Biomedical subjects
Publications and source records attributed to V Ramakrishnan.
A 3-year-old boy presented with a 3 x 3 cm dermoid cyst in the midline of the anterior chest wall. This was successfully removed, using an endoscopic-assisted technique, through a single incision placed in the anterior axillary fold, avoiding the need for a mid-sternal incision. This technique and its application to paediatric soft-tissue surgery are discussed.
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A retrospective study was conducted in 75 consecutive patients requiring postmastectomy breast reconstruction over a period of 30 months. Each woman was offered one of the following four reconstructive options: free transverse rectus abdominis musculocutaneous flap (total number of reconstructions, n = 34); latissimus dorsi musculocutaneous flap (with or without expander and implant, n = 14); endoscopically assisted harvest of the latissimus dorsi muscle (with expander and implant, n = 13); and application of expander and implant only (n = 12). Of those patients originally selected for retrospective study, six did not meet the short-term prognostic criteria, and concerted attempts to contact two others proved unsuccessful. The remaining 67 patients were examined for the clinically assessed aesthetic appearance of the reconstructed breast(s), the subjective self-assessment of patient satisfaction, and the possible development of postoperative complications. Of these patients, six required bilateral surgery, which accounts for a final sample size of 73 individual breast reconstructions. The 67 individual patients were assessed after a minimum time of 6 months postreconstruction and became the sampling units for analysis. The free transverse rectus abdominis musculocutaneous flap procedure was the preferred method of breast reconstruction in 34 of 73 patients (47 percent), provided that it was generally agreed that the patient could endure a prolonged operation and that there was sufficient unscarred abdominal tissue available. Thereafter, postmastectomy radiotherapy at the chest wall became the primary criterion for assignment of a patient to a particular surgical procedure. Whenever radiotherapy resulted in poor-quality skin at the chest wall, endoscopically assisted transfer of latissimus dorsi muscle flap was considered to be the optimal treatment (13 of 73 patients, or 18 percent). Body mass index and smoking were secondary factors that were taken into account when this alternative technique was being considered.In the absence of radiotherapy, and provided that the chest wall was minimally scarred, patients who were reluctant to have reconstruction with autologous tissue were treated with expander and implant only (12 of 73, or 16 percent). This third procedure is a physically less arduous ordeal for the patient and was therefore the choice for all patients for whom a prolonged operation was not a realistic option. The fourth (and final) surgical procedure, latissimus dorsi musculocutaneous flap (with or without expander and implant), was selected for all patients with a better quality of skin over the chest wall, those whose abdomen was extensively scarred, and those who were on a general surgeon's operating list to undergo immediate breast reconstruction after mastectomy (14 of 73, or 19 percent). Equally good aesthetic results could be demonstrated with each of the four treatment options, provided that the reconstructive procedure selected was optimal for the individual patient and in accordance with the criteria described above. A variety of potential risk factors were considered for association with postoperative complications, including prescribed medication, obesity, smoking behavior, use of radiotherapy, and the recorded aggregated operative time. Of these, only body mass index (p < 0.001) and use of steroids (p = 0.016) were identified as having statistically significant effects on the incidence of adverse events.Finally, the general level of satisfaction expressed by the patient was highly correlated with a good appearance of the reconstructed breast, the physical comfort experienced while wearing a brassiere, and the general mobility of the unsupported reconstruction.
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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.
Proteinase-activated receptor-2 (PAR-2) is a member of a family of G-protein-coupled, seven-transmembrane domain receptors that are activated by proteolytic cleavage. The receptor is expressed in a number of different tissues and potential physiological activators identified thus far include trypsin and mast cell tryptase. Acrosin, a trypsin-like serine proteinase found in spermatozoa of all mammals, was found to cleave a model peptide fluorescent quenched substrate representing the cleavage site of PAR-2. This substrate was cleaved with kinetics similar to those of the known PAR-2 activators, trypsin and mast cell tryptase. Acrosin was also shown to induce significant intracellular calcium responses in Chinese hamster ovary cells stably expressing intact human PAR-2, most probably due to activation of the receptor. Immunohistochemical studies using PAR-2 specific antibodies indicated that the receptor is expressed by mouse oocytes, which suggests that acrosin may play additional role(s) in the fertilization process via the activation of PAR-2 on oocytes.
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.
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.
