Biomedical subjects

N J Lamb

Publications and source records attributed to N J Lamb.

At least 37 records · Page 2Linked to original sources

PubMed1997

Cyclin dependent kinase 5, cdk5, is a positive regulator of myogenesis in mouse C2 cells.

We have examined the expression, activity and localization of cyclin dependent kinase 5 (cdk5), during myogenesis. Cdk5 protein was found expressed in adult mouse muscle. In murine C2 cells, both the protein level and kinase activity of cdk5 showed a marked increase during early myogenesis with a peak between 36 and 48 hours of differentiation, decreasing as myotubes fuse after 60 to 72 hours. This increase in cdk5 protein level was specific for differentiation and not simply related to cell cycle arrest since it was not observed in fibroblasts grown for 48 hours in low serum medium. Indirect immunofluorescence using monospecific purified anti-cdk5 antibodies showed a low level cytoplasmic staining in proliferative myoblasts, a rapid increase in nuclear staining during the initial 12 hours of differentiation and a predominant nuclear staining in myotubes. Microinjection of plasmids encoding wild-type cdk5 into C2 myoblasts enhanced differentiation as assessed by both myogenin and troponin T expression after 48 hours of differentiation. In contrast, microinjection of plasmids encoding a dominant negative mutant of cdk5 inhibited the onset of differentiation. These data imply a previously unsuspected role for cdk5 protein kinase as a positive modulator of early myogenesis.

Animals↗

PubMed1996

cdk5 expression and association with p35nck5a in early stages of rat cerebellum neurogenesis; tyrosine dephosphorylation and activation in post-mitotic neurons.

We have examined the expression of cyclin dependent kinase (cdk) 5 protein kinase and p35nck5a, its activator subunit, during postnatal neurogenesis in rat cerebellum, using mono-specific antibodies. Both cdk5 and p35nck5a are present and associated in proliferative stages, although cdk5-p35 kinase activity is barely detectable. Cdk5-p35 activity, but not the expression of either subunit, increases up to 6-fold during neuronal differentiation. Since we observe that cdk5 is phosphorylated on tyrosine in proliferative, but not in post-mitotic stages, we suggest that post-translational regulatory mechanisms control cdk5-p35 protein kinase activity during neurogenesis.

Animals↗

PubMed1996

Microinjection strategies for the study of mitogenic signaling in mammalian cells.

First used in the analysis of dynamic changes in cell structure, microneedle microinjection allows in situ study of individual living cells as opposed to large scale metabolic analysis of heterogeneous cell culture. In addition, microinjection also offers the possibility to examine in vivo regulated processes by modulating the intracellular levels and activity of key regulatory proteins and genes in both a specific and controlled manner. A number of different strategies have been developed over the past 5 years to examine the pathways and effectors that are involved in mitogenic signaling as well as in the regulation of gene expression during the proliferative response to growth factors by normal fibroblasts. These strategies include: 1. Direct in vivo competition for various trans-activating DNA binding activities by microinjection of double-stranded oligonucleotides, microinjection of monospecific antibodies against transcription factors and microinjection of dominant negative mutants of transcription factors based upon their DNA binding domain. 2. Microinjection of purified enzymes (kinases and phosphatases) or peptides and antibodies that specifically inhibit these activities. 3. Microinjection of expression plasmids which encode various normal and epitope-tagged regulatory molecules. In many of the experiments described below, c-fos gene expression was monitored as an early marker of mitogenic response. The c-fos gene belongs to a family of genes whose transcription is activated very early after addition of growth factor (1-4). For in vivo studies, the c-fos promoter offers several unique advantages. Primarily, it is easy to manipulate. In practical terms, when mammalian fibroblasts are made quiescent (by replacing the normal growth media, with growth factors-depleted media) and subsequently activated by re-adding mitogen (growth factors, serum), c-fos RNA expression is restored within 15 minutes and the protein is specifically detected in the nuclei of cells after 90 minutes, but is no longer detectable after 3 hours. Secondly, results obtained with the c-fos promoter are directly applicable to cell growth since expression of c-fos is itself a prerequisite for proliferation as demonstrated by microinjection of anti-fos antibodies which prevented proliferation in mammalian cells (5). Thirdly, the c-fos promoter is exquisitely sensitive to agents which cause cell stress. In this respect, heat-shock, poor microinjection or microinjection in the presence of heavy metals or chelating agents in the culture media all rapidly stimulate c-fos expression. However, when compared to c-fos expression in the proliferative response, stress mediated c-fos expression is induced both more rapidly and strongly, reverses more slowly (the protein is still detectable after 5-6 hours) and does not result in cell proliferation (unpublished observation). As such, it provides an excellent internal control for identifying poor treatment and manipulation of cells . Finally, the c-fos promoter is subject to several levels of auto-regulation enabling the analysis of not only components involved in transcriptional activation , but also various aspects of transcriptional down regulation and shut-off.

