Developmental and Hormonal Regulation of Genes Coding for Proline-Rich Proteins in Female Inflorescences and Kernels of Maize1

The pattern of expression of two genes coding for proteins rich in proline, HyPRP (hybrid proline-rich protein) and HRGP (hydroxyproline-rich glycoprotein), has been studied in maize (Zea mays) embryos by RNA analysis and in situ hybridization. mRNA accumulation is high during the first 20 d after pollination, and disappears in the maturation stages of embryogenesis. The two genes are also expressed during the development of the pistillate spikelet and during the first stages of embryo development in adjacent but different tissues. HyPRP mRNA accumulates mainly in the scutellum and HRGP mRNA mainly in the embryo axis and the suspensor. The two genes appear to be under the control of different regulatory pathways during embryogenesis. We show that HyPRP is repressed by abscisic acid and stress treatments, with the exception of cold treatment. In contrast, HRGP is affected positively by specific stress treatments.

Hormonal Control of Parthenocarpic Ovary Growth by the Apical Shoot in Pea1

The role of the apical shoot as a source of inhibitors preventing fruit growth in the absence of a stimulus (e.g. pollination or application of gibberellic acid) has been investigated in pea (Pisum sativum L.). Plant decapitation stimulated parthenocarpic growth, even in derooted plants, and this effect was counteracted by the application of indole acetic acid (IAA) or abscisic acid (ABA) in agar blocks to the severed stump. The treatment of unpollinated ovaries with gibberellic acid blocked the effect of IAA or ABA applied to the stump. [3H]IAA and [3H]ABA applied to the stump were transported basipetally, and [3H]ABA but not [3H]IAA was also detected in unpollinated ovaries. The concentration of ABA in unpollinated ovaries increased significantly in the absence of a promotive stimulus. The application of IAA to the stump enhanced by 2- to 5-fold the concentration of ABA in the inhibited ovary, whereas the inhibition of IAA transport from the apical shoot by triiodobenzoic acid decreased the ovary content of ABA (to approximately one-half). Triiodobenzoic acid alone, however, was unable to stimulate ovary growth. Thus, in addition to removing IAA transport from the apical shoot, the accumulation of a promotive factor is also necessary to induce parthenocarpic growth in decapitated plants.

Formate Dehydrogenase, an Enzyme of Anaerobic Metabolism, Is Induced by Iron Deficiency in Barley Roots1

To identify the proteins induced by Fe deficiency, we have compared the proteins of Fe-sufficient and Fe-deficient barley (Hordeum vulgare L.) roots by two-dimensional polyacrylamide gel electrophoresis. Peptide sequence analysis of induced proteins revealed that formate dehydrogenase (FDH), adenine phosphoribosyltransferase, and the Ids3 gene product (for Fe deficiency-specific) increased in Fe-deficient roots. FDH enzyme activity was detected in Fe-deficient roots but not in Fe-sufficient roots. A cDNA encoding FDH (Fdh) was cloned and sequenced. Fdh expression was induced by Fe deficiency. Fdh was also expressed under anaerobic stress and its expression was more rapid than that induced by Fe deficiency. Thus, the expression of Fdh observed in Fe-deficient barley roots appeared to be a secondary effect caused by oxygen deficiency in Fe-deficient plants.

Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. Rom1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and rom1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and rom1. The critical threshold for the combined abundance of rds and rom1 is ≈60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.

Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

Folliculostellate cell network: A route for long-distance communication in the anterior pituitary

All higher life forms critically depend on hormones being rhythmically released by the anterior pituitary. The proper functioning of this master gland is dynamically controlled by a complex set of regulatory mechanisms that ultimately determine the fine tuning of the excitable endocrine cells, all of them heterogeneously distributed throughout the gland. Here, we provide evidence for an intrapituitary communication system by which information is transferred via the network of nonendocrine folliculostellate (FS) cells. Local electrical stimulation of FS cells in acute pituitary slices triggered cytosolic calcium waves, which propagated to other FS cells by signaling through gap junctions. Calcium wave initiation was because of the membrane excitability of FS cells, hitherto classified as silent cells. FS cell coupling could relay information between opposite regions of the gland. Because FS cells respond to central and peripheral stimuli and dialogue with endocrine cells, the form of large-scale intrapituitary communication described here may provide an efficient mechanism that orchestrates anterior pituitary functioning in response to physiological needs.

