Diurnal and temporal variations of water-soluble dicarboxylic acids and related compounds in aerosols from the northern vicinity of Beijing: implication for photochemical aging during atmospheric transport.

Aerosol samples were collected in autumn 2007 on day- and nighttime basis in the northern receptor site of Beijing, China. The samples were analyzed for total carbon (TC) and water-soluble dicarboxylic acids (C2-C12), oxocarboxylic acids (C2-C9), glyoxal and methylglyoxal to better understand the photochemical aging of organic aerosols in the vicinity of Beijing. Concentrations of TC are 50% greater in daytime when winds come from Beijing than in nighttime when winds come from the northern forest areas. Most diacids showed higher concentrations in daytime, suggesting that the organics emitted from the urban Beijing and delivered to the northern vicinity in daytime are subjected to photo-oxidation to result in diacids. However, oxalic acid (C2), which is the most abundant diacid followed by C3 or C4, became on average 30% more abundant in nighttime together with azelaic, ω-oxooctanoic and ω-oxononanoic acids, which are specific oxidation products of biogenic unsaturated fatty acids. Methylglyoxal, an oxidation product of isoprene and a precursor of oxalic acid, also became 29% more abundant in nighttime. Based on a positive correlation between C2 and glyoxylic acid (ωC2) in nighttime when relative humidity significantly enhanced, we propose a nighttime aqueous phase production of C2 via the oxidation of ωC2. We found an increase in the contribution of diacids to TC by 3 folds during consecutive clear days. This study demonstrates that diacids and related compounds are largely produced in the northern vicinity of Beijing via photochemical processing of organic precursors emitted from urban center and forest areas.

Muscle development in the bamboo sole Heteromycteris japonicus with special reference to larval branchial levators.

Muscle development in the bamboo sole Heteromycteris japonicus was investigated, focusing primarily on the cranial muscles, using an improved whole mount immunohistochemical staining method with potassium hydroxide, hydrogen peroxide and trypsin. Larvae of H. japonicus had branchial levators, but not all of them were retained in adults, a condition also seen in the Japanese flounder Paralichthys olivaceus. In particular, larval branchial levators II and III disappeared during development, while I and IV remained to become the levator internus I and levator posterior, which were well-defined muscles in adults. In place of the atrophied muscles, levatores externi and levator internus II developed and regulated the branchial arches. The results showed that the muscle composition in the dorsal branchial arches changed to the adult form before metamorphosis in H. japonicus, as seen in P. olivaceus, and this transformation may be common to all members of that group.

Effects of a tropical cyclone on the distribution of hatchery-reared black-spot tuskfish Choerodon schoenleinii determined by acoustic telemetry.

The effects of a tropical cyclone on the distribution of hatchery-reared black-spot tuskfish Choerodon schoenleinii were examined using acoustic telemetry. Nine fish were released in Urasoko Bay, Ishigaki Island, Japan, in September 2006, and another nine were released in June to July 2007, before a cyclone's passing through the area in September 2007. Data for the fish released in 2006 were used as the cyclone-inexperienced group to compare their distribution pattern to that of the 2007 cyclone-experienced group. Both groups of fish were monitored for up to 150 days. Of the nine fish in each group, four (44%) and two (22%) were monitored for over 150 days in the cyclone-inexperienced and the cyclone-experienced groups, respectively. Three of the five fish that had settled in the monitoring area left the area within a few days of the cyclone event. To estimate the time of disappearance of the fish, maximum wind speed during a period of 7 days (indicating the occurrence and intensity of the tropical cyclone), fish size and release year were evaluated as explanatory variables using a Cox proportional hazards model with Akaike's information criterion. The best predictive model included the effect of maximum wind speed. One fish that left the monitoring area displayed movement patterns related to strong winds, suggesting that wind-associated strong currents swept the fish away. No relationships were found between the movement patterns of the other two fish and any physical environmental data. The daily detection periods of one of the two fish gradually decreased after the cyclone hit, and this fish eventually left the monitoring area within 3 days, suggesting that it shifted to a habitat outside the monitoring area. These results indicate that tropical cyclones have both direct and indirect effects on the distribution of hatchery-reared C. schoenleinii.

