Assessment of in situ host immunity to syngeneic tumors utilizing the multicellular spheroid model

By the 1g sedimentation method using discontinuous gradients of Ficoll solution (concentrations of 6 to 14%), keyhole limpet hemocyanin (KLH)-primed spleen cells of C3H/He or DBA/2 mice were fractionated into 4 to 10 populations after IgG antibody-coated erythrocytes (EA gamma) rosetting and then treatment with anti-Thy-1.2 + complement (C). No significant difference was observed in the distribution of isotype specificities of surface immunoglobulins on B cells in each population thus fractionated, when determined by indirect immunofluorescence staining. The mixture of the 12 and 14% Ficoll fractions contained 95% of B cells bearing Fc receptor for IgG (FcR+ gamma) and 3.58% of antigen-binding cells (ABC) for KLH, while the 8% Ficoll fraction included 15% of FcR+ gamma B cells and 1.53% of ABC. Nevertheless, the FcR- gamma B-cell-enriched populations caused intensive plaque-forming cell (PFC) responses to dinitrophenol (DNP), whereas FcR+ gamma B-cell-enriched populations generated weak responses. Noteworthy is that 4 days preculture of a population containing 95% FcR+ gamma B cells resulted in the appearance of precursor activity which was ascertained by a further 4 days culture of these cells with antigen, DNP-dextran. These findings suggest that FcR gamma bearing B cells intrinsically possess precursor activity for IgM/IgG antibody-forming cells, but lose it transiently by binding immune complexes (IC). Moreover, the titer of a factor suppressing anti-DNP PFC responses (suppressive B-cell factor, SBF) was higher in the 24-hr culture supernatants of the FcR+ gamma B-cell-enriched fraction than of the FcR- gamma B-cell-enriched fraction, suggesting that SBF is produced by FcR+ gamma B cells themselves. Thus, IC seems to play an important role for the negative feedback regulation of antibody production by stimulating FcR gamma bearing B cells.     

Effects of divalent metal ions on the fluorescence and glucose-quenching of yeast hexokinase isozymes

Titrations of the quenching of the tryptophan fluorescence of yeast hexokinase isozymes P-I and P-II by Mg²⁺, Mn²⁺, Ca²⁺, Cd²⁺, and Zn²⁺ ions and by glucose in the presence of each of these ions (10mM) were performed at pH 5.5 and 6.5 at 20°C. At the higher pH there was a reversal of the type of glucose-binding cooperativity for P-II from negative to positive when either Mn²⁺ or Ca²⁺ was present in the buffered isozyme solution before the glucose titration, whereas Mg²⁺ caused the glucose binding to become noncooperative. Zn²⁺ and Cd²⁺ decreased the glucose quenching of P-II fluorescence drastically at pH 5.5, from a value of 15% in buffer to only 4%. Thus, only these two ions, of the five studied, cause the conformation change that results in quenching of the glucose-quenchable cleft tryptophan of P-II. Glucose binding to the P-I isozyme exhibited positive cooperativity in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺, as well as in buffer alone, at both pH's. At the lower pH, Ca²⁺ enhanced the efficiency of glucose quenching of P-I fluorescence several-fold, while Mn²⁺ increased it only about 40% and Mg²⁺ not at all. Further, Ca²⁺ raised the degree of cooperativity (Hill coefficient) of glucose binding to P-I at this pH from the value of 1.42 in buffer and in the presence of Mg²⁺ and Mn²⁺ to 1.94, i.e., almost up to the highest possible value, 2, for dimeric hexokinase. However, at pH 6.5 the Ca²⁺ effect on the cooperativity was negligible, while Mg²⁺ and Mn²⁺ decreased the coefficient from 1.6 in buffer to about 1.4. The biological implications of these diverse metal ion effects are discussed.     

Plasma dihydroxyphenylglycol (DHPG) in the in vivo assessment of human neuronal norepinephrine metabolism

We investigated the utility of deaminated norepinephrine (NE) metabolites in the study of human sympathetic nervous pathophysiology. Plasma levels of the NE metabolite dihydroxyphenylglycol (DHPG) appear to be related to intraneuronal NE stores. Plasma DHPG increases when sympathetic nervous activity or circulating NE increase and decreases when neuronal NE is depleted or neuronal NE reuptake is blocked. Changes in plasma dihydroxymandelic acid (DOMA) related less closely to changes in plasma NE. The coupling of measurements of plasma NE with its deaminated metabolites and DHPG may improve understanding of human NE metabolism and neuronal NE reuptake.     

