Biomedical subjects

H F Kung

Publications and source records attributed to H F Kung.

At least 91 records · Page 5Linked to original sources

PubMed1997

Radioligand binding and immunoautoradiographic evidence for a lack of toxicity to dopaminergic nerve terminals in human cocaine overdose victims.

Radioligand binding to and immunolabeling of transport sites associated with monoamine-containing synaptic vesicles affords a novel approach for mapping the integrity of dopaminergic (DAergic) nerve terminals. The present study used [125I]iodovinyltetrabenazine ([125I]TBZ) and a fusion protein antibody directed at the large intraluminal loop of the neuronal vesicular monoamine transporter (hVMAT2-loop) as probes to assess the effects of chronic cocaine use on the integrity of DAergic nerve terminals in the striatum of cocaine fatalities. Visualization of [125I]TBZ binding in human brain revealed a distinct pattern of labeling throughout the rostral-caudal extent of the striatum. Saturation binding of [125I]TBZ in striatal membranes demonstrated a single high affinity site (Kd = 2.3 +/- 0.9 nM and Bmax = 55.5 +/- 8.1 pmol/g tissue) with a pharmacological profile (tetrabenazine > or = iodovinyltetrabenazine > ketanserin > or = reserpine > haloperidol > GBR 12909) consistent with the specific labeling of hVMAT2. Quantitative in vitro autoradiography demonstrated no significant alteration in the density of [125I]TBZ binding sites in the anterior and posterior sectors of the striatum in cocaine fatalities with and without preterminal excited delirium as compared to drug-free and age-matched control subjects. Similarly, the levels of hVMAT2-loop immunoreactivity were not significantly different across control and cocaine fatality groups. The results demonstrate the lack of an alteration in [125I]TBZ binding sites and hVMAT2 protein in the striatum from a young cohort of cocaine fatalities. Since striatal VMAT2 is primarily associated with DAergic nerve terminals, these results suggest that chronic cocaine use failed to affect the integrity of striatal DAergic nerve terminals.

Adolescent↗

PubMed1997

Synthesis and characterization of technetium-99m-labeled tropanes as dopamine transporter-imaging agents.

In the development of novel Tc-99m-labeled tropane derivatives as dopamine transporter (reuptake site)-imaging agents, a series of neutral and lipophilic complexes containing bis-(aminoethanethiol) as a neutral complexing moiety for a [99mTc]TcO3+ center core was successfully prepared. Biological evaluation of the Tc-99m-labeled complexes 13-16 as central nervous system (CNS) dopamine transporter-imaging agents was reported. Synthesis of the tropane derivatives was achieved by stepwise reactions adding two aminoethanethiol units. The final free thiol ligands were obtained by deblocking the 4-methoxybenzyl protecting group with Hg(OAc)2 to obtain trifluoroacetate salts. All of the Tc-99m complexes, with the exception of 16, displayed good initial brain uptake and selective uptake in the striatal area, where dopamine transporters are concentrated. One of the compounds, [2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo [3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethy] amino]ethanethiolato-(3-)-N2,N2',S2,S2'] oxo-[1R-(exo-exo)]- [99mTc]technetium,[99mTc]TRODAT-1 (13), displayed the highest initial uptake in rat brain (0.4% at 2 min post iv injection); the striatal/cerebellar (ST/ CB) ratio reached 2.8 at 60 min after an iv injection. The specific uptake in rat brain can be blocked by pretreating rats with a competing dopamine transporter binding agent, beta-CIT (RTI-55, 2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane; iv, 1 mg/kg), which reduced the regional brain uptake ratio (ST/CB) to 1.2. In contrast, the specific striatal uptake was not affected by pretreating rats with a noncompeting ligand, haldol (iv, 1 mg/kg). After an iv injection of 9 mCi of [99mTc]TRODAT-1 (13), in vivo images of baboon brain using single-photon emission-computed tomography exhibited excellent localization in striatum (basal ganglia), where dopamine neurons are known to be concentrated. This series of compounds may provide a convenient source of short-lived imaging agents for routine diagnosis of CNS diseases (i.e., Parkinson's disease) in which changes in the dopamine transporter concentration are implicated.