Animals↗

PubMed1996

Plasma fatty acid changes and increased lipid peroxidation in patients with adult respiratory distress syndrome.

OBJECTIVE: There is a strong evidence that adult patients with acute respiratory distress syndrome (ARDS) are under severe oxidative stress, which leads to molecular damage. Using gas chromatography-mass spectrometry, our objective was to sequentially monitor changes, in intensive care unit (ICU) patients, characteristic of the oxidative loss of plasma unsaturated fatty acids and formation of the highly specific oxidation product of linoleic acid, 4-hydroxy-2-nonenal. DESIGN: Prospective, nonintervention, descriptive study. Limited statistics were applied to facilitate interpretation of the data. SETTING: ICU of a postgraduate teaching hospital. PATIENTS: Eighteen critically ill patients with an established diagnosis of ARDS requiring high FIO2 administered by mechanical ventilation were compared with ten normal, healthy controls and ten patients pre- and postcardiopulmonary bypass surgery at risk for developing ARDS. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Sixty percent of the patients with ARDS included in this study survived. Major changes in the plasma concentrations of fatty acids occurred in all patients during their stay in the ICU. Percentage decreases in plasma linoleic acid concentrations were accompanied by increases in plasma oleic and palmitoleic acid concentrations. Circulating linoleic acid concentrations were significantly (p = .0001) lower in patients with ARDS than in the two control groups. The patients with ARDS who did not survive had lower (p = .0056) plasma oleic acid values than normal healthy controls and patients at risk for ARDs as a consequence of undergoing cardiopulmonary bypass surgery. Changes in palmitoleic acid, however, did not reach significance within the different groups studied. Patients with ARDS showed higher plasma concentrations of 4-hydroxy-2-nonenal (0.433 +/- 0.048 vs. 0.523 +/- 0.069 nmol/mL plasma for survivors and nonsurvivors, respectively) when compared with normal healthy controls (0.205 +/- 0.03 nmol/mL, p = 0.0001) and cardiopulmonary bypass patients at risk for developing ARDS (0.279 +/- 0.027 nmol/mL, p = .034 prebypass). CONCLUSIONS: During intensive care treatment, patients with ARDS decrease their percentage plasma concentrations of total plasma linoleic acid, but increase their percentage concentrations of oleic and palmitoleic acids. As plasma linoleic acid concentrations decreased, there was usually an increase in plasma 4-hydroxy-2-nonenal values, one of its specific peroxidation products, suggestive of severe oxidative stress leading to molecular damage to lipids.

Adolescent↗

PubMed1996

Disruption of the actin cytoskeleton in living nonmuscle cells by microinjection of antibodies to domain-3 of caldesmon.

