Characterization of human malignant melanoma cell lines. VII. Glycoprotein synthesis and shedding as revealed by [3H]glucosamine labeling

We have established conditions for the study of membrane glycoprotein synthesis and turnover in cultured human malignant melanoma cell lines using the labeled precursor [3H]glucosamine. Uptake of label increased parallel with cell growth, reaching a steady state in resting cultures. Fifteen to 30% of incorporated label can be released from the cells by trypsin treatment depending on the conditions of exposure to the enzyme, and about 50% of the incorporated label is spontaneously shed from the cells within 96 hr of incubation. Labeling in exhausted medium gave a 5- to 8-fold increase in uptake which was inhibited by addition of glucose (2 mg per ml) into the culture medium. The percentage of trypsin-releasable material was identical in fresh and exhausted medium; however, the percentage shed was less in cells initially labeled in exhausted medium. These data provide background information for further studies on the antigenic composition of the glycoproteins of cultured melanoma cells.     

Establishment and characterization of human ocular melanoma cell lines

Five continuous cell lines have been established from 29 ocular melanomas and maintained for periods ranging from 3 to 9 years in medium identical to that in which 3 concomitantly studied lines of cutaneous melanoma cells were cultured as controls. The long-term problems to be overcome in establishing uveal cell lines are related to cell-doubling times which ranged from 72 to 432 hr, and plating efficiency, which ranged from 0.5%-6.5%. Tumors and cell lines were found to contain melanosomes. The morphology of uveal cells during the early subcultures exhibited multiple changes. Two different established cell lines were obtained from one ciliary-body tumor. Biochemical studies revealed markers of melanogenesis and neuroendocrine compounds. Cytogenetic studies revealed chromosomal abnormalities that differed between uveal and conjunctival melanomas.     

Cations affect [3H]mazindol and [3H]WIN 35,428 binding to the human dopamine transporter in a similar fashion

The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn2+, Mg2+, Hg2+, Li+, K+, and Na+ were assessed on the binding of [3H]mazindol and [3H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21 degrees C. Zn2+ (30-100 microM) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [3H]mazindol; Mg2+ (0.1-100 microM) had no effect; Hg2+ at approximately 3 microM stimulated binding to membranes, with a relatively smaller effect for [3H]mazindol than [3H]WIN 35,428 at 0 degrees C, and at 30-100 microM inhibited both intact cell and membrane binding; Li+ and K+ substitution (30-100 mM) inhibited binding to membranes more severely than to intact cells; and Na+ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21 degrees C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn2+ at 0 and 21 degrees C and Hg2+ at 0 degrees C.     

Lobeline and nicotine evoke [3H]overflow from rat striatal slices preloaded with [3H]dopamine: differential inhibition of synaptosomal and vesicular [3H]dopamine uptake

Lobeline is currently being developed as a substitution therapy for tobacco smoking cessation. Activation of CNS dopamine (DA) systems results in the reinforcing properties of nicotine. The present study compared the effects of lobeline and nicotine on rat striatum. Both lobeline and nicotine evoked [3H]overflow from striatal slices superfused in the presence of pargyline and nomifensine in the buffer. Marked DA depletion (42-67%) and a concomitant 2-fold increase in dihydroxyphenylacetic acid (DOPAC) in slices superfused with high concentrations (30-100 microM) of lobeline were observed. The effect of nicotine (10 microM) was inhibited in a concentration-dependent manner by mecamylamine (1-100 microM). However, lobeline (0.1-100 microM)-evoked [3H]overflow was calcium-independent, and was not antagonized by mecamylamine (1-100 microM), suggesting a mechanism of action other than stimulation of nicotinic receptors. Lobeline inhibited [3H]DA uptake into synaptosomes (IC50 = 80 +/- 12 microM) and vesicles (IC50 = 0.88 +/- 0.001 microM), whereas nicotine (< or =100 microM) did not inhibit synaptosomal or vesicular [3H]DA uptake. In the absence of pargyline and nomifensine in the buffer, endogenous DA was detected in superfusate only in those slices exposed to the highest concentration (100 microM) of lobeline. However, endogenous DOPAC concentration was increased in a concentration-dependent manner, indicating that lobeline exposure resulted in increased cytosolic DA which was rapidly metabolized to DOPAC. Under these conditions, lobeline (10-100 microM) also significantly depleted (66-85%) DA content; however, no change in DOPAC content was observed. The results suggest that, unlike nicotine, lobeline increases DA release by potent inhibition of DA uptake into synaptic vesicles, and a subsequent alteration in presynaptic DA storage.     

