Biochemical analysis of the activation of adherent neutrophils in vitro

The role played by neutrophil oxidative responses in host defense and injury is an area of active investigation. In order to study neutrophil responses in vitro, methods are required for cell purification, enumeration, and quantification of activation responses, which mimic the in vivo situation as closely as possible. In this communication (and its companion paper, Albertine et al., 1988) improved methods for all of these tasks are described and applied to investigate neutrophil structure-function relationships in vitro and in vivo. Human neutrophils were purified by using a series of platelet-poor plasma-Percoll gradients (51, 62, 76 and 80% in Percoll). This modification of previously published procedures results in consistently successful neutrophil purification and has allowed us to purify neutrophils from bronchoalveolar lavage fluid as well as blood. Activation of human and sheep neutrophils (superoxide anion production) was quantitated by the reduction of ferricytochrome c using a microtiter plate reader to measure the increase in absorbance at 550 nm from adherent neutrophils. Adherence of neutrophils was quantitated by measurement of LDH in cells lysed with Triton X-100 using a new method which uses readily available commercial reagents and can quantitate the LDH content of as few as 5000 neutrophils (or the LDH released from 5% of 100,000 neutrophils). Assay conditions for superoxide anion were optimized, limitations both in assay design and instruments used to measure OD were explored and enumerated, and these methods were used to quantitate sheep and human neutrophil activation responses. Using methods described in Albertine et al. (1988) for fixing neutrophils in microtiter wells after assay of their functional capacity, we have studied the same cells functionally and morphologically. We have used these techniques to study blood and alveolar neutrophils from a patient with acute respiratory failure. His alveolar neutrophils displayed 67% of the activation response as peripheral neutrophils (4.31 +/- 0.12 nmol superoxide released per 250,000 neutrophils at 60 min vs. 6.38 +/- 0.18 in blood, P less than 0.01) and structural changes which suggested previous activation in vivo. These studies demonstrate that similar morphological changes are observed in neutrophils activated with phorbol myristate acetate in vitro, as are observed in cells which have been activated by pathophysiologic processes in vivo.     

Ultrastructure of the approximately 26S complex containing the approximately 20S cylinder particle (multicatalytic proteinase/proteasome).

We have isolated a large protein complex of approximately 26S from Xenopus laevis oocytes and eggs which is composed of the approximately 20S cylinder particle (multicatalytic proteinase/proteasome) and additional proteinaceous components. In its polypeptide composition and sedimentation coefficient this approximately 26S complex closely resembles the 26S ubiquitin-dependent protease, a high molecular weight multienzyme complex recently described in the literature. Specific antibodies directed against a single subunit of the approximately 20S cylinder particle retain, on affinity columns, the large approximately 26S complex, and on sucrose gradients up to approximately 50% of the approximately 20S cylinder particles present in oocyte extracts sedimented with approximately 26S, suggesting that a large proportion of the approximately 20S particles exists in the cell as a component of the approximately 26S complex. Electron microscopy reveals the approximately 26S complex to be a symmetrical elongated macromolecular assembly of at least three protein particles. The central core of the complex is formed by the approximately 20S cylinder particle to which two other large components are attached at the ends, yielding a dumbbell-shaped complex of approximately 40 nm in length. Dissociation of the approximately 26S complexes releases in addition to approximately 20S cylinder particles a novel type of a disc-shaped particle of approximately 15 nm diameter which may represent the attached components or subcomplexes of them. Based on its structural and biochemical properties we postulate that the approximately 26S complex identified here is identical to the ubiquitin-dependent protease.     

Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.     

Mapping of the structural gene for S-adenosyl homocysteine hydrolase to mouse Chromosome 2, and related sequences to Chromosomes 8 and X

Comparative mapping studies in human and mouse have shown that, to date, human Chromosome (Chr) 20 is completely syntenic with distal mouse Chr 2. The structural locus for S-adenosyl-l-homocysteine hydrolase (EC 3.3.1.1) in human, AHCY, maps to 20 qterq13.1, and we report here that the homologous locus in the mouse, Ahcy, maps to distal mouse Chr 2 with gene order Pcna-Ahcy-Ada. Analysis of 123 progeny of an interspecific backcross between a laboratory stock, AN, and Mus spretus using a rat cDNA probe revealed the presence of at least two other Ahcy-related sequences segregating independently in the mouse genome. One, Ahcy-rs1, was mapped to Chr 8 in the BXH recombinant inbred strains, and the other, Ahcy-rs2, shows a pattern of inheritance consistent with X-linkage.     

