Biomedical subjects

Weiming Li

Publications and source records attributed to Weiming Li.

35 records · Page 2Linked to original sources

PubMed2004

15Alpha-hydroxyprogesterone in male sea lampreys, Petromyzon marinus L.

There is growing evidence that sea lampreys, Petromyzon marinus L., produce gonadal steroids differing from those of other vertebrates by possessing an additional hydroxyl group at the C15 position. Here we demonstrate that sea lamprey testes produce 15alpha-hydroxyprogesterone (15alpha-P) in vitro when incubated with tritiated progesterone, that 15alpha-P is present in the plasma of sea lampreys, and that plasma concentrations of immunoreactive (ir) 15alpha-P rise dramatically in response to injections of gonadotropin-releasing hormone (GnRH). The identity of the tritiated 15alpha-P produced in vitro was confirmed by co-elution with standard 15alpha-P on high performance liquid chromatography, co-elution with standard and acetylated 15alpha-P on thin layer chromatography, and specific binding to antibodies raised against standard 15alpha-P. The in vitro conversion was used to produce tritiated 15alpha-P label for a radioimmunoassay (RIA), which is able to detect 15alpha-P in amounts as low as 2 pg per tube. The RIA has been used to measure the plasma concentrations of 15alpha-P in males given two serial injections, 24 h apart, of either lamprey GnRH I or GnRH III (50, 100, or 200 microg/kg) or saline control, with plasma being sampled 8 and 24 h after the second injection. Plasma concentrations of ir-15alpha-P rose from < 1 to 36 ng/ml (mean of all treatments) 8 h after injection and declined within 24 h. This is the first time that an RIA has detected such high steroid concentrations in lampreys. This finding is suggestive of a role for 15alpha-P in control of reproduction in the sea lamprey.

Animals↗

PubMed2004

15alpha-hydroxytestosterone induction by GnRH I and GnRH III in Atlantic and Great Lakes sea lamprey (Petromyzon marinus L.).

The sea lamprey (Petromyzon marinus L.) represents one of the two most ancient classes of vertebrates and possesses a functional hypothalamus-pituitary-gonadal axis. However, the presence and functionality of androgens in the sea lamprey remain elusive. Recently, 15alpha-hydroxytestosterone (15alpha-T) has been found in sea lamprey gonads and blood plasma. In this study we examined changes of circulatory concentrations of 15alpha-T in response to gonadotropin releasing hormone (GnRH) treatments. Plasma concentrations of 15alpha-T in sea lamprey increased 2-5 times for all GnRH-injected sea lamprey compared to controls (P < 0.001). However, there were no differences among responses: (1) to the two forms of GnRH (lamprey GnRH I or lamprey GnRH III), (2) to the doses delivered (50, 100, or 200 microg/kg), or (3) between post-injection sample intervals (8 or 24 h). Between lampreys from the Atlantic Ocean and Great Lakes sites, two of seven GnRH form and dosage comparisons showed between-site differences, but were not believed to represent an overall between-site difference. These are the first data to show a response of a C19 steroid to GnRH stimulation in sea lamprey.

Animals↗

PubMed2004

Brain pathways and behavioral responses to weak electric fields in parasitic sea lampreys (Petromyzon marinus).

The authors characterized the behavioral and brain responses of parasitic sea lampreys (Petromyzon marinus) to weak electric fields. Lampreys showed preferences for the cathodal end of the testing aquarium after electric stimulation. Within a range of cathodal fields (-0.1 to -30.0 mu-V/cm), lampreys exhibited increased active behaviors. In contrast, anodal fields decreased most active behaviors below baseline. Exposure to electric fields resulted in changes in Western blot patterns for the neuronal activity markers Fos, Fos-B. and Jun in whole-brain homogenate. Electric stimulation also increased Fos-B immunoreactivity in the octavolateral and the habenula-fasciculus retroflexus-interpeduncular systems. These results confirm that the octavolateral system is associated with electroreception and suggest that the habenula-fasciculus retroflexus-interpeduncular system may be pan of the electroreceptive network.

Animals↗

PubMed2004

Impaired nuclear transport and uncoating limit recombinant adeno-associated virus 2 vector-mediated transduction of primary murine hematopoietic cells.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

Active Transport, Cell Nucleus↗

PubMed2004

beta-naphthoflavone induction of CYP1A in brain of juvenile lake trout (Salvelinus namaycush Walbaum).