Two classes of affinity-purified polyclonal antibodies directed against the four different domains of gizzard caldesmon were prepared and their specificity was verified by immunochemical assays. One set of antibodies recognized exclusively domain-3 spanning residues 483-578 and the other one included IgG reactive toward the remaining domains-1, -2 and -4 corresponding to residues 1-482 and 581-756. Microinjection into cultured fibroblasts of each antibody preparation was employed to probe the functional importance of the interaction between caldesmon and tropomyosin within living nonmuscle cells. Low concentrations of the antibody to domain-3 caused a rapid, severe and reversible disassembly of the microfilament network. In contrast, the antibodies to domains-1, -2 and -4 were ineffective. The effects of the anti-domain-3 IgG were observed not only with the purified antibody but also when present in the whole caldesmon antiserum serving for its isolation. The microfilament disintegrating activity of this antibody was completely abolished upon preincubation with native caldesmon or a proteolytic caldesmon fragment encompassing amino acids 483-578. In enzyme-linked immunosorbent assay competition experiments, tropomyosin, but not F-actin, significantly decreased the binding of the domain-3 antibody to caldesmon. Consistent with recent mutational studies pinpointing to the contribution of domain-3 in the in vitro binding of cellular caldesmon to tropomyosin, the findings suggest that the microinjected domain-3 antibody selectively disrupts the association of tropomyosin with caldesmon domain-3, thereby destabilizing the protein complex and alleviating its known protective action against F-actin severing in the cell. Thus, our data further highlight the in vivo involvement of this particular domain in the attachment of non-muscle caldesmon to tropomyosin as well as the direct participation of the caldesmon-tropomyosin complex in the cytoskeletal organization of the microfilaments.

Actin Cytoskeleton↗

PubMed1995

Deregulation of translational control of the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A leads to multinucleated cells.

Efficient translation of the mRNA encoding the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A (PP2A) is prevented by an out of frame upstream AUG and a stable stem-loop structure (delta G = -55.9 kcal/mol) in the 5'-untranslated region (5'-UTR). Deletion of the 5'-UTR allows efficient translation of the PR65 alpha message in vitro and overexpression in COS-1 cells. Insertion of the 5'-UTR into the beta-galactosidase leader sequence dramatically inhibits translation of the beta-galactosidase message in vitro and in vivo, confirming that this sequence functions as a potent translation regulatory sequence. Cells transfected or microinjected with a PR65 alpha expression vector lacking the 5'-UTR, express high levels of PR65 alpha, accumulating in both nucleus and cytoplasm. PR65 alpha overexpressing rat embryo fibroblasts (REF-52 cells) become multinucleated. These data and previous results (Mayer-Jaekel, R. E., Ohkura, H., Gomes, R., Sunkel, C. E., Baumgartner, S., Hemmings, B. A., and Glover, D. M. (1993) Cell 72, 621-633) suggest that PP2A participates in the regulation of both mitosis and cytokinesis.

Animals↗

PubMed1995

Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression.

Protein phosphatase 2A (PP2A) appears to be involved in the regulation of many cellular processes. Control mechanisms that lead to the activation (and deactivation) of the various holoenzymes to initiate appropriate dephosphorylation events remain obscure. The core components of all PP2A holoenzymes are the catalytic (PP2Ac) and 63-65-kD regulatory (PR65) subunits. Monospecific and affinity-purified antibodies against both PP2Ac and PR65 show that these proteins are ubiquitously localized in the cytoplasm and the nucleus in nontransformed fibroblasts. As determined by quantitative immunofluorescence the core subunits of PP2A are twofold more concentrated in the nucleus than in the cytoplasm. Detailed analysis of synchronized cells reveals striking changes in the nuclear to cytoplasmic ratio of PP2Ac-specific immunoreactivity albeit the total amounts of neither PP2Ac nor PR65 in each compartment alters significantly during the cell cycle. Our results imply that differential methylation of PP2Ac occurs at the G0/G1 and G1/S boundaries. Specifically a demethylated form of PP2Ac is found in the cytoplasm of G1 cells, and in the nucleus of S and G2 cells. In addition nuclear PP2A holoenzymes appear to undergo conformational changes at the G0/G1 and G1/S boundaries. During mitosis PP2A is lost from the nuclear compartment, and unlike protein phosphatase 1 shows no specific association with the condensed chromatin.

