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Biomedical subjects

W J Cook

Publications and source records attributed to W J Cook.

At least 19 recordsLinked to original sources
PubMed2001

Microarrays for studying the host transcriptional response to microbial infection and for the identification of host drug targets.

It is essential to understand the molecular basis of a host's response to microbial infection so that disease and tissue damage can be prevented. Modulation of host RNA expression is a critical set of molecular changes that occur upon infection. Global analysis of gene expression should provide an understanding of host RNA transcriptional changes that occur upon host-pathogen interaction. This series of articles focuses on the use of microarrays for analyzing the transcriptional response of a host to microbial infection and for drug target identification.

Animals↗
PubMed2000

Durable donor-specific T and B cell tolerance in rhesus macaques induced with peritransplantation anti-CD3 immunotoxin and deoxyspergualin: absence of chronic allograft nephropathy.

Tolerance induction can prevent acute kidney allograft rejection without chronic immunosuppression. It is uncertain whether specific tolerance can prevent chronic allograft nephropathy (CAN), which involves both nonimmune and immune injury. This report provides evidence that immunologically tolerant macaques, induced with immunotoxin and deoxyspergualin, developed neither acute rejection nor CAN. Long survivors, bearing MHC-mismatched grafts without chronic immunosuppression for 0.8 to 3.4 years, exhibited general immune competence with donor-specific T and B cell tolerance and no functional or histological evidence of CAN. Stringent criteria for tolerance were satisfied by specific prolongation of donor skin grafts with rapid rejection of third-party skin, followed by indefinite acceptance of a second donor kidney graft and establishment of microchimerism. Primate tolerance with documented absence of CAN may give impetus to the clinical application of tolerance.

Animals↗
PubMed2000

Postransplant lymphoproliferative disorder localized near the allograft in renal transplantation.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD), a complication of immunosuppression, develops in approximately 1% of renal allograft recipients. Typically, PTLD is a proliferation of B-cells associated with Epstein-Barr virus (EBV) infection; it is said to be most often a systemic disease. Involvement occasionally is localized near the allograft. METHODS: This is a retrospective analysis of all cases of PTLD in recipients of 1474 renal transplants performed at University of Alabama at Birmingham between 1993 and 1997. RESULTS: Of 14 patients developing PTLD, 10 had disease localized near the allograft. The mean interval from transplantation to diagnosis was 221 +/- 70 days. All patients presented with renal dysfunction; an ultrasound examination revealed a hilar mass, with hydronephrosis in five and stenosis of renal vessels in eight. No patient had lymphadenopathy, according to computerized tomographic or magnetic resonance imaging findings. After reduction of immunosuppressive therapy, seven required a nephrectomy because of rejection, progressive dysfunction, or mass enlargement. Tissue recovered in four patients was consistent with PTLD; the tumors in the remaining three patients were unresectable and regressed. One patient died 1 month after a nephrectomy, and another died 4 years after surgery; neither had evidence of PTLD when they died. Three patients retain functional grafts without clinical or radiographical evidence of progression. All patients with disseminated disease died. CONCLUSIONS: In a large cohort of renal allograft recipients, PTLD affected 1%. Disease localized near the allograft was the most common variant. For most patients with localized disease, the outcome was graft loss, and the mortality was low. Localized PTLD should be considered in the differential diagnosis of allograft dysfunction in the 1st posttransplant year.

Adolescent↗
PubMed2000

Requirement of calmodulin binding by HIV-1 gp160 for enhanced FAS-mediated apoptosis.

