Blood: tests used to assess the physiological and immunological properties of blood.

The properties of blood and the relative ease of access to which it can be retrieved make it an ideal source to gauge different aspects of homeostasis within an individual, form an accurate diagnosis, and formulate an appropriate treatment regime. Tests used to determine blood parameters such as the erythrocyte sedimentation rate, hemoglobin concentration, hematocrit, bleeding and clotting times, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean cell volume, and determination of blood groups are routinely used clinically, and deviations outside the normal range can indicate a range of conditions such as anemia, pregnancy, dehydration, overhydration, infectious disease, cancer, thyroid disease, and autoimmune conditions, to mention a few. As these tests can be performed relatively inexpensively and do not require high levels of technical expertise, they are ideally suited for use in the teaching laboratory, enabling undergraduate students to link theory to practice. The practicals described here permit students to examine their own blood and that of their peers and compare these with clinically accepted normal ranges. At the end of the practicals, students are required to answer a number of questions about their findings and to link abnormal values to possible pathological conditions by answering a series of questions based on their findings.

The sympathetic release test: a test used to assess thermoregulation and autonomic control of blood flow.

When a subject is heated, the stimulation of temperature-sensitive nerve endings in the skin, and the raising of the central body temperature, results in the reflex release of sympathetic vasoconstrictor tone in the skin of the extremities, causing a measurable temperature increase at the site of release. In the sympathetic release test, the subject is gently heated by placing the feet and calves in a commercially available foot warming pouch or immersing the feet and calves in warm water and wrapping the subject in blankets. Skin blood flow is estimated from measurements of skin temperature in the fingers. Normally skin temperature of the fingers is 65-75°F in cool conditions (environmental temperature: 59-68°F) and rises to 85-95°F during body heating. Deviations in this pattern may mean that there is abnormal sympathetic vasoconstrictor control of skin blood flow. Abnormal skin blood flow can substantially impair an individual's ability to thermoregulate and has important clinical implications. During whole body heating, the skin temperature from three different skin sites is monitored and oral temperature is monitored as an index of core temperature. Students determine the fingertip temperature at which the reflex release of sympathetic activity occurs and its maximal attainment, which reflects the vasodilating capacity of this cutaneous vascular bed. Students should interpret typical sample data for certain clinical conditions (Raynaud's disease, peripheral vascular disease, and postsympathectomy) and explain why there may be altered skin blood flow in these disorders.

Molecular mechanism of target RNA transcript recognition by Argonaute-guide complexes.

Argonaute proteins participate in conferring all known functions of RNA-mediated gene silencing phenomena. However, prior to structural investigations of this evolutionarily conserved family of proteins, there was little information concerning their mechanisms of action. Here, we describe our crystallographic analysis of the PIWI domain of an archaeal Argonaute homolog, AfPiwi. Our structural analysis revealed that the Argonaute PIWI fold incorporates both an RNase-H-like catalytic domain and an anchor site for the obligatory 5' phosphate of the RNA guide strand. RNA-AfPiwi binding assays combined with crystallographic studies demonstrated that AfPiwi interacts with RNA via a conserved region centered on the carboxyl terminus of the protein, utilizing a novel metal-binding site. A model of the PIWI domain of Argonaute in complex with a small interfering RNA (siRNA)-like duplex is consistent with much of the existing biochemical and genetic data, explaining the specificity of the RNA-directed RNA endonuclease reaction and the importance of the 5' region of microRNAs (miRNAs) (the "seed") to nucleate target RNA recognition and provide high-affinity guide-target interactions.

Minimally invasive parathyroidectomy without intraoperative localization.

Minimally invasive parathyroidectomy (MIP) is gaining popularity as an alternative to traditional bilateral exploration for patients with primary hyperparathyroidism. The success of MIP relies on the ability of preoperative and intraoperative localization studies to guide a directed exploration for resection of a diseased gland. We hypothesize that excellent results can be achieved with MIP when only technetium-99m sestamibi (MIBI) is used for localization. We conducted a prospective analysis of all patients presenting with a biochemical diagnosis of primary hyperparathyroidism between January 1997 and November 2000. Patients meeting inclusion criteria were given a choice of MIP and directed exploration versus traditional bilateral exploration. Fifty patients chose MIP. Three patients who chose MIP had a negative MIBI, which left 47 patients in the primary study group. The MIBI correctly identified a parathyroid adenoma in 42 patients (89.3%). In two other patients MIBI was inaccurate; however, directed exploration was successfully converted to a bilateral exploration. Overall 44 of 47 (93.6%) patients in the study group were rendered normocalcemic after the initial operation. Three patients experienced persistent hypercalcemia and subsequently underwent successful bilateral exploration. Including those patients choosing a bilateral exploration, a total of 59 positive MIBI scans were evaluated. There were 54 true positives (positive predictive value 91.5%), and if all patients had chosen a MIP 94.9 per cent would have been successfully treated at the initial operation. Mean operative time for MIP was 54.6 minutes, and in 32 patients (68.1%) MIP was performed with local anesthesia and sedation. Twenty-six patients (55.3%) were discharged the same day of the procedure. There were no significant complications in any group analyzed. We conclude that MIP can be successfully performed on the basis of a positive MIBI scan. The present study highlighting many of the advantages of MIP questions the necessity of additional adjuncts such as intraoperative parathyroid hormone measurement and gamma-probe localization.