Animals↗

PubMed1997

Microdialysis and SPECT measurements of amphetamine-induced dopamine release in nonhuman primates.

The competition between endogenous transmitters and radiolabeled ligands for in vivo binding to neuroreceptors might provide a method to measure endogenous transmitter release in the living human brain with noninvasive techniques such as positron emission tomography (PET) or single photon emission computerized tomography (SPECT). In this study, we validated the measure of amphetamine-induced dopamine release with SPECT in nonhuman primates. Microdialysis experiments were conducted to establish the dose-response curve of amphetamine-induced dopamine release and to document how pretreatment with the dopamine depleter alpha-methyl-para-tyrosine (alpha MPT) affects this response. SPECT experiments were performed with two iodinated benzamides, [123I]IBZM and [123I]IBF, under sustained equilibrium condition. Both radio-tracers are specific D2 antagonists, but the affinity of [123I]IBZM (KD-0.4 nM) is lower than that of [123I]IBF (KD 0.1 nM). With both tracers, we observed a prolonged reduction in binding to D2 receptors following amphetamine injection. [123I]IBZM binding to D2 receptors was more affected than [123I]IBF by high doses of amphetamine, indicating that a lower affinity increases the vulnerability of a tracer to endogenous competition. With [123I]IBZM, we observed an excellent correlation between reduction of D2 receptor binding measured with SPECT and peak dopamine release measured with microdialysis after various doses of amphetamine. Pretreatment with alpha MPT significantly reduced the effect of amphetamine on [123I]IBZM binding to D2 receptors, confirming that this effect was mediated by intrasynaptic dopamine release. Together, these results validate the use of this SPECT paradigm as a noninvasive measurement of intrasynaptic dopamine release in the living brain.

Amphetamine↗

PubMed1997

P-[18F]-MPPF: a potential radioligand for PET studies of 5-HT1A receptors in humans.

The purpose of this study was to develop a radiopharmaceutical that could be used to selectively image 5-HT1A receptors with positron emission tomography (PET). No-carrier-added 4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p-[18F] fluorobenzamido]ethylpiperazine (p-[18F]-MPPF, 2) was synthesized by the nucleophilic substitution of the corresponding nitro precursor 1 with K[18F]/Kryptofix 2.2.2. in dimethyl sulfoxide (DMSO) at 140 degrees C for 20 min followed by purification with high-performance liquid chromatography (HPLC) in 10% yield in a synthesis time of 90 min from end of bombardment (EOB). Specific activity was 1-4 Ci/microM. Biodistribution studies in rats showed that the initial uptake of 2 in the brain was high (0.7% dose/g tissue at 2 min). It was then rapidly eliminated. Rates of elimination were significantly slower in brain regions with high concentrations of 5-HT1A receptors (hippocampus, cortex, and hypothalamus) than in control regions. The maximum hippocampal/cerebellar ratio was 5.6:1 at 30 min postinjection. Uptake values in serotonergic, but not in control, regions were significantly reduced by prior treatment with either (+/-)-8-OH-DPAT (2 mg/kg, i.v., 5 min prior) or WAY 100635 (1 mg/kg, i.v., 5 min prior). Radioactivity in the femur did not increase with time, suggesting that in vivo defluorination may not be the major route of metabol sm. PET studies of 2 in a monkey demonstrated selective uptake and retention of 2 in the hippocampus. The hippocampal/cerebellar ratio was 3:1 at 30 min postinjection. The ratio was reduced to 1:1 by administering (+/-)-8-OH-DPAT (2 mg/kg, i.v.) 23 min postinjection of 2. Analyses of arterial plasma by HPLC revealed that 20% of radioactivity in the plasma remained as the parent compound 2 at 30 min postinjection. The results suggest that p-[18F]-MPPF may be a useful radioligand for studying cerebral 5-HT1A receptors in humans with PET techniques.

Aminopyridines↗

PubMed1997

[99mTc]TRODAT-1: a novel technetium-99m complex as a dopamine transporter imaging agent.