Accelerated apoptosis is one mechanism proposed for the loss of CD4+ T-lymphocytes in human immunodeficiency virus type 1 (HIV-1) infection. The HIV-1 envelope glycoprotein, gp160, contains two C-terminal calmodulin-binding domains. Expression of gp160 in Jurkat T-cells results in increased sensitivity to FAS- and ceramide-mediated apoptosis. The pro-apoptotic effect of gp160 expression is blocked by two calmodulin antagonists, tamoxifen and trifluoperazine. This enhanced apoptosis in response to FAS antibody or C(2)-ceramide is associated with activation of caspase 3, a critical mediator of apoptosis. A point mutation in the C-terminal calmodulin-binding domain of gp160 (alanine 835 to tryptophan, A835W) eliminates gp160-dependent enhanced FAS-mediated apoptosis in transiently transfected cells, as well as in vitro calmodulin binding to a peptide corresponding to the C-terminal calmodulin-binding domain of gp160. Stable Tet-off Jurkat cell lines were developed that inducibly express wild type gp160 or gp160A835W. Increasing expression of wild type gp160, but not gp160A835W, correlates with increased calmodulin levels, increased apoptosis, and caspase 3 activation in response to anti-FAS treatment. The data indicate that gp160-enhanced apoptosis is dependent upon calmodulin up-regulation, involves the activation of caspase 3, and requires calmodulin binding to the C-terminal binding domain of gp160.

Alanine↗
PubMed2000

Crystal structure of adenosine kinase from Toxoplasma gondii at 1.8 A resolution.

Human infection with Toxoplasma gondii is an important cause of morbidity and mortality. Protozoan parasites such as T. gondii are incapable of de novo purine biosynthesis and must acquire purines from their host, so the purine salvage pathway offers a number of potential targets for antiparasitic chemotherapy. In T. gondii tachyzoites, adenosine is the predominantly salvaged purine nucleoside, and thus adenosine kinase is a key enzyme in the purine salvage pathway of this parasite. The structure of T. gondii adenosine kinase was solved using molecular replacement and refined by simulated annealing at 1.8 A resolution to an R-factor of 0.214. The overall structure and the active site geometry are similar to human adenosine kinase, although there are significant differences. The T. gondii adenosine kinase has several unique features compared to the human sequence, including a five-residue deletion in one of the four linking segments between the two domains, which is probably responsible for a major change in the orientation of the two domains with respect to each other. These structural differences suggest the possibility of developing specific inhibitors of the parasitic enzyme.

Adenosine↗
PubMed2000

Prolonged hepatitis B surface antigenemia after vaccination.

Infection with hepatitis B virus can lead to serious long-term complications including chronic hepatitis B virus infection leading to hepatocellular carcinoma, liver failure, and death. We report a case of prolonged hepatitis B antigenemia after routine vaccination with Engerix B. A positive hepatitis B surface antigen was found when the individual donated blood 18 days after vaccination. This resulted in rejection of the donated blood and permanent deferral from further donation. It also led to referral to a physician, creating anxiety in the individual and additional unnecessary testing. Additional studies are needed to identify the length to time of hepatitis B surface antigenemia after hepatitis B vaccination, and blood collection centers should be aware of the potential for donors to have a prolonged false-positive hepatitis B surface antigen after vaccination against hepatitis B. hepatitis B, hepatitis B vaccine, hepatitis B surface antigen, vaccine-induced positive hepatitis B surface antigen, Engerix B.

Adolescent↗
PubMed1999

Peritransplant tolerance induction in macaques: early events reflecting the unique synergy between immunotoxin and deoxyspergualin.

BACKGROUND: Day of transplant T cell depletion with anti-CD3 immunotoxin or F(Ab)2 immunotoxin induces stable tolerance to renal allografts in rhesus monkeys given 15-deoxyspergualin (DSG), a NF-kappaB inhibitor that suppresses proinflammatory cytokine (PC) production. Because PC and NF-kappaB are involved in dendritic cell (DC) maturation, we asked if impaired DC maturation and Th2-type cytokine deviation might be related to the synergistic effect of DSG in this novel model. METHODS: Immunosuppression was initiated 4 hr before transplanting a major histocompatibility complex mismatched renal allograft. Some groups received a supplemental 5-day course of cyclosporine A or DSG or a 15-day course of DSG. Peripheral lymph nodes were sequentially examined for presence of mature DC. In vitro effects of DSG on PC-induced maturation of DC were also examined. RESULTS: Allografts survived without rejection in 87% of recipients given immunotoxin or F(Ab)2 immunotoxin with DSG x 15 days, in 50% with DSG x 5 days, and 0% with cyclosporine A. The longest DSG survivors are >1000 days with normal graft function and tolerance validated, including acceptance of challenge second donor kidneys without treatment. DSG-treated recipients were unique in developing polarized Th2-type plasma cytokines. In DSG recipients, mature DC were significantly reduced in day +5 lymph node biopsies, with complete repopulation by 30 days. In vitro studies verified an inhibitory effect of DSG on DC maturation. CONCLUSIONS: The study suggests DSG arrests DC maturation. The unusual synergy of immunotoxin and DSG apparently involves coincidental reduction in lymph node T cell mass and mature DC, a transient circumstance favoring development of stable tolerance.

