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Biomedical subjects

D A Lawrence

Publications and source records attributed to D A Lawrence.

At least 37 records · Page 2Linked to original sources
PubMed2000

Partitioning of serpin-proteinase reactions between stable inhibition and substrate cleavage is regulated by the rate of serpin reactive center loop insertion into beta-sheet A.

The serpin family of serine proteinase inhibitors is a mechanistically unique class of naturally occurring proteinase inhibitors that trap target enzymes as stable covalent acyl-enzyme complexes. This mechanism appears to require both cleavage of the serpin reactive center loop (RCL) by the proteinase and a significant conformational change in the serpin structure involving rapid insertion of the RCL into the center of an existing beta-sheet, serpin beta-sheet A. The present study demonstrates that partitioning between inhibitor and substrate modes of reaction can be altered by varying either the rates of RCL insertion or deacylation using a library of serpin RCL mutants substituted in the critical P(14) hinge residue and three different proteinases. We further correlate the changes in partitioning with the actual rates of RCL insertion for several of the variants upon reaction with the different proteinases as determined by fluorescence spectroscopy of specific RCL-labeled inhibitor mutants. These data demonstrate that the serpin mechanism follows a branched pathway, and that the formation of a stable inhibited complex is dependent upon both the rate of the RCL conformational change and the rate of enzyme deacylation.

Amino Acids↗
PubMed2000

Central/peripheral nervous system and immune responses.

Maintenance of health is dependent on numerous regulatory interactions between organ systems. This review discusses interorgan communication between the nervous, endocrine, and immune systems and environmental and genetic influences on this neuroendocrine immune circuitry. Stresses of multiple types, including psychological and exposure to chemicals and infectious agents, may combine to enhance neuroimmunotoxicology. Altered nervous system functions can alter immunity which could result in exacerbation of infections, cancers or other immune-associated problems. Inversely, aberrant immune system activities could lead to pathologies associated with altered nervous activities, such as Alzheimer's disease, chronic fatigue, or multiple sclerosis. The nervous, endocrine and immune circuitry is multi-directional, and a chemical, physical or emotional stress could upset the homeostasis.

Animals↗
PubMed2000

Immunotoxic effects of inorganic lead on host resistance of mice with different circling behavior preferences.

We have observed differential immune responses in mice with different circling preferences, which are posited to reflect interindividual immune response differences influenced by brain laterality effects on neuroimmune circuits. In this study, we have investigated the influence of inorganic lead (Pb) and/or Listeria monocytogenes (LM) infection on the cytokine and corticosterone (CORT) levels of mice grouped by lateralized behavior. Pb increased the LM susceptibility of mice with both left (LC)- and right-circling (RC) preferences; however, Pb did not inhibit the host resistance of mice with no circling preference (NP mice). The basal serum IFNgamma levels were lowered in all groups after Pb exposure, which coincided with a decrease in host resistance in LC and RC mice, but not NP mice. Pb also altered the basal serum CORT levels, and these changes appear to correlate better with changes in the host resistance of all groups. The basal CORT levels were significantly lowered by Pb in mice with a circling preference, and Pb significantly suppressed the host resistance of mice with a circling preference. However, Pb slightly increased the serum CORT level of NP mice, and their host resistance was slightly improved by Pb. After infection, the increase in CORT levels was associated with an increase in the serum IL-6 levels, which may reflect cytokine influences on the hypothalamic-pituitary-adrenal axis. At 3 days after infection, the serum IL-6 level seems to be a good indicator of the severity of the infection. We suggest that environmental stressors can reorder the observed differential susceptibility to LM in mice with different circling preferences, in that relatively resistant mice (RC mice) become more susceptible than NP mice after exposure to Pb. The results suggest that environmental stressors may have differential effects among individuals with endogenous differences in their neuroimmune circuits, since brain laterality is known to influence immune functions.

Animals↗
PubMed2000

Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5.

Fas (APO-1/CD95) and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis by recruiting the apoptosis initiator caspase-8 through the adaptor FADD. Fas binds FADD directly, whereas TNFR1 binds FADD indirectly, through TRADD. TRADD alternatively recruits the NF-kappaB-inducing adaptor RIP. The TNF homolog Apo2L/TRAIL triggers apoptosis through two distinct death receptors, DR4 and DR5; however, receptor over-expression studies have yielded conflicting results on the ligand's signaling mechanism. Apo2L/TRAIL induced homomeric and heteromeric complexes of DR4 and DR5 and stimulated recruitment of FADD and caspase-8 and caspase-8 activation in nontransfected cells. TRADD and RIP, which bound TNFR1, did not bind DR4 and DR5. Thus, Apo2L/TRAIL and FasL initiate apoptosis through similar mechanisms, and FADD may be a universal adaptor for death receptors.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Neuronal cell death and reactive oxygen species.

