Biomedical subjects

J I Clark

Publications and source records attributed to J I Clark.

At least 37 records · Page 2Linked to original sources

PubMed2000

Small heat-shock proteins and their potential role in human disease.

The elevated expression of stress proteins is considered to be a universal response to adverse conditions, representing a potential mechanism of cellular defense against disease and a potential target for novel therapeutics, including gene therapy and chaperone-modulating reagents. Recently, a single mutation in the small heat-shock protein human alphaB-crystallin was linked to desmin-related myopathy, which is characterized by abnormal intracellular aggregates of intermediate filaments in human muscle. New findings demonstrate that the high level of expression of stress proteins can contribute to an autoimmune response and can protect proteins that contribute to disease processes.

Amino Acid Sequence↗

PubMed2000

Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy.

Expansion of a CTG trinucleotide repeat in the 3' UTR of the gene DMPK at the DM1 locus on chromosome 19 causes myotonic dystrophy, a dominantly inherited disease characterized by skeletal muscle dystrophy and myotonia, cataracts and cardiac conduction defects. Targeted deletion of Dm15, the mouse orthologue of human DMPK, produced mice with a mild myopathy and cardiac conduction abnormalities, but without other features of myotonic dystrophy, such as myotonia and cataracts. We, and others, have demonstrated that repeat expansion decreases expression of the adjacent gene SIX5 (refs 7,8), which encodes a homeodomain transcription factor. To determine whether SIX5 deficiency contributes to the myotonic dystrophy phenotype, we disrupted mouse Six5 by replacing the first exon with a beta-galactosidase reporter. Six5-mutant mice showed reporter expression in multiple tissues, including the developing lens. Homozygous mutant mice had no apparent abnormalities of skeletal muscle function, but developed lenticular opacities at a higher rate than controls. Our results suggest that SIX5 deficiency contributes to the cataract phenotype in myotonic dystrophy, and that myotonic dystrophy represents a multigenic disorder.

3' Untranslated Regions↗

PubMed2000

Fibroblast growth factor-2 selectively stimulates angiogenesis of small vessels in arterial tree.

There is a critical need for quantifiable models of angiogenesis in vivo, and in general, differential effects of angiogenic regulators on vascular morphology have not been measured. Because the potent angiogenic stimulators fibroblast growth factor (FGF)-2 (basic FGF) and vascular endothelial growth factor (VEGF) are reported to stimulate angiogenesis through distinct signaling pathways, we hypothesized that FGF-2 stimulates vascular morphology differently than does VEGF and that stimulation of angiogenesis by FGF-2 is directly correlated to FGF receptor density. FGF-2 was applied at embryonic day 7 (E7), E8, or E9 to the quail chorioallantoic membrane (CAM); subsequent response of the arterial tree was measured by the fractal dimension (D(f)), a mathematical descriptor of complex spatial patterns, and by several generational branching parameters that included vessel length density (L(v)). After application of FGF-2 at E7, arterial density increased according to D(f) as a direct function of increasing FGF-2 concentration, and FGF-2 stimulated the growth of small vessels, but not of large vessels, according to L(v) and other branching parameters. For untreated control specimens at E7, L(v) of small vessels and D(f) were 11.1+/-1.6 cm(-1) and 1.38+/-0.01, respectively; at E8, after treatment with 5 microgram FGF-2/CAM for 24 hours, L(v) of small vessels and D(f) increased respectively to 22.8+/-0.7 cm(-1) and 1. 49+/-0.02 compared with 16.3+/-0.9 cm(-1) and 1.43+/-0.02 for PBS-treated control specimens. Application of FGF-2 at E8 and E9 did not significantly increase arterial density. By immunohistochemistry, the expression of 4 high-affinity tyrosine kinase FGF receptors was significantly expressed at E7, when CAM vasculature responded strongly to FGF-2 stimulation, but FGF receptor expression decreased throughout the CAM by E8, when vascular response to FGF-2 was negligible. In conclusion, the "fingerprint" vascular pattern elicited by FGF-2 was distinct from vascular patterns induced by other angiogenic regulators that included VEGF(165), transforming growth factor-beta1, and angiostatin.