Sensitivity to ethanol across development in rats: comparison to [3H]zolpidem binding

Previous research has suggested that rats tested at 28 to 30 days of age show a marked subsensitivity to the sedative effects of ethanol. In the present study, rats of different ages were tested for aerial righting following acute ethanol (3 g/kg) treatment. These results were compared with the effects of the atypical benzodiazepine zolpidem (3 and 5 mg/kg) and pentobarbital (10 and 15 mg/kg). Animals tested at 25, 28, or 35 days of age were significantly less impaired by ethanol than preweanling rats (age 20 days) or older rats (age 65 to 75 days), whereas animals tested at 25 or 28 days of age were less impaired by the higher dose of zolpidem. With pentobarbital, the most distinct age-related trend was greater impairment in 20-day-old rats. Because ethanol may be active at the same type I GABA(A) receptor site selectively labeled by [3H]zolpidem, levels of [3H]zolpidem binding were determined for rats of different ages. Although some brain regions showed progressive increases in binding of [3H]zolpidem across development, other regions demonstrated increased binding from day 12 or 17 to day 20, then a plateau of binding levels across days 20, 25, and 28, with further increases occurring by day 36 or day 60. This pattern was observed in the cingulate cortex, medial septal nucleus, globus pallidus, inferior colliculus, red nucleus, and cerebellum. Overall, the results indicate that the period of subsensitivity to the sedative effects of ethanol is coincident with a change in the developmental pattern of GABA(A) receptor sites targeted by [3H]zolpidem.     

Laminar distribution of nicotinic receptor subtypes in human cerebral cortex as determined by [3H](-)nicotine, [3H]cytisine and [3H]epibatidine in vitro autoradiography

We have used gene disruption to isolate two talin (-/-) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of beta1 integrin, although levels of alpha5 and alphaV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (-/-) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (-/-) ES cells were able to assemble talin-containing focal adhesions. Both talin (-/-) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (-/-) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the beta1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for beta1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.     

Autoradiographic characterization of [3H]inositol (1,4,5) trisphosphate and [3H]inositol (1,3,4,5) tetrakisphosphate binding sites in human brain

Autoradiographic techniques were used to investigate the characteristics of tritiated inositol(1,4,5)trisphosphate ([3H]IP3) and inositol (1,3,4,5) tetrakisphosphate ([3H]IP4) binding to human brain. In brain sections [3H]IP3 exhibited a two-site binding with KD values of 87 nM and 9.3 microM respectively for the higher and lower affinity sites. [3H]IP4 also bound to two sites with KD values of 43 nM and 1.4 microM, respectively. With the conditions fixed in this study, [3H]IP3 and [3H]IP4 autoradiography in the cortex, caudate, hippocampus and cerebellum were performed. The most prominent [3H]IP3 binding among these regions was found in the cerebellum, particularly in the molecular layer. Within the hippocampus, the subiculum and the CA1 region showed much more prominent binding than the other subfields. [3H]IP4, binding was fairly homogeneous in the regions studied, with the exception of a slightly higher binding in the molecular layer of the cerebellum.     

Effects of handling and etherization on incorporation of [3H] lysine into protein of mouse brain and liver

An oxygen jet method ventilating patients during laryngoscopy has been applied to fiberoptic bronchoscopy. A 3.5 mm plastic tube 24.5 cm long was inserted into the trachea through the mouth. An intermittent jet of oxygen at 3.5 atm (50 psi) was applied to this tube using a 1.5 mm ID plastic catheter to ventilate the patient. Anesthesia was accomplished with intravenous thiamylal and Innovar. The patients were paralyzed with continuous succinylcholine. The technique has subsequently been used without complications in more than 1,000 patients. A fluidic ventilator was developed for delivering and controlling the oxygen jet. The airway pressure can be monitored continuously and, by the use of fluidic devices, the jet can be set to cut off automatically if the airway pressure is too high. The above techique for laryngoscopy with the fluidic ventilator was used in 28 patients undergoing fiberoptic bronchoscopy (Olympus 5.7 mm diameter). The airway pressure was continuously monitored with a line attached to the suction port of the bronchoscope. Arterial PCO2 ranged from 23 to 42 mmHg and PO2 from 105 to 325 mmHg. The high PO2 levels were maintained even during suctioning. General anesthesia for fiberoptic bronchoscopy can be performed using an endotracheal tube not smaller than 8 mm internal diameter (ID). The advantages of the oxygen jet technique are that it can be used in smaller patients and that the upper airway can be examined.     

Uptake of [3H]glycine and [3H]GABA by amacrine cells in the cat retina

After intravitreal injection, [3H]glycine accumulates in 3 distinct subpopulations of amacrine cells in the cat retina whereas [3H]GABA accumulates in 4 different subpopulations. Each labeled cell type can be distinguished on the basis of size and cytologic features. The density of label associated with each subpopulation serves as an additional distinguishing characteristic. [3H]Glycine is concentrated within the outer two-thirds of the inner plexiform layer (IPL). [3H]GABA is localized in two narrow bands in the outer half of the IPL and in a wider band adjacent to the ganglion cell layer.     

