Large-scale identification by shotgun proteomics of proteins expressed in porcine liver and salivary gland

Protein catalogs containing a large number of proteins expressed in a variety of organs can be powerful tools for stem-cell research, because this requires accurate knowledge about how cells differentiate. Salivary gland progenitor (SGP) cells are somatic stem cells isolated from the salivary gland that can differentiate into hepatic or pancreatic cell lineages. Their differentiation state has been assessed by the expression of major protein markers, but to use these cells in regenerative medicine, it will be necessary to establish additional means of quality assessment. We examined the use of shotgun proteomics for porcine salivary gland (a source of SGP cells) and liver (a destination of differentiated SGP cells) for determining the state of SGP cell differentiation. Protein complexes from each organ were digested into peptides and separated by two-dimensional liquid chromatography involving strong cation-exchange chromatography followed by reversed-phase liquid chromatography. The separated peptides were analyzed by on-line electrospray ionization tandem mass spectrometry using a quadrupole-time of flight mass spectrometer (ESI Q-TOF MS/MS), and the spectra obtained were processed to search peptides against a mammalian database for protein identification. Using this method, we identified 117 proteins in porcine salivary gland and 154 proteins in porcine liver. Of these, 72 and 109 were specific to salivary gland and liver, respectively, and some of these were previously shown to be organ specific. The current study can be utilized in the future as a basis to study the pattern of differentiation in protein expression by stem cells.     

Induction of serum amyloid A genes is associated with growth and apoptosis of HC11 mammary epithelial cells

In this study, we examined the expression and functions of serum amyloid A (SAA) isoforms during apoptosis of HC11 mammary gland epithelial cells. Expression of SAA mRNAs and apoptosis were increased in HC11 cells by serum withdrawal and gradually decreased upon the addition of serum, or epidermal growth factor (EGF). TNFalpha treatment of HC11 cells also induced expression of SAA genes, and the effect on SAA1 and SAA2 expression was suppressed by treatment with MG132, and in cells transfected with a dominant negative mutant form of IkappaBalpha. Similar results were observed in response to interleukin-1 (IL-1), IL-6 and interferon gamma (IFNgamma). Furthermore, overexpression of the SAA1 and SAA2 isoforms suppressed growth and accelerated apoptosis of HC11 cells by increasing caspase 3/7 and caspase 8 activities, but the apoptotic effect of tumor necrosis factor alpha (TNFalpha) on HC11 cells was not enhanced. We found that expression of SAA1 and SAA2, but not SAA3, was regulated by an NFkappaB-dependent pathway, and that overexpression of SAA isoforms accelerated the apoptosis of HC11 cells.     

Synthesis of 3'-acetamidoadenosine derivatives as potential A3 adenosine receptor agonists

On the basis of high binding affinity of 3'-aminoadenosine derivatives 2b at the human A3 adenosine receptor (AR), 3'-acetamidoadenosine derivatives 3a-e were synthesized from 1,2:5,6-di-O-isopropylidene-D-glucose via stereoselective hydroboration as a key step. Although all synthesized compounds were totally devoid of binding affinity at the human A3AR, our results revealed that 3'-position of adenosine can only be tolerated with small size of a hydrogen bonding donor like hydroxyl or amino group in the binding site of human A3AR.     

Validity of the BOD POD for assessing body composition in athletic high school boys

The purpose of this study was to validate the percentage of body fat (%BF) values estimated from the BOD POD (BP) with those obtained from hydrostatic weighing (HW) in athletic American high school boys. Additionally, the %BF values measured via near-infrared interactance (NIR), bioelectrical impedance (BIA), and skinfold (SF) were compared to HW to determine the validity of these measures. Thirty white boys (mean age +/- SD = 15.8 +/- 1.0 years) who where currently participating in organized sports volunteered to have their %BF estimated. Measurements were obtained from NIR, BP, BIA, and SF in random order and concluded with HW. The findings from the present study indicated that the NIR and BIA instruments produced significant (P < 0.008) constant error (CE) and total error (TE) values that were too large to be of practical value (TE > 4.0%BF). The BP produced a significantly (P < 0.008) higher CE with acceptable TE values compared to HW, but compared to all three SF estimations, the BP TE values were higher. Two of the SF equations were nonsignificant (P > 0.008) and had the lowest TE values compared to HW. These data suggest that the BP can produce acceptable body fat measures for athletic white boys but is not superior to estimates made by the SF equations used in this study.     

