Biomedical subjects

D A Schultz

Publications and source records attributed to D A Schultz.

At least 19 recordsLinked to original sources

PubMed2001

Suramin-induced neuropathy in an animal model.

Suramin is being used either alone, or in combination with other chemotherapeutic agents, in the treatment of hormone-refractory or metastatic prostate cancer. Use of this potentially valuable chemotherapy is limited by a dose-dependent polyneuropathy. It has been difficult in human studies to characterize peripheral suramin toxicity separately from cancer-related neuropathy. To characterize suramin-induced neuropathy in a rat model, adult rats were given either a single dose of 500 mg/kg (high dose) or 50 mg/kg (low dose) weekly suramin for 2 months. Electrophysiology and peroneal/sural nerve morphometry were performed. In high dose animals, neuropathy developed within 2 weeks, most severe in the digital sensory responses (DSR) (p<0.05) and tail and hind limb compound muscle action potential (p<0.001). Histologically, there was evidence of axonal degeneration and axon atrophy. With low dose suramin, the DSR (p<0.05) and tail distal sensory and motor responses (p<0.01) were most severely affected at 2 months. Axonal degeneration was seen in teased fibers from most animals. With TEM, there were abundant characteristic lysosomal inclusion bodies in DRG and Schwann cells. Electrophysiological and histological evidence of peripheral demyelination was rare, being observed in only one animal. Suramin induced a length, dose and time-dependent axonal sensorimotor polyneuropathy associated with axonal degeneration, atrophy, and accumulation of glycolipid lysosomal inclusions.

Animals↗

PubMed2000

An EF-hand phage display study of calmodulin subdomain pairing.

The interaction between the two EF-hands, EF3 and EF4, in the C-terminal domain of vertebrate calmodulin is addressed using an EF-hand phage display library. Significant specificity is observed in the presence of Ca(2+), as EF3-EF4 heterodimers are favored over EF3-EF3 and EF4-EF4 homodimers. Primarily EF4-type (and not EF3-type) amino acids are selected when an EF3 peptide is used as the target and vice versa. The results show that this specificity is promoted by several factors. There are three positions, corresponding to Phe89, Ala102, and Leu105, that are strongly selected as EF3-type hydrophobic residues with an EF4 target. When EF3 is the target peptide, EF4-type residues, Ile125, Tyr138 and Phe141, are selected. Remarkably, this subset consists of the same three residue positions in EF3 or EF4 and seems to be involved in specifying the heterodimer preference in both cases. In addition, electrostatic repulsion between the acidic monomers in an EF4 homodimer may further influence the preferred stability of heterodimers. This hypothesis is based on the observation that positively charged residues are strongly selected at four positions when EF4 is the target. A survey of EF-hand pairs suggests that charge separation is a common way to achieve efficient attraction of Ca(2+) without causing electrostatic repulsion between the subdomains. No significant specificity of binding is observed in the ion free state or in the presence of magnesium as no sequence is preferentially selected. The residues at the interface between the two EF-hands are thus highly optimized for the Ca(2+) bound state. At some residue positions, EF3-type amino acids are chosen with EF3-target in the presence of Ca(2+). These residues are not involved in the preference for heterodimer over homodimer formation, but represent key positions to mutate in the intact domain to stabilize its Ca(2+)-bound state.

Amino Acid Sequence↗

PubMed2000

Single-target molecule detection with nonbleaching multicolor optical immunolabels.

We introduce and demonstrate the use of colloidal silver plasmon-resonant particles (PRPs) as optical reporters in typical biological assays. PRPs are ultrabright, nanosized optical scatterers, which scatter light elastically and can be prepared with a scattering peak at any color in the visible spectrum. PRPs are readily observed individually with a microscope configured for dark-field microscopy, with white-light illumination of typical power. Here we illustrate the use of PRPs, surface coated with standard ligands, as target-specific labels in an in situ hybridization and an immunocytology assay. We propose that PRPs can replace or complement established labels, such as those based on radioactivity, fluorescence, chemiluminescence, or enzymatic/colorimetric detection that are used routinely in biochemistry, cell biology, and medical diagnostic applications. Moreover, because PRP labels are nonbleaching and bright enough to be rapidly identified and counted, an ultrasensitive assay format based on single-target molecule detection is now practical. We also present the results of a model sandwich immunoassay for goat anti-biotin antibody, in which the number of PRP labels counted in an image constitutes the measured signal.