Allantois↗

PubMed2000

New therapeutic strategies for the treatment and prevention of head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy that is now the sixth most common neoplasm in the world today. Approximately 50,000 cases in the United States and more than 500,000 cases worldwide will be diagnosed in 2000 [1]. Despite numerous advances in treatment utilising the most recent protocols for surgery, radiation and chemotherapy, the long-term survival has remained at less than 50% over the past 40 years [2]. This poor long-term survival is due to a number of variables including delayed diagnosis as well as the frequent development of multiple primary tumours. Therefore, in addition to early detection, continued emphasis must be placed on preventing the development of new primaries as well as establishing more effective treatments for individuals who present with advanced disease. This review will summarise some of the recent advances in the realms of chemotherapy and radiation therapy. In addition, it will discuss the present status of chemoprevention in HNSCC. Finally, we will discuss the rationale for the use of anti-angiogenic agents as one possible means of developing new chemopreventive protocols that result in reduced toxicity while maintaining similar clinical efficacies.

Animals↗

PubMed2000

Clinical significance of defective dendritic cell differentiation in cancer.

Defective dendritic cell (DC) function has been described previously in cancer patients and tumor-bearing mice. It can be an important factor in the escape of tumors from immune system control. However, the mechanism and clinical significance of this phenomenon remain unclear. Here, 93 patients with breast, head and neck, and lung cancer were investigated. The function of peripheral blood and tumor draining lymph node DCs was equally impaired in cancer patients, consistent with a systemic rather than a local effect of tumor on DCs. The number of DCs was dramatically reduced in the peripheral blood of cancer patients. This decrease was associated with the accumulation of cells lacking markers of mature hematopoietic cells. The presence of these immature cells was closely associated with the stage and duration of the disease. Surgical removal of tumor resulted in partial reversal of the observed effects. The presence of immature cells in the peripheral blood of cancer patients was closely associated with an increased plasma level of vascular endothelial growth factor but not interleukin 6, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, interleukin 10, or transforming growth factor-beta and was decreased in lung cancer patients receiving therapy with antivascular endothelial growth factor antibodies. These data indicate that defective DC function in cancer patients is the result of decreased numbers of competent DCs and the accumulation of immature cells. This effect may have significant clinical implications.

Adult↗

PubMed2000

[Analysis of cytoskeletal proteins in rat selenium cataract using two-dimensional electrophoresis].

BACKGROUND AND PURPOSE: Previous studies used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to characterize the modifications to lens proteins during opacification in the selenite model for cataract. To analyze the protein modifications in more detail, two dimensional electrophoresis (2 DE), which is more sensitive than 1 D electrophoresis, was used. RESULTS: The results of 2 DE demonstrated rapid changes in cytoskeletal proteins including spectrin and vimentin at the earliest stages of opacification. CONCLUSION: The results suggested that the mechanism of opacification might involve changes in cytoskeletal proteins during the earliest stages of cataract formation.

Animals↗

PubMed1999

The cardiomyopathy and lens cataract mutation in alphaB-crystallin alters its protein structure, chaperone activity, and interaction with intermediate filaments in vitro.

Desmin-related myopathy and cataract are both caused by the R120G mutation in alphaB-crystallin. Desmin-related myopathy is one of several diseases characterized by the coaggregation of intermediate filaments with alphaB-crystallin, and it identifies intermediate filaments as important physiological substrates for alphaB-crystallin. Using recombinant human alphaB-crystallin, the effects of the disease-causing mutation R120G upon the structure and the chaperone activities of alphaB-crystallin are reported. The secondary, tertiary, and quaternary structural features of alphaB-crystallin are all altered by the mutation as deduced by near- and far-UV circular dichroism spectroscopy, size exclusion chromatography, and chymotryptic digestion assays. The R120G alphaB-crystallin is also less stable than wild type alphaB-crystallin to heat-induced denaturation. These structural changes coincide with a significant reduction in the in vitro chaperone activity of the mutant alphaB-crystallin protein, as assessed by temperature-induced protein aggregation assays. The mutation also significantly altered the interaction of alphaB-crystallin with intermediate filaments. It abolished the ability of alphaB-crystallin to prevent those filament-filament interactions required to induce gel formation while increasing alphaB-crystallin binding to assembled intermediate filaments. These activities are closely correlated to the observed disease pathologies characterized by filament aggregation accompanied by alphaB-crystallin binding. These studies provide important insight into the mechanism of alphaB-crystallin-induced aggregation of intermediate filaments that causes disease.

