Genetic engineering of carbohydrate biosynthetic pathways in transgenic mice demonstrates cell cycle-associated regulation of glycoconjugate production in small intestinal epithelial cells.

Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.

Production of cyclic peptides and proteins in vivo

Combinatorial libraries of synthetic and natural products are an important source of molecular information for the interrogation of biological targets. Methods for the intracellular production of libraries of small, stable molecules would be a valuable addition to existing library technologies by combining the discovery potential inherent in small molecules with the large library sizes that can be realized by intracellular methods. We have explored the use of split inteins (internal proteins) for the intracellular catalysis of peptide backbone cyclization as a method for generating proteins and small peptides that are stabilized against cellular catabolism. The DnaE split intein from Synechocystis sp. PCC6803 was used to cyclize the Escherichia coli enzyme dihydrofolate reductase and to produce the cyclic, eight-amino acid tyrosinase inhibitor pseudostellarin F in bacteria. Cyclic dihydrofolate reductase displayed improved in vitro thermostability, and pseudostellarin F production was readily apparent in vivo through its inhibition of melanin production catalyzed by recombinant Streptomyces antibioticus tyrosinase. The ability to generate and screen for backbone cyclic products in vivo is an important milestone toward the goal of generating intracellular cyclic peptide and protein libraries.

Three small nucleolar RNAs that are involved in ribosomal RNA precursor processing

Three small nucleolar RNAs (snoRNAs), E1, E2 and E3, have been described that have unique sequences and interact directly with unique segments of pre-rRNA in vivo. In this report, injection of antisense oligodeoxynucleotides into Xenopus laevis oocytes was used to target the specific degradation of these snoRNAs. Specific disruptions of pre-rRNA processing were then observed, which were reversed by injection of the corresponding in vitro-synthesized snoRNA. Degradation of each of these three snoRNAs produced a unique rRNA maturation phenotype. E1 RNA depletion shut down 18 rRNA formation, without overaccumulation of 20S pre-rRNA. After E2 RNA degradation, production of 18S rRNA and 36S pre-rRNA stopped, and 38S pre-rRNA accumulated, without overaccumulation of 20S pre-rRNA. E3 RNA depletion induced the accumulation of 36S pre-rRNA. This suggests that each of these snoRNAs plays a different role in pre-rRNA processing and indicates that E1 and E2 RNAs are essential for 18S rRNA formation. The available data support the proposal that these snoRNAs are at least involved in pre-rRNA processing at the following pre-rRNA cleavage sites: E1 at the 5′ end and E2 at the 3′ end of 18S rRNA, and E3 at or near the 5′ end of 5.8S rRNA.

Growth hormone-releasing hormone: An autocrine growth factor for small cell lung carcinoma

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers in vivo. This effect is thought to be exerted through suppression of the pituitary growth hormone–hepatic insulin-like growth factor I (IGF-I) axis and direct inhibition of autocrine/paracrine production of IGF-I and -II in tumors. However, other evidence points to a direct effect of GHRH antagonists on tumor growth that may not implicate IGFs, although an involvement of GHRH in the proliferation of cancer cells has not yet been established. In the present study we investigated whether GHRH can function as an autocrine/paracrine growth factor in small cell lung carcinoma (SCLC). H-69 and H-510A SCLC lines cultured in vitro express mRNA for GHRH, which apparently is translated into peptide GHRH and then secreted by the cells, as shown by the detection of GHRH-like immunoreactivity in conditioned media from the cells cultured in vitro. In addition, the levels of GHRH-like immunoreactivity in serum from nude mice bearing H-69 xenografts were higher than in tumor-free mice. GHRH(1–29)NH2 stimulated the proliferation of H-69 and H-510A SCLCs in vitro, and GHRH antagonist JV-1–36 inhibited it. JV-1–36 administered s.c. into nude mice bearing xenografts of H-69 SCLC reduced significantly (P < 0.05) tumor volume and weight, after 31 days of therapy, as compared with controls. Collectively, our results suggest that GHRH can function as an autocrine growth factor in SCLCs. Treatment with antagonistic analogs of GHRH may offer a new approach to the treatment of SCLC and other cancers.

