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Biomedical subjects

J B Ross

Publications and source records attributed to J B Ross.

At least 37 records · Page 2Linked to original sources
PubMed1995

Cutaneous malignancies in renal transplant recipients from Nova Scotia, Canada.

Four hundred and seventy-four Nova Scotian renal transplant recipients were screened, using the records of the Nova Scotia Cancer Registry and the Victoria General Hospital, in a retrospective study, for the development of cutaneous malignancies. Sixteen patients developed a total of 60 squamous cell carcinomas (SCC) and 14 developed 28 basal cell carcinomas (BCC). The survival curve shows that within 10 years of transplant, less than 10% will develop SCC. This is a 16-fold increase above the rate of SCC in the general population of Nova Scotia, Canada. Age at time of transplantation appears to be a significant determinant of subsequent skin cancer risk. None of the previously reported HLA associations was found to hold in this small study. The role of ultraviolet radiation, due to both geographic and lifestyle exposure, is discussed when comparing to other transplant studies.

Adolescent↗
PubMed1994

Identification of the cysteine in the steroid-binding site of human corticosteroid binding globulin by site-directed mutagenesis and site-specific chemical modification.

Binding of corticosteroids by human corticosteroid binding globulin (hCBG) is thought to involve interaction with one of its two cysteine residues (Cys60 and Cys228). To identify which of the two cysteine residues mediates steroid binding, we have produced mutant hCBGs containing serine or alanine in place of Cys228 by site-directed mutagenesis. Alteration of Cys228 to serine or alanine does not change the steroid binding affinity of hCBG, demonstrating that Cys228 is not involved in the binding interaction. This finding strongly suggests that Cys60 is the functionally important cysteine. By modifying the wild-type and mutant hCBGs with the sulfhydryl-specific reagents N-ethylmaleimide, iodoacetamide, and sodium tetrathionate, we have demonstrated that Cys60 is present at the steroid binding site, and that it may be directly involved in steroid binding. This result also identifies Cys60 as the accessible cysteine reported in previous studies.

Base Sequence↗
PubMed1994

Use of an oriented transmembrane protein to probe the assembly of a supported phospholipid bilayer.

Planar-supported phospholipid bilayers formed by the adsorption of vesicles are increasingly used in the investigation of lipid-dependent reactions. We have studied the way in which these bilayers are formed with phospholipid vesicles containing the transmembrane protein Tissue Factor (TF). TF complexed with the serine protease, factor VIIa, is the primary initiator of blood coagulation by way of activation of the zymogen factor X. TF has been shown to orient randomly on the inner and outer leaflets of vesicles. We used proteolytic digestion to produce vesicles in which the extracellular domain of TF is located on the inner leaflet. These vesicles show no cofactor activity for factor VIIa as a result of the inability of the extracellular domain of TF to bind VIIa. After freeze/thawing, 50% of the cofactor activity was regained, indicating reorientation of the sequestered, inner leaflet TF. Adsorption of these vesicles to the inner surface of glass microcapillaries results in a continuous phospholipid bilayer. The microcapillaries were perfused with a solution of factors VIIa and X, and the effluent was monitored for factor Xa production, a sensitive measure of the activity of the TF-VIIa complex. For coatings produced with the digested vesicles, minimal TF-VIIa activity was observed, showing that the supported bilayer preserves the orientation of the leaflets in the vesicles, i.e., the outer leaflet of the vesicles forms the outer leaflet of the supported bilayer.

Adsorption↗
PubMed1994

Thermographic assessment of craniomandibular disorders: diagnostic interpretation versus temperature measurement analysis.

This study assessed electronic thermography as a diagnostic alternative for evaluation of temporomandibular disorders. The study populations consisted of 50 temporomandibular joint patients having internal derangement or osteoarthrosis and 30 normal temporomandibular joint subjects. An Agema 870 thermovision unit was used for analysis. Diagnostic evaluations by expert interpreters were made using standard procedures. Thermography measurements included mean absolute temperature measurements and right-left temperature differences for five anatomic zones and four spot areas. Statistical analysis of data included both linear discriminant analysis and classification-tree analysis. Results indicated that when differentiating between "abnormal" and "normal" temporomandibular joints using classification-tree analysis, correct classifications were made in 89% of the cases and observer diagnostic accuracy was 84%. When evaluating for specific diagnoses (eg, osteoarthrosis, internal derangement, or normal temporomandibular joint), correct classifications using classification-tree analysis were made in 73% of the cases and observer evaluation was correct in 59%. The three best temperature measures found were: (1) delta T of the zone immediately overlying the temporomandibular joint; (2) the zone temperature of the half-face; and (3) the spot temperature anterior to the external auditory meatus. Additional studies are needed before thermographic diagnosis of craniomandibular disorders is accepted clinically.

