Isolation of an auxin-regulated gene cDNA expressed during the transition from G0 to S phase in tobacco mesophyll protoplasts.

A cDNA clone for an auxin-regulated gene was isolated from a tobacco mesophyll protoplast cDNA library by differential screening. Nucleotide, sequence analysis showed that the deduced product of the gene, which we have designated par, is hydrophilic and is composed of 220 amino acids. No significant homology to other known proteins was detected. The mRNA of the par gene is approximately 900 bases long and its accumulation was detected in cultured mesophyll protoplasts as early as 30 min after the addition of 2,4-dichlorophenoxyacetic acid to the culture medium. The par mRNA was not detected in leaves or freshly prepared protoplasts or in protoplasts in the absence of 2,4-dichlorophenoxyacetic acid. Expression of the par gene was detected at a low level in actively dividing BY-2 tobacco suspension culture cells. The conspicuous accumulation of par mRNA before the initiation of DNA synthesis in tobacco mesophyll protoplasts suggests that the par gene product could play a role in the initiation of meristematic activity in differentiated mesophyll cells.     

Colony formation from mesophyll protoplasts of a cereal, oat

Differentiated leaf cells of gramineous plants, among them the cereals with their immense importance for human nutrition, are considered extremely recalcitrant to, if not incapable of, reentering the cell cycle. This recalcitrance is related to the poor wound response of the monocots—in contrast to most dicots—and the difficulties encountered in monocot tissue culture. We report here the highly reproducible induction of sustained divisions at high frequency (up to 95%) and colony formation from mesophyll protoplasts of a cereal, oat, demonstrating that—contrary to most earlier evidence—mesophyll cells of a gramineous plant have not irreversibly lost their potential for cell division.     

The dubious evidence for smokeless tobacco

Abstract. The argument put forward by West & Krafona that the continued availability of Skoal Bandits in the UK would have led to a decrease in tobacco-related diseases is not supported by the available evidence. It has not been established that young people take up snuff dipping instead of smoking. On the contrary, most studies suggest that both habits co-exist. That smokeless tobacco is less addictive than cigarette smoking is by no means proven—it may well be that both habits are equally addictive. The aggressive marketing techniques of a leading American smokeless tobacco manufacturer resulted in the creation of a new generation of snuff dippers in the USA; there is little to suggest that the same would not have happened in the UK and in other countries targeted by this and other manufacturers.     

Liposome-mediated delivery of tobacco mosaic virus RNA into tobacco protoplasts: A sensitive assay for monitoring liposome-protoplast interactions

Tobacco mosaic virus (TMV) RNA was encapsulated in large, unilamellar phospholipid vesicles (liposomes), and the encapsulated TMV RNA was shown to be infectious when incubated with tobacco protoplasts under appropriate conditions. Maximal virus production in protoplasts was observed after their incubation with TMV RNA entrapped in phosphatidylserine/cholesterol liposomes. Infection was dependent on the presence of polyalcohols in the incubation mixture. Other parameters, such as the extent of vesicle binding, the cell-induced leakage of vesicle contents, and the degree of liposome toxicity were shown to be important in determining the efficiency of infectivity. Liposome-mediated delivery offers an efficient and reproducible method for introducing RNA into plant protoplasts.     

Functional analysis of an olfactory receptor in Drosophila melanogaster

Fifty nine candidate olfactory receptor (Or) genes have recently been identified in Drosophila melanogaster, one of which is Or43a. In wild-type flies, Or43a is expressed at the distal edge of the third antennal segment in about 15 Or neurons. To identify ligands for the receptor we used the Gal4/UAS system to misexpress Or43a in the third antennal segment. Or43a mRNA expression in the antenna of transformed and wild-type flies was visualized by in situ hybridization with a digoxigenin-labeled probe. Electroantennogram recordings from transformed and wild-type flies were used to identify cyclohexanol, cyclohexanone, benzaldehyde, and benzyl alcohol as ligands for the Or43a. This in vivo analysis reveals functional properties of one member of the recently isolated Or family in Drosophila and will provide further insight into our understanding of olfactory coding.     

Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers

Little is known about the catalytic mechanism of the recently discovered ThyX family of flavin-dependent thymidylate synthases that are required for thymidylate (deoxythymidine 5'-monophosphate) synthesis in a large number of microbial species. Using a combination of site-directed mutagenesis and biochemical measurements, we have identified several residues of the Helicobacter pylori ThyX protein with crucial roles in ThyX catalysis. By providing functional evidence that the active site(s) of homotetrameric ThyX proteins is formed by three different subunits, our findings suggest that ThyX proteins have evolved through multimerization of inactive monomers. Moreover, because the active-site configurations of ThyX proteins, present in many human pathogenic bacteria, and of human thymidylate synthase ThyA are different, our results will aid in the identification of compounds specifically inhibiting microbial growth.     

Functional evidence for an inward rectifier potassium current in rat renal afferent arterioles

An inward rectifier potassium current, Kir, has been identified in cerebral and coronary resistance vessels, where it is considered to be an important determinant of resting membrane potential (RMP) and to play a role in blood flow regulation. We investigated the functional role of Kir in the renal afferent arteriole using the in vitro-perfused hydronephrotic rat kidney. Increasing external KCl from 5 to 15 mmol/L induced afferent arteriolar vasodilation. This response was inhibited by 10 to 100 micromol/L Ba(2+), concentrations selective for blockade of Kir, and by chloroethylclonidine (100 micromol/L) but was not blocked by glibenclamide (10 micromol/L) or ouabain (3 mmol/L). Reducing external KCl from 5 to 1.5 mmol/L to enhance rectification of Kir caused vasoconstriction at low renal arterial pressure (40 mm Hg) and vasodilation during myogenic vasoconstriction (120 mm Hg), suggesting that this current dominates RMP at low perfusion pressures. When administered to kidneys perfused at 40 mm Hg renal arterial pressure, 30 micromol/L Ba(2+) elicited afferent arteriolar depolarization, reducing RMP from -47+/-2 to -34+/-2 mV (n=10, P:<0.0001), and vasoconstriction, reducing diameters from 14.5+/-1 to 10.9+/-0.8 microm (n=10, P:=0.0016). Although Ba(2+) reduced resting diameter, blockade of Kir did not prevent myogenic signaling in this vessel. Our findings thus demonstrate the presence of Kir in rat renal afferent arterioles and suggest that this current is an important determinant of RMP in situ.     

Corticoid receptors in rat brain: evidence for an aldosterone receptor.

The existence of high and low affinity mineralocorticoid-binding macromolecules (receptors) has been demonstrated in vitro in cytosols derived from the adrenalectomized rat brain by the specific binding of [3H]aldosterone (3H-A). The high-affinity aldosterone sites can be distinguished from those sites which have a higher affinity for either [3H]dexamethasone (3H-DM) or [3H]corticosterone (3H-B) on the basis of selectivity for spirolactone SC-9420 or non-radioactive A, DM, and B. The binding of 3H-A to the receptors was maximal after 2 hours of incubation of 0-4C. No significant binding of 3H-A to the receptors could be demonstrated when incubations of the radioactive ligand were performed at either 20 or 37 C, indicating that the receptor is heat-liabile. Scatchard analysis of the 3H-A binding data over a 200-fold concentration range of 3H-A indicated that there are two binding sites for aldosterone, a high affinity component (a1) with a Kd approximately equal to 1.5 X 10(-9)M and a low-affinity component (a2) with a Kd approximately equal to 6.3 X 10(-8)M. A similar study using 3H-DM as the radioactive ligand demonstrated only one site for the 3H-DM binding with a Kd = 6.2 X 10(-9)M. The presence of specific aldosterone receptors in the brain with high affinity, limited capacity, and selectivity for aldosterone suggests a possible extra-renal mechanism of action of the hormone in or mediated through the CNS.     

