A PCR method for typing B-LβII family (class II MHC) alleles in broiler chickens

Certain haplotypes of the major histocompatibility (B) complex are strongly associated with resistance or susceptibility to several infectious diseases in Leghorn chickens. Identification of chicken haplotypes based on the nucleotide sequence of B complex loci could provide more precise identification of haplotypes than traditional serological methods. We report the development and application of polymerase chain reaction with sequence specific primers (PCR-SSP) to type broiler chicken B haplotypes based on the DNA sequence of B-L beta II family genes. Five well-defined standard B haplotypes from White Leghorns and 12 recently characterized B haplotypes from a broiler breeder line were used to develop the test system. The B-L beta II family loci were amplified from genomic DNA by B-L beta II family specific primers and then characterized by PCR-SSP. In total, ten pairs of primers, derived from the sequences of expressed B-L beta II family alleles, were used in the PCR typing test to discriminate the chicken B haplotypes identified previously by serological means. The PCR-SSP showed that each haplotype had a different amplification pattern, except those haplotypes known or suspected to have the same B-L beta alleles. Cloning and sequencing of the family specific PCR products indicated that two loci in the B-L beta II family, presumably B-L beta I and B-L beta II, were amplified. Finally, B-L beta PCR-SSP typing was used in combination with B-G RFLP analyses to characterize unusual (variant) B serotypes; the results indicate that some of these are natural recombinants within the B complex.     

Modulation of postjunctional cholinergic sensitivity of rat diaphragm muscle by cyclic adenosine monophosphate.

Addition of 2.5 mM cyclic adenosine monophosphate (cAMP) to the solution bathing a rat diaphragm muscle alters the magnitude of depolarization responses to iontophoretic pulses of acetylcholine (ACh) at neuromuscular endplates. Alterations are repeatable with small variability on a given preparation for initial and repeat experiments on both hemidiaphragms, but are different on each preparation. Five min after addition of the nucleotide solution, increases (potentiations) of up to 30% above control levels and decreases (attenuations) to 50% below control levels are observed. The effects on sensitivity to ACh of dibutyryl cAMP (1.25 mM), monobutyryl cAMP (0.25 mM), and cAMP (2.5 mM) in Ca++ -free solution are a function of whether the experiment is an initial one on that preparation or a repeat experiment after 10 or more minutes of perfusion flow. In all three cases, initial exposure attenuates sensitivity (means at 5 min: --30, --10, and --20%, respectively) and repeat exposure potentiates sensitivity (mean: 20% at 5 min, 15% at 5 min, and 10% at 2 min respectively). A concentration of dibutyryl cAMP (0.25 mM) which is without effect on sensitivity alone, produces a large, transient potentiation (mean: 45% at 1 min) in conjunction with 0.5 mM theophylline. A decrease in the rate of desensitization is observed during exposure to 0.25 mM cAMP. The results are interpreted in terms of a physiological mechanism whereby receptor activity at the postjunctional membrane is modulated by cAMP formed from prejunctionally released ATP.     

Solute distribution between vacuole and cytosol of sugarcane suspension cells: Sucrose is not accumulated in the vacuole

The compartmentation of solutes in suspension cells of Saccharum sp. during different growth phases in batch culture was determined using CuCl₂ to permeabilize the plasma membrane of the cells. The efflux of cytosolic and vacuolar pools of sugars, cations and phosphate was monitored, and the efflux data for phosphate were compared and corrected using data from compartmentation analysis of phosphate as determined by ³¹P-nuclear magnetic resonance spectroscopy. The results show that sucrose is not accumulated in the vacuoles at any phase of the growth cycle. On the other hand, glucose and fructose are usually accumulated in the vacuole, except at the end of the cell-culture cycle when equal distribution of glucose and fructose between the cytosol and the vacuole is found. Both Na⁺ and Mg²⁺ are preferentially located in the vacuoles, but follow the same tendency as glucose and fructose with almost complete location in the vacuole in the early culture phases and increasing cytosolic concentration with increasing age of the cell culture. Potassium ions are always clearly accumulated in the cytosol at a concentration of about 80 mM; only about 20% of the cellular K⁺ is located inside the vacuole. Cytosolic phosphate is little changed during the cell cycle, whereas the vacuolar phosphate pool changes according to total cellular phosphate. In general there are two different modes of solute compartmentation in sugarcane cells. Some solutes, fructose, glucose, Mg²⁺ and Na⁺, show high vacuolar compartmentation when the total cellular content of the respective solute is low, whereas in the case of ample supply the cytosolic pools increase. For other solutes, phosphate and K⁺, the cytosolic concentration tends to be kept constant, and only excess solute is stored in the vacuole and remobilized under starvation conditions. The behaviour of sucrose is somewhat intermediate and it appears to equilibrate easily between cytosol and vacuole.Abbreviation NMR nuclear magnetic resonance The very cooperative help by Dr. J. Reiner with the ³¹P-NMR measurements and the technical assistance by D. Keis are gratefully acknowledged. This research was supported by the Deutsche Forschungsgemeinschaft and by Fonds der Chemischen Industrie.     

Modulation of in vitro immune responses by monoclonal antibody to T200 antigen

The effects of monoclonal antibody to the T200 antigen on murine mixed-lymphocyte cultures (MLC) and on the generation of alloreactive cytotoxic T lymphocytes (CTL) are investigated. Addition of monoclonal anti-T200 without complement to MLC results in a late suppression of the proliferative response preceded in some cases by an early enhancement. These modulations require the presence of allogeneic stimulator cells; no effects are seen when antibody is added to responders alone. A similar effect is seen on the generation of CTL. Compared to controls without antibody, cultures carried out in the presence of anti-T200 show reduced levels of cytotoxicity measured against allogeneic targets by Day 5. The kinetics of the suppressive effects differ from those seen with anti-Lyt-2, and no suppressive effects are seen with monoclonal antibodies to other cell surface molecules.     

Hierarchical Classification I: Single-Linkage Method

This is the first part of a survey of hierarchical clustering algorithms using joining methods: the Single-Linkage algorithm. Complete-Linkage and general algorithms defined by d(A_i, B) = = α,d(A_i, A_r)±α_sd(A_i, A_s)±βd(A_r, A_s) will be discussed in two subsequent papers.