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There are two inheritance patterns, the X-linked recessive (XL) pattern and the autosomal recessive pattern. There is no information on the predominant inheritance pattern of male patients with chronic granulomatous disease (CGD) in Mexico.

Plastic microfluidic array platforms and synergistic multiplexed assay chemistries are under development for a variety of applications, including assays of gene expression, proteomics, genotyping, DNA sequencing and fragment analysis, sample prepraration and high-throughput pharmaceutical discovery. The low production costs of plastic substrates makes possible economical single-use device arrays, eliminating cleaning and sample-to-sample carryover contamination. Hundreds of microchannels and reservoirs are readily included on a single microtitre-plate-size substrate, enabling the manufacture of highly parallel fluidic array systems to increase throughput and speed.

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Optimal T cell activation requires the interactions of co-stimulatory molecules, such as those in the CD28 and B7 protein families. Recently, we described the co-stimulatory properties of the murine ligand to ICOS, which we designated as B7RP-1. Here, we report the co-stimulation of human T cells through the human B7RP-1 and ICOS interaction. This ligand-receptor pair interacts with a K:(D) approximately 33 nM and an off-rate with a t((1/2)) > 10 min. Interestingly, tumor necrosis factor (TNF)-alpha differentially regulates the expression of human B7RP-1 on B cells, monocytes and dendritic cells (DC). TNF-alpha enhances B7RP-1 expression on B cells and monocytes, while it inhibits it on DC. The human B7RP-1-Fc protein or cells that express membrane-bound B7RP-1 co-stimulate T cell proliferation in vitro. Specific cytokines, such as IFN-gamma and IL-10, are induced by B7RP-1 co-stimulation. Although IL-2 levels are not significantly increased, B7RP-1 co-stimulation is dependent on IL-2. These experiments define the human ortholog to murine B7RP-1 and characterize its interaction with human ICOS.

The narrowest feature of an integrated circuit is the silicon dioxide gate dielectric (3–5 nm). The viability of future CMOS technology is contingent upon thinning the oxide further to improve drive performance, while maintaining reliability. Practical limitations due to direct tunneling through the gate oxide may preclude the use of silicon dioxide as the gate dielectric for thicknesses less than 1.3 nm, however.

Granulocyte colony-stimulating factor (G-CSF) forms a tetrameric complex with its receptor, comprising two G-CSF and two receptor molecules. The structure of the complex is unknown, and it is unclear whether there are one or two binding sites on G-CSF and the receptor. The immunoglobulin-like domain and the cytokine receptor homologous module of the receptor are involved in G-CSF binding, and Arg288 in the cytokine receptor homologous module is particularly important. To identify residues in G-CSF that interact with Arg288, selected charged residues in G-CSF were mutated to Ala. To clarify whether there are two binding sites, a chimeric receptor was created in which the Ig domain was replaced with that of the related receptor gp130. This chimera bound G-CSF but could not transduce a signal, consistent with failure of dimerization and loss of one binding site. The G-CSF mutants had reduced mitogenic activity on cells expressing wild-type receptor. When tested with the chimeric receptor, all G-CSF mutants except one (E46A) showed reduced binding, suggesting that Glu46 is important for interaction with the Ig domain. On cells expressing R288A receptor, all the G-CSF mutants except E19A showed reduced mitogenic activity, indicating that Glu19 of G-CSF interacts with Arg288 of the receptor.

The recently introduced end-bridging (EB) Monte Carlo move is revisited, and a thorough analysis of its geometric formulation and numerical implementation is given. Detailed results are presented from applying the move, along with concerted rotation, in atomistic simulations of polyethylene (PE) melt systems with mean molecular lengths ranging from C78 up to C500, flat molecular weight distributions, and polydispersity indices I ranging from 1.02 to 1.12. To avoid finite system-size effects, most simulations are executed in a superbox containing up to 5000 mers and special neighbor list strategies are implemented. For all chain lengths considered, excellent equilibration is observed of the thermodynamic and conformational properties of the melt at all length scales, from the level of the bond length to the level of the chain end-to-end vector. In sharp contrast, if no end bridging is allowed among the Monte Carlo moves, no equilibration is achieved, even for the C78 system. The polydispersity index I is found to have no effect on the equilibrium properties of the melt. To quantify the efficiency of the EB Monte Carlo move, the CPU time t0 required for the chain center of mass to travel a distance equal to the root-mean-square end-to-end distance is estimated by simple analytical arguments. It is found that t0 should scale as n/(&Xmacr;Δ^2.5), where n is the total number of mers in the system, &Xmacr; is the average chain length, and Δ ≃ [3(I − 1)]^1/2 is the reduced width of the chain-length distribution function. This means that, if the size of the model system and the shape of the chain-length distribution are kept constant, systems of larger average molecular weight equilibrate faster, a remarkable attribute of the EB Monte Carlo method. The simulation results obey the estimated scaling of t0 with &Xmacr;, n, and Δ remarkably well in the range of chain lengths and polydispersities for which the premises of the analysis are not violated (mean chain lengths greater than C156 and polydispersity indices above about 1.07). Results for volumetric behavior, structure, and chain conformation at temperature T = 450 K and pressure P ranging from 1 to 800 atm are presented, using three different PE united atom models proposed in the recent literature. All three models are shown to overestimate the density by ca. 4% and also overestimate the stiffness of chains. The Yoon et al. model is in best agreement with experimental characteristic ratios. Simulation predictions for the structure factor and for the chain-length dependence of the density are in excellent agreement with experiment.