Putative receptor for the plant growth hormone auxin identified and characterized by anti-idiotypic antibodies

The molecular mechanism of action of auxins, which are an important class of plant growth regulators, is not known. While they are thought to influence cellular processes such as gene expression and membrane hyperpolarization by binding to specific receptors, no receptor mediating these processes has been unambiguously identified. We describe the identification of a putative auxin receptor by using anti-idiotypic antibodies. We have demonstrated that a 65-kilodalton protein found in a wide range of plants specifically binds auxins. Furthermore, since it is localized to the nucleus, it is a candidate for an auxin receptor that mediates some nuclear process.     

Functional epialleles at an endogenous human centromere

Human centromeres are defined by megabases of homogenous alpha-satellite DNA arrays that are packaged into specialized chromatin marked by the centromeric histone variant, centromeric protein A (CENP-A). Although most human chromosomes have a single higher-order repeat (HOR) array of alpha satellites, several chromosomes have more than one HOR array. Homo sapiens chromosome 17 (HSA17) has two juxtaposed HOR arrays, D17Z1 and D17Z1-B. Only D17Z1 has been linked to CENP-A chromatin assembly. Here, we use human artificial chromosome assembly assays to show that both D17Z1 and D17Z1-B can support de novo centromere assembly independently. We extend these in vitro studies and demonstrate, using immunostaining and chromatin analyses, that in human cells the centromere can be assembled at D17Z1 or D17Z1-B. Intriguingly, some humans are functional heterozygotes, meaning that CENP-A is located at a different HOR array on the two HSA17 homologs. The site of CENP-A assembly on HSA17 is stable and is transmitted through meiosis, as evidenced by inheritance of CENP-A location through multigenerational families. Differences in histone modifications are not linked clearly with active and inactive D17Z1 and D17Z1-B arrays; however, we detect a correlation between the presence of variant repeat units of D17Z1 and CENP-A assembly at the opposite array, D17Z1-B. Our studies reveal the presence of centromeric epialleles on an endogenous human chromosome and suggest genomic complexities underlying the mechanisms that determine centromere identity in humans.     