ATP-Binding Cassette Transporters↗

PubMed1997

A region of vitamin K-dependent protein S that binds to C4b binding protein (C4BP) identified using bacteriophage peptide display libraries.

Vitamin K-dependent protein S, a blood coagulation inhibitor, interacts with the C4b-binding protein (C4BP) in human plasma with high affinity (KD = 0.1 nM). Identification of a portion of protein S that binds to C4BP has been approached using random libraries of 6- and 15-mer peptides displayed on bacteriophage surfaces. Bacteriophage binding to the beta-chain of C4BP were selected in several rounds of affinity purification with intervening amplification in E. coli. Homology searches of the affinity purified peptide sequences against protein S led to the identification of four regions in protein S that were similar to several of the selected peptides. These regions were synthesized as linear peptides and tested in inhibition experiments. Only one distinct peak (around position 450) was observed when the homology scores versus human protein S sequence were averaged over all affinity purified peptides. A synthetic peptide comprising residues 439-460 in human protein S was found to inhibit protein S binding to C4BP. The same result was found with two overlapping peptides (residues 447-468 and 435-468, respectively) in a second set of synthetic peptides. Direct binding of the peptides to C4BP was inferred from titrations monitored by recording the near UV circular dichroism spectra or the polarization of tryptophan fluorescence. The results suggest that residues 447-460 constitute a portion of protein S that is important for the interaction with C4BP. These findings may have implications for patients suffering from thrombosis, due to the lack of free protein S, by directing the design of drugs that disrupt protein S binding to C4BP.

Amino Acid Sequence↗

PubMed1993

A ribonuclease S-peptide antagonist discovered with a bacteriophage display library.

From a filamentous phage library displaying random hexapeptides, we selected clones displaying peptides that bind S-protein, a 104-amino-acid (aa) fragment of bovine pancreatic ribonuclease (RNase). The selected peptides show a sequence motif, (F/Y)NF(E/V)(I/V)(L/V), that bears little resemblance to S-peptide, a 20-aa fragment of RNase that is S-protein's natural ligand. One of the displayed peptides, YNFEVL, was synthesized chemically and shown by isothermal titration calorimetry to bind S-protein with a dissociation equilibrium constant of 5.5 microM at 25 degrees C, an affinity comparable to that of previously studied S-peptide variants. The YNFEVL peptide is an antagonist of S-peptide, in that it blocks the ability of S-peptide to restore enzyme activity to S-protein. The S-protein/S-peptide system preserves the essential features of a pharmacologically significant receptor/hormone couple, and the S-peptide antagonist can therefore be regarded as a new RNase-specific 'drug'. This work illustrates the potential value of phage display libraries for discovering novel classes of pharmaceuticals.

Amino Acid Sequence↗

PubMed1992

Change in the frequency of apoptosis after low- and high-dose X-irradiation of human lymphocytes.

The purpose of the present study was to correlate the type and frequency of cell death in human lymphocytes receiving variable doses of X-irradiation. Monocyte-depleted lymphocyte fractions were exposed in vitro to variable doses of X-rays of 0-20 Gy (0-2000 rads) and incubated for 4 and 16 h. An assessment of the mode of cell death (apoptosis vs. classical necrosis) was carefully evaluated using a multidisciplinary approach using light, fluorescence and electron microscopy (EM), and dye exclusion assays. Eosin Y exclusion assays indicated the absence of classical necrosis occurring in short-term cultures (4 h postirradiation). An assessment of cell counts, however, revealed a mean decrease of 4% at 0 Gy and 13% at 10-20 Gy (1000-2000 rads). The predominant mode of cell death was apoptosis, but the percent apoptotic cells (determined by EM) did not parallel this increase in cell loss with increasing radiation and actually decreased at doses above 5 Gy (500 rads). The discrepancy between percent cell loss and percent apoptosis was explained by a proposed change in overall duration of the apoptotic process. In long-term cultures (16 h postirradiation), a combination of classical necrosis, classical apoptosis, and combined apoptosis and necrosis (secondary necrosis of apoptotic cells) was apparent and was associated with a marked decrease in viability. Irradiation effects on lymphocytes showing none of the morphologic features of apoptosis or classical necrosis in short-term culture were evidenced by an increase in nuclear lobation. The results of this study indicate that the vast majority of peripheral blood lymphocytes are radioresistant. The use of irradiation in an in vitro model to study the biochemical events of the apoptotic process is also evaluated.

Apoptosis↗

PubMed1992

Cis proline mutants of ribonuclease A. I. Thermal stability.

