Biomedical subjects

L L Johnson

Publications and source records attributed to L L Johnson.

At least 19 recordsLinked to original sources

PubMed2000

A rationalization of the acidic pH dependence for stromelysin-1 (Matrix metalloproteinase-3) catalysis and inhibition.

The pH dependence of matrix metalloproteinase (MMP) catalysis is described by a broad bell-shaped curve, indicating the involvement of two unspecified ionizable groups in proteolysis. Stromelysin-1 has a third pK(a) near 6, resulting in a uniquely sharp acidic catalytic optimum, which has recently been attributed to His(224). This suggests the presence of a critical, but unidentified, S1' substructure. Integrating biochemical characterizations of inhibitor-enzyme interactions with active site topography from corresponding crystal structures, we isolated contributions to the pH dependence of catalysis and inhibition of active site residues Glu(202) and His(224). The acidic pK(a) 5.6 is attributed to the Glu(202).zinc.H(2)O complex, consistent with a role for the invariant active site Glu as a general base in MMP catalysis. The His(224)-dependent substructure is identified as a tripeptide (Pro(221)-Leu(222)-Tyr(223)) that forms the substrate cleft lower wall. Substrate binding induces a beta-conformation in this sequence, which extends and anchors the larger beta-sheet of the enzyme. substrate complex and appears to be essential for productive substrate binding. Because the PXY tripeptide is strictly conserved among MMPs, this "beta-anchor" may represent a common motif required for macromolecular substrate hydrolysis. The striking acidic profile of stromelysin-1 defined by the combined ionization of Glu(202) and His(224) allows the design of highly selective inhibitors.

Binding Sites↗

PubMed2000

Structure-activity relationships and pharmacokinetic analysis for a series of potent, systemically available biphenylsulfonamide matrix metalloproteinase inhibitors.

A series of biphenylsulfonamide derivatives of (S)-2-(biphenyl-4-sulfonylamino)-3-methylbutyric acid (5) were prepared and evaluated for their ability to inhibit matrix metalloproteinases (MMPs). For this series of compounds, our objective was to systematically replace substituents appended to the biphenyl and alpha-position of 5 with structurally diverse functionalities to assess the effects these changes have on biological and pharmacokinetic activity. The ensuing structure-activity relationship (SAR) studies showed that biphenylsulfonamides substituted with bromine in the 4'-position (11c) significantly improved in vitro activity and exhibited superior pharmacokinetics (C(max), t(1/2), AUCs), relative to compound 5. Varying the lipophilicity of the alpha-position by replacing the isopropyl group of 11c with a variety of substituents, in general, maintained potency versus MMP-2, -3, and -13 but decreased the oral systemic availability. Subsequent evaluation of its enantiomer, 11c', showed that both compounds were equally effective MMP inhibitors. In contrast, the corresponding hydroxamic acid enantiomeric pair, 16a (S-isomer) and 16a' (R-isomer), stereoselectivity inhibited MMPs. For the first time in this series, 16a' provided nanomolar potency against MMP-1, -7, and -9 (IC(50)'s = 110, 140, and 18 nM, respectively), whereas 16a was less potent against these MMPs (IC(50)'s = 24, 78, and 84 microM, respectively). However, unlike 11c, compound 16a' afforded very low plasma concentrations following a single 5 mg/kg oral dose in rat. Subsequent X-ray crystal structures of the catalytic domain of stromelysin (MMP-3CD) complexed with inhibitors from closely related series established the differences in the binding mode of carboxylic acid-based inhibitors (11c,c') relative to the corresponding hydroxamic acids (16a,a').

Animals↗

PubMed2000

Autogenous tendon graft substitution for absent knee joint meniscus: a pilot study.

