Beta Process Research Articles

A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.

Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.

Formation of Acetyl‐Ser‐Asp‐Lys‐Pro, a New Regulator of the Hematopoietic System, through Enzymatic Processing of Thymosin β4a

The demonstration that AcSDKP, a new regulator of the hematopoietic system, is formed in the bone marrow by a one-step enzymatic maturation processing of thymosin beta 4 (T beta 4) is presented. AcSDKP and T beta 4 were both detected in bone marrow cells (BMC). Incubation of [3H]T beta 4 with either intact or lysed BMC led to the formation of [3H]AcSDKP, whereas the labeled tetrapeptide was not degraded under these conditions. Model enzymatic degradation of T beta 4 carried out with bacterial enzymes suggests that a mammalian endoproteinase Asp-N might be involved in the formation of AcSDKP through the specific cleavage of the Pro4-Asp5 peptidic bond of T beta 4. In contrast, alpha-prolyl-endopeptidase was ineffective in carrying out a similar processing.

Formation of intrachain disulfide bonds gives rise to two different forms of the murine IL-1 beta precursor.

IL-1 beta is an inflammatory cytokine produced by activated macrophages. Secretion of IL-1 beta comprises a biologic inactive precursor (molecular mass = 31 kDa) and a bioactive low molecular mass peptide (17 to 20 kDa). The questions of where IL-1 beta processing takes place and what is its relationship to secretion remain to be analyzed. Here we report the novel finding that lysates of murine macrophages contain two forms, with molecular masses of 31 and 35 kDa, of the IL-1 beta precursor that can be distinguished by their different electrophoretic mobilities under nonreducing conditions. The more rapid migration of the 31-kDa polypeptide was due to a disulfide-mediated protein folding, as concluded from the following evidence. The native 31-kDa polypeptide could be unfolded to a 35-kDa polypeptide by reduction, and it spontaneously refolded to the 31-kDa polypeptide when the reducing agent was removed. Refolding of the reduced 31-kDa polypeptide was blocked in the presence of iodoacetamide, indicating that alkylation of the protein prevented the re-oxidation of disulfides. Culture supernatants contained predominantly the 31-kDa polypeptide and the mature IL-1 beta with a low m.w. of 20,000. Little or no 35-kDa IL-1 beta was detected extracellularly. These data indicate that murine macrophages contain two populations of the IL-1 beta precursor, one of which can undergo a disulfide-mediated protein folding; they also suggest that oxidation of -SH groups may be critical for the proteolytic processing of the IL-1 beta precursor.

Tensile yield parameters for poly(vinyl chloride) blends with a methyl methacrylate/ethyl acrylate copolymer

Tensile yield measurements were made on blends of poly(vinyl chloride) (PVC) with a methyl methacrylate/ethyl acrylate 90/10 copolymer (CP-A) covering a blend range of 0 to 50 wt% CP-A, a temperature range of −50 to 50°C and a strain-rate range of 10 −3 to 10 0 s −1. Increasing CP-A level in PVC has the same effect on the brittle-to-ductile transition as does decreasing temperature or increasing strain rate in neat PVC. The maximum stresses observed for these blends tend to be independent of CP-A level over the low-concentration range, i.e. <25 wt%, but they do decrease over the 50 wt% range. The tensile yield stress variation with temperature and strain rate was represented in terms of the Ree-Eyring-Roetling rate model. This model represents the experimental data within experimental error. The tensile yield beta process parameters were compared to those determined from viscoelastic measurements on these same blends. The activation energies were found to be the same for the viscoelastic and tensile yield measurements, while the entropies of activation were found to be proportional.