BACKGROUND: X-ray crystallography has recently yielded much-improved electron-density maps of the bacterial ribosome and its two subunits and many structural details of bacterial ribosome subunits are now being resolved. One approach to complement the structures and elucidate the details of rRNA and protein packing is to determine structures of individual protein components and model these into existing intermediate resolution electron density. RESULTS: We have determined the solution structure of the ribosomal protein S16 from Thermus thermophilus. S16 is a mixed alpha/beta protein with a novel folding scaffold based on a five-stranded antiparallel/parallel beta sheet. Three large loops, which are partially disordered, extend from the sheet and two alpha helices are packed against its concave surface. Calculations of surface electrostatic potentials show a large continuous area of positive electrostatic potential and smaller areas of negative potential. S16 was modeled into a 5.5 A electron-density map of the T. thermophilus 30S ribosomal subunit. CONCLUSIONS: The location and orientation of S16 in a narrow crevice formed by helix 21 and several other unassigned rRNA helices is consistent with electron density corresponding to the shape of S16, hydroxyl radical protection data, and the electrostatic surface potential of S16. Two protein neighbors to S16 are S4 and S20, which facilitate binding of S16 to the 30S subunit. Overall, this work exemplifies the benefits of combining high-resolution nuclear magnetic resonance (NMR) structures of individual components with low-resolution X-ray maps to elucidate structures of large complexes.
Although hydrogens comprise half of the atoms in a protein molecule and are of great importance chemically and structurally, direct visualization of them by using crystallography is difficult. Neutron crystallography is capable of directly revealing the position of hydrogens, but its use on unlabeled samples faces certain technical difficulties: the large incoherent scattering of hydrogen results in background scattering that greatly reduces the signal to noise of the experiment. Moreover, whereas the scattering lengths of C, N, and O are positive, that of hydrogen is negative and about half the magnitude. This results in density for hydrogens being half as strong and close to the threshold of detection at 2.0-A resolution. Also, because of its opposite sign, there is a partial cancellation of the hydrogen density with that from neighboring atoms, which can lead to ambiguities in interpretation at medium resolution. These difficulties can be overcome by the use of deuterated protein, and we present here a neutron structure of fully deuterated myoglobin. The structure reveals a wealth of chemical information about the molecule, including the geometry of hydrogen bonding, states of protonation of histidines, and the location and geometry of water molecules at the surface of the protein. The structure also should be of broader interest because it will serve as a benchmark for molecular dynamics and energy minimization calculations and for comparison with NMR studies.
Single crystal electron paramagnetic resonance (EPR) of Cu(C5H5NO)6(BF4)2 is studied at 300 K. The spin Hamiltonian parameters obtained at 300 K are A11 = -149.8, A22 = -50.7 and A33 = -37.7 (all in units of 10(-4) cm(-1)), g11 = 2.3452, g22 = 2.0679 and g33 = 2.0695. Angular variation of hyperfine structure lines shows the presence of a single magnetic site. The low magnitude of the A11 value for Cu(C5H5NO)6(BF4)2 is rationalized in terms of a mixed d(x2-y2)/d(z2) ground state.
BACKGROUND: In this study, repeated measurements were made of levels of mold spores, bacteria, and dust-mite allergens over a 7-month period in the homes of asthmatics, and relationships with measures of asthma severity were evaluated. METHODS: A sample of 57 asthmatic individuals, living in 44 homes in East Moline, Illinois, and nearby communities, participated in a panel study. The homes were visited up to nine times during the study to collect air and dust samples. Asthma severity indicators were derived from questionnaire data and from the daily health records from the panel study. Associations between indoor levels of mold spores, bacteria, and dust-mite allergens were tested with several asthma severity indicators. RESULTS: There was evidence of associations between all asthma severity measures and levels of total and gram-negative bacteria, but mold-spore abundance was associated only with emergency room (ER) visits for asthma. No significant associations were found with house-dust-mite allergen and any of the asthma severity indicators, but the levels of dust-mite allergen were low, with median concentrations of 0.18 microg/g dust Der f 1 and 0.19 microg/g dust Der p 1. CONCLUSIONS: Some evidence was found for associations of increased concentrations of gram-negative bacteria and mold spores with asthma severity, particularly with ER visits. No association was found between house-dust-mite allergen and asthma severity indicators; however, the mite-allergen levels in the study homes were generally well below the proposed threshold level of 2 microg/g dust.
