Liquid chromatographic determination of caffeine in biologic fluids

A high-performance liquid chromatographic procedure was developed for the determination of caffeine in various biologic fluids and coffee. A reversed-phase column and UV detection at 254 nm were used to obtain a sensitivity of 0.1 μg/ml caffeine in serum and saliva using a sample volume of 0.1 ml. Caffeine metabolites and commonly ingested xanthines do not interfere with the assay. The within-day coefficients of variation were 9.8 and 9.9% at plasma caffeine concentrations of 2 and 10 μg/ml, respectively. The day-to-day coefficients of variation were 6.8 and 6.6% at plasma caffeine concentrations of 2 and 10 μg/ml, respectively. Serum and saliva caffeine concentrations were determined following a single oral dose of coffee and an intravenous infusion of caffeine in one subject. Computer estimates of caffeine pharmacokinetic parameters in one subject are in excellent agreement with previously published values.     

Murine transfer factor. I. Description of the model and evidence for specificity

A model for studying transfer of delayed-type sensitivity to mice with cellfree materials is described. The results with a particulate antigen (Candida) and 4 soluble protein antigens (PPD, ferritin, cytochrome c, and horseradish peroxidase) suggest that the phenomenon is antigen specific. Identical preparations from the spleens of insensitive donors were not active. This murine model should facilitate characterization of the immunologic and chemical properties of transfer factor.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Role of collagen and fibronectin in neural crest cell adhesion and migration

Cultured neural crest cells which are freshly trypsinized require serum or purified fibronectin to attach to collagen substrates of types I–V. Furthermore, neural crest cells migrate in a Boyden chamber in response to fibronectin, and a “checkerboard” analysis demonstrates that fibronectin is both chemotactic and chemokinetic for these cells. It is proposed that collagen serves as a substrate for neural crest cells as they migrate in the early embryo. By mediating the cells' attachment to collagen, fibronectin may influence the movement of the cells. Local differences in fibronectin concentration or availability in the matrix could affect the degree of attachment of the cells to the collagen substrate and could also direct their migration by serving as a chemoattractant.     

Chemotaxis of Spirochaeta aurantia: involvement of membrane potential in chemosensory signal transduction.

The effects of valinomycin and nigericin on sugar chemotaxis in Spirochaeta aurantia were investigated by using a quantitative capillary assay, and the fluorescent cation, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide was used as a probe to study effects of chemoattractants on membrane potential. Addition of a chemoattractant, D-xylose, to cells in either potassium or sodium phosphate buffer resulted in a transient membrane depolarization. In the presence of valinomycin, the membrane potential of cells in potassium phosphate buffer was reduced, and the transient membrane depolarization that resulted from the addition of D-xylose was eliminated. Although there was no detectable effect of valinomycin on motility, D-xylose taxis of cells in potassium phosphate buffer was completely inhibited by valinomycin. In sodium phosphate buffer, valinomycin had little effect on membrane potential or D-xylose taxis. Nigericin is known to dissipate the transmembrane pH gradient of S. aurantia in potassium phosphate buffer. This compound did not dissipate the membrane potential or the transient membrane depolarization observed upon addition of D-xylose to cells in either potassium or sodium phosphate buffer. Nigericin did not inhibit D-xylose taxis in either potassium or sodium phosphate buffer. This study indicates that the membrane potential but not the transmembrane pH gradient of S. aurantia is somehow involved in chemosensory signal transduction.     

Field evaluation of female sex pheromone components of the cotton bollworm,Heliothis armigera

(Z)-11-hexadecenal and (Z)-9-hexadecenal were ineffective lures forH. armigera males unless combined. Attraction depended upon perception of a 90%–99% combination of (Z)-11-hexadecenal with 1%–10% (Z)-9-hexadecenal. Increasing the level of (Z)-9-hexadecenal in the mixture to 26.2% reduced catches. Adding 2.3% (Z)-7-hexadecenal to the mixture did not enhance or reduce attraction, while adding 8.7% (Z)-11-hexadecenol significantly reduced male catches. The combination of (Z)-11-hexadecenal and (Z)-9-hexadecenal was effective only when released from rubber dispensers but not from polyethylene vials. A load of 2 mg of the mixture on rubber dispensers effectively attracted males for at least 31 days. TheH. zea lure which contained all the pheromonal components of that species was also effective in attractingH. armigera males. TheH. virescens lure attracted significantly fewerH. armigera males than theH. zea lure.