Genus-specific distribution and pathovar-specific variation of the glycinecin R gene homologs in <Emphasis Type="Italic">Xanthomonas</Emphasis> genomes

Xanthomonas axonopodis pv. glycines produces bacteriocins called glycinecin, and two glycinecin genes, glyA and glyR, were reported previously. In this paper, we describe genomic distribution and variation of the glyR gene revealed by extensive Southern hybridization analysis. In contrast to the glyA gene present only in X. axonopodis pv. glycines, the glyR gene was found to be distributed widely in all the pathovars of Xanthomas genus. It was also found that the glyR gene is a multigene family while the glyA is a single copy gene. Moreover, the copy number and the variation of the glyR multigene are unique to each pathovar of Xanthomonas. The uniqueness can be easily detected by the patterns resulted from Southern hybridization using the genomic digests. Thus, we suggest the glyR gene can serve as a useful genus-specific and pathovar-specific DNA marker for Xanthomonas. One of the glyR homologs was further isolated from X. axonopodis pv. glycines, and analyzed to be functional with strong inhibitory activity against several members of Xanthomonas.     

Molecular mass sorting of proteome using hollow fiber flow field-flow fractionation for proteomics

Hollow fiber flow field-flow fractionation (HF FlFFF) has been demonstrated as a tool for pre-fractionating proteomes by differences in molecular mass (Mr), where the resulting protein fractions are subsequently digested and analyzed by shotgun proteomics using two-dimensional liquid chromatography-electrospray ionization-tandem mass spectrometry (2D-LC-ESI-MS/MS). HF FlFFF is a separation device capable of fractionating proteins or cells by hydrodynamic radius, and protein fraction can be readily collected as intact conditions in aqueous buffer solutions. In this study, HF FlFFF was applied to fractionate the proteome of Corynebacterium glutamicum, a well known soil bacterium that has been widely used in bioindustry due to its remarkable ability to secrete high amounts of glutamic acid. The collected HF FlFFF fractions of different MW intervals were enzymatically digested for protein identification by 2D-LC-ESI-MS/MS. Experiments showed improvements in protein identification when HF FlFFF pre-fractionation was applied, due to decreases in the ionization suppression effect and the MS exclusion effect by spectral congestion. Pre-fractionation of C. glutamicum proteome allowed us to find 90 additional proteins by 2D-LC-ESI-MS/MS that were not found by a direct shotgun analysis without pre-fractionation. A total of 415 proteins were found overall with 203 proteins commonly found from experiments with and without pre-fractionation.     

Tattooing Dye as a Green Electrode Material for Lithium Batteries

Lawsone (2‐hydroxy‐1,4‐naphthoquinone), a naturally derived red‐orange dye, is investigated as a promising cathode material for next‐generation lithium batteries. Lithium cells based on lawsone cathode display a high discharge capacity of 280 mA h g^?1 (99% theoretical capacity), a high energy density of 664 W h kg^?1, and long life of 1000 cycles at 0.5 C along with good rate performance up to 5 C. These results represent significant improvements from previously reported organic cathode materials, and surpass those of conventional lithium batteries based on LiCoO₂ cathodes (140 mA h g^?1 and 520 W h kg^?1, respectively). Its success stems from the unique 2D planar packing of lawsone molecules, with maximized overlap of adjacent p orbitals for redox active sites. The result is the simultaneous enhancement of electrical and ionic conductivities that are an order of magnitude higher than those of other synthetic quinones. Given that lawsone is derived from the henna plant and has long been used as a dye for human hair and skin, this work may open a new chapter in the design of future green batteries.