From the Cover: Functional evidence for TCR-intrinsic specificity for MHCII

How T cells become restricted to binding antigenic peptides within class I or class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) via clonotypic T-cell receptors (TCRs) remains debated. During development, if TCR–pMHC interactions exceed an affinity threshold, a signal is generated that positively selects the thymocyte to become a mature CD4⁺ or CD8⁺ T cell that can recognize foreign peptides within MHCII or MHCI, respectively. But whether TCRs possess an intrinsic, subthreshold specificity for MHC that facilitates sampling of the peptides within MHC during positive selection or T-cell activation is undefined. Here we asked if increasing the frequency of lymphocyte-specific protein tyrosine kinase (Lck)-associated CD4 molecules in T-cell hybridomas would allow for the detection of subthreshold TCR–MHC interactions. The reactivity of 10 distinct TCRs was assessed in response to selecting and nonselecting MHCII bearing cognate, null, or “shaved” peptides with alanine substitutions at known TCR contact residues: Three of the TCRs were selected on MHCII and have defined peptide specificity, two were selected on MHCI and have a known pMHC specificity, and five were generated in vitro without defined selecting or cognate pMHC. Our central finding is that IL-2 was made when each TCR interacted with selecting or nonselecting MHCII presenting shaved peptides. These responses were abrogated by anti-CD4 antibodies and mutagenesis of CD4. They were also inhibited by anti-MHC antibodies that block TCR–MHCII interactions. We interpret these data as functional evidence for TCR-intrinsic specificity for MHCII.Positive and negative selection limit the αβT-cell repertoire to cells expressing clonotypic T-cell receptors (TCRs) that distinguish the antigenicity of peptides embedded within class I and class II major histocompatibility complex molecules (pMHCI or pMHCII, respectively) based on their source of origin (i.e., self or foreign) (1–4). Approximately 7.5% of CD4⁺CD8⁺ double-positive (DP) thymocytes express TCRs that interact with self-pMHC above an affinity threshold required for positive selection, whereas 7.5% cross a higher affinity threshold that mediates negative selection and the remaining TCRs fail to direct positive selection (5). The rules that restrict TCR recognition of antigenic peptides within MHCI or MHCII are unresolved.Two models have been proposed to explain MHC restriction. One posits that restriction is imposed by CD4 or CD8 during thymocyte development to eliminate TCRs that recognize non-MHC ligands (2, 6). Here, the CD4- and CD8-associated Src kinase, p56^Lck [lymphocyte-specific protein tyrosine kinase (Lck)], is sequestered away from the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR-associated CD3δε, CD3γε, and CD3ζζ signaling modules. Positively selecting signals are then generated in thymocytes expressing TCRs that bind MHCII or MHCI together with CD4 or CD8, respectively, as this localizes Lck to the ITAMs. Those thymocytes expressing TCRs that do not bind MHCI or MHCII would fail to localize Lck to the ITAMs and die. In the second model, germ line-encoded complementary determining regions (CDR) 1 and 2 allow each clonotypic TCR to bind distinct classes and alleles of MHC molecules via unique yet specific recognition codons that impose a canonical docking polarity and MHC restriction (1, 3, 4, 7, 8). Although it is not obvious that these models are mutually exclusive, the key distinction is that in the first model the randomly generated preselection TCR repertoire would contain TCRs that do and do not bind pMHC, whereas in the second model most if not all TCRs would have a specificity for MHC that is germ line-encoded, regardless of the class or allele of MHC.The canonical docking polarity of TCRs on MHCI or MHCII observed in crystal structures, and the CDR1 and CDR2 contacts therein, provides evidence for germ line-encoded TCR–MHC interactions for positively selected TCRs (1, 3, 4, 7, 8). But this is taken as supporting either model, as germ line-encoded contacts are likely to be required to allow the formation of a TCR–CD3–pMHC–CD4/CD8 macrocomplex that situates the CD3 ITAMs and Lck in a functionally mandated orientation (1–4, 6, 9, 10). Structural insights from positively selected TCRs thus do not allow the basis of MHC restriction to be cleanly addressed, and functional data that support either model have been reported (11–15).An open question that can shed light on the similarities and differences between the two models is whether TCRs participate in subthreshold scanning of MHC (4, 16). Scanning would allow a TCR to dock on MHC and survey its contents for peptides that increase the duration of TCR–pMHC interactions, via contacts with clonotypic CDR3s, and allow the formation of a TCR–CD3–pMHC–CD4/CD8 macrocomplex that generates signals (4, 10). In the co-receptor imposed model, a diverse preselection repertoire would contain TCRs with no intrinsic capacity to bind MHC, TCRs that interact with pMHC by atypical modalities, and TCRs that interact with a composite pMHC surface in a canonical modality in a lock-and-key manner akin to antibody–antigen recognition (2, 6). Once selected, this last group of TCRs would be predicted to scan composite pMHC with shapes (i.e., topology and chemical characteristics) related to the selecting pMHC—presumably the same MHC, or similar allelic variant, presenting related peptides. In the germ line-encoded recognition model, TCR scanning of MHC via recognition codons would be intrinsic to most if not all TCRs, regardless of the class of MHC, allelic variants, or the peptide sequence therein (4). At present, functional evidence for TCR scanning of MHC is lacking, regardless of whether it is MHC class-, allele-, and peptide sequence-dependent.Recently, the frequency of Lck-associated CD4 molecules was proposed to influence if a TCR–pMHC interaction is of sufficient duration to direct a specific cell fate decision, such as negative selection (17). We thus hypothesized that genetically increasing the frequency of CD4–Lck association should allow for the detection of subthreshold TCR–pMHC interactions that are normally of insufficient duration to elicit a functional response. Here we show that T-cell hybridomas expressing 10 distinct TCRs along with a CD4–Lck fusion make IL-2 in response to APCs expressing selecting or nonselecting MHCII, regardless of the sequence of the presented peptide. These responses were independent of positive selection on MHCII, as TCRs that were positively selected on MHCI, or generated in vitro and thus not thymically selected, yielded similar responses. These data provide functional evidence for subthreshold TCR scanning of MHCII that is independent of the class of MHC, the allele, or the peptide sequence therein.     

