. The now well-recognised homology between the Amt proteins, found in archaea, eubacteria, fungi and plants, and the Rhesus proteins, found characteristically in animals, also means that studies on E. coli AmtB can potentially shed light on structure/function relationships in the clinically important Rh proteins.

Glycerol dehydrogenase. structure, specificity, and mechanism of a family III polyol dehydrogenase.

BACKGROUND: Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) (glycerol:NAD(+) 2-oxidoreductase, EC 1.1.1.6) catalyzes the oxidation of glycerol to dihydroxyacetone (1,3-dihydroxypropanone) with concomitant reduction of NAD(+) to NADH. Analysis of the sequence of this enzyme indicates that it is a member of the so-called iron-containing alcohol dehydrogenase family. Despite this sequence similarity, GlyDH shows a strict dependence on zinc for activity. On the basis of this, we propose to rename this group the family III metal-dependent polyol dehydrogenases. To date, no structural data have been reported for any enzyme in this group. RESULTS: The crystal structure of B. stearothermophilus glycerol dehydrogenase has been determined at 1.7 A resolution to provide structural insights into the mechanistic features of this family. The enzyme has 370 amino acid residues, has a molecular mass of 39.5 kDa, and is a homooctamer in solution. CONCLUSIONS: Analysis of the crystal structures of the free enzyme and of the binary complexes with NAD(+) and glycerol show that the active site of GlyDH lies in the cleft between the enzyme's two domains, with the catalytic zinc ion playing a role in stabilizing an alkoxide intermediate. In addition, the specificity of this enzyme for a range of diols can be understood, as both hydroxyls of the glycerol form ligands to the enzyme-bound Zn(2+) ion at the active site. The structure further reveals a previously unsuspected similarity to dehydroquinate synthase, an enzyme whose more complex chemistry shares a common chemical step with that catalyzed by glycerol dehydrogenase, providing a striking example of divergent evolution. Finally, the structure suggests that the NAD(+) binding domain of GlyDH may be related to that of the classical Rossmann fold by switching the sequence order of the two mononucleotide binding folds that make up this domain.

Expression, refolding and crystallization of the OpcA invasin from Neisseria meningitidis.

OpcA is an integral outer membrane from the Gram-negative pathogen Neisseria meningitidis that plays a role in adhesion of meningococci to host cells. The protein was overexpressed in Escherichia coli in an insoluble form and a procedure developed for refolding by rapid dilution from denaturant into detergent solution. The refolded material was identical to native OpcA isolated from meningococci, as judged by overall molecular weight, migration on SDS-PAGE and reaction against monoclonal antibodies. Both native and recombinant OpcA crystallized under similar conditions to give an orthorhombic crystal form (P2(1)2(1)2), with unit-cell parameters a = 96.9, b = 46.3, c = 74.0 A. Complete data sets of reflections were collected from native and refolded OpcA to 2.0 A resolution.

Subchondral insufficiency fracture of the femoral head: histopathologic correlation with MRI.

OBJECTIVE: To correlate the magnetic resonance imaging (MRI) features with the histopathologic findings in subchondral insufficiency fracture (SIF) of the femoral head. DESIGN AND PATIENTS: This study was based on a retrospective review of the MRI features and histopathologic findings in seven patients with SIF who had had total hip replacement. Results. In all seven cases, MRI showed a bone marrow edema pattern in the femoral head, and a focal low-intensity band beneath the articular cartilage on some slices (not all) on the T1-weighted images. The shape of the low-intensity band varied: it was irregular and serpentine in four cases, well-delineated, smooth, and a mirror image to the articular surface in two cases, and parallel to the articular surface in one case. On histologic examination, the low-intensity band on MRI corresponded to a fracture line and its associated repair tissue. In all but one case, the band was not visible on T2-weighted or fat suppression images, and the proximal subchondral portion of the lesion had a homogeneous high signal intensity. This region of high signal intensity corresponded histopathologically to viable bone and marrow tissue with associated callus, edema, and vascular granulation tissue. CONCLUSIONS: SIF of the femoral head characteristically demonstrates a low-intensity band on T1-weighted images that corresponds, histopathologically, to a linear subchondral fracture and its associated repair tissue. In most cases, the subchondral portion of the lesion appears on T2-weighted images as an area of homogeneously high signal intensity.

[Osteonecrosis]. / L'ostéonécrose.

In orthopedic pathology, the pathologist is most commonly faced with the study of resected femoral heads for osteonecrosis. Such a study necessitates a knowledge of clinical findings, of physiopathology and chiefly of radiological findings. Osteonecrosis of the femoral head following fracture has been reported with four morphological stages which are very precisely described. In the same way, idiopathic, non traumatic, or primary osteonecrosis is covered, stressing the putative etiological factors and the most important findings of imaging. The review includes also the skeletal manifestations of decompression sickness as well as bone infarctions not associated with caisson disease, and spontaneous osteonecrosis of the knee. From the recruitment of the Hospital for Special Surgery, NY, it has been recently reported that a significant number of patients regarded as cases of osteonecrosis, either in the femoral head or in the knee, are actually cases of subchondral insufficiency fractures.