Many environmental pollutants induce expression of the cytochrome P450 (CYP) 1A subfamily of genes. We integrated cellular and molecular biological techniques to examine the effects of beta-naphthoflavone (BNF) exposure in lake trout brain CYP1A distribution and dynamics. Over a 32-day time-course, real time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) results showed that CYP1A mRNA induction in response to BNF exposure occurred rapidly and continued to rise in the BNF-treated lake trout after 4 h, with a peak at or after 2 days. Messenger RNA levels fell after 4 days, and this trend continued after 16 days of exposure. In situ hybridization indicated that CYP1A mRNA was universally elevated in the brain of BNF-exposed fish and was mainly expressed in the endothelia and occasionally in the glial cells. CYP1A immunoreactivity was induced in the olfactory bulb and valvula cerebelli of BNF-treated fish. Other brain areas showed constitutive CYP1A immunoreactivity in both control and BNF-treated fish. Some BNF-treated fish contained multifocal hemorrhages in the brain tissue, and these fish had overall depressed CYP1A immunoreactivity in the brain. The relationship between transcriptional and translational effects of BNF exposure in the brain of juvenile lake trout is discussed.

Animals↗

PubMed2003

Glomerular territories in the olfactory bulb from the larval stage of the sea lamprey Petromyzon marinus.

The goal of this study was to investigate the spatial organization of olfactory glomeruli and of substances relevant to olfactory sensory neuron activity in the developing agnathan, the sea lamprey Petromyzon marinus. A 45-kD protein immunoreactive to G(olf), a cAMP-dependent olfactory G protein, was present in the ciliary fraction of sea lamprey olfactory epithelium and in olfactory sensory neurons of larval and adult sea lampreys. This result implies that G(olf) expression was present during early vertebrate evolution or evolved in parallel in gnathostome and agnathostome vertebrates. Serial sectioning of the olfactory bulb revealed a consistent pattern of olfactory glomeruli stained by GS1B(4) lectin and by anterograde labeling with fluorescent dextran. These glomerular territories included the dorsal cluster, dorsal ring, anterior plexus, lateral chain, medial glomeruli, ventral ring, and ventral cluster. The dorsal, anterior, lateral, and ventral glomeruli contained olfactory sensory axon terminals that were G(olf)-immunoreactive. However, a specific subset, the medial glomeruli, did not display this immunoreactivity. Olfactory glomeruli in the dorsal hemisphere of the olfactory bulb, the dorsal cluster, dorsal ring, anterior plexus, lateral chain, and medial glomeruli, were seen adjacent to 5HT-immunoreactive fibers. However, glomeruli in the ventral hemisphere, the ventral ring, and ventral cluster did not display this association. The presence of specific glomerular territories and discrete glomerular subsets with substances relevant to olfactory sensory neuron activity suggest a spatial organization of information flow in the lamprey olfactory pathway.

Animals↗

PubMed2003

Primary endothelial cells isolated from the yolk sac and para-aortic splanchnopleura support the expansion of adult marrow stem cells in vitro.

The embryonic origin and development of hematopoietic and endothelial cells is highly interdependent. We hypothesized that primary endothelial cells from murine yolk sac and para-aortic splanchnopleura (P-Sp) may possess the capacity to expand hematopoietic stem cells (HSCs) and progenitor cells ex vivo. Using Tie2-GFP transgenic mice in combination with fluorochrome-conjugated monoclonal antibodies to vascular endothelial growth factor receptor-2 (Flk1) and CD41, we have successfully isolated pure populations of primary endothelial cells from 9.5-days after coitus (dpc) yolk sac and P-Sp. Adult murine bone marrow Sca-1+c-Kit+lin- cells were cocultured with yolk sac or P-Sp Tie2-GFP+Flk-1+CD41- endothelial cell monolayers for 7 days and the total number of nonadherent cells increased 47- and 295-fold, respectively, and hematopoietic progenitor counts increased 9.4- and 11.4-fold, respectively. Both the yolk sac and P-Sp endothelial cell cocultures facilitated long-term (> 6 months) HSC competitive repopulating ability (2.8- to 9.8-fold increases, respectively). These data suggest that 9.5-dpc yolk sac- and P-Sp-derived primary Tie2-GFP+Flk-1+CD41- endothelial cells possess the capacity to expand adult bone marrow hematopoietic progenitor cell and HSC repopulating ability ex vivo.