Purified preparations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and interleukin 3 (IL-3 or multi-CSF) alone and in combination, have been compared for their stimulatory effects on human granulocyte-macrophage colony forming cells (GM-CFC). In cultures of unseparated normal human bone marrow, the combinations of G-CSF plus IL-3 and GM-CSF plus IL-3 stimulated additive numbers of GM colonies, while GM-CSF plus G-CSF stimulated greater than additive numbers of GM colonies, compared with the sum of the colony formation obtained with each factor alone. Cultures of unseparated bone marrow, harvested from patients four to six days after administration of 5-fluorouracil (5-FU), resulted in additive GM colony formation with GM-CSF plus G-CSF, GM-CSF plus IL-3, and G-CSF plus IL-3. In order to address the possibility of secondary factor involvement in the synergistic interaction of GM-CSF and G-CSF, CD33+/CD34+ colony forming cells were separated from normal and post FU marrow by two color fluorescence activated cell sorting. In cultures of CD33+/CD34+ cells the combination of GM-CSF plus G-CSF stimulated a synergistic increase in GM colonies while GM-CSF plus IL-3 stimulated additive numbers of colonies. These results suggest that GM-CSF, G-CSF, and IL-3 stimulate distinct populations of GM-CFC. Furthermore GM-CSF and G-CSF interact synergistically and this action is a direct effect on progenitor cells not stimulated by GM-CSF or G-CSF alone.

The organization of projections from the principal sensory trigeminal nucleus (PSN) to the hypoglossal nucleus (XII) in the rat was investigated at the light and electron microscopic level with retrograde and anterograde axonal tracer techniques. Microiontophoretic injection of horseradish peroxidase (HRP) into XII resulted in retrograde labeling of neurons confined to the dorsal one-third of the PSN. Labeled neurons were found bilaterally, although a clear preponderance for ipsilateral distribution was evident. Most labeled neurons were found in the medial one-third and caudal two-thirds of the PSN. Labeled neurons were large (30–50 µm), round-to-pear shaped multipolar cells with dendrites oriented primarily in the mediolateral direction. At the electron microscopic level, HRP reaction product was found throughout the cytoplasm of soma and processes of PSN projection neurons. The ultrastructural characteristics of these cells included a round, centrally placed nucleus and invaginated nuclear envelope, sparse Nissl bodies, numerous free ribosomes, mitochondria, lysosomes and Golgi complexes. Three to four main stem dendrites gradually tapered from the cell body and numerous synaptic terminals impinged upon soma and dendrites of labeled PSN neurons. Microiontophoretic injection of tritiated amino acids or HRP into the dorsal one-third of the PSN resulted in moderately dense terminal labeling in XII bilaterally, although mainly ipsilaterally. Terminal labeling was found diffusely throughout all regions of XII. Fibers descended the brainstem in the dorsolateral reticular formation and entered XII ventrolaterally. At the electron microscopic level, boutons containing HRP reaction product were found to synapse on dendritic processes in XII. Labeled boutons were characterized by clear, spherical vesicles and an asymmetrical postsynaptic density. The significance of these results are discussed in relation to oro-lingual motor behavior.

The purpose of this study was to define the types and distribution of synaptic terminals in the hypoglossal nucleus (XII) of the rat. Based on differences in bouton and vesicle size and shape, synaptic specializations and association with postsynaptic organelles, five types of terminals were identified in XII. In order of decreasing frequency they were: 1) S-boutons (spherical vesicles with an asymmetrical synapse); 2) F-boutons (flattened vesicles with a symmetrical synapse); 3) P-boutons (pleomorphic admixture of flattened and spherical vesicles with a symmetrical synapse); 4) C-boutons (pleomorphic vesicles with a subsynaptic cistern); and 5) Tboutons (spherical vesicles with an asymmetrical synapse and subsynaptic dense bodies). S-boutons were the predominant type found on dendrites, while boutons containing flattened vesicles were more prevalent on motoneuron somata. C-boutons were restricted exclusively to cell bodies and large dendrites, and T-boutons were seen primarily on smaller dendritic profiles. These results are, in general, comparable to those previously described in the ventral horn and cranial nerve motor nuclei in several species. However, differences were noted. Specifically, large M-boutons and axo-axonic synapses were not observed in the present study. The functional significance of these findings are discussed in relation to oro-lingual behaviour.

Abstract—We develop a seismic model that characterises the sudden tensional failure of oil-well casings. The energy released by the rupture of a well casing is transformed into heat and seismic energy. The upper bound of the seismic efficiency of this process is estimated at about 3%. The static situation at the completion of a casing failure episode is modelled by calculating the static displacement field generated by two opposing forces separated by an arm. The azimuthal patterns of these displacements and the change in the strain and stress fields caused by the force couple are described. The dynamics of the failure episode are modelled as a dipole with a seismic moment equivalent to the product of the average drop in shear stress, the failure surface, and an arm. The radiated P and S waves have mean-square radiation pattern coefficients of 1/5 for P waves and 2/15 for S waves. The displacement field as a function of time during rupture and the spectral properties in the far field are derived. The most promising seismic parameters that can be used for distinguishing between casing failure events and other possible events are polarisation properties of S waves and S/P amplitude ratios. S-wave polarisation distinguishes between shear events and casing failure events. S/P amplitude ratios distin guish between tensile events and casing failure events.

