Phasing the 30S ribosomal subunit structure.

The methods involved in determining the 850 kDa structure of the 30S ribosomal subunit from Thermus thermophilus were in many ways identical to those that are generally used in standard protein crystallography. This paper reviews and analyses the methods that can be used in phasing such large structures and shows that the anomalous signal collected from heavy-atom compounds bound to the RNA is both necessary and sufficient for ab initio structure determination at high resolution. In addition, measures to counter problems with non-isomorphism and radiation decay are described.

Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: purification, crystallization and structure determination.

We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 A resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 A resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model.

Recognition of cognate transfer RNA by the 30S ribosomal subunit.

Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or "wobble," position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs.

Crystal structure of an initiation factor bound to the 30S ribosomal subunit.

Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis. In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3. Here we report the crystal structure of a complex of IF1 and the 30S ribosomal subunit. Binding of IF1 occludes the ribosomal A site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, burying them in pockets in IF1. The binding of IF1 causes long-range changes in the conformation of H44 and leads to movement of the domains of 30S with respect to each other. The structure explains how localized changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit.

Structure of the 30S ribosomal subunit.

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.

Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics.

The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.

The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit.

We have used the recently determined atomic structure of the 30S ribosomal subunit to determine the structures of its complexes with the antibiotics tetracycline, pactamycin, and hygromycin B. The antibiotics bind to discrete sites on the 30S subunit in a manner consistent with much but not all biochemical data. For each of these antibiotics, interactions with the 30S subunit suggest a mechanism for its effects on ribosome function.

Comparisons of urinary versus serum cortisol in older persons.

The present analysis compared urine- versus serum-based amounts of the stress hormone cortisol in two older adult samples, given that urine as a sample medium is a less expensive and less invasive method of determining cortisol amounts relative to serum. Seventy-three older adults provided urine samples as part of an ongoing study to assess levels of cortisol as a function of intellectual efficacy/performance; these data were compared to serum cortisol levels obtained from 96 older adults in a separate study examining health beliefs and cortisol levels. Analyses indicated that the cortisol levels did not differ across samples, i.e., cortisol amounts measured in serum or urine yielded similar, typical (within normal ranges) results. The data, though preliminary, indicate that urine may provide an alternative to serum when assessing cortisol in older persons.

MRA of the intracranial circulation in asymptomatic patients with sickle cell disease.

BACKGROUND: MR angiography (MRA) provides a mechanism for non-invasively studying blood flow, thus providing a new opportunity to study the intracranial circulation in asymptomatic sickle cell disease (SCD) patients. Although conventional angiography is the gold standard for the depiction of vascular anatomy, this is too invasive for an asymptomatic population. OBJECTIVE: To establish the range of appearances in asymptomatic SCD patients and to correlate brain MRI results (either sub-clinical abnormalities or normal brain parenchyma) with the MRA findings. MATERIALS AND METHODS: Brain MRI and MRA of the intracranial circulation was performed on 22 patients (13 male and 9 female, median age 7.5 years, range 1.3-20 years). Fourteen were homozygous SS and eight were SC. The median haematocrit at the time of MRI was 25.9 (range 13.8-33.3). RESULTS: On MR imaging, four patients had infarcts in eight vascular territories (six anterior and two posterior). In 3/4 of anterior vascular territories with infarction, long ( >/= 6 mm) segments of abnormal signal were seen at the internal carotid artery bifurcation with associated reduced distal flow. Short focal areas of abnormal signal were commonly seen where vessels branched, bifurcated or curved and were not associated with infarcts. These areas probably represent turbulence-related dephasing secondary to high velocity flow found in SCD. CONCLUSION: Long segments ( >/= 6 mm) of abnormal signal with reduced distal flow correlated with sub-clinical infarction.

Cerebral infarction in patients with AIDS.

PURPOSE: To establish the frequency, distribution, and pathogenesis of cerebral infarction as confirmed with MR imaging in a cohort of patients with acquired immunodeficiency syndrome (AIDS). METHODS: We reviewed all (71) abnormal cranial MR studies obtained at our institution in human immunodeficiency virus (HIV)-positive patients over a 2-year period and recorded the number and distribution of ischemic lesions, any associated abnormalities, and the MR angiographic findings, where available. Patients' charts were studied for relevant clinical data, biochemical and culture results, and potential etiologic factors. RESULTS: Twenty-two infarcts were seen in 13 of the 71 patients. Of these 22, the basal ganglia area was affected in 15, the middle cerebral artery territory in two, and the vertebrobasilar territory in five. Five patients had concomitant evidence of infection, six others used cocaine or were intravenous drug abusers. MR angiography was performed in eight patients; two of these had multiple lesions consistent with vasculitis, two had isolated lesions that corresponded with their parenchymal infarct, and four had normal findings. CONCLUSIONS: The frequency of infarction was 18%, higher than previously reported. The pathogenesis of infarction was multifactorial. Underlying infectious causes were identified in 39% of patients. Two patients had an idiopathic vasculitis.