The purpose of this pilot study was to explore the potential of an autogenous tendon graft to substitute for an absent human knee joint meniscus. Based on the results of animal studies and human reports, it was hypothesized that autogenous tendon tissue would substitute for human knee joint meniscus: maintain mechanical integrity, convert to fibrocartilage, preserve the joint compartment, and provide symptomatic relief for the patient. Five patients, 2 men and 3 women, average age 41 years, had surgical absence of the lateral meniscus, genu valgum, and severe degenerative arthritis of the lateral compartment, but a stable knee. All patients were offered alternative treatments: do nothing, medication, arthroscopic debridement, osteotomy, and knee replacement. The operations were performed by arthroscopy. An accompanying arthroscopic debridement procedure was performed in the same compartment. In 4 cases, the donor graft was the semitendinosus tendon. In 1, the patellar tendon was used because the semitendinosus had been previously used in an anterior cruciate ligament reconstruction. Four of the 5 patients had a second-look arthroscopy and biopsy between 9 and 24 months. There was partial physical integrity to the tendon graft. The tendon graft did not completely convert to fibrocartilage. The joint surface was not preserved. Only 1 patient had minimal clinical improvement; the others were not improved. No patient was made worse. One patient had a total knee replacement 1 year later. Another had a knee fusion after 4 years. All other patients are considering future reconstructive surgery. The autogenous tendon graft as used in this pilot study was not successful as a substitute for an absent meniscus. The hypothesis was not realized. The observations from this pilot study should be helpful in future study protocol design.

Adult↗

PubMed2000

The application of arthroscopic principles to bone grafting of delayed union of long bone fractures.

The purpose of this study was to explore the potential of applying arthroscopic techniques to autogenous bone grafting of long bone fracture delayed union. There were 9 patients in this initial series, including 4 patients (average age, 37 years) with humeral lesions and 5 patients (average age, 25 years) with tibial fractures. There were 6 men and 3 women. Techniques customarily employed in arthroscopy were used to visualize, expose, and deliver the onlay cancellous bone grafts. Bony union occurred in all but 1 patient in an average of 4 months. This patient had a fibrous union and sustained a reinjury that led to successful repeat open bone graft surgery. The arthroscopic approach for bone grafting of certain long bone delayed union appears to be a safe and effective procedure. The procedure is best suited for patients with mechanically stabilized fragments, and it lends itself to those with overlying skin or soft tissue compromise. There are some relative contraindications: grossly unstable fragments, severe malunion, and/or infection.

Adolescent↗

PubMed2000

B cells are essential for vaccination-induced resistance to virulent Toxoplasma gondii.

T lymphocytes and gamma interferon (IFN-gamma) are known mediators of immune resistance to Toxoplasma gondii infection, but whether B cells also play an important role is not clear. We have investigated this issue using B-cell-deficient (muMT) mice. If vaccinated with attenuated T. gondii tachyzoites, muMT mice are susceptible to a challenge intraperitoneal infection with highly virulent tachyzoites that similarly vaccinated B-cell-sufficient mice resist. Susceptibility is evidenced by increased numbers of parasites at the challenge infection site and by extensive mortality. The susceptibility of B-cell-deficient mice does not appear to be caused by deficient T-cell functions or diminished capacity of vaccinated and challenged B-cell-deficient mice to produce IFN-gamma. Administration of Toxoplasma-immune serum, but not nonimmune serum, to vaccinated B-cell-deficient mice significantly prolongs their survival after challenge with virulent tachyzoites. Vaccinated mice lacking Fc receptors or the fifth component of complement resist a challenge infection, suggesting that neither Fc-receptor-dependent phagocytosis of antibody-coated tachyzoites nor antibody-dependent cellular cytotoxicity nor antibody-and-complement-dependent lysis of tachyzoites is a crucial mechanism of resistance. However, Toxoplasma-immune serum effectively inhibits the infection of host cells by tachyzoites in vitro. Together, the results support the hypothesis that B cells are required for vaccination-induced resistance to virulent tachyzoites in order to produce antibodies and that antibodies may function protectively in vivo by blocking infection of host cells by tachyzoites.

Animals↗

PubMed2000

Myocardial uptake of a (99m)Tc-nitroheterocycle in a swine model of occlusion and reperfusion.

