Wild-type Escherichia coli grows on the chitin disaccharide, N,N′-diacetylchitobiose, by expressing the cel operon

We report here that wild-type Escherichia coli can grow on the chitin disaccharide, N,N′-diacetylchitobiose (GlcNAc)2, as the sole source of carbon. Transposon mutants were isolated that were unable to ferment (GlcNAc)2 but grew normally on the monosaccharide GlcNAc. One such mutant was used to screen a wild-type E. coli genomic cosmid library for restoration of (GlcNAc)2 fermentation. A partial sequence analysis of the isolated fragment mapped the clone to the (previously sequenced) E. coli genome between 39.0 and 39.2 min. The nucleotide ORFs at this region had been previously assigned to code for a “cryptic” cellobiose utilization (cel) operon. We report here, however, that functional analysis of the operon, including growth and chemotaxis, reveal that it encodes a set of proteins that are not cryptic, but are induced by (GlcNAc)2 and catabolize the disaccharide. We therefore propose to rename the cel operon as the chb (N,N′-diacetylchitobiose) operon, with the letter designation of the genes of the operon to be reassigned consistent with the nomenclature based on functional characterization of the gene products as follows: celA to chbB, celB to chbC, celC to chbA, celD to chbR, and celF to chbF. Furthermore, sequencing evidence indicates that the operon contains an additional gene of unknown function to be designated as chbG. Thus, the overall gene sequence is to be named chbBCARFG.

Subcellular Localization of Myosin-V in the B16 Melanoma Cells, a Wild-type Cell Line for the dilute Gene

The discovery that the dilute gene encodes a class V myosin led to the hypothesis that this molecular motor is involved in melanosome transport and/or dendrite outgrowth in mammalian melanocytes. The present studies were undertaken to gain insight into the subcellular distribution of myosin-V in the melanoma cell line B16-F10, which is wild-type for the dilute gene. Immunofluorescence studies showed some degree of superimposed labeling of myosin-V with melanosomes that predominated at the cell periphery. A subcellular fraction highly enriched in melanosomes was also enriched in myosin-V based on Western blot analysis. Immunoelectron microscopy showed myosin-V labeling associated with melanosomes and other organelles. The stimulation of B16 cells with the α-melanocyte-stimulating hormone led to a significant increase in myosin-V expression. This is the first evidence that a cAMP signaling pathway might regulate the dilute gene expression. Immunofluorescence also showed an intense labeling of myosin-V independent of melanosomes that was observed within the dendrites and at the perinuclear region. Although the results presented herein are consistent with the hypothesis that myosin-V might act as a motor for melanosome translocation, they also suggest a broader cytoplasmic function for myosin-V, acting on other types of organelles or in cytoskeletal dynamics.

Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specific in vitro priming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules for immunotherapeutical approaches.

Cytoplasmically sequestered wild-type p53 protein in neuroblastoma is relocated to the nucleus by a C-terminal peptide

Cytoplasmic sequestration of wild-type p53 protein occurs in a subset of primary human tumors including breast cancer, colon cancer, and neuroblastoma (NB). The sequestered p53 localizes to punctate cytoplasmic structures that represent large protein aggregates. One functional consequence of this blocked nuclear access is impairment of the p53-mediated G1 checkpoint after DNA damage. Here we show that cytoplasmic p53 from NB cells is incompetent for specific DNA binding, probably due to its sequestration. Importantly, the C-terminal domain of sequestered p53 is masked, as indicated by the failure of a C-terminally directed antibody to detect p53 in these structures. To determine (i) which domain of p53 is involved in the aggregation and (ii) whether this phenotype is potentially reversible, we generated stable NB sublines that coexpress the soluble C-terminal mouse p53 peptide DD1 (amino acids 302–390). A dramatic phenotypic reversion occurred in five of five lines. The presence of DD1 blocked the sequestration of wild-type p53 and relocated it to the nucleus, where it accumulated. The nuclear translocation is due to shuttling of wild-type p53 by heteroligomerization to DD1, as shown by coimmunoprecipitation. As expected, the nuclear heterocomplexes were functionally inactive, since DD1 is a dominant negative inhibitor of wild-type p53. In summary, we show that nuclear access of p53 can be restored in NB cells.

Independent evolution of the prochlorophyte and green plant chlorophyll a/b light-harvesting proteins

The prochlorophytes are oxygenic prokaryotes differing from other cyanobacteria by the presence of a light-harvesting system containing both chlorophylls (Chls) a and b and by the absence of phycobilins. We demonstrate here that the Chl a/b binding proteins from all three known prochlorophyte genera are closely related to IsiA, a cyanobacterial Chl a-binding protein induced by iron starvation, and to CP43, a constitutively expressed Chl a antenna protein of photosystem II. The prochlorophyte Chl a/b protein (pcb) genes do not belong to the extended gene family encoding eukaryotic Chl a/b and Chl a/c light-harvesting proteins. Although higher plants and prochlorophytes share common pigment complements, their light-harvesting systems have evolved independently.