Seabream GnRH: partial cDNA cloning, localization and stage-dependent expression in the ovary of snake head murrel, Channa striatus.

Vertebrate reproduction is under the neuroendocrine control of the hypothalamic decapeptide GnRH which synchronizes various reproductive events and influences other reproduction related aspects like spawning behavior and pheromonal action in fish. Multiple forms of GnRH peptides have been reported across diverse vertebrate and invertebrate classes. Here we report the partial seabream GnRH (sbGnRH) cDNA sequence cloned from the brain of Channa striatus (snake head murrel) a fresh water perciform with immense economic and medicinal value across Asiatic countries. sbGnRH mRNA was found in brain, gill and ovary of mature murrel with possible implications to the effect of GnRH on pheromonal phenomena and on reinitiation of oocyte meiosis. In keeping with the earlier reported role of GnRH in initiation of oocyte meiosis we here present evidence from RT-PCR, ICC demonstrating an increase in the level of sbGnRH mRNA in ovary from pre-vitellogenic to post-vitellogenic follicles.

Effect of photoperiod manipulation on the daily rhythms of melatonin and reproductive hormones in caged European sea bass (Dicentrarchus labrax).

Reproduction in fish is cyclical and timed to guarantee the survival of the offspring. Seasonal variations in reproductive hormones of fish have been deeply investigated in fish over the last years. However, there are few studies regarding the daily changes in reproductive hormone profiles in teleosts. The aim of the present research was to investigate the effects of photoperiod manipulation on melatonin and reproductive hormones (pituitary sbGnRH, pituitary LH and plasma LH, testosterone [T], and 11-ketotestosterone [11KT]) daily rhythms in male sea bass, kept in net cages under farming conditions in winter (9L:15D). Fish were distributed in two groups, one under constant long photoperiod (18L:6D) and the other under natural photoperiod. The photoperiod strongly influenced the daily melatonin profile, so that the duration of the nocturnal melatonin rise was longer in the control group than in the group exposed to the artificial photoperiod (18L:6D). A daily rhythm was observed in the pituitary sbGnRH profile in both groups, showing the lowest levels during the dark period. A daily rhythm of pituitary LH was detected in the control group, which was suppressed in the group under long photoperiod. Daily variations in plasma LH were observed, the highest levels being found in the dark phase in both groups, although this profile was significantly altered by artificial light, maintaining a fixed relationship between the first nocturnal rise of melatonin and the nocturnal peaks of plasma LH in both groups. Plasma T levels showed significant fluctuations in their daily cycle following a sinusoidal pattern with an acrophase around sunrise in both groups, without any influence of light regime. No significant daily variations in plasma levels of 11-KT were observed in none of the groups. Our results provide the first evidence of the presence of daily variations in pituitary sbGnRH content, pituitary and plasma LH, and plasma T in sea bass. Artificial lights suppressed the circulating melatonin and significantly affected the daily rhythm of LH storage and release.

Correlation between messenger RNA expression of cytochrome P450 aromatase and its enzyme activity during oocyte development in the red seabream (Pagrus major).

In teleosts, estradiol-17beta (E2) is an important hormone responsible for oocyte development. To elucidate the molecular mechanisms underlying E2 biosynthesis, we characterized the structure of red seabream (Pagrus major) cytochrome P450 aromatase (P450(arom)) that is directly involved in E2 biosynthesis and found changes in mRNA levels of P450(arom) during oocyte development induced by implantation of gonadotropin-releasing hormone analogue. A cDNA clone encoding P450(arom) is 1779 base pairs in length and encodes a protein of 519 amino acids in length, with a calculated molecular weight of 58.9 kDa. Northern blot analysis showed that P450(arom) mRNA levels increased gradually from Day 8, when oocytes reached the secondary yolk globule stage, and were maintained at high levels at the day of spawning (Day 15). The P450(arom) mRNA levels increased in association with an increase of the gonadosomatic index (gonad weight/body weight x 100%), serum E2, and P450(arom) enzyme activity (in vitro conversion of testosterone to E2 in the ovarian fragments). Furthermore, an increase in mRNA levels of the LHbeta, but not FSHbeta, correlated with increased P450(arom) mRNA levels during the course of ovarian development. In addition, the levels of P450(arom) mRNA increased in isolated ovarian follicles during the course of vitellogenic oocyte growth and became undetectable in follicles at the migratory nucleus and the mature stages. These findings, together with those of the previous studies, suggest that LH, not FSH, may regulate E2 biosynthesis via increased levels of P450(arom) mRNA during oocyte development of red seabream.

