Fc receptor-mediated binding and endocytosis by human mononuclear phagocytes: monomeric IgG is not endocytosed by U937 cells and monocytes.

Fc receptor-mediated endocytosis of monomeric IgG1 by human mononuclear phagocytes was evaluated under conditions where aggregated IgG and insulin readily undergo receptor-mediated internalization. U937 cells or normal human peripheral blood monocytes were incubated at 37 degrees C in the absence of free radioligand after having first bound 125I-IgG1 at 0 degrees C. To determine the amount of cell-associated IgG1 internalized after varying periods of 37 degrees C incubation, surface-bound IgG1 was removed by sequential exposure of cells at 0 degrees C to a nonspecific proteinase for 1 h and to acetic acid at pH 3.2 for 3 min. The failure to develop a proteinase- and acid-resistant fraction, similar to that seen over time at 37 degrees C in parallel experiments with 125I-insulin and 125I-aggregated IgG, and the lack of degradation of the IgG1 released into the medium from the same cells over time show that these cells do not endocytose and degrade monomeric IgG by an Fc receptor-specific mechanism and suggest that constitutive recycling without degradation is unlikely to be occurring. These data fulfill one prediction of the hypothesis that receptor-receptor interaction triggers Fc receptor-mediated endocytosis.

Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland.

Mitochondrial biogenesis in the parenchymal cell of the mouse mammary gland appears to occur in two distinct phases: replication during cell proliferation, and maturation during cell differentiation. This study of the mitochondrial maturation phase in the mouse gland demonstrates a significant increase in organelle density on isopycnic sucrose gradient centrifugation during the transition from late pregnancy to day 8 of lactation. Differential fragility to high sucrose concentrations or changes in mitochondrial lipid composition do not satisfactorily explain the density increases. When organelle densities were assessed by centrifugation under iso-osmotic conditions with Ficoll gradients in 0.25 M sucrose, the mitochondria from pregnant glands were observed to be more dense than those from lactating glands. The two mitochondrial populations were also found to differ in their response to changes in sucrose concentration in the Ficoll gradients. When sucrose concentration was increased, the density of both pregnant and lactating gland mitochondria increased nonlinearly, the increase being greater with the lactating gland organelles. By use of mathematical models, the differing response was interpreted as a change in the density and osmotic activity of the mitochondrial internal compartment (inner membrane plus matrix space). We have proposed that the changes reflect a large expansion of the inner mitochondrial membrane and perhaps the matrix material during the transition into lactation in the differentiating parenchymal cell.

PIP: Developmental changes in mitochondria during pregnancy and the transition into lactation in the parenchymal cell of the mouse mammary gland were studied. There were 2 apparent distinct phases in mitochondrial biogenesis: replication during cell proliferation and maturation during cell differentiation. Isopycnic sucrose gradient centrifugation during the transition from late pregnancy to Day 8 of lactation revealed a marked increase in organelle density. This increase in organelle density could not be explained by differential fragility to high sucrose concentrations or changes in mitochondrial lipid composition. Mitochondria from pregnant glands were more dense than those from lactating glands as determined by centrifugation under iso-osmotic conditions with Ficoll gradients in .25 M sucrose. These mitochondrial populations also differed in their response to changes in the concentration of sucrose in the Ficoll gradients. The density of both pregnant and lactating gland mitochondria increased nonlinearly when the sucrose concentration was increased, with the increase in density being greater in the latter. The difference in response was mathematically interpreted to reflect a change in the density and osmotic activity of the mitochondrial internal compartment (inner membrane plus matrix space). It is proposed that the developmental changes observed reflect a large expansion of the inner mitochondrial membrane and possibly the matrix material during the transition into lactation in the differentiating parenchymal cell.

Embryonic expression of the putative gamma subunit of the sodium pump is required for acquisition of fluid transport capacity during mouse blastocyst development.

