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Biomedical subjects

K Leung

Publications and source records attributed to K Leung.

At least 91 records · Page 5Linked to original sources
PubMed1988

HTLV-1 transactivator induces interleukin-2 receptor expression through an NF-kappa B-like factor.

Like other viruses that infect primate cells, the human T lymphotropic virus-I (HTLV-I) stimulates production of some host cell proteins. In particular, HTLV-I infected T cells synthesize interleukin-2 receptor alpha (IL-2R alpha) chain, which is probably induced through the mediation of the tat-I gene product of the virus. Activated T cells contain a trascription factor called NF-kappa B, which stimulates the expression of human immunodeficiency virus-1 (HIV-1) by binding to an 11-base-pair enhancer sequence called kappa B. We have now found evidence that a similar transcription factor is involved in the induction of IL-2R alpha expression by tat-I. We have identified a sequence upstream of IL-2R alpha which is the same as the kappa B site at 9 of 11 base pairs, competes for binding to the kappa B sequence, and serves as a tat-I responsive element when multiple copies are inserted upstream of a heterologous promoter. The tat-I product also induces kappa B and the IL-2R alpha kappa B binding activity in transfected Jurkat T lymphoid leukaemia cells. Both HTLV-I and HIV-1 thus interact with NF-kappa B-like transcription factors which might normally regulate expression of a growth factor receptor gene.

Base Sequence↗
PubMed1988

Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.

Animals↗
PubMed1988

Divergent changes in the concentrations of gonadotropin beta-subunit messenger ribonucleic acid during the estrous cycle of sheep.

Cyclic changes in the production of the pituitary gonadotrophic hormones, LH and FSH are essential events in the maintenance of the reproductive system of female mammals. While studies have examined changes in the secretion of LH and FSH during the estrous cycle and demonstrated the importance of these hormones in regulation of ovarian development and gametogenesis, considerably less is known concerning the regulation of the biosynthesis of these hormones. Although initial studies have examined changes in LH subunit mRNA concentrations during the rat and ovine estrous cycles, no information concerning the physiological regulation of FSH beta mRNA concentrations has been available. In the present study we have examined the relationship between pituitary concentrations of LH and FSH subunit mRNAs and the serum concentrations of these gonadotropins. The results demonstrate a very different pattern of change for FSH beta subunit mRNA than that observed for alpha and LH beta subunit mRNAs. In fact, FSH beta mRNA concentration decline substantially during the preovulatory period, reaching minimal values at a time when alpha and LH beta mRNA levels are near maximal. Furthermore, this decline in FSH beta mRNA amounts occurs when serum FSH concentrations are maximal. Thus, FSH beta mRNA concentrations follow a very different pattern than that of serum FSH. In contrast, LH beta mRNA and serum LH concentrations tend to increase at the same time. These findings provide evidence that concentrations of LH beta and FSH beta mRNAs are likely regulated by different mechanisms.

Animals↗
PubMed1987

Influence of Phosphate on the Growth and Nodulation Characteristics of Rhizobium trifolii.

The growth and nodulating characteristics of Rhizobium trifolii 6 and 36 differed under different external phosphate conditions. Under growth conditions designed to deplete the internal phosphate content of the rhizobia, strain 6 maintained a generation time of 5 h during the exponential phase over two cycles of growth in phosphate-depleted medium. In contrast, the generation time of strain 36 was extended from 3.5 to 9.8 h over two cycles of phosphate-depleted growth, although the organism eventually achieved the same cell density and cellular phosphate content as that of strain 6 at stationary phase. Phosphate-depleted strain 6 required 0.51 +/- 0.08 muM phosphate to commence proliferation, whereas phosphate-depleted strain 36 required 0.89 +/- 0.04 muM phosphate under the same conditions. Phosphate-depleted strain 6 maintained viability when exposed to external phosphate concentrations subcritical for growth to occur, whereas phosphate-depleted strain 36 lost viability within 48 h when exposed to medium containing phosphate at concentrations subcritical for growth. Phosphate-depleted strain 36 was inferior to phosphate-depleted strain 6 at nodulating subterranean clover (Trifolium subterraneum L. cv. Mt. Barker) by taking 2 to 4 days longer to develop nodules in phosphatedepleted plant grown medium at pH 5.5. Nodulation by phosphate-depleted strain 36 was accelerated either by including phosphate in the plant growth medium at pH 5.5 or by raising the solution pH of phosphate-depleted plant growth medium to pH 6.5. External phosphate and pH effects were not observed on the nodulating capabilities of phosphate-depleted strain 6 or on luxury phosphate-grown cells of either strain. Phosphatedepleted strains 6 and 36 proliferated to a similar extent on the rhizoplanes even under stringently low external P(i) concentrations. The phosphatase activities of both phosphate-depleted strains were significantly (P = 0.05) higher at pH 6.5 than at pH 5.5, and the activity of strain 6 was significantly higher (P = 0.05) than that of strain 36 at pH 5.5 and 5.0.