Alterations in the GnRH-LH system in relation to gonadal stage and Aroclor 1254 exposure in Atlantic croaker.

Exposure of Atlantic croaker (Micropogonias undulatus) to the polychlorinated biphenyl mixture (Aroclor 1254, PCB; 1 mg/kg body wt/day for 30 days) during the early-recrudescence phase of the gonadal cycle results in the impairment of LH secretion and gonadal growth. In order to determine whether impairment was due to disruption of the stimulatory GnRH neuroendocrine pathway, we compared various parameters of the GnRH-LH system in early recrudescing vs. spermiating (mature) fish. Seabream GnRH (GnRH) content in the preoptic anterior hypothalamic area (POAH) and pituitary, pituitary GnRH receptor concentrations, and basal and GnRH analog (GnRHa)-induced LH secretion were significantly higher in gonadally mature croaker compared to early-recrudescing fish. In a subsequent experiment, the effects of PCB on the same neuroendocrine indices were investigated during the gonadal recrudescence phase of croaker. PCB exposure during the period of testicular maturation prevented the natural increase in GnRH content in the POAH but not in the pituitary. This finding suggests that PCB may impair GnRH synthesis in the POAH. The number of pituitary GnRH receptors also remained significantly lower in the PCB-exposed group, which was likely due to an impairment of GnRH release. The GnRH content in the POAH, number of pituitary GnRH receptors, and LH secretion in the PCB-exposed group were comparable to those in early-recrudescing fish, suggesting an impairment of normal maturation of the GnRH-LH system during the gonadal recrudescence phase. This impairment may be due to a direct action of PCB on GnRH neurons and/or indirectly via interference with other neurotransmitter pathways that modulate GnRH function.

Effects of serotonin, GABA and neuropeptide Y on seabream gonadotropin releasing hormone release in vitro from preoptic-anterior hypothalamus and pituitary of red seabream, Pagrus major.

The effects of serotonin (5-HT), GABA and neuropeptide Y (NPY) on in vitro release of seabream (sb) gonadotropin releasing hormone (GnRH) from slices of the preoptic-anterior hypothalamus (P-AH) and pituitary of red seabream were studied. 5-HT, GABA and NPY all stimulated the release of sbGnRH from the P-AH but not from the pituitary of immature red seabream. They also stimulated sbGnRH release from the P-AH with a similar potency during the course of gonadal development. Specific agonists and/or antagonists of 5-HT, GABA and NPY showed that 5-HT and GABA utilize 5-HT(2) and GABAA receptor subtypes, respectively, to mediate their action, and that NPY employs at least NPY(Y1) and NPY(Y2) receptor subtypes to stimulate sbGnRH release. Combinations of different antagonists for 5-HT, GABA and noradrenaline/adrenaline did not block the stimulatory influence of NPY on release of sbGnRH, indicating that the action of NPY on the sbGnRH neuronal system is probably direct.

Human tissue distribution of TA02, which is homologous with a new type of aspartic proteinase, napsin A.

The N-terminal amino acid sequence of TA02 (molecular weight 35.0 kDa, isoelectric point 5.29), which is associated with primary lung adenocarcinoma, was determined and a fragment peptide was used to generate mouse monoclonal antibodies (mAbs) against TA02. The amino acid sequence suggested that TA02 might be homologous with napsin A, a new type of aspartic proteinase. In this context, we confirmed the expression of napsin A in primary lung adenocarcinoma using reverse-transcription polymerare chain reaction (RT-PCR) and showed that the TA02 mAbs reacted with glutathione-S-transferase (GST)-napsin A fusion protein. We concluded that TA02 is the same molecule as napsin A, and showed immunohistochemically that it is distributed mainly in type II pneumocytes, alveolar macrophages, renal tubules and exocrine glands and ducts in the pancreas. In particular, type II pneumocytes and alveolar macrophages showed high expression of TA02 among human normal tissues. In primary lung adenocarcinoma, 47 out of 58 (81.0%) primary lesions were positive. All well-differentiated adenocarcinomas except those of goblet cell type showed high expression of TA02. In addition, two out of seven (28.6%) large cell carcinomas showed low expression of TA02. The other histopathological types of primary lung cancer did not express TA02 at all. A few cases of renal cell cancer, pancreatic cancer, breast cancer, thyroid cancer, colon cancer and ovarian cancer showed low expression, but the staining patterns were completely different from that of primary lung adenocarcinoma, which showed a granular staining pattern. Our novel mAbs should be valuable for immunochemical detection of TA02/napsin A.

