Translocation of an intracellular antigen to the surface of medullary breast cancer cells early in apoptosis allows for an antigen-driven antibody response elicited by tumor-infiltrating B cells

Tumor-infiltrating lymphoplasmacytic cells are a key feature of medullary carcinoma of the breast (MCB), a distinct subtype of human breast cancer that, despite cytologically anaplastic characteristics, has a more favorable prognosis than other types of breast cancer. Since it has been proposed that the improved clinical outcome is due at least in part to the presence of a prominent lymphoplasmacytic cell infiltrate in the tumor stroma, we recently examined the tumor-infiltrating B cell response in MCB and showed that it is oligoclonal and directed against an intracellular protein translocated to the cell surface upon MCB cell apoptosis. Human Abs cloned from MCB lymphoplasmacytic infiltrate-derived phage display libraries and reflecting the dominant part of the response were used to identify the target Ag as actin. Here, we have characterized in detail the cloned human IgG Abs and the translocation process of actin to the cell surface of apoptotic MCB cells. Our analysis shows that the cloned Abs bind specifically and with high affinity to actin, as determined by ELISA and surface plasmon resonance. Sequence analysis revealed that the Abs are highly somatically mutated, with high replacement to silent ratios, indicative of an Ag-driven, affinity-matured response. Interestingly, the tumor-infiltrating B cells in half the MCB patients mainly exhibited an IgG2 response, while IgG1 dominated in the others. To gain insight to the molecular events that may elicit such an Ab response, we examined the translocation of actin to the cell surface of apoptotic MCB cells using flow cytometry and laser scanning cytometry. Our results show that actin becomes exposed on the cell surface of a large proportion of apoptotic MCB cells as an early apoptotic event. We propose that the Ab response against actin produced by tumor-infiltrating B lymphoplasmacytic cells is Ag-driven, affinity-matured, and elicited due to the increased rate of apoptosis occurring within the MCB tumor that facilitates the translocation and proteolytic fragmentation of intracellular proteins.     

Addressing protein localization within the nucleus

Bridging the gap between the number of gene sequences in databases and the number of gene products that have been functionally characterized in any way is a major challenge for biology. A key characteristic of proteins, which can begin to elucidate their possible functions, is their subcellular location. A number of experimental approaches can reveal the subcellular localization of proteins in mammalian cells. However, genome databases now contain predicted sequences for a large number of potentially novel proteins that have yet to be studied in any way, let alone have their subcellular localization determined. Here we ask whether using bioinformatics tools to analyse the sequence of proteins whose subnuclear localizations have been determined can reveal characteristics or signatures that might allow us to predict localization for novel protein sequences.     

Contiguous deletion of the X-linked adrenoleukodystrophy gene (ABCD1) and DXS1357E: a novel neonatal phenotype similar to peroxisomal biogenesis disorders

X-linked adrenoleukodystrophy (X-ALD) results from mutations in ABCD1. ABCD1 resides on Xq28 and encodes an integral peroxisomal membrane protein (ALD protein [ALDP]) that is of unknown function and that belongs to the ATP-binding cassette-transporter superfamily. Individuals with ABCD1 mutations accumulate very-long-chain fatty acids (VLCFA) (carbon length >22). Childhood cerebral X-ALD is the most devastating form of the disease. These children have the earliest onset (age 7.2 +/- 1.7 years) among the clinical phenotypes for ABCD1 mutations, but onset does not occur at <3 years of age. Individuals with either peroxisomal biogenesis disorders (PBD) or single-enzyme deficiencies (SED) in the peroxisomal beta-oxidation pathway--disorders such as acyl CoA oxidase deficiency and bifunctional protein deficiency--also accumulate VLCFA, but they present during the neonatal period. Until now, it has been possible to distinguish unequivocally between individuals with these autosomal recessively inherited syndromes and individuals with ABCD1 mutations, on the basis of the clinical presentation and measurement of other biochemical markers. We have identified three newborn boys who had clinical symptoms and initial biochemical results consistent with PBD or SED. In further study, however, we showed that they lacked ALDP, and we identified deletions that extended into the promoter region of ABCD1 and the neighboring gene, DXS1357E. Mutations in DXS1357E and the ABCD1 promoter region have not been described previously. We propose that the term "contiguous ABCD1 DXS1357E deletion syndrome" (CADDS) be used to identify this new contiguous-gene syndrome. The three patients with CADDS who are described here have important implications for genetic counseling, because individuals with CADDS may previously have been misdiagnosed as having an autosomal recessive PBD or SED     

Targeted proteomics of low-level proteins in human plasma by LC/MSn: using human growth hormone as a model system

