Μl Medium Research Articles

Solid-phase enzyme-linked assay for the screening of glycosidase activities and inhibitors

A solid-phase assay designed for the detection of glycosidase activity has been developed which detects 5 × 10–4 unit of α and β-galactosidases in 50 μL medium. The assay is highly stereo-specific. This assay is suitable for turbid or coloured fluids containing glycosidases and allows rapid and reliable Ki determination of glycosidase inhibitors. © Rapid Science Ltd. 1998

Intracellular events in response to GnRH causing gonadotropin release from pituitary cells of a channid fish, Channa punctatus (Bloch)

Addition of Channa punctatus GnRH (cGnRH, 2 μg/well) to suspended pituitary cell (6 × 10 4 cells/well) with 2 mM Ca 2+ caused about 14% increase in cellular Ca 2+ as compared to controls after 15 min. Ca 2+ remained at the elevated level for a further 45 min. A concurrent decrease of Ca 2+ in the medium from 2 to 1.25 mM/well (400 μl medium) was observed during the first 15 min of cGnRH incubation. Addition of verapamil (3 μM/well) significantly inhibited cellular Ca 2+ increase and medium Ca 2+ decrease ( p < 0.05). A sharp rise of gonadotropin (GtH) release from the pituitary cells in response to cGnRH plus Ca 2+ occurred between 5 and 15 min. GtH remained steady for an additional 15 min and was followed by a significant increase at 45 min ( p < 0.01). cGnRH stimulated GtH synthesis transiently. Cycloheximide (20 μg/well) blocked the increase of GtH release from 30 min onward but had no effect on stimulated GtH release at 15 min in response to cGnRH. Addition of 0.5 mM dibutyryl cAMP stimulated the release and synthesis of GtH from 45 min onward. cGnRH plus Ca 2+ resulted in a peak of cellular cAMP at 15 min, which followed by a sharp decline after 30 min. Baseline levels were reached after 60 min. cGnRH plus Ca 2+ lead to a significant increase in cellular calmodulin (CaM). Stimulation of phosphodiesterase (PDE) activity could only be detected at 30 min, but was increased significantly at 45 min. There is a clear correlation between cAMP level, Ca 2+, CaM, and PDE activity suggesting that a rise in cellular Ca 2+ results in an increase in CaM, which in turn stimulates the PDE activity and this affects degradation of cAMP. cGnRH plus Ca 2+ stimulated cellular calmodulin-dependent protein kinase II (CaM-kinase II) activity as early as 5 min. Increases in protein kinase A (PKA) activity were only noticed in the later (60 min) part of incubation. These results indicate two distinct phases of events in murrel pituitary cells in response to cGnRH: 1) cGnRH causes a rapid influx of extracellular Ca 2+ with a concurrent rise in cellular CaM-kinase II activity, resulting in an acute GtH release from the stored GtH, and 2) a sustained release of GtH, involving cAMP and PKA.

Caprine blastocyst formation following intracytoplasmic sperm injection and defined culture.

Experiments were undertaken to develop intracytoplasmic sperm injection (ICSI) to produce caprine embryos out of the normal breeding season. Oocytes were obtained from 2-6 mm ovarian follicles at slaughter. Selected oocytes with two to four layers of cumulus cells were incubated in 1 ml of H-TCM199 supplemented with 10 micrograms each of oFSH and bLH (NHPP, NIDDK, NICHD, USDA) and 20% fetal bovine serum (FBS) in a thermos (38.5 degrees C) for 4.5 h during transportation. Then, oocytes were transferred into 75 microliters of freshly prepared maturation medium under paraffin oil and a mixture of 5% O2, 5% CO2 and 90% N2. Approximately 26 h after recovery oocytes were denuded by incubation with hyaluronidase (100 IU/ml) and pipetting and held at 38.5 degrees C for 90 min. Spermatozoa frozen in egg yolk extender were thawed in a 37 degrees C water bath for 15 s. Motile fractions were selected by swim-up, then incubated for 90 min in TALP with 10 micrograms heparin/ml. Each oocyte was positioned with its first polar body at 6 or 12 o'clock by a holding pipette. Sperm (1 microliter) were added to 10 microliters medium containing 10% polyvinylpyrrolidone. A sperm cell was aspirated into a pipette, and then injected head-first into the cytoplasm of an oocyte maintained in H-TCM199 + 20% FBS at 37 degrees C. Injected oocytes were transferred to HM and, after 90 min, cultured in 50 microliters of BSA-free synthetic oviduct fluid plus polyvinyl alcohol, citrate and non-essential amino acids. Results demonstrate that caprine blastocysts can be produced outside the breeding season by the use of frozen-thawed semen and injection of sperm cells with broken tails into ova followed by culture in defined medium.

