Thermostatistics of a single particle on a granular dimer lattice: influence of defects.

We study the thermostatistical fluctuations of a single Delrin monomer on a granular lattice of dimer particles using both experiment and simulation. The goal is to examine the collision frequency, energy injection, and sidewall effects on a single second-layer particle in a bilayer granular gas experiment. Non-Gaussian velocity statistics are observed for the single particle of the top layer and result from the presence of defects in the first layer. These deviations are not directly due to the presence of the boundary wall, since the form of velocity distributions is quite spatially homogeneous, but are the consequence of the presence of a few mobile defects in the first layer.

Caspase cleavage of the APC tumor suppressor and release of an amino-terminal domain is required for the transcription-independent function of APC in apoptosis.

The adenomatous polyposis coli (APC) tumor suppressor is inactivated by mutation in most colorectal tumors. APC is a component of the Wnt signaling pathway and is best known for its ability to downregulate beta-catenin and consequent effects on transcriptional regulation. Previous work demonstrated that APC accelerates apoptosis-associated caspase activity independently of transcription, and suggested novel tumor suppressor functions of APC. In this work, we have mapped the APC apoptosis-accelerating region to amino acids (aa) 1-760 by testing a series of non-overlapping APC segments. Interestingly, this segment corresponds to a stable group II caspase cleavage product of APC released during apoptosis that includes the amino-terminal aa1-777. Mutation of the APC aspartic acid residue at position 777 to an alanine completely abolished in vitro cleavage of APC by a recombinant group II caspase and rendered the full-length protein unable to accelerate apoptosis in vitro. A truncated APC protein associated with familial and sporadic colorectal cancer, also unable to accelerate apoptosis in vitro and in vivo, is resistant to group II caspase cleavage. These results demonstrate that cleavage of APC and the subsequent release of an amino-terminal segment are necessary for the transcription-independent mechanism of APC-mediated apoptosis.

The cAMP responsive element and CREB partially mediate the response of the tyrosine hydroxylase gene to phorbol ester.

Tyrosine hydroxylase (TH) gene promoter activity is increased in PC12 cells that are treated with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). Mutagenesis of either the cAMP responsive element (CRE) or the activator protein-1 element (AP1) within the TH gene proximal promoter leads to a dramatic inhibition of the TPA response. The TH CRE and TH AP1 sites are also independently responsive to TPA in minimal promoter constructs. TPA treatment results in phosphorylation of cAMP responsive element binding protein (CREB) and activation of cAMP-dependent protein kinase (PKA) in PC12 cells; hence, we tested whether CREB and/or PKA are essential for the TPA response. In CREB-deficient cells, the response of the full TH gene proximal promoter or the independent response of the TH CRE by itself to TPA is inhibited. The TPA-inducibility of TH mRNA is also blocked in CREB-deficient cells. Expression of the PKA inhibitor protein, PKI, also inhibits the independent response of the TH CRE to TPA. Our results support the hypothesis that TPA stimulates the TH gene promoter via signaling pathways that activate either the TH AP1 or TH CRE sites. Both signaling pathways are dependent on CREB and the TH CRE-mediated pathway is dependent on PKA.

Evaluation of changes in trabecular bone architecture and mechanical properties of minipig vertebrae by three-dimensional magnetic resonance microimaging and finite element modeling.