NFATc1 Expression as a Prognosticator in Urothelial Carcinoma of the Upper Urinary Tract

We recently found that NFATc1, a member of the NFAT family and a key regulator of the immune response, could induce bladder carcinogenesis and cancer progression. In this study, we immunohistochemically stained for NFATc1 in upper urinary tract urothelial carcinoma (UUTUC) specimens and paired nonneoplastic urothelial tissues. NFATc1 was positive in 51 [52%; 40 (40%) weak (1+), 9 (9%) moderate (2+), and 2 (2%) strong (3+)] of 99 UUTUCs, which was significantly higher than in benign urothelium [30 (36%) of 83; 28 (34%) weak and 2 (2%) moderate] (0 vs 1+/2+/3+, P = .038; 0/1+ vs 2+/3+, P = .023). There were no significant associations between NFATc1 expression pattern and tumor grade or pT stage. However, the positive rates of NFATc1 expression tended to be higher in renal pelvic tumors (60%) than in ureteral tumors (42%; P = .080) as well as in pN+ tumors (75%) than in pN0 tumors (49%; P = .089). Kaplan-Meier and log-rank tests revealed that moderate (2+) to strong (3+) NFATc1 expression correlated with lower progression-free survival (P = .032) and cancer-specific survival (P = .005) rates in the 99 cases. Patients with high (2+/3+) NFATc1 muscle-invasive tumor (n = 9) also had a significantly higher risk of cancer-specific mortality (P = .021) compared to those with low (0/1+) NFATc1 muscle-invasive tumor (n = 53). Thus, compared with nonneoplastic urothelium, a significant increase in the expression of NFATc1 in UUTUC was seen, implying the involvement of NFATc1 signals in the development of UUTUC. The current results further suggest that NFATc1 overexpression serves as a predictor of poor prognosis in patients with UUTUC.     

Lipopolysaccharide interferes with the staining of lipoprotein on polyacrylamide gels

After electrophoresis of total membrane preparations of Escherichia coli B on sodium dodecyl sulfate polyacrylamide gels, and subsequent staining with Coomassie Brilliant blue, a band corresponding to the Braun lipoprotein fails to appear. This is in contrasr to similar preparations of E. coli K-12 which do display the lipoprotein uponm staining. Experiments described below indicate that failure to observe this protein in E. coli B is due to interference in the staining reaction by the lipopolysaccharide present in the membrane preparations.     

Behavior of lactating rats in a dual-chambered maternity cage

Videotape sampling of behavior in a dual-chambered apparatus indicates that the continuous monitoring of the amount of time a mother spends in the cage with its litter can be taken as a valid reflection of maternal behavior. Nursing is the principal behavior of lactating females while in the compartment with their litters; lying still, consummatory behavior, and activity occur with greater frequency in the cage away from the litter. Both the time that mothers spend with their litters and nursing behavior displayed a 24-hr rhythm with crest values occurring during the period of light.     

Cytotoxicity of human peripheral blood monocytes

Native tumoricidal activity of human peripheral blood mononuclear cells was examined before and after their separation by counterflow centrifugation elutriation (CCE). Tumoricidal activity was found in the subpopulation of small mononuclear cells but not within the relatively pure subpopulation of large monocytes. Addition of lymphokine and/or lipopolysaccharide demonstrated that large monocytes were resistant to activation for tumor killing, in contrast to small mononuclear cells. However, cryopreservation or simply exposure to dimethyl sulfoxide (DMSO) rendered the large monocytes sensitive to activating agents without altering their unstimulated tumoricidal activity. Cryopreservation was not detrimental to small or large monocytes either in number or tumoricidal function but did decrease the number of large granular lymphocytes (LGL). The small mononuclear cell fraction was enriched for small monocytes to 80% by combining CCE with Percoll gradient separation. HNK-1 mouse monoclonal antibody against human LGL was used with complement to remove virtually all LGL from cryopreserved cells as judged by morphology and tumoricidal activity against K-562 human lymphoblastoid cells. Such treatment actually augmented rather than suppressed tumoricidal activity against P-815 mastocytoma cells. Therefore, we conclude that small monocytes but not large monocytes possess native tumoricidal activity distinct from that attributed to LGL or natural killer lymphocytes. Further, small monocytes are readily activated for tumor killing and can be cryopreserved without loss of tumoricidal activity.     