Amino Acid Sequence↗

PubMed1994

Nuclear localization of p85s6k: functional requirement for entry into S phase.

Immunolocalization of a newly described isoform of p70s6k, termed p85s6k, demonstrated a predominantly nuclear location in rat embryo fibroblasts (REF-52), a compartment in which growth factor-mediated phosphorylation of S6 has recently been reported. Microinjection of expression vectors encoding either p85s6k or a fusion protein containing only the putative nuclear localization motifs led to the exclusive accumulation of both products in the nucleus. Consistent with such a localization, microinjection of affinity-purified anti-p85s6k IgG into the nucleus, but not the cytoplasm, blocked serum-induced initiation of DNA synthesis. Co-injection into the nucleus of the anti-p85s6k IgG with activated p70s6k, which lacks the antigenic epitope, rescued the S phase block, arguing that the antibody exerts its effects through inhibiting p85s6k function. The results indicate a novel role for S6 phosphorylation in the nucleus distinct from that in the cytoplasm, a role essential for mitogenesis.

Amino Acid Sequence↗

PubMed1994

Cloning and characterization of AKAP 95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase.

The subcellular location of the type II cAMP-dependent protein kinase is dictated by the interaction of the regulatory subunit (RII) with A-kinase anchor proteins (AKAPs). Using an interaction cloning strategy with RII alpha as a probe, we have isolated cDNAs encoding a novel 761-amino acid protein (named AKAP 95) that contains both RII- and DNA-binding domains. Deletion analysis and peptide studies revealed that the RII-binding domain of AKAP 95 is located between residues 642 and 659 and includes a predicted amphipathic helix. Zinc overlay and DNA binding studies suggest that the DNA-binding domain is composed of two CC/HH-type zinc fingers between residues 464 and 486 and residues 553 and 576. The AKAP was detected in a nuclear matrix fraction, and immunofluorescence using purified anti-AKAP 95 antibodies revealed a distinct nuclear staining in a variety of cell types. Direct overlay of fluorescein isothiocyanate-labeled RII alpha onto fixed rat embryo fibroblasts showed that high-affinity binding sites for RII exist in the nucleus and that these sites are blocked by an anchoring inhibitor peptide. Furthermore, AKAP 95 was detected in preparations of RII that were purified from cellular extracts using cAMP-agarose. The results suggest that AKAP 95 could play a role in targeting type II cAMP-dependent protein kinase for cAMP-responsive nuclear events.

A Kinase Anchor Proteins↗

PubMed1994

Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis.

Human cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G2-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34cdc2 protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G2 cells into premature prophase in a manner similar to microinjection of purified active p34cdc2 protein kinase. Together these data show that multisite phosphorylation of cdc25C by p34cdc2-cyclin B protein kinase occurs at the G2-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis.

Amino Acid Sequence↗

PubMed1993

p70s6k function is essential for G1 progression.

An essential step in the pathway by which growth factors trigger cellular proliferation is the induction of high levels of protein synthesis. This appears in part to be controlled by multiple phosphorylation of the ribosomal protein S6 (refs 4, 5). The main kinase responsible, p70s6k (refs 6-8), is activated through the phosphorylation of four sites clustered in a putative autoinhibitory domain, which is mediated by a signalling pathway distinct from those used by other well characterized mitogen-activated serine/threonine kinases (such as p42/p44mapk or p90rsk; refs 10, 11). Here we investigate the role of p70s6k in the mitogenic response. Microinjection of quiescent rat embryo fibroblasts with any of three distinct polyclonal antibodies to p70s6k abolishes serum-induced entry into S phase of the cell cycle. This effect is preceded by almost complete abrogation of the activation of protein synthesis and the expression of an essential immediate early gene product, c-fos. The inhibitory effect on DNA synthesis is also elicited by microinjection of the antibodies late in G1 phase, consistent with the finding that p70s6k activity remains high throughout G1.

Amino Acid Sequence↗

PubMed1993

Expression and purification of the DNA-binding domain of SRF: SRF-DB, a part of a DNA-binding protein which can act as a dominant negative mutant in vivo.

