Proton transfer from the bulk to the bound ubiquinone QB of the reaction center in chromatophores of Rhodobacter sphaeroides: Retarded conveyance by neutral water

The mechanism of proton transfer from the bulk into the membrane protein interior was studied. The light-induced reduction of a bound ubiquinone molecule QB by the photosynthetic reaction center is accompanied by proton trapping. We used kinetic spectroscopy to measure (i) the electron transfer to QB (at 450 nm), (ii) the electrogenic proton delivery from the surface to the QB site (by electrochromic carotenoid response at 524 nm), and (iii) the disappearance of protons from the bulk solution (by pH indicators). The electron transfer to QB− and the proton-related electrogenesis proceeded with the same time constant of ≈100 μs (at pH 6.2), whereas the alkalinization in the bulk was distinctly delayed (τ ≈ 400 μs). We investigated the latter reaction as a function of the pH indicator concentration, the added pH buffers, and the temperature. The results led us to the following conclusions: (i) proton transfer from the surface-located acidic groups into the QB site followed the reduction of QB without measurable delay; (ii) the reprotonation of these surface groups by pH indicators and hydronium ions was impeded, supposedly, because of their slow diffusion in the surface water layer; and (iii) as a result, the protons were slowly donated by neutral water to refill the proton vacancies at the surface. It is conceivable that the same mechanism accounts for the delayed relaxation of the surface pH changes into the bulk observed previously with bacteriorhodopsin membranes and thylakoids. Concerning the coupling between proton pumps in bioenergetic membranes, our results imply a tendency for the transient confinement of protons at the membrane surface.

Neuronal migration is retarded in mice lacking the tissue plasminogen activator gene

Neuronal migration is a critical phase of brain development, where defects can lead to severe ataxia, mental retardation, and seizures. In the developing cerebellum, granule neurons turn on the gene for tissue plasminogen activator (tPA) as they begin their migration into the cerebellar molecular layer. Granule neurons both secrete tPA, an extracellular serine protease that converts the proenzyme plasminogen into the active protease plasmin, and bind tPA to their cell surface. In the nervous system, tPA activity is correlated with neurite outgrowth, neuronal migration, learning, and excitotoxic death. Here we show that compared with their normal counterparts, mice lacking the tPA gene (tPA−/−) have greater than 2-fold more migrating granule neurons in the cerebellar molecular layer during the most active phase of granule cell migration. A real-time analysis of granule cell migration in cerebellar slices of tPA−/− mice shows that granule neurons are migrating 51% as fast as granule neurons in slices from wild-type mice. These findings establish a direct role for tPA in facilitating neuronal migration, and they raise the possibility that late arriving neurons may have altered synaptic interactions.

The Kinetics of Zeaxanthin Formation Is Retarded by Dicyclohexylcarbodiimide1

The de-epoxidation of violaxanthin to antheraxanthin (Anth) and zeaxanthin (Zeax) in the xanthophyll cycle of higher plants and the generation of nonphotochemical fluorescence quenching in the antenna of photosystem II (PSII) are induced by acidification of the thylakoid lumen. Dicyclohexylcarbodiimide (DCCD) has been shown (a) to bind to lumen-exposed carboxy groups of antenna proteins and (b) to inhibit the pH-dependent fluorescence quenching. The possible influence of DCCD on the de-epoxidation reactions has been investigated in isolated pea (Pisum sativum L.) thylakoids. The Zeax formation was found to be slowed down in the presence of DCCD. The second step (Anth → Zeax) of the reaction sequence seemed to be more affected than the violaxanthin → Anth conversion. Comparative studies with antenna-depleted thylakoids from plants grown under intermittent light and with unstacked thylakoids were in agreement with the assumption that binding of DCCD to antenna proteins is probably responsible for the retarded kinetics. Analyses of the DCCD-induced alterations in different antenna subcomplexes showed that Zeax formation in the PSII antenna proteins was predominantly influenced by DCCD, whereas Zeax formation in photosystem I was nearly unaffected. Our data support the suggestion that DCCD binding to PSII antenna proteins is responsible for the observed alterations in xanthophyll conversion.

