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Biomedical subjects

J Lai

Publications and source records attributed to J Lai.

At least 91 records · Page 5Linked to original sources
PubMed1997

Functional respiratory rhythm generating networks in neonatal mice lacking NMDAR1 gene.

N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission is implicated in activity-dependent developmental reorganization in mammalian brain, including sensory systems and spinal motoneuron circuits. During normal development, synaptic interactions important in activity-dependent modification of neuronal circuits may be driven spontaneously (Shatz 1990b). The respiratory system exhibits substantial spontaneous activity in utero; this activity may be critical in assuring essential and appropriate breathing movements from birth. We tested the hypothesis that NMDA receptors are necessary for prenatal development of central neural circuits underlying respiratory rhythm generation by comparing the responsiveness of control mice and mutant mice lacking the NMDA receptor R1 subunit (NMDAR1) gene to glutamate receptor agonists and antagonists and comparing endogenous respiratory-related oscillations generated in vitro by brain stem-spinal cord and medullary slice preparations from control and mutant mice. In control mice, local application of NMDA and the non-NMDA receptor agonist, (R,S)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid hydrobromide (AMPA), over the pre-Bötzinger Complex, the C4 cervical motor neuron pool, and the hypoglossal motor nucleus produced profound increases in inspiratory frequency, tonic discharge on C4 ventral nerve roots, and inward currents in inspiratory hypoglossal motoneurons, respectively. Responses of mutant mice to AMPA were similar. However, mutant mice were completely unresponsive to NMDA applications. Preparations from mutant mice generated a respiratory rhythm virtually identical to control. Results demonstrate that NMDA receptors are not essential for respiratory rhythm generation or drive transmission in the neonate. More importantly, they suggest that NMDA receptors are not obligatory for the prenatal development of circuits producing respiratory rhythm.

Animals↗
PubMed1997

Substance P receptor expression and cellular responses to substance P in prenatal rat spinal cord cells.

Substance P receptors (SPRs) are expressed by prenatal rat spinal cord neurons and glial cells early in their differentiation, and SPRs may mediate developmental influences in the developing spinal cord. In order to understand better early SPR expression, we quantified SPR mRNA in the rat spinal cord during prenatal development using a cDNA probe for the rat SPR in nuclease protection assays. SPR mRNA was present in the rat spinal cord at E14, the earliest stage examined, and the presence of specific binding sites for radiolabeled SP suggested that SPRs were expressed at the protein level as well. Comparisons of samples from rats at different prenatal ages showed that the relative abundance of SPR mRNA declined by about 75% from E14 through the remainder of prenatal development. Assays of the hydrolysis of phosphatidyl inositol performed on prenatal spinal cord cells in culture revealed that SP caused a small but significant stimulation. These results show that expression of SPRs is an early molecular event in the development of the rat spinal cord in vivo and that SPRs on young spinal cord cells can mediate functional responses at early developmental stages.

Animals↗
PubMed1997

Ras(leu61) blocks differentiation of transformable 3T3 L1 and C3H10T1/2-derived preadipocytes in a dose- and time-dependent manner.

To investigate the functional relationship between the transforming ability of Ras and its role as an integral component of the differentiation-promoting insulin signaling pathway, we introduced a leu61-activated ras gene into the Ras-transformable 3T3 L1 (ATCC CCL92.1) and a number of C3H10T1/2-derived preadipocytic cell lines. The results demonstrate a quantitative reciprocal regulation of differentiation and several transformation-associated properties in response to graded levels of ras gene expression, with the loss of differentiative capacity, morphological transformation, stimulation of proliferation, and anchorage-independent growth requiring increasing levels of Ras(leu61) protein. Furthermore, using novel, tightly regulatable 3T3 L1 transfectants, we demonstrated that Ras(leu61) effectiveness in blocking adipocytic differentiation is strictly dependent on the timing of its expression relative to cell growth arrest, with ras(leu61) expression being ineffective at inhibiting differentiation or inducing morphological transformation once the differentiative process has commenced. Moreover, rasleu61 induction failed to substitute for or enhance the c-Ras-dependent differentiative insulin signal, even under conditions in which it did not induce transformation. Therefore, although necessary for insulin signal transduction, the Ras signal alone is not sufficient to induce adipocytic differentiation in this system. Consistent with its established role as a downstream effector of Ras, v-Raf expression mirrored the Rasleu61 effects on adipocytic differentiation and transformation.