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Résumé Les (Z)-11-hexadecenal et (Z)-9-hexadecenal sont des attractifs sexuels in efficaces pour les mâles deH. armigera. L'attraction dépend de la perception d'un mélange de 90 à 99% de (Z)-11-hexadecenal avec 1 à 10% (Z)-9-hexadecenal, L'augmentation jusqu'à 26,2% de la teneur en (Z)-9-hexadecenal réduit les captures. L'addition de 2,3% de (Z)-7-hexadecenal au mélange ne modifie pas l'attractivité, tandis que celle de 8,7% de (Z)-11-hexadecenol, réduit significativement les captures de mâles. Le mêlange de (Z)-11-hexadecenal et de (Z)-9-hexadecenal n'a été efficace qu'avec des diffuseurs en caoutchouc, par contre il a été sans effet à partir de récipients de polyéthylène. Une charge de 2 mg de mélange dans des diffuseurs en caoutchouc attire effectivement les mâles pendant ou moins 31 jours. L'attractif sexuel deH. zea qui contient tous les constituants de la phéromone de cette espèce attire aussi efficacement les mâles deH. armigera. Celui deH. virescens attire significativement moins de mâles deH. armigera que l'attractif sexuel deH. zea.

     

Specificity of adoptive chemoimmunotherapy of established syngeneic tumors

To examine the specificity of adoptive chemoimmunotherapy (ACIT) of established syngeneic tumors, two noncross-reactive C57BL/6 tumors were studied: a Friend virus-induced tumor (FBL-3) and a chemically induced virus-negative tumor EL-4(G-). In vitro studies confirmed that these tumors are antigenically distinct by demonstrating that the cytotoxic responses of spleen cells from mice immunized in vivo and reexposed to tumor in vitro are immunologically specific. Studies of ACIT with cells from mice immunized in vivo demonstrated similar specificity. Mice receiving 5 x 10(6) FBL-3 on day 0 all died by day 13. Treatment on day 5 with cyclophosphamide (CY), 180 mg/kg, prolonged the median survival time (MST) to day 23. Treatment on day 5 with CY plus 2 x 10(7) normal nonimmune C57BL/6 cells or CY plus cells sensitized to EL-4(G-) had no additional effect on survival whereas 2 x 10(7) C57BL/6 cells sensitized to FBL-3 in vivo prolonged MST to day 64 and cured 13 of 32 mice. Similarly, mice given 2 x 10(5) EL-4(G-) on day 0 all died by day 16, and CY on day 5 prolonged the MST to day 22. As an adjunct to CY, 2 X 10(7) normal cells or cells sensitized to FBL-3 had a modest effect, prolonging the MST to days 37 and 36, respectively. However, treatment with CY plus 2 x 10(7) cells immune to EL-4(G-) cured 22 of 32 mice. The results demonstrate the immunologic specificity of ACIT of syngeneic tumors treated with immune syngeneic cells.     

Fibronectin delays the fusion of L6 myoblasts

The effect of fibronectin on myogenesis has been studied in vitro. The addition of purified fibronectin to the myogenic cell line L₆ blocks fusion and causes an increase in cell number. The effects of fibronectin could be prevented by the immunoprecipitation of fibronectin from solutions using affinity-purified antifibronectin antibodies. Mild trypsinization of the cells (10 μ/ml trypsin for 20 min) which removes surface fibronectin, causes the rate of fusion to increase when the trypsinization is done just before the cells begin to fuse, day 4 (after the plating of the cells), an inhibition when done on one day earlier, day 3, and has no effect when done after the cells have begun to fuse, day 5. By measuring the binding of rhodamine-labeled antifibronectin antibodies to intact cells, it was found that surface fibronectin increased from day 3 to day 4 and then decreased on day 5. The stimulating effect of trypsin on fusion, therefore, corresponds to the day surface fibronectin reaches a peak. Affinity-purified antifibronectin antibodies were also shown to be capable of enhancing fusion. It is concluded from these results that high levels of fibronectin stimulate events which reduce fusion, whereas the removal of surface fibronectin during critical times either stimulates or reduces fusion.     

Neural crest cells: temperature-dependent transformation by Rous sarcoma virus.

Cranial neural crest cells from chick embryos, when cultured under appropriate conditions, differentiate after approx. 1 week into pigmented cells. Neurol crest cells were infested with a mutant (RSV-BH-Ta) of the Bryan 'high titer' strain of Rous sarcoma virus on the second day of culture before the cells were morphologically differentiated, or later after they became pigmented. Cells infected and maintained at the temperature permissive for transformation (37 degrees C) proliferated rapidly compared to uninfected cells are produced extensive cytoplasmic vacuoles in a fashion similar to other types of cells transformed with RSV-BH-Ta at 37 degrees C. Cells infected and maintained at the non-permissive temperature for transformation (41 degrees C) also proliferated rapidly but did not become morphologically transformed. Transformation occurred reversibly following a shift of temperature. Infection of morphologically undifferentiated neural crest cells at either temperature prevented their differentiation into pigment cells, and infection of pigmented neural crest cells at either temperature led to a gradual loss of pigmentation. These results suggest that even at the non-permissive temperature the virus may regulate the state of differentiation of certain types of cells.