Journal Article↗
PubMed1987

Specific immune serum to the Epstein-Barr virus DNA polymerase.

Epstein-Barr virus (EBV) DNA polymerase was released from phorbol ester-treated tamarin (Saguinus oedipus) cells (B95-8) and prepared for use as an antigen by sequential column chromatography with DEAE-Sephadex A-25, DEAE-cellulose, phosphocellulose, and single-stranded DNA cellulose. Proteins from single-stranded DNA cellulose with DNA polymerase activity in 100 mM ammonium sulfate were mixed with complete Freund adjuvant and injected intradermally into rats and rabbits. Immune sera that were screened for specific antibody by indirect immunofluorescence procedures reacted with approximately 3% of the cells in EBV-producer cultures (B95-8 and P3HR-1) but not with EBV genome-negative cells (BJAB). In functional enzyme assays, immune sera or the immunoglobulin fraction inhibited the activity of purified EBV DNA polymerase 90%. Inhibition of enzyme activity was not affected by absorption of immune sera with insoluble matrices of proteins prepared with tamarin and human cells which lacked the EBV genome. Cellular DNA polymerase alpha was not inhibited by immune sera to the EBV enzyme.

Animals↗
PubMed1987

Growth of B95-8 cells and expression of Epstein-Barr virus lytic phase in serum-free medium.

Epstein-Barr virus (EBV)-transformed tamarin (Saguinus oedipus) cells (B95-8) were selected for growth in medium with reduced serum and then transferred to serum-free medium which consisted of RPMI 1640 supplemented with insulin, transferrin, and selenium. Serum-free cells in continuous passage for 1 year had a morphology, growth rate, and culture density which approached those of B95-8 cells grown with serum. The cells expressed virus-induced antigens, including the EBV-associated DNA polymerase. Cells exposed to EBV-inducing agents, n-butyric acid and phorbol 12-myristate-13-acetate, produced transforming virus with titers comparable to those of cultures grown with serum. These findings demonstrate that serum is neither required for the growth of B95-8 cells nor necessary for induction or full expression of the EBV lytic phase in these cells.

Animals↗
PubMed1987

Differential regulation of gonadotropin subunit messenger ribonucleic acids by gonadotropin-releasing hormone pulse frequency in ewes.

Changes in the frequency of GnRH and LH pulses have been shown to occur between the luteal and preovulatory periods in the ovine estrous cycle. We examined the effect of these different frequencies of GnRH pulses on pituitary concentrations of LH and FSH subunit mRNAs. Eighteen ovariectomized ewes were implanted with progesterone to eliminate endogenous GnRH release during the nonbreeding season. These animals then received 3 ng/kg body weight GnRH in frequencies of once every 4, 1, or 0.5 h for 4 days. These frequencies represent those observed during the luteal and follicular phases, and the preovulatory LH and FSH surge of the ovine estrous cycle, respectively. On day 4, the ewes were killed and their anterior pituitary glands were removed for measurements of pituitary LH, FSH, and their subunit mRNAs. Pituitary content of LH and FSH, as assessed by RIA, did not change (P greater than 0.10) in response to the three different GnRH pulse frequencies. However, subunit mRNA concentrations, assessed by solution hybridization assays and expressed as femtomoles per mg total RNA, did change as a result of different GnRH frequencies. alpha mRNA concentrations were higher (P less than 0.05) when the GnRH pulse frequency was 1/0.5 h and 1 h, whereas LH beta and FSH beta mRNA concentrations were maximal (P less than 0.05) only at a pulse frequency of 1/h. Additionally, pituitary LH and FSH secretory response to GnRH on day 4 was maximal (P = 0.05) when the pulse infusion was 1/h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Suppression of lymphocyte activation and functions by a leukemia cell-derived inhibitor.