Cardiomyopathies↗

PubMed1999

ATP and the core "alpha-Crystallin" domain of the small heat-shock protein alphaB-crystallin.

Electrospray ionization mass spectrometry (ESI-LC/MS) of tryptic digests of human alphaB-crystallin in the presence and absence of ATP identified four residues located within the core "alpha-crystallin" domain, Lys(82), Lys(103), Arg(116), and Arg(123), that were shielded from the action of trypsin in the presence of ATP. In control experiments, chymotrypsin was used in place of trypsin. The chymotryptic fragments of human alphaB-crystallin produced in the presence and absence of ATP were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Seven chymotryptic cleavage sites, Trp(60), Phe(61), Phe(75), Phe(84), Phe(113), Phe(118), and Tyr(122), located near or within the core alpha-crystallin domain, were shielded from the action of chymotrypsin in the presence of ATP. Chemically similar analogs of ATP were less protective than ATP against proteolysis by trypsin or chymotrypsin. ATP had no effect on the enzymatic activity of trypsin and the K(m) for trypsin was 0.031 mM in the presence of ATP and 0.029 mM in the absence of ATP. The results demonstrated an ATP-dependent structural modification in the core alpha-crystallin domain conserved in nearly all identified small heat-shock proteins that act as molecular chaperones.

Adenosine Triphosphate↗

PubMed1999

Site-directed mutations within the core "alpha-crystallin" domain of the small heat-shock protein, human alphaB-crystallin, decrease molecular chaperone functions.

Site-directed mutagenesis was used to evaluate the effects on structure and function of selected substitutions within and N-terminal to the core "alpha-crystallin" domain of the small heat-shock protein (sHsp) and molecular chaperone, human alphaB-crystallin. Five alphaB-crystallin mutants containing single amino acid substitutions within the core alpha-crystallin domain displayed a modest decrease in chaperone activity in aggregation assays in vitro and in protecting cell viability of E. coli at 50 degrees C in vivo. In contrast, seven alphaB-crystallin mutants containing substitutions N-terminal to the core alpha-crystallin domain generally resembled wild-type alphaB-crystallin in chaperone activity in vitro and in vivo. Size-exclusion chromatography, ultraviolet circular dichroism spectroscopy and limited proteolysis were used to evaluate potential structural changes in the 12 alphaB-crystallin mutants. The secondary, tertiary and quaternary structures of mutants within and N-terminal to the core alpha-crystallin domain were similar to wild-type alphaB-crystallin. SDS-PAGE patterns of chymotryptic digestion were also similar in the mutant and wild-type proteins, indicating that the mutations did not introduce structural modifications that altered the exposure of proteolytic cleavage sites in alphaB-crystallin. On the basis of the similarities between the sequences of human alphaB-crystallin and the sHsp Mj HSP16.5, the only sHsp for which there exists high resolution structural information, a three-dimensional model for alphaB-crystallin was constructed. The mutations at sites within the core alpha-crystallin domain of alphaB-crystallin identify regions that may be important for the molecular chaperone functions of sHsps.

Amino Acid Sequence↗

PubMed1999

Disruption of the Sparc locus in mice alters the differentiation of lenticular epithelial cells and leads to cataract formation.

