An Improved Diluent for Rubella Hemagglutination and Hemagglutination-Inhibition Tests

Rubella hemagglutinating (HA) antigen was prepared in BHK-21 tissue as 5% cell suspensions and from unconcentrated and 20× concentrated infected supernatant fluids. In some instances, unconcentrated fluids were treated with Tween 80 and ether; cell suspensions were treated with ether alone. Preparations were tested for HA activity in dextrose-gelatin-Veronal (DGV) buffer solutions; 0.85% NaCl; Sorenson's phosphate-buffered saline, pH 7.2; and a diluent of 0.9% NaCl, 0.1% CaCl₂ (anhydrous), and 0.1% MgSO₄·7H₂O. HA titers were consistently two- to fourfold higher in the saline with added Ca⁺⁺ and Mg⁺⁺ than in DGV. Hemagglutination-inhibition titers of paired human sera were the same in either diluent. It is suggested that the interaction between rubella HA antigen and the red cells of young (less than 1-day-old) chicks may be at least partially ion dependent and that titers are enhanced by increased quantities of divalent cations.     

Influence of cell culture conditions on aromatase activity in human genital skin fibroblasts

Summary Because the measurement of aromatase activity in cultured human genital skin fibroblasts has been proposed as a means of studying estrogen production in men, we investigated the influence of culture conditions on aromatase activity. Genital skin fibroblasts were seeded onto culture plates at a density of 1×10⁶ cells/plate and aromatase activity was determined over a 1-mo. period. Enzyme activity rose slowly over the first 14 d but then rose rapidly to a 10-fold higher plateau by Day 28. The rise in aromatase activity was similar whether activity was normalized for protein or for DNA content. When cells were seeded at the usual density of 1×10⁶ or at 0.25×10⁶ cells/plate, aromatase activity was consistently lower during the first 2 wk in cells plated at lower density, but thereafter the levels of enzyme activity in the two groups converged. In cells plated at the lower density, the lower activity observed in the first 2 wk was associated with a lower V _max . Preincubation of cells plated at one density with conditoned medium from cells plated at the other density did not change the relatve levels of activity in the two groups. By contrast, dihydrotestosterone (DHT) receptor binding and 5α-reductase activity were similar at all time points, despite differences in plating density. In additional experiments, the culture medium was replaced daily rather than every 3rd d, and aromatase activity was assayed on Day 7. In cells fed daily, DNA and protein content were twice that of cells fed every 3rd d. By contrast, aromatase activity declined to 30% of the in the latter group. DHT and dexamethasone receptor binding and 5α-reductase activity were similar in the two groups. In summary, factors such as plating density, culture density, and frequency of media replacement dramatically influence aromatase activity in cultured human genital skin fibroblasts. Therefore, the interpretation of aromatase activity data obtained from cultured cells in relation to physiologic or pathologic states should be viewed with appropriate caution. The work was supported in part by grants R01 DK 35339 and R01 DK 00180 from the National Institutes of Health, Bethesda, MD, and by RR 00035 from CLINFO Systems at the Johns Hopkins University School of Medicine, Baltimore, MD.     

In vitro shoot regeneration of Populus deltoides: effect of cytokinin and genotype

Treatment differences were observed in the in vitro adventitious shoot regeneration response from internodal explants of three genotypes of Populus deltoides cultured on media supplemented with five concentrations each of the cytokinins 6-benzyladenine, 2-isopentyladenine, and zeatin. For each of the three genotypes, the greatest number of shoots were consistently regenerated on media containing the cytokinin zeatin. Tissue necrosis resulted when explants from any of the three genotypes were cultured on media supplemented with 6-benzyladenine. A zeatin concentration by genotype interaction was also observed. Genotypic differences in shoot regeneration were observed for 16 genotypes of Populus deltoides when cultured on medium supplemented with 0.5 mgL^–1 zeatin. Six genotypes were highly recalcitrant and failed to regenerate shoots. The percent of explants regenerating was greater than 50% for four genotypes.Abbreviations WNA modified Woody Plant Media - BA N⁶-benzyladenine - 2-iP 2-isopentenyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAR photosynthetically active radiation Journal Series No. 8938, Agricultural Research Division, University of Nebraska     

Characterization of a Nerve Growth Factor-Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule-Associated Protein 2 and pp250

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.     

