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Biomedical subjects

S Sinha

Publications and source records attributed to S Sinha.

At least 19 recordsLinked to original sources
PubMed1992

Isolation and quantification of soluble Alzheimer's beta-peptide from biological fluids.

Cerebral deposition of the beta-amyloid peptide (A beta) is an invariant feature of Alzheimer's disease. Since the original isolation and characterization of A beta (ref. 1) and the subsequent cloning of its precursor protein, no direct evidence for the actual production of discrete A beta has been reported. Here we investigate whether A beta is present in human biological fluids using antibodies specific for an epitope within A beta that spans the site of normal constitutive cleavage. These antibodies were used to construct a sandwich-type enzyme-linked immunosorbent assay that detects A beta in cerebrospinal fluid, plasma and conditioned medium of human mixed-brain cells grown in vitro (see also ref. 14). By affinity chromatography, we have purified and sequenced A beta and a novel A beta fragment from human cerebrospinal fluid and conditioned medium of human mixed-brain cell cultures. These findings demonstrate that A beta is produced and released both in vivo and in vitro. These observations offer new opportunities for developing diagnostic tests for Alzheimer's disease and therapeutic strategies aimed at reducing the cerebral deposition of A beta.

Alzheimer Disease↗
PubMed1992

Evidence for a nonsecretory, acidic degradation pathway for amyloid precursor protein in 293 cells. Identification of a novel, 22-kDa, beta-peptide-containing intermediate.

We have analyzed the metabolic pathway of maturation of APP751 in stably transfected 293 cells, in the presence of either of the cysteine protease inhibitors leupeptin or E-64. Metabolic labeling, followed by immunoprecipitation at various times in the chase with a rabbit polyclonal antibody (anti-BX6) specific to the carboxyl-terminal end of amyloid precursor protein (APP), revealed the accumulation of a novel approximately 22-kDa carboxyl-terminal fragment (22-CTF) in the inhibitor-treated cells. This fragment, which was not detectable in untreated cells, was immunoprecipitated by four separate antibodies to the carboxyl-terminal region of APP as well as by polyclonal and monoclonal antibodies specific to the first 16 amino acids of the beta-peptide domain. Antibodies to the amino-terminal end of APP do not, however, recognize the fragment. Co-treatment of the inhibitor-treated cells with either of the lysosomotropic agents chloroquine or ammonium chloride completely blocked the generation of this fragment but did not significantly affect APP maturation or secretion. All, however, slowed the intracellular turnover of the cell-associated, approximately 9-kDa carboxyl-terminal fragment (c-CTF) produced during constitutive secretion. Densitometric analyses of these results suggest that this non-secretory pathway of APP degradation, mediated by cysteine proteases in an intracellular acidic compartment, accounts for approximately 70% of total APP metabolism and that a key processing intermediate in this pathway is a 22-kDa, beta-peptide-containing APP carboxyl-terminal fragment. It is possible that inefficient degradation of such an intermediate leads to the formation of aggregating beta-peptide.

Amyloid beta-Protein Precursor↗
PubMed1992

Three different polypeptides are necessary for DNA binding of the mammalian heteromeric CCAAT binding factor.

Full-length cDNA clones for the CBF-A and CBF-B subunits of the CCAAT binding mammalian heteromeric transcription factor (CBF) have previously been isolated from both rat and mouse. Whereas recombinant CBF-B binds to DNA after complementation with a highly purified CBF-A fraction, recombinant CBF-A was unable to bind to DNA after complementation with either purified CBF-B or recombinant CBF-B. However, when recombinant CBF-A, synthesized as a fusion protein with glutathione S-transferase was denatured together with a highly purified fraction containing CBF-A in the presence of 5.5 M guanidine hydrochloride and subsequently renatured, the recombinant CBF-A bound to DNA after complementation with CBF-B. This binding of recombinant CBF-A could not be detected if recombinant CBF-A was not mixed during the denaturation-renaturation process together with the purified fraction containing the 32-kDa CBF-A. Using a Southwestern blot we demonstrated that a polypeptide of approximately 40 kDa, present in the purified CBF-A fraction, bound to DNA after complementation with both recombinant CBF-A and CBF-B. After fractionation of the purified CBF-A preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a species of approximately 40 kDa was eluted from the gel and shown to have DNA binding activity after complementation with both recombinant CBF-A and CBF-B. Our results indicate that a third polypeptide, designated CBF-C, forms a tight complex with CBF-A. Together with CBF-A and CBF-B, CBF-C is required for the DNA binding activity of CBF.