A novel precursor recognition element facilitates posttranslational binding to the signal recognition particle in chloroplasts

Signal recognition particles (SRPs) in the cytosols of prokaryotes and eukaryotes are used to target proteins to cytoplasmic membranes and the endoplasmic reticulum, respectively. The mechanism of targeting relies on cotranslational SRP binding to hydrophobic signal sequences. An organellar SRP identified in chloroplasts (cpSRP) is unusual in that it functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins. In assays that reconstitute thylakoid integration of the light harvesting chlorophyll-binding protein (LHCP), stromal cpSRP binds LHCP posttranslationally to form a cpSRP/LHCP transit complex, which is believed to represent the LHCP form targeted to thylakoids. In this investigation, we have identified an 18-aa sequence motif in LHCP (L18) that, along with a hydrophobic domain, is required for transit complex formation. Fusion of L18 to the amino terminus of an endoplasmic reticulum-targeted protein, preprolactin, led to transit complex formation whereas wild-type preprolactin exhibited no ability to form a transit complex. In addition, a synthetic L18 peptide, which competed with LHCP for transit complex formation, caused a parallel inhibition of LHCP integration. Translocation of proteins by the thylakoid Sec and Delta pH transport systems was unaffected by the highest concentration of L18 peptide examined. Our data indicate that a motif contained in L18 functions in precursor recruitment to the posttranslational SRP pathway, one of at least four different thylakoid sorting pathways used by chloroplasts.

Adhesion to fibronectin stimulates proliferation of wild-type and bcr/abl-transfected murine hematopoietic cells

Cells of most tissues require adhesion to a surface to grow. However, for hematopoietic cells, both stimulation and inhibition of proliferation by adhesion to extracellular matrix components have been described. Furthermore, it has been suggested that progenitor cells from chronic myelogenous leukemia show decreased β1 integrin-mediated adhesion to fibronectin, resulting in increased proliferation and abnormal trafficking. However, we show here that the chronic myelogenous leukemia-specific fusion protein p210bcr/abl stimulates the expression of α5β1 integrins and induces adhesion to fibronectin when expressed in the myeloid cell line 32D. Moreover, proliferation of both p210bcr/abl-transfected 32D (32Dp210) cells and untransfected 32D cells is stimulated by immobilized fibronectin. Cell cycle analysis revealed that nonadherent 32D and 32Dp210 cells are arrested in late G1 or early S phase, whereas the adherent fractions continue cycling. Although both adherent and nonadherent p210bcr/abl-transfected and parental 32D cells express equal amounts of cyclin A, a protein necessary for cell cycle progression at the G1/S boundary, cyclin A complexes immunoprecipitated from 32D cells cultured on immobilized fibronectin were found to be catalytically inactive in nonadherent but not in adherent cells. In addition, as compared with untransfected 32D cells, cyclin A immunoprecipitates from 32Dp210 cells exhibited a greatly elevated kinase activity and remained partially active irrespective of the adhesion status. The lack of cyclin A/cyclin-dependent kinase (CDK) 2 activity in nonadherent 32D cells appeared to result from increased expression and cyclin A complex formation of the CDK inhibitor p27Kip1. Taken together, our results indicate that adhesion stimulates cell cycle progression of hematopoietic cells by down-regulation of p27Kip1, resulting in activation of cyclin A/CDK2 complexes and subsequent transition through the G1/S adhesion checkpoint.

Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts

Several groups have attempted to develop gene therapy strategies to treat cancer via introduction of the wild-type (wt) p53 cDNA into cancer cells. Unfortunately, these approaches do not result in regulated expression of the p53 gene and do not reduce expression of the mutant p53 that is overexpressed in cancerous cells. These shortcomings may greatly limit the utility of this gene replacement approach. We describe an alternative strategy with trans-splicing ribozymes that can simultaneously reduce mutant p53 expression and restore wt p53 activity in various human cancers. The ribozyme accomplished such conversion by repairing defective p53 mRNAs with high fidelity and specificity. The corrected transcripts were translated to produce functional p53 that can transactivate p53-responsive promoters and down-modulate expression of the multidrug resistance (MDR1) gene promoter. The level of wt p53 activity generated was significant, resulting in a 23-fold induction of a p53-responsive promoter and a 3-fold reduction in MDR1 promoter expression in transfected cancer cells. Once efficient delivery systems are developed, this strategy should prove useful for making human cancers more responsive to p53 activity and more sensitive to chemotherapeutic agents.