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.

Amino Acid Sequence↗

PubMed1992

Cis proline mutants of ribonuclease A. II. Elimination of the slow-folding forms by mutation.

Ribonuclease A is known to form an equilibrium mixture of fast-folding (UF) and slow-folding (US) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, USII, USI, and possibly also minor populations of other US species. The two cis proline residues, P93 and P114, are logical candidates for producing the major US species after unfolding, by slow cis <==> trans isomerization. Much work has been done in the past on testing this proposal, but the results have been controversial. Site-directed mutagenesis is used here. Four single mutants, P93A, P93S, P114A, and P114G, and also the double mutant P93A, P114G have been made and tested for the formation of US species after unfolding. The single mutants P114G and P114A still show slow isomerization reactions after unfolding that produce US species; thus, Pro 114 is not required for the formation of at least one of the major US species of ribonuclease A. Both the refolding kinetics and the isomerization kinetics after unfolding of the Pro 93 single mutants are unexpectedly complex, possibly because the substituted amino acid forms a cis peptide bond, which should undergo cis --> trans isomerization after unfolding. The kinetics of peptide bond isomerization are not understood at present and the Pro 93 single mutants cannot be used yet to investigate the role of Pro 93 in forming the US species of ribonuclease A. The double mutant P93A, P114G shows single exponential kinetics measured by CD, and it shows no evidence of isomerization after unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Evaluation of C. diff.-CUBE test for detection of Clostridium difficile-associated diarrhea.

The toxin B assay was used to evaluate C. diff.-CUBE, a new dot-immunobinding assay (DIA) for the laboratory diagnosis of Clostridium difficile-associated diarrhea. The widely used latex agglutination test was also included for comparison. Stools from 100 patients suspected of having C. difficile-associated diarrhea were tested. The toxin B assay, latex agglutination, and DIA tests were positive for 12%, 9%, and 22% of the specimens, respectively. The sensitivity, specificity, and positive and negative predictive values of the DIA test were 67%, 84%, 36%, and 95%, respectively, compared with the toxin B assay. The specificity (98%) and positive predictive value (78%) for the latex agglutination test were significantly higher than those of the DIA test. Of 13 specimens solely positive by the DIA test, 11 were cultured and none were positive. Clinical assessment supported only two of the 13 positive DIA results. When clinical assessment was included in the analysis, the DIA positive predictive value rose to 45%. Although the sensitivity and negative predictive values of the DIA test are comparable to the latex agglutination test, the low specificity and positive predictive values of the DIA test make it an inappropriate method to use for screening in a population with a low prevalence of true positives.

Bacterial Toxins↗

PubMed1992

Effects of capture on hematological values and plasma cortisol levels of free-range koalas (Phascolarctos cinereus).

Eight free-range koalas (Phascolarctos cinereus) were captured on Kangaroo Island (South Australia) and later transported to the Adelaide Zoological Gardens. Blood samples were collected at the time of capture and at 6 hr, 24 hr and 7 days later. Routine hematological analyses were performed and plasma cortisol levels determined. Significant differences were observed between sampling times for erythrocyte numbers, hemoglobin concentrations, packed cell volumes, mean cell volumes, mean cell hemoglobin concentrations, numbers of leukocytes, neutrophils, lymphocytes and lymphocyte:neutrophil ratio (P less than 0.05). No significant differences were detected between sampling periods in the numbers of monocytes (P = 0.707), eosinophils (P = 0.174) or plasma cortisol levels (P = 0.192).

Animals↗

PubMed1991

Activation of bovine rod outer segment phosphatidylinositol-4,5-bisphosphate phospholipase C by calmodulin antagonists does not depend on calmodulin.

Calmodulin antagonists stimulated phosphatidylinositol-4,5-bisphosphate phospholipase C in soluble and particulate fractions of bovine rod outer segments. Antagonists tested include trifluoperazine, melittin, calmidazolium, compound 48/80, W-13 [N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide], and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. All were effective, but W-7 was chosen for further characterization of the effect, which was most pronounced in the soluble fraction. Phospholipase C activity in the soluble fraction did not increase linearly with the quality of enzyme assayed, suggesting the presence of an endogenous inhibitor or an inhibitory self-association of the enzyme. W-7 appeared to counteract this inhibition, resulting in a linear activity-quantity relationship. Stimulation by W-7 was therefore largest when large amounts of crude enzyme were assayed and small or nil when small amounts were assayed. The effect of W-7 was also dependent on [Ca2+], with half-maximal stimulation occurring between 0.1 and 1 microM. W-7 and W-13 were much more effective than their nonchlorinated analogues W-5 and W-12 at increasing phospholipase C activity. While this pattern of effectiveness is typical of calmodulin-mediated processes, the absence of any effect by added calmodulin and the retention of W-7 sensitivity by purified CaM-free enzyme argue against regulation by CaM. Octyl glucoside, a nonionic detergent, mimicked some of the effects of CaM antagonists, suggesting that the antagonists act by interfering with protein-protein interactions. It appears likely that CaM antagonists prevent an inhibitory multimerization or aggregation of at least one form of ROS phospholipase C.

