A sensitive and specific radiometric method for the measurement of plasma histamine in normal individuals

A radioenzymatic method for assaying histamine using histamine-N-methyltransferase from guinea pig brain was modified to increase its sensitivity, precision, and specificity. This was achieved by incorporation of a thin-layer chromatography step and use of N_α-methyl histamine as an internal standard in each sample. No prior extraction of the plasma samples is required, permitting the assay of 30 samples in 1 working day. Histamine was detected in the plasma of 17 normal volunteers, at a level of 3.4 ± 0.69 nmol/liter (range 0.82 to 4.7 nmol/liter). In 19 asthmatics, a low-peak expiratory flow rate was found to be associated with a plasma histamine concentration above this “normal” range.     

A silver carbonate method for cell counts of neurons and glial elements on paraffin embedded brain tissue.

A modification of the Del Rio-Hortega method for the demonstration of central nervous system elements is presented. This silver impregnation technique is particularly useful for the classification of cell types for quantitative differential cell counts. Formalin fixed paraffin sections are immersed in formol-ammonium bromide for 1 1/2 hours; this solution is an excellent mordant for various silver nitrate stains. The samples are stained for 20 to 60 minutes in a silver carbonate solution (25 ml of 25% silver nitrate combined with 200 ml of 5% sodium carbonate) and then reduced in a 1% formaldehyde solution to which 20 drops of acetic acid have been added. Finally, the slides are fixed in sodium thiosulfate, rinsed in tap water, dehydrated, cleared, and mounted. This procedure will enable this investigator to identify neurons, oligodendroglia, and astrocytes on the basis of their nuclear staining as well as to demonstrate the laminae of brain tissue since the method allows differentiation of cell layers and fiber tracts.     

The visual pigment and visual cycle of the lobster,Homarus

Summary The visual pigment of the American lobster,Homarus americanus, has been studied in individual isolated rhabdoms by microspectrophotometry. Lobster rhodopsin has _max at 515 nm and is converted by light to a stable metarhodopsin with _max at 490 nm. These figures are in good agreement with corresponding values obtained by Wald and Hubbard (1957) in digitonin extracts. Photoregeneration of rhodopsin to metarhodopsin is also observed. The absorbance spectrum of lobster metarhodopsin is invariant with pH in the range 5.4–9, indicating that even after isomerization of the chromophore fromcis totrans, the binding site of the chromophore remains sequestered from the solvent environment. Total axial density of the lobster rhabdom to unpolarized light is about 0.7.As described for several other Crustacea, aldehyde fixation renders the metarhodopsin susceptible to photobleaching, a process that is faster at alkaline than at neutral or acid pH. Small amounts of a photoproduct with _max at 370 nm are occasionally seen. A slower dark bleaching of lobster rhabdoms (_1/2–2 h) also occurs, frequently through intermediates with absorption similar to metarhodopsin.The molar extinction coefficient of metarhodopsin is about 1.2 times greater than that of rhodopsin, each measured at their respective _max. Isomerization of the chromophore fromcis totrans is accompanied by a change in the orientation of the absorption vector of about 3°. The absorption vector of metarhodopsin is either tilted more steeply into the membrane or is less tightly oriented with respect to the microvillar axes.When living lobsters are kept at room temperature, light adaptation does not result in an accumulation of metarhodopsin. At 4 °C, however, the same adapting lights cause a reduction of rhodopsin and an increase in metarhodopsin. There is thus a temperature-sensitive regeneration mechanism that supplements photoregeneration. Following 1 ms, 0.1 joule xenon flashes that convert about 70% of the rhodopsin to metarhodopsin in vivo, dark regeneration occurs in the living eye with half-times of about 25 and 55 min at 22 °C and 15 °C respectively.This work was supported by USPHS research grant EY 00222 to Yale University. S.N.B. was aided by NIH Postdoctoral Fellowship EY 52378.     

Dark adaptation,sensitivity, and rhodopsin level in the eye of the lobster,Homarus

Summary Dark adaptation of living lobsters was measured by recording the ERG at several temperatures in the range 5–20 °C following adapting flashes that convert about 70% of the rhodopsin to metarhodopsin. Recovery of log threshold is rapid, and at 10–20° is nearly complete in 10 min. Only at 5 °C is dark adaptation significantly slowed. Comparison of dark adaptation with data on regeneration of pigment (Bruno et al., 1977) is consistent with the hypothesis that as rhodopsin concentration rises and falls, its only effect on sensitivity is to alter the probability of quantum catch. This interpretation is further bolstered by observations on winter lobsters that have a 70% deficiency of rhodopsin without the concomitant increase in metarhodopsin that accompanies light adaptation. No effect of metarhodopsin on sensitivity was detected. These experiments support the growing body of evidence indicating that the relationship between rhodopsin concentration and log threshold is fundamentally different in the rhabdomeric photoreceptors of invertebrates and the rods and cones of vertebrates.This work was supported by USPHS research grant EY 00222 to Yale University. S.N.B. was aided by NIH Postdoctoral Fellowship EY 52378, by funds made available through the Unidel Foundation, and by a grant from the University of Delaware Research Foundation.     

