Proteomic analysis of the potato tuber life cycle

The tuber of potato (Solanum tuberosum) is commonly used as a model for underground storage organs. In this study, changes in the proteome were followed from tuberization, through tuber development and storage into the sprouting phase. Data interrogation using principal component analysis was able to clearly discriminate between the various stages of the tuber life cycle. Moreover, five well-defined protein expression patterns were found by hierarchical clustering. Altogether 150 proteins showing highly significant differences in abundance between specific stages in the life cycle were highlighted; 59 of these were identified. In addition, 50 proteins with smaller changes in abundance were identified, including several novel proteins. Most noticeably, the development process was characterized by the accumulation of the major storage protein patatin isoforms and enzymes involved in disease and defense reactions. Furthermore, enzymes involved in carbohydrate and energy metabolism and protein processing were associated with development but decreased during tuber maturation. These results represent the first comprehensive picture of many proteins involved in the tuber development and physiology.     

Dinucleotide polyphosphates contribute to purinergic signalling via inhibition of adenylate kinase activity

Dinucleoside polyphosphates are well described as direct vasoconstrictors and as mediators with strong proliferative properties, however, less is known about their effects on nucleotide-converting pathways. Therefore, the present study investigates the effects of Ap(4)A (diadenosine tetraphosphate), Up(4)A (uridine adenosine tetraphosphate) and Ap(5)A (diadenosine pentaphosphate) and the non-selective P2 antagonist suramin on human serum and endothelial nucleotide-converting enzymes. Human serum and HUVECs (human umbilical vein endothelial cells) were pretreated with various concentrations of dinucleotide polyphosphates and suramin. Adenylate kinase and NDP kinase activities were then quantified radiochemically by TLC analysis of the ATP-induced conversion of [(3)H]AMP and [(3)H]ADP into [(3)H]ADP/ATP and [(3)H]ATP respectively. Endothelial NTPDase (nucleoside triphosphate diphosphohydrolase) activity was additionally determined using [(3)H]ADP and [(3)H]ATP as preferred substrates. Dinucleoside polyphosphates and suramin have an inhibitory effect on the serum adenylate kinase [pIC(50) values (-log IC(50)): Ap(4)A, 4.67+/-0.03; Up(4)A, 3.70+/-0.10; Ap(5)A, 6.31+/-0.03; suramin, 3.74+/-0.07], as well as on endothelial adenylate kinase (pIC(50) values: Ap(4)A, 4.17+/-0.07; Up(4)A, 2.94+/-0.02; Ap(5)A, 5.97+/-0.04; suramin, 4.23+/-0.07), but no significant effects on serum NDP kinase, emphasizing the selectivity of these inhibitors. Furthermore, Ap(4)A, Up(4)A, Ap(5)A and suramin progressively inhibited the rates of [(3)H]ADP (pIC(50) values: Ap(4)A, 3.38+/-0.09; Up(4)A, 2.78+/-0.06; Ap(5)A, 4.42+/-0.11; suramin, 4.10+/-0.07) and [(3)H]ATP (pIC(50) values: Ap(4)A, 3.06+/-0.06; Ap(5)A, 3.05+/-0.12; suramin, 4.14+/-0.05) hydrolyses by cultured HUVECs. Up(4)A has no significant effect on the endothelial NTPDase activity. Although the half-lives for Ap(4)A, Up(4)A and Ap(5)A in serum are comparable with the incubation times of the assays used in the present study, secondary effects of the dinucleotide metabolites are not prominent for these inhibitory effects, since the concentration of metabolites formed are relatively insignificant compared with the 800 mumol/l ATP added as a phosphate donor in the adenylate kinase and NDP kinase assays. This comparative competitive study suggests that Ap(4)A and Ap(5)A contribute to the purinergic responses via inhibition of adenylate-kinase-mediated conversion of endogenous ADP, whereas Up(4)A most likely mediates its vasoregulatory effects via direct binding-mediated mechanisms.     

A role for the androgen receptor in follicular atresia of estrogen receptor beta knockout mouse ovary.