Animals↗

PubMed1991

Cytomegalovirus testing: antibody determinations and virus cultures with recommendations for use.

Human cytomegalovirus is a ubiquitous virus with the property of becoming latent in the immunocompetent host. It can also cause devastating infections in the immunocompromised patient. Several different assays are available to detect either cytomegalovirus antibodies or cytomegalovirus itself, five of which are used at the University of Wisconsin Hospitals and Clinics and the Wisconsin State Laboratory of Hygiene. The latex agglutination (LA) test is used to detect antibody positivity in transplant donors, transplant recipients, and others. The complement fixation (CF) test is used to detect seroconversion or reactivation of infection in the CMV positive host. The immunofluorescent antibody-IgM (IFA-IgM) test is used to diagnose infection in congenitally or neonatally infected infants. The "routine" CMV culture is the "gold standard" for the detection of the cytomegalovirus in patient secretions. The shell vial assay, a rapid method of detecting virus, is most useful when contemplating therapy for active CMV infection. The advantages and limitations, as well as the timing and appropriateness of each test, are reviewed.

Antibodies, Viral↗

PubMed1990

Side-chain interactions in the C-peptide helix: Phe 8 ... His 12+.

Previous studies have demonstrated that His 12 plays a major role in the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A). Here, amino acid replacement experiments show that His 12+ stabilizes the C-peptide helix chiefly by interacting with Phe 8. The Phe 8 ... His 12+ ring interaction is specific for the protonated form of His 12 (His 12+) and the interaction is not screened significantly by NaCl, unlike the charged group ... helix dipole interactions studied earlier in C-peptide. Analogs of C-peptide that are unable to form the Phe 8 ... His 12+ interaction show large increases in helix content for Phe----Ala and His----Ala. Therefore, the helical tendencies of the individual residues Phe, His, and Ala are important in determining the result of a replacement experiment. Since the side chains of Phe 8 and His 12 probably interact within the N-terminal helix of ribonuclease A, the existence of the Phe 8 ... His 12+ interaction in the isolated C-peptide helix adds to the evidence that the C-peptide helix is an autonomous folding unit.

Amino Acid Sequence↗

PubMed1987

Ventilatory responses of the horse to exercise: effect of gas collection systems.

Experiments were undertaken to determine whether respiratory masks worn by horses exercising strenuously on a treadmill may interfere with normal gas exchange. Four collection systems, two flow-through systems and two incorporating one-way valve systems with subject-generated airflow were studied. Six horses performed standard treadmill exercise tests consisting of a 2-min warm up followed by galloping 1 min each at 8,9, and 10 m/s. Each horse exercised six times while wearing each of the four respiratory masks. Each flow-through system was used twice with flow rates of 2,360 and 3,840 l/min for one system, and 3,840 and 6,300 l/min for the other. Arterial blood gas tensions were measured during exercise at each speed for each system and were compared with values measured when the horses performed the same test without wearing a mask. Hypercapnia developed during exercise with each of the respiratory masks except with the 6,300-l/min flow-through system. All horses became hypoxemic during every exercise test, but it was most severe when systems incorporating one-way valves were used. This, plus the degree of hypercapnia observed and a suboptimal heart rate-O2 uptake relationship, indicated that such systems severely impede ventilation and suggest that experiments performed while utilizing them do not represent the normal exercise condition.

Animals↗

PubMed1986

Diagnosis of exercise-induced left bundle branch block at rest by scintigraphic phase analysis.