Abstract—We analyze source parameters of microseismic events (M < − 1) associated with high flow-rate water injections in a shale formation at a depth of 220 m. Two types of events were observed several hundred impulsive events with clear P- and S-wave arrivals, and continuous emissions with peaked spectra detected well into the experiment. For a representative collection of impulsive events, an <omega>⁻² model provided satisfactory fits to displacement spectra corrected for attenuation, and average quality factors of 34 and 15 were obtained for P and S waves. P-wave first motion analysis and ES /EP ratios indicated the existence of a non-double-couple component in some events, particularly early in the experiment. A clear difference was observed for estimates of stress release parameters as non-double-couple events had smaller stress drops and apparent stresses. The seismic efficiency of double-couple and non-double-couple events was limited to 0.9% and 0.05% respectively, with average values being 0.25% and 0.02%. A comparison of our results with those reported for a similar magnitude range in a hard-rock formation indicates considerably smaller estimates of stress drop and apparent stress in our case while seismic efficiencies are comparable.

Abstract—Imperial Oil Resources Limited uses cyclic steam stimulation to recover oil from their Cold Lake oil field in Alberta. This operation, in particular situations, can be associated with the failure of well casings in the Colorado shales above the oil-bearing formation. A number of fluid injection operations was undertaken at this site and the associated microseismicity was detected using two three-component geophones and fifteen hydrophones. The purpose of this experiment was to simulate the occurrence of a casing failure, determine the feasibility of monitoring in a shallow environment, and characterize the microseismic activity. A calibration survey provided values of 1786 ± 108 m/s for P-wave velocity, 643 ± 56 m/s for S-wave velocity and 0.428 ± 0.017 for Poisson’s ratio in the shale formation. Estimates of the quality factor QP were 15 for the horizontal direction and 38 for the vertical direction, corroborating the evidence of velocity anisotropy. Calibration shots were located to within 10 m of the actual shot points using triangulation and polarization techniques. Several hundred microseis mic events were recorded and 135 events were located. The results showed that microseismic activity was confined to depths within 10 meters of the injection depth. The experiment clearly established the feasibility of detecting microseismicity induced by fluid injection rates typical of casing failures in shales at distances over 100 m.

Abstract—This paper describes observations of seismicity and casing failures associated with steam stimulation operations at Imperial Oil Ltd.’s Cold Lake oil field in Alberta, Canada. A total of 11 oil-producing pads were monitored over a 1–2 year period using 3-component geophones cemented at depths ranging from 160 m to 400 m and data acquisition systems with a flat frequency response up to 1.5 kHz. Most of the seismicity was detected during the steaming operations and was located in the formation overlying the oil-bearing layer. Some activity was observed in the shales above, however, the reservoir itself showed almost no evidence of seismicity. The estimated seismic moment of the observed events was in the range 10⁵–10⁷ N·m (−2.7 < M < −1.3). According to a theoretical model (Talebi et al., 1998) and in situ observations, the seismic source corresponding to casing failure events should be well described by a dipole registering seismic moment in the order of 2 · 10⁶ N·m. Seismic signals of a total of four observed casing failures were analyzed. The partial failures produced seismic moments slightly lower than this value while total failures were stronger by about one order of magnitude. The use of the SV/SH amplitude ratio, in conjunction with accurate source locations, provided a robust technique for the detection of casing failures.

We examined the in vivo effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in primates (cynomolgus monkeys) treated with subcutaneous doses of rhG-CSF for 14-28 d. A dose-dependent increase in the peripheral white blood cells (WBC) was seen, reaching a plateau after 1 wk of rhG-CSF treatment. The elevation of WBC was due to an increase in the absolute neutrophil count. These results demonstrate that rhG-CSF is a potent granulopoietic growth and differentiation factor in vivo. In cyclophosphamide (CY)-induced myelosuppression, rhG-CSF was able to shorten the time period of WBC recovery in two treated monkeys to 1 wk, as compared to more than 4 wk for the control monkey. Its ability to significantly shorten the period of chemotherapy-induced bone marrow hypoplasia may allow clinicians to increase the frequency or dosage of chemotherapeutic agents. In addition, the increase in absolute numbers of functionally active neutrophils may have a profound effect in the rate and severity of neutropenia-related sepsis. Furthermore, the activities reported here indicate a potential role for rhG-CSF in the treatment of patients with myelodysplastic syndrome, congenital agranulocytosis, radiation-induced myelosuppression, and bone marrow transplantation.

