Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures

A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about one-third those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried material fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.     

Hyperthermia Induces the Synthesis of a Heat Shock Protein by Polysomes Isolated from the Fetal and Neonatal Mammalian Brain

Abstract: Analysis of the cell-free translation products of polysomes isolated from fetal brain and other organs indicates that elevation of maternal body temperature induces the synthesis of a heat shock protein of molecular weight 74,000 (74K). The newborn mammal is particularly sensitive to induction of the 74K protein. As early postnatal development proceeds, higher body temperatures are required to induce synthesis of the 74K heat shock protein.     

Effects of iron deficiency and exercise on myoglobin in rats

The effects of iron deficiency and endurance training on muscle myoglobin (Mb), body weights, and blood lactic acid concentration were studied in rats. Fifty animals were divided into four groups: anemic trained (AT), normal trained (NT), anemic sedentary (AS), and normal sedentary (NS). Following 5 weeks of dietary control, the mean hemoglobin values for the AT and AS rats were 0.013 +/- 0.002 mmol X l-1 (8.7 +/- 1.4 g X dl-1) and 0.014 +/- 0.003 mmol X l-1 (9.2 +/- 1.7 g X dl-1) respectively, and did not significantly change throughout the study. AT and NT rats were run on a motor driven treadmill 4 days/week for 6 weeks up to a pre-established time of 90 min. Following the training, body weights of the AT (157 +/- 13 g) and NT (153 +/- 13 g) rats were lower than their respective sedentary groups AS (172 +/- 9 g) and NS (176 +/- 15 g). Resting blood lactic acid concentration following training was lower in both trained groups, AT (3.3 +/- 2.0 mM) and NT (2.3 +/- 1.9 mM) compared to AS (8.2 +/- 2.6 mM) and NS (3.8 +/- 1.6 mM). Training increased Mb concentration in hearts of both the anemic and normal trained groups (AT, 0.66 +/- 0.13 mg X g-1; NT, 0.95 +/- 0.08 mg X g-1) compared to the sedentary groups (AS, 0.44 +/- 0.08 mg X g-1; NS, 0.70 +/- 0.13 mg X g-1). Only the AT rats showed an increase in skeletal muscle Mb. This study provides evidence that myoglobin may limit aerobic metabolism.     

Characterization of sequential immune complexes in infective endocarditis by Western blot analysis

A patient with cutaneous vasculitis during infective endocarditis due to Lactobacillus casei was studied. Immune complexes (IC) were isolated from serum at the time of diagnosis and after 4 wk of therapy. Purification of IC used differential polyethylene glycol precipitation and competitive binding to staphylococcal protein A. In situ radioiodination of IC was performed, followed by SDS-polyacrylamide gel electrophoresis (PAGE). Anti-IC antisera were raised in rabbits by immunization with purified IC. IC were characterized by SDS-PAGE followed by electrophoretic transfer to nitrocellulose, incubation with antiserum and then with 125I protein A, and autoradiography. Although early and late IC differed quantitatively, there were no differentiating immunochemical features. Both IC contained a 60,000 dalton component that did not react with preimmune serum nor with anti-normal human serum. This component reacted with antiserum rendered specific for L. casei by affinity chromatography. The restricted antigen-antibody representation in IC contrasted with a wider panel of antibody activity in patient serum. The Western blot analysis proves to be an ideal method for the characterization of IC because of its sensitivity, dissociative capability, and preservation of immunoreactivity. IC isolated at a time removed from the original antigenic challenge may provide insight into the nature of the inciting antigen.     

The effects of date of planting on field establishment of serotinous cape Proteaceae

Season of fire have marked effects on the germination and establishment of serotinous shrubs of the family Proteaceae in fynbos vegetation. To investigate reasons for this, we simulated the effects of different fire seasons by planting seeds into cleared fynbos and then followed their progress. Four species of Proteaceae were planted monthly at four sites over two and a half years. Exclosures were used to exclude rodent seed predators. Germination was confined largely to the three winter months (June–Aug.). Seeds planted from January–June had higher germination than those planted in the second half of the year. Higher levels of regeneration noted after fires in the first half of the year, were previously hypothesised to be results of predation. However, we obtained similar results despite the exclusion of seed predators. Monthly minimum temperature was strongly correlated with germination percentage but monthly rainfall was not. Loss of seed viability may be important, in determining post-fire seedling densities. Differential seedling mortality of earlier and late germinants appears to be unimportant in determining establishment levels. Our results nevertheless support the current practice of restricting management fires in fynbos to the summer-autumn period.     

