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Biomedical subjects

M A Cahill

Publications and source records attributed to M A Cahill.

At least 19 recordsLinked to original sources
PubMed2004

Differential and quantitative molecular analysis of ischemia complexity reduction by isotopic labeling of proteins using a neural embryonic stem cell model.

The analysis of rapid changes of protein expression in living systems in response to insults requires rigorous methods of complexity reduction. To control dynamic pattern of hundreds or even thousands of protein isoforms, we applied a novel method of differential molecular analysis to a cellular model which is suited to study ischemia. Neural derivatives of murine embryonic stem cells were exposed to chemical ischemia. The model was used to obtain starting material for a quantitative differential proteomics analysis. Fractionation of phosphoproteins from these samples and subsequent identification by mass spectrometry of differential proteins provide proof of principle of how novel molecular analytical tools provide new insight into the network of neuroprotective molecular events during specific situations of neuronal stress and related pharmaceutical intervention. Our results indicate a particular role of an isoform of the acidic calcium-independent phospholipase A2 in this type of insult.

Alkylation↗
PubMed2000

Improved sensitivity proteomics by postharvest alkylation and radioactive labelling of proteins.

We describe approaches to improve the detection of proteins by postharvest alkylation and subsequent radioactive labeling with either [3H]iodoacetamide or 125I. Database protein sequence analysis suggested that cysteine is not suitable for detection of the entire proteome, but that cysteine alkylating reagents can increase the number of proteins able to be detected by iodination chemistry. Proteins were alkylated with beta-(4-hydroxyphenyl)ethyl iodoacetamide, or with 1,5-l-AEDANS (the Hudson Weber reagent). Subsequent iodination using the Iodo-Gen system was found to be most efficient. The enhanced sensitivity obtainable by using these approaches is expected to be sufficient for visualization of the lowest copy number proteins from human cells, such as from clinical samples. However, we argue that significantly improved methods of protein separation will be necessary to resolve the large number of proteins expected to be detectable with this sensitivity.

Acetamides↗
PubMed1999

MAPKAP kinase 2 phosphorylates serum response factor in vitro and in vivo.

Several growth factor- and calcium-regulated kinases such as pp90(rsk) or CaM kinase IV can phosphorylate the transcription factor serum response factor (SRF) at serine 103 (Ser-103). However, it is unknown whether stress-regulated kinases can also phosphorylate SRF. We show that treatment of cells with anisomycin, arsenite, sodium fluoride, or tetrafluoroaluminate induces phosphorylation of SRF at Ser-103 in both HeLa and NIH3T3 cells. This phosphorylation is dependent on the kinase p38/SAPK2 and correlates with the activation of MAPKAP kinase 2 (MK2). MK2 phosphorylates SRF in vitro at Ser-103 with similar efficiency as the small heat shock protein Hsp25 and significantly better than CREB. Comparison of wild type murine fibroblasts with those derived from MK2-deficient mice (Mk(-/-)) reveals MK2 as the major SRF kinase induced by arsenite. These results demonstrate that SRF is targeted by several signal transduction pathways within cells and establishes SRF as a nuclear target for MAPKAP kinase 2.

3T3 Cells↗
PubMed1997

Antioxidants as well as oxidants activate c-fos via Ras-dependent activation of extracellular-signal-regulated kinase 2 and Elk-1.

Intracellular reactive oxygen intermediate (ROI) levels play an important role in numerous physiological and pathophysiological conditions. Apart from causing oxidative stress and damage, ROI changes differentially activate gene expression. However the proto-oncogene encoding the AP-1 transcription factor subunit c-Fos is induced by both prooxidants and antioxidants. Here, the transcription factor Elk-1 is identified as being responsible for c-fos serum response element (SRE) induction in response to changes in the cellular redox status induced by treatment with either the oxidant H2O2 or various structurally unrelated antioxidants. A temporal correlation is observed between changes in the phosphorylation status of Elk-1 and the activation of the mitogen-activated protein kinase 2 (ERK2) in response to cellular redox changes. Correspondingly, the transcriptional response of the SRE to redox fluctuations is attenuated upon mutation of critical ERK2 target residues within the Elk-1 transactivation domain to alanine. Signals elicited by antagonistic intracellular redox changes converge at or above the level of Ras or an effector of Ras, leading to similar activation of c-fos transcription, since an [N17]Ras mutant interfered with redox signaling. Hence components of signaling pathways are revealed to be shared by mitogenic and redox-dependent stimuli.

Antioxidants↗
PubMed1997

c-Fos transrepression revisited.

The c-fos proto-oncogene was discovered by homology to transforming viral genes, leading to speculation that transforming viruses had captured a cellular gene involved in cell cycle control. Indeed overexpression of c-Fos protein led to deregulated growth control, and c-Fos was thought to be so critically involved in cell cycle control that transcriptional transrepression of its own promoter was interpreted as a negative feedback mechanism. However, recent findings render this conclusion improbable, Fos transrepression being most parsimoniously explained as transcriptional squelching imposed by artificially elevated levels of exogenous Fos protein.

