Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.     

An electrochemical immunosensor for milk progesterone using a continuous flow system

An electrochemical biosensor for cow's milk progesterone has been developed and used in a competitive immunoassay under thin-layer, continuous-flow conditions. Single-use biosensors were fabricated by depositing anti-progesterone monoclonal antibody (mAb) onto screen-printed carbon electrodes (SPCEs). Three operational steps could be identified: (1) Competitive binding of sample/conjugate (alkaline-phosphatase-labelled progesterone, AP-prog) mixture, (2) establishment of a steady-state amperometric baseline current and (3), measurement of an amperometric signal in the presence of enzyme substrate (1-naphthyl phosphate, 1-NP). In the thin-layer cell, the enzyme product, 1-naphthol, showed electrochemical behaviour consistent with bulk conditions and gave a linear amperometric response under continuous-flow conditions (E_app=+0.3 V vs. Ag/AgCl) over the range 0.1–1.0 μg/ml. After pre-incubating biosensors with progesterone standards, signal generation within the cell (substrate CONCENTRATION=5 mM) was recorded amperometrically as rate (nA/s) or maximum current (i_max, nA). Response values for milk standards were approximately 50% of those prepared in buffer. In both cases, calibration plots over the range 0–50 ng/ml progesterone were obtained. By conducting sample binding under flowing conditions, only 7% of the previous response was obtained, even at a substrate concentration of 50 mM, resulting in low signal:noise ratio. Using a stop-flow arrangement (i.e. quiescent sample binding, followed by continuous flow), low-noise amperograms were obtained at [1-NP]=5 mM. Calibration plots were obtained over the range 0–25 ng/ml, with a coefficient of variation of 12.5% for five replicate real milk samples.     

Cytosolic O-glycosylation is abundant in nerve terminals

Phosphorylation plays a key role in regulating growth cone migration and protein trafficking in nerve terminals. Here we show that nerve terminal proteins contain another abundant post-translational modification: beta-N-acetylglucosamine linked to hydroxyls of serines or threonines (O-GlcNAc(1)). O-GlcNAc modifications are essential for embryogenesis and mounting evidence suggests that O-GlcNAc is a regulatory modification that affects many phosphorylated proteins. We show that the activity and expression of O-GlcNAc transferase (OGT) and N-acetyl-beta-D-glucosaminidase (O-GlcNAcase), the two enzymes regulating O-GlcNAc modifications, are present in nerve terminal structures (synaptosomes) and are particularily abundant in the cytosol of synaptosomes. Numerous synaptosome proteins are highly modified with O-GlcNAc. Although most of these proteins are present in low abundance, we identified by proteomic analysis three neuron-specific O-GlcNAc modified proteins: collapsin response mediator protein-2 (CRMP-2), ubiquitin carboxyl hydrolase-L1 (UCH-L1) and beta-synuclein. CRMP-2, which is involved in growth cone collapse, is a major O-GlcNAc modified protein in synaptosomes. All three proteins are implicated in regulatory cascades that mediate intracellular signaling or neurodegenerative diseases. We propose that O-GlcNAc modifications in the nerve terminal help regulate the functions of these and other synaptosome proteins, and that O-GlcNAc may play a role in neurodegenerative disease.     

Genetic mapping of Sorghum bicolor (L.) Moench QTLs that control variation in tillering and other morphological characters

Grain yield of Sorghum bicolor (L.) Moench is significantly influenced by genetically controlled variation in the number of tillers, plant height, time of anthesis, and various other morphological and physiological characters. In this study, a minimum of 27 unique QTLs that control variation in nine morphological traits, including the presence versus the absence and the height of basal tillers, were mapped, and the percentage of additive genetic variance explained by the QTLs was determined in a population of 137 recombinant inbred lines in two environments. Four QTLs explained from 86.3% to 48.9% (depending upon the environment) of the additive genetic variance in the number of basal tillers with heads, and seven QTLs explained from 85.9% to 47.9% of the additive genetic variance in panicle width. It is unlikely that different alleles were segregating in the mapping population at any of the major dwarfing loci, but five QTLs that explained from 65.8% to 52.0% of the additive genetic variance in main-culm height were mapped. QTLs controlling variation in height of the tallest basal tiller, number of basal tillers per basal-tillered plant, panicle length, leaf angle, maturity, and awn length also were mapped. Three or more QTLs were mapped in linkage groups A, E, G, and I, while none were mapped in linkage groups B and D. Several of the QTLs mapped in this study are likely candidates for marker-assisted selection in breeding programs. Received: 20 September 2000 / Accepted: 26 October 2000     

