Differentiating proteomic biomarkers in breast cancer by laser capture microdissection and MALDI MS

We assessed proteomic patterns in breast cancer using MALDI MS and laser capture microdissected cells. Protein and peptide expression in invasive mammary carcinoma versus normal mammary epithelium and estrogen-receptor positive versus estrogen-receptor negative tumors were compared. Biomarker candidates were identified by statistical analysis and classifiers were developed and validated in blinded test sets. Several of the m/ z features used in the classifiers were identified by LC-MS/MS and two were confirmed by immunohistochemistry.     

The in vitro biological effect of nerve growth factor is inhibited by synthetic peptides.

Nerve growth factor (NGF)1 is a neurotrophic polypeptide that acts via specific receptors to promote the survival and growth of neurons. To delineate the NGF domain(s) responsible for eliciting biological activity, we synthesized small peptides corresponding to three regions in NGF that are hydrophilic and highly conserved. Several peptides from mouse NGF region 26-40 inhibited the neurite-promoting effect of NGF on sensory neurons in vitro. Inhibition was sequence-specific and could be overcome by increasing the concentration of NGF. Moreover, peptide actions were specific for NGF-mediated events in that they failed to block the neurotrophic activity of ciliary neuronotrophic factor (CNTF) or phorbol 12-myristate 13-acetate (PMA). In spite of the inhibition of NGF activity, peptides did not affect the binding of radiolabeled NGF. These studies define one region of NGF that may be required for neurotrophic activity.     

Transport and incorporation of N-acetyl-D-glucosamine in Bacillus subtilis

Bacillus subtilis 168 has been found to possess a high-affinity transport system for N-acetyl-D-glucosamine (GlcNAC). The Km for uptake was approximately 3.7 microM GlcNAc, regardless of the nutritional background of the cells. Apparent increases in Vmax were noted when the bacteria were grown in the presence of GlcNAc. The uptake of GlcNAc by B. subtilis was highly stereoselective; D-glucose, D-glucosamine, N-acetyl-D-galactosamine, D-galactose, D-mannose, and N-acetylmuramic acid did not inhibit GlcNAc uptake. In contrast, glycerol was an effective inhibitor of [3H]GlcNAc transport and incorporation. Partial inhibition of GlcNAc uptake was observed with azide, fluoride, and cyanide anions, carbonyl cyanide-m-chlorophenyl hydrazone, methyltriphenylphosphonium bromide, N,N'-dicyclohexylcarbodiimide, gramicidin, valinomycin, monensin, and nigericin. Two anions, arsenite and iodoacetate, were potent inhibitors of the uptake of GlcNAc in B. subtilis. Results from paper chromatography showed that there was no intracellular pool of free GlcNAc and that the acetylamino sugar was probably phosphorylated during transport. A modification of the Park-Hancock cell fractionation scheme indicated that cells grown on glycerol or D-glucose incorporated [3H]GlcNAc primarily into the cell wall fraction. When GlcNAc was used as the sole carbon source, label could be demonstrated in fractions susceptible to protease and nuclease, as well as lysozyme, showing that the N-acetylamino sugar was utilized in macromolecular synthesis and energy metabolism.     

Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Longitudinal Impedance of Skinned Frog Muscle Fibers

Longitudinal impedance of skinned muscle fibers was measured with extracellular electrodes and an oil gap method in which a central longitudinal section of fiber is insulated by oil while the ends of the fiber are bathed in conducting pools of relaxing solution. Intact single fibers were isolated from frog semitendinosus muscle and the sarcolemma removed either by mechanical or chemical methods. Stray capacitance across the oil gap was measured after each experiment and its admittance subtracted from the admittance of the fiber and oil gap. Effects of impedance at the ends of the fiber were eliminated by measuring the impedance with two lengths of fiber in the oil gap and subtracting the impedance at the shorter length from that at the longer length. Longitudinal impedance so determined for mechanically and chemically skinned fibers exhibited zero phase shift from 1 to 10,000 Hz, i.e., the longitudinal impedance of skinned fibers is purely resistive. If we assume that our skinned fibers are a model of the sarcoplasm of muscle, we conclude that the equivalent circuit of the sarcoplasm is a resistor.     

Synergistic stimulation of thromboxane biosynthesis by calcium ionophore and phorbol ester or thrombin in human platelets

Phorbol ester PMA and low concentrations of calcium ionophore A-23187, which given separately have minimal effect in stimulating thromboxane synthesis in human platelets, showed marked synergism when given simultaneously. A similar synergism can be also demonstrated between thrombin or collagen and low concentrations of A-23187 but not of PMA. Simultaneous addition of thrombin and PMA results in less synthesis of thromboxane than that of thrombin alone. These studies suggest that protein kinase C activation by agonists may not only induce but also regulate thromboxane synthesis in human platelets.     

Propagation and perception of bioluminescence: factors affecting counterillumination as a cryptic strategy

Many deep-sea species, particularly crustaceans, cephalopods, and fish, use photophores to illuminate their ventral surfaces and thus disguise their silhouettes from predators viewing them from below. This strategy has several potential limitations, two of which are examined here. First, a predator with acute vision may be able to detect the individual photophores on the ventral surface. Second, a predator may be able to detect any mismatch between the spectrum of the bioluminescence and that of the background light. The first limitation was examined by modeling the perceived images of the counterillumination of the squid Abralia veranyi and the myctophid fish Ceratoscopelus maderensis as a function of the distance and visual acuity of the viewer. The second limitation was addressed by measuring downwelling irradiance under moonlight and starlight and then modeling underwater spectra. Four water types were examined: coastal water at a depth of 5 m and oceanic water at 5, 210, and 800 m. The appearance of the counterillumination was more affected by the visual acuity of the viewer than by the clarity of the water, even at relatively large distances. Species with high visual acuity (0.11 degrees resolution) were able to distinguish the individual photophores of some counterilluminating signals at distances of several meters, thus breaking the camouflage. Depth and the presence or absence of moonlight strongly affected the spectrum of the background light, particularly near the surface. The increased variability near the surface was partially offset by the higher contrast attenuation at shallow depths, which reduced the sighting distance of mismatches. This research has implications for the study of spatial resolution, contrast sensitivity, and color discrimination in deep-sea visual systems.     

Going the distance, or not, with neurotrophin signals

NGF and NT-3 both signal through TrkA receptors on the axons of developing sympathetic neurons, but while NGF supports survival and differentiation, NT-3 does not. In this issue of Cell, the difference is explained as the ability of NGF, but not NT-3, to induce internalization and retrograde transport of activated TrkA.     

Identification of a Proline-Rich Sequence in the CD2 Cytoplasmic Domain Critical for Regulation of Integrin-Mediated Adhesion and Activation of Phosphoinositide 3-Kinase

The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in β1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of β1 integrin activity. CD2-induced increases in β1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of β1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.