Serial circulating immune complex values and development of metastatic disease in breast cancer and malignant melanoma patients

Summary A polyethylene glycol precipitation technique was used to determine the levels of circulating immune complexes (CIC) in breast cancer and melanoma patients. All patients in the study had undergone surgery and were free of distant metastatic disease. CIC were measured at two to four time intervals, of 3 to 6 months each, over an average follow-up period of 13.5 months (range 7–20 months). In both groups of patients, metastatic disease developed with a higher frequency in patients who had undetectable CIC levels throughout the follow-up period or had become negative at the time metastases were discovered.     

Stability of tissue culture medium pH as a function of autoclaving,time, and cultured plant material

Autoclaving is a standard procedure for sterilizing nutrient media for plant tissue cultures. Most tissue cultures are grown at pH 5.2 to 5.8 with pH adjustments being made prior to autoclaving. This paper reports that there are significant differences between initial pH levels and pH levels following autoclaving, particularly in the pH range of 5.7 to 8.5. This effect is noted with and without agar. In addition, we report that with time the pH of the medium drifts into the acid range. When Cucumis callus was added to the medium, the pH was changed significantly within 48 hours. The amount and direction (increase or decrease of pH) was significantly correlated with the original pH. This suggests that researchers should be wary of the true pH situation in their medium. In addition, in publications authors should specify whether their medium pH value was determined before or after autoclaving.     

The amount and nature of glutathione transferases in rat liver microsomes determined by immunochemical methods

The amount and nature of glutathione transferases in rat liver microsomes were determined using immunological techniques. It was shown that cytosolic glutathione transferase subunits A plus C, and B plus L were present at levels of 2.4 ± 0.6 and 1.5 ± 0.1 μg/mg microsomal protein, respectively. These levels are 10-times higher than those for non-specific binding of cytosolic components judging from the distribution of lactate dehydrogenase, a cytosolic marker. The possibility that a portion of these glutathione transferases is functionally localized on the endoplasmic reticulum is discussed. A previously described microsomal glutathione transferase which is distinct from the cytosolic enzymes is present in an amount of 31 ± 6 μg/mg microsomal protein.     

Glucocorticoid-Induced Cleft Palate in the Mouse: Two Major Histocompatibility Complex, H-2, Loci with Different Mechanisms

Isolated cleft palate is induced in the progeny of pregnant mice that are given glucocorticoids. The incidence varies among inbred strains and with dose and stage of gestation when the drug is given. One chromosomal region responsible for strain-associated differences in sensitivity is the major histocompatibility complex, H-2. H-2^a is associated with susceptibility, H-2^b with resistance. There appear to be both maternal and embryonic genetic factors affecting the sensitivity to glucocorticoids. In experiments reported here congenic strains of mice with H-2^a, H-2^d and H-2^k haplotypes on a C57BL/10 genomic background were used. This allowed the determination of the effect on sensitivity by two H-2 subregions; the subregions are H-2K to I-E and I-C to H-2D. Methods included dose-response analysis and reciprocal cross analysis using dexamethasone given on day 12 of pregnancy. Results show that each subregion affects the strain's sensitivity to dexamethasone-induced cleft palate. The regression coefficients for B10.A-H-2^a (45.4 ± 4.13) were different from those for B10.BR-H-2^k (67.2 ± 10.8) and B10.D2-H-2^d (70.5 ± 9.74). The estimated mean arcsine% cleft palate at 160 mg/kg was different for each strain: B10.A- H-2^a, 53.1 ± 2.19; B10.BR-H-2^k, 33.1 ± 2.27; B10.D2-H-2^d, 25.0 ± 2.75. Different patterns of change in sensitivity were observed among the reciprocal crosses. In summary, the H-2K to I-E subregion seemed to influence both maternal and embryonic factors, whereas only embryonic factors were influenced by the I-C to H-2D subregion. These data suggest that the mechanisms affecting glucocorticoid sensitivity which are genetically encoded within each H-2 subregion are different, and there is an interaction between the alleles. The mode of interaction can be either complementation or epistasis.     

Ganglioside-Induced Neuritogenesis: Verification That Gangliosides Are the Active Agents, and Comparison of Molecular Species

Abstract: Gangliosides were previously reported to induce neuritogenesis in primary neuronal cultures and in some neurally derived cell lines. Because isolated gangliosides usually contain variable quantities of peptides, we investigated the possibility the neurite-stimulating activity could be caused by these contaminants. Ganglioside preparations from bovine brain and other sources were subjected to a three-step purification procedure that eliminated at least 95% of the contaminating peptides. These purfied preparations retained their capacity to induce extensive neurite growth in neuro-2A murine neuroblastoma. Proteolytic digestion and a number of additional procedures were used to reduce residual contamination further without loss of activity. Several crude ganglioside samples had negative effects on neurite development until freed of theri inhibitory factors, which were derived from the tissue and/or introduced during laboratory operations. This was particularly evident for bovine white matter gangliosides whose activity increased in proportion to peptide removal. When carefully purified, virtually all of 11 different gangliosides tested were highly active, with the possible exception of GM4, which demonstrated only moderate activity in a limited number of tests. All of the neutral glycolipids tested, as well as sulfatides and free sialic acid, were inactive.     