Transport of Tryptophan into Brain from the Circulating, Albumin-Bound Pool in Rats and in Rabbits

Tryptophan is the only amino acid in the circulation that is bound by albumin, and previous studies have suggested that the brain tryptophan supply is a function of either the free or the albumin-bound pool of tryptophan in blood. Since the albumin molecule per se does not cross the brain capillary wall, i.e., the blood-brain barrier (BBB), the transport of tryptophan from the circulating albumin-bound pool may involve enhanced dissociation of tryptophan from the albumin binding sites within the cerebral microcirculation. This hypothesis was confirmed in the present studies wherein the dissociation constant (KaD) of albumin binding of tryptophan in the rat or rabbit brain microcirculation was measured in vivo. Brain extraction data for [14C]tryptophan determined with the carotid artery injection technique were fit to the Kety-Renkin-Crone equation modified for protein-bound solute. The KaD of albumin binding in the rat or rabbit brain microcirculation under pentobarbital anesthesia was 1.7 +/- 0.1 and 3.9 +/- 1.0 mM, respectively, as compared to the KD value measured in vitro with equilibrium dialysis, 0.13 +/- 0.03 mM. In contrast, the KaD value of albumin binding of tryptophan in vivo in the rabbit brain microcirculation was reduced by ether anesthesia to a value of 2.1 +/- 0.4 mM. This reduction in the KaD under ether anesthesia was associated with a 2.5-fold increase in cerebral blood flow. In addition, dialyzed rabbit serum caused a statistically significant inhibition in [14C]tryptophan influx during ether, but not pentobarbital, anesthesia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serological response of non-human primates to human melanoma disialoganglioside GD3

Summary The immunogenicity of the disialoganglioside, GD3, a melanoma-tumor-associated antigen, has been evaluated in non-human primates. Sera from four chimpanzees and two monkeys were evaluated for anti-GD3 antibody activity by solid-phase radioimmunoassay using GD3 and control gangliosides as targets. Serum from one monkey, immunized with cells from a melanoma cell line, was strongly reactive with GD3, having a titer of >2500. In contrast, serum from this animal was non-reactive with several other gangliosides including the structurally similar GM3. Anti-GD3 reactivity was also demonstrable, albeit in low titer, in the sera of an additional monkey and a chimpanzee. Each of these animals had likewise been immunized using cells from melanoma cell lines. On the basis of these observations, suggestive of a primate anti-GD3 antibody response, we initiated a series of immunizations of chimpanzee using purified GD3 bound to Salmonella minnesota, R595. IgG reactive with melanoma cells in the cell-binding assay was first detected in sera collected after 4 immunizations and increased in titer against each reactive melanoma cell line during the immunizations. Reactivity of this serum with melanoma cell lines demonstrated a direct correlation with the expression of GD3 by the respective cell line. Anti-GD3 reactivity was evident in solid-phase radioimmunoassay against purified GD3 beginning with serum collected after 11 immunizations. By comparison with its binding to the control ganglioside panel, this serum demonstrated strong specificity for GD3 (titer=640) while having only marginal reactivity with GM3 (titer=40). Immune serum from this animal was also able specifically to block subsequent binding of a murine IgM anti-GD3 antibody (DMab7) to target GD3 in solid-phase radioimmunoassay. Together, these observations suggest that GD3, in the form of a purified molecule bound to a bacterial matrix or as part of the intact melanoma cell membrane, can be immunogenic in non-human primates, and is able to elicit an antibody response of appropriate specificity.Supported in part by grant CA32672 from the National Cancer Institute, Veterans Administration Program 821 and by the Yerkes Regional Primate Center, Atlanta, Georgia. The Yerkes Center is fully accredited by the American Association for Accreditation of Laboratory Animal Care     

Regionally selective alterations in enzymatic activities and metabolic fluxes during thiamin deficiency