UNLABELLED: The purpose of this study was to evaluate the window for scan positivity of the radiolabeled nitroheterocycle (99m)Tc-BRU-59-21 in the peri-ischemic period using a swine model of occlusion and reperfusion. METHODS: A balloon catheter was placed in the left anterior descending coronary artery in each of 19 domestic swine. Blood flow and hemodynamic measurements were made at baseline, during occlusion, and at 15 and 180 min after reperfusion. A dose of approximately 925 MBq (99m)Tc-BRU-59-21 was injected before a brief (6 min) period of coronary occlusion at the following times: 15 min (n = 2), 5 min (n = 2), and 2.2 min (n = 5). In 5 experiments the dose was injected 15 min after reperfusion. Animals underwent SPECT imaging 3 h later. Animals were then killed, and hearts were removed, sliced, stained with triphenyl tetrazolium chloride, and imaged on the detector. RESULTS: The risk region became ischemic during occlusion on the basis of severe reduction in blood flow and lactate production, but necrosis occurred in only 3 experiments. Focal tracer uptake was seen in the risk region in animals injected 5 and 2.2 min before occlusion but not in animals injected 15 min before occlusion and 15 min after reperfusion. CONCLUSION: The window for scan positivity for (99m)Tc-BRU-59-21 injected in the peri-ischemic period is short using this model of balloon occlusion and reperfusion in swine.

Animals↗

PubMed2000

In vivo uptake of radiolabeled antibody to proliferating smooth muscle cells in a swine model of coronary stent restenosis.

UNLABELLED: Z2D3 is a monoclonal chimeric antibody fragment that is directed against a protein expressed on the surface of proliferating smooth muscle cells. The purpose of this study was to investigate the uptake of 111In-labeled Z2D3 F(ab')2 in a swine model of coronary neointimal proliferation after overexpansion coronary stenting. METHODS: Twenty-two domestic swine underwent overexpansion coronary stenting of 2 vessels. Fifteen swine survived 2-4 wk, at which time they received an injection of 111In Z2D3 F(ab')2 and underwent planar imaging. After the swine were killed, the hearts were excised and imaged on the detector. The cross-sectional area of each stented vessel was measured with digital morphometry. RESULTS: Pathology could be correlated with imaging for 24 vessels. The cross-sectional area of stenosis comprising neointimal proliferation ranged from 8% to 95%, with a mean +/- SD of 41% +/- 21%. The maximal stenosis ranged from 13% to 95%, with a mean of 51% +/- 20%. Seventeen of 24 vessels (71%) showed focal uptake on in vivo imaging, and 7 of 24 (29%) did not. Twenty of 24 (83%) showed uptake on ex vivo imaging. Of 11 stented vessels with maximal vessel stenosis less than 50%, 7 (64%) showed uptake both in vivo and ex vivo, and of 13 stented vessels with maximal vessel stenosis greater than 50%, 10 (77%) showed uptake both in vivo and ex vivo. CONCLUSION: Uptake of a radiolabeled antibody directed against a component of proliferating neointimal tissue can be visualized in the coronary arteries on in vivo imaging using a scintillation gamma camera.

Animals↗

PubMed1999

Effect of species differences on stromelysin-1 (MMP-3) inhibitor potency. An explanation of inhibitor selectivity using homology modeling and chimeric proteins.

For an animal model to predict a compound's potential for treating human disease, inhibitor interactions with the cognate enzymes of separate species must be comparable. Rabbit and human isoforms of stromelysin-1 are highly homologous, yet there are clear and significant compound-specific differences in inhibitor potencies between these two enzymes. Using crystal structures of discordant inhibitors complexed with the human enzyme, we generated a rabbit enzyme homology model that was used to identify two unmatched residues near the active site that could explain the observed disparities. To test these observations, we designed and synthesized three chimeric mutants of the human enzyme containing the single (H224N and L226F) and double (H224N/L226F) mutations. A comparison of inhibitor potencies among the mutant and wild-type enzymes shows that the mutation of a single amino acid in the human enzyme, histidine 224 to asparagine, is sufficient to change the selectivity profile of the mutant to that of the rabbit isoform. These studies emphasize the importance of considering species differences, which can result from even minor protein sequence variations, for the critical enzymes in an animal disease model. Homology modeling provides a tool to identify key differences in isoforms that can significantly affect native enzyme activity.

Amino Acid Sequence↗

PubMed1999

Matrix metalloproteinase inhibition during the development of congestive heart failure : effects on left ventricular dimensions and function.