Animals↗
PubMed1992

Perturbations in morphogen gradients induce budding in hydra.

Lateral grafting of small pieces of midregion tissue into different levels of the hydra body column was done to assess the influence of the host hypostome and basal disc (or, of the underlying morphogenetic gradients) in inducing secondary structures in the transplanted tissue; and also to identify the role, if any, of the induced secondary structures (or, perturbed morphogen gradients) on the pattern of the host. The same midpiece tissue differentiated to a basal disc when grafted near the host hypostome, and to a small hypostome with tentacles when grafted near the host basal disc. Chimeras with induced secondary basal discs showed a phenomenal increase in budding compared to the controls and to the chimeras having induced hypostomes. These results indicate a positive cross-reaction between both organizing regions during patterning in hydra.

Animals↗
PubMed1992

Prospective study of the effectiveness of retrobulbar and peribulbar anesthesia for anterior segment surgery.

Peribulbar anesthesia was performed in 407 patients and retrobulbar anesthesia in 441 patients in a prospective study comparing the efficacy and complication rate of the two procedures. Eighteen of the peribulbar procedures (4.4%) required reblocks. There were no major complications in this group. Thirty of the retrobulbar procedures (6.8%) required reblocks. This group also had no major complications. The chi-square method of statistical analysis (chi-square = 2.245; P = .134) indicates there was no statistically significant difference between the efficacy of the two local anesthesia methods.

Anesthesia, Local↗
PubMed1992

Magnetic resonance image synthesis from analytic solutions of spin-echo and radio frequency-spoiled gradient-echo images.

RATIONALE AND OBJECTIVES: To synthesize magnetic resonance images (MRI) in real-time using a minimal data set obtained with routine clinical protocols and stored in a picture archiving and communication system (PACS) database. METHODS: Analytic solutions for T1 and T2 were obtained from a double and a single spin-echo set, with routine parameters. Analytic solutions from radio frequency-spoiled gradient-echo images, with TRs as low as 33 mseconds, also were used to synthesize gradient-echo images. RESULTS: Phantom studies showed that the errors in the synthesized images were significantly smaller than the errors in the T1- and T2-calculated images and similar to the source images. The gradient-echo images resulted in significant scan time savings. CONCLUSION: MRI synthesis from analytic solutions of T1, T2, and rho saves computational time and yields accurate values for the intrinsic parameters while allowing the use of routine clinical protocols. The availability of clinical images in the PACS database and the ability to synthesize images in real-time has allowed the development of a practical interactive teaching module.

Brain↗
PubMed1992

Silicone breast implants in vivo: MR imaging.

This study was designed to evaluate pulse sequences and patient positioning for MR imaging of silicone breast implants in patients. One hundred forty-three patients (281 silicone implants) underwent imaging over a 21-month period. The combination of a T2-weighted fast spin echo technique (SE), T2-weighted fast SE with water suppression, and T1-weighted SE with fat suppression is recommended to reliably differentiate silicone from other breast tissues and to identify intracapsular and extracapsular ruptures or leaks. Seventy of the 143 patients underwent removal of their silicone implants. The sensitivity for detection of silicone implant rupture was 76%, with a specificity of 97%. Positioning the patient prone improved image quality.

Adult↗
PubMed1992

MR imaging of joints: analytic optimization of GRE techniques at 1.5 T.

To clarify the choice of imaging parameters for optimal gradient-recalled echo MR scanning of joints, we analyzed the behavior of contrast-to-noise and signal-to-noise ratios for spoiled (i.e., fast low-angle shot [FLASH] or spoiled GRASS) and steady-state (i.e., gradient-recalled acquisition in the steady state [GRASS] or fast imaging with steady precession) techniques at 1.5 T. The analysis is based on tissue characteristics derived from spin-echo measurements of hyaline cartilage and synovial fluid signal in the patellofemoral joints of 11 volunteers. Separate analysis of contrast-to-noise and signal-to-noise ratios for multiplanar (long TR) acquisitions shows that these parameters are each improved compared with single-slice methods. At TRs greater than 250 msec, there is no significant difference in the contrast behavior of FLASH and GRASS. For optimal contrast-to-noise ratio (synovial fluid-cartilage), the best multiplanar sequence (for TE less than 23 msec) is with a short TE and a large flip angle (e.g., 400/9/73 degrees [TR/TE/flip angle]). If a single-scan or three-dimensional technique is desired, than a GRASS sequence at minimal TR and TE and intermediate flip angle (18/9/32 degrees) is best. For optimal signal-to-noise ratio (for both synovial fluid and hyaline cartilage), the best multiplanar sequence uses a short TE and an intermediate flip angle (e.g., 400/9/30 degrees). If a short TR, high signal-to-noise technique is desired, then GRASS (18/9/13 degrees) is superior to FLASH.