Technetium-99m is the most commonly used radionuclide in routine nuclear medicine imaging procedures. Development of 99mTc-labeled receptor-specific imaging agents for studying the central nervous system is potentially useful for evaluation of brain function in normal and disease states. A novel 99mTc-labeled tropane derivative, [99mTc]TRODAT-1, which is useful as a potential CNS dopamine transporter imaging agent, was evaluated and characterized. After i. v. injection into rats, [99mTc]TRODAT-1 displayed specific brain uptake in the rat striatal region (striatum-cerebellum/cerebellum ratio 1.8 at 60 min), where dopamine neurons are concentrated. The specific striatal uptake could be blocked by pretreating rats with a dose of competing dopamine transporter ligand, beta-CIT (or RTI-55, i.v., 1 mg/kg). However, the specific striatal uptake of [99mTc]TRODAT-1 was not affected by co-injection of excess free ligand (TRODAT-1, up to 200 microg per rat) or by pretreating the rats with haloperidol (i.v., 1 mg/kg). The specific uptake in striatal regions of rats that had prior 6-hydroxydopamine lesion in the substantia nigra area showed a dramatic reduction. The radioactive material recovered from the rat striatal homogenates at 60 min after i.v. injection of [99mTc]TRODAT-1 showed primarily the original compound (>95%), a good indication of in vivo stability in brain tissue. Similar and comparable organ distribution patterns and brain regional uptakes of [99mTc]TRODAT-1 were obtained for male and female rats. Ex vivo autoradiography results of rat brain sections further confirmed the high uptake and retention of [99mTc]TRODAT-1 in the striatal region. In vitro binding studies measuring the affinity to dopamine transporters for the free ligand, TRODAT-1, and a nonradioactive rhenium derivative, Re-TRODAT-1, showed Ki values of 9.7 nM and 14.1 nM, respectively. Behavioral studies in rats using the free ligand, TRODAT-1 and Re-TRODAT-1 indicated that, unlike other tropane derivatives, they displayed no effect on locomotor activity, suggesting low toxicity. These results strongly support the conclusions that this novel 99mTc radioligand binds selectively to dopamine transporters in the brain and that is is potentially useful for in vivo assessment of the loss of dopamine neurons in Parkinson's and other neurodegenerative diseases.

Animals↗

PubMed1997

Relationship between clinical features of Parkinson's disease and presynaptic dopamine transporter binding assessed with [123I]IPT and single-photon emission tomography.

IPT [N-(3-iodopropen-2-yl)- 2beta-carbomethoxy-3beta-(4-chlorophenyl) tropane] is a new cocaine analogue which allows the presynaptic dopamine transporters to be imaged with single-photon emission tomography (SPET) as early as 1-2 h post injection. In the present study [123I]IPT SPET was performed in patients with Parkinson's disease (PD) to analyse the relationship between specific dopamine tansporter binding and clinical features of the disease. Twenty-six PD patients (Hoehn and Yahr stages I-IV, age range 40-79 years) and eight age-matched controls were studied. SPET imaging was performed 90-120 min after injection of 160-185 MBq [123I]IPT using a triple-head camera. For semiquantitative evaluation of specific [123I]IPT binding, ratios between caudate, putamen and background regions were calculated. Specific [123I]IPT uptake was significantly reduced in PD patients compared to controls. Most patients showed a marked asymmetry with a more pronounced decrease in [123I]IPT binding on the side contralateral to the predominant clinical findings. The putamen was always more affected than the caudate. [123I]IPT binding was significantly correlated with disease duration (r=-0.7, P<0.0001) but not with the age of PD patients (r=-0.10, P=0. 61). Specific [123I]IPT uptake in the caudate and putamen, and putamen to caudate ratios, decreased with increasing Hoehn and Yahr stage. Our findings indicate that [123I]IPT SPET may be a useful technique to estimate the extent of nigrostriatal degeneration in PD patients. Close relationships between striatal [123I]IPT binding and clinical features of the disease suggest that this method can be used to objectively follow the course and progression of PD. The reduced putamen to caudate ratios observed even in patients with mild, newly recognized symptoms indicate that particularly this parameter may help to establish the correct diagnosis in the early course of PD.

Adult↗

PubMed1997

Characterization of a novel iodinated ligand, IPMPP, for human dopamine D4 receptors expressed in CHO cells.