1. We have investigated the role of reactive oxygen species (ROS) in cell death induced by ischemia or application of the excitatory amino acid agonist, N-methyl-D-aspartate (NMDA) or kainate (KA), in acutely isolated rat cerebellar granule cell neurons, studied by flow cytometry. Various fluorescent dyes were used to monitor intracellular calcium concentration, ROS concentration, membrane potential, and viability in acutely dissociated neurons subjected to ischemia and reoxygenation alone, NMDA or kainate alone, and ischemia and reoxygenation plus NMDA or kainate. 2. With ischemia followed by reoxygenation, ROS concentrations rose slightly and there was only a modest increase in cell death after 60 min. 3. When NMDA or kainate alone was applied to the cells there was a large increase in ROS and in intracellular calcium concentration but only a small loss of cellular viability. However, when NMDA or kainate was applied during the reoxygenation period there was a large loss of viability, accompanied by membrane depolarization, but the elevations of ROS and intracellular calcium concentration were not greater than seen with the excitatory amino acids alone. 4. These observations indicate that other factors beyond ROS and intracellular calcium concentration contribute to cell death in cerebellar granule cell neurons.

Animals↗
PubMed2000

Neuroserpin mutation S52R causes neuroserpin accumulation in neurons and is associated with progressive myoclonus epilepsy.

Mutations in the Neuroserpin gene have been reported to cause familial presenile dementia. We describe a new family in which the S52R Neuroserpin mutation is associated with progressive myoclonus epilepsy in 2 siblings. The proband presented myoclonus and epilepsy at age 24, his brother and mother presented a similar disorder when they were 25. A clinical diagnosis of progressive myoclonus epilepsy was made on the proband and his brother. Skin and liver biopsies did not reveal the presence of cytological alterations in the proband. His neurological status worsened over the subsequent 19 yr during which he became demented and had uncontrollable seizures. He died at 43 yr of age from aspiration pneumonia. Neuropathologically, eosinophilic bodies, which were positive for periodic acid-Schiff and immunoreactive with antibodies against human neuroserpin, were present in the perikarya and cell processes of the neurons. They were found in large numbers in the cerebral cortex and substantia nigra and to a lesser extent, in most subcortical gray areas, spinal cord, and dorsal root ganglia. By electron microscopy, the intracytoplasmic bodies were contained within the membranes of the rough endoplasmic reticulum. Occasionally neuroserpin immunopositivity was seen throughout the cytoplasm, even without the presence of well-defined bodies. Our study characterizes for the first time the neuropathologic phenotype associated with hereditary progressive myoclonus epilepsy caused by the S52R Neuroserpin mutation.

Adult↗
PubMed2000

The efficiency of maternal transfer of lead and its influence on plasma IgE and splenic cellularity of mice.

Exposure to the well known environmental toxicant lead is typically assessed by blood and/or bone levels and has been implicated in the onset of a variety of diseases affecting multiple human systems. However, there are conflicting data regarding the efficiency of in utero versus lactational transfer of lead to offspring, and the immunomodulatory effects of lead in early life have not been well defined. Pregnant BALB/c mice were exposed to lead acetate in their drinking water beginning at approximately day 15 of gestation, and cross-fostering of exposed/nonexposed litters was performed at parturition. Significant increases of blood lead levels of all exposed offspring were found at 1 week of age with evidence for both transplacental and lactational transfer. Additionally, mice exposed to lead continuously beginning at approximately 6 days prior to birth showed significant decreases in their blood lead levels 2 weeks after weaning, despite continued exposure as adults. This result suggests maternal transfer of lead is more efficient than oral adult exposure and that substantial lead transfer occurs both transplacentally and lactationally. The incidence of childhood atopic responses including asthma has risen considerably in recent years, particularly within areas containing higher levels of environmental pollutants. Plasma IgE levels of 2-week-old neonates exposed to lead before and/or after birth were measured as an index of atopy. Neonates exposed to lead transplacentally and/or lactationally had significantly higher plasma IgE levels, a biomarker of atopy, and lower splenic white blood cell numbers than age-matched controls. These results resemble the lag in immunocompetency and increase in serum IgE noted in atopic children and suggest a role for environmental toxicants and non-allergen-specific immunology in the prevalence of atopy and asthma in children.