An inhibitor isolated from the serum-free culture medium of human myeloid leukemic HL-60 cells was able to suppress mitogen- and alloantigen-stimulated proliferative responses of normal lymphocytes in a dose-dependent manner. In vitro production, concentration, and purification by column chromatography and electrophoresis revealed that the inhibitor was produced constitutively, required RNA synthesis, and had a molecular weight in the range of 40,000-60,000. The inhibitor was also produced in vitro by myeloid leukemia cells isolated from patients with acute myelogenous leukemia. In a similar manner, the inhibitory material suppressed proliferative responses of allogeneic and autologous lymphocytes. Suppression was accompanied by drastically reduced production of interleukin 2 and lymphokines, which regulate differentiation of myeloid leukemia cells, and suppression was reversed by addition of exogenous interleukin 2. The inhibitor did not suppress clonogenic proliferation of normal granulocytes and macrophages suggesting that inhibition of production or interference with interleukin 2 activity as a possible mechanism. These interactions between leukemia cells and lymphocytes have shed new light on the immunosuppression and growth advantage of leukemia cells. Inhibitory activity of HL-60 cells was diminished after they were induced to differentiate, indicating that differentiation induced by lymphokines may be an effective means of controlling leukemia.

Cell Differentiation↗
PubMed1986

Relationship between pituitary GnRH-binding sites and pituitary release of gonadotrophins in post-partum beef cows.

Thirty primiparous suckling beef cows were slaughtered on Day 7, 14, 28, 42 or 56 after parturition. Some had resumed oestrous cyclicity by the time they were slaughtered on Days 42 and 56. Amongst acyclic cows between Days 7 and 42, pituitary LH concentrations and basal and GnRH-induced release of LH from pituitary explants doubled. Pituitary FSH concentration and basal release in FSH increased only by 15-20%, while GnRH-induced release of FSH in vitro was unchanged. During postpartum anoestrus, overall mean concentrations of serum FSH did not change, whereas overall mean concentrations and pulse amplitudes of serum LH increased. Numbers and affinity constants of GnRH-binding sites in pituitary glands remained constant during the post-partum period studied. We conclude that, under these experimental conditions, numbers and affinity constants of GnRH-binding sites in the pituitary gland of post-partum beef cows do not limit the ability of the anterior pituitary gland to release gonadotrophins.

Animals↗
PubMed1986

Anovulation in postpartum suckled beef cows. I. Associations among size and numbers of ovarian follicles, uterine involution, and hormones in serum and follicular fluid.

Changes in sizes and numbers of ovarian antral follicles, uterine size and weight, serum hormones, and frequency and duration of suckling were examined during the postpartum anovulatory period in primiparous, suckled beef cows. Twenty-one anovulatory, suckled cows (n = 4 to 6/d) were slaughtered on d 7, 14, 28 and 42 to 56 after parturition. In addition, a total of 11 postpartum cows that had begun cyclic activity were slaughtered on d 28, 42 or 56. Blood was collected at 10-min intervals for 6 h 1 d before slaughter for measurement of prolactin, cortisol and progesterone in serum. Numbers of medium (4.0 to 7.9 mm) follicles increased fourfold (P less than .05) between d 7 and 42 to 56 in anovulatory cows, whereas numbers of small (1.0 to 3.9 mm) and large (greater than or equal to 8.0 mm) follicles did not change (P greater than .10). Uterine involution was complete by d 28. In anovulatory cows, a higher (P less than .05) proportion of largest (but not second-largest) follicles was opposite the ovary containing the corpus albicans from pregnancy (CAP). In addition, 90% of these largest follicles opposite the CAP had concentrations of estradiol greater than progesterone. In cyclic cows, however, first ovulations occurred with equal frequency on either ovary. Concentrations of prolactin or cortisol in serum or duration of suckling were not associated with changes in uterine or ovarian measurements. In conclusion, growth and function of the largest (but not second-largest) follicle were reduced when located on the ovary containing the CAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Anovulation in postpartum suckled beef cows. II. Associations among binding of 125I-labeled gonadotropins to granulosa and thecal cells, and concentrations of steroids in serum and various sized ovarian follicles.