Unique expression of gonadotropin-I and -II subunit genes in male and female red seabream (Pagrus major) during sexual maturation.

Two distinct gonadotropins (GTHs) have been demonstrated in a number of teleost fishes. Although the physiological roles of GTHs have been extensively studied in salmonids, little is known about their biological functions in nonsalmonid fishes. In this study, to elucidate the role of GTH-I and GTH-II in reproduction, we cloned the alpha-glycoprotein subunit (alphaGSU) and gonadotropin beta subunits (Ibeta and IIbeta) of red seabream using the 5'- and 3'-RACE methods and used these cDNA probes to reveal changes in mRNA levels of each subunit during sexual maturation of both male and female red seabream. The nucleotide sequences of alphaGSU, Ibeta, and IIbeta are 629, 531, and 557 base pairs long, encoding peptides of 117, 120, and 146 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high homology with those of other teleosts. Northern blot analysis showed that Ibeta mRNA levels of males increase in association with gonadal development, whereas those of females remain low throughout sexual maturation, indicating sexual dimorphism in the expression pattern of Ibeta. In contrast, IIbeta mRNA levels of both sexes are maintained at high levels from the beginning of gametogenesis to spawning season. These results are different than those of salmonids and suggest that GTH-I may have important roles in male, but not female, gametogenesis. GTH-II may be involved in regulation of early and late gametogenesis in both male and female red seabream.

Primary giant cell malignant fibrous histocytoma of the lung: a case report.

A rare case of malignant fibrous histiocytoma of giant cell type originating in the lung of a 46-year-old woman is presented. The patient complained of having a cough that had lasted for a few weeks. A chest X-ray photograph showed a tumor shadow on the left lung. Histological and cytological examination of the biopsy specimen revealed that the tumor was a kind of sarcoma. An operative procedure was selected because of tumor invasion into the trunk of the left pulmonary artery, which was discovered on computed tomography examination, and because metastatic tumor was excluded clinically. The tumor was almost encapsulated and 6 x 6 x 6 cm in size; however, it also showed invasion into the pulmonary artery and bronchial lumen. A histological survey of the tumor showed a wide range of patterns such as fibrous, pleomorphic, fascicular and osteoclast-like giant cell figures; however, the osteoclast-like giant cell area was predominant. Immunohistochemically, the tumor cells were positive for vimentin, CD68 for histiocytic marker and alpha1-antichymotrypsin, and negative for keratin, epithelial membrane antigen, S-100 protein, MT-1, desmin, myoglobin and lysosome. No primary tumor was found clinically in any part of the patient's body at 2 and 4 months after operation. Consequently, she was diagnosed as having primary giant cell malignant fibrous histiocytoma of the lung.

Distribution and seasonal variations in levels of three native GnRHs in the brain and pituitary of perciform fish.