Leukemia inhibitory factor induces differentiation of pituitary corticotroph function: an immuno-neuroendocrine phenotypic switch.

Leukemia inhibitory factor (LIF) promotes differentiated cell function in several systems. We recently reported LIF and LIF receptor expression in human fetal pituitary corticotrophs in vivo and demonstrated LIF stimulation of adrenocorticotrophin (ACTH) transcription in vitro, suggesting a role for LIF in corticotroph development. We therefore assessed the action of LIF on proliferating murine corticotroph cells (AtT20). LIF impairs proliferation of AtT20 cells (25% reduction versus control, P < 0.03), while simultaneously enhancing ACTH secretion (2-fold, P < 0.001) and augmenting ACTH responsiveness to corticotrophin-releasing hormone (CRH) action (4-fold, P < 0.001). This attenuation of cell growth is due to a block of cell cycle progression from G1 into S phase, as measured by flow cytometric analysis (24 +/- 0.8 versus 11.57 +/- 1.5, P < 0.001). Using bromodeoxyuridine incorporation assays, loss of cells in S phase was confirmed (25 +/- 0.08 to 9.4 +/- 1.4, P < 0.008). In contrast, CRH induced the G2/M phase (3.6 +/- 0.2 to 15.4 +/- 3, P < 0.001). This effect was blunted by LIF (P < 0.001 versus CRH alone). Cyclin A mRNA levels, which decline in S phase, were stimulated 3.5-fold by LIF and markedly suppressed by CRH. These results indicate a LIF-induced cell cycle block occurring at G1/S in corticotroph cells. Thus, LIF reduces proliferation, enhances ACTH secretion, and potentiates effects of CRH on ACTH secretion while blocking effects of CRH on the cell cycle. Responses of these three markers of differentiated corticotroph function indicate LIF to be a differentiation factor for pituitary corticotroph cells by preferential phenotypic switching from proliferative to synthetic.

APC mutations in colorectal tumors with mismatch repair deficiency.

We have investigated the influence of genetic instability [replication error (RER) phenotype] on APC (adenomatous polyposis coli), a gene thought to initiate colorectal tumorigenesis. The prevalence of APC mutations was similar in RER and non-RER tumors, indicating that both tumor types share this step in neoplastic transformation. However, in a total of 101 sequenced mutations, we noted a substantial excess of APC frameshift mutations in the RER cases (70% in RER tumors versus 47% in non-RER tumors, P < 0.04). These frameshifts were characteristic of mutations arising in cells deficient in DNA mismatch repair, with a predilection for mononucleotide repeats in the RER tumors (P < 0.0002), particularly (A)n tracts (P < 0.00007). These findings suggest that the genetic instability that is reflected by the RER phenotype precedes, and is responsible for, APC mutation in RER large bowel tumors and have important implications for understanding the very earliest stages of neoplasia in patients with tumors deficient in mismatch repair.

P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development.

A novel OTX-related homeodomain transcription factor has been identified on the basis of its ability to interact with the transactivation domain of the pituitary-specific POU domain protein, Pit-1. This factor, referred to as P-OTX (pituitary OTX-related factor), is expressed in primordial Rathke's pouch, oral epithelium, first bronchial arch, duodenum, and hindlimb. In the developing anterior pituitary, it is expressed in all regions from which cells with distinct phenotypes will emerge in the mature gland. P-OTX is able to independently activate and to synergize with Pit-1 on pituitary-specific target gene promoters. Therefore, P-OTX may subserve functions in generating both precursor and specific cell phenotypes in the anterior pituitary gland and in several other organs.