Polyphloretin phosphate, an antagonist of thyrotropin action: evidence for an interaction with the hormone rather than the receptor

Polyphloretin phosphate (PPP) is known to be an inhibitor of bovine TSH (bTSH)-induced stimulation of the thyroid in both in vivo and in vitro assays. The present studies were undertaken to delineate the mechanism of these effects. A high molecular weight PPP preparation strongly inhibited both the binding of 125I-labeled bTSH [( 125I]bTSH) to human thyroid membranes and the stimulation of adenylate cyclase evoked by bTSH therein. Inhibition of bTSH-induced adenylate cyclase activity by PPP was evident both in the absence and the presence of NaCl (150 mM) in the incubation medium. Incubation of membranes with PPP, followed by its removal, did not affect subsequent binding of [125I]bTSH, indicating that PPP did not bind firmly to or damage the TSH receptor. Gel chromatography on Sephadex G-100 revealed that [125I]bTSH incubated with PPP eluted earlier than [125I]bTSH alone, indicating that PPP had formed a higher molecular weight complex with [125I]bTSH. This effect could be prevented by the addition of an excess of unlabeled bTSH to the incubation mixture. Binding of [125I] bTSH in the higher molecular weight peak generated by incubation with PPP was less than half that in control specimens of [125I]bTSH. Studies with PPP were also conducted in a highly sensitive assay that employs cultured porcine thyroid cells and measures the cAMP response induced by bTSH. The inhibitory effect of PPP on bTSH-induced cAMP accumulation was also evident in this assay. However, the presence of divalent cations Ca++ and Mg++ in the assay medium greatly diminished the inhibitory effect of PPP. Similarly, addition of Ca++ and Mg++ to the incubation medium greatly reduced or abolished the inhibitory effect of PPP on [125I]bTSH binding. Both effects of these salts to lessen the inhibitory response to PPP were overcome by increasing the PPP concentration. Gel chromatographic studies revealed that Ca++ and Mg++ acted by inhibiting the formation of the high molecular weight complex of bTSH and PPP. From these findings, we conclude that PPP exerts its inhibitory effect on TSH-induced stimulatory responses in the thyroid, in vivo as well as in vitro, by forming a complex with the hormone. The complex either does not bind to TSH receptors or does so with much lower affinity.     

Direct evidence that ganglioside is an integral component of the thyrotropin receptor.

Gangliosides were extracted from purified human and porcine thyrotropin (TSH) receptors (TSH-R) and were detected by probing with an 125I-labeled sialic acid-specific lectin, Limax flavus agglutinin. Gangliosides copurified with human and porcine TSH-R migrated between monosialoganglioside GM1 and disialoganglioside GD1a. Ceramide glycanase digestion of the purified human TSH-R-associated glycolipid confirmed its ganglioside nature. It was resistant to Vibrio cholerae sialidase, which digests all gangliosides except GM1, but was sensitive to Arthrobacter ureafaciens sialidase, which digests all gangliosides including GM1. These findings indicate that the human TSH-R contains ganglioside that belongs to the galactosyl(beta 1----3)-N-acetylgalactosaminyl (beta 1----4)-[N-acetylneuraminyl(alpha 2----3)]galactosyl(beta 1----4) glucosyl(beta 1----1)ceramide (GM1) family. Its intimate association with receptor protein implies a key role for ganglioside in the structure and function of the TSH-R.     

Functional characterization of GPIIb- and GPIIIa-specific monoclonal antibodies. Further evidence for the existence of agonist-specific activated states of the platelet fibrinogen receptor