Maturational Changes in the NMDA Receptor in Fetal Sheep: Effect of Dexamethasone. 1741

Cerebral N-methyl-D-aspartate (NMDA)-type glutamate receptor activity plays a role in synapse formation and brain development and has been implicated as a mechanism of hypoxic brain injury. Dexamethasone (DX) may enhance organ maturation and decrease perinatal brain injury. To investigate the maturational changes in the NMDA receptor during development in fetal sheep, we determined MK-801 binding as an index of receptor ion channel activity in 1 fetal sheep at 60d gestation (term=154d), 6 at 84d, and 8 at 120d, 4 treated with placebo and 4 treated with DX. DX-treated fetuses were studied 12 hrs after DX, 6 mg IM q 12 hrs × 4 doses, was administered to the ewes.3[H]MK-801 binding was determined in a 200 μl medium containing 100μg membrane protein, 100 μM glutamate, 100 μM glycine, 10 mM HEPES/1 mM EDTA buffer (pH 7.0), and concentrations of 3[H]MK-801 from 0.5-50 nM. Nonspecific binding was determined in the presence of 10 mM unlabelled MK-801. Scatchard plots were constructed and Bmax (apparent receptor number) and Kd (dissociation constant) calculated. During development Bmax (mean± SD) increased from 0.51 pmoles/mg protein at 60d to 0.6±0.09 at 84d and 0.97±0.14 at 120d (p <0.005, 84d vs 120d). In contrast, Kd did not change significantly with increasing brain maturation (4.25, 5.07±0.38 and 4.90±0.81 nM at 60d, 84d, and 120d, respectively). Dexamethasone had no effect on either Bmax or Kd at 120d. Thus the apparent number of NMDA receptors increased with brain maturation but the affinity of the receptor for MK-801 was unchanged, similar to the results of our previous studies in fetal guinea pigs at mid- to late gestation. Dexamethasone did not enhance the maturational changes in the NMDA receptor MK-801 binding site at 120d, in contrast to its reported effects on other aspects of brain function. The apparent increase in number of NMDA receptors with maturation may be related to the timing of synaptogenesis in fetal brain and may enhance susceptibility to hypoxic injury. (NIH HD 20337, HD 34618)

Functional cross-reactivities of some locustamyotropins and Bombyx pheromone biosynthesis activating neuropeptide

Naturally occurring, recently isolated insect neuropeptides locustamyotropin II and III and Bombyx pheromone biosynthesis activating neuropeptide I (PBAN-I) bearing common carboxy terminals (Phe-X-Pro-Arg-Leu-NH 2) were studied for possible functional crossreactivities. On a Locusta oviduct and Leucophaea hindgut bioassay, synthetic Bombyx PBAN-I caused an increase in the spontaneous contractions at a threshold of 0.6 × 10 −8 and 1.45 ± 0.4 × 10 −8 M, respectively. In Bombyx mori, injection of 0.001 pmol of locustamyotropin II and 0.01 pmol of locustamyotropin III/female induced bombykol production and maximal production was attained at 0.1 or 1 pmol dose, respectively. In Spodoptera litura pheromone production increased in a dose-dependent manner when 1–100 pmol of peptide was injected into females. When pheromone glands were incubated in vitro in Grace's medium containing different doses of locustamyotropin II or III, maximal sex pheromone production was induced at 5 pmol of peptide/150 μl medium in B. mori and at 15 pmol peptide/50 μl in S. litura, respectively. Present results support the idea that the common C-terminal pentapeptide (Phe-X-Pro-Arg-Leu-NH 2) is responsible for both, myotropic and pheromonotropic activity.