Growth retardation and cysteine deficiency in gamma-glutamyl transpeptidase-deficient mice.

gamma-Glutamyl transpeptidase (GGT) is an ectoenzyme that catalyzes the first step in the cleavage of glutathione (GSH) and plays an essential role in the metabolism of GSH and GSH conjugates of carcinogens, toxins, and eicosanoids. To learn more about the role of GGT in metabolism in vivo, we used embryonic stem cell technology to generate GGT-deficient (GGTm1/GGTm1) mice. GGT-deficient mice appear normal at birth but grow slowly and by 6 weeks are about half the weight of wild-type mice. They are sexually immature, develop cataracts, and have coats with a gray cast. Most die between 10 and 18 weeks. Plasma and urine GSH levels in the GGTm1/GGTm1 mice are elevated 6-fold and 2500-fold, respectively, compared with wild-type mice. Tissue GSH levels are markedly reduced in eye, liver, and pancreas. Plasma cyst(e)ine levels in GGTm1/GGTm1 mice are reduced to approximately 20% of wild-type mice. Oral administration of N-acetylcysteine to GGTm1/GGTm1 mice results in normal growth rates and partially restores the normal agouti coat color. These findings demonstrate the importance of GGT and the gamma-glutamyl cycle in cysteine and GSH homeostasis.

Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase.

Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic. In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses. We report herein the x-ray crystal structure of human MIF to 2.6 angstrom resolution. The protein is a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has an additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three beta-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis. Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules. The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

Nerve growth factor in the anterior pituitary: localization in mammotroph cells and cosecretion with prolactin by a dopamine-regulated mechanism.

Nerve growth factor (NGF) is well characterized for its neurotrophic actions on peripheral sensory and sympathetic neurons and on central cholinergic neurons of the basal forebrain. Recent evidence, however, has shown high levels of NGF to be present in a variety of biological fluids after inflammatory and autoimmune responses, suggesting that NGF is a mediator of immune interactions. Increased NGF serum levels have been reported in both humans and experimental animal models of psychological and physical stress, thus implicating NGF in neuroendocrine interactions as well. The possible source(s) and the regulatory mechanisms involved in the control of serum NGF levels, however, still remain to be elucidated. We now report the presence of both NGF gene transcripts and protein in the anterior pituitary. Immunofluorescence analysis indicated that hypophysial NGF is selectively localized in mammotroph cells and stored in secretory granules. NGF is cosecreted with prolactin from mammotroph cells by a neurotransmitter-dependent mechanism that can be pharmacologically regulated. Activation of the dopamine D2 receptor subtype, which physiologically controls prolactin release, resulted in a complete inhibition of vasoactive intestinal peptide-stimulated NGF secretion in vitro, whereas the specific D2 antagonist (-)-sulpiride stimulated NGF secretion in vivo, suggesting that the anterior pituitary is a possible source of circulating NGF. Given the increased NGF serum levels in stressful conditions and the newly recognized immunoregulatory function of this protein, NGF, together with prolactin, may thus be envisaged as an immunological alerting signal under neuronal control.

Down-regulation of pituitary receptors for luteinizing hormone-releasing hormone (LH-RH) in rats by LH-RH antagonist Cetrorelix.

Antagonists of luteinizing hormone-releasing hormone (LH-RH), unlike the LH-RH agonists, suppress gonadotropins and sex steroid secretion immediately after administration, without initial stimulatory effects. [Ac-D-Nal(2)1,D-Ph(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-R H (SB-75; Cetrorelix) is a modern, potent antagonistic analog of LH-RH. In this study, the binding characteristics of receptors for LH-RH in membrane fractions from rat anterior pituitaries were investigated after a single injection of Cetrorelix at a dose of 100 microg per rat. To determine whether the treatment with Cetrorelix can affect the concentration of measurable LH-RH binding sites, we applied an in vitro method to desaturate LH-RH receptors by chaotropic agents such as manganous chloride (MnCl2) and ammonium thiocyanate (NH4SCN). Our results show that the percentages of occupied LH-RH receptors at 1, 3, and 6 h after administration of Cetrorelix were approximately 28%, 14%, and 10%, respectively, of total receptors. At later time intervals, we could not detect occupied LH-RH binding sites. Ligand competition assays, following in vitro desaturation, demonstrated that rat pituitary LH-RH receptors were significantly (P < 0.01) down-regulated for at least 72 h after administration of Cetrorelix. The lowest receptor concentration was found 3-6 h after Cetrorelix treatment and a recovery in receptor number began within approximately 24 h. The down-regulation of LH-RH binding sites induced by Cetrorelix was accompanied by serum LH and testosterone suppression. Higher LH-RH receptor concentrations coincided with elevated serum hormone levels at later time intervals. Our results indicate that administration of LH-RH antagonist Cetrorelix produces a marked down-regulation of pituitary receptors for LH-RH and not merely an occupancy of binding sites.