In Vitro Studies on the Immunological Memory for Antibody Response to Bovine Serum Albumin

Immunological memory for T and B cells was studied in an in vitro culture system with spleen cells from mice primed with bovine serum albumin (BSA). Spleen cells taken from mice immunized at various times previously with a single intravenous injection of alum-precipitated (AP) BSA and bacterial endotoxin (ET) were cultured in Marbrook's system with dinitrophenylated (DNP) BSA as the in vitro antigen. In the cultures of spleen cells obtained from mice primed more than 14 days previously, an IgG-predominant anti-BSA response was generated. However, no anti-BSA response was observed in the culture of spleen cells taken from mice primed 7 days previously (day 7 spleen cells). The failure of day 7 spleen cells to generate an antibody response in vitro was shown to be attributable to both the lack of B memory cells and the effect of “suppressive” macrophages induced by ET. On the other hand, anti-BSA memory in the spleen of mice primed with AP-BSA plus ET and 2 months later challenged with AP-BSA matured within 7 days and declined rather quickly by 30 days after the challenge. The difference in the time course of the generation of memory between the spleen cells from primary and from secondary immunized mice might be attributable to the difference in the maturation of memory B cells, since the time course of the development of memory T cells after the secondary immunization was similar to that observed after primary immunization.     

Characterization of adenylate and guanylate cyclases in rat peritoneal mast cells

Adenylate and guanylate cyclase activities were confirmed in crude homogenates from rat peritoneal mast cells. Both enzyme activities were associated with the 105, 000 X g particulate fractions, but not detected in the supernatant fractions. The optimal pH for both cyclase activities was 8.2. Mn⁺⁺ was essentially required for guanylate cylcase activity, while adenylate cyclase activity was observed in the presence of either Mg⁺⁺ or Mn⁺⁺. The apparent Km values of adenylate cyclase for Mn⁺⁺-ATP and Mg⁺⁺-ATP were 160 μM and 340 μM, respectively, whereas the value of guanylate cyclase for Mn⁺⁺-GTP was 100 μM. Adenylate cyclase was activated by 10 mM NaF. However, both adenylate and guanylate cyclase activities were neither stimulated nor inhibited by the addition of various kinds of agents which stimulate or inhibit the release of histamine from mast cells.     

Quantitative detection of bisphenol A and bisphenol A diglycidyl ether metabolites in human plasma by liquid chromatography–electrospray mass spectrometry

Due to the ubiquity of epoxy resin compounds and their potential role in increasing the risk for reproductive dysfunction and cancer, the need for an assessment of human exposure is urgent. Therefore, we developed a method for measuring bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE) metabolites in human blood samples using high-performance liquid chromatography–electrospray ionization mass spectrometry (LC–MS). Human blood samples were processed using enzymatic deconjugation of the glucuronides followed by a novel sample preparation procedure using a solid-phase-cartridge column. This selective analytical method permits rapid detection of the metabolites, free BPA and a hydrolysis product of BADGE (BADGE-4OH) with detection limits in the low nanogram per milliliter range (0.1 ng ml⁻¹ of BPA and 0.5 ng ml⁻¹ of BADGE-4OH). The sample extraction was achieved by Oasis HLB column on gradient elution. The recoveries of BPA and BADGE-4OH added to human plasma samples were above 70.0% with a standard deviation of less than 5.0%. This selective, sensitive and accurate method will assist in elucidating potential associations between human exposure to epoxy-based compounds and adverse health effects.     

Beta-sheet folding of fragment (16-36) of bovine pancreatic trypsin inhibitor as predicted by Monte Carlo simulated annealing.

