Endocytosis of cell-cell junctions and spontaneous cell disaggregation in a cultured human ovarian adenocarcinoma (COLO 316)

Although cultured COLO 316 human ovarian adenocarcinoma cells are joined by extensive tight junctions and numerous desmosomes in confluent monolayers, viable cells may be spontaneously released into the nutrient medium. Intracytoplasmic vesicles containing tight junctional and desmosomal elements were identified in freeze-fracture and thin section preparations of the released cells and some vesicles exhibited structural signs of degradation. Possible mechanisms for tight junctional and desmosomal interiorization and the possible relationship between junctional interiorization and certain malignant behaviors are discussed.     

Aspirin and exercise-induced asthma

In four subjects with exercise-induced asthma, aspirin and placebo were administered prior to exercise in a double blind study. Pulmonary function tests did not reveal any difference between the response after aspirin or placebo. We conclude that in these four subjects aspirin did not prevent the bronchoconstrictor response. This might suggest that prostaglandins have no significant role in exercise-induced asthma.     

Proton nmr spectroscopy of high-spin ferrous cytochrome P-450 models

Alkyl mercaptide complexes of both synthetic and natural-derivative iron(II) porphyrins have been characterized in DMSO solution by proton nmr spectroscopy. A single mercaptide ligand binds to form a high-spin iron(II) complex as determined by solution magnetic measurements and the nmr isotropic shift pattern. Ligand exchange is slow on the nmr time scale unlike corresponding 2-methyl imidazole exchange rates which are very rapid. Further comparison of mercaptide and 2-methyl imidazole adducts reveals a downfield bias in isotropic shift values for the mercaptide species, which may be explained by different signs in the dipolar shift term for the two complexes. This apparent magnetic anisotropy of the mercaptide complex is in the same direction, although smaller, than that observed for bacterial cytochrome P-450. Isotropic shift values of at least 250 ppm for methylene resonances of the coordinated mercaptide support a very efficient unpaired spin delocalization for this axial ligand.     

Lysosomal carboxypeptidase B from rabbit lung purification and characterization

Lysosomal carboxypeptidase B has been purified from rabbit lung acetone powder by acid precipitation and ammonium sulfate fractionation followed by further purification on Sephadex G-100, DEAE-Sephadex, Organomercurial-Sepharose, preparative isoelectric focusing, Sephadex G-75, and carboxymethyl-Sephadex. This procedure resulted in a homogeneous preparation as determined by polyacrylamide gel electrophoresis at pH 4.5, 8.3 and with sodium dodecyl sulfate. This enzyme has a molecular weight of 52,000, is composed of two subunits of approximately equivalent molecular weight, and is a glycoprotein with a carbohydrate content estimated to be 10% by weight. The amino acid composition is also reported. The enzyme is active on two synthetic substrates, α-N-benyoyl-l-arginineamide and hippuryl-l-arginine. With these two substrates, respectively, lysosomal carboxypeptidase B has pH optima of 5.7 and 5.0, temperature optima of 40 and 50 °C, and K_m values of 10 and 16 mm. With each substrate, the enzyme requires the presence of a reducing agent for maximal activity and is inhibited to the same extent with several inhibitors. The most potent inhibitors were leupeptin and antipain at low concentrations (1 μm). Iodoacetate and Ac-(Ala)₃-Ala-chloro-methyl ketone also inhibited at higher concentrations (10 μm). However, compounds such as leucyl-chloromethyl ketone, bestatin, pepstatin, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and α-1-antitrypsin did not inhibit. When tested with peptides as substrates, this proteinase exhibited strong carboxypeptidase activity on the tetrapeptide, ThrProArgLys, and on angiotensin I, AspArgValTyrIle HisProPheHisLeu, liberating Lys, and Leu, respectively. Substance P (containing 11 amino acids plus a C-terminal amide group) was virtually inactive as a substrate for this enzyme. However, with oxidized insulin B chain as substrate, lysosomal carboxypeptidase B exhibited significant carboxypeptidase and endopeptidase activities.     