3T3 Cells↗
PubMed1997

Antisense targeting of delta opioid receptors in NG 108-15 cells: direct correlation between oligodeoxynucleotide uptake and receptor density.

Antisense oligodeoxynucleotides (ODN) have been used to inhibit the function of a number of structurally defined neurotransmitter receptors in vivo by transiently disrupting their expression in the CNS. However, issues concerning the cellular and molecular mechanisms of these ODN often raise questions about the specificity of such ODN-mediated "knock-down" of target proteins. This study sought to extend our in vivo "knock-down" of the delta opioid receptor (DOR) by targeting this receptor in the NG 108-15 cells with an antisense ODN for the DOR and by using a polyclonal antibody raised against this receptor to determine the efficiency and selectivity of the antisense ODN in inhibiting expression of the DOR. By fluorescence tagging the ODN and immunofluorescence labeling the DOR, we monitored the uptake efficiency of the ODN and the DOR density in individual cells that had been treated with the antisense ODN or with a mismatch control. Quantitative fluorescence image analysis showed that the uptake of ODN by NG 108-15 cells was time- and concentration-dependent and that it was not uniform within a population. Treatment with the antisense ODN elicited an inverse correlation between DOR immunoreactivity and the ODN fluorescence in individual cells. No correlation was found in cells treated with the mismatch control. These findings suggest that the antisense ODN-mediated "knock-down" of the DOR is governed by the sequence specificity of the ODN and the efficiency of its uptake by the target cells in a time- and concentration-dependent manner. These data provide further evidence in support of the selectivity of antisense ODN targeting and the utility of these molecules as an effective tool in neuropharmacological studies.

Animals↗
PubMed1997

[Studies on purging leukemic cells by photosensitizer PSD-007 laser photoradiation in vitro].

The sensitivity to photosensitization mediated by the hematoporphyrin photosensitizer PSD-007 of acute promyelocytic leukemic cell line (HL-60) was compared with normal human hemopoietic progenitor cells. The results showed that the leukemic cells were more sensitive. After being treated with 10 micrograms.ml-1 PSD-007 followed by 2J.cm-2 copper vapor laser light irradiation, the clonogenic leukemic (HL-60) cells were reduced 98%, but the survival rate of human granulocyte-macrophage colony-forming units (CFU-GM) was 40 +/- 8%. Mixing of normal human marrow cells with leukemic (HL-60) cells (ratio 100:1) did not interfere with elimination of tumor cells. The ultrastructure changes of HL-60 cells treated by laser photoradiation was observed under the trans-electronic microscope. The mitochondria, endoplasmic reticulum and cell membrane were involved. This means that the cell biomembrane is the main target to be attacked. It is considered that PSD-007-laser photoradiation therapy is efficient for killing leukemic cells.

Adult↗
PubMed1996

Selective blockade of peripheral delta opioid agonist induced antinociception by intrathecal administration of delta receptor antisense oligodeoxynucleotide.

Previous studies have shown that intrathecal (i.t.) administration of antisense, but not mismatch, oligodeoxynucleotides (ODNs) to the cloned delta opioid receptor (DOR) can inhibit the antinociceptive actions of i.t. delta (delta), but not mu (mu) or kappa (kappa), opioid agonists. As a major portion of spinal opioid receptors are localized on the central terminals of the small afferent fibers, we hypothesized that the effects of antisense ODNs given i.t. might be the result of actions at the level of the cell body in the dorsal root ganglion (DRG). This possibility was investigated by assessing the antinociceptive actions of an i.t. or intrapaw (ipaw) administered mu (morphine), delta ([D-Ala2, Glu4]deltorphin) or kappa (CI977) opioid agonist in rats treated with i.t. saline or antisense or mismatch ODNs to the DOR (12.5 micrograms, twice-daily for 3 days). The opioid agonists produced significant antinociception in the 5% formalin-flinch test following either i.t. or ipaw administration. DOR antisense ODN treatment blocked the antinociceptive actions of both i.t. or ipaw [D-Ala2, Glu4]deltorphin without affecting the antinociceptive actions of i.t. or ipaw morphine or CI977. Radioligand binding studies with [3H]naltrindole (NTI), a delta selective antagonist, indicated an approximate 50% decrease in delta opioid receptors in the lumbar spinal cord following i.t. DOR antisense, but not mismatch, ODN treatment. DOR antisense or mismatch ODN treatment did not affect nu or kappa radioligand binding in lumbar spinal cord. These data suggest the possibility that peripheral proteins can be targeted with i.t. antisense ODNs providing significant opportunities for the exploration of the physiological and pathological significance of these substances.