This paper describes the profiling of human growth hormone (hGH) in human plasma in order to assess the dynamic range of the ion-trap mass spectrometer for proteomic studies of complex biological samples. Human growth hormone is an example of a low-level plasma protein in vivo, present at subfemtomole levels. This study was performed on a plasma sample in which hGH has been spiked at 10-fold above the natural level, that is approximately 16 pg/microL of plasma. Initially, the measurement was carried out without any sample enrichment and consisted of the following steps: the full set of plasma proteins were reduced, alkylated, and digested with trypsin, and the resulting peptides were separated on a capillary C-18 column and then detected by ion-trap mass spectrometry (1D LC/MS). In addition, this study provided a global view of the serum proteome with over 200 plasma proteins being preliminarily identified. In the MS/MS analysis, hGH was detected by characterization of the first tryptic peptide (T1). The initial identification was confirmed by alternative approaches, which also allowed the evaluation of different sample purification protocols. First, the plasma sample containing hGH was fractionated on a reversed-phase HPLC column and digested, and hGH could now be identified by MS/MS measurements of two tryptic peptides (T1 and T4) by the same 1D LC/MS protocol. In addition, the assignment of peptide identity was made with higher certainty (as measured by an algorithm score). The plasma sample was also fractionated by 1D and 2D gel electrophoresis, the selected bands were digested and analyzed again by the 1D LC/MS protocol. In both cases using the gel prepurifications, hGH was identified with additional peptides. Finally, the plasma sample was analyzed by 2D chromatography (ion exchange and reversed phase) on a new instrumental platform (ProteomeX), and hGH was identified by the observation of five tryptic peptides. In conclusion, these experiments were able to detect growth hormone in the low femtomole level with a dynamic range of 1 in 40 000 by several independent approaches. The amount of growth hormone, while 10-fold above normal in vivo levels, represents concentrations that may be present in disease states (such as acromegaly) and also in doping control measurements. These studies have demonstrated that shotgun sequencing approaches (LC/MS/MS) not only can profile high-abundance proteins in complex biological fluids but also have the potential to identify and quantitate low-level proteins present in such complex mixtures without extensive prepurification protocols. A key to such studies, however, is to use targeted approaches that reduce the complexity of the solute mixture that is presented to the mass spectrometer at a given time point. The various sample preparation protocols described here all improved the quality of the hGH measurement, although in this study the 2D chromatographic approach gave the greatest sequence coverage.     

Quantitation of the tumor-targeting properties of antibody fragments conjugated to cell-permeating HIV-1 TAT peptides

Human monoclonal antibodies are promising agents for the development of more selective anticancer therapeutics. However, the tumor-targeting efficiency of most anticancer antibodies is severely limited by their poor penetration into the tumor mass. Recent studies have shown that a peptide derived from the HIV TAT protein could improve the distribution of cytoplasmic reporter proteins when administered systemically as fusion proteins or cross-linked chimeras. In this article, we tested by quantitative biodistribtution analysis whether conjugation to TAT peptides could improve the tumor targeting properties of scFv(L19)-Cys: an engineered human antibody fragment specific for the ED-B domain of fibronectin, a marker located in the modified extracellular matrix surrounding tumor neovasculature. Our results show that TAT peptides, consisting either of L-amino acids or D-amino acids, can efficiently transduce target cells when conjugated to fluorophores and/or antibody fragments, suggesting a receptor-independent cell entry mechanism. However, conjugation of scFv(L19)-Cys to TAT peptides resulted in a severely reduced tumor targeting performance compared to the unconjugated antibody, as measured in murine F9 teratocarcinoma-bearing mice, after intravenous injection of the radiolabeled antibody preparations. Our results outline the usefulness of TAT peptides for the efficient in vitro transduction of cells with globular proteins. In particular, the use of TAT peptides composed of D-amino acids may significantly reduce proteolytic degradation. At the same time, the poor biodistribution properties of antibody-TAT conjugates cast doubts over the applicability of this methodology for the delivery of biopharmaceuticals in vivo.     

Changes in LPLa and reverse cholesterol transport variables during 24-h postexercise period

We investigated the time course of exercise-induced lipoprotein lipase activity (LPLa) and reverse cholesterol transport (RCT) during the 24-h postexercise period. Subjects were 10 sedentary normolipidemic males [NTG; fasting triglyceride (TG) = 89.1 +/- 8.6 mg/dl] and 6 hyperlipidemic males (HTG; fasting TG = 296.8 +/- 64.0 mg/dl). Each subject performed a control trial (no exercise) and 4 exercise trials. In the exercise trials, a subject jogged on a treadmill at 60% of his maximal O(2) consumption for 1 h. Pre- and postheparin blood samples were taken before exercise (baseline) and at 4, 8, 12, and 24 h after exercise. There was no group difference in LPLa (P > 0.05) over the time points. When the LPLa data from the two groups were combined, LPLa at 24 h after exercise was higher than baseline or at 4, 8, 12 h after exercise (P < 0.05). Plasma TG and lecithin-cholesterol acyltransferase activity (LCATa) were higher in HTG than in NTG, and the total high-density lipoprotein-cholesterol (HDL(tot)-Chol) was lower in HTG than in NTG (P < 0.05). HDL(2)-Chol, LCATa, and cholesterol ester transfer protein activity did not differ during the 24-h postexercise period (P > 0.05). These results suggest that LPLa is still increasing 24 h after an acute aerobic exercise and that the magnitude of the increase in exercise-induced LPLa in HTG was similar to that in NTG. Furthermore, in the sedentary population with or without HTG, the variables related to RCT do not change during the 24-h period after exercise.     

Use of spot measurements for assessing residential ELF magnetic field exposure: a validity study

The purpose of this study was to evaluate residential short term "spot" measurements as surrogates for long term personal magnetic field (MF) exposure. In an epidemiological study on birth weight and pregnancy delay, MF exposure was assessed by taking five spot measurements in each room. For a subsample of 30 subjects 24 h personal MF measurements were made, and the following exposure metrics were calculated: 24 h arithmetic mean, 24 h median, percentage of time above 0.15 microT, and percentage of time above 0.29 microT. The 24 h exposure metrics were used as gold standards, when evaluating the validity of various summary measures calculated from spot measurements for assessing personal exposure. Based on Spearman correlation coefficient (r), specificity and sensitivity, the average of the spot measurements of a residence resulted in least exposure measurement error (misclassification). Also the above bed spot value correlated better with the 24 h metrics than any room average. Spot measurements performed about equally well in predicting different types of exposure metrics.