The role of subchondral insufficiency fracture in rapid destruction of the hip joint: a preliminary report.

OBJECTIVE: The pathophysiology of rapidly destructive arthrosis of the hip joint (RDA) is unknown. The purpose of this study was to document cases of subchondral insufficiency fracture of the femoral head, which has similar clinicoradiologic features to those of early-stage RDA. METHODS: This study was based on a retrospective review of 11 cases of subchondral insufficiency fracture of the femoral head that were confirmed histopathologically and for which radiographs both at the onset of hip pain and at the time of surgery were available. RESULTS: All patients were > 60 years of age (range 61-78 years, mean age 69), and 9 were women. On plain radiographs, the normal joint space had undergone rapid narrowing and/or disappeared within 9 months (mean 5.8 months). Magnetic resonance imaging, available in 2 cases, showed a pattern of bone marrow edema from the upper portion of the femoral head to the intertrochanteric region, with an associated focal low-intensity band on T1 paralleling the articular surface. In all cases, evidence of subchondral insufficiency fracture was confirmed histopathologically. In addition, in the marrow space, there were several round-to-oval granulomatous foci, which consisted of amorphous debris, fragmented bone, and articular cartilage surrounded by reactive histiocytes and giant cells. All 11 patients were osteopenic, as shown both radiologically and histopathologically. CONCLUSION: Subchondral insufficiency fracture resulting from osteopenia may lead to a rapid breakdown of the hip joint.

The quarternary molecular architecture of TetA, a secondary tetracycline transporter from Escherichia coli.

TetA, a tetracycline cation/proton antiporter, was expressed in Escherichia coli with a C-terminal tag of six histidines, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two-dimensional crystals were obtained after reconstitution of purified protein with lipids. Electron microscopy of negatively stained crystals revealed a trigonal symmetry, from which we infer that this secondary transporter has a trimeric structure. An overall molecular envelope can be described by a triangle of side approximately 100 A enclosing a central stain-filled depression. These dimensions are consistent with those obtained from projection views of single, isolated TetA particles that also display a trimeric architecture, confirming that the threefold symmetry is not simply a consequence of crystal-packing interactions. These data represent the first direct view of the quarternary arrangement of any antibiotic efflux pump. They are fully consistent with biochemical data on TetA, which indicate that it functions as a multimer and that the monomer consists of two domains, one of which plays the major part in oligomerization interactions.

A clinicopathologic study of osteonecrosis in the osteoarthritic hip.

OBJECTIVE: To investigate the incidence and nature of secondary osteonecrosis observed in osteoarthritis (OA) of the femoral head. DESIGN: This study is based on a retrospective review of the histopathologic and radiologic materials obtained from 906 consecutive cases (1007 femoral heads) diagnosed as OA. RESULTS: Secondary osteonecrosis was recognized grossly and confirmed microscopically in 38.2% of the femoral heads. The lesions were categorized into two types based on shape, size and depth; 'shallow' flat lesion (median axis 3-10 mm, depth 2-3 mm) with or without cysts (368 cases, 36.5%), and 'deep, wedge-shaped' large lesion (more than 20 mm across and 10 mm in depth) with or without cyst (17 cases, 1.7%). In the 'shallow' flat lesion, the age ranged from 25 to 88 (average 66), the female/male ratio was 0.8, and the location of osteonecrosis correlated best with the direction of migration in OA. In the 'deep, wedge-shaped' lesion, the age ranged from 56 to 92 (average 70), the female/male ratio was 1.8, and the location of osteonecrosis was similar to that found in primary osteonecrosis. CONCLUSION: Two different types of osteonecrosis were observed in OA. 'Shallow' osteonecrosis may be pressure necrosis as a result of eburnation, while 'deep, wedge-shaped' osteonecrosis appears to be an independent phenomena presumably caused by similar causal factors to those in primary osteonecrosis.

Spontaneous osteonecrosis of the knee: the result of subchondral insufficiency fracture.