Crystallization and preliminary crystallographic analysis of human alanine:glyoxylate aminotransferase and its polymorphic variants.

The human hereditary disease primary hyperoxaluria type 1 is caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). In this study, the crystallization and preliminary crystallographic analysis of C-terminal His-tagged human AGT expressed in Escherichia coli is reported. At least two crystal forms were obtained using similar conditions for three different polymorphic variants, namely AGT, AGT[P11L] and AGT[P11L, I340M]. Complete data have been collected for all three AGT variants. The crystals of AGT[P11L] belong to space group P4(1)2(1)2 (or its enantiomorph), with unit-cell parameters a = b = 90.81, c = 142.62 A, and diffract to a resolution of 2.8 A.

Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis.

cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/L-dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and D-dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4x10(6) M(-1) x s(-1)) was comparable with that of human MIF, while that of p-hydroxyphenylpyruvate (9.1x10(4) M(-1) x s(-1)) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of L-dopachrome compared with non-esterified L-dopachrome (>87000-fold) and a high kcat (approximately 4x10(4) s(-1). The crystal structure, determined to 1.65 A (1 A=0.1 nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p-hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5 ng/ml-5 pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500 ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6 ng/ml (55 ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is partially orthologous to mammalian MIF.

Crystal structure of glycogen synthase kinase 3 beta: structural basis for phosphate-primed substrate specificity and autoinhibition.

Glycogen synthase kinase 3 beta (GSK3 beta) plays a key role in insulin and Wnt signaling, phosphorylating downstream targets by default, and becoming inhibited following the extracellular signaling event. The crystal structure of human GSK3 beta shows a catalytically active conformation in the absence of activation-segment phosphorylation, with the sulphonate of a buffer molecule bridging the activation-segment and N-terminal domain in the same way as the phosphate group of the activation-segment phospho-Ser/Thr in other kinases. The location of this oxyanion binding site in the substrate binding cleft indicates direct coupling of P+4 phosphate-primed substrate binding and catalytic activation, explains the ability of GSK3 beta to processively hyperphosphorylate substrates with Ser/Thr pentad-repeats, and suggests a mechanism for autoinhibition in which the phosphorylated N terminus binds as a competitive pseudosubstrate with phospho-Ser 9 occupying the P+4 site.

Stereotactic core-needle breast biopsy by surgeons: minimum 2-year follow-up of benign lesions.

OBJECTIVE: To evaluate the reliability of stereotactic core-needle breast biopsy (SCNB) performed by surgeons to detect histologically benign tissue. SUMMARY BACKGROUND DATA: Stereotactic core-needle breast biopsy is widely used to obtain tissue for definitive pathologic diagnosis of mammographically suspicious breast lesions. It has an incidence of malignancy detection similar to that of open biopsy. The potential for sampling error is a concern. Minimal data regarding follow-up and failure rate are available, especially from series performed exclusively by surgeons. METHODS: Pertinent medical records of all patients who underwent SCNB between April 1995 and October 1997 were reviewed. Breast lesions were classified by mammographic Breast Imaging-Reporting and Data Systems (BI-RADS) categories before SCNB. Benign biopsy specimens were classified as nonproliferative or proliferative. Malignant lesions and those with atypical histopathology by SCNB were excluded from this analysis. All lesions initially reported as benign were followed up mammographically for at least 2 years for any suspicious change requiring repeat biopsy. RESULTS: During the 31-month period, SCNB was performed on 694 lesions in 619 patients. Histologic evidence of malignancy was found in 112 lesions (16%). The initial histologic diagnosis for the remaining 582 lesions was benign. Four hundred lesions were available for follow-up; of these, 373 (93%) were mammographically categorized as BI-RADS 3 (probably benign) or 4 (suspicious). Three hundred forty-three lesions were categorized as nonproliferative and 151 as proliferative (94 had combined nonproliferative and proliferative histology). Follow-up ranged from 24 to 48 months (mean 33 months). During the follow-up period, 87 lesions (21.8%) underwent either image-guided or open biopsy. At the time of follow-up rebiopsy, ductal carcinoma in situ was found in four lesions and infiltrating ductal carcinoma was found in one, for an overall false-negative rate of 4.3% (5/117) and a negative predictive value of 98.8% (395/400). For the five false-negative cases, the interval from initial SCNB to definitive diagnosis ranged from 7 to 36 months. No correlation was found between the type of initial histopathology and development of malignancy. CONCLUSIONS: These results support SCNB as an alternative to open biopsy and show the reliability of SCNB when benign pathology is obtained. However, given the possibility of sampling error and the nature of breast disease, close mammographic and clinical follow-up is necessary. The false-negative rate and negative predictive value in this series compare favorably with those in other reports, supporting the fact that surgeons can confidently use SCNB in the evaluation and treatment of breast disease.

