Evidence that leukocyte function-associated antigen-1 is involved in recirculation and homing of human lymphocytes via high endothelial venules

We report the results of mAb inhibition studies of human lymphocyte-high endothelial venule interaction in vitro. These studies in which T cells from both normal donors and from a LFA-1-deficient patient were used indicate that in addition to a system of organ-specific 90-kDa "homing" receptors on lymphocytes, LFA-1 is also involved in lymphocyte recirculation and homing.     

Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region

Matrix-associated regions may be useful for studying the role of chromatin architecture in transgene activity of transformed plants. The chicken lysozyme A element was shown to have specific affinity for tobacco nuclear matrices, and its influence on the variability of transgene expression in tobacco plants was studied. T-DNA constructs in which this element flanked either the [beta]-glucuronidase (GUS) reporter gene or both reporter and selection gene were introduced in tobacco. The variation in GUS gene activity was reduced significantly among mature first-generation transgenic plants carrying the A element. Average GUS activity became somewhat higher, but the maximum attainable level of gene expression was similar for all three constructs. Transient gene expression assays showed that the A element did not contain general enhancer functions; therefore, its presence seemed to prevent the lower levels of transgene expression. The strongest reduction in variability was found in plants transformed with the construct carrying the A elements at the borders of the T-DNA. In this population, expression levels became copy number dependent. The presence of two A elements in the T-DNA did not interfere with meiosis.     

Polarized Growth of Fungal Hyphae Is Defined by an Alkaline pH Gradient

Robson, G. D., Prebble, E., Rickers, A., Hosking, S., Denning, D. W., Trinci, A. P. J., and Robertson, W. 1996. Polarized growth of fungal hyphae is defined by an alkaline pH gradient. Fungal Genetics and Biology 20, 289-298. Polarized cell growth is exhibited by a diverse range of eukaryotic and prokaryotic cells. The events which are responsible for this growth are poorly understood. However, the existence of ion gradients may play an important role in establishing and driving cell polarity. Using a pH-sensitive, ratiometric fluorescent dye to monitor intracellular pH in growing fungal hyphae, we report a gradient at the extending hyphal tip that is up to 1.4 pH units more alkaline than more distal regions. Both the magnitude and the length of the pH gradient were strongly correlated with the rate of hyphal extension and eradication of the gradient-arrested growth. These results suggest that alkaline pH gradients may be integral to hyphal extension in fungi.     

Activation of LFA-1 through a Ca2(+)-dependent epitope stimulates lymphocyte adhesion

The leukocyte function-associated molecule-1 (LFA-1) plays a key role in cell adhesion processes between cells of the immune system. We investigated the mechanism that may regulate LFA-1-ligand interactions, which result in cell-cell adhesion. To this end we employed an intriguing anti-LFA-1 alpha mAb (NKI-L16), capable of inducing rather than inhibiting cell adhesion. Aggregation induced by NKI-L16 or Fab fragments thereof is not the result of signals transmitted through LFA-1. The antibody was found to recognize a unique Ca2(+)-dependent activation epitope of LFA-1, which is essentially absent on resting lymphocytes, but becomes induced upon in vitro culture. Expression of this epitope correlates well with the capacity of cells to rapidly aggregate upon stimulation by PMA or through the TCR/CD3 complex, indicating that expression of the NKI-L16 epitope is essential for LFA-1 to mediate adhesion. However, expression of the NKI-L16 epitope in itself is not sufficient for cell binding since cloned T lymphocytes express the NKI-L16 epitope constitutively at high levels, but do not aggregate spontaneously. Based on these observations we propose the existence of three distinct forms of LFA-1: (a) an inactive form, which does not, or only partially exposes the NKI-L16 epitope, found on resting cells; (b) an intermediate, NKI-L16+ form, expressed by mature or previously activated cells; and (c) an active (NKI-L16+) form of LFA-1, capable of high affinity ligand binding, obtained after specific triggering of a lymphocyte through the TCR/CD3 complex, by PMA, or by binding of NKI-L16 antibodies.     