Animals↗

PubMed2003

The homeoprotein Hex is required for hemangioblast differentiation.

The first hematopoietic and endothelial progenitors are derived from a common embryonic precursor termed the hemangioblast. The genetic cascades that regulate the differentiation of the hemangioblast to hematopoietic and endothelial cells are largely unknown. In general, much of embryonic development is coordinately regulated by temporal and spatial expression of transcription factors, such as the Homeobox (Hox) gene family. We and others isolated a divergent homeobox gene termed Hex (or Prh) that is preferentially expressed in hematopoietic and endothelial cells. Using in vitro Hex-/- embryonic stem (ES) cell differentiation, in vivo yolk sac hematopoietic progenitor assays, and chimeric mouse analysis, we found that Hex is required for differentiation of the hemangioblast to definitive embryonic hematopoietic progenitors and to a lesser extent endothelial cells. Therefore, Hex is a novel regulator of hemangioblast differentiation to hematopoietic and endothelial cells.

Animals↗

PubMed2003

Male sea lampreys, Petromyzon marinus L., excrete a sex pheromone from gill epithelia.

During the period when they are producing sperm, male sea lampreys (Petromyzon marinus L.) release a sex pheromone 7alpha, 12alpha, 24-trihydroxy-5alpha-cholan-3-one-24-sulfate (3 keto-petromyzonol sulfate, 3ketoPZS) that induces search and preference behaviors in ovulating females. In this study, we conducted a series of experiments to demonstrate that release of this pheromone into water takes place exclusively through the gills. In a behavioral maze, water conditioned with the anterior region of spermiating males induced an increase of search and preference behaviors in ovulating females. Similar behavior was not elicited by water conditioned by the posterior region. The anterior region washings and whole-body washings from spermiating males also elicited large and virtually identical electro-olfactogram responses from female sea lampreys, while the posterior washings produced negligible responses. Further, mass spectrometry and immunoassay confirmed that virtually all the 3ketoPZS released into water was through the gills. Immunocytochemistry revealed some gill epithelial cells and hepatocytes from spermiating males contained dense immunoreactive 3ketoPZS, but not those from prespermiating males. These results demonstrate that 3ketoPZS is released through the gill epithelia and suggest that this pheromone or its precursor may be produced in the liver.

Animals↗

PubMed2003

Quantitative PCR analysis of CYP1A induction in Atlantic salmon (Salmo salar).

Environmental pollutants are hypothesized to be one of the causes of recent declines in wild populations of Atlantic salmon (Salmo salar); across Eastern Canada and the United States. Some of these pollutants, such as polychlorinated biphenyls and dioxins, are known to induce expression of the CYP1A subfamily of genes. We applied a highly sensitive technique, quantitative reverse transcription-polymerase chain reaction (RT-PCR), for measuring the levels of CYP1A induction in Atlantic salmon. This assay was used to detect patterns of CYP1A mRNA levels, a direct measure of CYP1A expression, in Atlantic salmon exposed to pollutants under both laboratory and field conditions. Two groups of salmon were acclimated to 11 and 17 degrees C, respectively. Each subject then received an intraperitoneal injection (50 mg kg(-1)) of either beta-naphthoflavone (BNF) in corn oil (10 mg BNF ml(-1) corn oil) or corn oil alone. After 48 h, salmon gill, kidney, liver, and brain were collected for RNA isolation and analysis. All tissues showed induction of CYP1A by BNF. The highest base level of CYP1A expression (2.56 x 10(10) molecules/microg RNA) was found in gill tissue. Kidney had the highest mean induction at five orders of magnitude while gill tissue showed the lowest mean induction at two orders of magnitude. The quantitative RT-PCR was also applied to salmon sampled from two streams in Massachusetts, USA. Salmon liver and gill tissue sampled from Millers River (South Royalston, Worcester County), known to contain polychlorinated biphenyls (PCBs), showed on average a two orders of magnitude induction over those collected from a stream with no known contamination (Fourmile Brook, Northfield, Franklin County). Overall, the data show CYP1A exists and is inducible in Atlantic salmon gill, brain, kidney, and liver tissue. In addition, the results obtained demonstrate that quantitative PCR analysis of CYP1A expression is useful in studying ecotoxicity in populations of Atlantic salmon in the wild.

Animals↗

PubMed2003

15 alpha-Hydroxytestosterone produced in vitro and in vivo in the sea lamprey, Petromyzon marinus.