Induced intensification: Agricultural change in Bangladesh with implications for Malthus and Boserup

Bangladesh is dominated by a small-holder agrarian economy under extreme stress. Production shortfalls, increasing economic polarization, and chronic malnutrition are persistent, but major famine has been diverted in part by significant growth in agriculture. This recent history is open to both Malthusian and Boserupian interpretations—a history we explore here through a test of the induced intensification thesis of agricultural change. This thesis, framed by variations in the behavior of small-holders, has grown from a simple demand-production relationship to a consideration of the mediating influences on that relationship. The induced intensification thesis is reviewed and tested for 265 households in 6 villages in Bangladesh from 1950–1986. A time-series analysis of an induced intensification model provides relatively high levels of explained variance in cropping intensity (frequency and land productivity) and also indicates the relative impacts of household class, environment, and cropping strategies. On average, the small-holders in question kept pace with the demands on production, although important class and village variations were evident and the proportion of landless households increased. These results, coupled with evidence that agricultural growth involved intensification thresholds, provide clues about Malthusian and Boserupian interpretations of Bangladesh, and suggest that small-holder agriculture there is likely to continue on a “muted” path of growth.

The small GTPase RalA targets filamin to induce filopodia

The Ras-related small GTPases Rac, Rho, Cdc42, and RalA bind filamin, an actin filament-crosslinking protein that also links membrane and other intracellular proteins to actin. Of these GTPases only RalA binds filamin in a GTP-specific manner, and GTP-RalA elicits actin-rich filopods on surfaces of Swiss 3T3 cells and recruits filamin into the filopodial cytoskeleton. Either a dominant negative RalA construct or the RalA-binding domain of filamin 1 specifically block Cdc42-induced filopod formation, but a Cdc42 inhibitor does not impair RalA’s effects, which, unlike Cdc42, are Rac independent. RalA does not generate filopodia in filamin-deficient human melanoma cells, whereas transfection of filamin 1 restores the functional response. RalA therefore is a downstream intermediate in Cdc42-mediated filopod production and uses filamin in this pathway.

Functional separation of pre-rRNA processing steps revealed by truncation of the U3 small nucleolar ribonucleoprotein component, Mpp10

The U3 small nucleolar ribonucleoprotein (snoRNP) is required for three cleavage events that generate the mature 18S rRNA from the pre-rRNA. In Saccharomyces cerevisiae, depletion of Mpp10, a U3 snoRNP-specific protein, halts 18S rRNA production and impairs cleavage at the three U3 snoRNP-dependent sites: A0, A1, and A2. We have identified truncation mutations of Mpp10 that affect 18S rRNA synthesis and confer cold-sensitivity and slow growth. However, distinct from yeast cells depleted of Mpp10, the mutants carrying these truncated Mpp10 proteins accumulate a novel precursor, resulting from cleavage at only A0. The Mpp10 truncations do not alter association of Mpp10 with the U3 snoRNA, nor do they affect snoRNA or protein stability. Thus, the role in processing of the U3 snoRNP can be separated into cleavage at the A0 site, which occurs in the presence of truncated Mpp10, and cleavage at the A1/A2 sites, which occurs only with intact Mpp10. These results strongly argue for a role for Mpp10 in processing at the A1/A2 sites.