Carotid MRA--what advantages do the turbo field-echo and 3D phase-contrast sequences offer?

Our purpose was to investigate some of the newer MR angiography (MRA) techniques for studying the carotid arteries. Forty-two arteries in seven asymptomatic, healthy volunteers were studied using five MRA sequences: two conventional time-of-flight sequences, 2D time-of-flight (2DTOF) and 3D time-of-flight (3DTOF); 2D and 3D magnetisation-prepared, segmented time-of-flight sequences (2DTFE and 3DTFE); and a 3D phase contrast angiography (3DPCA) sequence. A protocol that could be realistically employed in a routine clinical situation was chosen. 2DTOF had significantly (P < 0.05) better signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) than 2DTFE. 3DTOF demonstrated better SNR than 3DTFE but 3DTFE demonstrated better CNR than 3DTOF. 3DPCA provided maximal anatomical coverage. No one sequence provided optimal anatomical coverage, accurate demonstration of the carotid bulb and maximal SNR and CNR. The combination of 3DPCA and a 3D inflow sequence was best. 2DTOF sequences are useful when only one brief sequence is practicably feasible.

Fast spin echo vs conventional spin echo in cervical spine imaging.

The major attraction of fast-spin-echo (FSE) imaging is reduced acquisition time; however, careful review of the literature reveals many weaknesses: phase-encoded blurring, truncation artefact, bright fat signal, reduced magnetic susceptibility and increased motion artefact. Our aim was a prospective, blinded comparison of FSE and conventional spin echo (CSE) in the cervical spine. Both sequences were performed in 43 patients (19 males and 24 females; mean age 45 years, range 15-66 years). Twenty-eight patients were studied at 1.5 T and 15 at 0.5 T. Typical sequence parameters were: at 1.5 T, TR/TE 2000/90 CSE and 3000/120 FSE, and at 0.5 T, 2200/80 CSE and 2800/120 FSE. Time saved on the FSE was used to increase the matrix and the number of acquisitions. Two neuroradiologists evaluated the images for pathology, artefacts, disc signal intensity, thecal sac compression and image quality. Ten patients had cord lesions; 2 (20 %) were missed on CSE. In 4 of 10 patients with moderate/severe thecal sac compression, the degree of stenosis was apparently exaggerated on CSE. The mean degree of confidence for the CSE sequences was 1.8 and for the FSE 1.1, where 1 is optimal. For cervical spine imaging, FSE should be preferred to CSE.

MR appearances of the temporal evolution and resolution of infectious spondylitis.

OBJECTIVE: We undertook this study to document the MR appearances of evolving or resolving infectious spondylitis. MATERIALS AND METHODS: A retrospective review was carried out of all patients with infectious spondylitis who had undergone MR imaging from 1991-1993 at Boston University Hospital and Boston City Hospital Imaging Foundation. The study population consisted of 25 patients (seven females and 18 males). There was a bimodal age distribution with peaks at 34 and 59 years old (age range, 25-81 years old). The causative organism was isolated in 20. Sixteen had Staphylococcus aureus, two had mycobacterium tuberculosis, and two had gram-negative bacilli. Follow-up MR imaging was performed in 20. Nine had two studies, three had three, five had four, two had five, and one had six. The median length of follow-up was 8 weeks (range, 2-104 weeks). Follow-up MR appearances were correlated with clinical outcome. RESULTS: Early imaging revealed atypical appearances. Fourteen of 20 (70%) improved; the first sign of response to treatment was a reduction in the inflammatory soft tissue (8/14, 57%). Changes in the bones or discs concurrently progressed in six of eight patients (75%) including involvement of a new disc level in four (50%). A definitive sign of healing was a peripheral rim of high T1 signal in bone (5/14, 36%). Gadolinium enhancement persisted long after resolution of changes in the soft tissues, for up to a median of 17.5 weeks (range, 8-80 weeks). A subgroup of six IV drug users showed unique radiologic features. CONCLUSION: The early appearances of infectious spondylitis may be atypical. Resolution of soft-tissue change and fat deposition in the bone marrow are reliable signs of healing. Bone or disc changes can progress despite clinical improvement. Gadolinium enhancement can increase and persist after symptom resolution.