The ATPase cycle of Hsp90 drives a molecular 'clamp' via transient dimerization of the N-terminal domains.

How the ATPase activity of Heat shock protein 90 (Hsp90) is coupled to client protein activation remains obscure. Using truncation and missense mutants of Hsp90, we analysed the structural implications of its ATPase cycle. C-terminal truncation mutants lacking inherent dimerization displayed reduced ATPase activity, but dimerized in the presence of 5'-adenylamido-diphosphate (AMP-PNP), and AMP-PNP- promoted association of N-termini in intact Hsp90 dimers was demonstrated. Recruitment of p23/Sba1 to C-terminal truncation mutants also required AMP-PNP-dependent dimerization. The temperature- sensitive (ts) mutant T101I had normal ATP affinity but reduced ATPase activity and AMP-PNP-dependent N-terminal association, whereas the ts mutant T22I displayed enhanced ATPase activity and AMP-PNP-dependent N-terminal dimerization, indicating a close correlation between these properties. The locations of these residues suggest that the conformation of the 'lid' segment (residues 100-121) couples ATP binding to N-terminal association. Consistent with this, a mutation designed to favour 'lid' closure (A107N) substantially enhanced ATPase activity and N-terminal dimerization. These data show that Hsp90 has a molecular 'clamp' mechanism, similar to DNA gyrase and MutL, whose opening and closing by transient N-terminal dimerization are directly coupled to the ATPase cycle.

Structural adaptation to selective pressure for altered ligand specificity in the Pseudomonas aeruginosa amide receptor, amiC.

The AmiC protein in Pseudomonas aeruginosa is the negative regulator and ligand receptor for an amide-inducible aliphatic amidase operon. In the wild-type PAC1 strain, amidase expression is induced by acetamide or lactamide, but not by butyramide. A mutant strain of P. aeruginosa, PAC181, was selected for its sensitivity to induction by butyramide. The molecular basis for the butyramide inducible phenotype of P.aeruginosa PAC181 has now been determined, and results from a Thr-->Asn mutation at position 106 in PAC181-AmiC. In the wild-type PAC1-AmiC protein this residue forms part of the side wall of the amide-binding pocket but does not interact with the acetamide ligand directly. In the crystal structure of PAC181-AmiC complexed with butyramide, the Thr-->Asn mutation increases the size of the ligand binding site such that the mutant protein is able to close into its 'on' configuration even in the presence of butyramide. Although the mutation allows butyramide to be recognized as an inducer of amidase expression, the mutation is structurally sub-optimal, and produces a significant decrease in the stability of the mutant protein.

Crystal structure of the C-terminal SH2 domain of the p85alpha regulatory subunit of phosphoinositide 3-kinase: an SH2 domain mimicking its own substrate.