Production, specificity, and functionality of monoclonal antibodies to specific peptide–major histocompatibility complex class II complexes formed by processing of exogenous protein

Several unanswered questions in T cell immunobiology relating to intracellular processing or in vivo antigen presentation could be approached if convenient, specific, and sensitive reagents were available for detecting the peptide–major histocompatibility complex (MHC) class I or class II ligands recognized by αβ T cell receptors. For this reason, we have developed a method using homogeneously loaded peptide–MHC class II complexes to generate and select specific mAb reactive with these structures using hen egg lysozyme (HEL) and I-Ak as a model system. mAbs specific for either HEL-(46–61)–Ak or HEL-(116–129)–Ak have been isolated. They cross-react with a small subset of I-Ak molecules loaded with self peptides but can nonetheless be used for flow cytometry, immunoprecipitation, Western blotting, and intracellular immunofluorescence to detect specific HEL peptide–MHC class II complexes formed by either peptide exposure or natural processing of native HEL. An example of the utility of these reagents is provided herein by using one of the anti-HEL-(46–61)–Ak specific mAbs to visualize intracellular compartments where I-Ak is loaded with HEL-derived peptides early after antigen administration. Other uses, especially for in vivo tracking of specific ligand-bearing antigen-presenting cells, are discussed.

Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs

Antisense-mediated gene silencing (ASGS) and posttranscriptional gene silencing (PTGS) with sense transgenes markedly reduce the steady-state mRNA levels of endogenous genes similar in transcribed sequence. RNase protection assays established that silencing in tobacco plants transformed with plant-defense-related class I sense and antisense chitinase (CHN) transgenes is at the posttranscriptional level. Infection of tobacco plants with cucumber mosaic virus strain FN and a necrotizing strain of potato virus Y, but not with potato virus X, effectively suppressed PTGS and ASGS of both the transgenes and homologous endogenes. This suggests that ASGS and PTGS share components associated with initiation and maintenance of the silent state. Small, ca. 25-nt RNAs (smRNA) of both polarities were associated with PTGS and ASGS in CHN transformants as reported for PTGS in other transgenic plants and for RNA interference in Drosophila. Similar results were obtained with an antisense class I β-1,3-glucanase transformant showing that viral suppression and smRNAs are a more general feature of ASGS. Several current models hold that diverse signals lead to production of double-stranded RNAs, which are processed to smRNAs that then trigger PTGS. Our results provide direct evidence for mechanistic links between ASGS and PTGS and suggest that ASGS could join a common PTGS pathway at the double-stranded RNA step.

Transgenic plants for tropical regions: Some considerations about their development and their transfer to the small farmer

Biotechnological applications, especially transgenic plants, probably hold the most promise in augmenting agricultural production in the first decades of the next millennium. However, the application of these technologies to the agriculture of tropical regions where the largest areas of low productivity are located, and where they are most needed, remains a major challenge. In this paper, some of the important issues that need to be considered to ensure that plant biotechnology is effectively transferred to the developing world are discussed.

Colony-forming progenitors from mouse olfactory epithelium: evidence for feedback regulation of neuron production.

The mammalian olfactory epithelium (OE) supports continual neurogenesis throughout life, suggesting that a neuronal stem cell exists in this system. In tissue culture, however, the capacity of the OE for neurogenesis ceases after a few days. In an attempt to identify conditions that support the survival of neuronal stem cells, a population of neuronal progenitors was isolated from embryonic mouse OE and cultured in defined serum-free medium. The vast majority of cells rapidly gave rise to neurons, which died shortly thereafter. However, when purified progenitors were co-cultured with cells derived from the stroma underlying the OE, a small subpopulation (0.07-0.1%) gave rise to proliferative colonies. A morphologically identifiable subset of these colonies generated new neurons as late as 7 days in vitro. Interestingly, development of these neuronal colonies was specifically inhibited when purified progenitors were plated onto stromal feeder cells in the presence of a large excess of differentiated OE neurons. These results indicate that a rare cell type, with the potential to undergo prolonged neurogenesis, can be isolated from mammalian OE and that stroma-derived factors are important in supporting neurogenesis by this cell. The data further suggest that differentiated neurons provide a signal that feeds back to inhibit production of new neurons by their own progenitors.

A mechanism for intergenomic integration: abundance of ribulose bisphosphate carboxylase small-subunit protein influences the translation of the large-subunit mRNA.