Reciprocal regulation of fructose 1,6-bisphosphatase and phosphofructokinase by fructose 2,6-bisphosphate in swine kidney

The effect of fructose 2,6-P₂, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P₂ inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P₂ was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P₂ restored 50% of the activity of PFK. Fructose 2,6-P₂ also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P₂, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P₂ inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P₂ during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P₂ is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.     

Epinephrine: A Potential Neurotransmitter in Retina

Abstract: Dopamine (DA), norepinephrine (NE), and epinephrine (EPI) are present in rat retina. DA is the major catecholamine, whereas NE and EPI represent ∼5% of the DA content. DA is contained in a subpopulation of amacrine cells and has been the subject of numerous studies. We investigated the origin and properties of NE and EPI in retina. Following superior cervical ganglionectomy, there was a decrease in NE content, but no decrease in EPI or phenylethanolamine- N -methyltransferase (PNMT) activity. PNMT in retina has many of the substrate-specificity and inhibitor-sensitivity characteristics of other tissues. Enzyme activity is enhanced in newborn rats by treatment with dexamethasone. Exposure to a lighted environment increases retinal EPI in normal and superior cervical ganglionectomized rats. EPI content increased for more than 2 h in a lighted environment. We conclude that most of the NE is contained within the sympathetic neurons that innervate the eye from the superior cervical ganglion, whereas EPI is contained in retinal elements that are responsive to photic stimulation.     

Bile acid uptake and calcium flux in brush border membrane vesicles

Our laboratory has recently reported that intestinal bile acid malabsorption in cystic fibrosis (CF) is a primary mucosal cell defect. Others have suggested that elevated intracellular Ca⁺⁺ levels in other cell types in CF may represent a common primary dysfunction in Ca⁺⁺ efflux in these cells. We examined the possibility that intestinal bile acid absorption and Ca⁺⁺ efflux in mucosal cells may be linked physiologically. Brush border membrane vesicles (BBMV) prepared from guinea pig ileum served as the experimental model to test this hypothesis. Ca⁺⁺ (2.5×10^?3M) present in the incubation medium did not alter the uptake of taurocholic acid (TCA) by BBMV. Also, TCA uptake into BBMV preloaded with Ca⁺⁺ was not significantly different from that in BBMV not previously loaded with Ca⁺⁺. Furthermore, with TCA present in the incubation medium, Ca⁺⁺ efflux from preloaded BBMV was not altered. These data suggest that ileal TCA uptake, as measured by BBMV, is not dependent upon either intra- or extravesicular Ca⁺⁺. Also, Ca⁺⁺ efflux from BBMV is unaffected by TCA uptake. Although separate lines of evidence suggest that intestinal bile acid malabsorption and reduced plasma membrane Ca⁺⁺ flux are primary defects in CF, we conclude that in the normal intestine these functions are independent physiological processes.     

Assimilation of 13NH 4 + by Anthoceros grown with and without symbiotic Nostoc

The pathways of assimilation of ammonium by pure cultures of symbiont-free Anthoceros punctatus L. and the reconstituted Anthoceros-Nostoc symbiotic association were determined from time-course (5–300 s) and inhibitor experiments using ¹³NH ₄ ⁺ . The major product of assimilation after all incubation times was glutamine, whether the tissues were cultured with excess ammonium or no combined nitrogen. The ¹³N in glutamine was predominantly in the amide-nitrogen position. Formation of glutamine and glutamate by Anthoceros-Nostoc was strongly inhibited by either 1mM methionine sulfoximine (MSX) or 1 mM exogenous ammonium. These data are consistent with the assimilation of ¹³NH ₄ ⁺ and formation of glutamate by the glutamine synthetase (EC 6.3.1.2)-glutamate synthase (EC 1.4.7.1) pathway in dinitrogen-grown Anthoceros-Nostoc. However, in symbiont-free Anthoceros, grown with 2.5 mM ammonium, formation of glutamine, but not glutamate, was decreased by either MSX or exogenous ammonium. These results indicate that during short incubation times ammonium is assimilated in nitrogenreplete Anthoceros by the activities of both glutamine synthetase and glutamate dehydrogenase (EC 1.4.1.2). In-vitro activities of glutamine synthetase were similar in nitrogen-replete Anthoceros and Anthoceros-Nostoc, indicating that the differences in the routes of glutamate formation were not based upon regulation of synthesis of the initial enzyme of the glutamine synthetase-glutamate synthase pathway. When symbiont-free Anthoceros was cultured for 2 d in the absence of combined nitrogen, total ¹³NH ₄ ⁺ assimilation, and glutamine and glutamate formation in the presence of inhibitors, were similar to dinitrogen-grown Anthoceros-Nostoc. The routes of immediate (within 2 min) glutamate formation and ammonium assimilation in Anthoceros were apparently determined by the intracellular levels of ammonium; at low levels the glutamine synthetase-glutamate synthase pathway was predominant, while at high levels independent activities of both glutamine synthetase and glutamate dehydrogenase were expressed.