A novel radioiodinated ligand with a high specific activity (2,200 Ci/mmol), 3-[4-(4-iodophenyl)piperazin-1-yl]methyl-1H-pyrrolo(2,3-b)pyridine ([125I]IPMPP), was successfully prepared. Binding characteristics of [125I]IPMPP were evaluated using human dopamine D4 (D4.2 variant) receptors expressed in Chinese hamster ovary (CHO) cells. Saturation analysis revealed high-affinity binding sites for [125I]IPMPP (Kd = 0.39 +/- 0.18 nM). The number of D4 receptors labeled with [125I]IPMPP at room temperature was four times higher than that labeled with [125I]S(-)5-OH-PIPAT, a radioiodinated agonist ligand (572 fmol/mg protein vs. 125 fmol/mg protein). A significant decrease in the number of binding sites was observed with [125I]S(-)5-OH-PIPAT when assays were carried out at a higher temperature (37 degrees C vs. 25 degrees C). In contrast to [125I]S(-)5-OH-PIPAT, [125I]IPMPP labeled more D4 sites at 37 degrees C. Neither magnesium ion nor guanylimidodiphosphate (Gpp(NH)p) affected [125I]IPMPP binding. These data support the conclusion that [125I]IPMPP is an antagonist ligand. The potency of various compounds, including clozapine, to inhibit [125I]IPMPP binding is consistent with the rank order measured with other radioligands for D4 receptors. In addition, measuring D4 receptor stimulation of [35S]GTPgammaS binding further demonstrated the antagonist property of IPMPP.

Animals↗

PubMed1997

Characterization of [125I]S(-)5-OH-PIPAT binding to dopamine D2-like receptors expressed in cell lines.

Recently, [125I]S(-)5-OH-PIPAT [5-hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl)aminotetralin], a derivative of S(-)5-OH-DPAT (5-hydroxy-N,N-di-n-propyl-aminotetralin), was reported to be a better radioiodinated dopamine D2-like receptor ligand than the previously reported iodinated ligand, [125I]R(+)7-OH-PIPAT. Therefore, in the present study, the binding profile of [125I]S(-)5-OH-PIPAT to D2-like receptors expressed in cell lines was established. High binding affinity (Kd = 0.3-0.4 nM) and NaCl sensitivity were displayed with this ligand in membranes of human embryonic kidney (HEK293) cells expressing either human D2 or rat D3 receptors and in Chinese hamster ovary (CHO) cells expressing human dopamine D4 receptors. Specific binding to D2 and D4 receptors was significantly increased in the presence of 2 mM MgCl2 and decreased in the presence of 100 microM 5'-guanylyl-imidodiphosphate (GMP-PNP). This finding is consistent with reports that 2-aminotetralin compounds display agonist properties. The specific binding to D3 receptors however, was not affected by either MgCl2 or GMP-PNP. This lack of GMP-PNP sensitivity for D3 receptors may result from inadequate G protein-receptor coupling in this cell line. The rank order of potency for inhibition of [125I]S(-)5-OH-PIPAT binding with various dopamine agents was consistent with reported values for D2, D3 and D4 receptors. In membranes prepared from Spodoptera frugiperda (Sf9) cells infected with baculovirus that contains DNA encoding D3 receptors, [125I]S(-)5-OH-PIPAT recognized only 70% of the receptor population labeled by [125I]NCQ298. This new ligand offers several unique advantages, including high specific activity, high binding affinity and selectivity for D2-like receptors, that make it an excellent probe for the investigation and the characterization of dopamine D2-like receptors.

Animals↗

PubMed1997

Expression and characterization of a cytotoxic human-frog chimeric ribonuclease: potential for cancer therapy.

Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and last six of the 104 amino acid residues to a genomic clone that encoded the remaining amino acid residues. Additionally, the 15 N-terminal amino acid residues of onconase were replaced with the first 21 amino acid residues of the homologous human RNase, eosinophil-derived neurotoxin, EDN. Two versions of the hybrid EDN-onconase protein were cloned, expressed and purified. The chimera that contained a glycine in lieu of the aspartic acid present in native onconase (position 26 in the chimera) exhibited enzymatic activity more characteristic of EDN than native onconase and was considerably more active with respect to both RNase activity and cellular cytotoxicity than recombinant onconase. In contrast to native or recombinant onconase, the EDN chimera was recognized by anti-EDN polyclonal antibodies, demonstrating that the chimera also shared structural antigenic determinants to the human enzyme. These results demonstrate that a chimeric ribonuclease has cytotoxicity comparable to onconase in two out of four cell lines tested. The implications with regard to cancer therapy are presented.