Animals↗
PubMed1999

Remote telemedical interpretation of neonatal echocardiograms: impact on clinical management in a primary care setting.

OBJECTIVE: The purpose of this study was to evaluate the utility of telemedical echocardiographically assisted neonatal cardiovascular evaluation in a primary care setting. BACKGROUND: Neonates with congenital heart disease are frequently born far from pediatric subspecialty centers and can be clinically unstable at presentation. Recent advances in telecommunication technology have made it possible to transmit echocardiographic images over long distances. This technology may be beneficial to newborns with heart defects who are born in primary care centers. METHODS: A retrospective review of all telemedical echocardiograms obtained from neonates (aged 1 day to 30 days) was performed. A telemedical link was created using a T-1 transmission line and a standard voice telephone line between the Mayo Clinic, Rochester, Minnesota (pediatric cardiology site), and the Altru Clinic, Grand Forks, North Dakota (primary care site), which is a general pediatric practice 400 miles from Rochester. Neonates with possible cardiac disorders were identified by the general pediatricians, who then requested telemedical echocardiography. RESULTS: The 133 neonates had 161 T-1 echocardiograms. Median patient age was two days (range, one day to 29 days). One hundred thirty-two of 133 initial echocardiograms (99%) were obtained because of urgent indications. Transmitted images provided adequate diagnostic information in all patients. Seventy-nine neonates (59%) had a change in medical management or required cardiology follow-up. An immediate change in management occurred in 32 patients (24%), including seven in whom emergency transfer was either arranged or avoided. CONCLUSIONS: Telemedical echocardiography provides accurate diagnostic data in neonates. Rapid telediagnosis facilitates appropriate care of sick neonates with possible congenital heart disease in the primary care setting. Unnecessary long-distance transfers can be avoided with this technology.

Heart Defects, Congenital↗
PubMed1998

Structural and theoretical studies suggest domain movement produces an active conformation of thymidine phosphorylase.

Two new crystal forms of Escherichia coli thymidine phosphorylase (EC 2.4.2.4) have been found; a monoclinic form (space group P21) and an orthorhombic form (space group I222). These structures have been solved and compared to the previously determined tetragonal form (space group P43212). This comparison provides evidence of domain movement of the alpha (residues 1 to 65, 163 to 193) and alpha/beta (residues 80 to 154, 197 to 440) domains, which is thought to be critical for enzymatic activity by closing the active site cleft. Three hinge regions apparently allow the alpha and alpha/beta-domains to move relative to each other. The monoclinic model is the most open of the three models while the tetragonal model is the most closed. Phosphate binding induces formation of a hydrogen bond between His119 and Gly208, which helps to order the 115 to 120 loop that is disordered prior to phosphate binding. The formation of this hydrogen bond also appears to play a key role in the domain movement. The alpha-domain moves as a rigid body, while the alpha/beta-domain has some non-rigid body movement that is associated with the formation of the His119-Gly208 hydrogen bond. The 8 A distance between the two substrates reported for the tetragonal form indicates that it is probably not in an active conformation. However, the structural data for these two new crystal forms suggest that closing the interdomain cleft around the substrates may generate a functional active site. Molecular modeling and dynamics simulation techniques have been used to generate a hypothetical closed conformation of the enzyme. Analysis of this model suggests several residues of possible catalytic importance. The model explains observed kinetic results and satisfies requirements for efficient enzyme catalysis, most notably through the exclusion of water from the enzyme's active site.