BACKGROUND: Spontaneous osteonecrosis of the knee is a superficial subchondral lesion classically seen in the medial femoral condyle; in general, it is markedly different in its clinicopathological presentation from the classic wedge-shaped subchondral osteonecrotic lesions seen in the hip, knee, and other joints. Recent reports on subchondral insufficiency fracture of the femoral head, which has marked morphological similarities with spontaneous osteonecrosis of the knee, led us to reevaluate a series of patients who had had operative treatment because of a clinical and pathological diagnosis of spontaneous osteonecrosis of the knee. METHODS: We reviewed the cases of fourteen patients who had had operative treatment of spontaneous osteonecrosis of the knee in order to reevaluate the gross and histological morphology of this lesion. The patients included eight women and six men who ranged in age from fifty-nine to eighty-eight years. In all patients, the diagnosis of spontaneous osteonecrosis of the knee had been based on clinical presentation, imaging studies, and pathological findings. The appearance of the lesion on plain radiographs was categorized into four stages, which corresponded to the gross and histological findings. In stage 1, the radiographic appearance is normal; in stage 2, a radiolucent oval area is seen subchondrally or there is slight flattening of the convexity of the condyle, or both; in stage 3, the radiolucent area is expanded and is surrounded by a sclerotic halo; and in stage 4, secondary osteoarthritic changes are apparent. RESULTS: No patient had a stage-1 lesion. Three patients, all of whom had a stage-2 lesion, were considered to have a subchondral insufficiency fracture of the medial femoral condyle. Another six patients, all of whom had a stage-3 lesion, were considered to have a subchondral fracture and associated focal osteonecrosis that was confined to the area between the fracture line and the articular surface. The remaining five patients, three of whom had a stage-3 lesion and two of whom had a stage-4 lesion, had indeterminate findings because the lesion had become detached from the condyle. CONCLUSIONS: Our histopathological findings suggest that the primary event leading to spontaneous osteonecrosis of the knee is a subchondral insufficiency fracture and that the localized osteonecrosis seen in association with this disease is the result of a fracture.

Insufficiency subchondral fracture of the femoral head.

The authors recently encountered a 65-year-old osteoporotic woman who had had intractable pain in the hip joint that was diagnosed clinically as osteonecrosis. She was treated by total hip replacement. Histopathologically, the most striking finding was the presence of a subchondral fracture with associated callus formation and granulation tissue along both sides of the fracture line. There was no evidence of antecedent osteonecrosis. This case was diagnosed histopathologically as insufficiency subchondral fracture of the femoral head. This is the first case report to substantiate the presence of insufficiency subchondral fracture of the femoral head by both gross and microscopic examination. Because the treatment and management of insufficiency subchondral fracture are entirely different from osteonecrosis, it is important to differentiate between these two conditions.

E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy.

Hemolysin E (HlyE) is a novel pore-forming toxin of Escherichia coli, Salmonella typhi, and Shigella flexneri. Here we report the X-ray crystal structure of the water-soluble form of E. coli HlyE at 2.0 A resolution and the visualization of the lipid-associated form of the toxin in projection at low resolution by electron microscopy. The crystal structure reveals HlyE to be the first member of a new family of toxin structures, consisting of an elaborated helical bundle some 100 A long. The electron micrographs show how HlyE oligomerizes in the presence of lipid to form transmembrane pores. Taken together, the data from these two structural techniques allow us to propose a simple model for the structure of the pore and for membrane interaction.

Subchondral insufficiency fracture of the femoral head and medial femoral condyle.

This case report documents the clinical, radiographic, and histologic findings in a 69-year-old obese man, who had subchondral insufficiency fracture both in the femoral head and medial femoral condyle. On plain radiographs, both lesions underwent subchondral collapse. Magnetic resonance images of the left hip showed a bone marrow edema pattern with associated low-intensity band on T1-weighted images, which was convex to the articular surface. The histopathologic findings in the hip and knee were characterized by the presence of a subchondral fracture with associated callus and granulation tissue along both sides of a fracture line. There was no evidence of antecedent osteonecrosis. To our knowledge, this is the first case report to describe the multiple occurrence of collapsed subchondral insufficiency fracture.

Protuberant fibro-osseous lesions of the temporal bone: a unique clinicopathologic diagnosis.

OBJECTIVE: The objective of the study was to describe the clinical presentation and treatment of exophytic fibro-osseous temporal bone lesions, a clinical entity never previously reported, and to consider the differential diagnosis. STUDY DESIGN: The design of the study was a retrospective case review. SETTING: The setting was a tertiary referral center. PATIENTS: Two patients diagnosed with exophytic fibro-osseous temporal bone lesions were included in the study. INTERVENTION: The intervention used was surgical excision. MAIN OUTCOME MEASURE: The main outcome measures were clinical, radiographic, and histopathologic examination. RESULTS: There was no evidence of recurrence at 15 and 17 months after surgery. CONCLUSION: Although fibro-osseous lesions of the temporal bone have been previously reported, there have been no reported cases of an exophytic variant.

The prevalence and clinicopathological appearance of extension of osteonecrosis in the femoral head.

In about 50% of cases, osteonecrosis of the femoral head is known to occupy more than one site. There is controversy as to whether a single focus may increase in size. We have reviewed 606 consecutive femoral heads which had been surgically removed for osteonecrosis. Extension of osteonecrosis was observed in only two (0.3%) and was confirmed histopathologically by the enlargement of the necrotic segment beyond the repair zone formed for the primary necrosis into the adjacent, previously uninvolved bone. In both cases, the necrotic regions were wedge-shaped and occupied over 80% of the femoral head. It appears that an increase in size is extremely rare and that osteonecrosis is due to a single event. Our findings may be of value in assessing the use of joint-salvage procedures for osteonecrosis of the femoral head.