A tertiary structure prediction is described using Monte Carlo simulated annealing for the peptide fragment corresponding to residues 16-36 of bovine pancreatic trypsin inhibitor (BPTI). The simulation starts with randomly chosen initial conformations and is performed without imposing experimental constraints using energy functions given for generic interatomic interactions. Out of 20 simulation trials, seven conformations show a sheet-like structure--two strands connected by a turn--although this sheet-like structure is not as rigid as that observed in native BPTI. It is also shown that these conformations are mostly looped and exhibit a native-like right-handed twist. Unlike the case with the C-peptide of RNase A, no conspicuous alpha-helical structure is found in any of the final conformations obtained in the simulation. However, the lowest-energy conformation does not resemble exactly the native structure. This indicates that the rigid beta-sheet conformation of native BPTI merely corresponds to a local minimum of the energy function if the fragment with residues 16-36 is isolated from the native protein. A statistical analysis of all 20 final conformations suggests that the tendency for the peptide segments to form extended beta-strands is strong for those with residues 18-24, and moderate for those with residues 30-35. The segment of residues 25-29 does not tend to form any definite structure. In native BPTI, the former segments are involved in the beta-sheet and the latter in the turn. A folding scenario is also speculated from this analysis.     

Analgesic effects of dynorphin-A and morphine in mice

To investigate whether or not dynorphin-A is analgesic, the effect of this peptide was tested in comparison with that of morphine in mice. Dynorphin-A produced a potent analgesic effect in the acetic acid writhing and tail pinch tests, but a weak effect in the tail flick test when given by intracerebroventricular injection. In contrast, morphine caused a potent analgesia in all the tests. Dynorphin-A was more effective when given by intrathecal injection than by intracerebroventricular injection, whereas morphine was equipotent by both injection routes. The results suggest that dynorphin-A is analgesic and that its analgesia may be differentiated from that of morphine.     

Precise identification of gene products in hearts after in vivo gene transfection, using Sendai virus-coated proteoliposomes.

Both efficient gene transfer and the exact identification of gene product are required for gene therapy. Gene transfection of green fluorescence protein (GFP) might be useful for the reporter. After in vivo cotransfection of GFP and beta-galactosidase (beta-Gal) genes in Sendai virus-coated proteoliposomes to rat hearts, we compared the sensitivity and specificity of three methods: GFP detection, histochemical staining (HC) of beta-Gal activity, and immunostaining (IS) of the beta-Gal protein. Fluorescence microscopy and double staining of HC and IS revealed that both GFP and IS were equally sensitive and fourfold superior to HC at the peak of gene expression. However, different from skeletal muscle, the GFP of transfected cardiomyocytes showed two demerits: the fluorescence quenching due to the intense staining of beta-Gal activity, and nonspecific autofluorescence from myocardium. Thus, specific IS would be so far the most reliable to identify the gene product in heart.     

Diurnal Rhythm and Characteristics of Photosynthesis and Respiration in the Leaf and Root of a Phalaenopsis Plant

A hybrid Phalaenopsis plant was grown hydroponically on a nutrientsolution of Hyponex in the greenhouse. Typical daily patternsof CO₂ fixation, production of malic acid and citric acid andpH of the leaf and root were determined. The rate of primaryfixation of CO₂ in the leaf increased markedly in the evening,remained high until well into the afternoon, then decreasedsharply. The pattern of production of organic acids resultedfrom the fact that the rate of uptake of CO₂ gas was highestat night and fell during the day. A high correlation betweenpH value and level of malic acid in the leaves was observed,and an exponential relationship appeared to exist between theseparameters. These rhythmic sequences were not observed in theroot. The results suggest that metabolism in the leaf had characteristicsof CAM (Crassulacean Acid Metabolism), while the root did not. (Received May 6, 1988; Accepted October 25, 1988)     

Metabolic responses of chicken embryos to graded, prolonged alterations in ambient temperature

1. Chicken embryos aged 12, 16, 18 and 20 (externally pipped) days of incubation were exposed to graded reductions (2 degrees C) in ambient temperature from 38 to 28 degrees C, exposure to each temperature lasting up to 9 hr. 2. Oxygen uptake was measured first at 38 degrees C and then in the quasi-equilibrium state at lowered temperatures. The temperature coefficient (Q10) was calculated for each egg. 3. For mild cooling (32 degrees C), the Q10 in 18-day-old embryos was about 1.5, while 12- and 16-day-old embryos had a Q10 value of about 2, indicating that a feeble homeothermic metabolic response to cooling appears in late prenatal embryos. It became more marked in externally pipped embryos and further augmented in hatchlings.