3T3 Cells↗
PubMed1996

Crosslinking of SRF to the c-fos SRE CArG box guanines using photo-active thioguanine oligodeoxynucleotides.

6-Thioguanine (thioG) was chemically incorporated into 25-base oligodeoxynucleotides encoding the c-fos serum response element (SRE) at positions corresponding to each guanine of the CArG box, which only slightly impaired DNA binding by the Serum Response Factor (SRF). Upon exposure to long wavelength UV light each thioG-containing SRE could be crosslinked to SRF, with efficiencies ranging from < 1 to 25% of the complex depending on the position of thioG in the SRE and on the UV source used. Crosslinking was strongest to the 3' side of the CArG box, and to the outer rather than the inner CArG box guanines, consistent with hydrogen bonds formed between SRF and the outer guanines in the crystal structure [Pellegrini et al., Nature 376, 490, 1995]. The crosslinked product was found to be chemically unstable. Possible mechanisms of crosslink formation are discussed.

Cross-Linking Reagents↗
PubMed1996

CD95 (APO-1/Fas) induces activation of SAP kinases downstream of ICE-like proteases.

Triggering of CD95 (APO-1/Fas) on different T- and B-cell lines resulted in the induction of a number of kinases (35 kDa, 38 kDa, 46 kDa and 54 kDa) that phosphorylate c-Jun and to a lesser extent Histone H1. Activation of these kinases was independent of protein biosynthesis and preceded apoptotic DNA degradation. The kinase activation pattern was specific for CD95 triggering since a variety of physical or chemical inducers of T- and B-cell apoptosis activated different kinases. The kinase activities at 46 and 54 kDa contained members of the stress-activated family of protein kinases (JNK/SAPK). Activation of the CD95-specific set of kinases was prevented by treating cells with the ICE-inhibiting peptide N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) or by overexpression of the cow pox virus serpin CrmA. However, despite inhibition of ICE-like proteases the death signal was readily initiated at the cell membrane since a CD95 death-inducing signaling complex (DISC) was formed. Thus, our results demonstrate that ICE-like proteases in the CD95 pathway function downstream of the DISC but upstream of SAP kinases.

Animals↗
PubMed1996

Signalling pathways: jack of all cascades.

The transcription factors that bind the c-fos promoter element SRE are targeted by multiple, independent signalling cascades; the identities of these signalling pathways and their modes of activation are being elucidated.

Animals↗
PubMed1995

Fibroblasts at the transection site of the injured goldfish optic nerve and their potential role during retinal axonal regeneration.

The region at and around the site of optic nerve transection (ONS) in goldfish, topologically the equivalent of the glial scar in mammals, is reported to remain free of astrocytes over weeks, but its cellular constituents are unknown. To learn what type of cell occupies the site of injury and thus provides support for the rapidly regenerating retinal growth cones, immunostaining experiments at the light microscopic level and electron microscopic examinations were undertaken. Between 2 and 30 days after ONS, an area up to 150 micrograms wide at the transection site exhibits intense anti-fibronectin immunoreactivity. This site contained cells and processes with ultrastructural characteristics of fibroblasts and abundant collagen fibrils. Moreover, on fibroblast cultures derived from regenerating optic nerves, retinal axons grew to considerable density in vitro. Since fibroblasts are constituents of the interfascicular spaces and outer nerve sheath of the normal goldfish optic nerve, the present data imply that fibroblasts of either source migrate into the lesion. Judging form fibronectin immunostaining they remain there during the passage of regenerating axons, and thus may provide physical and perhaps molecular support for axon growth. The fibroblasts are again restricted to interfascicular spaces after restoration of the astrocytic glia limitans around regenerated fascicles.

Animals↗
PubMed1995

Transcriptional repression mediated by the serum response factor.

The serum response element (SRE) contributes to transcriptional repression of the c-fos proto-oncogene. We show that the transcription factor SRF is able to repress SRE-dependent transcription, apparently by sequestering a co-activator. Only the DNA-binding core region is required for this SRE-dependent repression. Furthermore the phosphorylation status at potential casein kinase II sites within an N-terminal repression domain affects SRE-independent transcription. SRF may thus pleiotropically influence cellular transcription, representing a novel aspect of SRF function.

3T3 Cells↗
PubMed1995

Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction.

Anisomycin↗
PubMed1994

Co-occurrence of CArG boxes and TCF sites within viral genomes.

The transcription factor SRF is involved in the transduction of extracellular signals into nuclear responses, often in conjunction with ternary complex factors (TCFs). Here we report the identification of CArG box SRF binding-sites, and neighboring TCF binding-sites, in viral genomes. SRF binds and recruits TCFs to CMV, RSV and HTLV-1 viral genomes. At least one of two specific CArG boxes occurred in cytomegaloviruses in the 5' proximal region of the major immediate early gene, one always accompanied by a TCF site. This conservation was striking since neither the flanking sequences nor the spacing to the CAP site were conserved. Thus the ubiquitous SRF and TCF molecules may control events in the life cycle of viruses.