Femtosecond dynamics of the forbidden carotenoid S1 state in light-harvesting complexes of purple bacteria observed after two-photon excitation

Time-resolved excited-state absorption intensities after direct two-photon excitation of the carotenoid S1 state are reported for light-harvesting complexes of purple bacteria. Direct excitation of the carotenoid S1 state enables the measurement of subsequent dynamics on a fs time scale without interference from higher excited states, such as the optically allowed S2 state or the recently discovered dark state situated between S1 and S2. The lifetimes of the carotenoid S1 states in the B800-B850 complex and B800-B820 complex of Rhodopseudomonas acidophila are 7 ± 0.5 ps and 6 ± 0.5 ps, respectively, and in the light-harvesting complex 2 of Rhodobacter sphaeroides ≈1.9 ± 0.5 ps. These results explain the differences in the carotenoid to bacteriochlorophyll energy transfer efficiency after S2 excitation. In Rps. acidophila the carotenoid S1 to bacteriochlorophyll energy transfer is found to be quite inefficient (φET1 <28%) whereas in Rb. sphaeroides this energy transfer is very efficient (φET1 ≈80%). The results are rationalized by calculations of the ensemble averaged time constants. We find that the Car S1 → B800 electronic energy transfer (EET) pathway (≈85%) dominates over Car S1 → B850 EET (≈15%) in Rb. sphaeroides, whereas in Rps. acidophila the Car S1 → B850 EET (≈60%) is more efficient than the Car S1 → B800 EET (≈40%). The individual electronic couplings for the Car S1 → BChl energy transfer are estimated to be approximately 5–26 cm−1. A major contribution to the difference between the energy transfer efficiencies can be explained by different Car S1 energy gaps in the two species.

A response regulator of cyanobacteria integrates diverse environmental signals and is critical for survival under extreme conditions

Microorganisms must sense their environment and rapidly tune their metabolism to ambient conditions to efficiently use available resources. We have identified a gene encoding a response regulator, NblR, that complements a cyanobacterial mutant unable to degrade its light-harvesting complex (phycobilisome), in response to nutrient deprivation. Cells of the nblR mutant (i) have more phycobilisomes than wild-type cells during nutrient-replete growth, (ii) do not degrade phycobilisomes during sulfur, nitrogen, or phosphorus limitation, (iii) cannot properly modulate the phycobilisome level during exposure to high light, and (iv) die rapidly when starved for either sulfur or nitrogen, or when exposed to high light. Apart from regulation of phycobilisome degradation, NblR modulates additional functions critical for cell survival during nutrient-limited and high-light conditions. NblR does not appear to be involved in acclimation responses that occur only during a specific nutrient limitation. In contrast, it controls at least some of the general acclimation responses; those that occur during any of a number of different stress conditions. NblR plays a pivotal role in integrating different environmental signals that link the metabolism of the cell to light harvesting capabilities and the activities of the photosynthetic apparatus; this modulation is critical for cell survival.

Collecting and harvesting biological data: the GPCRDB and NucleaRDB information systems

The amount of genomic and proteomic data that is entered each day into databases and the experimental literature is outstripping the ability of experimental scientists to keep pace. While generic databases derived from automated curation efforts are useful, most biological scientists tend to focus on a class or family of molecules and their biological impact. Consequently, there is a need for molecular class-specific or other specialized databases. Such databases collect and organize data around a single topic or class of molecules. If curated well, such systems are extremely useful as they allow experimental scientists to obtain a large portion of the available data most relevant to their needs from a single source. We are involved in the development of two such databases with substantial pharmacological relevance. These are the GPCRDB and NucleaRDB information systems, which collect and disseminate data related to G protein-coupled receptors and intra-nuclear hormone receptors, respectively. The GPCRDB was a pilot project aimed at building a generic molecular class-specific database capable of dealing with highly heterogeneous data. A first version of the GPCRDB project has been completed and it is routinely used by thousands of scientists. The NucleaRDB was started recently as an application of the concept for the generalization of this technology. The GPCRDB is available via the WWW at http://www.gpcr.org/7tm/ and the NucleaRDB at http://www.receptors.org/NR/.