Effect of a methylmethacrylate–butadiene–styrene modifier on the dynamics of the viscoelastic beta process in poly(vinyl chloride)

The viscoelastic properties of a methylmethacrylate–butadiene–styrene (MBS) modifier over the temperature range −140 to 20°C and frequency range 0.01592 (0.1 Rad/s) to 79.62 Hz (500 Rad/s) is represented in terms of a relaxation function proposed by Havriliak and Shortridge to represent the viscoelastic beta process of MBS-modified and unmodified poly(vinyl chloride) (PVC). The parameters of the expression as well as their temperature dependence were determined from the multiresponse methods developed by Havriliak and Watts. The complex modulus of PVC/MBS modifier blends at 8.26, 10.71, and 13.04 wt % modifier, over a temperature range −140–20°C, and at a frequency of 1 Hz can be represented, nearly within experimental error, in terms of the complex modulus of the neat components, the volume fraction of the modifier, and the blend equations of Uemura and Takayanagi. The small but persistent deviations that are observed are discussed in terms of hydrostatic stresses due to differential coefficients of expansion.

Molecular dynamics of the beta process in the polycarbonate of bisphenol-A

The experimental dielectric relaxation data of Pratt and Smith on the polycarbonate of bisphenol-A (PC) in the experimental range of its beta process, i.e. 0.1 to 10 6 Hz and −150 to 25°C, is represented in terms of an analytical expression proposed by Havriliak and Negami. The parameters and their dependence on temperature were determined by the multi-response techniques developed by Havriliak and Watts. These parameters are then compared with the corresponding viscoelastic beta process parameters for PC recently reported by Havriliak and Pogonowski. The parameters for the dielectric and viscoelastic process are similar and some of them are within the 95% confidence limits for the parameter estimates. Some of the differences that are observed may be due to proprietary difference in ostensibly the same generic material. The dynamics of the dielectric beta process is discussed in terms of the polarizability of the para-substituted phenyl groups in PC as they undergo ring flips as proposed by Perchak et al.

Nonparametric Bayes Estimators Based on Beta Processes in Models for Life History Data

Several authors have constructed nonparametric Bayes estimators for a cumulative distribution function based on (possibly right-censored) data. The prior distributions have, for example, been Dirichlet processes or, more generally, processes neutral to the right. The present article studies the related problem of finding Bayes estimators for cumulative hazard rates and related quantities, w.r.t. prior distributions that correspond to cumulative hazard rate processes with nonnegative independent increments. A particular class of prior processes, termed beta processes, is introduced and is shown to constitute a conjugate class. To arrive at these, a nonparametric time-discrete framework for survival data, which has some independent interest, is studied first. An important bonus of the approach based on cumulative hazards is that more complicated models for life history data than the simple life table situation can be treated, for example, time-inhomogeneous Markov chains. We find posterior distributions and derive Bayes estimators in such models and also present a semiparametric Bayesian analysis of the Cox regression model. The Bayes estimators are easy to interpret and easy to compute. In the limiting case of a vague prior the Bayes solution for a cumulative hazard is the Nelson-Aalen estimator and the Bayes solution for a survival probability is the Kaplan-Meier estimator.

Analysis of a multiple‐contact synapse missing a normally obligatory postsynaptic neuron: An Electron microscopic Study in the compound eye of musca domestica

In the compound eye of the adult female fly Musca domestica, photoreceptors form populations of multiple-contact output synapses, stereotypic in their architecture and in the identity of the four postsynaptic elements. Two postsynaptic elements, always originating from monopolar interneurons L1 and L2, lie side by side beneath the elongated presynaptic bar. Beneath each end of the bar, a further postsynaptic contact is located. These contacts most often are two processes either of amacrine cells or of epithelial glial cells. Monopolar cell L3 may be postsynaptic as well, with either an amacrine or a glial process completing the tetrad. To learn more about the factors determining connectivity and synaptic architecture, a three-dimensional reconstruction of serial electron microscopic sections was used to analyze a population of photoreceptor synapses at which one of the normally obligatory postsynaptic neurons, L1, was missing. In this abnormal case, the synapses make the normal four postsynaptic contacts in only 39% of the cases, otherwise making three (39%) or two (22%) contacts. Specificity of connectivity is preserved faithfully except that beta processes of T1 cells were postsynaptic at 2% of the synapses, where they do not normally contribute. In contrast to normal synapses, where amacrine and glial cell processes are mutual exclusive, such pairings could coexist in the aberrant synapse (27% of all synapses). All postsynaptic cells contributed the normal number of processes to a synaptic site, except for three synapses each with a supernumerary amacrine cell process. The postsynaptic cells therefore may be involved in regulating the number of their contacts made to a synaptic site.