The development of congestive heart failure (CHF) is associated with left ventricle (LV) dilation and myocardial remodeling. The matrix metalloproteinases (MMPs) play a significant role in extracellular remodeling, and recent studies have demonstrated increased MMP expression and activity with CHF. Whether increased MMP activity directly contributes to the LV remodeling with CHF remains unknown. Accordingly, this study examined the effects of chronic MMP inhibition (MMPi) on LV size and function during the progression of CHF. Pigs were assigned to the following groups: (1) CHF, rapid pacing for 3 weeks at 240 bpm (n=12); (2) CHF/MMPi, rapid pacing and concomitant MMPi (PD166793, 20 mg/kg per day [n=10]), and (3) control (n=11). With pacing CHF, LV fractional shortening was reduced (19+/-1 versus 45+/-1%), and end-diastolic dimension increased (5.67+/-0.11 versus 3.55+/-0.05 cm), compared with baseline values (P<0.05). In the CHF/MMPi group, LV endocardial shortening increased (25+/-2%) and the end-diastolic dimension was reduced (4.92+/-0.17 cm) compared with CHF-only values (P<0.05). LV midwall shortening was reduced to a comparable degree in the CHF-only and CHF/MMPi groups. LV peak wall stress increased 3-fold with pacing CHF compared with controls and was significantly reduced in the CHF/MMPi group. LV myocardial stiffness was unchanged with CHF but was increased in the CHF/MMPi group. LV myocyte length was increased with pacing CHF compared with controls (180+/-3 versus 125+/-4 microm, P<0.05) and was reduced in the CHF/MMPi group (169+/-4 microm, P<0.05). Basal-state myocyte shortening velocity was reduced with pacing CHF compared with controls (33+/-2 versus 66+/-1 microm/s, P<0.05) and was unchanged in the CHF/MMPi group (31+/-2 microm/s). Using an ex vivo assay system, myocardial MMP activity was increased with pacing CHF and was reduced with chronic MMPi. In summary, concomitant MMPi with developing CHF limited LV dilation and reduced wall stress. These results suggest that increased myocardial MMP activity contributes to LV myocardial remodeling in developing CHF.

Animals↗

PubMed1999

Bioabsorbable suture anchor (co-polymer 85/15 D,L lactide/glycolide) implanted in bone: correlation of physical/mechanical properties, magnetic resonance imaging, and histological response.

A novel bioabsorbable suture anchor has been introduced for shoulder rotator cuff surgical repair made of the co-polymer 85/15 D,L lactide/glycolide. Previous clinical reports on the use of this material in anterior cruciate ligament reconstruction have described intraosseous edema at various time intervals following implantation. The purpose of this study was to analyze the implant's loss of physical properties and to correlate magnetic resonance imaging (MRI) finding with gross and histological observations at various time intervals after intraosseous implantation in the experimental animal. Six drill holes were made in the tibias of 11 dogs. The spherical implant was placed in 5 of the drill holes and the sixth was preserved as a sham control. The dogs were killed at 3, 4, 6, 9, 12, and 26 weeks for gross and microscopic inspection. Correlative MRIs were taken from the 4-, 12-, and 26-week specimens. Gross inspection showed that the overlying soft tissue healed to bone in 3 weeks. The implants were surrounded by new bone by 6 weeks. The implants maintained gross physical integrity for 6 to 12 weeks. Histologically, there was minimal inflammatory response to the degrading implant. The implant site had been completely replaced by bone at 12 weeks. Correlative MRI showed edema adjacent to the implant sites, but there was no correlative inflammation or cyst formation through the time necessary for complete absorption of the implant. Correlative MRI identified and differentiated the image of the intact and degrading implant.

Absorbable Implants↗

PubMed1999

The effect of bone compaction on early fixation of porous-coated implants.

The effect of a bone compaction technique versus conventional drilling on the early fixation of porous-coated implants was examined in a canine model. Compaction dilation resulted in a significant increase in implant fixation stiffness (P < .01) and ultimate fixation strength (P < .01) at 0 and 3 weeks. Fixation stiffness remained significantly increased at 6 weeks (P < .01); however, the ultimate fixation strength was not statistically significant between the 2 techniques (P > .05). There was no significant difference in either fixation value at 9 weeks (P > .05). Histological examination of the bone-implant interface demonstrated an increase in the density of cancellous bone immediately adjacent to the implants placed in the compaction dilated holes. The results of this study suggest that the compaction method of host bone preparation may optimize the initial stability of the implant interface of porous-coated prostheses.