The study objective was to analyze the three-dimensional (3D) trabecular architecture and mechanical properties in vertebral specimens of young and mature Sinclair minipigs to assess the relative contribution of architecture to bone strength. We used 3D magnetic resonance microimaging (MRmicroI) and direct image analysis to evaluate a set of standard structural measurements and new architectural descriptors of trabecular bone in biopsy specimens from L2, L3, and L4 vertebrae (n = 16 in each group) from young (mean age, 1.2 years) and mature (mean age, 4.8 years) minipigs. The measurements included bone volume/tissue volume (BV/TV), marrow star volume (Ma.St.V), connectivity density (ConnD), and two new parameters, percent platelike trabeculae (% plate) and percent bone in the load direction (% boneLD). The % plate, calculated from surface curvature, allowed the delineation of plates from rods. The % boneLD quantified the percentage of bone oriented along the long axis of the vertebral body. We showed that 3D MRmicroI can detect the subtle changes in trabecular architecture between the two age groups. ConnD, star volume, % plate, % boneLD, and BV/TV were found to be more effective than the model-based, derived indices (trabecular thickness [Tb.Th], trabecular separation [Tb.Sp], and trabecular number [Tb.N]) in differentiating the structural changes. BV/TV, % plate, and % boneLD significantly increased (p < 0.05) in all three vertebral sites of the mature minipigs. The significant decrease in ConnD and star volume in the mature vertebra was consistent with the concurrent increase of platelike trabecular bone (p < 0.05). Overall, ConnD, star volume, % plate, and % boneLD provided a coherent picture of the architectural changes between the two age groups. Apparent modulus and maximum stress were determined experimentally on biopsy specimens from L2 vertebrae (n = 16). When apparent modulus was predicted using 3D MRmicroI data sets as input for finite element modeling (FEM), the results were similar to the experimentally determined apparent modulus (p = 0.12). Both methods were then used to compare the young and the mature animals; the experimental and predicted apparent modulus were significantly higher for the mature group (p = 0.003 and 0.012, respectively). The experimental maximum stress in the vertebra of the mature animals was twice as high as that for the young animals (p = 0.006). Bone quantity (BV/TV or bone mineral content [BMC]) alone could explain approximately 74-85% of the total variability in stress and modulus. The inclusion of either ConnD or % boneLD with BV/TV in a multiple regression analysis significantly improved the predictability of maximum stress, indicating that architecture makes additional contributions to compressive strength in normal minipig vertebra.

Bone architecture and image synthesis.

3-D bone architecture can now be measured by micro-computer tomography or micro-magnetic resonance imaging. The principles of the micro-computer technique is reviewed and new architectural parameters can be computed. In addition, the method allows the contruction of polymer models by stereolithography, a method that can be used to perform repetitive mechanical studies on the same bone sample. These non destructive methods are interesting in the pre-clinical studies on bone diseases and in the investigation of animal trials on new pharmacological compounds active on bone.

The levels of leukemia inhibitory factor mRNA in a Schwann cell line are regulated by multiple second messenger pathways.

Axotomy of sympathetic and sensory neurons leads to changes in their neuropeptide phenotypes. These changes are mediated in part by the induction of leukemia inhibitory factor (LIF) by nonneuronal cells. In the present study, we identified satellite/Schwann cells as a possible source of the injury-induced LIF. Using a Schwann cell line, SC-1 cells, we examined mechanisms of LIF induction. LIF mRNA levels increased rapidly when the cells were treated with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, phorbol 12-myristate 13-acetate (PMA), or A23187. Among these reagents, PMA was the most efficacious. Inhibition of protein kinase C (PKC) by GF-1 09203X significantly reduced the PMA-induced LIF mRNA levels. As PKC is known to activate the extracellular signal-regulated kinase (ERK) signaling pathway, the involvement of this pathway in the PMA-stimulated induction of LIF mRNA was examined. Phosphorylation of ERKs was increased following PMA treatment in SC-1 cells. Moreover, inhibition of ERK kinase activity by PD98059 dramatically reduced PMA-stimulated phosphorylation of ERKs and induction of LIF mRNA. These results indicate that LIF mRNA levels can be regulated by ERK activation via stimulation of PKC in Schwann cells.

Long-term effects of amphetamine neurotoxicity on tyrosine hydroxylase mRNA and protein in aged rats.

Four injections (intraperitoneal) of 3 mg/kg amphetamine (2 hr apart) produced pronounced hyperthermia and sustained decreases in dopamine levels and tyrosine hydroxylase (TH) protein levels in the striatum of 15-month-old male rats. A partial recovery of striatal dopamine levels was observed at 4 months after amphetamine. In contrast, TH mRNA and TH protein levels in the midbrain were unaffected at all time points tested up to 4 months after amphetamine treatment. The number of TH-immunopositive cells in the midbrain was also unchanged at 4 months after amphetamine, even though the number of TH-positive axons in the striatum remained dramatically decreased at this time point. Interestingly, TH-immunopositive cell bodies were observed 4 months after amphetamine in the lateral caudate/putamen, defined anteriorly by the genu of the corpus collosum and posteriorly by the junction of the anterior commissures; these striatal TH-positive cells were not observed in saline- or amphetamine-treated rats that did not become hyperthermic. In addition, low levels (orders of magnitude lower than that present in the midbrain) of TH mRNA were detected using reverse transcription-polymerase chain reaction in the striatum of these amphetamine-treated rats. Our results suggest that even though there is a partial recovery of striatal dopamine levels, which occurs within 4 months after amphetamine treatment, this recovery is not associated with increased TH gene expression in the midbrain. Furthermore, new TH-positive cells are generated in the striatum at this 4-month time point.