Binding Sites↗
PubMed1998

Calf spleen purine nucleoside phosphorylase complexed with substrates and substrate analogues.

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway, which provides an alternative to the de novo pathway for the biosynthesis of purine nucleotides. PNP catalyzes the reversible phosphorolysis of 2'-deoxypurine ribonucleosides to the free bases and 2-deoxyribose 1-phosphate. Absence of PNP activity in humans is associated with specific T-cell immune suppression. Its key role in these two processes has made PNP an important drug design target. We have investigated the structural details of the PNP-catalyzed reaction by determining the structures of bovine PNP complexes with various substrates and substrate analogues. The preparation of phosphate-free crystals of PNP has allowed us to analyze several novel complexes, including the ternary complex of PNP, purine base, and ribose 1-phosphate and of the completely unbound PNP. These results provide an atomic view for the catalytic mechanism for PNP proposed by M. D. Erion et al. [(1997) Biochemistry 36, 11735-11748], in which an oxocarbenium intermediate is stabilized by phosphate and the negative charge on the purine base is stabilized by active site residues. The bovine PNP structure reveals several new details of substrate and inhibitor binding, including two phosphate-induced conformational changes involving residues 33-36 and 56-69 and a previously undetected role for His64 in phosphate binding. In addition, a well-ordered water molecule is found in the PNP active site when purine base or nucleoside is also present. In contrast to human PNP, only one phosphate binding site was observed. Although binary complexes were observed for nucleoside, purine base, or phosphate, ribose 1-phosphate binding occurs only in the presence of purine base.

Acyclovir↗
PubMed1998

Peritransplant tolerance induction with anti-CD3-immunotoxin: a matter of proinflammatory cytokine control.

BACKGROUND: Tolerance is gaining momentum as an approach to reduce lifelong immunosuppressive therapy while improving transplant longevity. Anti-CD3 immunotoxin (IT), FN18-CRM9, has potential to induce tolerance owing to its exceptional ability to deplete sessile lymph node T cells. However, if initiated at the time of transplantation, alpha-CD3-IT alone elicits a proinflammatory cytokine response, precluding establishment of tolerance. METHODS: Four groups of rhesus monkeys received kidney allografts and immunosuppression. Three groups received alpha-CD3-IT alone or alpha-CD3-IT supplemented with 15-deoxyspergualin (DSG) and/or methylprednisolone (MP). One group received alpha-CD3-monoclonal antibody with DSG and MP. Cytokines were measured by enzyme-linked immunosorbent assay. RESULTS: Supplementing peritransplant alpha-CD3-IT treatment with a brief course of DSG and MP promoted rejection-free kidney allograft acceptance in 75% of macaques followed for up to 550 days. Among those given alpha-CD3-IT alone or with MP, none were long-term survivors. Tolerance developed after alpha-CD3-IT, DSG, and MP treatment, but not when the unconjugated a-CD3 monoclonal antibody was substituted for IT. Systemic production of proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha induced after peritransplant alpha-CD3-IT was prevented only in animals given DSG. Despite high levels of interleukin (IL)-12 in the first month after transplant, tolerant recipients exhibited IL-12 resistance, as evidenced by baseline plasma levels of IFN-gamma but elevated IL-4. DSG was shown to inhibit IL-12-driven IFN-gamma production by a mechanism associated with inhibition of nuclear factor kappa-B. CONCLUSIONS: In this model, peritransplant induction of tolerance is promoted by efficient elimination of sessile lymph node T cells and control of the proinflammatory IFN-gamma response by a mechanism that appears to involve resistance to IL-12.

Animals↗
PubMed1998

Importance of the herpes simplex virus UL24 gene for productive ganglionic infection in mice.

