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Biomedical subjects

W W Li

Publications and source records attributed to W W Li.

At least 55 records · Page 3Linked to original sources
PubMed1996

Enkephalin positive sites in the developing human cerebellum.

Enkephalin (ENK) positive sites in the developing human cerebellum (gestation ages 18 weeks to 30 weeks) were studied by immunohistochemistry (ABC method). Positive reactions were registered as early as 20 weeks of gestation and initially in the deep cerebellar nuclei. By 23 weeks some mossy fibers exhibited positivity and by 27 weeks some climbing fibers as well as a few Purkinje basket, golgi and granule cells were also positive. This result indicated that the human cerebellum possesses ENK positive fibers and neurons before birth.

Cerebellar Cortex↗
PubMed1996

Apoptosis in human retinal degenerations.

PURPOSE: This paper examined the role of apoptosis in human retinal degenerations including pathologic myopia, age-related macular degeneration, serous retinal detachment, retinal lattice, and paving stone degenerations. METHOD: Thirty-seven enucleated human eyes with 1 of the above-mentioned retinal degenerations were studied by histopathology and by TdT-mediated biotin-dUTP nicked-end labelling (TUNEL) technique. RESULTS: Tunnel labelling characteristic DNA fragmentation of apoptosis was observed in photoreceptor cells in 2 of the 4 eyes with pathologic myopia and in 4 of 16 eyes with age-related macular degeneration, 2 of which were exudative and 2 of which were atrophic. However, only a few scattered photoreceptor cells were labelled in 4 of 8 eyes with serous retinal detachment secondary to malignant melanoma of the choroid. Moreover, none of the photoreceptors cells in the 4 eyes with retinal lattice degeneration and 6 eyes with retinal paving stone degeneration were labelled. CONCLUSIONS: Apoptosis is 1 of the important pathways of photoreceptor cell degeneration in pathologic myopia and age-related macular degeneration.

Aged↗
PubMed1995

Common genetic variation in the promoter of the human apo CIII gene abolishes regulation by insulin and may contribute to hypertriglyceridemia.

Overexpression of plasma apolipoprotein CIII (apo CIII) causes hypertriglyceridemia in transgenic mice. A genetically variant form of the human apo CIII promoter, containing five single base pair changes, has been shown to be associated with severe hypertriglyceridemia in a patient population. In animals and in cultured cells the apo CIII gene is transcriptionally downregulated by insulin. In this study we demonstrate that, unlike the wild-type promoter, the variant promoter was defective in its response to insulin treatment, remaining constitutively active at all concentrations of insulin. The loss of insulin regulation was mapped to polymorphic sites at -482 and -455, which fall within a previously identified insulin response element. Loss of insulin regulation could result in overexpression of the apo CIII gene and contribute to the development of hypertriglyceridemia. The variant apo CIII promoter is common in the human population and may represent a major contributing factor to the development of hypertriglyceridemia.

Alleles↗
PubMed1995

Ligand-induced growth cone collapse: amplification and blockade by variant GAP-43 peptides.

Growth cones are powerful amplifiers for signals from the microenvironment. Their collapse can be triggered by cell surface components of myelin and brain membranes, as well as by soluble ligands, including neurotransmitters. GAP-43 is a protein concentrated on the inner surface of the growth cone membrane. Assayed in isolation, it interacts with the heterotrimeric protein, G(o), and in oocytes it amplifies the effects of ligand-triggered G protein activation. We wished to examine whether GAP-43 serves to amplify signals at the growth cone. The G(o) stimulating region of GAP-43 is encoded in the 10 amino acids (MLCCMRRT-KQ) of the first exon. We examined the effect of this peptide upon chick dorsal root ganglion growth cone collapse and neurite retraction triggered by brain membranes or myelin, as well as by serotonin. We find that application of the GAP-43 1-10 peptide amplifies the effects of all three ligands. The amplification is greater when GAP-43 1-10 is injected intracellularly. Peptides with amino acid substitutions for the two cysteine residues manifest parallel changes in growth cone collapse and G(o) stimulation. In particular, tyrosine or methionine substitutions cause the peptide to inhibit G(o) and to block induced growth cone collapse. The GAP-43 peptides therefore regulate the sensitivity of growth cones to extrinsic signals. The modified peptides serve as a starting point for the design of reagents to enhance CNS regeneration.