Animals↗

PubMed1999

X-ray structure of human stromelysin catalytic domain complexed with nonpeptide inhibitors: implications for inhibitor selectivity.

Effective inhibitors of matrix metalloproteinases (MMPs), a family of connective tissue-degrading enzymes, could be useful for the treatment of diseases such as cancer, multiple sclerosis, and arthritis. Many of the known MMP inhibitors are derived from peptide substrates, with high potency in vitro but little selectivity among MMPs and poor bioavailability. We have discovered nonpeptidic MMP inhibitors with improved properties, and report here the crystal structures of human stromelysin-1 catalytic domain (SCD) complexed with four of these inhibitors. The structures were determined and refined at resolutions ranging from 1.64 to 2.0 A. Each inhibitor binds in the active site of SCD such that a bulky diphenyl piperidine moiety penetrates a deep, predominantly hydrophobic S'1 pocket. The active site structure of the SCD is similar in all four inhibitor complexes, but differs substantially from the peptide hydroxamate complex, which has a smaller side chain bound in the S'1 pocket. The largest differences occur in the loop forming the "top" of this pocket. The occupation of these nonpeptidic inhibitors in the S'1 pocket provides a structural basis to explain their selectivity among MMPs. An analysis of the unique binding mode predicts structural modifications to design improved MMP inhibitors.

Binding Sites↗

PubMed1998

Hydropic vacuolation in the liver of three species of fish from the U.S. West Coast: lesion description and risk assessment associated with contaminant exposure.

Hydropic vacuolation (HydVac) of biliary epithelial cells and hepatocytes is described for 3 species of U.S. West Coast bottom fishes. Risk assessment analyses are also conducted to determine if the prevalence of this lesion increases in association with contaminant exposure and site of capture. The morphology of HydVac in starry flounder Platichthys stellatus, white croaker Genyonemus lineatus and rock sole Lepidopsetta bilineata was similar to that described in winter flounder Pleuronectes americanus from the U.S. Atlantic Coast, especially in that HydVac most commonly affected biliary epithelial cells. Hydropic vacuolation was the most prevalent liver lesion in starry flounder and white croaker captured from contaminated environments. Risk assessment analyses confirmed that the relative risk for HydVac increased with the presence of aromatic and chlorinated hydrocarbons in sediment, fish bile, and fish liver for these species. Hydropic vacuolation also frequently occurred in rock sole, but the lesion showed no clear association with contaminant exposure in this species. The types of liver lesions that were useful biomarkers of contaminant effects in fish depended on the species and this factor must be taken into account when evaluating histopathological biomarkers of response to contaminant exposure.

Age Factors↗

PubMed1998

An enzyme-linked immunosorbent assay (ELISA) for measuring vitellogenin in English sole (Pleuronectes vetulus): development, validation and cross-reactivity with other pleuronectids.

Vitellogenin (Vtg) is a yolk protein produced in the liver of oviparous animals in response to estrogen. Vitellogenesis is normally observed only in sexually mature females, but it can be induced in male and juvenile animals by exposure to exogenous estradiol (E2) or substances that mimic estrogens. The abnormal production of Vtg by males can, therefore, be used as a biological indicator for exposure to xenoestrogens. In this study, an enzyme-linked immunosorbent assay (ELISA) for measuring Vtg in English sole (Pleuronectes vetulus) was developed and validated. Plasmatic Vtg was purified from E2-injected male English sole using DEAE ion-exchange and Sepharose size-exclusion chromatography, and polyclonal antibodies against the purified Vtg protein were generated in rabbits. In this assay, a competition for the Vtg antibody was established between Vtg coated onto microtiter plate wells and free Vtg. Detection of adsorbed antigen-antibody complexes was achieved using a horseradish peroxidase conjugated anti-rabbit secondary antibody whose enzyme activity was revealed with 3,3',5,5'-tetramethyl benzidine (TMB) substrate. Assay conditions provided a detectable Vtg range of 10-450 ng ml-1 (85-20% of binding) of diluted sample. Plasma dilution curves from vitellogenic female and E2-treated male English sole showed parallelism with the standard dilution curve. We are presently conducting field and laboratory studies to investigate estrogenic and anti-estrogenic activity resulting from exposure to contaminants.