Tyrosine hydroxylase gene promoter activity is regulated by both cyclic AMP-responsive element and AP1 sites following calcium influx. Evidence for cyclic amp-responsive element binding protein-independent regulation.

Membrane depolarization of PC12 cells using 50 mM KCl leads to induction of tyrosine hydroxylase (TH) mRNA. This induction of TH mRNA is apparently due to increased TH gene promoter activity mediated by the influx of Ca2+. In PC12 cells transiently transfected with a chimeric gene expressing chloramphenicol acetyltransferase (CAT) driven by the proximal TH gene 5'-flanking region, 50 mM KCl increases TH gene promoter activity 3-4-fold. Promoter analysis utilizing TH-CAT constructs containing mutagenized sequences indicates that this response to the depolarization-mediated influx of Ca2+ is primarily dependent on both the TH cAMP-responsive element (CRE) and TH activating protein-1 (AP1) site. Minimal promoter constructs that contain a single copy of either the TH CRE or TH AP1 site fused upstream of the TH gene basal promoter are only modestly responsive or nonresponsive, respectively, to depolarization. However, both these constructs are strongly responsive to the calcium ionophore, A23187. Gel shift assays indicate that TH AP1 complex formation is dramatically increased after treatment with either 50 mM KCl or A23187. Using antibodies to transcription factors of the Fos and Jun families, we show that the nuclear proteins comprising the inducible TH AP1 complex include c-Fos, c-Jun, JunB, and JunD. In cAMP-responsive element binding protein (CREB)-deficient cell lines that express antisense RNA complementary to CREB mRNA, the response of the TH gene promoter to cyclic AMP is dramatically inhibited, but the response to A23187 remains robust. This result indicates that transcription factors other than CREB can participate in the Ca2+-mediated regulation of the TH gene. In summary, our results support the hypothesis that regulation of the TH gene by Ca2+ is mediated by mechanisms involving both the TH CRE and TH AP1 sites and that transcription factors other than or in addition to CREB participate in this response.

The role of exposed tryptophan residues in the activity of the cardiotonic polypeptide anthopleurin B.

Scorpion and sea anemone venoms contain several polypeptides that delay inactivation of voltage-sensitive sodium channels via interaction with a common site. In this report, we target exposed hydrophobic residues at positions 33 and 45 of anthopleurin B (ApB) by polymerase chain reaction mutagenesis to ascertain their contribution to toxin activity. Nonconservative replacements are not permitted at position 33, indicating that Trp-33 may play an important structural role. Strikingly, the relatively conservative substitution of Trp-33 by phenylalanine results in major reductions in binding affinity for both the cardiac and neuronal channel isoforms as measured by ion flux, whereas substitution with tyrosine is tolerated and exhibits near wild-type affinities, suggesting that either the ability to form a hydrogen bond or the amphiphilic nature of the side chain are important at this position. Electrophysiological analysis of W33F indicates that its diminished affinity is primarily due to a decreased association rate. Analysis of a panel of mutants at Trp-45 shows only modest changes in apparent binding affinity for both channel isoforms but significant effects on Vmax. In neuronal channels, the maximal levels of uptake for W45A/S/F are about 50% those seen with ApB. This effect is also observed for W45A and W45S in the cardiac model, wherein W45F is normal. These results suggest that a hydrophobic contact is involved in toxin-induced stabilization of the open conformation of the cardiac sodium channel. We conclude that Trp-33 contributes significantly to apparent affinity, whereas Trp-45 does not appear to affect binding per se. Furthermore, W33F is the first ApB mutant that displays a significantly altered association rate and may prove to be a useful probe of the channel binding site.

The ferret as a small animal model with BMU-based remodeling for skeletal research.