Isometric force development, isotonic shortening, and elasticity measurements from Ca(2+)-activated ventricular muscle of the guinea pig

Isometric tension and isotonic shortening were measured at constant levels of calcium activation of varying magnitude in mechanically disrupted EGTA-treated ventricular bundles from guinea pigs. The results were as follows: (a) The effect of creatine phosphate (CP) on peak tension and rate of shortening saturated at a CP concentration more than 10 mM; below that level tension was increased and shortening velocity decreased. We interpreted this to mean that CP above 10 mM was sufficient to buffer MgATP(2-) intracellularly. (b) The activated bundles exhibited an exponential stress-strain relationship and the series elastic properties did not vary appreciably with degree of activation or creatine phosphate level. (c) At a muscle length 20 percent beyond just taut, peak tension increased with Ca(2+) concentration over the range slightly below 10(-6) to slightly above 10(-4)M. (d) By releasing the muscle length-active tension curves were constructed. Force declined to 20 percent peak tension with a decrease in muscle length (after the recoil) of only 11 percent at 10(-4)M Ca(2+) and 6 percent at 4x10(-6)M Ca(2+). (e) The rate of shortening after a release was greater at lower loads. At identical loads (relative to maximum force at a given Ca(2+) level), velocity at a given time after the release was less at lower Ca(2+) concentrations; at 10 M(-5), velocity was 72 percent of that at 10(-4)M, and at 4x10(-6)M, active shortening was usually delayed and was 40 percent of the velocity at 10(-4) M. Thus, under the conditions of these experiments, both velocity and peak tension depend on the level of Ca(2+) activation over a similar range of Ca(2+) concentration.     

Effects of a training program on resting plasma 2-hydroxycatecholestrogen levels in eumenorrheic women

De Crée, Carl, Peter Ball, BärbelSeidlitz, Gerrit Van Kranenburg, Peter Geurten, and Hans A. Keizer.Effects of a training program on resting plasma2-hydroxycatecholestrogen levels in eumenorrheic women.J. Appl. Physiol. 83(5):1551-1556, 1997.Catecholestrogens (CE) represent a majormetabolic pathway in estrogen metabolism. Previous information on CEand training is limited to two cross-sectional studies that did notinvolve standardized training. Our purpose, by means of a prospective design, was to evaluate the effects of a brief, exhaustive training program on resting plasma concentrations of 2-hydroxy CE. The experimental design spanned two menstrual cycles: a control cycle and atraining cycle. The subjects were nine previously untrained, eumenorrheic women [body fat: 24.8 ± 1.0 (SE) %]. Datawere collected during the follicular (FPh) and the luteal phases (LPh).Posttraining FPh and LPh tests were held the day after the last day ofa 5-day period of training on a cycle ergometer. Total2-hydroxyestrogens (2-OHE) averaged 200 ± 29 pg/ml during the FPhand 420 ± 54 pg/ml during the LPh(P < 0.05). Levels of total2-methoxyestrogens (2-MeOE) were 237 ± 32 pg/ml during the FPh and339 ± 26 pg/ml during the LPh (P < 0.05). After training, although the plasma levels of 2-OHEsignificantly decreased (21%;P < 0.05) during the LPh, the actualCE formation (as estimated from the 2-OHE-to-total estrogens ratio)increased (+29%; P < 0.05). CE activity, as expressed by the 2-MeOE-to-2-OHE ratio, showedsignificantly higher values in both phases (FPh, +14%; LPh, +13%;P < 0.05). At the same time, restinglevels of norepinephrine (NE) were increased by 42%(P < 0.05). CE strongly inhibitbiological decomposition of NE by catechol-O-methyltransferase (COMT).Results of the present study suggest that, in response to training, CEare increasingly competing with the enzyme COMT, thus preventingpremature NE deactivation.