Estrogen receptor beta (ERbeta) is highly expressed, but ERalpha is not detectable in granulosa cells in the mouse ovary. In ERbeta knockout (BERKO) mice, there is abnormal follicular development and very reduced fertility. At 3 wk of age, no significant morphologic differences were discernable between wild type (WT) and BERKO mouse ovaries, but by 5 mo of age, atretic follicles were abundant in BERKO mice and there were very few healthy late antral follicles or corpora lutea. At 2 yr of age, unlike the ovaries of their WT littermates, BERKO mouse ovaries were devoid of healthy follicles but had numerous large, foamy lipid-filled stromal cells. The late antral and atretic follicles in BERKO mice were characterized by a high level of expression of the androgen receptor (AR) and IGF-1 receptor. These proteins were abundantly expressed in granulosa cells of preantral and early antral follicles in both genotypes, but their expression was extinguished in late antral follicles of WT mice. Healthy late antral follicles and corpora lutea were restored in BERKO ovaries after 15 days of treatment of mice with the antiandrogen flutamide. The results suggest that in the absence of ERbeta there was a loss of regulation of AR. Because androgens enhance recruitment of primordial follicles into the growth pool and cause atresia of late antral follicles, the inappropriately high level of AR probably is related to the follicular atresia and to the early exhaustion of follicles in BERKO mice.     

Systems pathology analysis identifies neurodegenerative nature of age‐related vitreoretinal interface diseases

Aging is a phenomenon that is associated with profound medical implications. Idiopathic epiretinal membrane (iEMR) and macular hole (MH) are the major vision‐threatening vitreoretinal diseases affecting millions of aging people globally, making these conditions an important public health issue. iERM is characterized by fibrous tissue developing on the surface of the macula, which leads to biomechanical and biochemical macular damage. MH is a small breakage in the macula and is associated with many ocular conditions. Although several individual factors and pathways are suggested, a systems pathology level understanding of the molecular mechanisms underlying these disorders is lacking. Therefore, we performed mass spectrometry‐based label‐free quantitative proteomics analysis of the vitreous proteomes from patients with iERM and MH to identify the key proteins, as well as the multiple interconnected biochemical pathways, contributing to the development of these diseases. We identified a total of 1,014 unique proteins, many of which are linked to inflammation and the complement cascade, revealing the inflammation processes in retinal diseases. Additionally, we detected a profound difference in the proteomes of iEMR and MH compared to those of diabetic retinopathy with macular edema and rhegmatogenous retinal detachment. A large number of neuronal proteins were present at higher levels in the iERM and MH vitreous, including neuronal adhesion molecules, nervous system development proteins, and signaling molecules, pointing toward the important role of neurodegenerative component in the pathogenesis of age‐related vitreoretinal diseases. Despite them having marked similarities, several unique vitreous proteins were identified in both iERM and MH, from which candidate targets for new diagnostic and therapeutic approaches can be provided.     

Differential Regulation and Function of CD73, a Glycosyl-Phosphatidylinositol–linked 70-kD Adhesion Molecule, on Lymphocytes and Endothelial Cells

CD73, otherwise known as ecto-5′-nucleotidase, is a glycosyl-phosphatidylinositol–linked 70-kD molecule expressed on different cell types, including vascular endothelial cells (EC) and certain subtypes of lymphocytes. There is strong evidence for lymphocyte CD73 having a role in several immunological phenomena such as lymphocyte activation, proliferation, and adhesion to endothelium, but the physiological role of CD73 in other cell types is less clear. To compare the biological characteristics of CD73 in different cell types, we have studied the structure, function, and surface modulation of CD73 on lymphocytes and EC. CD73 molecules on lymphocytes are shed from the cell surface as a consequence of triggering with an antiCD73 mAb, mimicking ligand binding. In contrast, triggering of endothelial CD73 does not have any effect on its expression. Lymphocyte CD73 is susceptible to phosphatidylinositol phospholipase, whereas only a small portion of CD73 on EC could be removed by this enzyme. Furthermore, CD73 on EC was unable to deliver a tyrosine phosphorylation inducing signal upon mAb triggering, whereas triggering of lymphocyte CD73 can induce tyrosine phosphorylation. Despite the functional differences, CD73 molecules on lymphocytes and EC were practically identical structurally, when studied at the protein, mRNA, and cDNA level. Thus, CD73 is an interesting example of a molecule which lacks structural variants but yet has a wide diversity of biological functions. We suggest that the ligand- induced shedding of lymphocyte CD73 represents an important and novel means of controlling lymphocyte– EC interactions.     