Magnetization transfer contrast MR in lesions of the head and neck.

PURPOSE: To compare lesion-to-background contrast with and without magnetization transfer (MT) in lesions of the head and neck. METHODS: Twenty lesions (16 malignant, 4 benign) were evaluated in 17 patients (11 men, 6 women; mean age, 58 years; age range, 39-76 years). In 13 patients, MR imaging was performed at 0.1 T with continuous-wave, off-resonance MT; in 4 patients, MR imaging was performed at 1.5 T with on-resonance, binomial MT prepulses. Fifteen sequences were conducted before the administration of gadopentetate dimeglumine; 13 were conducted after the administration of that contrast material. The ratio of signal intensity with the MT pulses (Ms) to signal intensity without the MT pulses (Mo) was calculated, as were the lesion-to-background contrast and the contrast-to-noise ratios. RESULTS: Ms/Mo showed both wide variability and considerable overlap among different lesion types. Images from MT sequences showed better contrast than those from non-MT sequences in 23 of 28 lesions (12 of 15 before and 11 of 13 after the administration of contrast material). The mean contrast improvement percentages (+/- standard deviation) were 165.5% (+/- 58%) on unenhanced images and 186.6% (+/- 84.8%) on contrast-enhanced images. The mean improvements in contrast-to-noise ratios were 156% (+/- 60%) on unenhanced images and 171.6% (+/- 98.1%) on contrast-enhanced images. CONCLUSION: MT improved contrast between nodes or tumors showing an MT effect and background tissue (usually fat) not showing an MT effect. MT also improved contrast between contrast-enhanced neoplastic lesions and background tissue that showed an MT effect.

Clinical utility of a new contrast option from magnetization transfer contrast.

Two magnetization transfer (MT) contrast effects, a T2-like effect and the improved contrast observed when gadolinium is used with MT, are combined in a single sequence. Forty patients (22 males:18 females; mean age, 45 years (23-87)) with suspected intracranial pathology underwent MRI on a 1.5 Tesla system. Of 46 lesions; seven were ischemic, five infective, seven neoplastic, four hemorrhagic, four multiple sclerosis, seven human immunodeficiency virus (HIV) leukoencephalopathy, nine normal/miscellaneous, and three gliosis. A conventional spin-echo sequence (TR 900 TE 15) was used with on-resonance binomial MT pulses. The sequence was performed postgadolinium +/- MT. The signal intensity ratios +/- MT were: white matter, 0.62 +/- 0.03; gray matter, 0.75 +/- 0.04; ischemia, edema, and demyelination, 0.75 (0.57-0.86); and gadolinium/methemoglobin, 0.85 (0.81-0.98). Areas which exhibited MT had T2-like contrast and those that did not maintained expected contrast for the given parameters. The result was a combination of T2-like contrast, gadolinium enhancement, and dark cerebrospinal fluid (CSF) providing both increased sensitivity to lesions which exhibited both contrast features and improved delineation of periventricular lesions. Furthermore, the differential signal between T2-like contrast of edema and gadolinium enhancement in neoplastic or infective lesions was maintained.

Is transcranial Doppler a worthwhile addition to screening tests for cerebrovascular disease?

PURPOSE: Carotid duplex imaging has become the standard diagnostic evaluation for patients with suspected cerebrovascular disease. Transcranial Doppler ultrasonography expands the noninvasive diagnostic capabilities to the intracranial circulation. The purpose of this study was to evaluate the results of routine transcranial Doppler studies on patients referred for noninvasive cerebrovascular evaluation. METHODS: A total of 670 patients had routine transcranial Doppler examinations as part of their noninvasive cerebrovascular evaluation. Patients were categorized clinically and according to their severity of extracranial internal carotid artery stenosis (< 50%, 50% to 79%, 80% to 99%, occlusion). Transcranial Doppler examinations were classified as normal or abnormal (intracranial stenosis, collateral pathway, > 30% velocity difference normal or abnormal (intracranial stenosis, collateral pathway, > 30% velocity difference between sides, flow reversal, and velocities +/- SD from normal). RESULTS: Forty-eight percent of the patients were women, and 52% were men. The average age was 65.5 years. Fifty-four percent of the patients were white, 42% were black, 3% were Hispanic, and 1% were other. Forty-eight percent presented with hemispheric symptoms, 34% had no symptoms, and 18% had nonhemispheric symptoms. Forty-five percent (304 of 670) had an interpretable transcranial Doppler examination. The ability to insonate the basal cerebral arteries through the temporal bone was significantly reduced in women (p < 0.0001), black patients (p < 0.0001), and older patients (p < 0.0001). The results of forty-four percent of interpretable examinations were normal, 19% demonstrated side-to-side velocity differences, 13% showed collateral pathways, 11% showed velocities +/- 2 SD, 10% showed an intracranial stenosis, and 4% showed reversed flow pattern. Although 56% of the patients had notable findings, no patient had their diagnostic or therapeutic plan altered by the transcranial Doppler results. CONCLUSION: Less than 50% of the patients referred for first-time cerebrovascular examination had access for an interpretable transcranial Doppler examination. Though the number of positive findings is reasonably high, no material impact on diagnostic or treatment plans was seen in the patients in this series. These results indicate that selection criteria for examination of the intracranial arteries should be refined and that transcranial Doppler scanning should not be incorporated as part of the "routine" noninvasive cerebrovascular examination.