The UL24 gene of herpes simplex virus overlaps the viral thymidine kinase (tk) gene. Most previous studies of UL24 have examined UL24 mutants that have also contained tk and sometimes other mutations. To address the importance of UL24 for viral replication in cell culture and in infections of a mammalian host, we constructed a mutant virus containing a UL24 nonsense mutation that does not affect TK activity and a second mutant that contains clustered point mutations in UL24 and a mutation in tk that does not by itself affect the ability of the virus to replicate acutely in mouse ganglia or to reactivate from latent infection following corneal inoculation of mice. Both mutant viruses replicated in cells in culture and in the mouse eye, albeit less efficiently than wild type or control viruses. Both mutants were much more severely impaired for acute replication in trigeminal ganglia and for reactivation from latency following explant of these ganglia. Viral DNA and latency-associated transcripts were present, albeit at lower levels in ganglia infected with the nonsense mutant. These results indicate that UL24 is especially important for productive infection of mouse sensory ganglia and may have implications for the behaviors of certain tk mutants in pathogenesis.

Amino Acid Sequence↗
PubMed1998

Crystal structure of the cyanobacterial metallothionein repressor SmtB: a model for metalloregulatory proteins.

SmtB from Synechococcus PCC7942 is a trans-acting dimeric repressor that is required for Zn(2+)-responsive expression of the metallothionein SmtA. The structure of SmtB was solved using multiple isomorphous replacement techniques and refined at 2.2 A resolution by simulated annealing to an R-factor of 0.218. SmtB displays the classical helix-turn-helix motif found in many DNA-binding proteins. It has an alpha + beta topology, and the arrangement of the three core helices and the beta hairpin is similar to the HNF-3/fork head, CAP and diphtheria toxin repressor proteins. Although there is no zinc in the crystal structure, analysis of a mercuric acetate derivative suggests a total of four Zn2+ binding sites in the dimer. Two of these putative sites are at the opposite ends of the dimer, while the other two are at the dimer interface and are formed by residues contributed from each monomer. The structure of the dimer is such that simultaneous binding for both recognition helices to DNA would require either a bend in the DNA helix or a conformational change in the dimer. The structure of Synechococcus SmtB is the first in this family of metal-binding DNA repressors.

Amino Acid Sequence↗
PubMed1998

The role of herpes simplex virus ICP27 in the regulation of UL24 gene expression by differential polyadenylation.

Herpes simplex virus specifies two sets of transcripts from the UL24 gene, short transcripts (e.g., 1.4 kb), processed at the UL24 poly(A) site, and long transcripts (e.g., 5.6 kb), processed at the UL26 poly(A) site. The 1.4- and 5.6-kb transcripts initiate from the same promoter but are expressed with early and late kinetics, respectively. Measurements of transcript levels following actinomycin D treatment of infected cells revealed that the 1.4- and 5.6-kb UL24 transcripts have similar stabilities, consistent with UL24 transcript kinetics being regulated by differential polyadenylation rather than by differential stabilities. Although the UL24 poly(A) site, which gives rise to short transcripts, is encountered first during processing, long transcripts processed at the UL26 site are equally or more abundant; thus, operationally, the UL24 site is weak. Using a series of viral ICP27 mutants, we investigated whether ICP27, which has been suggested to stimulate the usage of weak poly(A) sites, stimulates 1.4-kb transcript accumulation. We found that accumulation of 1.4-kb transcripts did not require ICP27 during viral infection. Rather, ICP27 was required for full expression of 5.6-kb transcripts, and the decrease in 5. 6-kb transcripts relative to 1.4-kb transcripts was not due solely to reduced DNA synthesis. Our results indicate that temporal expression of UL24 transcripts can be regulated by differential polyadenylation and that although ICP27 is not required for processing at the operationally weak UL24 poly(A) site, it does modulate 5.6-kb transcript levels at a step subsequent to transcriptional initiation.

Animals↗
PubMed1998

Human thymidine kinase can functionally replace herpes simplex virus type 1 thymidine kinase for viral replication in mouse sensory ganglia and reactivation from latency upon explant.