Amino Acid Sequence↗
PubMed1995

The c Fos immunoreactivities in the developing and adult rat cerebella.

c Fos immunoreactivities were studied by immunohistochemistry in the developing and adult rat cerebella. c Fos immunoreactivities were observed in purkinje cells early in postnatal development (P26 d) and reached a peak by P40-P60 d, followed by a decline in the 3-month-old adult. The granule cells revealed c Fos immunoreactivities by P26 d but declined until P50 d. Then c Fos immunoreactivities increased in the granule layer by P50-P60 d and decreased again in the adult. The deep nuclei showed c Fos immunoreactivities by P40 d and persisted in the adult. In spite of the difference in the patterns of location in c Fos immunoreactivities from the early postnatal period to adulthood, no decrease in the amount of c Fos immunoreactivity was evident from immunoblotting studies. Thus, it is concluded that c Fos immunoreactivities change from development to adult, but this does not affect the total amount of c Fos protein expression in the cerebellum.

Age Factors↗
PubMed1995

Molecular mechanisms of resistance to antifolates, a review.

Methotrexate (MTX) is a clinically important antifolate that has been used in combination with other chemotherapeutic agents in the treatment of malignancies including acute lymphocytic leukemia, osteosarcoma, carcinomas of the breast, head and neck, choriocarcinoma and non-Hodgkin's lymphoma. The primary target of MTX is the enzyme dihydrofolate reductase (DHFR) which catalyzes the reduction of folate and 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate. Understanding of MTX action has revealed how cells acquire resistance to this drug. The four known mechanisms of MTX resistance are a decrease in the uptake of the drug, a decrease in the retention of the drug due to defective polyglutamylation or an increase in polyglutamate breakdown, an increase in the enzyme activity and a decrease in the binding of MTX to DHFR. The molecular basis for some of these mechanisms has been elucidated in MTX resistant cell lines; in particular the occurrence of gene amplification resulting in increased DHFR and point mutations resulting in altered DHFR with reduced affinity for MTX. Cloning of the human folylpolyglutamate synthase gene and the reduced folate transport gene have been reported recently and should facilitate the identification of the molecular basis of these resistant phenotypes. DHFR protein has been shown to regulate its synthesis by exerting an inhibitory influence on its own translation. Addition of MTX relieves this inhibition thus providing a possible molecular explanation for the rapid rise in DHFR activity noted in some cells after MTX administration. Alterations in genes involved in regulating the cell cycle such as cyclin D1 and the retinoblastoma (Rb) gene have also been shown to influence cellular response to MTX. Overexpression of cyclin D1 in HT1080, a human fibrosarcoma cell line, results in decreased MTX sensitivity. The molecular basis of this observation is under investigation. Abnormalities in the Rb gene may also have profound effects on MTX sensitivity. Rb interacts with the family of transcription factors called E2F reducing transcription of genes that contain E2F binding sites in the promoter regions e.g. DHFR. When Rb is deleted or rendered nonfunctional levels of "free" or unbound E2F are high resulting in enhanced transcription of genes such as DHFR. This results in increased DHFR protein and may lead to MTX resistance. As the knowledge regarding mechanisms of resistance increases newer approaches to circumvent such resistance or to target resistant cells can be undertaken.

Antineoplastic Agents↗
PubMed1995

Antitumor activity of antifolate inhibitors of thymidylate and purine synthesis in human soft tissue sarcoma cell lines with intrinsic resistance to methotrexate.