In this work human platelet aggregation induced in vitro by ADP, collagen, arachidonic acid and U-46619 (a thromboxane A analogue) was used as a functional test to characterize 19 anti-GPIIb (M series) and anti2 GPIIIa (P series) monoclonal antibodies whose epitope location is known for most of them. Additionally, flow cytofluorimetry was applied to study the epitope expression of these antibodies in resting, EDTA-treated and SFLLRN peptide (thrombin receptor agonist)-activated platelets. Antibodies M6 (epitope located at GPIIbH 657-665), P23-7 (GPIIIa 114-122) and P40 (GPIIIa 262-303) bind weakly to only 43%, 70% and 66%, respectively, of the resting platelet population. This binding was enhanced in EDTA-treated and in activated platelets. Platelet activation enhances the apparent binding of most of the other antibodies. Further evidence on the existence of agonist-specific activated states of GPIIb/IIIa was provided by the agonist-dependent immunochemical inhibition in vitro of platelet aggregation by some of the anti-subunit antibodies studied here. The most notable cases are those of P40 and M6, which at 140 nM inhibit most, the platelet aggregation induced by arachidonic acid and U-46619. On the other hand, three of the most strong and agonist-independent inhibitors, P37 (GPIIIa 101-109), P97 and P95-2 (GPIIIa N-terminal half) bind to resting platelets with high affinity (5-8 nM), compete with each other for binding to GPIIb-IIIa and their epitopes are located at the N-terminal domain of GPIIIa, where the receptor ligand binding site(s) have been found. Given that the formation of activated GPIIb-IIIa (GPIIb-IIIa*) is the first step at which the anti-subunit antibodies can intervene as inhibitors and that agonist-specific inhibitors should block only agonist-specific steps, while nonspecific inhibitors should block steps common to all the agonists, then our present work support the hypothesis that there are different agonist-specific GPIIb-IIIa*s or, alternatively, different receptor environments, that can be specifically blocked by some of the antibodies. These results add to earlier evidence on agonist-dependent ligand specificity and activated states found for this and other integrins. Finally, the correlation between the in vitro inhibition of platelet aggregation and the antithrombotic activity in vivo is discussed for these antibodies.     

Functional characterization of GPIIb- and GPIIIa-specific monoclonal antibodies. Further evidence for the existence of agonist-specific activated states of the platelet fibrinogen receptor

In this work human platelet aggregation induced in vitro by ADP, collagen, arachidonic acid and U-46619 (a thromboxane A(2) analogue) was used as a functional test to characterize 19 anti-GPIIb (M series) and anti2 GPIIIa (P series) monoclonal antibodies whose epitope location is known for most of them. Additionally, flow cytofluorimetry was applied to study the epitope expression of these antibodies in resting, EDTA-treated and SFLLRN peptide (thrombin receptor agonist)-activated platelets. Antibodies M6 (epitope located at GPIIbH 657-665), P23-7 (GPIIIa 114-122) and P40 (GPIIIa 262-303) bind weakly to only 43%, 70% and 66%, respectively, of the resting platelet population. This binding was enhanced in EDTA-treated and in activated platelets. Platelet activation enhances the apparent binding of most of the other antibodies. Further evidence on the existence of agonist-specific activated states of GPIIb/IIIa was provided by the agonist-dependent immunochemical inhibition in vitro of platelet aggregation by some of the anti-subunit antibodies studied here. The most notable cases are those of P40 and M6, which at 140 nM inhibit most, the platelet aggregation induced by arachidonic acid and U-46619. On the other hand, three of the most strong and agonist-independent inhibitors, P37 (GPIIIa 101-109), P97 and P95-2 (GPIIIa N-terminal half) bind to resting platelets with high affinity (5-8 nM), compete with each other for binding to GPIIb-IIIa and their epitopes are located at the N-terminal domain of GPIIIa, where the receptor ligand binding site(s) have been found. Given that the formation of activated GPIIb-IIIa (GPIIb-IIIa*) is the first step at which the anti-subunit antibodies can intervene as inhibitors and that agonist-specific inhibitors should block only agonist-specific steps, while nonspecific inhibitors should block steps common to all the agonists, then our present work support the hypothesis that there are different agonist-specific GPIIb-IIIa*s or, alternatively, different receptor environments, that can be specifically blocked by some of the antibodies. These results add to earlier evidence on agonist-dependent ligand specificity and activated states found for this and other integrins. Finally, the correlation between the in vitro inhibition of platelet aggregation and the antithrombotic activity in vivo is discussed for these antibodies.