The binding properties of Src homology-2 (SH2) domains to phosphotyrosine (pY)-containing peptides have been studied in recent years with the elucidation of a large number of crystal and solution structures. Taken together, these structures suggest a general mode of binding of pY-containing peptides, explain the specificities of different SH2 domains, and may be used to design inhibitors of pY binding by SH2 domain-containing proteins. We now report the crystal structure to 1.8 A resolution of the C-terminal SH2 domain (C-SH2) of the P85alpha regulatory subunit of phosphoinositide 3-kinase (PI3 K). Surprisingly, the carboxylate group of Asp2 from a neighbouring molecule occupies the phosphotyrosine binding site and interacts with Arg18 (alphaA2) and Arg36 (betaB5), in a similar manner to the phosphotyrosine-protein interactions seen in structures of other SH2 domains complexed with pY peptides. It is the first example of a non-phosphate-containing, non-aromatic mimetic of phosphotyrosine binding to SH2 domains, and this could have implications for the design of substrate analogues and inhibitors. Overall, the crystal structure closely resembles the solution structure, but a number of loops which demonstrate mobility in solution are well defined by the crystal packing. C-SH2 has adopted a binding conformation reminiscent of the ligand bound N-terminal SH2 domain of PI3K, apparently induced by the substrate mimicking of a neighbouring molecule in the crystal.

New insights into structure-function relationships in nitrogenase: A 1.6 A resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein.

The X-ray crystal structure of Klebsiella pneumoniae nitrogenase component 1 (Kp1) has been determined and refined to a resolution of 1.6 A, the highest resolution reported for any nitrogenase structure. Models derived from three 1.6 A resolution X-ray data sets are described; two represent distinct oxidation states, whilst the third appears to be a mixture of both oxidized and reduced states (or perhaps an intermediate state). The structures of the protein and the iron-molybdenum cofactor (FeMoco) appear to be largely unaffected by the redox status, although the movement of Ser beta90 and a surface helix in the beta subunit may be of functional significance. By contrast, the 8Fe-7S P-cluster undergoes discrete conformational changes involving the movement of two iron atoms. Comparisons with known component 1 structures reveal subtle differences in the FeMoco environment, which could account for the lower midpoint potential of this cluster in Kp1. Furthermore, a non-proline- cis peptide bond has been identified in the alpha subunit that may have a functional role. It is within 10 A of the FeMoco and may have been overlooked in other component 1 models. Finally, metal-metal and metal-sulphur distances within the metal clusters agree well with values derived from EXAFS studies, although they are generally longer than the values reported for the closely related protein from Azotobacter vinelandii. A number of bonds between the clusters and their ligands are distinctly longer than the EXAFS values, in particular, those involving the molybdenum atom of the FeMoco.

Crystal structure and induction mechanism of AmiC-AmiR: a ligand-regulated transcription antitermination complex.

Inducible expression of the aliphatic amidase operon in Pseudomonas aeruginosa is controlled by an antitermination mechanism which allows production of the full-length transcript only in the presence of small-molecule inducers, such as acetamide. Ligand-regulated antitermination is provided by AmiC, the ligand-sensitive negative regulator, and AmiR, the RNA-binding positive regulator. Under non-inducing or repressing growth conditions, AmiC and AmiR form a complex in which the activity of AmiR is silenced. The crystal structure of the AmiC-AmiR complex identifies AmiR as a new and highly unusual member of the response-regulator family of bacterial signal transduction proteins, regulated by sequestration rather than phosphorylation. Comparison with the structure of free AmiC reveals the subtle mechanism of ligand-induced release of AmiR.

Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin.

The cellular activity of several regulatory and signal transduction proteins, which depend on the Hsp90 molecular chaperone for folding, is markedly decreased by geldanamycin and by radicicol (monorden). We now show that these unrelated compounds both bind to the N-terminal ATP/ADP-binding domain of Hsp90, with radicicol displaying nanomolar affinity, and both inhibit the inherent ATPase activity of Hsp90 which is essential for its function in vivo. Crystal structure determinations of Hsp90 N-terminal domain complexes with geldanamycin and radicicol identify key aspects of their nucleotide mimicry and suggest a rational basis for the design of novel antichaperone drugs.

Crystal structure of an octameric RuvA-Holliday junction complex.

Holliday junctions occur as intermediates in homologous recombination and DNA repair. In bacteria, resolution of Holliday junctions is accomplished by the RuvABC system, consisting of a junction-specific helicase complex RuvAB, which promotes branch migration, and a junction-specific endonuclease RuvC, which nicks two strands. The crystal structure of a complex between the RuvA protein of M. leprae and a synthetic four-way junction has now been determined. Rather than binding on the open surface of a RuvA tetramer as previously suggested, the DNA is sandwiched between two RuvA tetramers, which form a closed octameric shell, stabilized by a conserved tetramer-tetramer interface. Interactions between the DNA backbone and helix-hairpin-helix motifs from both tetramers suggest a mechanism for strand separation promoted by RuvA.

ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.