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein that regulates cellular adhesion and proliferation. In this report, we show that SPARC protein is restricted to epithelial cells of the murine lens and ends abruptly at the equatorial bow region where lens fiber differentiation begins. SPARC protein was not detected in the lens capsule or in differentiated lens fibers. SPARC-null mice developed cataracts at approximately 3-4 months after birth, at which time posterior subcapsular opacities were observed by slit lamp ophthalmoscopy. Histological analyses of ocular sections from 3-month old animals revealed several microscopic abnormalities present in the SPARC-null mice but absent from the wild-type animals. Fiber cell elongation was incomplete posteriorly and resulted in displacement of the lenticular nucleus to the posterior of the lens. Nuclear debris was present in the posterior subcapsular region of the lens, an indication of the abnormal migration and elongation of either fetal or anterior epithelial cells, and the bow region was disrupted and vacuolated. In the anterior lens, the capsule appeared to be thickened and was lined by atypical, plump cuboidal epithelium. Moreover, anterior cortical fibers were swollen. Polyacrylamide gel electrophoresis of the epithelial, cortical and nuclear fractions of wild-type and SPARC-null lenses indicated no significant differences among the alpha-, beta-, and gamma-crystallins. Expression of alphaB-crystallin appeared similar in fiber cells of wild-type and SPARC-null lenses, although the distribution of alphaB-crystallin was asymmetric in SPARC-null lenses as a result of abnormal lens fiber differentiation. No evidence of atypical extracellular matrix deposition in areas other than the capsule was detected in wild-type or SPARC-null lens at 3 months of age. We conclude that the disruption of the Sparc locus in mice results in the alteration of two fundamental processes of lens development: differentiation of epithelial cells and maturation of fiber cells.

Animals↗

PubMed1999

Theoretical and experimental basis for the inhibition of cataract.

Aggregation of the lens proteins to form high molecular weight clusters is a major contributing factor in age-onset nuclear cataract [Benedek, G. B. (1971) Theory of transparency of the eye. Appl. Optics, 10, 459-473]. This aggregation occurs continually throughout life and contributes to an exponential increase, as a function of age, in the intensity of the light backscattered out of the lens. The time constant deltaT for this exponential increase in human populations is a valuable index, helpful for conducting clinical trials. In-vitro studies have identified reagents capable of inhibiting high molecular weight aggregate formation, as well as the non-covalent interprotein interactions responsible for phase separation. These reagents are also found experimentally to be effective cataract inhibitors in animal model systems in vivo. We believe that the stage is now set for human clinical trials of putative cataract inhibitors. We present rough quantitative estimates of the trial parameters needed to assure an unambiguous determination of efficacy in a trial population. Such a trial simply requires a measurement of the time constant deltaT in the treated population relative to the untreated population. A successful outcome of the trial is indicated if deltaT increases by 20% over that found for the untreated population. Our estimates suggest efficacy could be determined in a two year trial involving about 300 subjects in the treated group.

Animals↗

PubMed1999

Lens cytoskeleton and transparency: a model.

The function of the cytoskeleton in lens was first considered when cytoplasmic microtubules were observed in elongating fibre cells of the chick lens nearly 40 years ago. Since that time, tubulin, actin, vimentin and intermediate filaments have been identified and found to function in mitosis, motility and cellular morphology during lens cell differentiation. A role for the cytoskeleton in accommodation has been proposed and modification of the cytoskeletal proteins has been observed in several cataract models. Recently, a progressive increase in protein aggregation and lens opacification was found to correspond with the loss of cytoskeletal protein in the selenite model for cataract. In the present report a model is proposed for the role of tubulin, actin, vimentin, spectrin and the lens-specific filaments, filensin and CP49, in the establishment and maintenance of transparent lens cell structure.

Animals↗

PubMed1999

AlphaB-crystallin selectively targets intermediate filament proteins during thermal stress.

PURPOSE: AlphaB-Crystallin is a small heat shock protein (sHsp) expressed at high levels in the lens of the eye, where its molecular chaperone functions may protect against cataract formation in vivo. The purpose of this study was to identify protein targets for the sHsp alphaB-crystallin in lens cell homogenates during conditions of mild thermal stress. METHODS: The authors report the use of a fusion protein, maltose-binding protein alphaB-crystallin (MBP-alphaB), immobilized on amylose resin as a novel method for isolating endogenous alphaB-crystallin-binding proteins from lens cell homogenates after mild thermal stress. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western immunoblot analyses showed selective interactions in lens cell homogenates between MBP-alphaB and endogenous alphaA- and alphaB-crystallins, the lens-specific intermediate filament proteins phakinin (CP49) and filensin (CP115), and vimentin during a mild 20-minute heat shock at 45 degrees C. No interactions were observed with the beta- or gamma-crystallins, or the cytoskeletal proteins actin, alpha-tubulin, and spectrin, although these proteins were present in lens cell homogenates. In contrast, gamma-crystallin and actin interacted with MBP-alphaB at 45 degrees C only in their purified states. The results obtained with MBP-alphaB were confirmed by immunoprecipitation reactions in which immunoprecipitation of native bovine alphaB-crystallin from heat-shocked lens cell homogenates resulted in the coprecipitation of phakinin and filensin. CONCLUSIONS: In the lens the sHsp alphaB-crystallin may selectively target intermediate filaments for protection against unfolding during conditions of stress.

