Comprehensive proteome analysis of ovarian cancers using liquid phase separation, mass mapping and tandem mass spectrometry: a strategy for identification of candidate cancer biomarkers

A two-dimensional (2-D) liquid phase separation method, liquid isoelectric focusing followed by nonporous reversed-phase high performance liquid chromatography (HPLC), was used to separate proteins from human ovarian epithelial whole cell lysates. HPLC eluent was interfaced on-line to an electrospray ionization (ESI) time of flight (TOF) mass spectrometer to obtain accurate intact protein molecular weights (Mr). 2-D protein expression maps were generated displaying protein isoelectric point (pI) versus intact protein Mr. Resulting 2-D images effectively displayed quantitative differential protein expression in ovarian cancer cells versus non-neoplastic ovarian epithelial cells. Protein peak fractions were collected from the HPLC eluent, enzymatically digested, and analyzed by matrix-assisted laser desorption/ionization (MALDI) TOF-mass spectrometry (MS) peptide mass fingerprinting and by MALDI-quadrupole TOF tandem mass spectrometry peptide sequencing. Interlysate comparisons of differential protein expression between two ovarian adenocarcinoma cell lines, ES2 and MDAH-2774, and ovarian surface epithelial cells was performed. Five pI fractions from each sample were selected for comparative study and over 300 unique proteins were positively identified from the 2-D liquid expression maps using MS, which covered around 60% of proteins detected by on-line ESI-TOF-MS. This represents one of the most comprehensive proteomic analyses of ovarian cancer samples to date. Protein bands with significant up- or down-regulation in one cell line versus another as viewed in the 2-D expression maps were identified. This strategy may prove useful in identifying novel ovarian cancer marker proteins.     

Wound-induced proteinase inhibitors from tomato leaves. II. The cDNA-deduced primary structure of pre-inhibitor II

A cDNA containing the complete amino acid-coding region of wound-induced tomato Inhibitor II was constructed in the plasmid pUC9. The open reading frame codes for 148 amino acids including a 25-amino acid signal sequence preceding the N-terminal lysine of the mature Inhibitor II. The Inhibitor II sequence exhibits two domains, one domain having a trypsin inhibitory site and the other a chymotrypsin inhibitory site, apparently evolved from a smaller gene by a process of gene duplication and elongation. The amino acid sequence of tomato leaf Inhibitor II exhibits homology with two small proteinase inhibitors isolated from potato tuber and an inhibitor from eggplant. The small potato tuber inhibitors are homologous with 33 amino acids of the N-terminal domain and 19 amino acids from the C-terminal domain. Two identical nucleotide sequences of Inhibitor II cDNA in the 3' noncoding region were present that were also found in an Inhibitor I cDNA. These include an atypical polyadenylation signal, AATAAG, and a 10-base palindromic sequence, CATTATAATG, for which no function is yet known.     

PERMANENT GENETIC RESOURCES: Development of microsatellite markers and analysis of their inheritance in the Australian reptile tick, Bothriocroton hydrosauri

Despite long-term study, the mechanism explaining the parapatric distribution of two Australian reptile tick species is not understood. We describe the development of primers amplifying 10 microsatellite Bothriocroton hydrosauri loci, for the study of population structure and dispersal patterns of this tick. The numbers of alleles per locus ranged from two to seven in ticks from the study site, and the observed heterozygosity between 0.28 and 0.69. Pedigree analysis indicates that one locus is inherited in a non-Mendelian manner in three families, which was not explained by null allele presence.     

Identification of Novel Cryptosporidium Genotypes from the Czech Republic

Isolates of Cryptosporidium from the Czech Republic were characterized from a variety of different hosts using sequence and phylogenetic analysis of the 18S ribosomal DNA and the heat-shock (HSP-70) gene. Analysis expanded the host range of accepted species and identified several novel genotypes, including horse, Eurasian woodcock, rabbit, and cervid genotypes.     