An analysis of developmental timing in Dictyostelium discoideum

A new method has been developed to assess the minimum complexity and relationships of those pathways (developmental timers) which time the consecutive stages of a developing system (Soll, 1983). This method has been applied to the morphogenetic program of Dictyostelium discoideum and has resulted in (1) a minimum estimate of the number of components comprising the timers for the first seven stages of morphogenesis, (2) a characterization of the temperature sensitivities of these components including demonstration of a reversible timer component, (3) detailed temporal definition of a number of transition points between rate-limiting components including a major branch point for the onset of several independent timer components coincident with the onset of aggregation, and (4) a temporal model for the relationships between the timers of the seven consecutive morphogenetic stages, including several examples of parallel timers.     

The relationship of age,sex, and reproductive state to assimilation efficiency in the adult milkweed bug, Oncopeltus fasciatus

The influence of age, sex, and reproductive state on efficiency of assimilation was examined in the milkweed bug Oncopeltus fasciatus. Amounts of food ingested and excreted decline with age in virgin males and females. Mated females laying eggs ingest significantly more food than virgin animals of either sex and also maintain a consistently higher assimilation efficiency. A qualitative analysis of the level of assimilation required for reproductive effort is discussed.     

A new method for examining the complexity and relationships of “timers” in developing systems

Simple methods are developed for analyzing the rate-limiting pathways, or “developmental timers,” for consecutive stages in a developing system. Two conditions are first defined for short and long timing to a developmental stage. Shifts are then performed at time intervals from short to long and long to short conditions. The total time to the stage (time under first condition plus time under second condition) is scored and plotted as a function of the time of shift, resulting in two plots, one for shifts from the short to long condition, and the other for shifts from the long to short condition. Each plot is then analyzed for the number of components, slopes of components, absolute times of origins and termini of components, and discontinuities between components. This information is then used (1) to distinguish between single- and multiple-component timers, (2) to assess the sensitivity of each timer component to the change in the environmental condition employed in the method, including reversibility, (3) to test for the addition of a new timer component under long conditions, and (4) to test for an identity change of a timer component between short and long conditions. These interpretations in turn provide a minimum estimate of the complexity of the rate-limiting pathway to a developmental stage, temporally define major transition points between timer components, and provide some insight into the nature of timer components. By characterizing the rate-limiting pathway from the origin of a developmental program for each consecutive stage in that program, distinctions can also be made between single, parallel, sequential, and branching timer relationships. From these interpretations, a detailed temporal “map” of the rate-limiting program can be generated for any developmental system in which consecutive stages can be reproducibly monitored with time.     

Characterization of fragments produced by clostripain digestion of proteoglycans from the Swarm rat chondrosarcoma

Rat chondrosarcoma proteoglycan aggregate samples were digested with the protease clostripain (from Clostridium histolyticum) for various times. The progress of digestion was studied by Sepharose 2B chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After complete digestion, the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid was separated from the chondroitin sulfate-peptide clusters released by the enzyme. In this limit complex, the M_r of the link protein was 42,000, slightly smaller than the M_r of 45,000 observed for intact link protein. The chondroitin sulfate-peptides contained an average of about seven to eight polysaccharide chains per peptide and, after chondroitinase ABC digestion, were found to consist of two size classes of peptides. By comparison, chondroitin sulfate-peptides isolated from trypsin digests contained four to five chains per peptide and contained primarily the smaller size class of peptides. At early digestion times with clostripain, several distinct molecular weight intermediates containing hyaluronic acid-binding sites were identified on sodium dodecyl sulfate-polyacrylamide gels. These intermediates, with M_r, values of about 125,000, 100,000, and 85,000, decreased with increasing digestion time to yield a limit polypeptide (M_r = 67,000). Procedures are described for purifying this limit polypeptide and the link protein for further characterization. The results indicate that clostripain can be used to fragment proteoglycan molecules selectively to define different functional regions for study.     