Prior research has shown that the testes of lampreys are able to synthesize 15-hydroxylated steroid hormones in vitro. Here we show that testes of the sea lamprey Petromyzon marinus L. are able to convert tritiated testosterone into tritiated 15alpha-hydroxytestosterone (15alpha-T) in high yield. The identity of the tritiated 15alpha-T has been confirmed by: co-elution with standard 15alpha-T on high performance liquid chromatography (HPLC); co-elution on thin layer chromatography (TLC); co-elution of acetylated tritiated and standard 15alpha-T on TLC; and strong binding to an antiserum developed against 15alpha-T. The strong reaction between the tritiated 15alpha-T and the antiserum has been used to develop a radioimmunoassay (RIA). The RIA operates over the range of 500-2pg per tube; and can be applied directly to plasma samples. This assay has been used to demonstrate that 15alpha-T is present in blood plasma of the sea lamprey. The concentrations of 15alpha-T in captive lamprey were found to be as follows (pg/ml; mean+/-SEM, n): parasitic stage (reproductively immature), <20, n=7; pre-ovulatory females, 156+/-30, n=8; ovulated females, 62+/-9, n=5; pre-spermiating males, 275+/-19, n=8; spermiating males, 216+/-48, n=8. When spermiating male plasma was fractionated on HPLC, immunoreactivity was found exclusively in the expected elution position of 15alpha-T. The biological significance of this steroid has yet to be established.

Animals↗

PubMed2003

Pheromones of the male sea lamprey, Petromyzon marinus L.: structural studies on a new compound, 3-keto allocholic acid, and 3-keto petromyzonol sulfate.

This study reports the results of chemical and chromatographic studies which establish the presence of 3-keto allocholic acid (3kACA) in water extracts from spermiating male sea lamprey, Petromyzon marinus. This is the second compound to be isolated and identified from these extracts. The first was 3-keto petromyzonol sulfate (3kPZS), which was shown to act as strong pheromonal attractant for ovulated females. Some new characterization data on 3kPZS (utilizing an only recently available synthetic preparation of the compound) is also included. The possibility that a mixture of 3kACA and 3kPZS might be a more potent pheromonal attractant than either compound alone is discussed.

3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)↗

PubMed2003

HPLC and ELISA analyses of larval bile acids from Pacific and western brook lampreys.

Comparative studies were performed on two native lamprey species, Pacific lamprey (Lampetra tridentata) and western brook lamprey (Lampetra richardsoni) from the Pacific coast along with sea lamprey (Petromyzon marinus) from the Great Lakes, to investigate their bile acid production and release. HPLC and ELISA analyses of the gall bladders and liver extract revealed that the major bile acid compound from Pacific and western brook larval lampreys was petromyzonol sulfate (PZS), previously identified as a migratory pheromone in larval sea lamprey. An ELISA for PZS has been developed in a working range of 20 pg-10 ng per well. The tissue concentrations of PZS in gall bladder were 127.40, 145.86, and 276.96 micro g/g body mass in sea lamprey, Pacific lamprey, and western brook lamprey, respectively. Releasing rates for PZS in the three species were measured using ELISA to find that western brook and sea lamprey released PZS 20 times higher than Pacific lamprey did. Further studies are required to determine whether PZS is a chemical cue in Pacific and western brook lampreys.

Animals↗

PubMed2003

Adeno-associated virus type 2-mediated gene transfer: role of cellular T-cell protein tyrosine phosphatase in transgene expression in established cell lines in vitro and transgenic mice in vivo.