Cartilage, Articular↗
PubMed1992

A PACS-based interactive teaching module for radiologic sciences.

This article describes an interactive teaching module, linked to a picture archiving and communications system (PACS) data base, for teaching radiology. The module is currently tailored to MR images but can be adapted to any other imaging technique. An algorithm has been developed that allows the use of MR images acquired with routine clinical protocols and stored in the data base to yield, in real time, images at any other arbitrary TE and TR. In the browse mode, the user can study either the effect of different scan parameters or clinical cases on synthesized or acquired images. The quiz mode has multiple-choice questions and answers, accompanied by images. In the teaching mode, the instructor has access to the clinical data base and WRITE privileges for setting up the browse or quiz mode. The module achieves considerable flexibility when linked to the PACS, with access to all archived images and the ability to subsequently synthesize MR images at arbitrary TE and TR values in real time. The module is also "dynamic" in character, in that the instructor can easily add new cases and comments to the teaching files, both to enhance its clinical aspects and to reflect advances in technology.

Algorithms↗
PubMed1992

Analysis of circulating immune complexes from leprosy patients for Mycobacterium leprae antigens.

Circulating immune complexes (CICs) from 31 leprosy patients (16 tuberculoid, 15 lepromatous) and 12 healthy volunteers, precipitated by 3.5% polyethylene glycol, were individually subjected to SDS-PAGE and immunoblotting using a variety of monoclonal and polyclonal antibodies against Mycobacterium leprae. A common mycobacterial antigen of an apparent molecular size of 65 kDa was identified in CICs from about 40% of the patients. No correlation was observed between the positivity for this antigen and any of the following parameters: bacterial index, M. leprae-specific antibody titers, motor nerve involvement, duration of disease or treatment. Nevertheless, patients with a relatively recent and massive infection were more frequently positive for antigen than the others.

Antibodies, Bacterial↗
PubMed1992

Detection of gene deletion in patients of Duchenne muscular dystrophy/Becker muscular dystrophy using polymerase chain reaction.

Polymerase chain reaction (PCR) was used to study the presence of gene deletion (the most prominent type of mutations) in some families afflicted by Duchenne muscular dystrophy/Becker muscular dystrophy (DMD/BMD). The results clearly demonstrate deletion in the central part of the DMD gene in two of the three families studied. This information can be useful for genetic counselling with particular reference to prenatal diagnosis and carrier analysis.

Base Sequence↗
PubMed1991

Conversion of the Alzheimer's beta-amyloid precursor protein (APP) Kunitz domain into a potent human neutrophil elastase inhibitor.

Site-specific mutagenesis techniques have been used to construct active site variants of the Kunitz-type protease inhibitor domain present in the Alzheimer's beta-amyloid precursor protein (APP-KD). Striking alteration of its protease inhibitory properties were obtained when the putative P1 residue, arginine, was replaced with the small hydrophobic residue valine. The altered protein was no longer inhibitory toward bovine pancreatic trypsin, human Factor XIa, mouse epidermal growth factor-binding protein, or bovine chymotrypsin, all of which are strongly inhibited by the unaltered APP-KD (Sinha, S., Dovey, H. F., Seubert, P., Ward, P. J., Blacher, R. W., Blaber, M., Bradshaw, R. A., Arici, M., Mobley, W. C., and Lieberburg, I. (1990) J. Biol. Chem. 265, 8983-8985). Instead, the P1-Val-APP-KD was a potent inhibitor of human neutrophil elastase, with a Ki = 0.8 nM, as estimated by the inhibition of the activity of human neutrophil elastase measured using a chromogenic substrate. It also inhibited the degradation of insoluble elastin by the enzyme virtually stoichiometrically. Replacement of the P1' (Ala) and P2' (Met) residues of P1-Val-MKD with the corresponding residues (Ser, Ile) from alpha 1-proteinase inhibitor resulted in an inactive protein, underscoring the mechanistic differences between the serpins from the Kunitz-type protease inhibitor family. These results confirm the importance of the P1 arginine residue of APP-KD in determining inhibitory specificity, and are also the first time that a single amino acid replacement has been shown to generate a specific potent human neutrophil elastase inhibitor from a human KD sequence.

Amino Acid Sequence↗