Amino Acid Sequence↗

PubMed1997

Ethanol down-regulates the transcription of microsomal triglyceride transfer protein gene.

Microsomal triglyceride transfer protein (MTP) plays a central role in the assembly and secretion of apoB-containing lipoproteins. In this study, we investigated the effect of ethanol on the expression of the large subunit of MTP in a human liver hepatoma cell line, the HepG2 cells. Exposure of HepG2 cells to low concentrations of ethanol reduced MTP mRNA levels in a concentration- and time-dependent manner. The level of MTP mRNA decreased significantly (P<0.05, -26% relative to pretreatment control) when the concentration of ethanol in the culture medium was 50 ppm (0.005%, v/v). Maximal suppression (-50%) was observed at 100 ppm ethanol; the MTP mRNA levels remained at 50% of control when the ethanol concentration was raised to 10,000 ppm. Furthermore, a 10-day ethanol treatment caused a significant 50% decrease in the MTP activity and apoB secretion rate in HepG2 cells. To investigate the molecular mechanisms underlying this phenomenon, we examined the effect of ethanol on the promoter activity of the MTP gene. Transient transfection analysis of human MTP promoter-driven luciferase gene expression showed that ethanol down-regulates MTP promoter activity in a manner parallel to that observed for mRNA levels. Deletion analysis suggested that the MTP promoter sequence contains a negative ethanol response element -612 to -142 bp upstream of the transcription start site. To evaluate the in vivo relevance of the effect of ethanol on MTP mRNA levels, rats were given a single oral dose of ethanol, with hepatic and intestinal MTP mRNA measured 3 h after dosing. Rats receiving 1 or 3 g/kg of ethanol exhibited substantially lower hepatic and intestinal MTP mRNA levels. Taken together, these results strongly suggest that ethanol can modulate the secretion of apoB-containing lipoproteins by down-regulating the expression of MTP large subunit, primarily through inhibiting the transcription of the MTP gene.

Animals↗

PubMed1997

Differential regulation of neurogenesis by the two Xenopus GATA-1 genes.

Previously, we have shown that the ventralizing factor bone morphogenetic protein 4 (BMP-4) can inhibit Xenopus neurogenesis. The erythroid transcription factor GATA-1 functions downstream of the BMP-4 signaling pathway and mediates BMP-4-induced erythropoiesis. We have found that similar to BMP-4, GATA-1b inhibits neuralization of Xenopus animal cap (AC) cells. The neural inhibition is not seen with GATA-1a, although both GATA-1a and GATA-1b RNAs are translated at the same efficiency and induce globin expression equally in AC cells. GATA-1b RNA injection into AC cells neither induces expression of Xbra (a general mesoderm marker) nor affects expression of XK81 (epidermal keratin) or BMP-4 and Xvent-1 (two ventral markers). These data suggest that GATA-1b retains the epidermal fate of the AC. Intact GATA-1b protein is required for both inhibition of neurogenesis and induction of globin expression. Our findings indicate that GATA-1b can function in ectoderm to specifically regulate neural inducing mechanisms, apparently related to the expression of chordin, a neuralizing gene. Furthermore, tadpole stage embryos injected with GATA-1b are devoid of all dorsoanterior structures including neural tissue. This report provides evidence that the two transcription factors, derived from a recent genome duplication, share a common biological activity (stimulation of erythropoiesis) while also exhibiting a distinct function (inhibition of neurogenesis).

Animals↗

PubMed1997

Human prostate carcinogenesis.

