Chemical agents that inhibit pollen development: effects of the phenyl-cinnoline carboxylates SC-1058 and SC-1271 on the ultrastructure of developing wheat anthers (Triticum aestivum L. var Yecora rojò)

Summary Phenylcinnoline carboxylate compounds SC-1058 and SC-1271 cause complete male sterility in wheat when applied at suitable dosages at the pre-meiotic stage of anther development. Anthers from treated and untreated plants were compared using light and electron microscopy from the pre-meiotic stage through the formation of nearly mature pollen. Overall anther development is gradually slowed in treated plants and pollen development is generally arrested in the late prevacuolate or early vacuolate microspore stage, although the first pollen mitosis does sometimes occur. The sporopollenin-containing exine walls are thinner, and show abnormally developed foot and tectum layers with sparse connecting baculi. Microspore cytoplasm degenerates and the cells eventually collapse. At the early, prevacuolate, free microspore stage treated tapetal cells hypertrophy, expanding into the locule. They contain abnormally large vacuoles that appear to form from the fusion of secretory vesicles, and some vacuoles contain electrondense deposits. The sporopollenin-containing orbicular wall and Ubisch bodies are retarded in their development and are structurally deformed. Acetolysis of whole anthers and of thick sections shows that the sporopollen-in-containing structures of treated materials are greatly reduced in thickness and are less rigid than in the control. We conclude that application of these compounds causes interference with the secretory function of tapetal cells which supplies sporopollenin cell-wall polymers to the exine of the microspores and to the tapetal orbicular wall and associated Ubisch bodies. Interference with the tapetal secretion of other nutrients required for microspore development is strongly suggested.     

Delta-Cytomembranen und lamelläre Cytosomen

Zusammenfassung Im Epithel der Kiemenbüschel von Axolotl (Amblystoma mexicanum) findet sich ein besonderer Typ von cytoplasmatischen Membranen, den wir mit -Cytomembran bezeichnen. -Cytomembranen sind schichtweise in Schleifen oder Spiralen angeordnet und bestehen aus einer 30–45 Å dicken osmiophilen Schicht mit einem 60 Å breiten intermembranösen Intervall. Die -Cytomembranen differenzieren sich im perinucleären Bereich des Cytoplasmas aus einer homogenen, elektronendichten Substanz und stellen die Vorstufen der lamellären Cytosomen dar. Die -Cytomembranen und die lamellären Cytosomen sind aus einem Kohlenhydrat-Protein-Komplex mit möglicher Bindung an Phospholipoide zusammengesetzt. Wir glauben, daß diese besondere Gruppe von submikroskopischen Strukturen wichtige Funktionen für die Synthese der Mucopolysaccharide im allgemeinen und für die Schleimsekretion im speziellen hat.Stipendiat der Deutschen Forschungsgemeinschaft.     

A mixed-ligand iron-sulfur cluster (C556SPaB or C565SPsaB) in the Fx-binding site leads to a decreased quantum efficiency of electron transfer in photosystem I.

The proposed structure of Photosystem I depicts two cysteines on the PsaA polypeptide and two cysteines on the PsaB polypeptide in a symmetrical environment, each providing ligands for the interpolypeptide Fx cluster. We studied the role of Fx in electron transfer by substituting serine for cysteine (C565SPsaB and C556SPsaB), thereby introducing the first example of a genetically engineered, mixed-ligand [4Fe-4S] cluster into a protein. Optical kinetic spectroscopy shows that after a single-turnover flash at 298 K, the contribution of A1- (lifetime of 10 microseconds, 40% of total and lifetime of 100 microseconds, 20% of total) and Fx- (lifetime of 500-800 microseconds, 10-15% of total) to the overall P700+ back reaction have increased in C565SPsaB and C556SPsaB at the expense of the back reaction from [FA/FB]-. The electron paramagnetic resonance spectrum of Fx shows g-values of 2.04, 1.94, and 1.81 in both mutants and a similarly decreased amount of FA and FB reduced at 15 K after a single-turnover flash. These results indicate that the mixed-ligand (3 cysteines, 1 serine) Fx cluster is an inefficient electron carrier, but that a small leak through Fx still permits FA and FB to be reduced quantitatively when the samples are frozen during continuous illumination. The data confirm that Fx is a necessary intermediate in the electron transfer pathway from A1 to FA and FB in Photosystem I.     

High-resolution mapping of a linkage group on mouse chromosome 8 conserved on human chromosome 16Q

