Biomedical subjects

S Hakki

Publications and source records attributed to S Hakki.

9 recordsLinked to original sources

PubMed1997

Comparative study of monoclonal antibody scan in diagnosing orthopaedic infection.

When clinical data are insufficient to diagnose infection of bone or joints, nuclear scanning becomes crucial in making an accurate diagnosis. The efficacy of (99m)technetium antigranulocyte monoclonal antibody Fab' fragment (LeukoScan) is prospectively compared with (111)indium white blood cell and (99m)technetium methylene diphosphonate bone scans in 74 patients with suspected musculoskeletal infections. They were grouped according to site of suspected infection: 33 long bones, 23 prosthetic joints, and 18 diabetic feet. Sixty-two of these 74 patients had surgical verification with histopathology or culture. The remaining 12 patients had clinical followup as proof of absence of infection. The overall sensitivity of LeukoScan, (111)indium white blood cell, and (99m)technetium methylene diphosphonate bone scans was 93%, 85% and 92%, respectively. Specificity was 89%, 75% and 52%, and accuracy was 90%, 79% and 74%, respectively. The conclusion from this study is that LeukoScan is more accurate in detecting osteomyelitis, with better sensitivity and specificity in prosthetic joints. Compared with (111)indium white blood cell scans, LeukoScan++ gives superior images, and results are obtained in 1 to 6 hours without biohazard risk from handling blood products.

Aged↗

PubMed1994

Use of technetium antigranulocyte monoclonal antibody Fab' fragments for the detection of osteomyelitis.

Accurate early diagnosis of osteomyelitis is critical for optimal clinical management. Conventional radiology (X-rays, CT) and nuclear medicine scans (bone, gallium, and technetium/indium white blood cell [WBC]) have limitations and drawbacks. The monoclonal antibody (MAb) ImmuRAID-MN3 (Immunomedics Inc., Morris Plains, NJ), a 99m-Tc Antigranulocyte Fab' fragment, recognizes a surface glycoprotein NCA-90/95 shared by granulocytes, carcino-embryonic antigen (CEA), and meconium antigen (MA). Intravenous injection of radiolabeled MAb enables in vivo labeling of human granulocytes and targets infected lesions in the bone and throughout the body. Technetium labeled Fab' fragments rapidly clear the blood pool and high-quality images can be obtained the same day, as early as 1 h postinjection. Results at our institution on 13 patients with clinically suspected osteomyelitis of infected long bones, prostheses, and diabetic foot ulcers were compared with the surgical/bacteriological verification of the presence or absence of infection. The MAb scan showed six true positives, six true negatives, and one false negative (very low grade infection). The procedure was safe, no clinical or laboratory adverse reactions were encountered. The MAb fragments are markedly less immunogenic than whole IgG, resulting in lower induction of human antimouse antibody (HAMA) titers. No HAMA to this MAb fragment has been detected in 24 patients (data from multiple institutions). Our preliminary results suggest that 99m-Tc ImmuRAID-MN3 is highly accurate for detection of osteomyelitis. This study is part of an ongoing multiinstitutional project sponsored by Immunomedics, Inc. to evaluate the efficacy and safety of this radiopharmaceutical.

Adult↗

PubMed1993

A 56 kDa binding protein for Escherichia coli heat-stable enterotoxin isolated from the cytoskeleton of rat intestinal membrane does not possess guanylate cyclase activity.

Proteins binding Escherichia coli heat-stable enterotoxin were isolated from the cytoskeleton of intestinal membranes using an affinity matrix of biotinylated ST immobilized on monomeric avidin-agarose. ST binding proteins were purified 343-fold using this affinity technique, with 7% of the initial binding activity recovered in these preparations. ST binding proteins isolated by affinity chromatography possessed a native and subunit molecular mass of 56 kDa. These preparations exhibited both high- and low-affinity binding sites for ST. Guanylate cyclase in extracts of the intestinal membrane cytoskeleton was completely recovered in fractions which did not associate with the affinity matrix. In addition, ST binding proteins isolated by affinity chromatography were devoid of guanylate cyclase activity. These data, taken together with those obtained previously with crude and partially purified receptors, suggest that ST binds to different proteins in intestinal membranes, some of which do not possess guanylate cyclase activity.