Prostate cancer is a major medical problem that is expected to affect over 300,000 American men and cause over 40,000 deaths in 1997. Despite its widespread prevalence and because of the difficulties in clinical diagnosis and treatment of the disease, the etiological mechanism underlying prostate carcinogenesis remains poorly understood. Elucidation of the mechanism of prostate tumorigenesis has been slowed by a lack of tumor tissues and the limited number of human cell lines available for study. In vitro human cell models to study the molecular biology of prostate cancer progression are urgently needed. Normal human prostate cells require immortalization to provide a practical system for transformation studies. Neoplastic transformation of human prostate epithelial cells in culture has been achieved recently in a stepwise fashion--immortalization of primary cells in culture and conversion of the immortalized cells to a tumorigenic state. Reviewed here are the steps involved in the neoplastic transformation of human prostate cells. To provide an insight into the molecular and genetic mechanisms involved in the conversion of normal cells to a neoplastic state of growth, the authors have attempted to put into perspective the history of human prostate epithelial cell transformation by a combination of carcinogenic agents, and to discuss the current state-of-the-art in transformation of human prostate epithelial cells in culture.

Animals↗

PubMed1997

Simplified quantification and reproducibility studies of dopamine D2-receptor binding with iodine-123-IBF SPECT in healthy subjects.

UNLABELLED: The purpose of this study was to assess the feasibility of a simplified SPECT scan protocol to quantify D2-receptor binding using [123I]iodobenzofuran (IBF) and to evaluate reproducibility of quantitative IBF-SPECT imaging without blood data. METHODS: Twenty healthy volunteers participated in the study, six had test/retest studies separated by 1 wk. Scans were acquired every 5 min for 180 min using a triple-headed SPECT camera after a bolus injection of IBF (292 MBq). The receptor parameter was determined by using our previously proposed variation of graphical analysis that derives the distribution volume ratio (Rv = V3/V2) from multiple scan data without blood data. Rv' was determined from three 20-min scan data obtained at 0-20, 50-70 and 160-180 min postinjection and compared with Rv as determined from scans obtained at 0-180 min. RESULTS: The mean Rv' (2.93 +/- 0.59) underestimated the mean Rv (3.10 +/- 0.50) by 5%. The mean variability (mean percent absolute difference) between Rv' and Rv was low (10%) with excellent reliability (intraclass correlation coefficient, p = 0.90). The relationship between Rv' and Rv was linear (r = 0.95, p < 10(-5)). The mean test/retest Rv and mean test/retest Rv' were (3.19 +/- 0.70/3.18 +/- 0.80) and (3.16 +/- 0.81/3.01 +/- 0.94), respectively, and these measures were not significantly different between test/retest studies. The mean test/retest variability of Rv was low (5%) with excellent reliability (p = 0.98). In addition, the mean test/retest variability of Rv' was low (10%) with excellent reliability (p = 0.94). CONCLUSION: Three short (20 min) IBF-SPECT scans allowing for rest periods between scans permit reliable measurements of the dopamine D2-receptor parameter V3/V2. Quantitative IBF-SPECT imaging without blood data is reliable and reproducible.

Adult↗

PubMed1996

Preliminary in vitro growth cycle and transmission studies of HIV-1 in an autologous primary cell assay of blood-derived macrophages and peripheral blood mononuclear cells.

