High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease.

Infantile Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside phosphotransferase and dihydrofolate reductase genes. Following transfection in dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.

Spectral diffusion and the energy landscape of a protein

We present a novel type of spectral diffusion experiment in the millikelvin range to characterize the energy landscape of a protein as compared with that of a glass. We measure the time evolution of spectral holes for more than 300 hr after well-defined initial nonequilibrium conditions. We show that the model of noninteracting two-level systems can describe spectral diffusion in the glass, but fails for the protein. Our results further demonstrate that randomness in the energy landscape of a protein shows features of organization. There are “deep minimum” states separated by barriers, the heights of which we are able to estimate. The energy landscape of a glass is featureless by comparison.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA′-′lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Autoregulation at the level of mRNA 3′ end formation of the suppressor of forked gene of Drosophila melanogaster is conserved in Drosophila virilis

The Drosophila melanogaster Suppressor of forked [Su(f)] protein shares homology with the yeast RNA14 protein and the 77-kDa subunit of human cleavage stimulation factor, which are proteins involved in mRNA 3′ end formation. This suggests a role for Su(f) in mRNA 3′ end formation in Drosophila. The su(f) gene produces three transcripts; two of them are polyadenylated at the end of the transcription unit, and one is a truncated transcript, polyadenylated in intron 4. Using temperature-sensitive su(f) mutants, we show that accumulation of the truncated transcript requires wild-type Su(f) protein. This suggests that the Su(f) protein autoregulates negatively its accumulation by stimulating 3′ end formation of the truncated su(f) RNA. Cloning of su(f) from Drosophila virilis and analysis of its RNA profile suggest that su(f) autoregulation is conserved in this species. Sequence comparison between su(f) from both species allows us to point out three conserved regions in intron 4 downstream of the truncated RNA poly(A) site. These conserved regions include the GU-rich downstream sequence involved in poly(A) site definition. Using transgenes truncated within intron 4, we show that sequence up to the conserved GU-rich domain is sufficient for production of the truncated RNA and for regulation of this production by su(f). Our results indicate a role of su(f) in the regulation of poly(A) site utilization and an important role of the GU-rich sequence for this regulation to occur.

B7–CTLA4 interaction enhances both production of antitumor cytotoxic T lymphocytes and resistance to tumor challenge

Expression of B7-family costimulatory molecules CD80 (B7–1) and CD86 (B7–2) on tumor cells enhances host immunity. However, the role of the two B7 receptors, CD28 and CTLA4 (CD152), on T cells in antitumor immune response has not been clearly elucidated. Based on the effects of anti-CD28 and anti-CTLA4 mAbs on T cell response, it was proposed that CD28-B7 interaction promotes antitumor immunity, whereas B7-CTLA4 interaction down-regulates it. A critical test for the hypothesis is whether selective engagement of CTLA4 receptors by their natural ligands CD80 and CD86 enhances or reduces antitumor immunity. Here we used tumors expressing wild-type and mutant CD80, as well as mice with targeted mutation of CD28, to address this issue. We report that in syngeneic wild-type mice, B7W (W88>A), a CD80 mutant that has lost binding to CD28 but retained binding to CTLA4, can enhance the induction of antitumor cytotoxic T lymphocytes (CTL); B7Y (Y201>A), which binds neither CD28 nor CTLA4, fails to do so. Consistent with these observations, B7W-transfected J558 plasmocytoma and EL4 thymoma grow significantly more slowly than those transfected with either vector alone or with B7Y. Optimal tumor rejection requires wild-type CD80. Moreover, expression of a high level of CD80 on thymoma EL4 cells conveys immunity in mice with a targeted mutation of CD28 gene. Taken together, our results demonstrate that B7-CTLA4 interaction enhances production of antitumor CTL and resistance to tumor challenge and that optimal enhancement of antitumor immunity by CD80 requires its engagement of both CD28 and CTLA4.

Cell-Specific Production and Antimicrobial Activity of Naphthoquinones in Roots of Lithospermum erythrorhizon1

Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on “noninducing” medium, whereas induction of additional pigment production by abiotic (CuSO4) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and β-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed “hairy-root” cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.

Two Arabidopsis Mutants That Overproduce Ethylene Are Affected in the Posttranscriptional Regulation of 1-Aminocyclopropane-1-Carboxylic Acid Synthase1

The Arabidopsis mutants eto1 (ethylene overproducer) and eto3 produce elevated levels of ethylene as etiolated seedlings. Ethylene production in these seedlings peaks at 60 to 96 h, and then declines back to almost wild-type levels. Ethylene overproduction in eto1 and eto3 is limited mainly to etiolated seedlings; light-grown seedlings and various adult tissues produce close to wild-type amounts of ethylene. Several compounds that induce ethylene biosynthesis in wild-type, etiolated seedlings through distinct 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) isoforms were found to act synergistically with eto1 and eto3, as did the ethylene-insensitive mutation etr1 (ethylene resistant), which blocks feedback inhibition of biosynthesis. ACS activity, the rate-limiting step of ethylene biosynthesis, was highly elevated in both eto1 and eto3 mutant seedlings, even though RNA gel-blot analysis demonstrated that the steady-state level of ACS mRNA was not increased, including that of a novel Arabidopsis ACS gene that was identified. Measurements of the conversion of ACC to ethylene by intact seedlings indicated that the mutations did not affect conjugation of ACC or the activity of ACC oxidase, the final step of ethylene biosynthesis. Taken together, these data suggest that the eto1 and eto3 mutations elevate ethylene biosynthesis by affecting the posttranscriptional regulation of ACS.

Relatedness threshold for the production of female sexuals in colonies of a polygynous ant, Myrmica tahoensis, as revealed by microsatellite DNA analysis.

The genetic relationships of colony members in the ant Myrmica tahoensis were determined on the basis of highly polymorphic microsatellite DNA loci. These analyses show that colonies fall into one of two classes. In roughly half of the sampled colonies, workers and female offspring appear to be full sisters. The remaining colonies contain offspring produced by two or more queens. Colonies that produce female sexuals are always composed of highly related females, while colonies that produce males often show low levels of nestmate relatedness. These results support theoretical predictions that workers should skew sex allocation in response to relatedness asymmetries found within colonies. The existence of a relatedness threshold below which female sexuals are not produced suggests a possible mechanism for worker perception of relatedness. Two results indicate that workers use genetic cues, not queen number, in making sex-allocation decisions. (i) The number of queens in a colony was not significantly correlated with either the level of relatedness asymmetry or the sex ratio. (ii) Sex-ratio shifts consistent with a genetically based mechanism of relatedness assessment were seen in an experiment involving transfers of larvae among unrelated nests. Thus workers appear to make sex-allocation decisions on the basis of larval cues and appear to be able to adjust sex ratios long after egg laying.