We have developed an approach which allows functional in vivo examination of DNA-binding proteins through microinjection of polypeptides containing the DNA-binding domain into living fibroblasts. The present analysis utilizes serum response factor (SRF), a transcription factor that binds to the serum response element. We have expressed in bacteria a 30-kDa portion of this protein (amino acids 113 to 265) containing the DNA-binding domain of SRF (SRF-DB) and purified it to homogeneity by a single DNA affinity chromatography step using the high-affinity SRF-binding site (ACT.L). We have tested the efficiency of SRF-DB to prevent endogenous SRF function through analysis of c-fos expression and DNA synthesis stimulated by fetal calf serum, two events known to require SRF. Injection of purified SRF-DB into rat embryo fibroblasts inhibits c-fos induction by growth factors. Moreover, DNA synthesis, induced after serum addition, is also suppressed by SRF-DB injection. This implies that overproduction of SRF-DB makes the cell deficient in the function of wild-type SRF and that SRF-DB acts as a dominant negative mutant. These data show that, for the study of DNA-binding proteins, expressing and using portions of the protein that corresponds to the DNA-binding domain present a useful method for generating dominant negative mutants and illustrate the potential application of the DNA-binding region to facilitate the study of events at the DNA/protein level.

Animals↗

PubMed1992

Protein phosphatase type 1 in mammalian cell mitosis: chromosomal localization and involvement in mitotic exit.

We have examined the role of protein phosphatase type 1 (PP-1) in mammalian cell mitosis. Immunofluorescence using anti-PP-1 antibodies revealed that PP-1, which is mainly localized in the cytoplasm of G1 and S phase cells, accumulates in the nucleus during G2 phase and intensely colocalizes with individual chromosomes at mitosis. This increase in nuclear PP-1 in G2/M cells was confirmed by immunoblotting on subcellular fractions. Microinjection of neutralizing anti-PP-1 antibodies before division blocked cells at metaphase, whereas injection of PP-1 in one pole of an anaphase B cell accelerated cytokinesis and the reflattening of the injected cell. These results reveal a specific cell cycle-dependent redistribution of PP-1 and its involvement in reversing p34cdc2-induced effects after mid-mitosis in mammalian cells.

Animals↗

PubMed1992

cdc25 is a nuclear protein expressed constitutively throughout the cell cycle in nontransformed mammalian cells.

A family of proteins homologous to the cdc25 gene product of the fission yeast bear specific protein tyrosine phosphatase activity involved in the activation of the p34cdc2-cyclin B kinase. Using affinity-purified antibodies raised against a synthetic peptide corresponding to the catalytic site of the cdc25 phosphatase, we show that cdc25 protein is constitutively expressed throughout the cell cycle of nontransformed mammalian fibroblasts and does not undergo major changes in protein level. By indirect immunofluorescence, cdc25 protein is found essentially localized in the nucleus throughout interphase and during early prophase. Just before the complete nuclear envelope breakdown at the prophase-prometaphase boundary, cdc25 proteins are redistributed throughout the cytoplasm. During metaphase and anaphase, cdc25 staining remains distributed throughout the cell and excludes the condensed chromosomes. The nuclear locale reappears during telophase. In light of the recent data describing the cytoplasmic localization of cyclin B protein (Pines, J., and T. Hunter. 1991. J. Cell Biol. 115:1-17), the data presented here suggest that separation in two distinct cellular compartments of the cdc25 phosphatase and its substrate p34cdc2-cyclin B may be of importance in the regulation of the cdc2 kinase activity.

Amino Acid Sequence↗

PubMed1992

Role of fos-AP-1 binding sequence (FAP) in the induction of c-fos expression by purified C-kinase and in c-fos down-regulation following serum induction.