Scaling of the marsupial middle ear and its functional significance

The marsupial middle ear performs an anatomical impedance matching for acoustic energy travelling in air to reach the cochlea. The size of the middle ear sets constraints for the frequencies transmitted. For generalized placental mammals, it has been shown that the limit for high-frequency hearing can be predicted on the basis of middle ear ossicle mass, provided that the ears fulfil requirements of isometry. We studied the interspecific size variation of the middle ear in 23 marsupial species, with the following measurable parameters: skull mass, condylobasal length, ossicular masses for malleus, incus and stapes, tympanic membrane area, oval window area, and lever arm lengths for malleus and incus. Our results show that the middle ear size grows with negative allometry in relation to body size and that the internal proportions of the marsupial middle ear are largely isometric. This resembles the situation in placental mammals and allows us to use their isometric middle ear model to predict the high-frequency hearing limit for marsupials. We found that the isometry model predicts the high-frequency hearing limit for different marsupials well, indicating that marsupials can be used as auditory models for general therian mammalian hearing. At very high frequencies, other factors, such as the inner ear, seem to constrain mammalian hearing.     

Isolation and Characterization of a Hantavirus from Lemmus sibiricus: Evidence for Host Switch during Hantavirus Evolution

A novel hantavirus, first detected in Siberian lemmings (Lemmus sibiricus) collected near the Topografov River in the Taymyr Peninsula, Siberia (A. Plyusnin et al., Lancet 347:1835-1836, 1996), was isolated in Vero E6 cells and in laboratory-bred Norwegian lemmings (Lemmus lemmus). The virus, named Topografov virus (TOP), was most closely related to Khabarovsk virus (KBR) and Puumala viruses (PUU). In a cross focus reduction neutralization test, anti-TOP Lemmus antisera showed titers at least fourfold higher with TOP than with other hantaviruses; however, a rabbit anti-KBR antiserum neutralized TOP and KBR at the same titer. The TOP M segment showed 77% nucleotide and 88% amino acid identity with KBR and 76% nucleotide and 82% amino acid identity with PUU. However, the homology between TOP and the KBR S segment was disproportionately higher: 88% at the nucleotide level and 96% at the amino acid level. The 3' noncoding regions of KBR and the TOP S and M segments were alignable except for 113- and 58-nucleotide deletions in KBR. The phylogenetic relationships of TOP, KBR, and PUU and their respective rodent carriers suggest that an exceptional host switch took place during the evolution of these viruses; while TOP and KBR are monophyletic, the respective rodent host species are only distantly related.     

Basic FGF regulates interstitial collagenase gene expression in human smooth muscle cells

Basic fibroblast growth factor (bFGF) is a mitogenic factor that is implicated in smooth muscle cell growth in atherosclerosis and vascular restenosis. In this study, we examined the effect of bFGF on the expression of the interstitial collagenase gene in human vascular smooth muscle cells. Results from Northern transfer analysis showed that bFGF increased collagenase mRNA levels greater than threefold as early as 24 h. Collagenase pre-mRNA levels were elevated approximately threefold by bFGF, according to RT-PCR analysis. Transient transfections of the smooth muscle cells with a 4.4-kb human collagenase promoter-CAT reporter gene, however, failed to show upregulation of the promoter activity by bFGF. Interestingly, transfections with deleted fragments containing promoter sequences from -1047 to -2271 resulted in modest stimulation of the collagenase-CAT promoter activity by bFGF. bFGF did not alter the stability of the collagenase mRNA, as demonstrated by degradation studies. The enhanced collagenase mRNA levels elicited by bFGF were reflected in increased amounts of collagenase protein that were detected by Western blot analysis. In summary, bFGF upregulates the interstitial collagenase expression, resulting in turnover of the extracellular matrix, an event that could facilitate smooth muscle cell migration and proliferation during the early stages of atherosclerosis and restenosis. J. Cell. Biochem. 65:32–41. © 1997 Wiley-Liss, Inc.