The C-Terminal Fragment of Prostate-Specific Antigen,a 2331 Da Peptide,as a New Urinary Pathognomonic Biomarker Candidate for Diagnosing Prostate Cancer

Background and Objectives

Prostate cancer (PCa) is one of the most common cancers and leading cause of cancer-related deaths in men. Mass screening has been carried out since the 1990s using prostate-specific antigen (PSA) levels in the serum as a PCa biomarker. However, although PSA is an excellent organ-specific marker, it is not a cancer-specific marker. Therefore, the aim of this study was to discover new biomarkers for the diagnosis of PCa.

Materials and Methods

We focused on urine samples voided following prostate massage (digital rectal examination [DRE]) and conducted a peptidomic analysis of these samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MSⁿ). Urinary biomaterials were concentrated and desalted using CM-Sepharose prior to the following analyses being performed by MALDI-TOF/MSⁿ: 1) differential analyses of mass spectra; 2) determination of amino acid sequences; and 3) quantitative analyses using a stable isotope-labeled internal standard.

Results

Multivariate analysis of the MALDI-TOF/MS mass spectra of urinary extracts revealed a 2331 Da peptide in urine samples following DRE. This peptide was identified as a C-terminal PSA fragment composed of 19 amino acid residues. Moreover, quantitative analysis of the relationship between isotope-labeled synthetic and intact peptides using MALDI-TOF/MS revealed that this peptide may be a new pathognomonic biomarker candidate that can differentiate PCa patients from non-cancer subjects.

Conclusion

The results of the present study indicate that the 2331 Da peptide fragment of PSA may become a new pathognomonic biomarker for the diagnosis of PCa. A further large-scale investigation is currently underway to assess the possibility of using this peptide in the early detection of PCa.     

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Fractionation of oligosaccharides liberated by hydrazinolysis and structural studies of the neutral oligosaccharides

The asparagine-linked sugar chains of the plasma membrane glycoproteins of rat erythrocytes were released as oligosaccharides by hydrazinolysis and labeled by NaB³H₄ reduction. The radioactive oligosaccharides were separated into a neutral and at least four acidic fractions by paper electrophoresis. The neutral oligosaccharide fraction was separated into at least 11 peaks upon Bio-Gel P-4 column chromatography. Structural studies of them by sequential exoglycosidase digestion in combination with methylation analysis revealed that they were a mixture of three high mannose-type oligosaccharides and at least 11 complex type oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc as their cores and Galβ1 → 4GlcNAc, Galβ1 → 3Galβ1 → 4GlcNAc, and various lengths of Galβ1 → 4GlcNAc repeating chains in their outer chain moieties. Most of the complex-type Oligosaccharides were biantennary, and the tri- and tetraantennary Oligosaccharides contain only the Galβ1 → 3Galβ1 → 4GlcNAc group in their outer chain moieties.     

TRPA1 agonists--allyl isothiocyanate and cinnamaldehyde--induce adrenaline secretion

Thermosensitive transient receptor potential (TRP) channels, especially TRPV1 and TRPA1, are activated by the pungent compounds present in spices. TRPV1 activation by the intake of capsaicin, the irritant in hot pepper, induces adrenaline secretion and increases energy consumption. TRPV1 is mainly expressed in the sensory neurons and coexpressed with TRPA1 at a high frequency. However, the mechanism underlying adrenaline secretion by TRPA1 agonists such as allyl isothiocyanate (AITC) and cinnamaldehyde (CNA), the pungent ingredients in mustard and cinnamon, is not known. We examined whether AITC and CNA could induce adrenaline secretion in anesthetized rats. An intravenous injection of AITC or CNA (10 mg/kg) increased adrenaline secretion. These responses disappeared completely in capsaicin-treated rats with an impaired sensory nerve function. Moreover, pretreatment with cholinergic blockers (hexamethonium and atropine) attenuated the AITC- or CNA-induced adrenaline secretion. These results suggest that TRPA1 agonists activate the sensory nerves and induce adrenaline secretion via the central nervous system.     

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.     

Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells

Using a flow cytometry-based screen of commercial antibodies, we have identified cell-surface markers for the separation of pancreatic cell types derived from human embryonic stem (hES) cells. We show enrichment of pancreatic endoderm cells using CD142 and of endocrine cells using CD200 and CD318. After transplantation into mice, enriched pancreatic endoderm cells give rise to all the pancreatic lineages, including functional insulin-producing cells, demonstrating that they are pancreatic progenitors. In contrast, implanted, enriched polyhormonal endocrine cells principally give rise to glucagon cells. These antibodies will aid investigations that use pancreatic cells generated from pluripotent stem cells to study diabetes and pancreas biology.     