Administration of recombinant canine granulocyte-macrophage colony- stimulating factor (rcGM-CSF) to normal dogs in previous studies induced an increase in peripheral blood neutrophils and a dose- dependent decrease in platelet counts. In six dogs that received the highest tested dose of rcGM-CSF (50 micrograms/kg/d) for a minimum of 12 days, the mean nadir of the platelet count was 46,000/microL (range, 4,000 to 91,000/microL) on day 9 +/- 1.1 after starting therapy, compared with a mean baseline platelet count of 398,000/microL (range, 240,000 to 555,000/microL). In three dogs, survival of autologous 111In- labeled platelets was reduced from a mean of 4.9 days to 1.3 days during the administration of rcGM-CSF. Biodistribution studies with gamma camera imaging indicated that there was an increase in mean hepatic uptake during the administration of rcGM-CSF, from 15% to 44% of the total injected 111In-labeled platelets at 2 hours, whereas splenic uptake was not significantly changed. In contrast, in two evaluable dogs who were recipients of 111In-labeled platelets from matched allogeneic donors receiving rcGM-CSF, platelet survival was not reduced and no increased hepatic uptake was noted. A third dog became alloimmunized to the matched donor platelets and was not evaluable. Immunohistologic studies of liver and spleen were performed with monoclonal antibodies specific for canine gpIIb/IIIa and P-selectin in dogs treated with rcGM-CSF and compared with untreated controls. On treatment, a marked reduction of platelets in the red pulp of the spleen was evident, and in general, the presence of platelet antigen in the liver was unchanged. Therefore, platelets were not being sequestered, but destroyed in the liver and spleen. The platelet antigens, P-selectin and gpIIb/IIIa, were identified in association with Kupffer cells in the liver, but no difference in the number of distribution of these Kupffer cells was found between controls and rcGM- CSF-treated dogs. In the spleen during rcGM-CSF treatment, most platelet antigens were associated with large mononuclear cells in the marginal zone. During administration of rcGM-CSF, CD1c and CD11c expression was increased on Kupffer cells. Platelet P-selectin expression and binding of leukocytes to circulating platelets were unchanged from baseline studies with rcGM-CSF treatment. In conclusion, during the administration of rcGM-CSF to dogs, a local process in the liver and spleen is induced resulting in thrombocytopenia.(ABSTRACT TRUNCATED AT 400 WORDS)

Megakaryocyte growth and development factor (MGDF) is a novel cytokine that binds to the c-mpl receptor and stimulates megakaryocyte development in vitro and in vivo. This report describes the ability of recombinant human (r-Hu) MGDF to affect megakaryocytopoiesis in normal nonhuman primates. r-HuMGDF was administered subcutaneously to normal, male rhesus monkeys once per day for 10 consecutive days at dosages of 2.5, 25, or 250 micrograms/kg of body weight. Bone marrow and peripheral blood were assayed for clonogenic activity and peripheral blood counts were monitored. Circulating platelet counts increased significantly (P < .05) for all doses within 6 days of r-HuMGDF administration and reached maximal levels between day 12 and day 14 postcytokine administration. The 2.5, 25.0, and 250.0 micrograms/kg/d doses elicited peak mean platelet counts that were 592%, 670%, and 449% of baseline, respectively. Bone marrow-derived clonogenic data showed significant increases in the concentration of megakaryocyte (MEG)- colony-forming unit (CFU) and granulocyte-erythroid-macrophage- megakaryocyte (GEMM)-CFU, whereas that of granulocyte-macrophage (GM)- CFU and burst-forming unit-erythroid (BFU-e) remained unchanged during the administration of r-HuMGDF. These data show that r-HuMGDF is a potent stimulator of thrombocytopoiesis in the normal nonhuman primate.

The physiological mechanisms by which growth hormone (somatotropin) exerts its several metabolic activities remain poorly understood. In particular, there is disagreement as to whether the diabetogenic and lipolytic activities of the hormone are intrinsic properties of the molecule or are the result of contamination with other pituitary components. The availability of recombinant-DNA-derived bovine growth hormone (rebGH) presented an opportunity to compare the biological activities of rebGH and pituitary bGH in the absence of pituitary contaminants. Pituitary bGH and rebGH were immunologically identical in the radioimmunoassay for bGH, and good agreement was obtained for the potency of the latter measured by radioimmunoassay (1.6 units/mg) and the dwarf-mouse bioassay (1.4 units/mg). The lipolytic activity in vitro was examined by measuring the release of glycerol from rat epididymal fat maintained in the presence of dexamethasone (0.2 microgram/ml) and the material to be tested (0.1 and 0.2 mg/ml). Although two preparations of pituitary bGH stimulated a significant (P less than 0.01) increase in glycerol production, neither rebGH nor recombinant-DNA-derived chicken GH was lipolytic. However, when rebGH was intravenously injected into three sheep (0.15 mg/kg), the increase in plasma nonesterified fatty acids was similar to that measured with the same dose of pituitary bGH. Insulin-tolerance tests were conducted in sheep before and after treatment with rebGH and pituitary bGH (four subcutaneous injections of 0.15 mg/kg). Although the effect of rebGH was less than that of the pituitary hormone, both significantly impaired the ability of insulin to lower the concentration of plasma glucose. These data suggest that the lipolytic and diabetogenic activities of bGH are intrinsic properties of the molecule. However, the lipolytic activity may only become apparent after either modification of the molecule in vivo or activation of a lipolytic intermediate.