Effect of a methylmethacrylate-ethylacrylate copolymer on the dynamics of the viscoelastic beta process in poly(vinyl chloride)

Viscoelastic properties of blends of poly(vinyl chloride) (PVC) with a methylmethacrylate-ethylacrylate copolymer (CP) at various levels (0 to 25 wt%) were determined over a frequency range of 0.1 to 500 rad s −1 and a temperature range of −140 to 20°C. This experimental range is sufficient to characterize the dynamics of the viscoelastic beta process in PVC in terms of a five parameter relaxation function proposed by Havriliak and Shortridge 1. The parameters of the relaxation function and their dependence on temperature were determined by the multi-response statistical techniques developed by Havriliak and Watts 13,14. The parameters are then represented in terms of CP concentration in PVC. The dependence of the parameters on concentration is discussed in terms of the general relaxation mechanism of Mansfield 17, the more specific ring flip process of Perchank 16 in polycarbonate, or the segment-flip model in PVC.

Inhibition of pro-IL1 beta processing by protease inhibitors and a synthetic IL1-peptide

Interleukin 1 (IL1), a key mediator in the cytokine network, alters many pathophysiologically important functions of blood vessel wall cells. Vascular cells, such as endothelial cells and smooth muscle cells (SMC) can themselves transcribe IL1 genes, raising the possibility that IL1 regulates blood vessel wall functions by local autocrine or paracrine mechanisms. However, IL1 lacks a recognizable signal sequence and it is still unclear how vascular cells might release IL1 or if IL1 derived from vascular cells can actually produce autocrine or paracrine effects. We explored these issues in human vascular SMC, the most numerous cell type in arteries and veins, using cultured SMC and short term organoid cultures. SMC treated with lipopolysaccharide recombinant tumor necrosis factor (recTNF), or recIL1 itself (“activated SMC”) elaborated thymocyte costimulatory activity, a biological activity traditionally ascribed to IL1. However, neutralization experiments with monospecific antibodies disclosed that the more recently recognized cytokine IL6 rather than IL1 accounted for most of the soluble thymocyte costimulatory activity released by activated SMC. Using the D10S assay that distinguishes IL1 from IL6 and TNF we found that the culture supernatant of activated SMC contained little or no IL1, but that the cytosol and surface of these cells did exhibit this activity. Antiserum to recIL1α inhibited stimulation of D10S cells by surface-associated IL1 of activated SMC, while treatment with acid to elute receptor- or nonspecifically bound IL1 did not abrogate this D10S proliferation. Short term organoid cultures of both normal veins and human arteriosclerotic plaque also expressed tissue-associated IL1 activity upon stimulation with LPS but did not release significant soluble IL1 activity. To establish further the biological functions of cell-associated IL1, we incubated stimulated or unstimulated SMC that were fixed with paraformaldehyde and washed extensively (fixed SMC) with overlayered viable SMC of the same donor (responder SMC). Contact with fixed SMC that bore surface IL1 following TNF or IL1 stimulation evoked up to 20-fold higher IL6 release from responder SMC than did exposure to unstimulated SMC (57 vs 1052 ng/ml/day). Addition of anti-IL1 antibody inhibited the release of IL6 from the responder SMC. These results demonstrate that cytokine-activated SMC express biologically active IL1 on their cell surface and illustrate how these cells might actually participate in autocrine and paracrine signaling in the vessel wall. The requirement for direct intercellular contact for IL1 effects could facilate local information exchange among vascular wall cells and/or infiltrating leukocytes and permit costimulation while limiting undue propagation of inflammatory stimuli.