Multimeric protein complexes in chloroplasts and mitochondria are generally composed of products of both nuclear and organelle genes of the cell. A central problem of eukaryotic cell biology is to identify and understand the molecular mechanisms for integrating the production and accumulation of the products of the two separate genomes. Ribulose bisphosphate carboxylase (Rubisco) is localized in the chloroplasts of photosynthetic eukaryotic cells and is composed of small subunits (SS) and large subunits (LS) coded for by nuclear rbcS and chloroplast rbcL genes, respectively. Transgenic tobacco plants containing antisense rbcS DNA have reduced levels of rbcS mRNA, normal levels of rbcL mRNA, and coordinately reduced LS and SS proteins. Our previous experiments indicated that the rate of translation of rbcL mRNA might be reduced in some antisense plants; direct evidence is presented here. After a short-term pulse there is less labeled LS protein in the transgenic plants than in wild-type plants, indicating that LS accumulation is controlled in the mutants at the translational and/or posttranslational levels. Consistent with a primary restriction at translation, fewer rbcL mRNAs are associated with polysomes of normal size and more are free or are associated with only a few ribosomes in the antisense plants. Effects of the rbcS antisense mutation on mRNA and protein accumulation, as well as on the distribution of mRNAs on polysomes, appear to be minimal for other chloroplast and nuclear photosynthetic genes. Our results suggest that SS protein abundance specifically contributes to the regulation of LS protein accumulation at the level of rbcL translation initiation.

Characterization of small nontranslated polyadenylylated RNAs in vaccinia virus-infected cells.

Host protein synthesis is selectively inhibited in vaccinia virus-infected cells. This inhibition has been associated with the production of a group of small, nontranslated, polyadenylylated RNAs (POLADS) produced during the early part of virus infection. The inhibitory function of POLADS is associated with the poly(A) tail of these small RNAs. To determine the origin of the 5'-ends of POLADS, reverse transcription was performed with POLADS isolated from VV-infected cells at 1 hr and 3.5 hr post infection. The cDNAs of these POLADS were cloned into plasmids (pBS or pBluescript II KS +/-), and their nucleotide composition was determined by DNA sequencing. The results of this investigation show the following: There is no specific gene encoding for POLADS. The 5' ends of POLADS may be derived from either viral or cellular RNAs. Any RNA sequence including tRNAs, small nuclear RNAs and 5'ends of mRNAs can become POLADS if they acquire a poly(A) tail at their 3' ends during infection. This nonspecific polyadenylylation found in vaccinia virus-infected cells is probably conducted by vaccinia virus poly(A)+ polymerase. No consensus sequence is found on the 5' ends of POLADS for polyadenylylation. The 5' ends of POLADS have no direct role in their inhibitory activity of protein synthesis.

Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a spectrum of exoproducts many of which have been implicated in the pathogenesis of human infection. Expression of some of these factors requires cell-cell communication involving the interaction of a small diffusible molecule, an "autoinducer," with a positive transcriptional activator. In P. aeruginosa PAO1, LasI directs the synthesis of the autoinducer N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), which activates the positive transcriptional activator, LasR. Recently, we have discovered a second signaling molecule-based modulon in PAO1, termed vsm, which contains the genes vsmR and vsmI. Using HPLC, mass spectrometry, and NMR spectroscopy we now establish that in Escherichia coli, VsmI directs the synthesis of N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone (HHL). These compounds are present in the spent culture supernatants of P. aeruginosa in a molar ratio of approximately 15:1 and their structures were unequivocally confirmed by chemical synthesis. Addition of either BHL or HHL to PAN067, a pleiotropic P. aeruginosa mutant unable to synthesize either of these autoinducers, restored elastase, chitinase, and cyanide production. In E. coli carrying a vsmR/vsmI'::lux transcriptional fusion, BHL and HHL activated VsmR to a similar extent. Analogues of these N-acyl-L-homoserine lactones in which the N-acyl side chain has been extended and/or oxidized at the C-3 position exhibit substantially lower activity (e.g., OdDHL) or no activity (e.g., dDHL) in this lux reporter assay. These data indicate that multiple families of quorum sensing modulons interactively regulate gene expression in P. aeruginosa.