Effect of polyamines on the electrokinetic properties of red blood cells.

At the physiological pH 7.4, the zeta potential of the normal red blood cell in 1.5% glycine buffer was found to be ?52 mv, whereas that of sickling erythrocytes is ?45 mv. Addition of spermidine to normal red blood cells reduced the zeta potential by approximately 20 mv. In sickling red blood cells, where the polyamine content is determined to be 5 to 6 times greater than in the normal erythrocyte, addition of spermidine reduced the zeta potential by only 5 mv, indicating that little more polyamine binding occurs. The polyamine content of whole blood taken from 24 patients having sickle cell anemia was found to be more than ten times that of whole blood from normal donors. Binding of polyamines to the normal red blood cell was analyzed from the surface charge potential variation as a function of polyamine concentration and the apparent binding constant determined to be 130 d1/g. The difference in the electrokinetic properties of normal and sickling red blood cells in this system may be attributed, in part, to a variation in the polyamine content of the two types of erythrocytes.     

Chemotherapy and Radiofrequency-Induced Mild Hyperthermia Combined Treatment of Orthotopic Pancreatic Ductal Adenocarcinoma Xenografts

Patients with pancreatic ductal adenocarcinomas (PDAC) have one of the poorest survival rates of all cancers. The main reason for this is related to the unique tumor stroma and poor vascularization of PDAC. As a consequence, chemotherapeutic drugs, such as nab-paclitaxel and gemcitabine, cannot efficiently penetrate into the tumor tissue. Non-invasive radiofrequency (RF) mild hyperthermia treatment was proposed as a synergistic therapy to enhance drug uptake into the tumor by increasing tumor vascular inflow and perfusion, thus, increasing the effect of chemotherapy. RF-induced hyperthermia is a safer and non-invasive technique of tumor heating compared to conventional contact heating procedures. In this study, we investigated the short- and long-term effects (~20 days and 65 days, respectively) of combination chemotherapy and RF hyperthermia in an orthotopic PDAC model in mice. The benefit of nab-paclitaxel and gemcitabine treatment was confirmed in mice; however, the effect of treatment was statistically insignificant in comparison to saline treated mice during long-term observation. The benefit of RF was minimal in the short-term and completely insignificant during long-term observation.     

Control of cell differentiation during proliferation. I. Monocytic differentiation of HL-60 promyelocytes

The cell proliferation relating an uncommitted precursor cell to a differentiated terminal cell has been quantitated. HL-60 promyelocytes, a bipotent precursor cell capable of differentiating along either the myeloid or monocytic pathway, were induced by a human lymphocyte-conditioned medium (CM) to differentiate into macrophage-like cells. The promyelocytes had a generation time of approx. 42 h. Most promyelocytes which differentiated became macrophage-like cells after only one cell division. Some, a minority, underwent more than one division. The time between induction of differentiation and expression of differentiated characteristics could thus be very short. Labelled S-phase promyelocytes could differentiate after traversing S. G2 and undergoing mitosis. Some, approx. 21%, required a subsequent complete cell cycle before differentiating. The data suggest a model in which cells must undergo a S-phase-specific differentiation control event in the presence of CM in order to differentiate in the subsequent G1 phase. This model proposes that a discrete time in S phase exists when cells are susceptible to exogenous regulation directing them to yield differentiated daughter cells.     

Antigen-induced in vitro lymphocyte blastogenesis in the rabbit: a T-cell response.

In vitro lymphocyte stimulation of sensitized rabbit lymphocytes to specific antigen (ovalbumin) was found to depend on thymic-dependent lymphocytes. This conclusion is based on an enhanced response upon enrichment with T lymphocytes by passage of lymphocytes through nylon wool, and on the elimination of the response after treatment of lymphocytes with complement and an antiserum to rabbit thymus cells prepared in a goat. Specificity of the antiserum was demonstrated by elimination of in vitro T-cell function and retention of in vitro B-cell functions.     