To determine if specific binding of 125I-labeled gonadotropins to granulosa and thecal cells, or concentrations of steroids in ovarian follicles change during the postpartum anovulatory period, 21 suckled beef cows were slaughtered on d 7, 14, 28, 42 or 56 after parturition (n = 4 to 6 per d). After slaughter, 10 to 15 follicles were dissected from each pair of ovaries and categorized by diameter: small (1.0 to 3.9 mm), medium (4.0 to 7.9 mm) or large (greater than or equal to 8 mm). Progesterone (221 to 612 ng/ml), androstenedione (48 to 94 ng/ml) and estradiol (2.7 to 23.9 ng/ml) did not change (P greater than .10) in fluid of small or medium follicles from d 7 to 42 to 56 after parturition. Similarly, specific binding of human chorionic gonadotropin (125I-hCG) or follicle stimulating hormone (125I-oFSH) to homogenates of small, medium or large follicles did not change (P greater than .05). In contrast, progesterone in fluid of large follicles increased (P less than .05) 3.4-fold between d 7 and 14, but decreased (P less than .05) 55% between d 14 and 28. Concentrations of androstenedione in fluid of large follicles did not change (P greater than .10) from d 7 to 42 to 56. Concentrations of estradiol in fluid of large follicles remained constant between d 7 and 14, but increased (P less than .05) 4.2-fold between d 14 and 28. We conclude that during the postpartum anovulatory period, there is no change in steroidogenic capabilities of small or medium follicles, both of which predominantly produce progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗
PubMed1986

C1q content of serum and lung lavage fluid of rats exposed to toxic levels of oxygen.

We used a sensitive hemolytic assay to measure the level of C1q, a subcomponent of the first complement protein in the classic pathway, in bronchoalveolar lavage fluid and serum of rats exposed to air and hyperoxia. The serum level was 125 +/- 5 micrograms/ml and the lavage level was 41 +/- 17 ng/ml in rats breathing air. In rats exposed to acute oxygen toxicity (95% O2 for 66 hr), the serum level was at 107 +/- 10 micrograms/ml, but the level in lavage fluid increased to 2457 +/- 400 ng/ml (p less than 0.05 compared to air). Administration of the proline analogue cis-4-hydroxy-L-proline to air- and O2-exposed rats reduced the serum C1q level by 28% and 34%, respectively (both p less than 0.05), presumably by interfering with metabolism of the collagen-like sequence of C1q. The level of C1q in bronchoalveolar lavage fluid is a sensitive marker of acute lung injury.

Animals↗
PubMed1986

Radial partition immunoassay applied to automated quantification of human choriogonadotropin with use of two monoclonal antibodies.

We describe a novel application of radial partition immunoassay to quantification of human choriogonadotropin (hCG). In this "sandwich"-type assay, two monoclonal antibodies, specific for different sites on the intact molecule are used. The solid phase consists of tabs of glass-fiber filter paper containing a pre-immobilized antibody specific for the alpha subunit of hCG. The patient's sample is first applied directly to the central "reaction zone" of the tab, allowing hCG to bind to the solid-phase antibody. Then a buffered solution containing enzyme-labeled Fab' fragments of a monoclonal antibody specific for the beta subunit of hCG is applied, initiating "sandwich" formation. Finally, a wash buffer containing a fluorogenic substrate is applied, eluting unbound conjugate to the tab periphery. Bound enzyme conjugate is quantified by measuring the rate of increase in fluorescence. Rates are converted to clinical units by comparison with a stored calibration curve. Elapsed time from sample application to results is less than 8 min. Specific performance characteristics of this assay are reported.