     

Plasma 2-hydroxycatecholestrogen responses to acute submaximal and maximal exercise in untrained women

De Crée, Carl, Peter Ball, Bärbel Seidlitz,Gerrit Van Kranenburg, Peter Geurten, and Hans A. Keizer. Plasma2-hydroxycatecholestrogen responses to acute submaximal and maximalexercise in untrained women. J. Appl.Physiol. 82(1): 364-370, 1997.Exercise-induced menstrual problems are accompanied by an increase in catecholestrogen (CE) formation. It has been hypothesized that hypoestrogenemia may besecondary to an increased turnover from estrogens to CE, which then maydisrupt luteinizing hormone release. In addition, the strong affinityof CE for the catecholamine-deactivating enzyme catechol-O-methyltransferase (COMT)has led to speculations about their possible role in safeguardingnorepinephrine from premature decomposition during exercise. Weinvestigated whether acute exercise on a cycle ergometer produces anychanges in CE homeostasis. Nine untrained eumenorrheic women (body fat,24.8 ± 3.1%) volunteered for this study. Baseline plasma CEaverages for total 2-hydroxyestrogens (2-OHE) were 218 ± 29 (SE)pg/ml during the follicular phase (FPh) and 420 ± 58 pg/ml duringthe luteal phase (LPh). 2-Methoxyestrogens (2-MeOE) measured 257 ± 17 pg/ml in the FPh and 339 ± 39 pg/ml in the LPh. Duringincremental exercise, total estrogens (E) increased, but 2-OHE and2-MeOE levels did not significantly change in either phase. The 2-OHE/Eratio (measure of CE turnover) decreased during exercise in bothmenstrual phases, whereas the 2-MeOE/2-OHE ratio (correlates with COMTactivity) did not significantly change. These findings suggest thatthere is insufficient evidence to conclude that brief incrementalexercise in untrained eumenorrheic females acutely produces increasedCE formation.

     

(Over)training effects on quantitative electromyography and muscle enzyme activities in standardbred horses

Too intensive training may lead to overreaching or overtraining. To study whether quantitative needle electromyography (QEMG) is more sensitive to detect training (mal)adaptation than muscle enzyme activities, 12 standardbred geldings trained for 32 wk in age-, breed-, and sex-matched fixed pairs. After a habituation and normal training (NT) phase (phases 1 and 2, 4 and 18 wk, respectively), with increasing intensity and duration and frequency of training sessions, an intensified training (IT) group (phase 3, 6 wk) and a control group (which continued training as in the last week of phase 2) were formed. Thereafter, all horses entered a reduced training phase (phase 4, 4 wk). One hour before a standardized exercise test (SET; treadmill), QEMG analysis and biochemical enzyme activity were performed in muscle or in biopsies from vastus lateralis and pectoralis descendens muscle in order to identify causes of changes in exercise performance and eventual (mal)adaptation in skeletal muscle. NT resulted in a significant adaptation of QEMG parameters, whereas in muscle biopsies hexokinase activity was significantly decreased. Compared with NT controls, IT induced a stronger adaptation (e.g., higher amplitude, shorter duration, and fewer turns) in QEMG variables resembling potentially synchronization of individual motor unit fiber action potentials. Despite a 19% decrease in performance of the SET after IT, enzyme activities of 3-hydroxyacyl dehydrogenase and citrate synthase displayed similar increases in control and IT animals. We conclude that 1) QEMG analysis is a more sensitive tool to monitor training adaptation than muscle enzyme activities but does not discriminate between overreaching and normal training adaptations at this training level and 2) the decreased performance as noted in this study after IT originates most likely from a central (brain) rather than peripheral level.     

Direct action of estradiol-17β on the atrial action potential

The effect of the two C-17 isomers of estradiol on the shape of the action potential of rat atrial tissue was studied by means of classical glass electrodes for different concentrations of estradiol. Resting potential and upstroke were not affected by estradiol, but the duration of the action potential was reduced. Only estradiol-17β exhibits an effect in a concentration dependent way, while estradiol-17α has no effect at all. The ionic mechanism was studied by adding specific ionic blockers to the perfusate. Since the effect was much less pronounced when a slow inward current blocker was added, it was concluded that estradiol-17β acts mainly via the slow inward current channel. Only a small part of the interaction takes place via the potassium outward channel.