The sodium/potassium pump, Na+,K+-ATPase, is generally understood to function as a heterodimer of two subunits, a catalytic alpha subunit and a noncatalytic, glycosylated beta subunit. Recently, a putative third subunit, the gamma subunit, was cloned. This small protein (6.5 kD) coimmunoprecipitates with the alpha and beta subunits and is closely associated with the ouabain binding site on the holoenzyme, but its function is unknown. We have investigated the expression of the gamma subunit in preimplantation mouse development, where Na+, K+-ATPase plays a critical role as the driving force for blastocoel formation (cavitation). Using reverse transcriptase-polymerase chain reaction, we demonstrated that the gamma subunit mRNA accumulates continuously from the eight-cell stage onward and that it cosediments with polyribosomes from its time of first appearance. Confocal immunofluorescence microscopy revealed that the gamma subunit itself accumulates and is localized at the blastomere surfaces up to the blastocyst stage. In contrast with the alpha and beta subunits, the gamma subunit is not concentrated in the basolateral surface of the polarized trophectoderm layer, but is strongly expressed at the apical surface as well. When embryos were treated with antisense oligodeoxynucleotide complementary to the gamma subunit mRNA, ouabain-sensitive K+ transport (as indicated by 86Rb+ uptake) was reduced and cavitation delayed. However, Na+, K+-ATPase enzymatic activity was unaffected as determined by a direct phosphorylation assay ("back door" phosphorylation) applied to plasma membrane preparations. These results indicate that the gamma subunit, although not an integral component of Na+,K+-ATPase, is an important determinant of active cation transport and that, as such, its embryonic expression is essential for blastocoel formation in the mouse.

Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. II. Membrane marker enzymes and membrane ultrastructure.

The activity of cytochrome oxidase (an inner mitochondrial membrane marker) in mouse mammary gland homogenates was found to increase five- to sixfold from late pregnancy to day 8 of lactation, while that of monoamine oxidase (an outer membrane marker) increased only about 25%. The specific activity of cytochrome oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased fivefold. This reflects the fact that both cytochrome oxidase and mitochondrial protein are increasing at a much greater rate than is monoamine oxidase activity. Mixing experiments preclude the possibility that the release or removal of an inhibitor or stimulator produces the changes in enzymatic activity. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. In each preparation the pattern was the same with little change in the ratio until late pregnancy; and then a three- to fourfold increase occurred and the values reached a maximum by day 8 of lactation. These experiments were interpreted as demonstrating that the observed enzymatic changes are reflective of alterations in the mitochondria of the mammary parenchymal cell population. Electron micrographs of mid-pregnant and mid-lactating mammary parenchymal cells in situ were prepared, and distinct changes in the mitochondrial morphology noted. The most significant and obvious change is the large increase in the number of inner membrane cristae and an increase in matrix density in the lactating gland cell. Therefore, both enzymatic and morphological studies support the concept of an expansion of the mitochondrial inner membrane during presecretory differentiation in the mouse mammary parenchymal cell.

PIP: The enzyme markers for mitochondrial inner and outer membranes throughout the pregnancy-lactation cycle in the mouse were compared. The ultrastructural changes of the organelle during the transitions were studied by electron microscopy. The activity of cytochrome oxidase in mouse mammary gland homogenates was found to increase 5- to 6-fold from late pregnancy to Day 8 of lactation, while that of monoamine oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased 5-fold. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. The pattern was the same in each preparation with little change until late pregnancy and then a 3- to 4-fold increase occurred and values reached a maximum by Day 8 of lactation. Electron micrographs of midpregnant and midlactating mammary parenchymal cells in situ were prepared, and changes in the mitochondrial morphology noted. The most significant change is the large increase in number of inner membrane cristae and an increase in matrix density in the lactating gland cell.

A pilot randomised controlled trial of a home-based exercise programme aimed at improving endurance and function in adults with neuromuscular disorders.