UNLABELLED: The need exists for a small animal model with bone metabolic unit (BMU)-based remodeling to mimic the bone loss found in postmenopausal women. The purpose of this investigation was to evaluate the ferret as a potential model for skeletal research. Specifically, we determined whether the ferret: 1) exhibits evidence of BMU-remodeling, 2) has a skeletal response to parathyroid hormone (PTH) similar to other remodeling species, and 3) loses bone in response to reduced estrogen levels. METHODS: Using three sets of experiments, we determined the response of female ferrets to ovariectomy/light cycle manipulation or to administration of PTH. Scanning electron microscopy, light microscopy, determination of estrogen levels and/or single-photon absorptiometry (SPA) were used for evaluation. RESULTS: The ferret was found to exhibit BMU-based remodeling, and may therefore provide a small animal remodeling species for skeletal research. Ferrets reach skeletal maturity between four and seven months of age as evidenced by closure of the growth plate and maturation of trabecluae from thin rods to thick rods and plates. PTH treatment resulted in a marked increase in bone mass accompanied by the PTH-induced tunneling phenomenon known to occur in dogs and humans but not rats. The response to PTH supports the use of the ferret in studies of bone anabolic agents. Bone mass in the proximal tibia was significantly reduced when estrogen depletion was induced by either bilateral ovariectomy or short light/dark cycles (8 hour light, 16 hour dark). Maintenance of intact ferrets under short-light conditions mimiced ovariectomy in terms of serum estrogen levels, uterine weights, and tibial BMD.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional progress toward managed care.

Managed care's continual rise will impact each Virginia area market uniquely. Although almost all physicians will be affected in some way by managed care, the level and type of impact will vary greatly depending on the physician's geographic location, specialty, practice type, practice size, hospital affiliations and existing managed care affiliations. Staying abreast of the constant changes in the public and private sector will become increasingly crucial for all of Virginia's physicians as management of resources and care plays a larger role in the health care delivery system.

Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia.

The microsomal triglyceride transfer protein (MTP), which catalyses the transport of triglyceride, cholesteryl ester and phospholipid between phospholipid surfaces, is a heterodimer composed of the multifunctional protein, protein disulphide isomerase, and a unique large subunit with an apparent M(r) of 88K (refs 1-3). It is isolated as a soluble protein from the lumen of the microsomal fraction of liver and intestine. The large subunit of MTP was not detectable in four unrelated subjects with abetalipoproteinaemia, a rare autosomal recessive disease characterized by a defect in the assembly or secretion of plasma lipoproteins that contain apolipoprotein B (ref. 6). We report here the isolation and sequencing of complementary DNA encoding the large subunit of MTP. A comparison of this sequence to corresponding genomic sequences from two abetalipoproteinaemic subjects revealed a homozygous frameshift mutation in one subject and a homozygous nonsense mutation in the other. The results indicate that a defect in the gene for the large subunit of MTP is the proximal cause of abetalipoproteinaemia in these two subjects, and that MTP is required for the secretion of plasma lipoproteins that contain apolipoprotein B.

Effects of probucol on plasma lipids, lipoproteins and parameters of high density lipoprotein metabolism.

Eight patients with primary hypercholesterolemia were treated with probucol for 17 weeks. Plasma total cholesterol, low density lipoprotein (LDL)-cholesterol, and high density lipoprotein (HDL)-cholesterol decreased by 16.6, 15.0 and 25.7%, respectively, in response to probucol treatment. Plasma levels of apolipoprotein B and apolipoprotein A-I also decreased, while apolipoprotein A-II concentrations were unchanged. The decrease in HDL-cholesterol levels was associated with a reduction in HDL particle size. No changes in the plasma lecithin:cholesterol acyltransferase activity or mass occurred in response to probucol treatment. In contrast, a significant 25% increase in plasma cholesteryl ester and triglyceride transfer activity occurred following probucol treatment. There was a positive correlation (R = 0.94) between cholesterol ester and triglyceride transfer. We propose that the increase in lipid transfer activity may in part explain the changes in HDL concentration and size, as well as the previously reported effect probucol has on reducing atherosclerosis in animal models.

Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal triglyceride transfer protein.