We have performed a high-resolution linkage analysis for the conserved segment on distal mouse Chromosome (Chr) 8 that is homologous to human Chr 16q. The interspecific backcross used involved M. m. molossinus and an M. m. domesticus line congenic for an M. spretus segment from Chr 8 flanked by phenotypic markers Os (oligosyndactyly) and e, a coat colormarker. From a total of 682 N₂ progeny, the 191 animals revealing a recombination event between these phenotypic markers were typed for 23 internal loci. The following locus order with distances in cM was obtained: (centromere)–Os–4.1–Mmp2–0.2–Ces1,Es1, Es22–1.2–Mt1,D8Mit15–2.2–Got2, D8Mit11–3.7–Es30–0.3–Es2, Es7–0.9–Ctra1,Lcat–0.3–Cdh1, Cadp, Nmor1, D8Mit12–0.2–Mov34–2.5–Hp,Tat–0.2–Zfp4–1.6–Zfp1,Ctrb–10.9–e. In a separate interspecific cross involving 62 meioses, Dpep1 was mapped together with Aprt and Cdh3 at 12.9 cM distal to Hp, Tat, to the vicinity of e. Our data give locus order for markers not previously resolved, add Mmp2 and Dpep1 as new markers on mouse Chr 8, and indicate that Ctra1 is the mouse homolog for human CTRL. Comparison of the order of 17 mouse loci with that of their human homologs reveals that locus order is well conserved and that the conserved segment in the human apparently spans the whole long arm of Chr 16. Received: 30 July 1996 / Accepted: 15 November 1996     

Crystallization and preliminary X-ray crystallographic analysis of squalene-hopene cyclase from Alicyclobacillus acidocaldarius.

The membrane-associated protein squalene-hopene cyclase from Alicyclobacillus acidocaldarius was overexposed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Crystals of three interrelated forms were grown by vapor diffusion under identical conditions. The crystals diffract to about 2.3 A resolution, but they are unstable in the X-ray beam. An interpretable heavy-atom derivative was obtained.     

Cooperative Interception of Neuronal Apoptosis by BCL-2 and BAG-1 Expression: Prevention of Caspase Activation and Reduced Production of Reactive Oxygen Species

Abstract: Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D- and cycloheximide-sensitive caspase (ICE-like) activity. The peptide inhibitor of caspase activity, N -acetyl-Asp-Glu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone in preventing NGF withdrawal-induced apoptosis, suggesting an important role for caspase-3 (CPP32)-like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert -butyl-α-phenylnitrone, and the antioxidant, N -acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL-2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone.     

The CD4-independent tropism of human immunodeficiency virus type 2 involves several regions of the envelope protein and correlates with a reduced activation threshold for envelope-mediated fusion.

Several human immunodeficiency virus type 2 (HIV-2) strains have been shown to infect some CD4-negative cell lines (P. R. Clapham, A. McKnight, and R. A. Weiss, J. Virol. 66:3531-3537, 1992). Using molecular clones of HIV-2 with a CD4-independent tropism, we have identified critical amino acid residues in the envelope protein which are required for CD4-independent infection. Mutations located immediately upstream of a proposed coiled coil domain in the transmembrane protein (A526T or I528M) and flanking the base of the V4 loop (L378F and K403R) are crucial for the CD4-independent phenotype. Of several mutations conferring a positive charge in V1, V2, and V3, only the change in V3 (Q310K) helped to enhance the CD4-independent phenotype but could not mediate it on its own. These mutations reduce the amount of soluble CD4 required to trigger CD4-independent cell-cell fusion, suggesting that they lower the activation threshold for the fusion process. After binding to cell surface-anchored CD4, a CD4-independent recombinant envelope protein showed an increased binding of anti-envelope protein antibodies, suggesting either an enhanced binding to cell surfaces or more extensive conformational changes in CD4-independent compared to CD4-dependent envelope proteins. The reduced activation threshold of CD4-independent envelope proteins may thus enable them to utilize a membrane molecule for entry which is not as efficient as CD4 in triggering the conformational changes required for the membrane fusion process. CD4-independent HIV-2 variants may be conceptually similar to influenza virus variants capable of fusing at a higher than normal pH (R. S. Daniels, J. C. Downie, J. A. Hay, M. Knossow, J. J. Skehel, M. L. Wang, and D. C. Wiley, Cell 40:431-439, 1985).     

Unbiased research and the human spirit: the challenges of randomized controlled trials.

Research by Klein and associates provides useful information on the relation between episiotomy and outcomes such as perineal trauma, but the methodologic implications of their work are especially fascinating. Physicians who participated in their randomized controlled trial (RCT) were supposed to adhere to a policy of either liberal or restrictive use of episiotomy according to the study arm to which each patient was assigned. However, some used the procedure for approximately 90% of patients regardless of allocation. Klein and associates'' post-hoc study (see pages 769 to 779 of this issue) sheds light on the relation between physician attitudes and the practice of episiotomy. The author contends that the noncompliance encountered by Klein and associates reflects the fact that randomized trials are anathema to the human spirit. He offers suggestions for making RCTs more meaningful and stresses that, although RCTs are indispensible to the advancement of medical knowledge, they necessitate assiduous attention to matters of design and implementation.     

Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco

cDNAs encoding three proteins from barley ( Hordeum vulgare ), a class-II chitinase (CHI), a class-II β-1,3-glucanase (GLU) and a Type-I ribosome-inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani , which infects a range of plant species including tobacco. To create a situation similar to 'multi-gene' tolerance, which traditional breeding experience has shown to provide crops with a longer-lasting protection, several of these antifungal genes were combined and protection against fungal attack resulting from their co-expression in planta was evaluated. Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co-expressing the barley transgenes GLU/ CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level. The data indicate synergistic protective interaction of the co-expressed anti-fungal proteins in vivo .     

Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies

S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2). The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor 1-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 microM and 22 microM respectively. After prolonged incubation, 1-chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.