MR angiography of normal pelvic arteries: comparison of signal intensity and contrast-to-noise ratio for three different inflow techniques.

OBJECTIVE: Two-dimensional (2D) time-of-flight (TOF) MR angiography has been the standard technique for evaluating arteries of the lower extremity. However, this technique is limited by artifacts resulting from vessel pulsation, as well as by relatively poor vessel-to-background contrast. The purpose of this study was to evaluate two cardiac-gated inflow techniques to determine whether they exhibited better contrast and signal performance than the standard technique of 2D TOF MR angiography of the iliac arteries. SUBJECTS AND METHODS: Fourteen subjects who had no clinical evidence of vascular disease had standard 2D TOF, gated 2D TOF, and gated 2D turbo field-echo MR angiography. Images were evaluated for signal-intensity ratio, signal-to-noise ratio, and contrast-to-noise ratio, in addition to qualitative evaluation. RESULTS: Turbo field-echo MR angiography exhibited significantly higher signal-intensity, signal-to-noise, and contrast-to-noise ratios than did either gated or standard MR angiography for all vessel segments. We found no significant difference between gated and standard 2D TOF techniques for any vessel segment. Qualitative features of turbo field-echo MR angiography included improved visualization of horizontal vessel segments compared with the standard 2D TOF technique, less effective venous saturation compared with either the gated or standard 2D TOF technique, and increased ghosting artifacts compared with the gated 2D TOF technique. CONCLUSION: Two-dimensional turbo field-echo MR angiography exhibits improved signal and contrast for evaluation of normal iliac segments compared with standard or gated 2D TOF MR angiography. This technique should replace standard 2D TOF MR angiography for evaluation of the iliac arteries.

The piezoelectric pulse sensor device: a prospective evaluation.

The goal of this prospective study of the piezoelectric pulse sensor device was to determine its technical applications and its ability to detect lower extremity occlusive arterial disease. Ten extremities (five volunteers) were evaluated to assess the ability to place the sensor in the correct anatomic position on a foot without a palpable pulse during cuff occlusion so that pulsatile flow would be detected following cuff deflation; its sensitivity as an end-point detector for pulsatile perfusion; and whether there is a linear qualitative pulse wave response with increasing perfusion pressures. Forty extremities (20 patients) with suspected occlusive arterial disease were studied to evaluate its capability of detecting perfusion as compared with the presence of a palpable pulse, an audible Doppler signal, and a foot volume waveform. The placement of the sensor on 10 normal limbs with temporary arterial occlusion resulted in a recordable waveform following cuff deflation in 100% of the dorsalis pedis arteries and in 10% of the posterior tibial arteries. The piezoelectric pulse sensor was as sensitive for detecting pulsatile perfusion as an audible Doppler signal and demonstrated a linear change in the waveform's amplitude and shape with incremental changes in perfusion pressure. In the 40 extremities with ankle/brachial indices ranging from 0.00 to 1.35, there was uniform agreement between pulse volume and Pulse Check waveforms. The piezoelectric pulse sensor is a sensitive method for monitoring lower extremity arterial perfusion when supplied by the dorsalis pedis artery; however, it is inadequate for the posterior tibial artery.(ABSTRACT TRUNCATED AT 250 WORDS)

A new approach to alcoholism detection in primary care.

This article describes a model that brings together the chemical dependency, mental health, and primary care services of a staff model HMO for the purpose of establishing a primary care clinic-based program to assist physicians in early detection of chemical dependency and frequent psychiatric disorders. The model creates a partnership between a master's-level professional social worker (MSW) and a designated family physician from the clinic. Their focus is on provider education, consultation, and on assisting patients with referrals to the appropriate services. Parameters of success include changes on referral patterns, use of the MSW's services, and clinic satisfaction. In addition, there are indications that early intervention has had a positive impact on subsequent use of other health care system's resources.