Summary: An ultrastructural study of the hypoglossal nucleus (XII) in the rat has revealed two distinct neuronal populations. Hypoglossal motoneurons comprised the largest population of neurons in XII and were identified following injection of horseradish (HRP) into the tongue. Motoneurons were large (25–50 mgr), multipolar in shape and distributed throughout XII. The nucleus was large, round and centrally located, and the cytoplasm was characterized by dense lamellar arrays of rough endoplasmic reticulum. In contrast, a second population of small (10–18 mgr), round to oval shaped neurons was found restricted to the ventral and dorsolateral regions of XII. The nucleus was markedly invaginated and eccentric, the cytoplasm scant and filled with free ribosomes, and the absence of lamellar arrays of rough endoplasmic reticulum was conspicuous. Neurons of this type were never found to contain HRP reaction product. These results demonstrate that the hypoglossal nucleus does not consist solely of motoneurons, but includes a distinctly separate, presumably non-motoneuronal pool. Arguments are presented in favor of this second neuron population being interneurons. The functional significance of these findings in relation to tongue control is discussed.

The interaction of nucleotides with recombinant human granulocyte/macrophage colony stimulating factor (rhGM-CSF) has been investigated. Utilizing nucleotide photoaffinity probes [γ32P]-8N₃ATP and [β'32P]-8N₃Ap₄A, an analog of alarmone, the specificity of interaction was demonstrated by saturation of photoinsertion by these analogs and protection of photoinsertion by these analogs in the presence of natural nucleotide. The site of photoinsertion was tentatively determined to be Ser9. The photolabeled cytokine has lost most of its biological activity in a cellular proliferation assay, indicating a possible physiological role for this interaction.Copyright 1995, 1999 Academic Press. Inc.

The purification and characterization of a metalloproteinase inhibitor (MI) from bovine aortic endothelial cells, and the demonstration that it is related to, but distinct from, tissue inhibitor of metalloproteinase (TIMP), have previously been reported [De Clerck, Y. A., Yean, T.-D., Ratzkin, B. J., Lu, H.S. & Langley, K. E. (1989) J. Biol. Chem. 264, 17445-17453]. The cDNA cloning of the bovine MI and its human homolog is now reported. The bovine cDNA cloning used probes designed on the basis of NH2-terminal amino acid sequence of bovine MI. The human cDNA cloning in turn used probes representing parts of the bovine cDNA nucleotide sequence. Both cDNAs encode leader sequences of 26 amino acids and mature protein sequences of 194 amino acids. The amino acid sequences of the mature proteins are 94% identical. The human MI cDNA was expressed in Escherichia coli, and a preparation containing anticollagenase activity was recovered. The amino acid sequence of mature human MI is 38% identical to the sequence for human TIMP, and the 12 cysteines in MI and TIMP are aligned almost identically. Thus MI and TIMP comprise an inhibitor family.

Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (Ka) of 2-4 x 10(8) M-1, using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization Ka, greater than 90% of the circulating SCF would be in the monomeric form. When 125I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10-100 ng/ml, consistent with the Ka determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer Ka values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.

The receptor for glial cell line-derived neurotrophic factor (GDNF) consists of GFRalpha-1 and Ret. Neurturin is a GDNF-related neurotrophin whose receptor is presently unknown. Here we report that neurturin can bind to either GFRalpha-1 or GFRalpha-2, a novel receptor related to GFRalpha-1. Both GFRalpha-1 and GFRalpha-2 mediate neurturin-induced Ret phosphorylation. GDNF can also bind to either GFRalpha-1 or GFRalpha-2, and activate Ret in the presence of either binding receptor. Although both ligands interact with both receptors, cells expressing GFRalpha-1 bind GDNF more efficiently than neurturin, while cells expressing GFRalpha-2 bind neurturin preferentially. Cross-linking and Ret activation data also suggest that while there is cross-talk, GFRalpha-1 is the primary receptor for GDNF and GFRalpha-2 exhibits a preference for neurturin. We have also cloned a cDNA that apparently codes for a third member of the GFRalpha receptor family. This putative receptor, designated GFRalpha-3, is closely related in amino acid sequence and is nearly identical in the spacing of its cysteine residues to both GFRalpha-1 and GFRalpha-2. Analysis of the tissue distribution of GFRalpha-1, GFRalpha-2, GFRalpha-3, and Ret by Northern blot reveals overlapping but distinct patterns of expression. Consistent with a role in GDNF function, the GFRalphas and Ret are expressed in many of the same tissues, suggesting that GFRalphas mediate the action of GDNF family ligands in vivo.

A novel human chemokine STCP-1 (stimulated T cell chemotactic protein) was isolated from an activated macrophage cDNA library. The chemokine has four cysteines positioned in a manner that identifies STCP-1 as a member of the CC chemokine family. The amino acid sequence shows 34% identity with RANTES. The gene consists of 3 exons and 2 introns with the position of intron/exon boundaries similar to that of RANTES. The gene is expressed as a 3.4-kilobase transcript on lymph node, thymus, and Appendix. STCP-1 induces Ca2+ mobilization in a small percentage of primary activated T lymphocytes, but on repeated stimulation the percentage of T lymphocytes that respond to STCP-1 increases. The chemokine STCP-1 does not induce Ca2+ mobilization in monocytes, dendritic cells, neutrophils, eosinophils, lipopolysaccharide-activated B lymphocytes, and freshly isolated resting T lymphocytes. Similarly, STCP-1, while acting as a mild chemoattractant for primary activated T lymphocytes, is a potent chemoattractant for chronically activated T lymphocytes but has no chemoattractant activity for monocytes, neutrophils, eosinophils, and resting T lymphocytes. As STCP-1 acts specifically on activated T lymphocytes, it may play a role in the trafficking of activated/effector T lymphocytes to inflammatory sites and other aspects of activated T lymphocyte physiology.