High affinity specific [3H] (+)PN 200-110 binding to dihydropyridine receptors associated with calcium channels in rat cerebral cortex and heart

The binding properties of the 1,4-dihydropyridine calcium channel antagonist, [³H](⁺)PN 200-110, were studied in rat cerebral cortical and cardiac homogenates (37°C, Krebs phosphate buffer). Specific binding of [³H](⁺)PN 200-110 was saturable, reversible, and of high affinity (K_d values are 35 and 64 pM for the cerebral cortex and heart, respectively). In parallel studies with [³H](⁺)PN 200-110, the dissociation constant of [³H]nitrendipine was 10–12 times higher. Substituted dihydropyridine calcium channel antagonists and agonists competitively inhibited specific [³H](⁺)PN 200-110 binding, but d-cis diltiazem enhanced and verapamil incompletely inhibited [³H](⁺)PN 200-110 binding in both the cerebral cortex and the heart. The effects of diltiazem and verapamil on [³H](⁺)PN 200-110 binding were due mainly to alterations in the dissociation constant (K_d), without alterations in the binding density (B_max). The new [³H](⁺)PN 200-110 receptor binding assay is remarkable for its low degree of nonspecific binding as compared to [³H]nitrendipine at physiological temperatures. [³H(⁺)PN 200-110 is a useful ligand for the further analysis of the dihydropyridine binding sites associated with calcium channels.     

Amplification of lymph node cell tube leukocyte adherence inhibition (LAI) reactivity by leukocyte adherence inhibition factor (LAIF)

This investigation examines the immunologic basis for specific antigen-induced tube leukocyte adherence inhibition (LAI) reactivity of draining lymph node cells (LNC) from dogs with canine transmissible venereal sarcoma (CTVS). CTVS regressor LNC, macrophage-depleted LNC, and enriched T lymphocyte fractions, but not enriched B lymphocyte fractions, were specifically reactive to CTVS antigen extract in direct tube LAI. In addition, regressor LNC amplified tube LAI responses by generating supernatants with leukocyte adherence inhibition factor (LAIF) activity for normal dog indicator LNC and enriched peripheral blood mononuclear cells (PBMC) in an indirect tube LAI assay. However, macrophage-depleted LNC and enriched T lymphocyte fractions failed to generate supernatants with LAIF activity, suggesting that macrophage accessory cells play a central role in the amplification of tube LAI. Interestingly, CTVS regressor peripheral blood leukocytes (PBL) and PBMC, which were specifically reactive in direct tube LAI, also failed to generate supernatants with LAIF activity. These findings demonstrate a distinction between LAIF-mediated amplification and direct tube LAI reactivity, and suggest that leukocyte populations with differing cellular proportions and from different immunologic compartments may participate in tube LAI via different mechanisms.     

The inhibitory actions of progesterone: effects on male and female sexual behavior of the hamster

A series of three experiments compared the inhibitory effects of progesterone on estrogen- or androgen-induced sexual behavior in male and female hamsters. In the first experiment chronic progesterone treatment was found to have no effect on male copulatory behavior maintained after castration with testosterone propionate or estradiol benzoate. However, testosterone propionate was more effective at maintaining male behavior than estradiol benzoate. In the second experiment progesterone was found to have a slight inhibitory effect on the rate of the restoration of the intromission response after androgen treatment in males which had been castrated for 8 weeks. In the final experiment, chronic progesterone treatment markedly inhibited sexual receptivity in male and female hamsters which had been given 4 weeks of androgen or estrogen treatment and a single pretest injection of progesterone. Thus, progesterone was shown to be a potent inhibitor of androgen- or estrogen-induced estrus in both male and female hamsters. Due to the large difference in effectiveness on these two behavioral systems, we suggest that progesterone affects steroid-induced male copulatory behavior and female receptivity by different mechanisms of action.