Retardation of skeletal development and cervical abnormalities in transgenic mice expressing a dominant-negative retinoic acid receptor in chondrogenic cells

Skeletal formation is a fundamental element of body patterning and is strictly regulated both temporally and spatially by a variety of molecules. Among these, retinoic acid (RA) has been shown to be involved in normal skeletal development. However, its pleiotropic effects have caused difficulty in identifying its crucial target cells and molecular mechanisms for each effect. Development of cartilage primordia is an important process in defining the skeletal structures. To address the role of RA in skeletal formation, we have generated mice expressing a dominant-negative retinoic acid receptor (RAR) in chondrogenic cells by using the type II collagen α1 promoter, and we have analyzed their phenotypes. These mice exhibited small cartilage primordia during development and retarded skeletal formation in both embryonic and postnatal periods. They also showed selective degeneration in their cervical vertebrae combined with homeotic transformations, but not in their extremities. The cervical phenotypes are reminiscent of phenotypes involving homeobox genes. We found that the expression of Hoxa-4 was indeed reduced in the cartilage primordia of cervical vertebrae of embryonic day 12.5 embryos. These observations demonstrate that endogenous RA acts directly on chondrogenic cells to promote skeletal growth in both embryonic and growing periods, and it regulates the proper formation of cervical vertebrae. Furthermore, RA apparently specifies the identities of the cervical vertebrae through the regulation of homeobox genes in the chondrogenic cells. Great similarities of the phenotypes between our mice and reported RAR knockout mice revealed that chondrogenic cells are a principal RA target during complex cascades of skeletal development.

RNA folding kinetics regulates translation of phage MS2 maturation gene

The gene for the maturation protein of the single-stranded RNA coliphage MS2 is preceded by an untranslated leader of 130 nt, which folds into a cloverleaf, i.e., three stem–loop structures enclosed by a long distance interaction (LDI). This LDI prevents translation because its 3′ moiety contains the Shine–Dalgarno sequence of the maturation gene. Previously, several observations suggested that folding of the cloverleaf is kinetically delayed, providing a time window for ribosomes to access the RNA. Here we present direct evidence for this model. In vitro experiments show that ribosome binding to the maturation gene is faster than refolding of the denatured cloverleaf. This folding delay appears related to special properties of the leader sequence. We have replaced the three stem–loop structures by a single five nt loop. This change does not affect the equilibrium structure of the LDI. Nevertheless, in this construct, the folding delay has virtually disappeared, suggesting that now the RNA folds faster than ribosomes can bind. Perturbation of the cloverleaf by an insertion makes the maturation start permanently accessible. A pseudorevertant that evolved from an infectious clone carrying the insertion had overcome this defect. It showed a wild-type folding delay before closing down the maturation gene. This experiment reveals the biological significance of retarded cloverleaf formation.

Pleiotropic defects in ataxia-telangiectasia protein-deficient mice

We have generated a mouse model for ataxia-telangiectasia by using gene targeting to generate mice that do not express the Atm protein. Atm-deficient mice are retarded in growth, do not produce mature sperm, and exhibit severe defects in T cell maturation while going on to develop thymomas. Atm-deficient fibroblasts grow poorly in culture and display a high level of double-stranded chromosome breaks. Atm-deficient thymocytes undergo spontaneous apoptosis in vitro significantly more than controls. Atm-deficient mice then exhibit many of the same symptoms found in ataxia-telangiectasia patients and in cells derived from them. Furthermore, we demonstrate that the Atm protein exists as two discrete molecular species, and that loss of one or of both of these can lead to the development of the disease.

Blastic transformation of p53-deficient bone marrow cells by p210bcr/abl tyrosine kinase

Blastic transformation of chronic myelogenous leukemia (CML) is characterized by the presence of nonrandom, secondary genetic abnormalities in the majority of Philadelphia1 clones, and loss of p53 tumor suppressor gene function is a consistent finding in 25–30% of CML blast crisis patients. To test whether the functional loss of p53 plays a direct role in the transition of chronic phase to blast crisis, bone marrow cells from p53+/+ or p53−/− mice were infected with a retrovirus carrying either the wild-type BCR/ABL or the inactive kinase-deficient mutant, and were assessed for colony-forming ability. Infection of p53−/− marrow cells with wild-type BCR/ABL, but not with the kinase-deficient mutant, enhanced formation of hematopoietic colonies and induced growth factor independence at high frequency, as compared with p53+/+ marrow cells. These effects were suppressed when p53−/− marrow cells were coinfected with BCR/ABL and wild-type p53. p53-deficient BCR/ABL-infected marrow cells had a proliferative advantage, as reflected by an increase in the fraction of S+G2 phase cells and a decrease in the number of apoptotic cells. Immunophenotyping and morphological analysis revealed that BCR/ABL-positive p53−/− cells were much less differentiated than their BCR/ABL-positive p53+/+ counterparts. Injection of immunodeficient mice with BCR/ABL-positive p53−/− cells produced a transplantable, highly aggressive, poorly differentiated acute myelogenous leukemia. In marked contrast, the disease process in mice injected with BCR/ABL-positive p53+/+ marrow cells was characterized by cell infiltrates with a more differentiated phenotype and was significantly retarded, as indicated by a much longer survival of leukemic mice. Together, these findings directly demonstrate that loss of p53 function plays an important role in blast transformation in CML.

