Identification of two human sperm populations using flow and image cytometry

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder (“marginal population”). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide. © 1994 Wiley-Liss, Inc.     

How Many Nucleotides Are Required to Resolve a Phylogenetic Problem? The Use of a New Statistical Method Applicable to Available Sequences

The evolution of bootstrap proportions (BP) with sequence length was studied using a 28S ribosomal RNA data set. For different sequence lengths, informative sites were jackknifed several times. Bootstrapping was subsequently performed on each of these subsamples. For each node, BPs so obtained were plotted against sequence length, showing the evolution of the robustness with increasing number of informative sites. For robust nodes (BP of 100%), the pattern of BPs is unvarying and is described by a simple function BP = 100(1− e^{−b(x − x′)}), where x is the number of informative sites and b and x′ are two parameters estimated using a nonlinear regression procedure. When a node has a BP <100% and the pattern of BPs fits this function, it is possible to estimate the number of informative sites required to obtain a given average BP. The method also identifies nonrobust nodes (nonascending clusters of BP dots), for which it seems to be more cost effective and fruitful to turn to other species and/or genes rather than to continue sequencing longer gene lengths from the same species to reach a BP of 95%.     

IPG (inositolphosphate glycan) as a cellular signal for TGF-β1 modulation of chondrocyte cell cycle

The knowledge of transforming growth factor (TGF)-β receptors has greatly progressed in the recent years. TGF-β receptors type I and II have been implicated in the modulation of cell proliferation, whereas type III (betaglycan) may act as a component presenting TGF-β to its signaling receptors. In addition, four other proteins that bind TGF-β1 or TGF-β2 have been recently identified in some cell lines, three being anchored to the membrane through a glycosylphosphatidylinositol (GPI). Despite this knowledge, the molecular mechanism of signal transduction through the TGF-β receptors remain an enigma. TGF-β family does not signal via any of the classical pathways. As GPI anchors of membrane proteins have been implicated in the transduction of some hormonal effects, we investigated the putative role of GPI in signaling the TGF-β effects on the proliferation of rabbit articular chondrocytes (RAC). We previously showed that TGF-β1 increased DNA replication rate of RAC, with a recruitment of cells in G2/M followed by a subsequent mitosis wave. Here, we find that the factor causes specific GPI hydrolysis, with correlated increase of inositolphosphate glycan (IPG). This effect was specifically inhibited by antibodies that bind TGF-β1. Using [³H]-inositol labeling and Triton X-114 extraction, we demonstrate that a hydrophobic material from the membrane is cleaved by treatment of cell cultures with phosphatidylinositol specific phospholipase C (PI-PLC) or by exposure to TGF-β, supporting that a PI-anchored molecule gives rise to IPG by TGF-β-induced hydrolysis. The biological relevance of this hydrolysis was demonstrated by the enhancing effect of purified IPG on the DNA synthesis rate, which mimicked the TGF-β action. These results demonstrate that IPG could be an early messenger in the cellular signaling that mediates the effect of TGF-β on RAC growth. © 1993 Wiley-Liss, Inc.     

In Xenopus laevis, the product of a developmentally regulated mRNA is structurally and functionally homologous to a Saccharomyces cerevisiae protein involved in translation fidelity.

We have performed a differential screen of a Xenopus egg cDNA library and selected two clones (Cl1 and Cl2) corresponding to mRNA which are specifically adenylated and recruited into polysomes after fertilization. Sequence analysis of Cl1 reveals that the corresponding protein is 67.5% identical (83% similar) to the product of the Saccharomyces cerevisiae SUP45 (also called SUP1 or SAL4) gene. This gene, when mutated, is an omnipotent suppressor of nonsense codons. When expressed in a sup45 mutant, the Xenopus Cl1 cDNA was able to suppress sup45-related phenotypes, showing that the structural homology reflects a functional homology. Our discovery of a structural and functional homolog in Xenopus cells implies that the function of SUP45 is not restricted to lower eukaryotes and that the SUP45 protein may perform a crucial cellular function in higher eukaryotes.     

Electrically induced calcium elevation,activation, and parthenogenetic development of bovine oocytes