We examined the antitumor effects of two antifolate inhibitors of thymidylate synthesis, N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-theno yl-L-glutamic acid (D1694; Tomudex) and 1843U89 as well as a folate-based inhibitor of purine synthesis, 5, 10-dideazatetrahydrofolic acid (DDATHF) on human soft tissue sarcoma cell lines having intrinsic resistance to methotrexate (MTX) due to impaired accumulation of polyglutamates of MTX (HS-16 and HS-42 cells) and to increased levels of dihydrofolate reductase and thymidylate synthase activity (HS-18 cells). Growth inhibition studies showed that ED50 values for D1694 and 1843U89 after a 24-h exposure were 11-19-fold and 22-222-fold lower, respectively, than those for MTX in HT-1080, a MTX-sensitive cell line, and the three MTX-resistant cell lines. In contrast, DDATHF was less cytotoxic than MTX in both the MTX-sensitive and the three resistant sarcoma cell lines. Uptake of D1694, 1843U89, or DDATHF was 2.5-4.5-fold higher than MTX in these sarcoma cell lines. However, D1694 and 1843U89, unlike MTX, accumulate in HS-16 and HS-42 cells as polyglutamate forms, reaching 70% of the total intracellular drug level after 24 h. DDATHF polyglutamates (9.4-24%) were less in the same cell lines. Much lower Km values for D1694 and 1843U89 as compared to MTX for folylpolyglutamate synthase were measured in the sarcoma cell lines, with Vmax values equal to or slightly higher than those obtained with MTX. D1694 and 1843U89 are significantly more cytotoxic than MTX in intrinsically MTX-resistant sarcoma cell lines as a result of extensive formation of polyglutamates. These two thymidylate synthase inhibitors should be evaluated in patients with soft tissue sarcomas.

Antimetabolites, Antineoplastic↗
PubMed1994

Axonal sprouting in the hemisected adult rat spinal cord.

The morphological and biochemical changes were studied in adult Sprague-Dawley rats after hemisection at the L3 spinal cord level. After survival periods of one, two and three months, fluorescent tracers, FluoroGold or rhodamine B, were implanted into the dorsal white columns of these rats at the positions of the corticospinal tract below the lesion. Following uptake of the tracer, the rats were killed and the motor cortices and spinal cords of both control and hemisected rats were analysed for positively labelled neurons. The highest number of labelled cells were found two months after hemisection. They were present in both sides of the cortices, particularly in the contralateral cortex, and also in the gray matter of the spinal cord above the hemisection. A few rats which were subjected to complete transection of the spinal cord also showed labelling of neurons in the motor cortex two months after lesion. The Protargol silver technique and the [3H]choline uptake study confirmed the presence of nerve fibres traversing the lesion site in the hemisected spinal cord. Furthermore, when the rats that had been hemisected two months earlier were subjected to a second cut at the same site, chromatolytic neurons were observed in the spinal cord as well as in the motor cortices of both sides. The hemisected rats demonstrated limited recovery in limb movement. The evidence of this study clearly shows that sprouting of nerve fibres has occurred in the lesioned adult rat spinal cord.

Animals↗
PubMed1994

An immunohistochemical study of the c-fos protooncogene in the developing human retina.

The localization and distribution of the protooncogene c-fos were studied immunohistochemically in the retina of human fetuses ranging in age from 15 to 40 weeks of gestation. The highest levels of immunoreactivity were observed in the retinae of younger fetuses, decreasing in intensity with increasing age. At 15 weeks of gestation intense immunoreactivity was observed in the inner nuclear layer while the photoreceptor cells exhibited moderate staining. At 26 weeks of gestation, immunoreactivity in the inner nuclear layer was reduced. The ganglion cells, amacrine cells and photoreceptor cells showed moderate immunopositivity throughout the 26-40 weeks period. The role of c-fos in development is discussed in the light of its other known functions.

Embryonic and Fetal Development↗
PubMed1994

Immunohistochemical localization of neuropeptide Y and somatostatin in human fetal retina.

The localization of neuropeptide Y and somatostatin in the retina of six prenatal human specimens was determined by the utilization of light microscopic immunoperoxidase and immunofluorescence methods. The age of the prenatal fetuses ranged from 15 to 40 weeks of gestation. At 15 weeks, round or pear-shaped neuropeptide Y-immunopositive cells were observed in the inner nuclear layer and somatostatin immunoreactivity was only detected in the ganglion cell layer. Positive neuropeptide Y ganglion cells were observed by 17 weeks of gestation and by 28 weeks neuropeptide Y-immunopositive cells were demonstrated in the ganglion cell layer and inner nuclear layer. At 26-28 weeks, neuropeptide Y-immunopositive cells exhibited approximately the same shape as at 15 weeks of gestation, but the appearance of one or two processes was detected extending from the somata. By 38-40 weeks of gestation, neuropeptide Y-immunopositive cells were detected in the ganglion cell layer, inner nuclear layer and an immunopositive fibrous configuration in the inner plexiform layer. During this same period (38-40 weeks), somatostatin-positive cells were located in the ganglion cell layer, inner nuclear layer and the inner segments layers. Due to the difficulty of obtaining fetal materials, the exact time of initiation in the expression of neuropeptide Y and somatostatin is presently hard to delineate; however, it is safe to state that the peptides appear early in development.