Analgesics, Opioid↗
PubMed1996

Tramadol, M1 metabolite and enantiomer affinities for cloned human opioid receptors expressed in transfected HN9.10 neuroblastoma cells.

Tramadol hydrochloride is a centrally acting synthetic analgesic in widespread clinical use. Despite different degrees of opioid-like characteristics in preclinical tests, it is characterized by lack of full naloxone reversibility or naloxone-precipitated withdrawal in humans. To investigate this apparent discrepancy, the present study measured the affinity of tramadol (and its enantiomers) and an active O-desmethyl metabolite (M1) (and its enantiomers) to cloned human opioid receptors of the mu, delta and kappa type stably expressed in HN9.10 neuroblastoma cells. At mu sites, the Ki values for tramadol, its (+) and (-) enantiomers, M1, and its (+) and (-) enantiomers were 17000, 15700, 28800, 3190, 153 and 9680 nM, respectively, compared to 7.1 nM for morphine. These results are consistent with the suggestion of a non-opioid contribution to the clinical profile of tramadol.

Brain Neoplasms↗
PubMed1996

Immunofluorescence analysis of antisense oligodeoxynucleotide-mediated 'knock-down' of the mouse delta opioid receptor in vitro and in vivo.

We have previously used antisense oligodeoxynucleotides (ODN) to the cloned delta opioid receptor (DOR) to inhibit the antinociceptive response to spinally administered delta opioid receptor selective agonists in mice. Here we have examined the effect of DOR antisense ODN treatment on the level of DOR expressed in NG 108-15 cells and the spinal cord, through immuno-fluorescence microscopy, to determine the efficiency and selectivity of the antisense ODN-mediated "knock-down' of the DOR in these tissues. Antisense ODN, but not mismatch control, treatment resulted in a significant reduction in DOR immunoreactivity (-ir) in NG 108-15 cells and spinal cord. Thus, the inhibition of antinociceptive response to intrathecal delta selective agonists by DOR antisense ODN correlates with the loss of DOR-ir in the superficial layers of the dorsal horn of the spinal cord.

Animals↗
PubMed1996

Regulation by prostaglandin E2 of interleukin release by T lymphocytes in mucosa.

Regulation of immune cell activation in lymphocyte-bearing human tissues is a pivotal host function, and metabolites of arachidonic acid (prostaglandin E2 in particular) have been reported to serve this function at non-mucosal sites. However, it is unknown whether prostaglandin E2 is immunoregulatory for the large lymphocyte population in the lamina propria of intestine; whether low (nM) concentrations of prostaglandin E2 modulate immune responses occurring there; and whether adjacent inflammation per se abrogates prostaglandin E2's regulatory effects. To address these issues, intestine-derived lymphocytes and T hybridoma cells were assessed, T cell activation was monitored by release of independently quantitated lymphokines, and dose-response studies were performed over an 8-log prostaglandin E2 concentration range. IL-3 release by normal intestinal lamina propria mononuclear cells was reduced (up to 78%) in a dose-dependent manner by prostaglandin E2, when present in as low a concentration as 10(-10) M. PGE2 also inhibited (by > or = 60%) mucosal T lymphocytes' ability to destabilize the barrier function of human epithelial monolayers. Further, with an intestine-derived T lymphocyte hybridoma cell line, a prostaglandin E2 dose-dependent reduction in IL-3 and IL-2 (90 and 95%, respectively) was found; this was true for both mitogen- and antigen-driven T cell lymphokine release. Concomitant [3H] thymidine uptake studies suggested this was not due to a prostaglandin E2-induced reduction in T cell proliferation or viability. In contrast, cells from chronically inflamed intestinal mucosa were substantially less sensitive to prostaglandin E2, e.g., high concentrations (10(-6) M) of prostaglandin E2 inhibited IL-3 release by only 41%. We conclude that prostaglandin E2 in nM concentrations is an important modulator of cytokine release from T lymphocytes derived from the gastrointestinal tract, and it may play a central role in regulation of lamina propria immunocyte populations residing there.