To further elucidate the molecular basis of the selective damage to various brain regions by thiamin deficiency, changes in enzymatic activities were compared to carbohydrate flux through various pathways from vulnerable (mammillary bodies and inferior colliculi) and nonvulnerable (cochlear nuclei) regions after 11 or 14 days of pyrithiamin-induced thiamin deficiency. After 11 days,large decreases (–43 to –59%) in transketolase (TK) occurred in all 3 regions; 2-ketoglutarate dehydrogenase (KGDHC) declined (–45%), but only in mammillary bodies; pyruvate dehydrogenase (PDHC) was unaffected. By day 14, TK remained reduced by 58%–66%; KGDHC was now reduced in all regions (–48 to –55%); PDHC was also reduced (–32%), but only in the mammillary bodies. Thus, the enzyme changes did not parallel the pathological vulnerability of these regions to thiamin deficiency.¹⁴CO₂ production from¹⁴C-glucose labeled in various positions was utilized to assess metabolic flux. After 14 days, CO₂ production in the vulnerable regions declined severely (–46 to 70%) and approximately twice as much as those in the cochlear nucleus. Also by day 14, the ratio of enzymatic activity to metabolic flux increased as much as 56% in the vulnerable regions, but decreased 18 to 30% in the cochlear nuclei. These differences reflect a greater decrease in flux than enzyme activities in the two vulnerable regions. Thus, selective cellular responses to thiamin deficiency can be demonstrated ex vivo, and these changes can be directly related to alterations in metabolic flux. Since they cannot be related to enzymatic alterations in the three regions, factors other than decreases in the activity of these TPP-dependent enzymes must underlie selective vulnerability in this model of thiamin deficiency.Abbreviations KGDHC 2-ketoglutarate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.61, EC 1.6.4.3. - PDHC pyruvate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.12, EC 1.6.4.3 - TK transketolase (EC 2.2.1.1) - TPP thiamin pyrophosphate     

K+-evoked taurine efflux from cerebellar astrocytes: On the roles of Ca2+ and Na+

The ionic requirements for K⁺-evoked efflux of endogenous taurine from primary cerebellar astrocyte cultures were studied. The Ca²⁺ ionophore A23187 evoked taurine efflux in a dose-dependent fashion with a time-course identical to that of K⁺-induced efflux. The Ca²⁺-channel antagonist nifedipine had no effect upon efflux induced by 10 or 50 mM K⁺. In addition, verapamil did not antagonize 50 mM K⁺-evoked efflux except at high, non-pharmacological concentrations (>100 M), and preincubation with 2 M -conotoxin had no effect on 50 mM K⁺-evoked efflux. Similarly, preincubation with 1 mM ouabain had no effect on the amount of taurine released by K⁺ stimulation, but did accelerate the onset of efflux by 2–4 min. Although 2 M tetrodotoxin had no effect on K⁺-evoked release, replacing Na⁺ with choline abolished the taurine efflux seen in response to K⁺ stimulation. Together, these findings suggest that neuronal N- and L-type Ca²⁺- and voltage-dependent Na⁺-channels are not involved in the influx of Ca²⁺ which appears to be necessary for K⁺-evoked taurine efflux, and that in addition to Ca²⁺, extracellular Na⁺ is also required.     

Different long-term effects of bilateral and unilateral nucleus basalis lesions on rat cerebral cortical neurotransmitter content

Young adult rats received either unilateral or bilateral ibotenic acid infusions in their nucleus basalis, destroying most of the cholinesterase-staining neurons in that region. Cerebral cortex levels of choline acetyltransferase, somatostatin, neuropeptide Y, and monoamines were then assayed 2.5 and 10 months after bilateral lesions, or, 2.5, 10, and 14 months after unilateral lesions. Entorhinal and cerebral cortex levels of several amino acid transmitters were also measured. As expected, choline acetyltransferase activity was decreased in the frontal cortex ipsilateral to the ibotenic acid infusion in unilaterally or bilaterally lesioned animals. Parietal cortex concentrations of somatostatin and neuropeptide Y were altered by lesioning in a complicated, time-dependent manner. Thus, while unilateral lesions transiently decreased or had no effect on these neuropeptide levels, bilateral lesions elevated the level of each neuropeptide by over 100% at 10 months. Other cortical transmitter systems investigated appeared to be less affected by nucleus basalis-lesions. Unilateral lesions had no effect on prefrontal cortex norepinephrine, serotonin, or dopamine content at 14 months post-lesioning. These different neurochemical effects of unilateral and bilateral nucleus basalis lesions may be important for developing a model for the trans-synaptic effects of cortical cholinergic deafferentation.