Sulfated asparagine-linked sugar chains of hen egg albumin

The fraction of hen egg albumin glycopeptides mixture, which passes through a Dowex 50-H+ column, contains two sulfate-containing glycopeptides. Based on the structural studies of oligosaccharides released from the glycopeptides by hydrazinolysis, their structures were elucidated as follows. (formula; see text)     

Difference in the sugar chains of two subunits and of isozymic forms of rat kidney gamma-glutamyltranspeptidase

A comparative study by gel-permeation chromatographic analysis of oligosaccharides released from the heavy and the light subunits of rat kidney gamma-glutamyltranspeptidase has revealed that high-mannose-type sugar chains are found only in the heavy subunit, and the nonsialylated and nonfucosylated biantennary complex-type sugar chains are included only in the light subunit. By the same analysis of the oligosaccharide fractions obtained from four isozymic forms of rat kidney gamma-glutamyltranspeptidase, it was found that all these enzymes contain 2 mol of neutral sugar chains but different numbers of acidic sugar chains. The total numbers of sialic acid residues showed a reciprocal relationship to the isoelectric point of each isozymic form, and an increase of 1 mol of sialic acid residue corresponds to a decrease of 0.5 in the value of the isoelectric point.     

Structural studies on the carbohydrate moieties of rat liver cathepsins B and H

Cathepsins B and H from rat liver contain one asparagine-linked sugar chain in each molecule. The sugar chains were liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Paper electrophoresis of the radioactive oligosaccharide fractions revealed that they were mixtures of neutral oligosaccharides only. After fractionation by gel filtration the structure of each oligosaccharide was studied by sequential exoglycosidase digestion in combination with methylation analysis. The sugar chain of cathepsin H was a high mannose type oligosaccharide which varied in size from 5 to 9 mannose residues; on the other hand the major oligosaccharide of cathepsin B was a tetrasaccharide whose structure was Manalpha 1----6Manbeta 1----4GlcNAcbeta 1----4GlcNAc.     

Expression and function of a 90-kilodalton homodimeric molecule (10D1 antigen) on human thymocytes

The 10D1 Ag is a 90-kDa homodimeric molecule specifically expressed on a subpopulation of human T cells, and is involved in an alternative pathway of T cell activation. In the present study, we have examined the expression and function of the 10D1 Ag on human thymocytes. Three-color FMF analysis showed that the 10D1 Ag was highly expressed on minor but distinct subpopulations of double-negative and CD4 single-positive thymocytes, and weakly on a part of double-positive thymocytes, but not on CD8 single-positive thymocytes. In double-negative thymocytes, the vast majority of 10D1+ cells were immature thymocytes of CD7+2+3- phenotype. Interestingly, 10D1 mAb could induce the proliferation of CD4 single-positive thymocytes in the presence of goat anti-mouse Ig to cross-link the 10D1 Ag. The treatment of thymocytes with OKT4 mAb plus C but not with OKT8 mAb plus C totally abrogated the proliferative response induced by 10D1 mAb, indicating that the 10D1-responsible thymocytes were of CD4+8- phenotype. This 10D1 mAb-induced thymocyte proliferation was perfectly dependent on the endogenous IL-2/IL-2R system since a complete inhibition was observed with anti-IL-2 and anti-IL-2R mAb. The proliferating CD4 single positive thymocytes predominantly expressed the IL-2R alpha (p55) but not a detectable level of the IL-2R beta (p75). These results indicate that, although the 10D1 Ag can be detected on the CD7+2+3-4-8- thymocytes, its functional expression is restricted to a minor more mature CD4+ thymocyte population as well as in peripheral blood T cells, and the implications of these findings are discussed.     

Neutral oligosaccharide structures linked to asparagines of porcine zona pellucida glycoproteins

N-Linked sugar chains were liberated by hydrazinolysis from porcine zona pellucida glycoproteins obtained from ovarian follicular oocytes. Neutral sugar chains were separated from acidic ones by paper electrophoresis and fractionated with a serial lectin column chromatography and Bio-Gel P-4 column chromatography. Their structural analysis by sequential glycosidase digestion in combination with methylation analysis revealed that the neutral sugar chains are of bi-, tri-, and tetraantennary complex type with a fucosylated trimannosyl core. Twenty-six percent of the sugar chains contain N-acetyllactosamine repeating structures in their outer chain moieties. Only linear N-acetyllactosamine repeats, the maximum size of which is hexasaccharide, are detected. A characteristic feature is that 39% of the sugar chains contain N-acetylglucosamine residues at their nonreducing termini in spite of the absence of bisected sugar chains. This study provided, for the first time, the substantial information about the sugar chain structures of mammalian zona pellucida glycoproteins.