AIM: To investigate the feasibility and effect of a home-based exercise programme on walking endurance, muscle strength, fatigue and function in people with neuromuscular disorders (NMDs). METHODS: 20 adults with NMDs recruited to a control (n = 11) or exercise (n = 9) group were assessed by blinded assessors at baseline and at week 8. Walking and strengthening exercises were given to the exercise group in an 8-week home exercise programme. A 2-min walk distance was the main outcome measurement; isometric muscle strength, fatigue and function were secondary measurements. RESULTS: 2-min walk distances were not found to change in either group (p>0.05; control: mean 14.50 (SD 22.06) m; exercise: mean 2.88 (SD 20.08) m), and no difference was observed in the change scores between groups (p>0.05). Leg muscle strength increased in the exercise group (p<0.05) but not in the control group (p>0.05). Significance was reached between the groups with respect to the difference in change in muscle strength scores in the right quadriceps (p<0.05; control: mean -2.82 (SD 4.87) kg; exercise: mean -7.08 (SD 2.82) kg). No change was observed in fatigue or function scores (p<0.05). CONCLUSIONS: A home-based approach aimed at improving endurance in adults with NMDs is feasible and further investigation on a larger sample is warranted.

Application of Maximum Entropy reconstruction to PISEMA spectra.

Maximum Entropy reconstruction is applied to two-dimensional PISEMA spectra of stationary samples of peptide crystals and proteins in magnetically aligned virus particles and membrane bilayers. Improvements in signal-to-noise ratios were observed with minimal distortion of the spectra when Maximum Entropy reconstruction was applied to non-linearly sampled data in the indirect dimension. Maximum Entropy reconstruction was also applied in the direct dimension by selecting sub-sets of data from the free induction decays. Because the noise is uncorrelated in the spectra obtained by Maximum Entropy reconstruction of data with different non-linear sampling schedules, it is possible to improve the signal-to-noise ratios by co-addition of multiple spectra derived from one experimental data set. The combined application of Maximum Entropy to data in the indirect and direct dimensions has the potential to lead to substantial reductions in the total amount of experimental time required for acquisition of data in multidimensional NMR experiments.

Relative mitogenic activities of wild-type and retinoblastoma binding-defective SV40 T antigens in serum-deprived and senescent human diploid fibroblasts.

A novel gene transfer approach was used to investigate whether the retinoblastoma (Rb)-binding domain of simian virus 40 (SV40) T antigen is required for efficient T antigen-mediated stimulation of DNA synthesis is quiescent or senescent human embryo fibroblasts. In senescent cells, comparison between wild-type T antigen and a mutant defective in Rb binding (Glu-107-->Lys) revealed the latter to have approximately 15-fold lower activity. In contrast, comparison of wild-type and Rb- T antigens in serum-deprived quiescent cells revealed a much smaller (1.8-fold) difference. Surprisingly, an 18-fold differential could be induced by treating quiescent cells with the differentiating agent sodium butyrate. These results suggest that the role of Rb in control of the cell cycle is strongly dependent on the physiological state of the cell and the mechanism of growth arrest.

Activity of L-phenylalanine ammonia-lyase in organic solvents.

L-Phenylalanine ammonia-lyase (EC 4.3.1.5), (PAL) was shown to be active in a monophasic non-aqueous medium for the first time. Ultraviolet absorbance spectra of trans-cinnamic acid were shown to be similar in both water and n-octanol. High catalytic rates were observed only when the enzyme was placed in solvents containing high concentrations of water. PAL forward reaction was observed only when the water concentration in n-octanol exceeded 2.0% (v/v), which corresponds to a value of 0.8 in thermodynamic water activity (aw) terms. In n-octanol containing either 2.0 or 3.5% (v/v) H2O (and 2 mM L-phenylalanine), lyophilized and aw = 0.113 pre-equilibrated PAL powder exhibited catalytic rates 0.02 and 1.75% of the value observed in aqueous solution respectively. A freshly lyophilized (non-equilibrated) PAL preparation incubated in water-saturated n-octanol (measured [H2O] = 3.6% (v/v), L-phenylalanine concentration approximately 6.8 mM) gave catalytic activity values 17% of those observed in aqueous solution. This is the first demonstration of catalytic activity of an amino acid ammonia-lyase in monophasic organic solvent.

Sequence-related behaviour of transmembrane domains from class I receptor tyrosine kinases.