Animals↗
PubMed1996

Opioid receptor types and subtypes: the delta receptor as a model.

Since the discovery of opioid receptors over two decades ago, an increasing body of work has emerged supporting the concept of multiple opioid receptors. Molecular cloning has identified three opioid receptor types--mu, delta, and kappa--confirming pharmacological studies that previously postulated the existence of these three receptors. The cloned opioid receptors are highly homologous and belong to the family of seven-transmembrane, G protein-coupled receptors. With the development of novel opioid ligands, subtypes of the mu, delta, and kappa receptors have been proposed, although the molecular basis of these subtypes has not been elucidated. In this review, we present the pharmacological data supporting the concept of multiple delta opioid receptor subtypes and offer hypothetical mechanisms which might generate these "subtypes."

Alternative Splicing↗
PubMed1996

Prostaglandin F2 alpha induces cardiac myocyte hypertrophy in vitro and cardiac growth in vivo.

Several prostaglandins [prostaglandin (PG) A2, -B2, -D2, -E2, -F2 alpha, and -I2 and carbaprostacyclin] and the thromboxane analogue U-46619 were analyzed for the ability to induce hypertrophy of rat neonatal cardiac ventricular myocytes. Myocyte hypertrophy was induced specifically by PGF2 alpha. Myocytes exposed to this prostanoid in culture increased in size and protein content. The contractile fibrils within the cells became organized into parallel arrays, and the cells tended to cluster and beat spontaneously. PGF2 alpha also induced the expression of c-fos, atrial natriuretic factor (ANF), and alpha-skeletal actin in these cells. The effects of PGF2 alpha were compared with several known cardiac myocyte hypertrophy factors (phenylephrine, endothelin-1, leukemia inhibitory factor, cardiotrophin-1, and angiotensin II). PGF2 alpha was found to be intermediate in potency among the factors but induced a level of ANF production that was approximately 10-fold higher than any of the other effectors. Responsiveness to PGF2 alpha was not limited to neonatal cardiocytes. Ventricular myocytes isolated from adult rats also responded specifically to PGF2 alpha with a morphological change similar to that observed with phenylephrine and by producing ANF. In rats, chronic administration of fluprostenol, a potent agonist analogue of PGF2 alpha, resulted in a dose-dependent increase in heart weight- and ventricular weight-to-body weight ratios. The amount of PGF2 alpha extractable from the hearts of rats with cardiac hypertrophy induced by myocardial infarction was also found to be greater than that in sham-operated control rats. These results indicate that PGF2 alpha may play an important role in inducing cardiac hypertrophy.

Aging↗
PubMed1996

On the safety of 5-[125I]iodo-2'-deoxyuridine--Preclinical evaluation in swine.

To increase tumor incorporation and minimize hepatic degradation of radio-IUdR, compartmental administration routes are being considered as an alternative to intravenous (i.v.) injections. Although there are significant data on the biodistribution and some reports on radiotoxicity of i.v.-administered 125IUdR, similar results for other routes of delivery are not available. We have undertaken a series of experiments intended to examine radiation effects of 125IUdR after intravesical (3 swine; eight 3 mCi doses at 4-day intervals), intracarotid (3 swine; two 10 mCi doses at 2-week intervals), and intra-aortic (5 swine, single dose of 10 mCi) administration in a swine model. Liver, renal functions, and complete blood counts were monitored throughout the duration of the experiment. Pharmacokinetics, systemic distribution of radioactivity and metabolites were measured. The normal tissue 125IUdR uptake and histology were determined after necropsy. No adverse systemic effects were identified. Clinical observations, laboratory data, and necropsy results were within normal range.