Aging↗
PubMed1999

Familial dementia caused by polymerization of mutant neuroserpin.

Aberrant protein processing with tissue deposition is associated with many common neurodegenerative disorders; however, the complex interplay of genetic and environmental factors has made it difficult to decipher the sequence of events linking protein aggregation with clinical disease. Substantial progress has been made toward understanding the pathophysiology of prototypical conformational diseases and protein polymerization in the superfamily of serine proteinase inhibitors (serpins). Here we describe a new disease, familial encephalopathy with neuroserpin inclusion bodies, characterized clinically as an autosomal dominantly inherited dementia, histologically by unique neuronal inclusion bodies and biochemically by polymers of the neuron-specific serpin, neuroserpin. We report the cosegregation of point mutations in the neuroserpin gene (PI12) with the disease in two families. The significance of one mutation, S49P, is evident from its homology to a previously described serpin mutations, whereas that of the other, S52R, is predicted by modelling of the serpin template. Our findings provide a molecular mechanism for a familial dementia and imply that inhibitors of protein polymerization may be effective therapies for this disorder and perhaps for other more common neurodegenerative diseases.

Biopolymers↗
PubMed1999

The active conformation of plasminogen activator inhibitor 1, a target for drugs to control fibrinolysis and cell adhesion.

BACKGROUND: Plasminogen activator inhibitor 1 (PAI-1) is a serpin that has a key role in the control of fibrinolysis through proteinase inhibition. PAI-1 also has a role in regulating cell adhesion processes relevant to tissue remodeling and metastasis; this role is mediated by its binding to the adhesive glycoprotein vitronectin rather than by proteinase inhibition. Active PAI-1 is metastable and spontaneously transforms to an inactive latent conformation. Previous attempts to crystallize the active conformation of PAI-1 have failed. RESULTS: The crystal structure of a stable quadruple mutant of PAI-1(Asn150-->His, Lys154-->Thr, Gln319-->Leu, Met354-->Ile) in its active conformation has been solved at a nominal 3 A resolution. In two of four independent molecules within the crystal, the flexible reactive center loop is unconstrained by crystal-packing contacts and is disordered. In the other two molecules, the reactive center loop forms intimate loop-sheet interactions with neighboring molecules, generating an infinite chain within the crystal. The overall conformation resembles that seen for other active inhibitory serpins. CONCLUSIONS: The structure clarifies the molecular basis of the stabilizing mutations and the reduced affinity of PAI-1, on cleavage or in the latent form, for vitronectin. The infinite chain of linked molecules also suggests a new mechanism for the serpin polymerization associated with certain diseases. The results support the concept that the reactive center loop of an active serpin is flexible and has no defined conformation in the absence of intermolecular contacts. The determination of the structure of the active form constitutes an essential step for the rational design of PAI-1 inhibitors.

Binding Sites↗
PubMed1999

Growth stimulation of murine fibroblasts by TGF-beta1 depends on the expression of a functional p53 protein.

Transforming Growth Factor-beta1 (TGF-beta1) inhibits the proliferation of most cells, but stimulates some mesenchymal cell types, including murine NIH3T3 fibroblasts. We show here that TGF-beta1 growth stimulation of NIH3T3 fibroblasts is reversed when these cells are transformed by SV40 or are transfected with a plasmid encoding the SV40 Large T antigen. Inversion of the TGF-beta1 growth stimulation of NIH3T3 cells is not observed when these cells are transfected with plasmids expressing either a mutant Large T, unable to bind P53, or the E1A adenovirus oncoprotein which binds the retinoblastoma protein pRB but not P53. But when the TGF-beta1-growth stimulated cells are transfected with a plasmid expressing a mutant form of Large T capable of binding to P53, but not to pRB, or with one expressing the E1B-55 kD adenovirus oncoprotein, which also binds to P53 but not to pRB, the cells are growth-inhibited by TGF-beta1. The cdk inhibitor p21Waf is decreased in TGF-beta1-stimulated NIH3T3 fibroblasts and increased in TGF-beta1-inhibited SV40-transformed cells. Finally, we show that T12 fibroblasts, from a P53 knockout mouse, are growth inhibited by TGF-beta1 and that they remain so upon transfection with a P53 which is mutant at restrictive temperature, but become growth-stimulated by this factor at permissive temperature when P53 is functional. These data strongly suggest that growth-stimulation of fibroblasts by TGF-beta1 depends on the presence of a functional P53 protein and that inversion of this response occurs if P53 is absent or inactivated.