2H NMR spectroscopy and freeze-fracture electron microscopy were used to compare the transmembrane domains of two Class I protein receptor tyrosine kinases (the EGF receptor and Neu/erbB-2) regarding overall behaviour in fluid lipid bilayer membranes. The 34-residue peptide, EGFRtm, was synthesised to contain the 23 amino acid hydrophobic stretch (Ile622 to Met644) thought to span the membrane of the human EGF receptor, plus the first 10 amino acids (Arg645 to Thr654) of the cytoplasmic domain. Deuterium probes replaced selected 1H nuclei at sites corresponding to Ala623, Met644, and Val650. The 38-residue peptide, Neutm, was synthesised having the 21 residue hydrophobic stretch (Ile660 to Ile680) calculated to span the membrane in rat Neu/erbB-2, plus residues Lys681 to Thr691 of the contiguous cytoplasmic domain. Deuterium probes replaced selected 1H nuclei at Ala661, Leu667, and Val676. A third peptide, Neutm*, was also prepared, corresponding to the transmembrane domain of a constitutively-activating Neu/erbB-2 transformant in which Val664 is replaced by Glu: it was deuterated in a manner identical to Neutm. Peptides were studied by 2H NMR spectroscopy at 1 mol% and 6 mol% in unsonicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and in POPC containing 33 mol% cholesterol, over the range 12 degrees to 65 degreesC. Overall motion was found to be different for each of the three peptides under a given set of conditions. EGFRtm spectra were characteristic of axially symmetric motion in membranes of POPC alone, and in POPC/cholesterol at 35 degreesC and above. In contrast, spectra of the transmembrane peptides, Neutm and Neutm*, were characteristic of significantly axially asymmetric motion under all conditions studied (and regardless of sample preparation method). Addition of 33% cholesterol to membranes was accompanied by spectral changes consistent with increased formation of peptide dimers/oligomers in all cases. The transformant peptide, Neutm*, showed greater spectral evidence of immobilisation than did the wild type - probably reflecting a greater tendency to form large oligomers. Sequence-related details within the transmembrane domains of Class I receptor tyrosine kinases appear to exert important control over their associations within membranes. Freeze-fracture electron microscopy of the NMR samples demonstrated their liposomal nature. Peptide-related intramembranous particles (IMPs) were present which likely represent oligomers of the transmembrane peptide. IMP size and distribution were similar under a given set of conditions for all three peptides, suggesting that the differences seen by NMR spectroscopy reflect structures smaller than the 2 nm resolution limit of freeze-fracture EM and peptide relationships within its 20 nm accuracy of identifying lateral position.

Diabetic complications in a patient with coexisting anterior hypopituitarism.

We present a patient who developed insulin-requiring diabetes several years after the onset of symptoms of anterior hypopituitarism. It is likely that the hypopituitarism protected him against the development of diabetic retinopathy and glomerular basement membrane thickening but not against neuropathy and atheroma. The significance of this in relation to the growth-hormone-microangiopathy hypothesis is discussed.

NMR experiments on aligned samples of membrane proteins.

NMR methods can be used to determine the structures of membrane proteins. Lipids can be chosen so that protein-containing micelles, bicelles, or bilayers are available as samples. All three types of samples can be aligned weakly or strongly, depending on their rotational correlation time. Solution NMR methods can be used with weakly aligned micelle and small bicelle samples. Solid-state NMR methods can be used with mechanically aligned bilayer and magnetically aligned bicelle samples.

Expression and structural analysis of 14-3-3 proteins.

The 14-3-3 family of proteins plays a role in a wide variety of cellular functions including regulation of protein kinase C and exocytosis. Using antisera specific for the N termini of 14-3-3 isoforms described previously and an additional antiserum specific for the C terminus of epsilon isoform, protease digestion of intact 14-3-3 showed that the N-terminal half of 14-3-3 (a 16 kDa fragment) was an intact, dimeric domain of the protein. Two isoforms of 14-3-3, tau and epsilon, were expressed in E. coli and their secondary structure was shown by circular dichroism to be identical to wild-type protein, and expression of N-terminally-deleted epsilon 14-3-3 protein showed that the N-terminal 26 amino acids are important for dimerization. Intact 14-3-3 is a potent inhibitor of protein kinase C, but the N-terminal domain does not inhibit PKC activity. Site-specific mutagenesis of several regions in the tau isoform of 14-3-3, including the mutation of a putative pseudosubstrate site to a potential substrate sequence, did not alter its inhibitory activity. Intact 14-3-3 proteins are phosphorylated by protein kinase C with a low stoichiometry, but truncated isoforms are phosphorylated much more efficiently by this kinase. This may imply that the proteins may adopt a different structural conformation, possibly upon binding to the membrane, which could modulate their activity. 14-3-3 proteins are found at high concentration on synaptic plasma membranes and this binding is mediated through the N-terminal 12 kDa of 14-3-3.