Sodium gradient-stimulated transport of L-carnitine into renal brush border membrane vesicles: kinetics, specificity, and regulation by dietary carnitine

L-Carnitine transport by rat renal brush border membrane vesicles was stimulated by a Na+ gradient (extravesicular greater than intravesicular). Total carnitine entry was 2.7 and 3.2 times higher at 15 S in the presence of a 100 mM NaCl gradient than when the vesicles were incubated isoosmotically in buffered 100 mM KCl or buffered mannitol, respectively. Specific carnitine transport (total entry minus contribution from diffusion) was stimulated 3.6- and 5.7-fold, respectively. An "overshoot" was observed for total carnitine entry in the presence of a Na+ gradient but not in the presence of a K+ gradient or in the absence of an ion gradient. L-Carnitine transport was saturable. KT and Vmax for total carnitine transport were 0.11 mM and 11.6 pmol S-1 mg protein-1, respectively, and for Na+-gradient-dependent carnitine transport, 0.055 mM and 5.09 pmol S-1 mg protein-1, respectively. The transport process was structure-specific for a quaternary nitrogen and carboxyl groups attached by a 4- to 6-carbon chain, but without other charged functional groups. Other evidence for a carrier-mediated process included trans-stimulation of transport by intravesicular carnitine and a peak of activity at near physiological temperature. Kinetic data derived from this study, coupled with data from previous physiological studies from this laboratory, suggests that carnitine transport by the brush border membrane is not limiting for carnitine reabsorption. Dietary carnitine (1% of diet for 10 days) reduced by 52% the rate of carnitine transport across the brush border membrane in vitro, without affecting rates of D-glucose, L-lysine, L-glutamic acid, or L-alanine transport. Down-regulation of carnitine transport may prevent excessive or toxic accumulation of L-carnitine in renal tubular cells exposed to high extracellular carnitine concentrations.     

The calibration and use of a calcium ion-specific electrode for kinetic studies of mitochondrial calcium transport.

A calcium ion-specific electrode has been used to study calcium transport by isolated,hepatic mitochondria. The methodology used requires only a sensitive pH meter operated in the millivolt mode with the electrode. Free calcium ion concentrations may be followed continuously. Using incubation conditions which cause release of intramitochondrial calcium, the calcium electrode system may also be used to determine total. intramitochondrial calcium. Techniques for the calibration of the electrode response are discussed. Free calcium ion concentrations have been calculated from total calcium concentrations and the association constants for the binding species present in the assay medium. The observation that the electrode response is linear to submicromolar concentrations allows calculation of a linear least-squares fit of millivolt reading to computed free calcium ion concentration. A computer program written in BASIC for these computations is included in Appendix material. The half-maximal rate constant for mitochondrial calcium uptake has been found to occur at a free calcium ion concentration of 6.5 μm. The interaction or Hill coefficient for the process is 2.3, indicating positive cooperativity.     

Purification and properties of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

Blue crab muscle (Callinectes danae) glycogen phosphorylase a was purified by adsorption of a crude extract on a starch column, elution with a dilute glycogen solution, selective precipitation with ammonium sulfate, dialysis against a solution containing ammonium sulfate and ethylenediaminetetraacetate, followed by centrifugation and chromatography on Sephadex G-25 (sp act 64.5 IU, recovery of 53.8%, and a purification factor of 189). The lyophilized preparation is stable for several months. Disc electrophoresis of the purified phosphorylase yields two protein bands, both with enzymatic activity of the a form. One of the protein bands represents about 10% of the total amount of protein present in the two bands. The molecular weight of the enzyme is 176,000 as determined by ultracentrifugation in a sucrose density gradient and 180,000 as determined by discontinuous polyacrylamide gel electrophoresis. The molecular weight found by disc electrophoresis corresponds to the main protein band. Crab muscle phosphorylase a is not associated under electrophoretic conditions in which rabbit muscle phosphorylase a shows association behavior. Subunit studies by continuous SDS-gel electrophoresis suggest that crab muscle phosphorylase a possesses only one subunit. Pyridoxal-5′-phosphate is a cofactor of the enzyme.     