Animals↗

PubMed1993

Solubilization and characterization of functionally coupled Escherichia coli heat-stable toxin receptors and particulate guanylate cyclase associated with the cytoskeleton compartment of intestinal membranes.

1. Particulate guanylate cyclase and receptors for E. coli heat-stable enterotoxin were solubilized from the rat intestinal cytoskeletal compartment using Lubrol-PX and KCl. 2. Thirty to forty percent of the ST receptor and guanylate cyclase activities were extracted from the lipid layer with Lubrol-PX alone. 2. Seventy percent of the remaining activities were solubilized from the cytoskeleton with Lubrol-PX and KCl. 3. Guanylate cyclase solubilized from either compartment exhibited similar reaction kinetics. 4. Both high- and low-affinity classes of ST receptors were solubilized from the lipid and cytoskeleton compartments. 5. In the presence of ATP gamma S, ST selectively activated the guanylate cyclase solubilized from the cytoskeleton compared to that solubilized from the lipid bilayer. 6. Crosslinking experiments demonstrated a preferential solubilization of the 130 kDa receptor subunit from the cytoskeleton and the 56 kDa subunit from the lipid bilayer. 7. Development of a procedure to solubilize ST receptors and guanylate cyclase from the intestinal membrane cytoskeleton will permit purification and further detailed studies of the coupling of these activities.

Adenosine Triphosphate↗

PubMed1991

Identification and characterization of a new family of high-affinity receptors for Escherichia coli heat-stable enterotoxin in rat intestinal membranes.

Novel high-affinity, low-capacity binding sites in intestinal membranes for the heat-stable toxin produced by Escherichia coli have been defined. The appearance of these sites is observed in the presence of physiological concentrations of NaCl in binding reactions. Scatchard analyses of equilibrium binding in the absence of NaCl demonstrated a single class of binding sites with KD = 1.9 x 10(-9) M and Bmax = 0.75 pmol/mg of protein. In contrast, similar experiments in the presence of NaCl demonstrated, in addition to the previously described low-affinity site, a high-affinity site with a KD of 2.1 x 10(-11) M and a Bmax of 73 fmol/mg of protein. Confirmation of the presence of high- and low-affinity sites was obtained in studies of the kinetics of ST binding. These sites exhibited similar dissociation but markedly different association kinetics. Determination of the association and dissociation constants permitted calculation of the KD's for the high- and low-affinity sites, which were 1.15 x 10(-11) M and 1.89 x 10(-9) M, respectively. These data agree closely with those obtained in studies of equilibrium binding. Furthermore, similar values for the KD's of these sites were obtained in experiments of competitive displacement of labeled ST, confirming the presence of two receptors for this toxin. Binding of ST to high-affinity sites is completely reversible and does not appear to be coupled to activation of particulate guanylate cyclase. In contrast, binding of ST to low-affinity sites appears to be partially reversible and may be coupled to activation of guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Interactions of nucleotide analogues with rod outer segment guanylate cyclase.