Panhandle PCR: a technical advance to amplify MLL genomic translocation breakpoints.

Translocations involving a breakpoint cluster region of the MLL gene at chromosome band 11q23 are the most common molecular abnormalities in acute leukemias of infants and acute leukemias related to chemotherapy with DNA topoisomerase II inhibitors. Molecular cloning of MLL genomic breakpoints by PCR has previously been difficult because MLL has many translocation partners and several breakpoints involve unknown partner genes. We review a new approach to MLL genomic breakpoint cloning called panhandle PCR. By adding an oligonucleotide sequence to the unknown 3' partner gene that is complementary to a known 5' MLL sequence, we have been able to generate a genomic template with an intrastrand loop for PCR schematically shaped like a pan with a handle. The intrastrand loop contains the translocation breakpoint and unknown partner DNA, while the handle contains the known 5' sequence from MLL and a complement to that sequence. Primers both derived from MLL are used to amplify the breakpoint by panhandle PCR. Panhandle PCR offers the advantage of having specificity for the strand of interest at both primer annealing sites without requiring specific primers for the many partner genes of MLL. Panhandle PCR is a straightforward method that represents a technical advance in MLL genomic breakpoint cloning.

Quantitation of intracellular Mac-1 (CD11b/CD18) pools in human neutrophils.

The adhesive glycoprotein Mac-1 (CD11b/CD18) of the CD11/CD18 complex contributes to multiple neutrophil inflammatory functions. Activation of neutrophils by chemotactic stimuli results in a rapid, protein synthesis-independent increase in surface Mac-1 derived from incompletely defined intracellular compartments. Therefore, we developed a novel quantitative lectin immunoblot technique to define intracellular pools of Mac-1 in subcellular neutrophil fractions resolved on discontinuous Percoll gradients. In cavitates of unstimulated neutrophils, 30% and 26% of total Mac-1 was identified in beta [1.10 gm/ml; vitamin B12 binding protein (vit B12 B.P.)-rich] or pre-gamma (1.07 gm/ml; vit B12 B.P.-poor) granular fractions, respectively, whereas 24% was associated with the plasma membrane-rich gamma (1.06 gm/ml) fractions. N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation (10(-8) M, 15 min, 37 degrees C) significantly diminished Mac-1 in pre-gamma (-18% of total, P less than 0.05) but not beta fractions (+6% of total). Under these conditions, the content of Mac-1 in gamma fractions increased 13% in association with four- to eightfold increase in surface Mac-1 expression (OKM-1 binding). These findings suggest that chemotactic stimuli increase plasma membrane and/or surface Mac-1 on human neutrophils by mobilizing a novel intracellular granule pool.

Rotational osteotomies for congenital radioulnar synostosis.

In five children, six forearms with a fixed pronation deformity secondary to congenital radioulnar synostosis were treated by a derotation osteotomy of the distal radius and the midshaft of the ulna. There were three boys and two girls with a mean age of 4.9 years (3.5 to 8.25) who were followed up for a mean of 29 months (18 to 43). The position of the forearm was improved from a mean pronation deformity of 68 degrees (40 degrees to 80 degrees ) to a pre-planned position of 10 degrees of supination in all cases. Bony union was achieved by 6.3 weeks with no loss of correction. There was one major complication involving a distal radial osteotomy which required exploration for a possible compartment syndrome.

Biodegradable microparticles as a delivery system for measles virus cytotoxic T cell epitopes.