This paper presents a case study of using a multicompartment isoelectric focusing apparatus to determine the isoelectric points and focus preparative quantities of brain derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3). A separation of PEGylated from unPEGylated forms of granulocyte colony stimulating factor (G-CSF) is described as well. Both BDNF and NT3 have substantially higher pI values in this system than is predicted from sequenced based modeling. Although PEGylated forms of G-CSF can be separated from the unPEGylated forms, separation of protein with differing degrees of PEGylation was not achieved. The paper additionally demonstrates that this technique can be used simultaneously as an analytical and preparative tool, eliminating the need for analytical IEF gels.

The administration of cyclophosphamide (50 to 100 mg/kg) at 48 to 72 h before removal of murine lung or spleen mononuclear cells for culture rendered DBA/2 mice incapable of generating an effective cytotoxic T-lymphocyte response to influenza A virus-infected cells. The cytotoxic T-lymphocyte precursor frequency to influenza A virus in lung and spleen cells from cyclophosphamide-treated mice was significantly decreased when compared with that of normal littermate controls. The low cytotoxic T-lymphocyte activity in the lungs and spleens of cyclophosphamide-treated mice could be partially restored in vitro by human interleukin 2.

Canine granulocyte-macrophage colony-stimulating factor (caGM-CSF) was cloned and expressed to allow further investigation of GM-CSF in a large animal model. The cDNA is 850 base pairs (bp) long and encodes a peptide of 144 amino acids. The nucleotide and amino acid sequence homology between caGM-CSF and human GM-CSF (hGM-CSF) is 80% and 70%, respectively. A mammalian expression vector pCMV/CAGM was constructed and used to transfect COS cells for expression of caGM-CSF. Supernatant from transfected COS cells enriched with caGM-CSF was shown to have significant stimulating activity in granulocyte-macrophage colony forming unit (CFU-GM) assays of canine marrow. caGM-CSF, expressed from bacteria, was used to treat seven dogs at varying doses twice daily subcutaneously (sc) for 14 to 16 days. Circulating blood neutrophils and monocytes increased significantly. The increase in circulating eosinophils was variable. Thrombocytopenia developed during administration of caGM-CSF but corrected rapidly after cessation of treatment. Evaluation of survival times of 51Cr-labeled autologous platelets suggested increased consumption as the primary reason for thrombocytopenia. A species-specific GM-CSF will be a useful tool for hematologic or immunologic studies in dogs.

Recombinant canine granulocyte-macrophage colony-stimulating factor (rcGM-CSF) was studied in normal dogs and in dogs receiving otherwise lethal total body irradiation (TBI) without marrow transplant. Five normal dogs receiving 25 micrograms/kg of rcGM-CSF by subcutaneous (SC) injection twice daily (BID) for 14 days showed increases in peripheral blood neutrophil counts of three to five times the baseline. Platelet counts decreased during administration of rcGM-CSF to a mean nadir of 52,800. Ten dogs received 400 cGy TBI at 10 cGy/min from two opposing 60Co sources and no marrow graft. Within 2 hours of TBI, rcGM-CSF was begun at a dose of 50 micrograms/kg SC BID for 5 doses and then continued at 25 micrograms/kg SC BID for 21 days. Only 1 of the 10 dogs receiving rcGM-CSF survived with complete and sustained recovery of hematopoiesis. One of 13 historical control dogs survived after 400 cGy with no hematopoietic growth factor or marrow infusion. Results with rcGM-CSF were compared with previous and concurrent data with G-CSF studied in the same model. Of 10 dogs receiving G-CSF, 8 survived with complete and sustained hematopoietic recovery, a significantly better survival than that seen with rcGM-CSF (P = .006). Neutrophil counts were sustained at higher levels after TBI for the first 18 days in the G-CSF group (P < .016) and the neutrophil nadirs were higher. No differences in neutrophil nadirs were noted between the rcGM-CSF and control groups. Dogs treated with rcGM-CSF experienced a more rapid decline of platelet counts than G-CSF-treated or control dogs over the first 18 days (P < .001). The nadir of the platelet count was higher in the control group than in either the G-CSF or rcGM-CSF group and no significant difference was observed between the G-CSF and rcGM-CSF groups. After otherwise lethal TBI (400 cGy) in dogs, rcGM-CSF was not effective in promoting hematopoietic recovery or improving survival.

Colony-stimulating factors are a category of glycoproteins that are instrumental in the regulation of hematopoiesis and inflammation. This investigation documented the clinical bone marrow and peripheral blood responses to short-term and long-term administration of a recombinant bovine granulocyte colony-stimulating factor (rb-GCSF) and an analog, where the cysteine at position 17 was substituted with a serine (rb-GCSF ser17). The colony-stimulating factors produced the expected changes in the hematologic findings of the bovine subjects in the study, and there was a cell-specific response to the compounds. The sustained neutrophilia in the long-term study indicates that the bovine species can tolerate the administration of recombinant forms of bovine GCSF for extended periods of time without detectable adverse side effects. The neutrophils from the short-term study revealed no apparent fluctuation, either as enhanced or reduced capability to reduce nitro blue tetrazolium as compared to pretreatment neutrophils. The administration of both recombinant forms of GCSF produced large increases in the bone marrow myeloid: erythroid (M:E) ratio concomitantly with the neutrophilias. This is the first preliminary report documenting the bone marrow response of cattle to the native and recombinant (rb-GCSF ser17) forms of bovine GCSF.