In vivo incorporation of [9,10-3H]-palmitate into a rat metastatic brain-tumor model

Lipid metabolism of an intracerebrally implanted brain tumor and normal brain was investigated in awake Fischer 344 rats using intravenously injected [9,10(-3)H]-palmitate as a probe. A suspension of Walker 256 carcinosarcoma cells (250 cells in 5 microliters medium), with or without 1% low-melting-point agar, was implanted into the caudate nucleus of rats 8 to 9 weeks old. Control animals received an intracerebral injection without tumor cells. Seven days after implantation, awake rats were infused intravenously for 5 minutes with [9,10(-3)H]-palmitate (6.4 mCi/kg). The rats were killed 20 minutes after initiation of the infusion and coronal brain slices were obtained for quantitative autoradiography and light histological study. Tumor cell masses were histologically well demarcated from the surrounding brain tissue. Tumor tissue incorporation of [9,10(-3)H]-palmitate was heterogeneous, ranging on average from 3.1- to 6.1-fold greater than in the corresponding contralateral brain. In addition, incorporation corresponded to regional tumor cell density. The incorporation rate constant of [9,10(-3)H]-palmitate in tumor was significantly increased compared to control brain and was independent of tumor size. Necrotic areas within tumors showed no incorporation of radiolabeled palmitate. Brain surrounding the tumors and control injection sites showed reactive gliosis, and possessed 30% greater incorporation of [9,10(-3)H]-palmitate than contralateral normal brain. These results suggest that [9,10(-3)H]-palmitate can be used to image brain tumors in vivo, measuring turnover and/or synthesis of tumor and brain lipid.

Allatinhibin, a neurohormonal inhibitor of juvenile hormone biosynthesis in Manduca sexta

The corpora allata (CA) of Manduca sexta larvae become incapable of synthesizing juvenile hormone (JH) early in the wandering stage of the last larval stadium. They then switch to the synthesis and release of JH acids. This change in CA activity is induced by an inhibitory factor—allatinhibin (AI)—from the brain. AI activity is present in the fifth (Vth) instar hemolymph from about Day 4 (day of wandering) until Day 7 (early prepupa). CA of early fifth instar larvae (uninhibited) incubated in vitro with brain-corpora cardiaca-corpora allata (Br-CC-CA) complexes or brain alone from wandering larvae are inhibited as demonstrated by bioassay. On the basis of these observations, an in vitro-in vivo assay for AI was developed. Br-CC-CA or Br alone were first incubated in tissue culture medium overnight. Day 0 (0d) Vth instar CA incubated for 16 hr in such medium will lose the ability to induce a larval molt in allatectomized 0d IVth instar larvae if the medium contained AI activity. The highest AI activity was exhibited by the medium obtained from incubations of brain from wandering larvae whereas the medium from incubation of 0d Vth and 0d pupal brains showed no AI activity. Dose-response data show that AI is active at 0.03 brain equivalents/200 μl medium. CA must be exposed to AI for 12–16 hr for manifestation of inhibition. AI causes a stable inhibition of CA. AI is heat-labile, protease sensitive, has a molecular size between 1.0 and 3.5 kDa, and is clearly distinct from the allatostatins described by others.