Animals↗

PubMed1999

Daily subcutaneous ultra-low-dose interleukin 2 with daily low-dose interferon-alpha in patients with advanced renal cell carcinoma.

A limited institution Phase II pilot study was performed using a very low-dose combination of daily s.c. interleukin (IL)-2 with IFN-alpha-2b in patients with advanced renal cancer in an attempt to duplicate or increase the response documented with higher dose schedules without the attendant toxicity profile. We selected a dose of IL-2 with documented immunological activity and combined it with clinically active low-dose IFN. Between August 1994 and September 1996, 19 patients with metastatic renal cell carcinoma, who had been judged incapable of tolerating high-dose i.v. IL-2, were treated with IL-2 (1 million units/m2/day) and IFN (1 million units/day), administered s.c. daily. All treatments were administered on an outpatient basis. Virtually all patients had bulky tumor burden with multiple sites of involvement, including five patients with bone metastases. No major objective responses were observed; however, one patient experienced a minor response lasting 13 months, with an associated improvement in performance status. Median survival was 6 months, and 1-year survival was 16%. Toxicity was generally mild and consisted almost entirely of constitutional symptoms. No serious grade 3 or 4 toxicity was observed, although two patients withdrew from treatment due to treatment-related fatigue. On therapy, mild eosinophilia but no lymphocytosis was noted; in fact, peripheral lymphocyte counts decreased, only to rebound after treatment was discontinued. No toxic deaths occurred. Despite the reasonable tolerability of this daily low-dose s.c. regimen, we conclude that this regimen is an ineffective treatment in metastatic renal cell carcinoma patients who are incapable of tolerating high-dose i.v. IL-2.

Aged↗

PubMed1998

ATP-enhanced molecular chaperone functions of the small heat shock protein human alphaB crystallin.

We report direct experimental evidence that human alphaB-crystallin, a member of the small heat shock protein family, actively participates in the refolding of citrate synthase (CS) in vitro. In the presence of 3.5 mM ATP, CS reactivation by alphaB-crystallin was enhanced approximately twofold. Similarly, 3.5 mM ATP enhanced the chaperone activity of alphaB-crystallin on the unfolding and aggregation of CS at 45 degrees C. Consistent with these findings, cell viability at 50 degrees C was improved nearly five orders of magnitude in Escherichia coli expressing alphaB-crystallin. SDS/PAGE analysis of cell lysates suggested that alphaB-crystallin protects cells against physiological stress in vivo by maintaining cytosolic proteins in their native and functional conformations. This report confirms the action of alphaB-crystallin as a molecular chaperone both in vitro and in vivo and describes the enhancement of alphaB-crystallin chaperone functions by ATP.

Adenosine Triphosphate↗

PubMed1998

A novel assay of angiogenesis in the quail chorioallantoic membrane: stimulation by bFGF and inhibition by angiostatin according to fractal dimension and grid intersection.