The influence of electrical stimulation on the level of intracellular Ca²⁺ in bovine oocytes, as well as activation and extent of parthenogenetic development, was investigated. Mature oocytes were electrically stimulated at 29 hr of maturation, and intracellular Ca²⁺ concentration was determined with the Ca²⁺ indicator fura-2 dextran (fura-2 D). The Ca²⁺ response of oocytes to a given electrical pulse was variable. Oocytes responded with either no Ca²⁺ rise from baseline (≈? 12 nM), a short-duration Ca²⁺ rise (from 12 nM to 300 nM) that returned to baseline within 2 min of the pulse, or a long-duration Ca²⁺ rise (from 12 nM to 1,000–2,000 nM) that never returned to baseline during the 8 min period over which the oocytes were monitored. In these oocytes, Ca²⁺ level returned to baseline when oocytes were removed from 0.30 M mannitol and placed in an ionic medium. Increasing field strength or pulse duration tended to increase the proportion of oocytes displaying a Ca²⁺ rise, and at 1.0 kVcm^?1 for 40 μsec, all oocytes displayed a long-duration Ca²⁺ elevation. Direct transfer of oocytes from culture medium to mannitol also triggered a Ca²⁺ rise. Multiple stimulations, either electrical or by transferring to mannitol, produced multiple Ca²⁺ rises. This mannitol-induced Ca²⁺ rise could be inhibited by first washing the oocytes in medium containing equal parts of 0.30 M mannitol and phosphate buffered saline (PBS). The level of Ca²⁺ stimulation affected activation and development of oocytes. Insufficient, or, conversely, excessive Ca²⁺ stimulation impaired development. Optimum development was obtained with (1) three pulses of 0.2 kVcm^?1 for 20 μsec, each pulse 22 min apart, after direct transfer of oocytes from culture medium to mannitol (22% blastocysts) or (2) three pulses of 1.0 kVcm^?1 for 20 μsec after transfer of oocytes from culture medium to medium containing equal parts mannitol and PBS, then to mannitol (24% blastocysts). This procedure avoided induction of a Ca²⁺ rise prior to the pulse. The results indicate that the level of Ca²⁺ stimulation can be regulated by incubation conditions prior to the pulse and, to some extent, by field strength and pulse duration. The level of electrical stimulation influenced oocyte Ca²⁺ response, activation, and parthenogenetic development. © 1993 Wiley-Liss, Inc.     

Clinical pharmacology of the new COMT inhibitor CGP 28 014

CGP 28 014 is a specific inhibitor of catechol-O-methyltransferase (COMT) in vivo. In humans, the inhibition was assessed by measuring urinary excretion of isoquinolines and with the levodopa test. Following administration of CGP 28 014, urinary excretion of isoquinolines was significantly increased. In rats, CGP 28 014 reduced plasma and striatal concentrations of 3-O-methyldopa (30MD) in a dose-dependent manner. Acute and subchronic administration of CGP 28 014 alone or in combination with the peripherally acting decarboxylase inhibitor benserazide decreased plasma 30MD as an index of COMT inhibition by about 50%. There seems to be a close relationship between the time-course of plasma concentrations of CGP 28 014 and the extent of COMT inhibition assessed by the 30MD/DOPA ratio in plasma.     

In vitro study of the proteolytic activity of rumen anaerobic fungi

Abstract To better define the antigenic structure of the outer cell membranes of Legionellae, a panel of 6 monoclonal antibodies was raised against partially purified outer membranes of Legionella pneumophila serogroup 1, Corby strain. This study describes the purification and characterization of one of these monoclonal antibodies reacting with a 135-kDa protein, which was shown to be common to all 14 serogroups of Legionella pneumophila . It shows no cross-reactivity with 20 other Legionella species, or 9 other Gram-negative species tested by SDS-PAGE and Western blotting procedures. The epitope would appear to be predominantly surface exposed and, from preliminary detergent extraction studies, not peptidoglycan-associated.     

Trade-offs and synergies between ecosystem productivity and stability in temperate grasslands

Aim

It is crucial to monitor how the productivity of grasslands varies with its temporal stability for management of these ecosystems. However, identifying the direction of the productivity–stability relationship remains challenging because ecological stability has multiple components that can display neutral, positive or negative covariations. Furthermore, evidence suggests that the direction of the productivity–stability relationship depends on the biotic interactions and abiotic conditions that underlie ecosystem productivity and stability. We decipher the relationships between grassland productivity and two components of its stability in four habitat types with contrasting environments and flora.

Location

France.

Time period

2000–2020.

Major taxa

Grassland plant species.

Methods

We used c. 20,000 vegetation plots spread across French permanent grasslands and remotely sensed vegetation indices to quantify grassland productivity and temporal stability. We decomposed stability into constancy (i.e., temporal invariability) and resistance (i.e., maximum deviation from average) and deciphered the direct and indirect effects of abiotic (namely growing season length and nitrogen input) and biotic (namely plant taxonomic diversity, trait diversity and community-weighted mean traits) factors on productivity–stability relationships using structural equation models.

Results

We found a positive relationship between productivity and constancy and a negative relationship between productivity and resistance in all habitats. Abiotic factors had stronger effects on productivity and stability compared with biotic factors. A longer growing season enhanced grassland productivity and constancy. Nitrogen input had positive and negative effects on grassland productivity and resistance, respectively. Trait values affected the constancy and resistance of grassland more than taxonomic and trait diversity, with effects varying from one habitat to another. Productivity was not related to any biotic factor.

Main conclusions

Our findings reveal how vital it is to consider both the multiple components of stability and the interaction between environment and biodiversity to gain an understanding of the relationships between productivity and stability in real-world ecosystems, which is a crucial step for sustainable grassland management.