Deoxyribokinase from Salmonella typhimurium. Purification and properties

Deoxyribokinase, which catalyzes the ATP-dependent phosphorylation of 2-deoxy-d-ribose to 2-deoxy-d-ribose-5-P as the first step in the inducible fermentation pathway for this sugar in Salmonella typhimurium, was purified approximately 600-fold from deoxyribose-grown cells. Apparent K_m′s for 2-deoxy-d-ribose and ATP were 0.1 and 0.5 mm, respectively. The enzyme had an absolute requirement for divalent cations which was best satisfied by Mg²⁺. Optimal activity was obtained in the presence of 0.5 m NH₄⁺ or Cs⁺. Rb⁺ and K⁺ also stimulated enzyme activity whereas Na⁺ and Li⁺ inhibited. d-Ribose and 2-deoxy-d-ribitol could replace 2-deoxy-d-ribose as phosphoryl acceptor, and several ribo- and deoxyribonucleotides could replace ATP as phosphoryl donor. Molecular weight determinations gave values of 67,800 for the native enzyme and 33,500 for the dissociated enzyme, suggesting the probable existence of two subunits of similar size.     

Immunoregulation in disseminated histoplasmosis: Characterization of splenic suppressor cell populations

Potent immunosuppressor cell activity was induced during the course of disseminated histoplasmosis in C3H/Anf mice. Spleen cells from infected mice severely suppressed the primary antibody response in vitro of normal syngeneic spleen cells to both a T-dependent antigen (sheep red blood cells) and a T-independent antigen (trinitrophenyl-lipopolysaccharide) at Weeks 1 and 3 of infection, respectively. Likewise, marked suppressor cell activity was present within lymph nodes. In a kinetic study, suppressor activity was detected first on Day 2 and increased to the maximum level on Day 4 after inoculation of Histoplasma capsulatum. Two populations of spleen cells express suppressor function in this model. One population, identified as T cells, was nonadherent to nylon wool columns; its suppressor capacity was abolished by anti-Thy 1 and reduced greatly by low-dosage X-irradiation (500 R). Cells of the second suppressor population had macrophage-like properties; although poorly adherent to plastic surfaces, they adhered to nylon wool columns and could be removed from spleen cell suspensions by carbonyl iron treatment; high-dosage X-irradiation (3000 R) and mitomycin C treatment failed to abrogate suppression by these cells.     

Characterization of cell membrane and sarcoplasmic reticulum from bovine uterine smooth muscle

Subcellular fractions, enriched in sarcoplasmic reticulum or in cell membrane, were separated from one another. Starting material was a microsomal pellet (15–40 × 1000g) obtained by differential centrifugation from the uteri of close-to-term pregnant cows. A microsomal fraction enriched in ATP-dependent calcium accumulation was shown to contain sarcoplasmic reticulum and cell membrane. Only 8% or less of the protein in this fraction could be recovered, using affinity chromatography on Sepharose 6MB wheat germ agglutinin. The small yields did not allow extensive characterization. A method was developed to separate sarcoplasmic reticulum from cell membrane using discontinuous sucrose density gradient centrifugation. Protein was collected at the 24–28, the 28–33, and the 33–45% sucrose interfaces. Characterization was by enzyme assays and by specific receptor assay. ATP-dependent calcium accumulation was fourfold greater in the 24–28% sucrose layer than in the 33–45% layer. In contrast, 5′-nucleotidase was more than threefold as high in the 33–45% sucrose layer as in the 24–28% layer. Ouabain-inhibited p-nitrophenylphosphatase doubled and ouabain-inhibited Na,K-ATPase tripled in the 28–33% layer, compared with the 24–28% layer, specific ouabain binding was also doubled in the 28–33% sucrose layer. ¹²⁵I-Labeled wheat germ agglutinin binding was greatest in the 33–45% sucrose layer. It is concluded that the 24–28% layer consists primarily of sarcoplasmic reticulum, whereas the 28–33 and the 33–45% layers are concentrated in the cell membrane. Specific prostaglandin (PGE₂) binding was found to be a property of the cell membrane.     

Synergistic functions of phorbol ester and calcium in serotonin release from human platelets

In human platelets, thrombin activates Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and mobilizes Ca2+ concomitantly, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA) may be intercalated into membranes and directly activates protein kinase C without mobilization of Ca2+ in sufficient quantities. A series of experiments with TPA and Ca2+-ionophore (A23187) indicates that activation of protein kinase C is a prerequisite requirement for release of serotonin, and that this enzyme activation and Ca2+ mobilization act synergistically to elicit a full cellular response. Both cyclic AMP and cyclic GMP inhibit activation of protein kinase C by prohibiting the signal-dependent breakdown of inositol phospholipid to produce diacyl-glycerol, but none of these cyclic nucleotides prevents the TPA-induced activation of this enzyme.     