Phenotypic alterations in insulin-deficient mutant mice

Two mouse insulin genes, Ins1 and Ins2, were disrupted and lacZ was inserted at the Ins2 locus by gene targeting. Double nullizygous insulin-deficient pups were growth-retarded. They did not show any glycosuria at birth but soon after suckling developed diabetes mellitus with ketoacidosis and liver steatosis and died within 48 h. Interestingly, insulin deficiency did not preclude pancreas organogenesis and the appearance of the various cell types of the endocrine pancreas. The presence of lacZ expressing β cells and glucagon-positive α cells was demonstrated by cytochemistry and immunocytochemistry. Reverse transcription-coupled PCR analysis showed that somatostatin and pancreatic polypeptide mRNAs were present, although at reduced levels, accounting for the presence also of δ and pancreatic polypeptide cells, respectively. Morphometric analysis revealed enlarged islets of Langherans in the pancreas from insulin-deficient pups, suggesting that insulin might function as a negative regulator of islet cell growth. Whether insulin controls the growth of specific islet cell types and the molecular basis for this action remain to be elucidated.

Rump white inversion in the mouse disrupts dipeptidyl aminopeptidase-like protein 6 and causes dysregulation of Kit expression

The mouse rump white (Rw) mutation causes a pigmentation defect in heterozygotes and embryonic lethality in homozygotes. At embryonic day (E) 7.5, Rw/Rw embryos are retarded in growth, fail to complete neurulation and die around E 9.5. The Rw mutation is associated with a chromosomal inversion spanning 30 cM of the proximal portion of mouse chromosome 5. The Rw embryonic lethality is complemented by the W19H deletion, which spans the distal boundary of the Rw inversion, suggesting that the Rw lethality is not caused by the disruption of a gene at the distal end of the inversion. Here, we report the molecular characterization of sequences disrupted by both inversion breakpoints. These studies indicate that the distal breakpoint of the inversion is associated with ectopic Kit expression and therefore may be responsible for the dominant pigmentation defect in Rw/+ mice; whereas the recessive lethality of Rw is probably due to the disruption of the gene encoding dipeptidyl aminopeptidase-like protein 6, Dpp6 [Wada, K., Yokotani, N., Hunter, C., Doi, K., Wenthold, R. J. & Shimasaki, S. (1992) Proc. Natl. Acad. Sci. USA 89, 197–201] located at the proximal inversion breakpoint.

Elucidation of a PTS–Carbohydrate Chemotactic Signal Pathway in Escherichia coli Using a Time-resolved Behavioral Assay

Chemotaxis of Escherichia coli toward phosphotransferase systems (PTSs)–carbohydrates requires phosphoenolpyruvate-dependent PTSs as well as the chemotaxis response regulator CheY and its kinase, CheA. Responses initiated by flash photorelease of a PTS substrates d-glucose and its nonmetabolizable analog methyl α-d-glucopyranoside were measured with 33-ms time resolution using computer-assisted motion analysis. This, together with chemotactic mutants, has allowed us to map out and characterize the PTS chemotactic signal pathway. The responses were absent in mutants lacking the general PTS enzymes EI or HPr, elevated in PTS transport mutants, retarded in mutants lacking CheZ, a catalyst of CheY autodephosphorylation, and severely reduced in mutants with impaired methyl-accepting chemotaxis protein (MCP) signaling activity. Response kinetics were comparable to those triggered by MCP attractant ligands over most of the response range, the most rapid being 11.7 ± 3.1 s−1. The response threshold was <10 nM for glucose. Responses to methyl α-d-glucopyranoside had a higher threshold, commensurate with a lower PTS affinity, but were otherwise kinetically indistinguishable. These facts provide evidence for a single pathway in which the PTS chemotactic signal is relayed rapidly to MCP–CheW–CheA signaling complexes that effect subsequent amplification and slower CheY dephosphorylation. The high sensitivity indicates that this signal is generated by transport-induced dephosphorylation of the PTS rather than phosphoenolpyruvate consumption.