Adult↗
PubMed1994

Thermographic characterization of internal derangement of the temporomandibular joint.

This study assessed the ability of electronic thermography to identify internal derangement of the temporomandibular joint. The study population consisted of 30 patients with internal derangement verified by temporomandibular joint arthrotomography. Electronic thermography was conducted using an Agema 870 thermographic unit. Thermal assessments included: (1) pattern recognition; (2) pattern symmetry; (3) absolute temperature measurements; (4) delta T measurements; and (5) mean temperature measurements and differences within five designated anatomic zones. Results indicated: (1) low levels of thermal symmetry in patients with internal derangement of the temporomandibular joint, and (2) absolute temperature measurements and mean temperature zone measurements showing large delta T values (0.4 degrees C to 0.8 degrees C). Demonstration of characteristic thermal temporomandibular joint changes suggests that electronic thermography may have potential for assessing internal derangement of the temporomandibular joint. However, more extensive studies are needed before thermographic procedures can be accepted clinically.

Adult↗
PubMed1993

The primary self-assembly reaction of bacteriophage lambda cI repressor dimers is to octamer.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. It has long been believed that cooperativity is mediated by self-assembly of repressor dimers to form tetramers which can then bind simultaneously to adjacent operator sites. As a first step in defining the individual energy contributions to binding cooperativity, sedimentation equilibrium and steady-state fluorescence anisotropy methods have been used to study the higher order assembly reactions of the free repressor in solution. Wild-type repressor with 5-hydroxytryptophan (5-OHTrp) substituted for the native tryptophan [Ross et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12023-12027] and two mutant repressor proteins that bind cooperatively to OR but have altered dimerization properties were also studied. We report here that the primary assembly mode of all four proteins is dimer to octamer. It is not dimer to tetramer as previously assumed. While tetramer does form as an assembly intermediate, dimer-octamer assembly is a concerted process so that tetramer is never a predominant species in solution. Sedimentation velocity experiments suggest that the octamer is highly asymmetric, consistent with an elongated shape. This conformation could allow octamers to bind simultaneously to all three operator sites at either OR or OL. Examination of tetramer and octamer concentrations suggests that both species could be involved in cooperative repressor-operator interactions. Our previous work used the unique spectral properties of 5-OHTrp to demonstrate that octamer binds single-operator DNA and is not dissociated to tetramer [Laue et al. (1993) Biochemistry 32, 2469-2472]. Taken together with the results presented here, octamers as well as tetramers must be considered in developing models to explain the cooperativity of lambda cI repressor binding to operator DNA.

5-Hydroxytryptophan↗
PubMed1993

Human factor VIIa and its complex with soluble tissue factor: evaluation of asymmetry and conformational dynamics by ultracentrifugation and fluorescence anisotropy decay methods.

Ultracentrifugation and fluorescence anisotropy decay measurements were used to evaluate the asymmetry and conformational dynamics of human blood clotting enzyme VIIa (VIIa) and the complex it forms with a soluble truncation mutant of human tissue factor (sTF) which acts as an essential cofactor for VIIa. Sedimentation velocity experiments showed that both VIIa and the sTF.VIIa complex are highly asymmetric. In each case, the friction ratio f/fsphere, is consistent with a family of general elliposids ranging from prolate to oblate. Fluorescence anisotropy decay experiments were used to limit the family of elliposids which can describe the hydrodynamic behavior of VIIa and sTF.VIIa. For both VIIa and the sTF.VIIa complex, the oblate ellipsoid of revolution was eliminated. In addition, the fluorescence anisotropy decay data clearly show that upon binding sTF.VIIa loses a segmental motion involving a domain containing the active site of the enzyme. This suggests that sTF causes a stabilization of a limited range of VIIa conformations. This stabilization may be important for proper recognition of the TF.VIIa substrate, factor X.

Chemical Phenomena↗
PubMed1993

5-hydroxytryptophan as a new intrinsic probe for investigating protein-DNA interactions by analytical ultracentrifugation. Study of the effect of DNA on self-assembly of the bacteriophage lambda cI repressor.