3T3 Cells↗
PubMed1999

Differential immune responses in mice with left- and right-turning preference.

Humoral and cell-mediated immune responses of inbred BALB/c male mice were assayed for differential reactivities associated with behavioral sidedness, which was evaluated by spontaneous rotational behavior in a circular cage model system. Mice with left-turning preference had lower in vivo primary IgM and IgG anti-Keyhole Limpet Hemocyanin (KLH) antibody responses, delayed-type hypersensitivity (DTH) responses, and host-resistance against the intracellular bacteria, Listeria monocytogenes, than mice with right-turning preference. The only immune parameter not shown to be associated with turning preference was the secondary humoral immune response to KLH. The weak innate immune response of left-turners for clearance of Listeria showed close intercorrelation with elevated serum IL-6 levels. Serum corticosterone and splenic norepinephrine levels were differentially increased and decreased by infection, respectively. We suggest that the observed differential immune reactivities of individual animals with same age, gender, and genetic background are associated with functional asymmetries within the brain, that the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic innervation are involved in the regulatory brain: immune interconnection after infection, and that the HPA axis and sympathetic nervous system are involved in the brain laterality effects on immune responses.

Adjuvants, Immunologic↗
PubMed1999

Evidence for control of nitric oxide synthesis by intracellular transforming growth factor-beta1 in tumor cells. Implications for tumor development.

Transforming growth factor-beta1 (TGF-beta1) has been shown to down-regulate NO synthesis in a variety of normal cells. In the present study, we investigated the influence of TGF-beta1 upon NO production in tumor cells and its consequences for tumor development. During the growth of PROb colon carcinoma cells intraperitoneally injected in syngeneic BDIX rats, intratumoral concentration of TGF-beta1 increases while NO concentration stays very low. Tumor regression induced by intraperitoneal injections of a lipid A is associated with a decrease in TGF-beta1 and an increase in NO intratumoral concentration. In these tumors, PROb tumor cells are the NO- and TGF-beta1-secreting cells. Using PROb cells transfected with an expression vector coding for TGF-beta1 antisense mRNA, we demonstrate in vitro that there is an inverse correlation between the amount of TGF-beta1 secreted and the ability of PROb cells to secrete NO. As the same results were obtained in the presence of an anti-TGF-beta type II receptor neutralizing antibody, and as exogenous TGF-beta1 is without any effect on NO secretion by PROb cells, TGF-beta1 apparently down-regulates NO synthesis in PROb cells by an intracellular mechanism. These results suggest that endogenous TGF-beta1 constitutes a potential target in a search for new antitumoral agents.

Activin Receptors, Type I↗
PubMed1999

Familial encephalopathy with neuroserpin inclusion bodies.

We report on a new familial neurodegenerative disease with associated dementia that has presented clinically in the fifth decade, in both genders, and in each of several generations of a large family from New York State-a pattern of inheritance consistent with an autosomal dominant mode of transmission. A key pathological finding is the presence of neuronal inclusion bodies distributed throughout the gray matter of the cerebral cortex and in certain subcortical nuclei. These inclusions are distinct from any described previously and henceforth are identified as Collins bodies. The Collins bodies can be isolated by simple biochemical procedures and have a surprisingly simple composition; neuroserpin (a serine protease inhibitor) is their predominant component. An affinity-purified antibody against neuroserpin specifically labels the Collins bodies, confirming their chemical composition. Therefore, we propose a new disease entity-familial encephalopathy with neuroserpin inclusion bodies (FENIB). The conclusion that FENIB is a previously unrecognized neurodegenerative disease is supported by finding Collins bodies in a small kindred from Oregon with familial dementia who are unrelated to the New York family. The autosomal dominant inheritance strongly suggests that FENIB is caused by mutations in the neuroserpin gene, resulting in intracellular accumulation of the mutant protein.

Amino Acid Sequence↗
PubMed1999

Safety and antitumor activity of recombinant soluble Apo2 ligand.

TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.

Animals↗
PubMed1999

Altered serum cytokine and immunoglobulin levels in the workers exposed to antimony.

1 Antimony (Sb), an industrial and environmental toxicant, is known to cause dermatitis and pulmonary inflammations, but the immunomodulatory effects of environmental or occupational exposure to Sb have not been reported. To initiate investigation of Sb-induced alterations of the immune system, the concentrations of the IgG subclasses, IgE, interleukin-2, interferon-gamma, and interleukin-4 in sera obtained from workers exposed to Sb through antimony trioxide manufacture were determined and compared with those of control subjects. 2 The serum levels of IgG1, an immunoglobulin involved in host defense against many microbial infections, were significantly lower in the sera of Sb-exposed individuals than the controls. The serum concentrations of IgE, an immunoglobulin mediating allergic hypersensitivity, also were lower in the Sb-exposed workers than the controls. In addition, the levels of interleukin-2 and interferon-gamma, multifunctional cytokines for T-cell mediated immunity, were lowered in the factory workers. 3 A significant positive correlation between IgG4 and urine Sb levels was found among the Sb-exposed workers, indicating a possible role of IgG4 in Sb-mediated pulmonary or skin pathogenesis. 4 The present study provides the first report on immune alterations induced by occupational exposure to Sb and suggests that Sb exposure disturbs immunohomeostasis in humans observed as a function of aberrant serum cytokine and immunoglobulin levels, which could influence health.

Adult↗
PubMed1999

New Zealand mixed mice: a genetic systemic lupus erythematosus model for assessing environmental effects.

Systemic lupus erythematosus (SLE) is a multiphenotypic autoimmune disease. The hallmark of SLE is the production of anti-double-stranded DNA autoantibodies and the deposition of immune complexes in target tissues such as the kidney, skin, and brain. Additional phenotypic traits are the presence of arthritis, anemia, central nervous system involvement, and a variety of autoantibodies. Females of childbearing age are particularly at risk. Recent genetic analysis of murine SLE shows that susceptibility is under complex polygenic control. It is also apparent that environmental factors contribute to the induction and exacerbation of SLE. We describe here the genotypic and phenotypic characterization of a group of recombinant inbred strains of SLE-prone mice that were derived from NZB and NZW progenitors, the parental strains of the classic female F1 hybrid lupus model. Recombination and reassortment of these ancestral genomes resulted in the NZM (New Zealand mixed) strains with strain-specific patterns of renal disease penetrance and other autoimmune traits such as Coombs positive anemia and neurologic deficits. Multiple susceptibility loci of the ancestral strains demonstrate that SLE is inherited as a threshold trait. Because some of these loci co-localize with the susceptibility loci of the insulin-dependent diabetes of nonobese diabetic strain, it is apparent that there are disease-specific as well as autoimmunity-promoting genes. It is proposed that the NZM strains, particularly those with reduced disease penetrance or partial genotypes, provide an improved genetic model for assessment of the effects of environmental agents on SLE and autoimmunity.

Animals↗
PubMed1999

Altered complex formation between p21waf, p27kip and their partner G1 cyclins determines the stimulatory or inhibitory transforming growth factor-beta1 growth response of human fibroblasts.

TGF-beta1 stimulates proliferation of WI38 human embryo fibroblasts but inhibits that of their SV40-transformed counterparts, VA13 cells. Protein expression levels of cyclins A, D1, E and that of cdk2 and cdk4 were not affected by TGF-beta1 in either of these cells. However, TGF-beta1-treatment increases cdk2 kinase activity in WI38 cells and reduces it in VA13 cells. The same treatment reduces the amount of p21waf present in complexes with cyclins D1 and E in growth-stimulated WI38 cells, but the reverse applies in growth-inhibited VA13 cells. Mitogenic stimulation of WI38 fibroblasts correlated with decreased expression of p27kip protein and reduced amounts of it in complex with cyclin E. In contrast, proliferative inhibition of VA13 fibroblasts by TGF-beta1 caused a reduction of p27kip in complexes with cyclin D1, but increased it in complexes with cyclin E, without affecting the overall level of p27kip protein expression. Thus, in this human fibroblast model, TGF-beta1-mediated stimulation or inhibition of proliferation depends on modulation in the amounts of p21waf and p27kip in complexes with cyclins D1 and E.

CDC2-CDC28 Kinases↗