A severe persistent neutropenia developed in a rabbit that was injected intradermally with 120, 60, 60, and 120 μg of recombinant canine granulocyte colony-stimulating factor (cG-CSF) on days 1, 22, 31, and 44, respectively. The neutropenia was present from day 44 to day 205. The nadir of the neutropenia (60 cells/μl) occurred in conjunction with peak antibody titer (640,000) to cG-CSF on day 58. The immune antiserum from this rabbit reacted positively for cG-CSF on Western blot analysis. The immune antiserum also neutralized the activity of cG-CSF. On day 160, examination of the bone marrow showed marked granulocytic hypoplasia and mild erythroid hyperplasia. On day 205, the rabbit was still neutropenic (430 cells/μl), even though the last injection of cG-CSF was given 161 days previously. Necropsy on day 205 showed that there was still mild granulocytic hypoplasia with mild erythroid hyperplasia. Because of the lack of any inflammatory foci found at necropsy and the granulocytic hypoplasia, it was thought that the neutropenia was most likely due to decreased production and was not a consumptive process. It is hypothesized that the antibody that was produced to cG-CSF neutralized the effect of endogenous rabbit granulocyte colony-stimulating factor and prevented the normal proliferation and maturation of the rabbit neutrophils.

We develop and test a new elementary Monte Carlo move for use in the efficient simulation of polymer systems. The move consists of a concerted rotation around up to seven adjacent skeletal bonds that leaves the rest of the chain unaffected. No assumption is made concerning the backbone geometry other than that bond lengths and bond angles are held constant during the elementary move. Special sampling techniques are needed because the new move involves a correlated change in seven degrees of freedom along the chain backbone. We use the new move in conjunction with reptation in an isothermal-isobaric Monte Carlo simulation of a bulk tetracosane melt system and find that it improves computational efficiency relative to a purely reptation-based Monte Carlo scheme. Comparisons are also made between a concerted rotation-based Monte Carlo simulation and a molecular dynamics simulation of an oligomer of atactic polypropylene.

Osmotic pumps containing Escherichia coli-derived recombinant human granulocyte colony-stimulating factor (rhG-CSF) were attached to indwelling jugular vein catheters and implanted subcutaneously into Golden Syrian hamsters. Within 3 days, peripheral granulocyte counts had increased greater than 10-fold with a concomitant 4-fold increase in total leukocytes. Microscopic examination of Wright-Giemsa-stained blood smears from rhG-CSF hamsters showed that only the neutrophil subpopulation of granulocytes had increased. No significant changes in lymphocyte or monocyte counts were observed during the course of continuous rhG-CSF treatment. After subcutaneous injection at rhG-CSF doses of up to 10 micrograms X kg-1 X day-1 only granulocyte counts were affected. However, at higher dose levels, a transient thrombocytopenia was noted. Erythrocyte had lymphocyte/monocyte counts remained unaffected by rhG-CSF over the entire dose range (0.3-300 micrograms X kg-1 X day-1) studied. Total leukocyte counts increased 3-fold within 12 hr after a single s.c. injection of rhG-CSF. This early effect was associated with an increase in the total number of colony-forming cells and the percent of active cycling cells in the marrow. A sustained elevation of peripheral leukocyte and marrow progenitor counts was observed following seven daily s.c. injections of rhG-CSF. The ability of rhG-CSF to increase the production and release of granulocytes from the marrow may underlie the beneficial effect it produced on the restoration of peripheral leukocyte counts in hamsters made leukopenic by treatment with 5-fluorouracil.

The L-arabinose operon in Escherichia coli is a model system for the study of the control of gene expression. Maximal expression of the araBAD operon requires two positive control components: the araC protein-L-arabinose complex and the cyclic AMP receptor protein-cyclic AMP complex. Both araC protein and cyclic AMP receptor protein are required for the initiation of transcription of araBAD mRNA. We have used the plasmid pBR322 as a vector for cloning DNA fragments that contain the araBAD promoter. The cloned ara fragments were identified by both physical and genetic tests. A restriction map was constructed and the DNA sequence of the promoter was determined. The promoter contains a site that is similar to the RNA polymerase recognition sites in the galactose and lactose operons. It also contains a region similar to the known cyclic AMP receptor protein binding sites in the galactose and lactose operons.

Nedzel and Arnold1report experimental evidence that massive absorption of viable bacteria may take place from the normal canine intestinal contents into the blood stream, an appreciable “cyclic circulation” of their test bacteria (B. prodigiosus) to and from certain mucous cavities. They report that this normal “circulation” is increased as a result of the local action of egg-white.In attempting to confirm their experimental data, we have been forced to the conclusion that while massive duodenal absorption of their test microorganisms may occasionally take place through the presumably normal intestinal mucosa, in approximately 90% of all normal dogs thus far tested by us, the intestinal absorption was much less rapid than their data would indicate. In our hands only an occasional femoral blood sample showed one or more viable microorganisms per cubic centimeter. (Table I.)Counts approaching their femoral artery data, however, were obtained from periodic or continuous blood samples withdrawn from the portal vein. (Dogs 11 to 14, Table II.) These samples were obtained by means of a glass cannula inserted into one of the portal collaterals. No very material increase in this normal portal count was observed, however, by substituting egg-white for Ringer's solution in the injection mass (Dogs 15 to 18, Table II).We have made a few introductory tests of the effects of certain local pathological conditions on the normal duodenal absorption rate. Much to our surprise, slight mechanical abrasions to the duodenal mucosa did not increase the usual portal count, nor were uniform changes in absorption rate produced by previous sensitization or immunization with B. prodigiosusor its autolysate. Work on the effects of specific immunization, however, is being continued.A marked increase in normal duodenal absorption was noted, however, as a result of local passive congestion (mechanical obstruction of portal vein).