Hsp90 is an abundant molecular chaperone essential to the establishment of many cellular regulation and signal transduction systems, but remains one of the least well described chaperones. The biochemical mechanism of protein folding by Hsp90 is poorly understood, and the direct involvement of ATP has been particularly contentious. Here we demonstrate in vitro an inherent ATPase activity in both yeast Hsp90 and the Escherichia coli homologue HtpG, which is sensitive to inhibition by the Hsp90-specific antibiotic geldanamycin. Mutations of residues implicated in ATP binding and hydrolysis by structural studies abolish this ATPase activity in vitro and disrupt Hsp90 function in vivo. These results show that Hsp90 is directly ATP dependent in vivo, and suggest an ATP-coupled chaperone cycle for Hsp90-mediated protein folding.

Initial cervical exploration for parathyroidectomy is not benefited by preoperative localization studies.

Published data is controversial as to the ability of preoperative localization studies (PLS) to enhance the outcome of initial cervical exploration in patients with primary hyperparathyroidism (PHPT). One surgeon's experience was reviewed to compare surgical success, operative time, and morbidity of initial cervical exploration for PHPT in patients who had undergone PLS versus those who had not. From August 1991 to September 1997, 95 patients who had not undergone prior central cervical exploration presented for surgical management of PHPT. Sixty-seven patients underwent initial cervical exploration without any PLS having been performed (Group A). Twenty-eight patients underwent PLS, either alone or in combination, before surgical intervention (Group B). Analysis of intergroup variability was conducted upon the data available using a two-tailed t test for independent samples. In addition, the sensitivities and positive predictive values of the PLS were calculated using study reports and operative and histologic findings. There was no statistically significant difference in surgical success between those patients who had PLS and those that did not undergo PLS. Sixty-four of 67 patients (95.5%) not having PLS were cured with initial surgery, while 27 of 28 patients (96.4%) who had PLS were surgically cured. Mean postoperative calcium and intact parathormone levels were similar between the two groups, and the mean operative time did not differ. Permanent hypocalcemia occurred in one patient, and five patients had transient hoarseness. Thirty-six total PLS were obtained at an average cost of $752.68/patient, and seven patients underwent multiple tests. Overall, sestamibi scan had the highest positive predictive value (81%). For adenomatous disease alone, sestamibi scan was the most sensitive (83%). Our study shows that for matched groups limited to age, sex, and clinical diagnosis, the use of PLS did not shorten operative time, decrease complication frequency, nor alter the success of the operation as measured by postoperative calcium and parathormone levels. Therefore, routine use of preoperative localization studies before initial cervical exploration for PHPT cannot be recommended.

Stereotactic and ultrasound core needle breast biopsy performed by surgeons.

BACKGROUND: The authors evaluated outcomes and treatment costs of stereotactic core needle biopsy (SCNB) and ultrasound core needle biopsy (UCNB), and needle localization biopsy (NLB) in managing patients with mammographic abnormalities presenting to the surgeon. METHODS: Data for all patients with mammographic lesions who underwent SCNB or UCNB since their introduction at this institution were prospectively collected over 17 months. Mean inclusive costs of the three procedures were accumulated and compared. RESULTS: Stereotactic core needle biopsy was performed for 342 lesions in 319 women, for a malignancy rate of 19%; UCNB was performed for 157 lesions in 144 patients, yielding a malignancy rate of 17%. With a mean follow-up of 13.5 months, 1 patient with in situ carcinoma was diagnosed late. Absolute cost savings for the period studied was $721,963. CONCLUSIONS: Minimally invasive breast biopsy procedures can safely and reliably be performed by surgeons in clinical practice with increased patient convenience and decreased costs.

Potassium and ventilation during exercise above and below the ventilatory threshold.

Increases in arterial plasma potassium during exercise may provide an important drive to ventilation. We examined the changes in arterialized venous plasma potassium concentration ([K +]av) and ventilation that occur during sustained exercise at workloads above and below the ventilatory threshold (Vt) in young health humans. After the onset of exercise at a workload below-Vt, [K +]av rose by 0.3 (+/- 0.1) mmol l-1 (mean +/- SEM). Following 30 min of exercise at this intensity [K +]av had fallen (P < 0.05, ANOVA) by an amount approximately equal to one third of the initial increase. While [K +]av fell, ventilation remained stable. At 5 min after the onset of sustained exercise above the Vt [K +]av had risen by 0.7 (+/- 0.1) mmol l-1 and thereafter remained constant. Ventilation slowly increased throughout the above-Vt protocol. These results show significant differences in the time course of the changes in [K +]av and ventilation. They do not support the hypothesis that changes in [K +]a during moderate exercise cause linearly related changes in ventilation.