Chlorophyll and carotenoid binding in a simple red algal light-harvesting complex crosses phylogenetic lines

The membrane proteins of peripheral light-harvesting complexes (LHCs) bind chlorophylls and carotenoids and transfer energy to the reaction centers for photosynthesis. LHCs of chlorophytes, chromophytes, dinophytes, and rhodophytes are similar in that they have three transmembrane regions and several highly conserved Chl-binding residues. All LHCs bind Chl a, but in specific taxa certain characteristic pigments accompany Chl a: Chl b and lutein in chlorophytes, Chl c and fucoxanthin in chromophytes, Chl c and peridinin in dinophytes, and zeaxanthin in rhodophytes. The specificity of pigment binding was examined by in vitro reconstitution of various pigments with a simple light-harvesting protein (LHCaR1), from a red alga (Porphyridium cruentum), that normally has eight Chl a and four zeaxanthin molecules. The pigments typical of a chlorophyte (Spinacea oleracea), a chromophyte (Thallasiosira fluviatilis), and a dinophyte (Prorocentrum micans) were found to functionally bind to this protein as evidenced by their participation in energy transfer to Chl a, the terminal pigment. This is a demonstration of a functional relatedness of rhodophyte and higher plant LHCs. The results suggest that eight Chl-binding sites per polypeptide are an ancestral trait, and that the flexibility to bind various Chl and carotenoid pigments may have been retained throughout the evolution of LHCs.

Protective Function of Chloroplast 2-Cysteine Peroxiredoxin in Photosynthesis. Evidence from Transgenic Arabidopsis1

2-Cysteine peroxiredoxins (2-CPs) constitute a ubiquitous group of peroxidases that reduce cell-toxic alkyl hydroperoxides to their corresponding alcohols. Recently, we cloned 2-CP cDNAs from plants and characterized them as chloroplast proteins. To elucidate the physiological function of the 2-CP in plant metabolism, we generated antisense mutants in Arabidopsis. In the mutant lines a 2-CP deficiency developed during early leaf and plant development and eventually the protein accumulated to wild-type levels. In young mutants with reduced amounts of 2-CP, photosynthesis was impaired and the levels of D1 protein, the light-harvesting protein complex associated with photosystem II, chloroplast ATP synthase, and ribulose-1,5-bisphosphate carboxylase/oxygenase were decreased. Photoinhibition was particularly pronounced after the application of the protein synthesis inhibitor, lincomycin. We concluded that the photosynthetic machinery needs high levels of 2-CP during leaf development to protect it from oxidative damage and that the damage is reduced by the accumulation of 2-CP protein, by the de novo synthesis and replacement of damaged proteins, and by the induction of other antioxidant defenses in 2-CP mutants.

The Xanthophyll Cycle Modulates the Kinetics of Nonphotochemical Energy Dissipation in Isolated Light-Harvesting Complexes, Intact Chloroplasts, and Leaves of Spinach1

We analyzed the kinetics of nonphotochemical quenching of chlorophyll fluorescence (qN) in spinach (Spinacia oleracea) leaves, chloroplasts, and purified light-harvesting complexes. The characteristic biphasic pattern of fluorescence quenching in dark-adapted leaves, which was removed by preillumination, was evidence of light activation of qN, a process correlated with the de-epoxidation state of the xanthophyll cycle carotenoids. Chloroplasts isolated from dark-adapted and light-activated leaves confirmed the nature of light activation: faster and greater quenching at a subsaturating transthylakoid pH gradient. The light-harvesting chlorophyll a/b-binding complexes of photosystem II were isolated from dark-adapted and light-activated leaves. When isolated from light-activated leaves, these complexes showed an increase in the rate of quenching in vitro compared with samples prepared from dark-adapted leaves. In all cases, the quenching kinetics were fitted to a single component hyperbolic function. For leaves, chloroplasts, and light-harvesting complexes, the presence of zeaxanthin was associated with an increased rate constant for the induction of quenching. We discuss the significance of these observations in terms of the mechanism and control of qN.

What was natural in the coastal oceans?

Humans transformed Western Atlantic coastal marine ecosystems before modern ecological investigations began. Paleoecological, archeological, and historical reconstructions demonstrate incredible losses of large vertebrates and oysters from the entire Atlantic coast. Untold millions of large fishes, sharks, sea turtles, and manatees were removed from the Caribbean in the 17th to 19th centuries. Recent collapses of reef corals and seagrasses are due ultimately to losses of these large consumers as much as to more recent changes in climate, eutrophication, or outbreaks of disease. Overfishing in the 19th century reduced vast beds of oysters in Chesapeake Bay and other estuaries to a few percent of pristine abundances and promoted eutrophication. Mechanized harvesting of bottom fishes like cod set off a series of trophic cascades that eliminated kelp forests and then brought them back again as fishers fished their way down food webs to small invertebrates. Lastly, but most pervasively, mechanized harvesting of the entire continental shelf decimated large, long-lived fishes and destroyed three-dimensional habitats built up by sessile corals, bryozoans, and sponges. The universal pattern of losses demonstrates that no coastal ecosystem is pristine and few wild fisheries are sustainable along the entire Western Atlantic coast. Reconstructions of ecosystems lost only a century or two ago demonstrate attainable goals of establishing large and effective marine reserves if society is willing to pay the costs. Historical reconstructions provide a new scientific framework for manipulative experiments at the ecosystem scale to explore the feasibility and benefits of protection of our living coastal resources.