The use of adeno-associated virus type 2 (AAV) vectors has gained attention as a potentially useful alternative to the more commonly used retrovirus and adenovirus vectors for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein (FKBP52), interacts with the single-stranded D sequence within the AAV inverted terminal repeats, inhibits viral second-strand DNA synthesis, and consequently limits high-efficiency transgene expression (K. Qing, J. Hansen, K. A. Weigel-Kelley, M. Tan, S. Zhou, and A. Srivastava, J. Virol., 75: 8968-8976, 2001). FKBP52 can be phosphorylated at both tyrosine and serine/threonine residues, but only the phosphorylated forms of FKBP52 interact with the D sequence. Furthermore, the tyrosine-phosphorylated FKBP52 inhibits AAV second-strand DNA synthesis by greater than 90%, and the serine/threonine-phosphorylated FKBP52 causes approximately 40% inhibition, whereas the dephosphorylated FKBP52 has no effect on AAV second-strand DNA synthesis. In the present study, we have identified that the tyrosine-phosphorylated form of FKBP52 is a substrate for the cellular T-cell protein tyrosine phosphatase (TC-PTP). Deliberate overexpression of the murine wild-type (wt) TC-PTP gene, but not that of a cysteine-to-serine (C-S) mutant, caused tyrosine dephosphorylation of FKBP52, leading to efficient viral second-strand DNA synthesis and resulting in a significant increase in AAV-mediated transduction efficiency in HeLa cells in vitro. Both wt and C-S mutant TC-PTP expression cassettes were also used to generate transgenic mice. Primitive hematopoietic stem/progenitor cells from wt TC-PTP-transgenic mice, but not from C-S mutant TC-PTP-transgenic mice, could be successfully transduced by recombinant AAV vectors. These studies corroborate the fact that tyrosine phosphorylation of the cellular FKBP52 protein strongly influences AAV transduction efficiency, which may have important implications in the optimal use of AAV vectors in human gene therapy.

Animals↗

PubMed2003

CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo.

The platelet glycoprotein IIb (alpha(IIb); CD41) constitutes the alpha subunit of a highly expressed platelet surface integrin protein. We demonstrate that CD41 serves as the earliest marker of primitive erythroid progenitor cells in the embryonic day 7 (E7.0) yolk sac and high-level expression identifies essentially all E8.25 yolk sac definitive hematopoietic progenitors. Some definitive hematopoietic progenitor cells in the fetal liver and bone marrow also express CD41. Hematopoietic stem cell competitive repopulating ability is present in CD41(dim) and CD41(lo/-) cells isolated from bone marrow and fetal liver cells, however, activity is enriched in the CD41(lo/-) cells. CD41(bright) yolk sac definitive progenitor cells co-express CD61 and bind fibrinogen, demonstrating receptor function. Thus, CD41 expression marks the onset of primitive and definitive hematopoiesis in the murine embryo and persists as a marker of some stem and progenitor cell populations in the fetal liver and adult marrow, suggesting novel roles for this integrin.

Animals↗

PubMed2002

Bile Acid secreted by male sea lamprey that acts as a sex pheromone.

We show that reproductively mature male sea lampreys release a bile acid that acts as a potent sex pheromone, inducing preference and searching behavior in ovulated female lampreys. The secreted bile acid 7alpha,12alpha,24-trihydroxy-5alpha-cholan-3-one 24-sulfate was released in much higher amounts relative to known vertebrate steroid pheromones and may be secreted through the gills. Hence, the male of this fish species signals both its reproductive status and location to females by secreting a pheromone that can act over long distances.

Animals↗

PubMed2002

Development and application of an ELISA for a sex pheromone released by the male sea lamprey (Petromyzon marinus L.).

An enzyme-linked immunosorbent assay (ELISA) has been developed for a conjugated bile acid, 7alpha,12alpha,24-trihydroxy-5alpha-cholan-3-one 24-sulfate (commonly referred to as 3-keto petromyzonol sulfate [3kPZS]), a pheromone released by reproductively mature male sea lampreys to attract sexually mature females. A polyclonal antiserum against the pheromone was raised by injecting 3-keto petromyzonol 24-hemisuccinate (3kPZ-HS) conjugated to bovine serum albumin into rabbits. The enzyme label was prepared by conjugating 3kPZ-HS to acetylcholinesterase. The standard curve had a working range of 20 pg-10 ng/well. Intra- and inter-assay variations were less than 5 and 12%, respectively. The antiserum had 100% cross-reaction with 3-keto petromyzonol and 3-keto allocholic acid but less than 0.2% cross-reaction with petromyzonol, allocholic acid, cholic acid, and taurolithocholic acid sulfate. The assay was applied to water which had been conditioned for 4h by either larvae, parasitic juveniles, ovulating females, pre-spermiating males, or spermiating males. Immunoactive material (average 200 ng/ml, which is equivalent to 500 microg animal/h) was only found in water from the reproductively mature males and diluted parallel with the standard curve. Assay of water samples collected from male lampreys in bisected aquaria also established that 99.6% of the immunoactive material emanated from the front end of the fish. This assay has applications in both physiological and ecological aspects of sea lamprey reproduction.

Animals↗