Molecular heterochrony in the early development of Drosophila

Heterochrony, the relative change of developmental timing, is one of the major modes of macroevolutionary change; it identifies temporally disassociated units of developmental evolution. Here, we report the results of a fine-scale temporal study for the expression of the developmental gene hairy and morphological development in three species of Drosophila, D. melanogaster, D. simulans, and D. pseudoobscura. The results suggest that between and among closely related species, temporal displacement of ontogenetic trajectory is detected even at the earliest stage of development. Overall, D. simulans shows the earliest expression, followed by D. melanogaster, and then by D. pseudoobscura. Setting D. melanogaster as the standard, we find the approximate time to full expression is accelerated by 13 min, 48 s in D. simulans and retarded by 24 min in D. pseudoobscura. Morphologically, again with D. melanogaster setting the standard, initiation of cellularization is faster in D. simulans by 15 min, 42 s; and initiation of morphogenesis is faster in D. simulans by 18 min, 7 s. These results seem to be consistent with the finding that the approximate time to full expression of hairy is accelerated by 13 min, 48 s in D. simulans. On the other hand, the same morphological events are delayed by 5 min, 32 s, and by 11 min, 32 s, respectively, in D. pseudoobscura. These delays are small, compared with the 24-min delay in full expression. The timing changes, in total, seem consistent with continuous phyletic evolution of temporal trajectories. Finally, we speculate that epigenetic interactions of hairy expression timing and cell-cycle timing may have led to morphological differences in the terminal system of the larvae.

A mouse CD8 T cell-mediated acute autoimmune diabetes independent of the perforin and Fas cytotoxic pathways: Possible role of membrane TNF

Double transgenic mice [rat insulin promoter (RIP)-tumor necrosis factor (TNF) and RIP-CD80] whose pancreatic β cells release TNF and bear CD80 all develop an acute early (6 wk) and lethal diabetes mediated by CD8 T cells. The first ultrastructural changes observed in β cells, so far unreported, are focal lesions of endoplasmic reticulum swelling at the points of contact with islet-infiltrating lymphoblasts, followed by cytoplasmic, but not nuclear, apoptosis. Such double transgenic mice were made defective in either the perforin, Fas, or TNF pathways. Remarkably, diabetes was found to be totally independent of perforin and Fas. Mice lacking TNF receptor (TNFR) II had no or late diabetes, but only a minority had severe insulitis. Mice lacking the TNF-lymphotoxin (LTα) locus (whose sole source of TNF are the β cells) all had insulitis comparable to that of nondefective mice, but no diabetes or a retarded and milder form, with lesions suggesting different mechanisms of injury. Because both TNFR II and TNF-LTα mutations have complex effects on the immune system, these data do not formally incriminate membrane TNF as the major T cell mediator of this acute autoimmune diabetes; nevertheless, in the absence of involvement of the perforin or Fas cytotoxic pathways, membrane TNF appears to be the likeliest candidate.

Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells

Epithelial (E)-cadherin and its associated cytoplasmic proteins (α-, β-, and γ-catenins) are important mediators of epithelial cell–cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin–catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.

Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae bile resistance, virulence factor expression, and intestinal colonization

The transmembrane transcriptional activators ToxR and TcpP modulate expression of Vibrio cholerae virulence factors by exerting control over toxT, which encodes the cytoplasmic transcriptional activator of the ctx, tcp, and acf virulence genes. However, ToxR, independently of TcpP and ToxT, activates and represses transcription of the genes encoding two outer-membrane porins, OmpU and OmpT. To determine the role of ToxR-dependent porin regulation in V. cholerae pathogenesis, the ToxR-activated ompU promoter was used to drive ompT transcription in a strain lacking OmpU. Likewise, the ToxR-repressed ompT promoter was used to drive ompU transcription in a strain lacking both ToxR and OmpT. This strategy allowed the generation of a toxR+ strain that expresses OmpT in place of OmpU, and a toxR− strain that expresses OmpU in place of OmpT. Growth rates in the presence of bile salts and other anionic detergents were retarded for the toxR+ V. cholerae expressing OmpT in place of OmpU, but increased in toxR− V. cholerae expressing OmpU in place of OmpT. Additionally, the toxR+ V. cholerae expressing OmpT in place of OmpU expressed less cholera toxin and toxin-coregulated pilus, and this effect was shown to be caused by reduced toxT transcription in this strain. Finally, the toxR+ V. cholerae expressing OmpT in place of OmpU was ≈100-fold reduced in its ability to colonize the infant-mouse intestine. Our results indicate that ToxR-dependent modulation of the outer membrane porins OmpU and OmpT is critical for V. cholerae bile resistance, virulence factor expression, and intestinal colonization.