Effect of methylmethacrylate‐butadiene‐styrene modifier level on the viscoelastic beta process in poly(vinyl chloride)

AbstractViscoelastic measurements were made on test specimens cut from plaques made from milled, compression molded blends of poly(vinyl chloride) (PVC) and methylmethacrylate‐butadiene‐styrene modifier (MBS). The experimental frequency range was from 0.0159 to 79.6 Hz. and the temperature range was from −140 to 20°C. The MBS modifier level studied was 8.26, 10.71, and 13.04 weight percent. This experimental range covers the beta relaxation process of PVC and the alpha relaxation process of the rubbery regions of the modifier. The complex compliance was represented in terms of a function originally proposed by Havriliak and Negami to represent the dielectric relaxation processes in polymers. The parameters of this expression as well as their dependence on temperature were determined by the multi‐response statistical techniques developed by Havriliak and Watts. Addition of MBS modifier to PVC appears to enhance the already present beta relaxation process in PVC. In addition the temperature dependence of the relaxation time has been increased so that the process is faster at room temperature. The α and β parameters of the relaxation function have changed so that the process is narrower at room temperature. There is no significant viscoelastic evidence supporting a second phase. This observation suggests significant impedance matching of the two phases which may be an important criteria for impact modification. Similarity of the two processes suggests a possible mechanism for the PVC beta process.

Effect of K‐value on the tensile yield properties of poly(vinyl chloride)

AbstractTensile measurements were made on milled compression molded test specimens of PVC of different K values from strain rates of 0.0001–0.1 s−1 and temperatures of −40 to 25°C. The dependence of yield stress on temperature and strain rate was treated in terms of the Ree–Eyring model as proposed by Roetling for poly(methyl methacrylate) and Bauwens‐Crowet et al. for poly(vinyl chloride). The data reduction was achieved using sophisticated and unbiased statistical techniques that not only provided parameter estimates but their confidence intervals. The yield stress parameters for the alpha and beta processes were found to be independent of K value and within experimental error the same as those reported by Bauwens‐Crowet et al. The activation energy for the beta process from these yield stress studies (15.1 ± 0.3 kcal) is within experimental error the same as that determined from viscoelastic measurements (14.1 ± 1.2 kcal) reported by the authors. This observation supports the contention that beta process in the viscoelastic and yield stress measurements are closely related mechanisms.

A Pre-aspartate-specific Protease from Human Leukocytes That Cleaves Pro-interleukin-1 β

Interleukin-1 beta is a 17.4-kilodalton hormone derived from a 33-kilodalton inactive precursor produced by monocytes. We used the precursor as a substrate to detect proteolytic activities in peripheral blood mono-nuclear cell-conditioned medium that might be involved in interleukin-1 beta processing. We found that the conditioned medium, following passage through DEAE-Sephacel, generates a biologically active fragment from the precursor that runs slightly higher than the mature hormone in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The responsible activity behaved as a single protein in ion exchange chromatography. It was completely inhibited by metal ion chelators and not by inhibitors of serine, cysteine, or aspartate proteases, and it was dependent on both calcium (or magnesium) and zinc. The enzyme was not inhibited by three substrate-based metalloprotease inhibitors, phosphoramidon, benzyloxycarbonyl-Gly-Leu-NH2, and N-(2-carboxy-3-phenylpropionyl)-Leu. NH2-terminal sequence analysis showed that cleavage of the precursor occurred between a histidine and an aspartate residue, and digestion of synthetic peptides indicated that the protease is specific for pre-aspartate cleavages.