Specific and sensitive radioimmunoassays (RIAs) were newly developed for two types of gonadotropin-releasing hormone (GnRH), namely, seabream (sb) GnRH and chicken (c) GnRH-II. We employed these two RIAs together with a previously reported RIA for salmon (s) GnRH to study the presence and regional distribution of these three GnRHs in the brains and pituitaries of four perciform fishes (red seabream, Pagrus major; black seabream, Acanthopagrus schlegeli; striped knifejaw, Oplegnathus fasciatus; and Nile tilapia, Oreochromis niloticus), as well as clarify seasonal changes in levels of these GnRHs in the brain and pituitary of red seabream. All three GnRHs were found in brains of all fishes examined, with regional distributions in the brains of the three GnRHs being rather similar. sbGnRH was abundant in telencephalon and hypothalamus. cGnRH-II was concentrated from the middle to posterior part of the brain and distributed throughout the brain. sGnRH was concentrated in the olfactory bulb and distributed all over the brain, as was cGnRH-II. The dominant form of GnRH in the pituitary was sbGnRH, with levels 500- to 2400-fold higher than those of sGnRH, while cGnRH-II was undetectable in all four species. In the brain and pituitary of female red seabream, levels of both brain and pituitary sbGnRH increased from October (immature phase) and reached a peak in April (spawning phase), reflecting the increase in gonadosomatic index and vitellogenesis. However, levels of sbGnRH remained high only in the pituitary of completely regressed fish in June. Levels of both sGnRH and cGnRH-II in the brain were higher in the regressed phase and remained lower during the spawning phase. From these and previous results, it appears that sbGnRH is physiologically the most important form of GnRH in reproduction in red seabream and, probably, in other perciforms also.

Cryopreservation of sperm from the endangered formosan landlocked salmon (Oncorhynchus masou formosanus).

The Formosan landlocked salmon (Oncorhynchus masou formosanus) are at a high risk of extinction, and the sustained maintenance of the population will soon depend on aquaculture systems, which use cryopreservation of spermatozoa to increase genetic diversity. We investigated the effectiveness of dimethyl sulfoxide (DMSO), dimethyl-acetamide (DMA), and methanol as cryoprotectants in combination with 300 mM glucose as extender on the freezing of Formosan landlocked salmon spermatozoa. We also evaluated the morphological changes of Formosan landlocked salmon spermatozoa after their immediate dilution in the 300 mM glucose-DMSO extender and after freeze-thawing. The spermatozoa frozen with DMSO as a cryoprotectant showed significantly higher post-thaw motility and fertility than spermatozoa frozen with DMA or methanol. The fertilization capacity of frozen-thawed Formosan landlocked salmon was comparable to that of fresh spermatozoa. Intersubspecies fertilization trials between cryopreserved Formosan landlocked salmon spermatozoa and Amago salmon eggs showed high fertilization rates. Based on the findings, the potential value of using sperm bank to safeguard this endangered species is discussed.

Levels of the native forms of GnRH in the pituitary of the gilthead seabream, Sparus aurata, at several characteristic stages of the gonadal cycle.

Brains of the gilthead seabream, Sparus aurata, contain three different forms of gonadotropin-releasing hormone (GnRH): seabream (sb) GnRH, chicken (c) GnRH-II, and salmon (s) GnRH. In the present study, we developed three specific enzyme-linked-immunosorbent assays (ELISA) for sbGnRH, cGnRH-II, and sGnRH and used them to measure the levels of each GnRH form in the pituitary of male and female seabream at different stages of gametogenesis. The sensitivity was 6 pg/well for the sbGnRH assay, 7 pg/well for the cGnRH-II assay, and 2 pg/well for the sGnRH assay. Levels of each of the three GnRH forms were measured in pituitaries from fish sampled at the beginning of gonadal recrudescence and during the spawning season. Of the three forms, only sbGnRH and cGnRH-II were detected in the pituitary, irrespective of reproductive state or sex. Recrudescent fish had similar levels of sbGnRH and cGnRH-II in the pituitary. In sexually mature fish, the levels of sbGnRH were higher than those in recrudescent fish while pituitary cGnRH-II content remained unchanged. Consequently, sbGnRH levels were 3- to 17-fold higher than cGnRH-II levels in mature fish. Positive correlations also existed between pituitary sbGnRH content and pituitary and plasma gonadotropin (GtH) II levels. Surprisingly, mature 1-year-old males had significantly higher levels of sbGnRH in the pituitary than mature 3-year-old males, while pituitary and plasma GtH II levels were similar between these two groups. Although the reason for this difference in sbGnRH levels is unclear, a possible role of sbGnRH in the processes of puberty or sex-inversion is implied. Based on the present results, it can be suggested that in the gilthead seabream, sbGnRH is the most relevant form of GnRH in the control of reproduction.

GTH II but not GTH I induces final maturation and the development of maturational competence of oocytes of red seabream in vitro.