Recent interest focused on the dynamics of HIV-1 replication in primary monocytes/macrophages and T-lymphocytes of the immune system, as well as the standardization of virological and immunological in vitro assays with primary isolates, provided the impetus for these studies. These types of studies have never been performed as they would occur in vivo, i.e., where the envelope of the virus and cell membranes of the two cell types of the same host origin. Therefore, the biological and physicochemical properties of an uncloned, primary dual-tropic isolate HIV-1ADA during the initial lag, log, and stationary phases of viral replication were studied in an autologous donor cell assay in peripheral blood mononuclear cells (PBMC) and blood monocyte-derived macrophages (MDM). Similar total numbers (10(9) virus particles/ml) were produced by both cell types during the stationary period. On a per cell per day basis, during peak stationary periods, 0.92 x 10(3) virions/day for MDMs and 5.31 x 10(3) virions/day for PBMCs were produced. Interestingly, virus replicating from MDMs during the log-growth phase demonstrated the greatest infectious fraction which was 3 logs greater than virus replicating in PBMCs. Despite constant virus particle production in MDMs, the infectious fraction was found to fall 3 to 4 logs over a 10-day period. Due to an infectious fraction less than 1 (0.053 infectious unit/cell/24 hr), virus spread in PBMCs during the rapid log phase could only have occurred by cell-to-cell contact, whereas in MDMs with an infectious fraction of about one infectious particle (approximately 1/cell/24 hr), cell-free transmission could account for the observed results. Most of the MDMs (> 90%) became productively infected, whereas only 5-10% of the total PBMCs were found replicating virus. The period of peak stationary virus production (i.e., stationary phase) was at minimum 4 to 5 times longer in MDMs than PBMCs. Whereas the majority of p24, RT, and gp 120 found to be associated with MDM-derived virions, no increased dissociation of these components was observed in PBMC-derived virions. The virion-associated gp 120 was 3 to 4 times more stable on both PBMC- and MDM-derived virus (> 96 hr) and present at 10-25 times the concentration per virion than that observed for a T-cell-line-adapted laboratory strain of HIV-1 replicating in T-cell lines. These in vitro results suggest that important differences exist between MDMs and PBMCs with regard to the viral dynamics of infection and replication which should provide for a qualitative and quantitative basis to estimate virus replication on a per-cell basis for other known cellular targets of HIV-1. Studying the multiple biophysicochemical characteristics and viral replication dynamics as described herein provides an autologous in vitro model of additional quantifiable parameters for analysis and understanding of virus/host factor(s) and/or antivirals which influence them.

Cell Separation↗

PubMed1996

The role of BMP-4 and GATA-2 in the induction and differentiation of hematopoietic mesoderm in Xenopus laevis.

Vertebrate embryonic blood formation is regulated by factors that participate in dorsal-ventral patterning and mesoderm induction. The GATA-binding transcription factors are required for normal hematopoiesis and are expressed during gastrulation when ventral mesoderm (VM) is induced to form blood. Based on the recent demonstration that bone morphogenetic protein (BMP-4) is a potent ventralizing factor and inducer of hematopoietic tissue, we hypothesized that GATA-2 could be induced or activated by BMP-4. Here we demonstrate that BMP-4 can stimulate GATA-2 expression, and that expression of a dominant negative BMP-4 receptor can suppress GATA-2 induction by BMP-4 in ventral mesoderm. Over-expression of GATA-2 in ventral mesoderm leads to increased globin production and forced expression of GATA-2 in primitive ectoderm adjacent to ventral mesoderm also stimulates globin expression. Our results suggest that BMP-4 and GATA-2 can function in two adjacent germ layers, mesoderm and ectoderm, to participate in blood cell formation during embryogenesis.

Animals↗

PubMed1996

Enhancement of vincristine cytotoxicity in drug-resistant cells by simultaneous treatment with onconase, an antitumor ribonuclease.