Effect of catecholamine-induced cardiac hypertrophy on the force–interval relationship

Cardiac hypertrophy was induced in adult female Wistar rats following 12 days of daily subcutaneous injections of isoproterenol (ISO). The left atria responded with a 13-14% increase in tissue growth, while the ventricles achieved a 34-39% increased tissue mass. Maximum force generation and twitch characteristics in 1.0 mM external Ca2+ for the left atria or the right papillary muscle were unchanged in the ISO-treated animals. The force-interval relation was determined at 26 degrees C between 0.5 and 120 s. The development of maximum force clearly passed through two phases identified as alpha and beta. To characterize these two processes the data were fitted to a two-term linear combination of exponentials (two-compartment model). The time constant and capacity of each process to contribute to the whole force-interval curve was determined by a four-parameter least square fit method. In control atrial muscle the time constants for the alpha and beta processes were 0.47 and 11.23 s, respectively. The contribution of each process to the total force curve in control atrial muscle was approximately 50% alpha and 50% beta. Following ISO-induced growth the time constants were 0.38 and 13.33 s with a shift of contributions towards 60% alpha and 40% beta. Control papillary muscle from the right ventricle had a similar alpha time constant of 0.49 s compared with atrial muscle but possessed a considerably slower beta time constant of 26.17 s. The contribution of each process to interval-dependent force development was 44.5 and 55.5%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of Pituitary Luteinizing Hormone Secretion by Gonadotropin-Releasing Hormone is Not Coupled to β-Luteinizing Hormone Gene Transcription

The physiological stimulation and inhibition of release of several peptide and protein hormones appears to be associated with transcription of the respective peptide- and protein hormone-encoding genes. In the current studies we investigated whether this was also true for the rat pituitary LH system. Using an anterior pituitary primary tissue culture system, we have analyzed the effects of 10(-9) M GnRH on beta LH gene transcription using a transcription run-on assay, and on nuclear beta LH RNA levels using a highly sensitive solution hybridization-S1-nuclease protection assay. GnRH-stimulated release of LH does not appear to be coupled to a significant change in the rate of beta LH gene transcription, but is associated with both a 2- to 3-fold increase in levels of the beta LH primary transcript and processing intermediates and a rapid decrease in the levels of fully processed beta LH mRNA in the nucleus. No significant change in beta LH cytoplasmic mRNA levels, however, was associated with GnRH-stimulated release of LH, in vitro. Our findings suggest that unlike several other peptide and protein hormone systems, stimulated release of beta LH by GnRH in vitro is not associated with an increase in beta LH gene transcription or cytoplasmic mRNA levels.

A molecular model for tensile shear banding and the brittle‐ductile transition in poly(vinyl chloride)

AbstractRoetling's adaptation of the Ree‐Eyring model to represent tensile shear banding of polymers in terms of two relaxation processes and Mansfield's model to describe alpha and beta relaxation processes in terms of polymer segment mobility are combined with the particulate structure of poly(vinyl chloride) (PVC) to give a more detailed connection between the macroscopic yielding process and the microscopic mobility of polymer segments. Mansfield's model for relaxation processes is cast in terms of intra‐ and inter‐molecular interactions that serve to hinder orientational motions of polymer segments. The viscoelastic beta process in PVC is interpreted in terms of this model to be a hindered rotation of a segment about its main chain axis. About half of the activation energy of the beta process can be estimated from the barriers hindering rotation in simple alkyl halides. According to tensile yield studies it is the beta process that governs the tensile yield behavior of PVC at high loading rates or low temperatures, i.e. the brittle to ductile transition. Viscoelastic measurements were made on milled compression molded powder blends of PVC of differing K‐values (55–69), over a frequency range of 0.1 to 500 Rad/sec. and a temperature range of −140°C to 20°C. The dependence of the complex compliance on frequency and temperature was represented in terms of a five parameter model proposed by Havriliak and Negami to represent the dielectric dispersions of polymers. The five parameters and their dependence on temperature were estimated using the multi‐response statistical techniques developed by Havriliak and Watts. The parameters are interpreted in terms of Mansfield segmental jumping model.