Eye Proteins↗
PubMed1994

Stress induction of the mammalian GRP78/BiP protein gene: in vivo genomic footprinting and identification of p70CORE from human nuclear extract as a DNA-binding component specific to the stress regulatory element.

GRP78, also known as BiP, is one of the better-characterized molecular chaperones. It has been implicated in protein folding and also calcium sequestration in the endoplasmic reticulum. When the cells are subjected to endoplasmic reticulum stress, in particular the depletion of stored calcium and/or the accumulation of abnormal proteins, the rate of transcription of grp78 is enhanced. Previous studies have shown that the core region of the rat grp78 promoter (-170 to -135), which is 95% conserved with the human grp78 core (-133 to -98), is one of the key regulatory elements. Using ligation-mediated PCR, we have found that there are specific changes in factor occupancy after stress induction and the major changes occur within a cluster of bases located in the 3' half of the grp core, whereas other regulatory elements are constitutively occupied. This inducible binding to the 3' half of the human grp78 core region is observed under diverse stress signals, suggesting a common mechanism for the grp stress response. Nonetheless, the lack of constitutive in vivo protection at this region is not due to the absence of a binding factor in nuclear extracts. Using in vitro gel mobility shift assays, we detected a constitutive binding activity which exhibits specificity and affinity to the stress-inducible region. Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis size fractionation and renaturation analysis, the activity is found in polypeptides with molecular sizes of 65 to 75 kDa. After a three-step purification scheme including core affinity column chromatography, we purified p70CORE, which is about 70 kDa in its monomeric form. The purified p70CORE is sufficient to form a complex specific to the stress-inducible region.

Base Sequence↗
PubMed1994

Bilateral retinal responses during the acute phase (4-14 days) after traumatization of a single eye in the mouse.

The infiltration of T lymphocytes and macrophages was examined in the retinae of traumatized and non-traumatized contra-lateral eyes of mice. Traumatization was produced by channeling a small hole in the limbic region of a single eye in each animal while it was under Nembutal anesthesia and a small piece of iron wire was inserted into the eye. After the T lymphocytes and macrophages were made apparent by immuno-histochemistry, they were counted on sectioned strips of the peripheral retinae 250 micrometer in length in both traumatized and non-traumatized eyes. During the first two weeks after the induced disturbance, both the retinae of the traumatized and non-traumatized eyes showed infiltration of T lymphocytes and macrophages. Although this increase in T lymphocyte infiltration was observed in both damaged eye and the undamaged one, the former displayed an enhanced increase. The contrary was noticed for macrophages infiltrating the undamaged eye. A bilateral response was therefore evident through the cellular infiltration in the traumatized and un-traumatized eyes. Morphometrical observations also revealed nuclear size changes in the retina during this acute phase of traumatization.

Acute Disease↗
PubMed1993

Transactivation of the grp78 promoter by Ca2+ depletion. A comparative analysis with A23187 and the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin.