Antibodies, Monoclonal↗
PubMed1985

Human leukemia cell maturation induced by a T-cell lymphokine isolated from medium conditioned by normal lymphocytes.

Human myelogeneous leukemia cells in liquid culture can be induced to mature along the monocyte/macrophage pathway by a maturation inducer derived from the conditioned medium of activated human T lymphocytes. Serum-free conditioned medium was used for the isolation of the T-cell lymphokine. The maturation inducer was purified approximately equal to 6000-fold by ammonium sulfate precipitation, low-salt elution from DEAE-Sepharose CL-6B, gel filtration on Bio-Gel A-0.5m, and NaDodSO4/PAGE under nonreducing conditions. The molecular weight of the maturation inducer was 36,000-58,000 on NaDodSO4/PAGE. Terminal differentiation associated with inhibition of leukemia cell proliferation and expression of mature cell properties was observed with the isolated maturation inducer, identical to the activity observed with the unfractionated conditioned medium. Cell-cycle analysis revealed that the proportion of replicating S-phase cells was reduced from 40% to 7% after initial interaction of the maturation inducer with cells. The differentiating cells simultaneously acquired monocyte antigen, membrane complement receptors, phagocytic function, and monocyte/macrophage morphology. The maturation-inducing activity is dose-dependent, with more inducer causing the development of more mature cells in a shorter time period. The maturation inducer was shown to be stable after pH 2 treatment, independent of interleukin 2 and colony-stimulating factor, devoid of alpha-, beta-, and gamma-interferon, and not affected by antibody to interferon. The maturation inducer may play a role as a physiological regulator of monocytic and leukemia cell development.

Cell Cycle↗
PubMed1985

Rat Clq: isolation and purification from normal serum and development of a sensitive hemolytic assay.

Rat Clq was isolated and purified from normal rat serum by a two-step procedure: affinity chromatography on a human IgG-Sepharose 4B column and Bio-Gel A 5m column chromatography. From 1.8 l of normal serum, the procedure yielded 4.5 mg of homogeneous and hemolytically active rat Clq as shown by three bands of peptide in SDS polyacrylamide gel electrophoresis under reducing conditions and by a single precipitin line between rat Clq and rabbit anti-rat Clq antibody. Rabbit anti-rat Clq antibody prepared by immunizing a rabbit with 225 micrograms of rat Clq was specific as shown by a single precipitin line between the antibody and normal rat serum and rat Clq with complete identity. The amino acid composition of rat Clq was very similar to that of human Clq. The purification procedure also yielded Clq-depleted rat serum which was used with the homogeneous Clq to establish a sensitive hemolytic assay: 4.5-13.5 ng of rat Clq can be reproducibly quantitated. The concentration of Clq in Brown Norway rats was estimated to be 41.5 +/- 3.0 micrograms/ml serum.

Amino Acids↗
PubMed1984

Relationship between pituitary responsiveness to Gn-RH and number of Gn-RH-binding sites in pituitary glands of beef cows.

Changes in the ability of Gn-RH to induce gonadotrophin release with time after synchronization of oestrus was determined in 4 groups of 6 cows each. Cows were given Gn-RH at 40-min intervals for 6 h beginning at -24, 0, 18 or 36 h (time 0 = removal of progestagen implant). Changes in concentration (ng/ml) of serum LH after Gn-RH averaged 2.9, 6.2, 6.4 and 33.4, whereas serum FSH averaged 25.7, 35.8, 35.8 and 97.3. Thus the responsiveness of the pituitary to Gn-RH had increased by 36 h after implant removal. Other groups of cows subjected to the same synchronization scheme were slaughtered at 0 h, 24 h or at various times after onset of oestrous behaviour. Gn-RH binding to crude pituitary membrane preparations was assessed. There was no apparent change in the affinity constant of Gn-RH-binding sites with time after synchronization. The number of Gn-RH-binding sites remained unchanged until the period of oestrus when a significant decline with time was detected. We conclude that the increase in pituitary responsiveness to Gn-RH that occurs before the preovulatory gonadotrophin surge was not directly associated with changes in number or affinity of pituitary Gn-RH-binding sites in crude pituitary membrane preparations.

Animals↗