A new death receptor 3 isoform: expression in human lymphoid cell lines and non-Hodgkin's lymphomas

Two isoforms encoding the full-length transmembrane death receptor 3 (DR3) were isolated from mRNAs of a panel of human cell lines and tumor tissues obtained from patients with follicular non-Hodgkin's lymphoma. A new DR3 variant (DR3 beta) was characterized by 2 insertions of respectively 20- and 7-base pairs (bp) which result in a predictive translated polypeptide differing from the described DR3 molecule by a 28 amino-acid stretch in the extracellular domain. DR3 was shown to be expressed in all cell lines and lymphoma samples tested, whereas DR3 beta expression was restricted to lymphoid T-cell and immature B-cell lines and to selected cases with follicular lymphoma. These data provide new insight into the molecular heterogeneity of DR3, suggesting the presence of several receptor isoforms that can participate in lymphoid cell homeostasis.     

Changes in apparent in vivo binding of [3H]raclopride and [3H]N-methylspiperone induced by oxotremorine

The effects of muscarinic agonist, oxotremorine (0.3 mg/kg), and antagonist, scopolamine (0.5 mg/kg), on in vivo [3H]raclopride (RAC) and [3H]N-methylspiperone (NMSP) binding were investigated. Following tracer administration to control or pretreated mice, binding potentials, and the rate constants k3 and k4 were determined by kinetic analysis. Oxotremorine resulted in a 70% increase in striatal RAC binding potential compared with controls. RAC and NMSP showed almost identical decreases in k3 (40%), whereas k4 for RAC was unexpectedly decreased by 64%. Scopolamine resulted in no significant changes in RAC or NMSP binding. These results, in combination with previous data obtained in reserpinized mice, show that 1) competition by endogenous ligand may not be the only factor influencing the magnitude of apparent in vivo receptor binding, and 2) interneuronal communication may be partly mediated by changes in the rates of ligand-receptor binding.     

Age-related changes of sodium-dependent D-[3H]aspartate and [3H]FK506 binding in rat brain

We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependent D-[3H]aspartate and [3H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependent D-[3H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [3H]FK506 binding was significantly lower in only the hippocampus, thalamus and cerebellum. 3[H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependent D-[3H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependent D-[3H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10-25% reduction in sodium-dependent D-[3H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

Comparison of thymidine and folinic acid protection from methotrexate toxicity in human lymphoid cell lines

Hypoxanthine, thymidine, and folinic acid protection from methotrexate cytotoxicity were compared in human lymphoid cell lines variably sensitive to thymidine-induced growth suppression. Hypoxanthine protection differed among the cell lines, and the dose-response relationship for protection occurred within the physiologic bone marrow hypoxanthine concentration range. The slopes of the thymidine protection curves were virtually superimposable in a B cell line versus a T cell line, but threefold to fourfold higher thymidine concentrations were required to maximally protect the B versus the T cell line. Thymidine (plus hypoxanthine) protection was superior to that of folinic acid in both the B and the T cell lines when protection was delayed. As the interval between methotrexate and folinic acid exposure was progressively delayed, there was a linear decline in the degree of maximum protection in both cell lines. However, as thymidine exposure was progressively delayed, maximal protection was maintained, except at 12 hours in the B cell line. The influence of methotrexate on thymidine-induced growth suppression was studied. Methotrexate enhanced thymidine-induced growth suppression in sensitive cells as manifested by a significant shift of the dose-response curve. Deoxycytidine protection from thymidine toxicity was superior in the presence of methotrexate. The results of these studies indicate that thymidine protection and folinic acid protection against methotrexate toxicity produce different effects, which in part are dependent on the cell type. The complexity of these interactions points out the need for further studies.     

Fucosyltransferase activities in human pancreatic tissue: comparative study between cancer tissues and established tumoral cell lines

This review focuses on polymerase chain reaction (PCR) sexing of bovine embryos in commercial situations with emphasis on new developments. Simplifications of the biopsy technique is one of the major simplifications over the last few years. The stabilization of the embryo by means of protein-free medium or scratches produced on the bottom of the Petri dish makes it possible to perform a biopsy with a single microinstrument. The traditional PCR sexing approach utilizes electrophoresis, which involves the risk of deoxyribonucleic acid (DNA) contamination of subsequent assays. Such contamination, resulting in females misdiagnosed as males, is avoided efficiently by using a non-electrophoretic method in which the sex is determined based on fluorescence of unopened tubes. However, female samples cannot be distinguished from blank samples in the non-electrophoretic assay, which thus relies on accurate transfer of biopsy into tubes. Nevertheless, an accuracy of about 95% can be reached with both approaches. High pregnancy rates (50-70%) can be reached with biopsied Grade 1 embryos, but there is evidence that pregnancy rates with Grade 2 embryos is 15-20% lower. Recent data indicate that pregnancy rates of 50% can be achieved with frozen-thawed biopsied Grade 1 embryos. In conclusion, recent developments in biopsy techniques, detection systems and freezing should increase interest in PCR sexing.