The calcium ionophore A23187 has been shown to induce the expression of a set of glucose-regulated protein (GRP) genes through the depletion of Ca2+ from the intracellular Ca2+ stores. Here we demonstrate that thapsigargin, which inhibits specifically the endoplasmic reticulum Ca(2+)-ATPase and causes a discharge of the intracellular Ca2+ store, is able to induce the transcription of two grp genes (grp78/BiP and grp94) with kinetics and magnitude similar to that of A23187. The induction of the grp genes by both reagents requires several hours of sustained treatment, in contrast to the rapid induction previously described for c-jun and c-fos. The transactivation of the rat grp78 promoter by A23187 is mediated through sequences spanning -154 to -130 and -99 to -90. Further, simultaneous mutation of two 10-base pair regions, spanning -139 to -130 and -99 to -90, severely reduced the A23187 response. The induction by thapsigargin is also partially mediated through these same promoter elements, without the involvement of the TRE and CRE-like elements of the grp78 promoter. The Ca2+ response elements are further defined by their ability to confer Ca2+ stress inducibility to a heterologous promoter. We show that subdomains of the grp78 promoter are capable of conferring the Ca2+ stress response. In particular, two copies of a 50-base pair region spanning -159 to -110, when cloned in either orientation, can confer a 5- and 9-fold induction by A23187 and thapsigargin, respectively. Our results lend support to the hypothesis that the induction of grp78 by A23187 and thapsigargin following ER Ca2+ discharge acts through a novel pathway in which a Ca2+ signal is transduced through redundant elements containing CCAAT box-like motifs flanked by GC-rich regions.

Animals↗
PubMed1993

Intrinsic resistance of cervical squamous cell carcinoma cell lines to methotrexate (MTX) as a result of decreased accumulation of intracellular MTX polyglutamates.

The causes of intrinsic of intrinsic MTX resistance in four human cervical squamous cell carcinoma cell lines (SiHa, C-33A, ME-180, C-4I) with different sensitivities to methotrexate (MTX) were determined. The in situ or whole-cell assay for thymidylate synthase (TS) was used to screen for known causes of MTX resistance, including increased dihydrofolate reductase (DHFR), altered DHFR, impaired MTX uptake, and decreased formation of MTX polyglutamates. While all four cell lines displayed initial sensitivity to MTX as demonstrated by a TS activity of < 20% following a 3-hr incubation with MTX, TS activity recovered in three of the four cell lines following a 4-hr incubation in drug-free media. Determination of MTX-polyglutamate accumulation in the four cell lines following a 24-hr incubation with 10 microM [3H]MTX revealed that a higher total intracellular level of MTX polyglutamates was achieved in the sensitive cell line (SiHa), with 54% occurring as long-chain tri-, tetra-, or pentaglutamates. The three resistant cell lines were found to contain less total MTX polyglutamates, with only 38, 14, and 13% occurring as long-chain MTX polyglutamates. Intrinsic MTX resistance in some cervical squamous cell carcinoma cell lines appears to be associated with a diminished accumulation of long-chain MTX polyglutamates, which are preferentially retained intracellularly.

Adult↗
PubMed1993

Mutations leading to antifolate resistance in Chinese hamster ovary cells after exposure to the alkylating agent ethylmethanesulfonate.

Chinese hamster ovary cells with a single allele for dihydrofolate reductase were used as a model system to study the effect of exposure to an alkylating agent, ethylmethanesulfonate, on rates and types of mutations at the dihydrofolate reductase locus leading to antifolate resistance. After overnight exposure to 400 micrograms/ml ethylmethanesulfonate, cells were allowed to recover for 3 days, and resistant colonies were selected in 8 x 10(-8) M trimetrexate. Trimetrexate, rather than methotrexate, was used as the selecting agent to increase the probability of obtaining mutations in dihydrofolate reductase, rather than in the reduced folate transport carrier protein. Seven of several hundred surviving colonies were selected at random, and cell lines were established. Cell lines 1-3 were maintained in culture in the presence of 8 x 10(-8) M trimetrexate and were 66-170-fold resistant to the drug. Cell lines 4-7 were initially expanded in 8 x 10(-8) M trimetrexate but were then maintained in the absence of the drug. These cell lines were 4.4-26-fold resistant to the drug, compared with the parental cell line. Cell line 1 was found to have an increase in dihydrofolate reductase activity, a corresponding increase in mRNA for dihydrofolate reductase, and amplification of this gene. Cell lines 2 and 6 had a mutated dihydrofolate reductase with altered trimetrexate- and methotrexate-binding properties. Cell line 3 had a 3-fold increase in dihydrofolate reductase activity. In cell lines 4, 5, and 7 the mechanisms of resistance to trimetrexate remain unknown.

Alkylating Agents↗