Cytotoxic T-cell (CTL) responses are likely to be important for the clearance of a measles virus (MV) infection. To induce CTL responses. replicating vectors have generally been used but the use of such vectors in humans mav be problematic, and immunization with synthetic peptides may be more appropriate. We have investigated the potential of poly(lactide-co-glycolide)(PLG) microparticles as a delivery system for a CTL epitope representing residues 51-59 from MV nucleoprotein. After a single intraperitoneal injection in saline of the encapsulated epitope, CTL responses to the homologous peptide and MV were detected over a period of 4 months. Responses reached a maximum 30 days after priming and were maintained at high levels for 120 days. These responses were higher than those observed when the CTL epitope was administered in saline or as an emulsion in Incomplete Freund's Adjuvant. The pronounced immunostimulatory effect of microparticles, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic peptide immunogens.

Pilin C-terminal peptide binds asialo-GM1 in liposomes: a 2H-NMR study.

Wideline 2H-NMR observations are described demonstrating the interaction of a synthetic peptide (PAK), representing residues 128-144 of the binding domain of pilin surface protein from Pseudomonas aeruginosa, with a complex glycosphingolipid thought to be its natural receptor. The receptor glycolipid (asialo-GM1) carried 2H probe nuclei on the terminal and next-to-terminal carbohydrate residues and was present as a minor component in fluid phosphatidylcholine liposomes. The peptide induced spectral changes that could be understood as arising from receptor motional changes, without receptor immobilization on the NMR time scale of 10(4) s-1. Spectral effects were reversed by reduction of the single peptide disulfide bond--a structural feature previously shown to be a determinant of PAK conformation (Campbell AP, McInnes C, Hodges RS, Sykes BD. 1995. Biochemistry 34:16255-16268). This is the first demonstration of PAK interaction with its epithelial cell receptor in liposomes.

The mouse Na+-K+-ATPase gamma-subunit gene (Fxyd2) encodes three developmentally regulated transcripts.

The Na(+)-K(+)-ATPase is understood to function as a hetero-oligomer of alpha- and beta-subunits, but a third subunit, gamma, has been proposed to influence the enzyme's catalytic function. Recently, two variants of the gamma-subunit have been described in kidney, raising the possibility of multiple gamma-subunits with diverse functions. We now report the cloning and sequencing of the mouse gamma-subunit gene (Fxyd2). Analysis of the structure of the gene shows that it encodes three mRNAs that have distinct NH(2)-terminal (extracellular) encoding sequences but common transmembrane and COOH-terminal-encoding sequences resulting from differential splicing and, probably, alternate promoter usage. The three mRNAs have tissue-specific expression patterns. The existence of three different extracellular domains of the gamma-variants and how they may interact with the sodium pump to alter its cation transport properties must now be taken into account for future understanding of the modulation of the Na(+)-K(+)-ATPase by its gamma-subunit.

Stability and detection of recombinant pre-pro-concanavalin A after cytoplasmic expression in Escherichia coli.

The cDNA for pre-pro-concanavalin A (pre-pro-Con A) from Canavalia ensiformis was used to construct two cytoplasmic expression vectors: pKconA (no transcription terminator) and pTKconA (containing the cry transcription terminator). The latter produced 2- to 3-fold greater amounts of pre-pro-Con A. This product containing the plant signal can be detected by Western blotting only after electrophoretic transfer in the presence of SDS, indicating reduced solubility. The signal is not removed and pre-pro-Con A is clearly stable after expression in E. coli JM109. The protein is not cleaved and ligated as in the plant, in contrast to a recent report.

Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: implications for function as adapter proteins.

14-3-3 proteins play a role in many cellular functions: they bind to and regulate several proteins which are critical for cell proliferation and differentiation. 14-3-3 proteins exist as dimers, and in this study we have shown that diverse 14-3-3 proteins can form both homo- and heterodimers in vitro (by cross-linking studies) and in vivo (by coimmunoprecipitation and Western blot analysis); this interaction is mediated solely through the N-terminal domain of the proteins. The composition of 14-3-3 dimers within a cell may play a key part in the role of this family of proteins as modulators or adapters which facilitate the interaction of distinct components of signalling pathways.