Recovery of the pyrrolic nucleus of pyrrolizidine alkaloid metabolites from sulphur conjugates in tissues and body fluids

Reactive pyrrolic metabolites formed when pyrrolizidine alkaloids are given to rats can alkylate soluble and tissue-bound thiol groups. Pyrrolic thioethers thus formed are relatively stable, and may persist in tissues for long periods. A simple procedure has been developed for recovering the nucleus of the pyrrolic metabolite from such S-binding, whether in solution or attached to solid tissues, in an easily identifiable form. The thioether bond was broken by silver nitrate and the pyrrolic moiety allowed to react with ethanol or methanol to form an ethoxy or methoxy derivative. The chemical basis of the procedure was established by model experiments on a preparative scale, but for small scale recovery from tissues, pyrrolic ethers were extracted and identified by TLC, HPLC, capillary GC and mass spectrometry. Because the pyrrolic derivatives thus formed were easily recognised and unrelated to any physiological compound, the recovery method described, especially when applied to blood samples, provided a way to monitor animals for previous exposure to toxic pyrrolizidine alkaloids.     

Ferritin in normal human peripheral blood T-lymphocyte subpopulations

Six peripheral blood lymphoid fractions (total lymphocytes, non-T, T, Tar (autologous rosette-forming T cells/precursor), T mu (helper), and T gamma (suppressor) lymphocytes) isolated through rosetting procedures were examined for the presence of ferritin by a direct immunofluorescence technique. Although ferritin was present in all lymphoid fractions studied, a significantly higher proportion of ferritin-containing cells were detected in the T-cell fraction than in the non-T-cell fraction, (mean +/- SD = 7.9 +/- 1.6% and 5.0 +/- 1.2%, respectively). T mu- and T gamma-cell fractions showed a twofold increase in the number of ferritin-positive cells (14.1 +/- 1.4% and 15.4 +/- 2.6%, respectively), as compared with Tar (7.0 +/- 0.9%)-and total lymphocyte (6.9 +/- 1.3%)-cell fractions. These results indicate that ferritin is preferentially distributed in T mu and T gamma lymphocytes and may constitute the basis for explaining some of the roles exercised by these cells in the control of other biological systems.     

The critical weed-free period in organically-grown winter wheat

Two experiments were conducted in central southern England between September 1994 and August 1996 to identify the critical weed-free period in organically grown winter wheat (Triticum aestivum, cv. Mercia). In competition with a mixed weed infestation of predominately Alopecurus myosuroides and Tripleurospermum inodorum it was found that wheat yield decreased as the duration of the weed-infested period increased and that the crop needed to be kept free of weeds from sowing in order to completely avoid any yield loss. Also, weeds emerging in the wheat crop (predominately T. inodorum) during the growing season had a significant and detrimental effect on yield. The existence of the critical period, therefore, depends on the imposition of an acceptable yield loss. If a 5% yield loss gives a marginal benefit compared with the cost of weed control, the critical period will begin at 506°C days after sowing (November) and end at 1023°C days after sowing (February). This information could be used by farmers to target mechanical weeding operations to control weeds at a time that will have maximum benefit to the crop.     

In vitro propagation of the medicinal plant Plumbago zeylanica L. Through nodal explants

Summary A protocol for rapid in vitro propagation using nodal explants obtained from 2-yr-old, field-grown medicinal plants of Plumbago zeylanica L. belonging to the family Plumbaginaceae is described. High frequency bud break and fast development of shoots were induced on Murashige and Skoog's basal medium supplemented with 27.2 μM adenine sulfate +2.46 μM indole-3-butyric acid (IBA). Induction of rooting was achieved by transferring the shoots to the same basal medium containing 4.92 μM IBA. Using our protocol from one twig of P. zeylanica (eight responsive nodes per explant shoot) within a period of 5 mo., eight plantlets could be raised. After a hardening period of 4 wk, there was a 90% transplantation success in the field compared to the 60–65% survival of plantlets recorded in the experiments of previous workers. The plantlets derived through in vitro propagation mimic the growth and morphological characteristics of the donor plants.