Recombinant derived human interleukin-2 and an analog in which cysteine 125 has been replaced with alanine have been crystallized in a form suitable for x-ray diffraction. The crystals are triclinic, space group P1, with two protein molecules in the unit cell; unit cell parameters are a = 55.8 A, b = 40.1 A, c = 33.7 A, alpha = 90.0 degrees, beta = 109.3 degrees, gamma = 93.2 degrees. The interleukin-2 structure has been solved to 5.5 A resolution using heavy atom isomorphous replacement methods. The resultant low resolution model reveals a significant fraction of alpha helical secondary structure and outlines the overall tertiary structure of the molecule.

For certain organ transplantations it has been necessary for us to find two distinct protein antigens toward which dogs may be rendered equally and practically invariably hypersensitive. We have found a very wide variation in canine susceptibility to different proteins, wholly unexpected from our experience with other animal species. The following are typical results:(a) Horse Serum: Dogs injected subcutaneously with 0.5 cc. horse serum per kilogram of body weight, followed 24 hours later by an intravenous injection with the same dose, are almost invariably hypersensitive if tested after an incubation period of about 21 days. Fully 95 per cent of them are thrown into severe anaphylactic shock on intravenous injection with 1 cc. horse serum per kilogram of body weight, with fatalities in about 20 per cent of the cases. With 2 cc. horse serum per kilogram of body weight the fatalities are increased to about 40 per cent.(b) Goat Serum:Dogs injected with the same doses of goat serum and tested after the same incubation period are usually not demonstrably hypersensitive. Only about 30 per cent of them give recognizable anaphylactic reactions on intravenous injection with 2 cc. goat serum per kilogram of body weight.(c) Egg White:Dogs injected with the same doses of 50 per cent egg white (Ringer's solution) and tested after the same incubation period, give no suggestion of anaphylaxis, even on intravenous injection with doses as large as 5 cc. 50 per cent egg white per kilogram of body weight.There is evidently a determining factor in canine protein sensitization of which we are at present wholly ignorant.

Dogs injected intraperitoneally with 0.5 cc. 50 per cent horse erythrocyte suspension per kilogram of body weight followed 24 hours later by an intravenous injection with the same dose, are almost invariably hypersensitive if tested after an incubation period of about 20 days. The shock thus produced, however, differs materially from the shock in canine serum anaphylaxis.In serum anaphylaxis, the characteristic precipitous fall in arterial blood pressure and the characteristic contraction of the urinary bladder do not begin till at least 45 to 60 seconds after commencing the intravenous protein injection. This is the time necessary for the formation or liberation of histamine-like depressor substances by the hypersensitive liver and their transmission to other parts of the body. In erythrocyte anaphylaxis (Fig. 1) both reactions begin within 15 to 20 seconds after commencing the intravenous corpuscle injection, the same time relations as those observed on intravenous injection with histamine.It is evident, therefore, that the characteristic reactions in erythrocyte anaphylaxis do not depend upon hepatic function, but are presumably due to immediate humoral or vaso-motor reactions.The above tracing represents the maximum reaction we have thus far obtained in canine erythrocyte anaphylaxis, corresponding roughly with the transcent clinical symptoms reported by Kritschewsky and Friede.1

In the preceding paper1we report evidence that marked chemical alterations take place in specific foreign proteins on injection into the animal body, and that certain important immunological adaptations are due to these altered or denaturized proteins, rather than to the primary proteins originally injected. We have obtained further evidence in support of this by a study of the altered anaphylactic toxicity of specific foreign proteins on intravenous injection into normal, hypersensitive and immune dogs.Measured quantities of horse serum were injected intravenously into these animals, and at varying intervals after this injection quantitative blood transfusions were made into partially exsanguinated anaphylactic recipients. The following is a summary of our results to date:(1) Normal Dogs.Quantitative transfusions into anaphylactic recipients at any time within 6 hours after intravenous injection of 2 cc. horse serum per kg. of body weight into normal dogs invariably show the circulating blood to have a greater anaphylactic toxicity than that of control amounts of unaltered horse serum. This increase suggests the possibility that the initial change in the injected horse serum in normal dogs is an increase in the number of specific protein molecules by hydrolysis or colloidal dispersion, though other explanations of this increased toxicity are of course possible.Transfusions at the end of 24 hours show a toxicity approximately equal to that of the control horse serum dose. A slight reduction in toxicity is noted by the end of 48 hours. A toxicity equal to about a quarter of the control dose is noted at the end of 3 days. There is no recognizable anaphylactic toxicity at the end of 4 days.Quantitative titrations with rabbit precipitin show that there is little or no reduction in the amount of circulating horse protein in the injected normal dog at the end of 4 days.