Protein disulfide isomerase (PDI) is a component of the microsomal triglyceride transfer protein (MTP) complex. This study was initiated to help elucidate the role of PDI in MTP. The 88-kDa polypeptide of MTP (88K) was dissociated from PDI by using chaotropic agents (NaClO4 and KSCN), low concentrations of a denaturant (guanidine hydrochloride) or a nondenaturing detergent (octyl glucoside). As assessed by fluorescence and circular dichroism spectroscopy, these three different approaches appeared to dissociate the components of MTP under mild, nondenaturing conditions. The dissociating agents were diluted or removed by dialysis, and the free PDI and 88K were further characterized. In all cases, the dissociation coincided with the loss of triglyceride transfer activity. The free 88-kDa polypeptide readily aggregated, suggesting that it is a hydrophobic peptide. Even in the presence of chaotropic agents, when 88K was not aggregated, transfer activity was not expressed. These results suggest that the association of PDI with 88K is necessary to maintain the catalytically active form of the triglyceride transfer protein and prevent the aggregation of 88K.

Response to treatment with the leukocyte-derived immunomodulator IMREG-1 in immunocompromised patients with AIDS-related complex. A multicenter, double-blind, placebo-controlled trial.

OBJECTIVE: To determine if a 6-month course of therapy with IMREG-1, a leukocyte-derived immunomodulator, slows disease progression in patients with AIDS-related complex. DESIGN: Randomized, double-blind trial. SETTING: Five academic- and three community-based clinics. PATIENTS: Immunocompromised patients (143) with HIV. INTERVENTIONS: IMREG-1 or placebo every 2 weeks (13 doses). MAIN RESULTS: Twelve of forty-eight patients on placebo and 5 of 95 patients on IMREG-1 experienced adverse events (AIDS-defining opportunistic infection or neoplasm, wasting syndrome, HIV-associated encephalopathy, or peripheral sensory neuropathy). Based on an intention-to-treat analysis, Kaplan-Meier event probabilities were 26% for the placebo group and 6% for the IMREG-1 group (P less than 0.001); based on the Cox proportional hazards model, the relative risk for patients on placebo compared with patients on IMREG-1 was 5.1 (95% CI, 1.8 to 14.8). The frequency of symptoms significantly increased from baseline in patients receiving placebo. The mean decrease in CD4+ cells from baseline was 80 x 10(6) cells/L in the placebo group and 29 x 10(6) cells/L in patients on IMREG-1, with 20% (8) and 38% (32) of patients, respectively, showing a trend toward an increase (P = 0.04). In patients receiving IMREG-1, the size and rate of delayed hypersensitivity responses were larger than in the placebo group. CONCLUSIONS: Patients with AIDS-related complex experienced fewer adverse events and constitutional symptoms after IMREG-1 treatment. The slower loss of CD4+ cells and increased size and rate of delayed hypersensitivity responses most likely reflect the effect of IMREG-1 on the immune system. No toxicity related to IMREG-1 administration was observed.

Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex.

A bovine liver protein which catalyzes the transfer of triglyceride between membranes has previously been isolated from the lumen of the microsomal fraction. When further purified about 100-fold, two polypeptides of molecular mass 58,000 and 88,000 were identified (Wetterau, J. R., and Zilversmit, D. B. (1985) Chem. Phys. Lipids 38, 205-222). We demonstrate here that the two polypeptides (referred to as 58-kDa and 88-kDa, respectively) are associated in a protein-protein complex, and that the triglyceride transfer activity is associated with this complex. Antibodies specific for either polypeptide immunoprecipitated both the 58-kDa and 88-kDa polypeptides as well as the lipid transfer activity. The 58-kDa subunit of the microsomal transfer protein complex was identified as protein disulfide-isomerase (PDI) (EC 5.3.4.1) by 1) a comparison of the amino-terminal sequence of PDI and the 58-kDa subunit of the transfer protein, 2) a comparison of the reverse phase high performance liquid chromatography peptide maps of CNBr digests of PDI and the lipid transfer protein, 3) immunoprecipitation competition experiments in which PDI was found to compete with the lipid transfer protein for immunoprecipitation by the anti-58-kDa polyclonal antibodies, 4) immunological cross-reactivity of the microsomal triglyceride transfer protein complex with polyclonal antibodies raised against PDI, and 5) the appearance of protein disulfide isomerase activity following the dissociation of purified microsomal transfer protein complex with guanidine HCl. In conclusion, the microsomal triglyceride transfer protein has a multi-subunit structure which is unique compared to other intracellular lipid transfer proteins which have been described to be single polypeptides. The unexpected finding that PDI is a component of the microsomal triglyceride transfer protein complex suggests a new previously undescribed role for protein disulfide isomerase.