Pairwise cooperativity between proteins bound to DNA is believed to be important in governing the transcriptional regulation of numerous genes. However, the spectral overlap of normal proteins and DNA has blocked the study of these interactions by many physical methods. As shown recently by Ross et al. (in press), lambda cI repressor spectrally enhanced by 5-hydroxytryptophan (5-OHTrp), expressed in vivo using an Escherichia coli tryptophan auxotroph, exhibits dimer formation and DNA binding properties identical with those of the wild-type repressor. Moreover, the 5-OHTrp provides a spectral signal that allows monitoring of the protein concentration without interference from DNA. In this article, the ability to selectively detect 5-OHTrp-labeled repressor during analytical ultracentrifugation is used to study the higher order assembly of repressor dimers in the absence and in the presence of operator DNA. Contrary to the expectation that tetramer might be the limiting oligomer, lambda cI repressor undergoes a definite association to octamer. The relatively narrow concentration range over which transition from predominantly dimer to predominantly octamer occurs makes it unlikely that significant levels of tetramer are formed in the absence of DNA. Moreover, mass measurements reveal that an OR1 oligonucleotide binds to octameric repressor and does not dissociate it to tetramers. The use of the 5-OHTrp spectral enhancement opens a promising new avenue for the exploration of protein-protein and protein-nucleic acid interactions by analytical ultracentrifugation.

5-Hydroxytryptophan↗
PubMed1993

Steric requirements at position B12 for high biological activity in insulin.

The alpha-helix formed by the amino acid residues 9-19 of the B-chain of insulin is involved in the stabilization of its three-dimensional structure. We have shown that modification at positions B9, B10, B12, and B16 results in analogues possessing biological activities ranging from ca. 0.2% to ca. 500% relative to that of natural insulin. The lowest potency was displayed by [B12 Asn]insulin, in which the hydrophobic B12 Val residue was replaced by the hydrophilic Asn residue. We now report the synthesis of four insulin analogues in which hydrophobicity is retained, and only the spatial arrangement of atoms in the B12 region is altered. Substitution of B12 Val with alpha-aminoisobutyric acid (Aib), D-Ala, and Phe led to analogues possessing biological activities, in lipogenesis assays, of 8.5%, 2%, and 0.2%, respectively, relative to that of natural insulin. Inversion of the B11-B12 sequence, -Leu-Val-, led to an analogue displaying 3.3% activity. A synthetic B-chain in which the B11 Leu-B12 Val sequence was replaced by B11 Ala-B12 Ile was incapable of combining with the natural A-chain. We conclude that the Val residue in the B12 position in insulin fulfills special side-chain packing requirements involved in the stability of the structure of insulin. Even slight steric alteration at position B12 results in a distortion of the overall conformation of the B-chain which affects its ability to combine with the natural A-chain. This distortion is retained in the corresponding analogue, which is reflected in diminished biological potency.

Amino Acid Sequence↗
PubMed1993

The effect of placement of tryptophan residues in selected A-chain positions on the biological profile of insulin.

In continuation of our efforts to study the solution structure and conformational dynamics of insulin by time-resolved fluorescence spectroscopy, we have synthesized and examined the biological activity of five insulin analogues in which selected naturally occurring residues in the A-chain have been replaced with the strongly fluorescent tryptophan residue. The potency of these analogues was evaluated in lipogenesis assays in isolated rat adipocytes, in receptor binding assays using rat liver plasma membranes, and in two cases, in receptor binding assays using adipocytes. [A3 Trp]insulin displays a potency of 3% in receptor binding assays in both liver membranes and in adipocytes, but only 0.06% in lipogenesis assays as compared to porcine insulin. [A10 Trp]insulin displays a potency of ca. 40% and ca. 25% in rat liver receptor binding and lipogenesis assays, respectively. [A13 Trp]insulin displays a potency of ca. 39% in rat liver receptor binding assays, but only ca. 9% in receptor binding in adipocytes; in lipogenesis assays, [A13 Trp]insulin displays a potency of ca. 12%, comparable to its potency in adipocyte receptor binding assays. [A15 Trp]insulin exhibits a potency of 18% and 9% in rat liver receptor binding and lipogenesis assays, respectively. The doubly substituted analogue, [A14 Trp, A19 Trp] insulin, displays a potency of ca. 0.7% in both rat liver receptor binding assays and lipogenesis assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Familial cold urticaria.