Animals↗

PubMed1998

Cancellous bone grafting of large osteochondral defects: an experimental study in dogs.

Autogenous cancellous bone was evaluated as a material to repair large osteochondral defects in 20 adult mongrel dogs. In one knee, the bone graft was used to fill an osteochondral cylindrical defect (10 mm diameter x 10 mm deep) created in the femoral trochlea. A similar lesion was created in the contralateral knee but was left untreated for spontaneous healing. Four animals were killed at each of five periods (2, 4, 8, 12, and 24 weeks), and the healing response of the defects was evaluated by gross anatomic inspection, plain film radiography, high-resolution radiography, and histology. The results of this study suggest that the use of a cancellous bone graft accelerates the repair of large osteochondral defects and produces more uniform filling of the defect than the ungrafted control. The reparative surface of the grafted lesions also differed from that of controls, having uniform coverage with histochemical-positive staining fibrocartilage at 8 weeks, a finding not observed in any control defect through the length of this study, 24 weeks.

Animals↗

PubMed1998

Matrix metalloproteinases.

Matrix metalloproteinases are a family of highly regulated peptidases that are collectively responsible for the degradation of extracellular matrix during tissue remodeling. Dysregulated activity has long been implicated in the pathologies of cancer and arthritis, and the number of diseases more recently associated with these enzymes has been increasing. In the past year, new transgenic models of matrix metalloproteinase knockouts have been described, allowing the direct assessment of specific enzyme activity in particular disease models. In addition, more selective inhibitors with improved pharmacokinetic profiles have entered clinical trials, allowing the assessment of the safety and efficacy of this strategy.

Animals↗

PubMed1998

Decreased resistance to primary intravenous Cryptococcus neoformans infection in aged mice despite adequate resistance to intravenous rechallenge.

It is often stated that impaired immune functions in the aged underlie their greater susceptibility to infections. Indeed, in many experimental settings, T-cell responses in aged mice have been shown to be deficient compared with those from young adults. Nonetheless, there are very few examples where a greater susceptibility to infection in aged mice has been demonstrated to result from impaired T-cell function. The clinical importance of understanding the basis for increased susceptibility to infection that accompanies advanced age dictates a need for experimental models with which to study the effect that aging has on immunological resistance to infection. This study was undertaken to investigate whether aged mice were less resistant than young adult control mice to infection with the fungus Cryptococcus neoformans. After a primary intravenous challenge with yeast, aged mice died sooner and developed higher organ burdens of yeast than did young adults. Deficient in vitro responses were observed in T cells from aged mice; however, greater susceptibility to intravenous infection appeared not to result from less effective T-cell-dependent resistance in vivo. In fact, T-cell-replete aged mice were more susceptible to intravenous cryptococcal infection than were T-cell-depleted young adults. Furthermore, aged mice were as resistant to primary pulmonary challenge with Cryptococcus as were young adults. Similarly, vaccinated aged mice were as resistant to rechallenge as were young adult counterparts. Therefore, despite demonstrably deficient in vitro responses of T cells from aged mice, their T-cell-dependent resistance to C. neoformans is as effective as that of young adults.

Aging↗

PubMed1998

Treatment of bacterial biofilms on polymeric biomaterials using antibiotics and ultrasound.

New studies on the effect of frequency and duration of exposure upon the ultrasonic enhanced action of gentamicin against biofilms of Escherichia coli are presented in this paper. Ultrasound was applied at clinical levels which would not harm human tissue. The study on frequency indicated that lower frequencies produce higher levels of killing. The results of the timed experiment indicate that complete sterilization of a 14-h biofilm can be achieved after 6 h of exposure. These findings are significant because they show that biofilms can be reduced to zero reproductive ability as assessed by plate counting.

Biocompatible Materials↗