In a novel assay of angiogenesis in the quail chorioallantoic membrane (CAM), we measured vascular pattern and angiogenic rate after homogeneous exposure of the entire vascular tree to recognized modulators of vessel growth. In comparison to phosphate-buffered saline (PBS)-treated controls, the vascular stimulator, basic fibroblast growth factor (bFGF or FGF-2), increased the rate of angiogenesis by a maximum of 72%, whereas a recently discovered angiogenic inhibitor, angiostatin, decreased the rate of vascular growth by a maximum of 68%. The perturbants were applied in PBS to the CAM of 7-day-old embryos (E7) cultured in petri dishes, and the embryos were cultured further until fixation at E8 or E9. For morphometry of the quasi-two-dimensional CAM vasculature, digital images of arterial endpoints from the middle region of the CAM were acquired in grayscale at a magnification of 10x, binarized to black/white, and skeletonized. The pattern of vessel branching was assessed by measurement of the fractal dimension (Df), and vessel density (rhov), with the method of grid intersection. Correlations between these two statistical techniques were linear (r2 ranged from 0.967 to 0.985). For skeletonized images at E9, Df and rhov of bFGF-treated samples were 1.55 +/- 0.01 and 782 +/- 26/cm2, respectively (relative to 1.49 +/- 0.02 and 583 +/- 60/cm2 for controls), and of angiostatin-treated samples, 1.43 +/- 0.02 and 424 +/- 74/cm2 (relative to 1.50 +/- 0.02 and 616 +/- 59/cm2 for controls). To establish normalization values for rates of angiogenesis, we analyzed untreated CAMs of E6 to E12. From E7 to E10 in skeletonized images, Df increased linearly from 1.37 +/- 0.01 to 1.54 +/- 0.01 and rhov from 311 +/- 67 to 746 +/- 124/cm2 (in both cases, r2 = 1.000). Thus, the rates of normal angiogenic growth as measured by Df and rhov were 0.06/day and 138/cm2-day, respectively. From E10 to E12, Df and rhov declined slightly. Differences between the vasculature of untreated and PBS-treated CAMs were statistically insignificant. In conclusion, vascular branching pattern and density in the quail CAM were stimulated by bFGF and inhibited by angiostatin. We quantified these changes with statistical significance by Df and rhov, which are expressed relative to the rates of normal developmental angiogenesis measured for the two parameters in untreated quail embryos.

Allantois↗

PubMed1998

SPARC deficiency leads to early-onset cataractogenesis.

PURPOSE: To determine the role of SPARC (secreted protein, acidic, and rich in cysteine) in cataractogenesis by examining mice deficient in a matricellular protein SPARC. METHODS: Mice were rendered SPARC-deficient by a targeted disruption of the gene. Slit-lamp microscopy and histology were used to examine the eyes of SPARC-null and wild-type mice from birth to 14 months of age. RESULTS: SPARC-null mice developed opacities in the posterior cortex of the eye as early as 1.5 months after birth. The diffuse cataracts appeared to progress toward the anterior cortex and reached maturity in many animals by 3.5 months of age. Early stages of cataractogenesis in SPARC-null mice included inhibition of normal lens fiber cell differentiation, degeneration of fiber cells, vacuole formation at the equator, and liquefaction of the cortex. No cataracts were detected in wild-type mice up to the age of 8 months. CONCLUSIONS: The early onset of cataracts in SPARC-null mice establishes that the gene is essential to the maintenance of lens transparency.

Animals↗

PubMed1997

Human alphaB-crystallin. Small heat shock protein and molecular chaperone.

The polymerase chain reaction was used to amplify a cDNA sequence encoding the human alphaB-crystallin. The amplified cDNA fragment was cloned into the bacterial expression vector pMAL-c2 and expressed as a soluble fusion protein coupled to maltose-binding protein (MBP). After maltose affinity chromatography and cleavage from MBP by Factor Xa, the recombinant human alphaB-crystallin was separated from MBP and Factor Xa by anion exchange chromatography. Recombinant alphaB-crystallin was characterized by SDS-polyacrylamide electrophoresis (PAGE), Western immunoblot analysis, Edman degradation, circular dichroism spectroscopy, and size exclusion chromatography. The purified crystallin migrated on SDS-PAGE to an apparent molecular weight (Mr approximately 22,000) that corresponded to total native human alpha-crystallin and was recognized on Western immunoblots by antiserum raised against human alphaB-crystallin purified from lens homogenates. Chemical sequencing, circular dichroism spectroscopy, and size exclusion chromatography demonstrated that the recombinant crystallin had properties similar or identical to its native counterpart. Both recombinant alphaB-crystallin and MBP-alphaB fusion protein associated to form high molecular weight complexes that displayed chaperone-like function by inhibiting the aggregation of alcohol dehydrogenase at 37 degrees C and demonstrated the importance of the C-terminal domain of alphaB-crystallin for chaperone-like activity.

Blotting, Western↗