Ultrastructural changes in major organelles during spermatial differentiation inBangia (Rhodophyta)

Summary The major organelles within the cells of maleBangia atropurpurea (Roth) C. Ag. filaments undergo a series of ultrastructural transformations during the production of spermatia. Initially, thylakoids within the large axial chloroplast develop a reticulate pattern commencing at the central pyrenoid region. Subsequent changes involve loss of lobes and diminution of volume through division; chloroplasts in final stages contain a few dilated, distorted thylakoids and many plastoglobuli. During differentiation the large nucleolus disappears from the nucleus and four masses of chromatin aggregate near the nuclear envelope. Furrows originating from the nuclear envelope form double membranes around each of the chromatin masses and most of the nucleoplasm is eliminated. Several types of fibrillar vesicles are formed during the process and large floridean starch reserves are utilized. Multilamellar bodies and microbody-like structures occur within the cells during certain phases of spermatiogenesis.     

A dye mixture (Neurobiotin and Alexa 488) reveals extensive dye-coupling among neurons in leeches; physiology confirms the connections

Although the neuronal circuits that generate leech movements have been studied for over 30 years, the list of interneurons (INs) in these circuits remains incomplete. Previous studies showed that some motor neurons (MNs) are electrically coupled to swim-related INs, e.g., rectifying junctions connect IN 28 to MN DI-1 (dorsal inhibitor), so we searched for additional neurons in these behavioral circuits by co-injecting Neurobiotin and Alexa Fluor 488 into segmental MNs DI–1, VI–2, DE–3 and VE–4. The high molecular weight Alexa dye is confined to the injected cell, whereas the smaller Neurobiotin molecules diffuse through gap junctions to reveal electrical coupling. We found that MNs were each dye-coupled to approximately 25 neurons, about half of which are likely to be INs. We also found that (1) dye-coupling was reliably correlated with physiologically confirmed electrical connections, (2) dye-coupling is unidirectional between MNs that are linked by rectifying connections, and (3) there are novel electrical connections between excitatory and inhibitory MNs, e.g. between excitatory MN VE-4 and inhibitory MN DI-1. The INs found in this study provide a pool of novel candidate neurons for future studies of behavioral circuits, including those underlying swimming, crawling, shortening, and bending movements.     

Proteinase inhibitors I and II in fruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal

The juice of unripe fruit from a wild species of tomato, Lycopersicon peruvianum (L.) Mill., LA 107, contains over 50% of its soluble proteins as the sum of two proteinase inhibitors. These are the highest levels of proteinase inhibitors and highest percentage of soluble proteins as proteinase inhibitors of any plant or animal tissue found to date. Fruit of the modern tomato, L. esculentum Mill., contains only negligible quantities of the two inhibitors. The two proteinase inhibitors in the fruit of L. peruvianum are members of the Inhibitor I and II families previously found in potato tubers and in leaves of wounded potato and tomato plants. The levels of the two inhibitors in the unripe fruit decrease significantly during ripening. Unripe fruit from other wild Lycopersicon species such as L. parviflorum Rick, Kesicki, Fobes et Holle, L. hirsutum Humb. et Bonpe., L. pimpinellifolium Mill., and other lines of L. peruvianum contain moderate levels of the inhibitors that also decrease during ripening. Another wild tomato species, L. pennellii Corr., is similar to L. esculentum in not containing the two proteinase inhibitors in either unripe or ripe fruit. The transient levels of the inhibitors in fruit of wild species indicate that they are present in unripe fruit as defensive chemicals against insects, birds or small mammals and their disappearance during ripening may render them edible to facilitate seed dispersal. High levels of mRNAs coding for Inhibitors I and II in unripe fruit of L. peruvianum, LA 107, indicate that strong promoters may regulate the developmentally expressed proteinase-inhibitor genes in tomato fruit that may have a substantial potential for use in genetic-engineering experiments to enhance the production of large quantities of proteinase inhibitors or other proteins in field tomatoes.Abbreviations poly(A)⁺ mRNA polyadenylated mRNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis Project 1791, College of Agriculture and Home Economics Research Center, Washington, State University