Administration, Intravesical↗
PubMed1996

Colon-specific prodrugs of 5-radioiodo-2'-deoxyuridine.

Two glycoside-based prodrugs, 125IUdR-5'-beta-D-glucopyranoside and 125IUdR-5'-beta-D-galactopyranoside, were synthesized. This selection was dictated by the abundance of appropriate enzymes in the GI tract of mice and similar levels of beta-D-glycosidases in human and rodent large intestine. Studies to establish the ability of colonic microflora to release 125IUdR were conducted in vitro and in Swiss Webster mice. Both prodrugs released 125IUdR in the presence of the corresponding enzymes or the GI content homogenates in vitro, and in vivo. Luminal enzymes in the proximal and distal small intestine in mice degraded less than 10% of each prodrug whereas enzymes from the colonic/caecal lumen of mice released nearly 100% of 125IUdR. 125IUdR freed by bacterial glycosidases was stable in the GI content. No significant amounts of other metabolites or deiodination products were observed. Total radioactivity recovered as by-products was less than 10%. The efflux of prodrugs from the GI tract after oral administration in mice was slow and limited. Unlike 125IUdR, prodrugs were not dehalogenated in vivo as indicated by biodistribution and imaging studies.

Administration, Oral↗
PubMed1996

Characterization of antinociception to opioid receptor selective agonists after antisense oligodeoxynucleotide-mediated "knock-down" of opioid receptor in vivo.

Pharmacological studies in vivo and in vitro have suggested the existence of subtypes of the delta opioid receptor termed delta1 and delta2 (delta1 and delta2). The hypothesis of subtypes of delta receptors was further explored by assessing the effects of administration of antisense or mismatch oligodeoxynucleotides (ODN) in vivo to the cloned DOR, or to a conserved region of the cloned opioid receptors, on the antinociceptive responses elicited by selective mu, ku and delta opioid receptor agonists in mice. Additionally, the density of opioid delta receptors in brain after delta opioid receptor (DOR) ODN treatment was investigated. Repeated twice daily intracerebroventricular (i.c.v.) administration of DOR antisense, but not mismatch, ODN, produced a dose- and time-related blockade of i.c.v. [D-Ala2, Glu4]deltorphin (delta2 agonist), but not [D-Pen2, D-Pen5]enkephalin (delta1 agonist), antinociception. The antinociceptive responses to selective mu and kappa opioid agonists were unaffected by DOR antisense or mismatch ODN treatments. The antinociceptive effect of an A90 dose of [D-Ala2, Glu4]deltorphin was significantly reduced by the third day of DOR antisense ODN administration and persisted over a treatment period of 6 days with recovery by the third posttreatment day. Saturation studies in mouse whole brain preparations with the selective delta-radioligand [3H]naltrindole showed that DOR antisense, but not mismatch, ODN treatment produced a significant time-related reduction in Bmax values of approximately 30 to 40% by day 6, without changing the Kd value. The reduction in DOR density was reversible and returned to control levels within 3 days after cessation of antisense ODN treatment. The i.c.v. administration of an antisense, but not mismatch, ODN directed to a conserved region of the cloned opioid receptors, termed common opioid receptor antisense ODN, inhibited the antinociceptive effects of i.c.v. mu, kappa and delta agonists, including [D-Pen2, D-Pen5]enkephalin. These data further support the hypothesis of subtypes of opioid delta receptors.

Analgesics↗
PubMed1996

Opioid antagonists and antisera to endogenous opioids increase the nociceptive response to formalin: demonstration of an opioid kappa and delta inhibitory tone.