Familial cold urticaria (FCU) is a rare autosomal dominant condition, first described in 1940. The onset is in early life in all reported cases. Symptoms are triggered by generalized exposure to cold air, particularly in damp and windy weather. The cutaneous lesions consist of erythematous macules or plaques, urticarial lesions and sometimes petechiae. Associated fever, chills, joint pains, nausea, stiffness and swelling of the hands and feet frequently occur. The symptoms are variable, ranging from mild to incapacitating. The pathogenesis of FCU remains unknown. To our knowledge only 10 pedigrees have been published, seven from the USA and one each from Holland, France and South Africa. We wish to report another extensive pedigree after having had the opportunity to investigate one member of the family in detail. A short form of this pedigree has been published elsewhere.

Cold Temperature↗
PubMed1992

Spectral enhancement of proteins: biological incorporation and fluorescence characterization of 5-hydroxytryptophan in bacteriophage lambda cI repressor.

We have used a tryptophan-requiring Escherichia coli auxotroph to replace the three tryptophan residues of lambda cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp). By using a nonleaky promoter, we have achieved > 95% replacement of tryptophan in the repressor. We show that the absorbance and fluorescence properties of 5-OHTrp-lambda cI are clearly distinct from lambda cI repressor and that the fluorescence of 5-OHTrp-lambda cI repressor can be observed selectively in the presence of exogenous tryptophan. We also show that the 5-OHTrp-lambda cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor. Based on these results, we anticipate that the availability of spectrally enhanced proteins will significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems.

5-Hydroxytryptophan↗
PubMed1992

Tissue factor and its extracellular soluble domain: the relationship between intermolecular association with factor VIIa and enzymatic activity of the complex.

We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.

Amino Acid Chloromethyl Ketones↗
PubMed1992

Correlation of tryptophan fluorescence intensity decay parameters with 1H NMR-determined rotamer conformations: [tryptophan2]oxytocin.

While the fluorescence decay kinetics of tyrosine model compounds [Laws, W. R., Ross, J. B. A., Wyssbrod, H. R., Beechem, J. M., Brand, L., & Sutherland, J. C. (1986) Biochemistry 25, 599-607] and the tyrosine residue in oxytocin [Ross, J. B. A., Laws, W. R., Buku, A., Sutherland, J. C., & Wyssbrod, H. R. (1986) Biochemistry 25, 607-612] can be explained in terms of heterogeneity derived from the three ground-state chi 1 rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from chi 2 rotations. In this paper, the time-resolved and steady-state fluorescence properties of [tryptophan2]oxytocin at pH 3 are presented and compared with 1H NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength and a global analysis of these decay curves for common emission wavelength-independent decay constants, only three exponential terms are required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the 1H NMR-determined chi 1 rotamer populations of the indole side chain. 15N was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the chi 1 rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two chi 2 populations. Depending upon the degree of correlation between chi 2 and chi 1, there may be from three to six side-chain conformations for the tryptophan residue. The combined fluorescence and NMR results are consistent with a rotamer model in which either (i) the chi 2 rotations are fast compared to the fluorescence intensity decay of the tryptophan residue, (ii) environmental factors affecting fluorescence intensity decay properties are dominated by chi 1 interactions, or (iii) the chi 2 and chi 1 rotations are highly correlated.

Amino Acid Sequence↗
PubMed1992

A therapeutic trial of the use of penicillin V or erythromycin with or without rifampin in the treatment of psoriasis.

BACKGROUND: After the publication of an uncontrolled trial of nine patients with streptococcus-associated psoriasis who appeared to benefit from a course of oral penicillin or erythromycin with the addition of rifampin in the last 5 days, we wished to confirm or refute the validity of this observation. OBJECTIVE: Our purpose was to confirm the effectiveness of antibiotics in the treatment of streptococcus-associated psoriasis. METHODS: Twenty patients were placed randomly into two groups. One group was given penicillin or erythromycin for 14 days with a placebo added during the last 5 of the 14 days. The other group received the same medication with the addition of rifampin in the last 5 days. RESULTS: Although all the patients studied met the criteria of the reported preliminary study, we were unable to detect any evidence of improvement in their psoriasis. CONCLUSION: There was no apparent benefit for patients with streptococcus-associated psoriasis from a course of oral penicillin or erythromycin with the addition of rifampin in the last 5 days in a 14-day trial.

Adolescent↗