BACKGROUND: Onconase, a protein isolated from oocytes and early embryos of the frog Rana pipiens, shares extensive homology with bovine pancreatic ribonuclease (RNase A) and possesses similar enzyme activity. Onconase is cytotoxic toward cancer cells in vitro and exhibits antitumor activity in animal models. In addition, Onconase has been shown to enhance the cytotoxic activity of some chemotherapeutic agents in vitro. PURPOSE: We studied interactions between the cytotoxic effects of Onconase and the chemotherapeutic agent vincristine (VCR) in the treatment of drug-sensitive and multidrug-resistant human colon carcinoma cells in vitro and in mice. METHODS: Transplantable human colon carcinoma cells (HT-29par cells) were infected with a retrovirus containing human mdr1 (also known as MDR1 and PGY1) complementary DNA (encoding P-glycoprotein [P-gp]), and clones that were cross-resistant to colchicine, doxorubicin, and vinblastine were selected (HT-29mdr1 cells). Drug-resistant HT-29mdr1 cells and drug-sensitive HT-29par parental cells were treated with Onconase and/or VCR in vitro at varying concentrations to measure the effects on protein synthesis and cell viability. The impact of Onconase on VCR accumulation in both types of cells was determined in the presence or absence of MRK-16, an anti-P-gp monoclonal antibody capable of reversing the multidrug-resistant phenotype. The antitumor effects of Onconase and/or VCR treatment were assessed in nude mice bearing established HT-29par or HT-29mdr1 intraperitoneal tumors. IC50 values (drug concentrations resulting in 50% inhibition of protein synthesis or cell viability) for Onconase and VCR were determined from semilogarithmic dose-response curves; interactions between the cytotoxic effects of these two agents were evaluated using data from protein synthesis inhibition experiments and a two-way analysis of variance. Survival distributions from in vivo experiments were compared using Cox proportional hazards models. RESULTS: The combination of Onconase and VCR yielded enhanced cytotoxicity in vitro that was independent of P-gp expression. Evaluation of the effects of these two compounds on protein synthesis over a wide range of drug concentrations indicated possible synergistic interactions (i.e., greater than additive effects) in both drug-resistant and drug-sensitive cells. The enhancement of VCR cytotoxicity was dependent on Onconase enzyme activity and was not associated with increased intracellular levels of VCR. Simultaneous treatment of mice bearing HT-29par tumors with Onconase and VCR did not extend their median survival time (MST) significantly (MST with VCR = 66 days; MST with VCR plus Onconase = 69 days; two-tailed P = .57); however, the MST of mice with HT-29mdr1 tumors was extended significantly by this treatment (MST with VCR = 44 days; MST with VCR plus Onconase = 66 days; two-tailed P<.001). CONCLUSION: Combined administration of Onconase and VCR yields enhanced cytotoxicity in vitro and in vivo against human colon carcinoma cells that overexpress the mdr1 gene.

Animals↗

PubMed1996

A nuclear matrix-specific factor that binds a specific segment of the negative regulatory element (NRE) of HIV-1 LTR and inhibits NF-kappa(B) activity.

The negative regulatory element (NRE) of human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) is a defined region that has been reported to downregulate LTR-directed HIV gene expression. However, information on the precise role of this region in regulating HIV gone transcription is lacking. We have investigated the possibility that these NRE sequences regulate HIV transcription by a mechanism mediated through a nuclear matrix-specific DNA-protein interaction. We find a nuclear matrix attachment region (MAR) present within the NRE of the HIV-1 LTR that recognizes a sequence-specific DNA-binding protein present in the nuclear matrix of HIV infected cells. Moreover, we also show that the purified DNA-binding nuclear matrix protein (NMP) specifically represses the DNA-binding activity of NF-kappaB. It is likely that the MAR and MAR-enriched specific DNA-binding NMP are brought into juxtaposition by the non-chromatin scaffolding of the nucleus, thus influencing NF-kappaB (and other nuclear proteins) DNA-binding activity through protein-protein and protein-DNA interactions. Our date suggest that one possible role of the NRE could be to act as a matrix attachment site in the nuclear matrix, thus, allowing interaction with a sequence-specific trans-acting factor. The negative effect on NF-kappaB activity due to this MAR-NMP-specific interaction provides a mechanism by which the NRE downregulates HIV gene expression.

Antigens, Nuclear↗

PubMed1996

PLC gamma 1 Src homology domain induces mitogenesis in quiescent NIH 3T3 fibroblasts.

Previously, we demonstrated that microinjection of phosphoinositide-specific phospholipase C gamma 1 (PLC gamma 1) and lipase-defective mutants of PLC gamma 1 induced G(0) growth arrested NIH 3T3 fibroblasts to enter S phase of the cell cycle. These experiments suggested that regions other than the catalytic domain of PLC gamma 1 may be responsible for inducing mitogenesis. To test other regions of PLC gamma 1 for DNA synthesis inducing activity, cDNA fragments encoding Src homology (SH) and pleckstrin homology (PH) domains were subcloned into the bacterial expression plasmid pGEX-2TK, and the GST fusion proteins were purified. The complete PLC gamma l SH domain peptide was found to induce DNA synthesis after microinjection into growth arrested fibroblasts. Peptides containing a single SH3 domain or two SH2 domains induced a partial response that was restored to full activity if they were co-injected. The PH domain peptide did not induce DNA synthesis. Thus, both SH3 and SH2 activity combine to give maximum DNA synthesis induction, demonstrating that non-catalytic structural domains of PLC gamma 1 have pronounced effects on mitogenic signaling pathways.

3T3 Cells↗