The present experiments explored the role of endogenous opioids in the behavioral response to a formalin-induced nociceptive stimulus in the rat. Flinching was taken as a measure of the intensity of the nociceptive stimulus after the administration of formalin into the dorsal surface of the paw of control animals, or in animals receiving i.p. administration of receptor-selective doses of opioid antagonists including naloxone, naltrindole (delta opioid antagonist), nor-binaltorphimine (kappa opioid antagonist) or beta-funaltrexamine (mu opioid antagonist). Additionally, antisera to [Leu5]enkephalin, [Met5]enkephalin and dynorphin A (1-13) (dynorphin) were administered intrathecally before formalin to explore the contribution of endogenous opioids in modulation of the flinching response. Formalin-induced flinching was increased significantly by naloxone, and receptor selective doses of naltrindole and nor-binaltorphimine, but not beta-funaltrexamine. Additionally, antisera to [Leu5]enkephalin and dynorphin also resulted in a significant increase in formalin-induced flinching, whereas antisera to [Met5]enkephalin had no effect. On the basis of significant increases in formalin-induced flinching produced by 1) receptor-selective doses of delta and kappa, but not mu, opioid antagonists and 2) antisera to [Leu5]enkephalin and dynorphin A, but not [Met5]enkephalin, these data suggest the presence of an opioid inhibitory tone which acts to limit the intensity of the pain signal. This tone appears to be mediated via activation of delta and kappa receptors, possibly by a [Leu5]enkephalin- and dynorphin-like substance, respectively.

Animals↗
PubMed1996

Restriction fragment length polymorphism (RFLP) analysis on DNA from human compact bone.

DNA typing techniques primarily identify specific genetic markers that are highly polymorphic within a population and have found great utility in forensic science. The established DNA identification protocol, termed restriction fragment length polymorphism (RFLP), has been admitted as physical evidence in the investigation of crimes such as assault, sexual assault, and homicide. The limitation associated with this procedure concerns the integrity of the genetic material. This study sought to evaluate human bone as a source material for DNA identification following exposure to common forensic field conditions. Often, with the onset of decomposition and eventual disarticulation of a body, soft tissues, hair and teeth may not be recovered. The significance of this study lies in the fact that, within forensic anthropology, human bone represents the most biologically stable evidence and is sometimes all that remains after periods of exposure. Genomic DNA was extracted from human bone following exposure to surface deposit, shallow burial, and fresh water immersion. Samples were collected over a three month time course and analyzed by spectrophotometry and agarose gel electrophoresis as well as RFLP analysis. The data suggest that high molecular weight DNA may indeed be extracted from human bone and typed by RFLP analysis for use in forensic identification. Under simulated forensic field conditions, the severity of DNA degradation was in the order of fresh water immersion > shallow burial > surface deposit. Genomic DNA from bone deposited on the desert surface for up to 4 weeks was detected by RFLP analysis. No spurious bands were detected in any specimens, and to the extent that bands were still present, the RFLP patterns matched. These findings demonstrate that human bone can be a reliable source of genomic DNA, and that bone recovered from surface deposit is the most desirable for use in forensic identification.

Bone and Bones↗
PubMed1995

Attenuation of gastrin-induced gastric acid secretion by antisense oligonucleotide to the CCKB/gastrin receptor.

The effects of treatment with CCK receptor antagonists or administration of an antisense oligonucleotide to the gastrin receptor, on gastrin-I and cholecystokinin-8-induced acid secretion in mouse stomach were evaluated. Administration of gastrin-I (1 microM) or cholecystokinin-8 (30 nM) stimulated acid output at the rates of 2.6 +/- 0.27 and 1.0 +/- 0.21 microEq h-1, respectively. Gastrin-I-induced acid output was significantly blocked by pretreatment of stomachs with 3R[+]-N-[2,3-dihydro-1-methyl-2-oxo-5- phenyl-1H-1,4-benzodiazepin-3-yl]-N[3-methylphenyl[urea (L-365,260; 1 microM), but not by devazepide (L-364,718; 1 microM). Cholecystokinin-8-induced acid output, on the other hand, was sensitive to both L-365,260 (100 nM) and L-364,718 (100 nM). Administration of antisense, but not mismatch, oligonucleotide significantly reduced gastrin-induced acid output, while antisense oligonucleotide treatment had no effect on cholecystokinin-8-induced acid output. These results of antagonist and antisense oligonucleotide studies suggest that gastrin-I and cholecystokinin-8 may involve different receptor subtypes in stimulating gastric acid secretion in mice, and that antisense oligonucleotide administration may serve an useful tool in characterizing CCK/gastrin receptor subtypes.

Animals↗