Adrenergic desensitization in leukocytes of normal and asthmatic subjects.

Cyclic AMP was measured in leukocytes of normal and asthmatic subjects before and after one week of treatment with equal amounts of ephedrine. During the control and placebo periods, the measurements of cyclic AMP in leukocytes of asthmatic subjects were similar to those of normal individuals. After one week of treatment with ephedrine, both groups exhibited suppression of the leukocyte cyclic AMP response to adrenergic stimulation in vitro: however, the suppression of response was significantly greater in asthmatic subjects (p less than .u1). Subcutaneous administration of epinephrine was followed by further suppression of the leukocyte cyclic AMP response to in vitro stimulation which was similar in both groups during all treatment periods. The results indicate that in vivo exposure to adrenergic medications is followed by desensitization of the leukocyte responses to subsequent adrenergic stimulation in vitro. After administration of small doses of medication, the severity and/or duration of desensitization is significantly greater in asthmatic leukocytes.     

Some observations in tetraclita squamosa rufotincta Pilsbry

Aspects of the biology of Tetraclita squamosa rufotincta Pilsbry were investigated. Although a tropical species, on the shores at Elat, Israel, its breeding is virtually restricted to the colder winter months. The causal factors are considered and the results of some experimental work relative to temperature control given.For a cirripede, T.s. rufotincta has a remarkably large ‘egg’ — unusual in a tropical or even warm-water species. The egg size is compared with that of other species and the survival value of large size considered; excess nutrients are carried over from the embryo to at least the first planktonic larva, and may aid survival in a relatively nutrient poor environment.The animals do not become sexually mature until the second year after their settlement. Growth takes place largély in the colder winter months and is seasonally separated from the period when gonadal tissue is developed.     

Inhibition of sugar uptake in adenosine-treated 3T3 cells

Addition of 5 to 250 micromolar adenosine to the culture medium resulted in a 30–80% inhibition of the rate of uptake of 2-deoxyglucose or 3–0-methylglucose by sparse or confluent 3T3 cells within three hours. The inhibition of deoxyglucose uptake could be reversed partially by changing the cells to medium without adenosine for two hours and could be prevented completely by the addition of persantin, an inhibitor of nucleoside uptake. The adenosine effect is not due to inhibition of pyrimidine synthesis, since it is not prevented by uridine. It is not seen in 3T6 cells lacking adenosine kinase. The inhibition could be observed on confluent cells whose deoxyglucose uptake was stimulated by insulin, epidermal growth factor (EGF), calf serum or calcium phosphate. Although the percentage stimulation over control by these factors varied, the percentage inhibition by addition of adenosine of the stimulated rates, as well as the unstimulated rate, was relatively constant. EGF, insulin and calcium phosphate caused little or no stimulation of deoxyglucose uptake by sparse cells, whether adenosine treated or untreated. The results suggest that adenosine acts intracellularly after phosphorylation to regulate sugar uptake through a mechanism which is independent of the regulation by hormones and cell density.     

Growth and sporulation of Beauveria bassiana and Metarrhizium anisopliae on media containing various amino acids

Natural isolates of two entomogenous fungi, Beauveria bassiana and Metarrhizium anisopliae, were cultured in liquid culture media containing 24 amino acids and KNO₃ to determine their effect on growth and sporulation. In addition, the growth and medium pH changes for each isolate grown on an asparagine-containing medium were compared. Tryptophan and alanine were most effective for growth and sporulation of B. bassiana, although glutamine and KNO₃ also produced large numbers of regularly shaped spores. Tryptophan, glutamic acid, and histidine were all well utilized for both growth and sporulation of M. anisopliae. Nitrogen sources containing sulfur were poorly utilized for sporulation by M. anisopliae. In general, B. bassiana produces greater mycelial mass and much larger numbers of spores than M. anisopliae. Both fungi attained nearly the same growth maximum on asparagine medium though B. bassiana exhibited an initially more rapid growth rate. In both fungi this rapid growth phase was accompanied by a decline in medium pH followed by a rise in pH during the decline phase of growth.     

Associations of like and unlike polysaccharides: Mechanism and specificity in galactomannans,interacting bacterial polysaccharides,and related systems

Chiroptical, rheological, and n.m.r.-relaxation evidence is presented, to identify interactions of two types between different polysaccharides: (1) mutual exclusion of incompatible molecules, with consequent increase in the effective concentration of both; and (2) energetically favourable association of structurally and sterically regular chain-segments. β-1,4-linked plant polysaccharides interact by association of unsubstituted backbone regions, either with like chians, or with sterically compatible, unlike molecules. Extracellular polysaccharides (xanthans) of Xanthomonas plant pathogens maintain their ordered native conformation in solution, and this accounts for their industrially valuable, rheological peculiarities. These materials bind strongly to the plant glycans. Random-coil bacterial gums show no such interactions, although dextran enhances autogelation of galactomannans by exclusion. Extracellular polysaccharides from Arthrobacter species also have ordered native conformations in solution, but do not share the specific interactions of xanthan. Native xanthan shows marked specificity in its interactions with plant glycans, indicating a possible biological role in host-pathogen recognition.