Microinjection of purified calcium phospholipid-dependent protein kinase (C-kinase) resulted in the rapid and transient induction of c-fos in quiescent rat embryo fibroblats. This C-kinase-induced expression of c-fos was prevented by in vivo competition using co-injection of oligonucleotides corresponding to the sequence of either the serum response element (SRE) or the fos AP-1 binding sequence (FAP) adjacent to SRE. This indicates that both these sequences must be involved in the binding/activation of protein factors required for the induction of c-fos by C-kinase. In contrast, the induction of c-fos by serum or by casein kinase II microinjection, which is also inhibited by injection of SRE oligonucleotides, is only delayed and then markedly prolonged by injecting TRE/FAP sequence, demonstrating that the FAP site plays a prominent role in vivo in the down-regulation of the endogenous c-fos gene expression.

Animals↗

PubMed1991

Cyclin A is required for the onset of DNA replication in mammalian fibroblasts.

Cyclin A protein is synthesized and localized into the nucleus at the onset of S phase in nontransformed mammalian fibroblasts. Inhibition of cyclin A synthesis or activity through microinjection of plasmids encoding antisense cyclin A cDNA or affinity-purified anti-cyclin A antibodies during G1 phase was shown to abolish the nuclear staining for cyclin A in plasmid-injected cells, and both procedures led to inhibition of DNA synthesis. No similar effect was observed with injection of other antisense vectors including antisense cyclin B, and reinjection of purified human cyclin A protein into cyclin A antisense-injected cells effectively relieved this inhibition of DNA synthesis. Taken together, these data suggest that cyclin A plays a major role in the control of DNA replication in mammalian cells.

Animals↗

PubMed1991

Inhibition of cAMP-dependent protein kinase plays a key role in the induction of mitosis and nuclear envelope breakdown in mammalian cells.

Inhibiting cAMP-dependent protein kinase (A-kinase) in mammalian fibroblasts through microinjection of a modified specific inhibitor peptide, PKi(m) or the purified inhibitor protein, PKI, resulted in rapid and pronounced chromatin condensation at all phases of the cell cycle. Together with these changes in chromatin, a marked reorganization of microtubule network occurred, accompanied in G2 cells by extensive alterations in cell shape which have many similarities to the premitotic phenotype previously observed after activation of p34cdc2 kinase, including the lack of spindle formation and the persistence of a nuclear envelope. In order to examine whether A-kinase inhibition and p34cdc2 kinase form part of the same or different inductive pathways, PKI and p34cdc2 kinase were injected together. Co-injection of both components resulted in nuclear envelope disassembly, an event not observed with injection of either component alone. This result implies that p34cdc2 and A-kinase inhibition have complementary and additive effects on the process of nuclear envelope breakdown in living fibroblasts, a conclusion further supported by our observation of a pronounced dephosphorylation of lamins A and C in cells after injection of PKi(m). Taken together, these data suggest that down-regulation of A-kinase is a distinct and essential event in the induction of mammalian cell mitosis which co-operates with the p34cdc2 pathway.

Animals↗

PubMed1991

Casein kinase II induces c-fos expression via the serum response element pathway and p67SRF phosphorylation in living fibroblasts.

Elevation of intracellular casein kinase II (CKII) levels through microinjection of purified CKII results in the rapid and transient induction of c-fos in quiescent rat embryo fibroblasts, and activation of quiescent cells by serum is accompanied by the nuclear relocation of endogenous CKII. The induction of c-fos by CKII is inhibited by coinjection of oligonucleotides corresponding to the sequence of the serum response element (SRE) present in the c-fos promoter, indicating that competitive displacement of positive factors from the endogenous c-fos SRE prevents c-fos induction by CKII. Furthermore, the expression of c-fos induced by either CKII injection or serum activation is also inhibited by microinjection of antibodies against the 67 kDa serum response factor (p67SRF) indicating the absolute requirement of p67SRF in this process. Finally, we show the specific phosphorylation of p67SRF in vivo following microinjection of CKII into quiescent cells. Together, these data strongly support that CKII induces c-fos expression through binding/activation of the phosphorylated p67SRF at the SRE sequence.

Animals↗