Herpes simplex virus type 1 thymidine kinase exhibits a strikingly broad substrate specificity. It is capable of phosphorylating deoxythymidine and deoxyuridine as does human thymidine kinase, deoxycytidine as does human deoxycytidine kinase, the cytosolic kinase whose amino acid sequence it most closely resembles, and thymidylate as does human thymidylate kinase. Following peripheral inoculation of mice, viral thymidine kinase is ordinarily required for viral replication in ganglia and for reactivation from latency following ganglionic explant. To determine which activity of the viral kinase is important for replication and reactivation in mouse ganglia, recombinant viruses lacking viral thymidine kinase but expressing individual human kinases were constructed. Each recombinant virus expressed the appropriate kinase activity with early kinetics following infection of cultured cells. The virus expressing human thymidine kinase exhibited thymidine phosphorylation activity equivalent to approximately 5% of that of wild-type virus in a quantitative plaque autoradiography assay. Nevertheless, it was competent for ganglionic replication and reactivation following corneal inoculation of mice. The virus expressing human thymidylate kinase was partially competent for these activities despite failing to express detectable thymidine kinase activity. The virus expressing human deoxycytidine kinase failed to replicate acutely in neurons or to reactivate from latency. Therefore, it appears that low levels of thymidine phosphorylation suffice to fulfill the role of the viral enzyme in ganglia and that this role can be partially fulfilled by thymidylate kinase activity alone.

Animals↗
PubMed1997

The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology.

BACKGROUND: Purine nucleoside phosphorylase (PNP) from Escherichia coli is a hexameric enzyme that catalyzes the reversible phosphorolysis of 6-amino and 6-oxopurine (2'-deoxy)ribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate. In contrast, human and bovine PNPs are trimeric and accept only 6-oxopurine nucleosides as substrates. The difference in the specificities of these two enzymes has been utilized in gene therapy treatments in which certain prodrugs are cleaved by E. coli PNP but not the human enzyme. The trimeric and hexameric PNPs show no similarity in amino acid sequence, even though they catalyze the same basic chemical reaction. Structural comparison of the active sites of mammalian and E. coli PNPs would provide an improved basis for the design of potential prodrugs that are specific for E. coli PNP. RESULTS: The crystal structure of E. coli PNP at 2.0 A resolution shows that the overall subunit topology and active-site location within the subunit are similar to those of the subunits from human PNP and E. coli uridine phosphorylase. Nevertheless, even though the overall geometry of the E. coli PNP active site is similar to human PNP, the active-site residues and subunit interactions are strikingly different. In E. coli PNP, the purine- and ribose-binding sites are generally hydrophobic, although a histidine residue from an adjacent subunit probably forms a hydrogen bond with a hydroxyl group of the sugar. The phosphate-binding site probably consists of two main-chain nitrogen atoms and three arginine residues. In addition, the active site in hexameric PNP is much more accessible than in trimeric PNP. CONCLUSIONS: The structures of human and E. coli PNP define two possible classes of nucleoside phosphorylase, and help to explain the differences in specificity and efficiency between trimeric and hexameric PNPs. This structural data may be useful in designing prodrugs that can be activated by E. coli PNP but not the human enzyme.

Binding Sites↗
PubMed1997

Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution.

Ubiquitin C-terminal hydrolases catalyze the removal of adducts from the C-terminus of ubiquitin. We have determined the crystal structure of the recombinant human Ubiquitin C-terminal Hydrolase (UCH-L3) by X-ray crystallography at 1.8 A resolution. The structure is comprised of a central antiparallel beta-sheet flanked on both sides by alpha-helices. The beta-sheet and one of the helices resemble the well-known papain-like cysteine proteases, with the greatest similarity to cathepsin B. This similarity includes the UCH-L3 active site catalytic triad of Cys95, His169 and Asp184, and the oxyanion hole residue Gln89. Papain and UCH-L3 differ, however, in strand and helix connectivity, which in the UCH-L3 structure includes a disordered 20 residue loop (residues 147-166) that is positioned over the active site and may function in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain-like enzymes, we propose a model describing the binding of ubiquitin to UCH-L3. The UCH-L3 active site cleft appears to be masked in the unliganded structure by two different segments of the enzyme (residues 9-12 and 90-94), thus implying a conformational change upon substrate binding and suggesting a mechanism to limit non-specific hydrolysis.

Amino Acid Sequence↗