Light activation of cyclic GMP hydrolysis in rod outer segments is mediated by a G-protein which is active in the GTP-bound form. Substitution of GTP with a nonhydrolyzable GTP analogue is thought to leave the G-protein in a persistently activated state, thereby prolonging the hydrolysis of cyclic GMP. Restoration of cyclic GMP concentration in the cell also depends upon GTP since it is the substrate for guanylate cyclase, but little is known about the effects of GTP analogues on this enzyme. We report here the effects of the analogues of GTP and ATP as inhibitors and substrates of rod disk membrane guanylate cyclase. The rate of cyclic GMP synthesis from GTP in rod disk membranes was about 50 pmol min-1 (nmol of rhodopsin)-1. Analogues of GTP and adenine nucleotides competitively inhibited the cyclase activity. The order of inhibition, with magnesium as metal cofactor, was ATP greater than GMP-PNP greater than AMP-PNP approximately GTP-gamma-S; with manganese, AMP-PNP was more inhibitory than GTP-gamma-S. The inhibition constants, with magnesium as cofactor, were 0.65-2.0 mM for GTP-gamma-S, 0.4-0.8 mM for GMP-PNP, 1.5-2.3 mM for AMP-PNP, and 0.07-0.2 mM for ATP. The fraction of cyclase activity inhibited by analogues was similar at 1 and 0.03 microM calcium. Besides inhibition of cyclase, the analogues also served as its substrates. GTP-gamma-S substituted GTP with about 85% efficiency while GMP-PNP and ATP were about 5 and 7% as efficient, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Guanylate cyclase from bovine rod outer segments: solubilization, partial purification, and regulation by inorganic pyrophosphate.

In spite of its pivotal role in visual transduction, very little is known about guanylate cyclase of retinal photoreceptor cells. The enzyme has not yet been purified principally because of the difficulty in solubilizing it. We report here a simple method for solubilization of 67% of the cyclase activity from the retinal rod disk membranes (RDM). With Nonidet P-40 as detergent, the solubilization of cyclase is favored by a high concentration of KCl and exclusion of manganese. The solubilized and the residual insoluble enzymes are both highly unstable but could be partially stabilized by dithiothreitol. They were both insensitive to calcium, calmodulin, and atrial natriuretic factor. They also responded similarly to varying the manganese concentration in the assay. For the activity in both fractions, the Km for GTP was about 230 microM, Line-weaver-Burk plots showed that substrate binding was cooperative, and Hill plots suggested that there are two substrate binding sites. Cumulatively, these observations showed that while the entire activity could not be solubilized, the solubilized and the residual insoluble activities probably belonged to the same enzyme. Partial purification resolved the solubilized enzyme into two activities refered to as enzymes 1 and 2. Both had substrate saturation kinetics similar to the solubilized enzyme and were inhibited competitively by inorganic pyrophosphate, one of the products of the cyclase reaction. The Ki for PPi for enzyme 1 was 70-100 microM and 150-200 microM for enzyme 2. cGMP at concentrations up to 800 microM had no influence on the activity of either enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

The overlooked, retained Double J stent.

A series of 4 patients with long overlooked, retained ureteral stents is presented to illustrate the variable, unpredictable, and at times, hazardous course of such patients. These cases are cited to re-emphasize the need for careful documentation, observation, and follow-up of patients in whom stents are placed.

Adolescent↗

PubMed1990

Photoreceptor rod outer segment 48-kDa protein has ATPase activity.

The role of 48-kDa protein in visual transduction remains unresolved. Two hypotheses for its role in quenching the light activation of cyclic GMP cascade suggest that the protein binds to either phosphodiesterase or phosphorylated rhodopsin. Since the protein is also reported to bind ATP, we anticipated that the protein may have ATP hydrolyzing activity, and in analogy with the GTP-binding protein of the rod outer segments, such activity may be greatly enhanced by the elements of transduction cyclic GMP cascade, permitting the protein to function cyclically as GTP-binding protein does. We found that purified 48-kDa protein hydrolyzes ATP but at a slow rate of 0.04-0.05 per min. The Km for ATP is about 45-65 microM. The activity is inhibited noncompetitively by ADP with a Ki of about 50 microM. The ATPase activity of 48-kDa protein is not affected by rhodopsin, bleached rhodopsin, phosphorylated rhodopsin, unactivated cyclic GMP phosphodiesterase, or phosphodiesterase (PDE) activated by GMP PNP-bound G-protein. These data show that although 48-kDa protein has ATPase activity, lack of regulation of this activity by the elements of visual transduction makes it unlikely for this activity to have a role in quenching the light activation of cyclic GMP cascade.

Adenosine Diphosphate↗