Chemosensory responses in isolated olfactory receptor neurons from Necturus maculosus

Olfactory receptor neurons were isolated without enzymes from the mudpuppy, Necturus maculosus, and tested for chemosensitivity. The cells responded to odorants with changes in firing frequency and alterations in excitability that were detected with tight-seal patch electrodes using on-cell and whole-cell recording conditions. Chemosensitive cells exhibited two primary response characteristics: excitation and inhibition. Both types of primary response were observed in different cells stimulated by mixtures of amino acids as well as by the single compound L-alanine, suggesting that there may be more than one transduction pathway for some odorants. Using the normal whole-cell recording method, the chemosensitivity of competent cells washed out rapidly; a resistive whole-cell method was used to record odorant responses under current-clamp conditions. In response to chemical stimulation, excitability appeared to be modulated in several different ways in different cells: odorants induced hyperpolarizing or depolarizing receptor potentials, elicited or inhibited transient, rhythmic generator potentials, and altered excitability without changing the membrane potential or input resistance. These effects suggest that olfactory transduction is mediated through at least three different pathways with effects on four or more components of the membrane conductance. Polychotomous pathways such as these may be important for odor discrimination and for sharpening the "odor image" generated in the olfactory epithelium.     

Effect of heparin on the binding affinity of acidic FGF for the cloned human FGF receptors, flg and bek.

Heparin potentiates the mitogenic activity of acidic fibroblast growth factor (aFGF) by 20-100 fold but mechanisms detailing this potentiation have not yet been fully elucidated. We report that heparin increases the binding affinity of aFGF for the two cloned and overexpressed human FGF receptors, flg and bek, by 2-3 fold. This increase in binding affinity, together with previous data demonstrating a 3-5 fold increase in the stability of aFGF, are likely to account for a significant portion of heparin's potentiation of aFGF activity observed in biological assay systems.     

The gene for human acidic fibroblast growth factor encodes two upstream exons alternatively spliced to the first coding exon

We have isolated two cDNA clones encoding human acidic fibroblast growth factor (aFGF) which represent the utilization of alternative upstream exons in aFGF mRNA. Isolation and sequence analysis of genomic clones spanning the first coding exon and each of the upstream sequences confirms that the divergent 5' sequences are separate exons, spliced alternatively to the first coding exon 34 nucleotides upstream of the initiator AUG codon. Restriction mapping of the genomic clones provides a minimum size estimate of 45 kilobase pairs for the aFGF locus.     

A novel bufadienolide synthesis

A simple two-step synthesis of bufadienolides is reported. It consists in the addition of the dimethyl acetal of chloroketene to a steroidal 20-methylene 21-aldehyde and in the treatment of the resulting 2,2-dimethoxy 3-chloro 2,3-dihydropyran with sodium methoxide in dimethylsulfoxide. This method is exemplified by the synthesis of 3β-hydroxy-5α,14α-bufadienolide from 3β-acetoxy-20-methylene-5α-pregnan-21-al, prepared from 3β-acetoxy-5α-androstan-17-one. The new procedure represents the most efficient bufadienolide synthesis yet known.     

Antigen presentation of a modified tumor-derived peptide by tumor infiltrating lymphocytes

CD8+ T-lymphocytes recognize peptides in the context of major histocompatibility complex (MHC) class I antigens. Upon activation, these cells differentiate into effector cytotoxic T lymphocytes (CTL) and no longer require formal antigen presentation by professional antigen presenting cells (APC). Subsequently, any cell expressing MHC class I/cognate peptide can stimulate CTL. Using TIL specific for a melanoma antigen-derived peptide, IMDQVPFSV (g209 2M), we sought to determine whether these CTL could present peptide to each other. Our findings demonstrate that peptide presentation of the g209 2M peptide epitope by TIL is comparable to conventional methods of using T2 cells as APC. We report here that CTL are capable of self-presentation of antigenic peptide to neighboring CTL resulting in IFN-gamma secretion, proliferation, and lysis of peptide-loaded CTL. These results demonstrate that human TIL possess both APC functions as well as cytotoxic functions and that this phenomenon could influence CTL activity elicited by immunotherapy.     

NuMA is a component of an insoluble matrix at mitotic spindle poles

NuMA associates with microtubule motors during mitosis to perform an essential role in organizing microtubule minus ends at spindle poles. Using immunogold electron microscopy, we show that NuMA is a component of an electron-dense material concentrated at both mitotic spindle poles in PtK1 cells and the core of microtubule asters formed through a centrosome-independent mechanism in cell-free mitotic extracts. This NuMA-containing material is distinct from the peri-centriolar material and forms a matrix that appears to anchor microtubule ends at the spindle pole. In stark contrast to conventional microtubule-associated proteins whose solubility is directly dependent on microtubules, we find that once NuMA is incorporated into this matrix either in vivo or in vitro, it becomes insoluble and this insolubility is no longer dependent on microtubules. NuMA is essential for the formation of this insoluble matrix at the core of mitotic asters assembled in vitro because the matrix is absent from mitotic asters assembled in a cell-free mitotic extract that is specifically depleted of NuMA. These physical properties are consistent with NuMA being a component of the putative mitotic spindle matrix in vertebrate cells. Furthermore, given that NuMA is essential for spindle pole organization in vertebrate systems, it is likely that this insoluble matrix plays an essential structural function in anchoring and/or stabilizing microtubule minus ends at spindle poles in mitotic cells.     

BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis

Plant steroid hormones, brassinosteroids (BRs), are perceived by the plasma membrane-localized leucine-rich-repeat-receptor kinase BRI1. Based on sequence similarity, we have identified three members of the BRI1 family, named BRL1, BRL2 and BRL3. BRL1 and BRL3, but not BRL2, encode functional BR receptors that bind brassinolide, the most active BR, with high affinity. In agreement, only BRL1 and BRL3 can rescue bri1 mutants when expressed under the control of the BRI1 promoter. While BRI1 is ubiquitously expressed in growing cells, the expression of BRL1 and BRL3 is restricted to non-overlapping subsets of vascular cells. Loss-of-function of brl1 causes abnormal phloem:xylem differentiation ratios and enhances the vascular defects of a weak bri1 mutant. bri1 brl1 brl3 triple mutants enhance bri1 dwarfism and also exhibit abnormal vascular differentiation. Thus, Arabidopsis contains a small number of BR receptors that have specific functions in cell growth and vascular differentiation.     

First cleavage of the mouse embryo responds to change in egg shape at fertilization

Although mouse development is regulative, the cleavage pattern of the embryo is not random. The first cleavage tends to relate to the site of the previous meiosis. Sperm entry might provide a second cue, but evidence for and against this is indirect and has been debated. To resolve whether sperm entry position relates to the first cleavage, we have followed development from fertilization by time-lapse imaging. This directly showed cytokinesis passes close to the site of the previous meiosis and to both the sperm entry site and trajectory of the male pronucleus in a significant majority of eggs. We detected asymmetric distribution of Par6 protein in relation to the site of meiosis, but not sperm entry. Unexpectedly, we found the egg becomes flattened upon fertilization in an actin-mediated process. The sperm entry position tends to lie at one end of the short axis along which cleavage will pass. When we manipulated eggs to change their shape, this repositioned the cleavage plane such that eggs divided along their experimentally imposed short axis. Such manipulated eggs were able to develop to term, emphasizing the regulative nature of their development.