3T3 Cells↗
PubMed1994

Regulatory squelching.

An important function of transcription factors may be to sequester coactivators or corepressors of transcription. In this manner transcription factors could regulate in trans the activity of promoters to which they do not bind. This may be of widespread significance as a mechanism to control cell cycle-dependent and differentiation-specific transcriptional activity within eukaryotic cells. Therefore squelching in vivo may be important than hitherto appreciated.

Animals↗
PubMed1993

Sensory and neurosecretory innervation of leech nephridia is accomplished by a single neurone containing FMRFamide.

The neural control of the excretory system of the medicinal leech Hirudo medicinalis has been characterized morphologically and chemically using light and electron microscopy, immunocytochemistry and biochemistry. Immunoreactivity against RFamide-like peptides revealed elaborate neuronal aborizations of a neurone in the nephridium, around the urinary bladder sphincter and in the central nervous system. The processes arose from the nephridial nerve cell (NNC), a previously identified receptor neurone. Using a combination of reverse-phase high pressure liquid chromatography, radioimmunoassay and subsequent Edman degradation and mass spectrometry, authentic FMRFamide has been identified as the major peptide of the NNC. Sensory and neurosecretory innervation of the nephridia is thus accomplished by a single neurone, which is thought to modulate nephridial performance.

Amino Acid Sequence↗
PubMed1989

A phase I clinical trial of recombinant interleukin-2 by periodic 24-hour intravenous infusions.

Recombinant interleukin-2 (rIL-2) (NSC# 600664; Hoffmann-La Roche, Inc., Nutley, NJ) was studied in a phase I clinical trial in 33 patients with advanced, measureable cancer of the colon or malignant melanoma, Eastern Cooperative Oncology Group (ECOG) performance status O-1, and no prior chemotherapy or radiotherapy. The goal of the study was to identify a dose and schedule of IL-2 to generate maximal immune modulation with tolerable toxicity. Such a regimen might allow the addition of other treatment modalities and/or prolonged treatment duration in later trials. Each patient received IL-2 as a continuous 24-hour infusion once weekly for 4 weeks and then twice weekly for 4 weeks. Five treatment groups received from 10(3) U/m2 to 3 x 10(7) U/m2 per 24-hour infusion. The maximal tolerated dose was 3 x 10(7) U/m2/d twice weekly. Patients treated twice weekly at 1 x 10(7) and 3 x 10(7) U/m2/d had immune modulation in terms of lymphocytosis, eosinophilia, increased natural killer (NK) activity, and elevated numbers of peripheral blood mononuclear cells expressing CD16, OKT10/Leu-17, and Leu-19 surface markers. Endogenous generation of peripheral blood lymphokine-activated killer (LAK) activity was demonstrated by lysis of NK-resistant Daudi targets, in patients treated at 3 x 10(7) U/m2/d. Biochemical and hematological abnormalities were moderate and reversible. Clinical toxicity included hypotension, myalgia, arthralgia, stomatitis, fever, fatigue, nausea, headache, chills, diarrhea, and oliguria at high doses. Cardiovascular toxicity was tolerable for most patients and reversed after IL-2 was stopped. Two of six melanoma patients at 3 x 10(7) U/m2/d achieved partial responses by the end of the eighth week. This IL-2 schedule appears to produce potentially clinically useful immune enhancement with tolerable toxicity.

Adolescent↗
PubMed1982

The innervation pattern of crustacean skeletal muscle. Morphometry and ultrastructure of terminals and synapses.

The innervation pattern of distal muscle fibers of the opener muscle of walking legs of crayfish (Astacus leptodactylus) was investigated using methylene-blue staining, cobalt infiltration, and electron microscopy. A quantitative analysis of the entire innervation of single muscle fibers was attempted. It was found that instead of the generally assumed parallel array of numerous excitatory and inhibitory terminals, innervation consists of only a few branched terminals. The branches of excitatory and inhibitory terminals lie side-by-side. Both types are characterized by numerous varicosities (see Fig. 9B). The aggregate length of excitatory as well as inhibitory terminals on one muscle fiber is, on the average, about 1,500 micrometer with a total of 152 varicosities spaced about 10 micrometer apart. The average diameter of the varicosities is 4.26 micrometer, that of the connecting thin segments about 0.5 micrometer. Total terminal surface of motor or inhibitory terminals amounts to about 10,000 micrometers2 per muscle fiber. There are approximately 2,000 motor synapses on each muscle fiber, but their average total area is only about 6% of the terminal membrane area, or 0.06% of the (idealized) muscle fiber surface. There are conspicuous differences in the postsynaptic specializations associated with excitatory and inhibitory terminals; these are described in detail. The results are discussed in a functional context and with regard to design and results of electrophysiological experiments.

Animals↗