The effects of red seabream gonadotropins (PmGTH I and PmGTH II) on the induction in vitro of germinal vesicle breakdown (GVBD) and the development of maturational competence (responsiveness to maturation-inducing steroid) were examined in the oocytes of red seabream. PmGTH II was highly effective in inducing GVBD in both maturationally incompetent (45.6 +/- 3.2% GVBD at a concentration of 300 ng/ml) and competent oocytes (42.3 +/- 0.4% GVBD at a concentration of 300 ng/ml). 17,20beta-Dihydroxy-4-pregnen-3-one (DHP, 10 ng/ml) increased the frequency of GVBD induced by PmGTH II. PmGTH I (33, 100, 300, and 900 ng/ml) was unable to induce GVBD at any tested concentration in the presence or absence of DHP. Actinomycin D (1 microgram/ml) and cycloheximide (1 microgram/ml) totally inhibited the PmGTH II-induced GVBD in the presence and in the absence of DHP. Both PmGTH I and PmGTH II stimulated in vitro production of 11-ketotestosterone in sliced testes of red seabream in a similar potency. These results indicate that PmGTH II, but not PmGTH I, induces the final maturation of oocytes, as well as the development of the maturational competence of oocytes, in red seabream.

Immunocytochemical identification of two distinct gonadotropic cells (GTH I and GTH II) in the pituitary of bluefin tuna, Thunnus thynnus.

Immunocytochemical identification of GTH I and GTH II cells in the pituitary of the bluefin tuna (Thunnus thynnus) was performed using antisera specific for the common alpha-subunit and the two distinct beta-subunits of tuna (Thunnus obesus) GTH I and GTH II. Cells of the dorsal part of the proximal pars distalis (PPD), in close association with somatotrophs, displayed immunoreactivity of GTHIbeta. GTH IIbeta immunoreactivity was present in cells of the central part of the PPD and the external border of the pars intermedia. Anti-GTHalpha immunostained both GTH Ibeta- and GTH IIbeta-immunoreactive cells and also thyrotrophs. Both GTH Ibeta- and GTH IIbeta-immunoreactive cells were observed in immature bluefin tuna, although there were greater numbers of GTH IIbeta immunoreactive cells. These results suggest that GTH I and GTH II are synthesized in separate cells in the pituitary of the bluefin tuna. The localization and appearance of the two distinct gonadotropic cells of the tuna are compared with the salmonid arrangement.

Distinct efficacies for two endogenous ligands on a single cognate gonadoliberin receptor.

A cDNA encoding a putative gonadoliberin receptor was cloned from the pituitary of the African catfish. Conceptual translation predicts a protein of 379 amino acids which shows typical characteristics of GTP-binding-protein-coupled receptors. The isolated cDNA was stable expressed in human embryonic kidney (HEK) 293 cells which were used for studies on gonadoliberin-activated second messenger systems (inositol phosphate production; increase in cAMP and/or intracellular Ca2+). The isolated cDNA encoded a functional receptor, designated catfish gonadoliberin receptor (cfGnRH-R), which had an amino acid sequence similarity of 38% with mammalian gonadoliberin receptors. In contrast to its mammalian counterparts which lack an intracellular carboxy-terminal domain, the cfGnRH-R contains an additional 49 amino acid residues. From the two endogenous gonadoliberins in African catfish, chicken gonadoliberin-II had a several hundredfold higher potency than catfish gonadoliberin to activate cfGnRH-R-associated second messenger systems in transfected HEK 293 cells. This is in line with the previously determined higher gonadotropin-release capacity of chicken gonadoliberin-II in catfish. Stimulation of second messenger systems with chicken gonadoliberin-II, but not with catfish gonadoliberin, resulted in a biphasic effect and chicken gonadoliberin-II led to a higher maximum stimulation than catfish gonadoliberin. Challenging cfGnRH-R simultaneously with chicken gonadoliberin-II and catfish gonadoliberin did not lead to additive effects. In contrast, two types of mutual inhibitory effects were recorded. These data indicate that a single cognate cfGnRH-R couples with distinct efficacies to signal transduction systems upon stimulation by the two endogenous gonadoliberins which, in addition, may interact negatively.