Oxidative phosphorylation by membrane vesicles from Bacillus alcalophilus

ADP and P_i-loaded membrane vesicles from l-malate-grown Bacillus alcalophilus synthesized ATP upon energization with $\text{ascorbate}\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenylenediamine}$. ATP synthesis occurred over a range of external pH from 6.0 to 11.0, under conditions in which the total protonmotive force was as low as ?30 mV. The phosphate potentials (ΔG_p) were calculated to be 11 and 12 kcal/mol at pH 10.5 and 9.0, respectively, whereas the values in vesicles at these two pH values were quite different (?40 ± 20 mV at pH 10.5 and ?125 ± 20 mV at pH 9.0). ATP synthesis was inhibited by KCN, gramicidin, and by N,N′-dicyclohexylcarbodiimide. Inward translocation of protons, concomitant with ATP synthesis, was demonstrated using direct pH monitoring and fluorescence methods. No dependence upon the presence of Na⁺ or K⁺ was found. Thus, ATP synthesis in B. alcalophilus appears to involve a proton-translocating ATPase which functions at low .     

Use of preformed cell aggregates and layers to measure tissue-specific differences in intercellular adhesion

The binding of aggregates formed from various 7-day chick embryo tissues to cultured cell layers was analyzed 24 hr following trypsin dissociation of the tissues. The proprotion of aggregates binding is independent of the number of aggregates added, and changes with time over 60 min in a manner consistent with a first-order process. The adhesive parameter measured, the percentage of aggregates binding to cell layers per unit time, varies slightly with aggregate size but is not dependent upon the probability of collision of the aggregate with the layer. The rate of binding and the effect of modifiers of binding (temperature, inhibitors of oxidative metabolism and glutaraldehyde) are substantially different for neural retina interactions than for liver or heart interactions, suggesting that retina cells may form intercellular bonds via a mechanism distinct from that of liver or heart cells. The rate of binding between like tissue types is, with one exception, greater than between unlike types. Glutaraldehyde treatment of only one of the reactants abolishes this adhesive specificity. Aggregate binding provides a means of quantitatively assessing intercellular adhesion which has the advantage of reducing the effects of trypsinization on measurements of adhesion, and therefore lends itself to the investigation of cellular consequences of adhesion.     

Initiation of the activation potential by an increase in intracellular calcium in eggs of the frog,Rana pipiens

The membrane potential of the frog egg undergoes a transient positive shift at fertilization which is a block to polyspermy. This paper addresses the question of how a sperm elicits this “fertilization potential.” Iontophoretic injection of Ca²⁺ activates Rana pipiens eggs to develop and initiates a transient, positive-going shift in the membrane potential (the activation potential) which is like the sperm-induced fertilization potential in amplitude, duration, and Cl^? dependence. Activation potentials are elicited by Ca² injection into both animal and vegetal regions of the egg, but the rate of the initial depolarization is much less when Ca²⁺ is injected into the vegetal region. Injections of K⁺, Na⁺, Cl^?, or Mg²⁺ do not result in activation potentials, but the Ca²⁺ analogs, Sr²⁺ and Ba²⁺, can substitute for Ca²⁺. Treatment of eggs with the divalent cation ionophore, A23187, also initiates a transient, positive-going depolarization. Because injection of Ca²⁺ is sufficient to elicit a response almost identical to a fertilization potential, the ion transport mechanisms necessary to produce a fertilization potential must preexist in the unfertilized eggs; the sperm contributes only the stimulus to activate these mechanisms. The results reported here suggest that the stimulus may be a rise in free Ca²⁺.     

Concurrent inhibition of the low-affinity Ca2+-stimulated ATPase and MgATP-dependent endocytosis in erythrocyte ghosts by N-naphthylmaleimide and carbonylcyanide-m-chlorophenylhydrazone.

Energy-dependent endocytosis and the low Ca²⁺ affinity Ca²⁺-stimulated ATPase activity of erythrocyte ghosts were inhibited concurrently by two inhibitors, carbonylcyanide-m-chlorophenylhydrazone (CCCP) and N-naphthylmaleimide. The conditions required to observe 50% inhibition of this Ca²⁺-stimulated ATPase activity with either inhibitor were the same conditions required to observe this level of inhibition of endocytosis. Under these conditions, none of the other ATPase activities measured were inhibited more than 20% by either of these reagents. This concurrence of inhibition of endocytosis and the low-affinity Ca²⁺-stimulated ATPase and the possible involvement of this ATPase in the mechanism by which endocytosis occurs is discussed.