Accurate diagnosis of diseases of the ventricular conducting system is essential for their appropriate therapy. Some conduction abnormalities, such as exercise-induced left bundle branch block (EX-LBBB), are not apparent on resting electrocardiograms. Phase analysis of rest and exercise radionuclide ventriculograms (RVG's) was used to compare four EX-LBBB patients with six normal controls. All patients had normal resting electrocardiograms, ejection fractions, and visually normal wall motion. First harmonic phase images were generated reflecting the timing of ventricular contraction. Dynamic phase displays were reviewed and graded in a blinded fashion by three independent experienced observers. Phase angle histograms of the right and left ventricle were determined for both resting and exercise images. The mean phase angle and standard deviation were also calculated for each ventricle. Visual grading of the resting phase images failed to show a significant difference between normal patients and patients with EX-LBBB. Quantitative analysis, however, revealed a significant difference in mean phase angle differences (LV-RV) in resting studies: 0.8 degrees (+/- 1.9 degrees SEM) in normals versus 9.3 degrees (+/- 2.3 degrees SEM) in EX-LBBB patients (P less than 0.03). Exercise accentuated the phase angle differences: 1.8 degrees in normals vs. 31.2 degrees in EX-LBBB patients (P less than 0.001). Quantitative phase analysis of resting RVG's permits the diagnosis of cardiac conduction disease that is not apparent on the resting EKG and may result in better monitoring and treatment.

Bundle-Branch Block↗

PubMed1986

Scintigraphic phase analysis of left anterior hemiblock.

Left anterior hemiblock (LAHB) is a relatively common disturbance of ventricular conduction which can be an indicator of early conduction system disease. In an effort to better understand this condition, phase analysis of resting radionuclide ventriculograms (RVG) was used to evaluate five patients with LAHB and six normal patients with particular reference to the phase angle difference between the septum and the postero-lateral wall. All patients had normal ejection fractions and visually normal wall motion on RVG. Visual analysis of phase images showed significant differences between the LAHB and normal patients' LV contraction synergy (p less than 0.03) with a delay in septal contraction versus the postero-lateral wall. Four of five patients with LAHB were outside 2 standard deviations of the normal range. Regional quantitative analysis of phase angle differences between posterolateral and septal walls tended to show this difference between normals and LAHB (p = 0.08) as well. Three of five patients with LAHB were outside 2 standard deviations from the normals' mean. There were no significant differences between the standard deviations, skewness, or kurtoses of phase angle histograms of LAHB versus normal patients. Phase analysis can identify some patients with LAHB by both visual and quantitative analysis. The ability to detect and possibly quantitate subtle conduction abnormalities such as LAHB may result in a better understanding of such conduction system diseases.

Electrocardiography↗

PubMed1985

Tomographic renal cortical scintigraphy: correlation with intravenous urography, computed tomography, ultrasonography, angiography, and nuclear magnetic resonance imaging.

This study evaluates single-photon renal tomoscintigraphy (SPECT) in the evaluation of renal masses and correlates this modality, where indicated, with computed tomography (CT), ultrasonography (US), angiography (ANGIO) and nuclear magnetic resonance imaging (NMR). Eight patients with renal cortical lesions detected on intravenous urography (IVP) were evaluated by SPECT and planar nuclear imaging using Tc-99m glucoheptonate (GH). Three of these patients were felt particularly likely to have renal tumors and were additionally evaluated with US, CT, ANGIO and NMR. The five patients with nodules on IVP that were not particularly suggestive of malignancy had functioning, benign, renal tissue accounting for their IVP lesions. Four of five were found by planar-GH nuclear imaging, five/five by SPECT-GH. In addition, SPECT-GH allowed better "confidence" in the normal renal tissue diagnosis in three/five cases. Of the three renal lesions that were highly suggestive of malignancy, two were hypernephromas and one was hypertrophied functioning cortical tissue. All three were correctly identified prospectively on SPECT-GH; however, one hypernephroma was missed on planar-GH. NMR, CT, and ANGIO detected only one of two hypernephromas prospectively (US detected both); all four modalities incorrectly diagnosed the hypertrophied tissue suggestive of malignancy.

Angiography↗

PubMed1985

Delayed rupture of the spleen. A case report.

The diagnosis of delayed rupture of the spleen is demonstrated and discussed by presenting a case in which the patient had significant thoracoabdominal trauma with an initially normal liver/spleen scintigram and benign hospital course. Twenty-three days after the trauma, the patient developed an acute abdominal crisis that brought him to the emergency room where an emergency ultrasound revealed hemoperitoneum and a subcapsular hematoma. At laparotomy, a splenic rupture was found. A splenectomy was not performed. Postoperative liver/spleen scintigram showed a persistent defect which was not present on the initial liver/spleen scintigram.

Abdominal Injuries↗