Relationship between TA01 and TA02 polypeptides associated with lung adenocarcinoma and histocytological features.

TA01 (molecular weight 35.0 kDa, isoelectric point 5.45) and TA02 (molecular weight 35.0 kDa, isoelectric point 5.29) polypeptides were detected using two-dimensional polyacrylamide gel electrophoresis (2-DE). A previous study has shown that these polypeptides are distributed in primary adenocarcinomas and some large-cell carcinomas of the lung. However, various expression levels of TA01 and TA02 polypeptides were demonstrated in adenocarcinoma, while large-cell carcinoma expressed low levels. To evaluate the relationship between the expression of TA01 and TA02 polypeptides and the histocytological features of primary adenocarcinoma of the lung, these two polypeptides were analysed by 2-DE combined with a non-enzymatic sample preparation technique, and their expression levels were compared with the histocytological features of primary lung adenocarcinoma. Out of 57 primary lung adenocarcinoma cases, 46 cases (80.7%) and 52 cases (91.2%) expressed TA01 and TA02 polypeptides respectively. Furthermore, the expression levels of TA01 and TA02 polypeptides correlated with the degree of cellular atypia, structural atypia and histocytological differentiation of primary lung adenocarcinoma. On the other hand, these two polypeptides were not detected in adenocarcinoma of the lung, metastatic from the colon and mammary glands. High expression of TA01 and TA02 polypeptides reflected the differentiation of primary adenocarcinoma in the lung. These two polypeptides are valuable in determining the histocytological differentiation of primary lung adenocarcinoma as well as in distinguishing between primary and metastatic adenocarcinoma of the lung.

Analysis of polypeptide expression in benign and malignant human breast lesions: down-regulation of cytokeratins.

Malignant progression of tumour cells is caused by the accumulation of genetic defects, which when combined will generate a large phenotypic diversity. Simultaneous quantitation of a large number of gene products in tumour cells is desirable, but difficult to achieve. We have here quantitated the levels of a number of abundant polypeptides in human breast carcinoma cells using two-dimensional gel electrophoresis (2-DE; PDQUEST). For this purpose, tumour cells were prepared from the tissue of 17 breast carcinomas. Fibroadenoma tissue was used as reference for benign cells. An increase of the spot density of the PCNA polypeptide was observed in rapidly proliferating tumour cells, confirming the validity of the procedures used. In the set of 24 polypeptide spots with known identity, decreases in cytokeratin and tropomyosin levels were observed. The levels of all cytokeratin forms resolved (CK7, CK8, CK15 and CK18) were significantly lower in carcinomas than in fibroadenomas. The levels of tropomyosin 2 and 3 were lower in carcinomas than in fibroadenomas. In contrast, the levels of some members of the stress protein family (pHSP60, HSP90 and calreticulin) were higher in carcinomas. Furthermore, changes in the expression of lactate dehydrogenase and GT-pi, but not in nm23, were observed. We conclude that simultaneous analysis of multiple polypeptides in human carcinomas can be achieved by 2-DE and may be useful in prognostic studies, and that malignant progression of breast carcinomas results in the decreased expression of cytokeratin polypeptides. This phenomenon must be considered in studies where cytokeratins are used as markers to identify the epithelial cell compartment in breast carcinomas.

Assessment of homogeneity in polypeptide expression in breast carcinomas shows widely variable expression in highly malignant tumors.

We describe the results from a protein-based approach to the study of heterogeneity in gene expression between human tumors. Cell preparations from 5 benign breast lesions, 5 potentially weakly malignant and 4 potentially highly malignant invasive ductal breast carcinomas were examined by 2-dimensional gel electrophoresis (2-DE) gels. Qualitative and quantitative differences were recorded by computerized analysis. Analysis of samples from different areas of the same tumor showed a high degree of similarity in the pattern of polypeptide expression. Analysis of 2 tumors and their metastases revealed similar 2-DE profiles. In contrast, variations between different lesions with comparable histological characteristics were considerable. Greater differences in polypeptide expression were observed between potentially highly malignant carcinomas compared with comparisons of less malignant lesions. Our results show that malignant human breast carcinomas may be highly heterogeneous in their patterns of gene expression.