Duck lymphocytes—III. Transformation responses to some common mitogens

The lymphocyte transformation (LT) test was performed using duck blood lymphocytes stimulated with phytohaemagglutinin (PHA), concanavalin A (Con A), lentil lectin (LC), Roman snail lectin (HP), peanut agglutinin (PNA), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), horseshoe crab lectin (HSC), pokeweed mitogen (PWM) and E. coli lipopolysaccharide (LPS). Cells were cultured in microtitre trays, at 41.6°C, 8 × 10 5 cells in 200 μl medium (=4 × 10 6 cells/ ml) supplemented with 10% pooled duck serum. Mitogens were added at final concentrations of 0.1–100 μg/ml and triplicate cultures at each concentration were harvested daily for scintillation counting 6 hr after addition of 1 μCi [ 3H]thymidine. Three patterns of response were observed. The responses to Con A, LC, HP and HSC were greatest at high mitogen concentrations (40–100 μg/ml) throughout the 7 days of culture. With PHA, PNA, WGA and LPS maximum stimulation was obtained at 3–5 days, at which time the cells were responding to lower concentrations of mitogen than were required at other times during the experiment. The response to BSS and PWM showed increasing sensitivity to lower concentrations of mitogen during the first 3 days of culture and then stimulated most strongly at 2–10 μg/ml in cultures harvested after 4–7 days. Cells from two ducks were cultured for 3 and 5 days with selected concentrations of these mitogens; the results confirmed the variation in response to different mitogens. It is possible that these patterns of response are the outcome of stimulating different populations of duck lymphocytes.

Solute Uptake by Dictyostelium discoideum and its Inhibition

A study has been made of the uptake of radioactively-labelled solutes by starved myxamoebae of the cellular slime mould Dictyostelium discoideum. Inulin uptake was energy-dependent, inhibited by cycloheximide, and the rate was proportional to extracellular inulin concentration. The ability of the cells to take up inulin did not change during starvation. Uptake of four other solutes, β-alanine, glucose, protein hydrolysate and uracil occurred at a similar rate to inulin uptake when expressed as an endocytic index (μl medium taken up h−1 per 106 cells). All the results were consistent with uptake occurring by fluid-phase pinocytosis. Pinocytic activity thus occurred in starving cells and was found to be affected by some agents that act as developmental inhibitors. Uptake of inulin and putrescine (previously shown to be taken up by adsorptive pinocytosis) was inhibited by chloroquine, KCl and NH4Cl, although ammonium ions also blocked developmental changes in the ability of myxamoebae to take up putrescine. Sodium ions were less inhibitory. ε-Aminocaproic acid inhibited solute uptake in a temperature-dependent, reversible manner.

Thyroxine to 3,5,3′-triiodothyronine conversion by rat anterior pituitary and liver

To evaluate the process of conversion of T 4 to T 3 in pituitary tissue in vitro, fragments of rat anterior pituitary were incubated for 3–4 hr in the presence of 125I T 4 and 131I T 3. The nuclei were isolated and the specifically bound 131I and 125I-labelled T 3 quantitated. No specifically bound nuclear 125I T 4 was found. The nuclear/medium ratio for 131I T 3 was 0.16 ± 0.02 (mean ± SD), cpm [μg DNA] −1/cpm [μl media] −1 after 3–4 hr. The 125I T 4 present contained 0.43% ± 0.04% 125I T 3 and this did not increase during the incubation. The estimated nuclear/media ratio for 125I T 3 was 1.5 ± 0.2, approximately ninefold higher than that for 131I T 3. These results indicate that pituitary tissue deiodinates T 4 to T 3. During incubation of pituitary homogenates with 1 μg T 4/ml, increases in radioimmunoassayable T 3 were not consistently observed. In the presence of 5 m M dithiothreitol (DTT) however, there was an eight-tenfold stimulation of T 4-T 3 conversion (18–37 ng T 3/g wet weight/20 min). Similar studies of liver homogenates showed considerably higher basal T 4 to T 3 conversion (49–56 ng T 3/g wet weight/15 min) but DTT-stimulated T 4 to T 3 conversion was only two-threefold higher than that in pituitary (62–63 ng T 3/g wet weight/15 min). Taken together, the present results indicate that the rat anterior pituitary deiodinates T 4 to T 3 and that sulfhydryl-containing compounds markedly stimulate this process. These in vitro results support our previous in vivo studies suggesting that intrapituitary T 4 to T 3 conversion contributes substantially to the nuclear T 3 in this tissue.