Effect of methylmethacrylate-butadiene-styrene modifiers on the viscoelastic beta process in poly(vinyl chloride)

Tensile yield studies on poly(vinyl chloride) (PVC) indicate that the high-strain-rate or low-temperature behaviour may be determined by its beta process. In a previous study we examined the viscoelastic beta process of unmodified PVC over the temperature range −140 to 20°C and frequency range 0.1 to 30 Hz. In this study we extend these measurements to methylmethacrylate-butadiene-styrene (MBS) modified PVC over the same frequency and temperature ranges. In both studies the complex compliance can be represented in terms of an expression that takes the form originally proposed by Havriliak and Negami for the complex dielectric dispersions observed in polymers. These parameters and their dependence on temperature were determined using the multi-response statistical techniques developed by Havriliak and Watts. In either case the complex compliance for the beta process of PVC can be represented with this expression to within experimental error. MBS modification of PVC appears to increase the magnitude of the beta process (i.e. J 0 − J ∞ is larger), decrease the relaxation time at room temperature, and decrease the width of the distribution of relaxation times, so that it is more intense in the estimated impact time region at room temperature.

Human interleukin 1 beta is not secreted from hamster fibroblasts when expressed constitutively from a transfected cDNA.

To understand the secretion and processing of interleukin-1 (IL-1), a Chinese hamster fibroblast cell line (R1610) was transfected with a human IL-1 beta cDNA under the control of the SV40 early promoter and linked to the gene for neomycin resistance. After selecting for transfected cells resistant to G418, two clones were found to constitutively express the IL-1 beta 31-kD precursor which was almost exclusively located in the cytosol. Pulse-chase experiments failed to show any secretion of IL-1 and very little IL-1 activity was detectable in cell supernatants. Furthermore, surface membrane IL-1 activity could not be detected, although low levels of activity could be released upon brief trypsin treatment. Therefore, unlike monocytes, these fibroblast cells lack the mechanism for secreting and processing of IL-1 beta.

Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor.

Interleukin-1 beta (IL-1 beta) is derived from an inactive precursor by proteolytic cleavage. To study IL-1 beta processing, we expressed the precursor in Escherichia coli, partially purified it, and used it as a substrate for various potentially relevant protease preparations. The precursor alone was virtually inactive, but incubation with membranes from human monocytes or myeloid cell lines yielded a 500-fold increase in IL-1 bioactivity. Western blot analysis of the incubated material showed that the 31,000-Da precursor is broken down to three major products, ranging from 17,400 to about 19,000 Da. The most active of these products is the smallest one, and it co-migrates during electrophoresis with mature IL-1 beta. Four purified known proteases were also tested for their effect on precursor IL-1 beta, and none of these products co-migrated with the mature protein. Chymotrypsin and Staphylococcus aureus protease yielded slightly larger products, which were highly active. Elastase and trypsin yielded substantially larger products, and these had little IL-1 activity. The products of three of the known proteases were identified by NH2-terminal sequencing. These results show conclusively that proteolysis of precursor IL-1 beta generates biological activity and that the cleavage must occur close to the mature NH2 terminus.

Effect of catecholamine induced cardiac hypertrophy on the force-interval relationship

Cardiac hypertrophy was induced in adult female Wistar rats following 12 days of daily subcutaneous injections of isoproterenol (ISO). The left atria responded with a 13–14% increase in tissue growth, while the ventricles achieved a 34–39% increased tissue mass. Maximum force generation and twitch characteristics in 1.0 mM external Ca2+ for the left atria or the right papillary muscle were unchanged in the ISO-treated animals. The force–interval relation was determined at 26 °C between 0.5 and 120 s. The development of maximum force clearly passed through two phases identified as alpha and beta. To characterize these two processes the data were fitted to a two-term linear combination of exponentials (two-compartment model). The time constant and capacity of each process to contribute to the whole force–interval curve was determined by a four-parameter least square fit method. In control atrial muscle the time constants for the alpha and beta processes were 0.47 and 11.23 s, respectively. The contribution ...