Dynamic changes in prey choice by stickleback during simultaneous encounter with large prey

When a three-spined stickleback Gasterosteus aculeatus encountered prey simultaneously the probability of hanging and the median pursuit time were greater than when prey were encountered sequentially. During simultaneous prey encounter fish did not choose to attack the more profitable prey but instead the nearer prey was handled first except when the difference between the two prey sizes was large. No difference was found in the level of total energetic intake by the fish regardless of prey size pairing. Fish that handled and ate the first prey of a pair in <5 s attacked the second prey with a high probability of success, demonstrating an opportunistic feeding strategy. Importantly however, the fish did not choose to maximize long term energy intake rate by eating both prey, but rather short-term considerations over the course of feeding took precedence. With an empty stomach, the probability of a fish eating ( P _eat) the first prey handled was high regardless of prey size. As stomach fullness increased, the P _eat the first prey handled decreased if it was the larger prey. Hence, the fish were unselective when the stomach was empty but thereafter there was a shift in preference towards the smaller prey. The decision of which prey to attack and eat appeared to be based on short-term energy considerations and the level of stomach fullness. This study demonstrates that feeding on a short-term scale is a crucial factor to take account of when analysing fish feeding during simultaneous prey encounter.     

Cutting edge: blockade of the CD28/B7 costimulatory pathway inhibits intestinal allograft rejection mediated by CD4+ but not CD8+ T cells.

The effect of blocking the CD28/B7 costimulatory pathway on intestinal allograft rejection was examined in mice. Murine CTLA4Ig failed to prevent the rejection of allografts transplanted into wild-type or CD4 knockout (KO) mice but did inhibit allograft rejection by CD8 KO recipients. This effect was associated with decreased intragraft mRNA for IFN-gamma and TNF-alpha and increased mRNA for IL-4 and IL-5. This altered pattern of cytokine production was not observed in allografts from murine CTLA4Ig-treated CD4 KO mice. These data demonstrate that blockade of the CD28/B7 pathway has different effects on intestinal allograft rejection mediated by CD4+ and CD8+ T cells and suggest that these T cell subsets have different costimulatory requirements in vivo. The results also suggest that the inhibition of CD4+ T cell-mediated allograft rejection by CTLA4Ig may be related to down-regulation of Th1 cytokines and/or up-regulation of Th2 cytokines.     

Ability of monovalent cations to replace potassium during stimulation of lymphoid cells

Stimulation of hamster thymocytes, splenocytes, or lymph node cells occurred to a minimal extent in the absence of K⁺. This observation was found for stimulation by T-cell mitogens (phytohemagglutinin and concanavalin A), A B-cell mitogen (lipopolysaccharide), or antigen (KLH). Marginal restoration of the responses to these stimulants occurred in the presence of 0.1 mM K⁺ and responsiveness returned to near maximal levels on addition of 1 mM K⁺ to the cultures. Attempts to restore the responsiveness with other monovalent cations revealed an order of effectiveness of K⁺ ≥ Rb⁺ ? NH₄⁺ ≥ Li⁺. At the 1 mM level K⁺ and Rb⁺ were equally effective in supporting stimulation by phytohemagglutinin while all concentrations of Li⁺ tested (0.1–10 mM) would not support stimulation. However, addition of Li⁺ to cultures reconstituted with 1 mM K⁺ or Rb⁺ revealed that this ion could enhance the phytohemagglutinin response by approximately 100% in the presence of K⁺ and only 30% in the presence of Rb⁺. These data support the hypotheses that the Na,K ATPase must be active for lymphocyte stimulation to occur and that some of the biological effects of Li⁺ on lymphocyte stimulation are mediated at the level of the Na,K ATPase.