Mouse Chromosome 7

Chair of Committee for Mouse Chromosome 7     

Pneumocystis carinii shows DNA homology with the ustomycetous red yeast fungi

Pneumocystis carinii causes life-threatening pneumonia in T-lymphocyte-immunodeficient subjects in transplant and oncology units or with acquired immune deficiency syndrome (AIDS). Recent DNA homology studies show P. carinii to be a fungus. To investigate the biology and epidemiology of this parasite further, we elected to determine for it a more precise taxonomic assignment within the fungal kingdom. We screened a wide range of organisms representing the major orders of fungi using DNA amplification and subsequently sequenced a portion of the mitochondrial gene encoding the large subunit ribosomal RNA. Our data show that the opportunistic pulmonary pathogen P. carinii is closely related to the ustomycetous red yeast fungi, a group which includes organisms that are extensively distributed throughout the environment and which release many widely dispersed airborne spores.     

The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen.

We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport.     

Quantitative analysis of immunogold labelling for ferritin in liver from control and iron-overloaded rats

Summary The distribution of ferritin antigenicity in control and iron-loaded rat hepatocytes was investigated with an immunogold-ferritin antibody technique. Antibody to horse spleen ferritin showed immunoreactivity as determined by dot blotting with immunogold/silver staining with purified rat liver ferritin but not with rat haemosiderin. The initial site of ferritin degradation was studied by analysing the density of gold labelling in the cytosol and lysosomes in combination with pre-embedding acid phosphatase cytochemistry.Immunoreactive ferritin was present in the cytosol, cytosolic clusters and lysosomes of normal hepatocytes. After iron-loading, the labelling density increased over tenfold in parenchymal cell cytosol with a smaller increase in Kupffer cells. Ferritin clusters contained substantially more immunoreactive ferritin than equivalent areas of lysosomes or cytosol. Analysis of the labelling density in hepatocyte lysosomes showed that, despite a striking increase in iron content, one-quarter of the lysosomes showed less immunolabelled ferritin than the cytosol. The existence of a wide range of ferritin labelling densities in the lysosomes with a large proportion unlabelled suggests that the ferritin protein shell is not degraded at a significant rate either in the cytosol or in clusters but only after incorporation into lysosomes.     

Age-related changes in secretion of luteinizing hormone and metabolism of hypothalamic amines in bull calves prior to puberty

Effects of age and castration on secretion of luteinizing hormone (LH) and metabolism of hypothalamic monoamines were determined in Holstein bulls. Calves were assigned to be intact or castrated and killed at 8, 12, or 24 wk of age. Animals were castrated and bled every 10 min for 6 h at 96 and 24 h prior to slaughter, respectively. The stalk median eminence (SME), medial basal (MBH), and anterior-preoptic (AHA-POA) hypothalamic regions were obtained at slaughter and assayed for norepinephrine (NE), dopamine (DA), dihydroxy-phenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-HT), and 5-hydroxyindole-acetic acid (5-HIAA) using high performance liquid chromatography with electrochemical detection (HPLC-EC). Concentrations of LH and testosterone in plasma were determined by radioimmunoassay (RIA). In intact calves, LH pulse frequency (pulses/6 h) increased between 8 and 12 wk (1.4 vs. 3.4) and then declined (1.6 at 24 wk of age). Frequency of LH discharges did not change during the first 72 h post-castration in calves 8 (1.4 vs 1.0) and 12 (3.4 vs. 3.8) wk of age, but increased in 24-wk-old calves during this time (1.6 vs. 6.4). The amplitude of LH pulses increased with age (p less than 0.05) and after castration (p less than 0.05). There were marked regional differences in concentrations of monoamines. However, effects of age and castration on concentrations of monoamines were observed only within the SME where DA, DOPAC and NE increased significantly with age. Plasma concentrations of testosterone were correlated with concentrations of NE and DOPAC within the SME. Changes in 5-HT with age were biphasic; at each age, 5-HT increased after castration. From these data, it is concluded that 1) different mechanisms regulate LH pulse frequency and amplitude in calves as early as 8 wk of age, and 2) differences in hypothalamic metabolism of monoamines may be related to maturational changes in secretion of LH in bull calves.     

Studies on the role of transferrin and endocytosis in the uptake of Fe3+ from Fe-nitrilotriacetate by mouse duodenum

Addition of iron-binding proteins (human serum transferrin, mouse serum transferrin, human lactoferrin) to the luminal fluid in tied-off segments of mouse intestine in vivo led to reduced 59Fe3+ absorption from 59Fe3+-nitrilotriacetate when compared to 59Fe3+-nitrilotriacetate alone. Assay of transferrin in luminal fluid from tied segments revealed only trace amounts of immunoreactivity. The levels of luminal transferrin are unaltered in chronic hypoxia where iron absorption is significantly enhanced. Studies in vitro revealed that NH4Cl, dansylcadavarine, para-chloromercuribenzoate and trinitrobenzenesulphonate have no effect on initial 59Fe3+ uptake rates from 59Fe3+-nitrilotriacetate, while N-ethylmaleimide (1 mM) caused a 40% inhibition. In vivo 59Fe3+ uptake was unaffected by preincubation of tied-off segments with colchicine (5 mM) for up to 2 h. These results suggest that receptor-mediated endocytosis of transferrin is not a significant mechanism in the uptake of luminal Fe3+ by mouse duodenum.