Systemic lupus erythematosus is a non-organ specific, autoimmune disease characterised by antinuclear antibodies and vasculitis. This may manifest itself with areas of cutaneous necrosis which may require extensive excision and reconstruction. Major areas of skin and soft tissue loss may require coverage with either local or microvascular free tissue transfer. This presents potential difficulties in patients with generalised vasculitis. We present the case of a professional cellist with extensive soft tissue loss in her dominant hand, successfully reconstructed with a free latissimus dorsi muscle flap, treated with peri- and postoperative anticoagulation and corticosteroids.
Certain patients requesting breast reconstruction may be described as having a high-risk chest wall with regard to implant loss and well-documented high complication rates. Such patients have a combination of one or more of the following: previous chest wall radiotherapy, heavy smoking, and thin, tethered chest wall flaps. If autologous transfer is not appropriate for such patients then reconstruction may be difficult. In this specific patient group the assistance of endoscopy has been used to raise the latissimus dorsi muscle to cover an expander placed within an endoscopically created chest wall pocket. The first 12 consecutive cases using this technique are discussed, showing an expander loss rate of 8% for the primary implant placement.
Lesions in the genes for GPIb alpha, GPIb beta or GPIX result in a bleeding diathesis, the Bernard-Soulier syndrome (BSS), which associates a platelet adhesion defect with thrombocytopenia, giant platelets and abnormal megakaryocytes (MK). The role of GPV, also absent in BSS, was recently addressed by gene targeting in mice. While a negative modulator function for GPV on thrombin-induced platelet responses was found in one model, the absence of GP V had no effect on GPIb-IX expression or platelet adhesion. Our study extends previous results and reports that electron microscopy of bone marrow from the GPV knockout mice revealed a normal MK ultrastructure and development of the demarcation membrane system (DMS). There was a usual presence of MK fragments in the bone marrow vascular sinus. Immunogold labelling of MK from the knockout mice showed a normal distribution of GPIb-IX in the DMS and on the cell surface. The distribution of fibrinogen, vWF and P-selectin was unchanged with, interestingly, P-selectin also localised within the DMS in both situations. Thus GPV is not crucial to MK development and platelet production, consistent with the fact that no mutation in the GPV gene has as yet been described in BSS.
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We report the crystal structure of the yeast protein Hpa2 in complex with acetyl coenzyme A (AcCoA) at 2.4 A resolution and without cofactor at 2.9 A resolution. Hpa2 is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, a family of enzymes with diverse substrates including histones, other proteins, arylalkylamines and aminoglycosides. In vitro, Hpa2 is able to acetylate specific lysine residues of histones H3 and H4 with a preference for Lys14 of histone H3. Hpa2 forms a stable dimer in solution and forms a tetramer upon binding AcCoA. The crystal structure reveals that the Hpa2 tetramer is stabilized by base-pair interactions between the adenine moieties of the bound AcCoA molecules. These base-pairs represent a novel method of stabilizing an oligomeric protein structure. Comparison of the structure of Hpa2 with those of other GNAT superfamily members illustrates a remarkably conserved fold of the catalytic domain of the GNAT family even though members of this family share low levels of sequence homology. This comparison has allowed us to better define the borders of the four sequence motifs that characterize the GNAT family, including a motif that is not discernable in histone acetyltransferases by sequence comparison alone. We discuss implications of the Hpa2 structure for the catalytic mechanism of the GNAT enzymes and the opportunity for multiple histone tail modification created by the tetrameric Hpa2 structure.
A role for glycoprotein (GP)V in platelet function has been proposed on the basis of observations that GP V is the major thrombin substrate on intact platelets cleaved during thrombin-induced platelet aggregation, and that GP V promotes GP Ib-IX surface expression in heterologous cells. We tested the hypotheses that GP V is involved in thrombin-induced platelet activation, in GP Ib-IX expression, and in other platelet responses by generating GP V null mice. Contrary to expectations, GP V -/- platelets were normal in size and expressed normal amounts of GP Ib-IX that was functional in von Willebrand factor binding, explaining why defects in GP V have not been observed in Bernard-Soulier syndrome, a bleeding disorder caused by a lack of functional GP Ib-IX-V. Moreover, in vitro analysis demonstrated that GP V -/- platelets were hyperresponsive to thrombin, resulting in increased fibrinogen binding and an increased aggregation response. Consistent with these findings, GP V -/- mice had a shorter bleeding time. These data support a role for GP V as a negative modulator of platelet activation. Furthermore, they suggest a new mechanism by which thrombin enhances platelet responsiveness independent of activation of the classical G-protein-coupled thrombin receptors.