Cultivation of Peptidiphaga gingivicola from subgingival plaque: The first representative of a novel genus of Actinomycetaceae.

A novel bacterium was isolated from the subgingival plaque of a patient with periodontal disease. Bacterial strain BA112T is a facultative Gram-positive coccus. It metabolizes alanine, arginine, glycine, histidine, leucine, proline, serine and tyrosine, but does not appear to use carbohydrates. Urease, esculin, indole, catalase and nitrate reduction tests were all negative. Major cellular fatty acids were C18:0 , C12:0 , C16:0 , C18:1 w9c and C20:0 . The genome was sequenced and is 2.4 Mbp in length and has 64% GC content. Based on phylogenetics of the 16S rRNA sequence and concatenated alignments of 37 conserved proteins, BA112T belongs to the family Actinomycetaceae but is located on a branch of the tree without currently named members. Based on our phenotypic and phylogenetic studies, we propose that BA112T is the first known representative of a new genus, for which the name Peptidiphaga gingivicola gen. nov., sp. nov. is proposed. The type strain is BA112T .

Transfer of the feline erythropoietin gene to cats using a recombinant adeno-associated virus vector.

Chronic renal failure and the associated erythropoietin-responsive anemia afflicts over 2 million domestic cats in the United States, resulting in morbidity that can affect the owner-pet relationship. Although treatment of cats with recombinant human erythropoietin (Epo) protein can be effective, response to the drug often dissipates over time, probably due to the development of antibodies reactive with the human protein. As an alternate approach to the treatment of this disease, we have developed a recombinant adeno-associated virus vector containing the feline erythropoietin gene (rAAV/feEpo). This vector, when administered intramuscularly to normal healthy cats, caused a dose-related increase in hematocrit over a 7-week period after injection. Thus, the rAAV/feEpo vector holds promise as a simple, safe and effective therapy for the anemia of chronic renal failure in domestic cats.

Functional nonequivalence of Drosophila actin isoforms.

We show that different Drosophila actin isoforms are not interchangeable. We sequenced the six genes that encode conventional Drosophila actins and found that they specify amino acid replacements in 27 of 376 positions. To test the significance of these changes we used directed mutagenesis to introduce 10 such conversions, independently, into the Act88F flight muscle-specific actin gene. We challenged these variant actins to replace the native protein by transforming germline chromosomes of a Drosophila strain lacking flight muscle actin. Only one of the 10 reproducibly perturbed myofibrillar function, demonstrating that most isoform-specific amino acid replacements are of minor significance. In order to establish the consequences of multiple amino acid replacements, we substituted portions of the Drosophila Act88F actin gene with corresponding regions of genes encoding other isoforms. Only one of five constructs tested engendered normally functioning flight muscles, and the severity of myofibrillar defects correlated with the number of replacements within the chimeric genes. Finally, we completely converted the flight muscle actin-encoding gene to one specifying a nonmuscle isoform, a change entailing a total of 18 amino acid replacements. Transformation of flies with this construct resulted in disruption of flight muscle structure and function. We conclude that actin isoform sequences are not equivalent and that effects of the amino acid replacements, while minor individually, collectively confer unique properties.

Expression of human monocyte chemoattractant protein-1 in the yeast Pichia pastoris.

The human monocyte chemoattractant protein-1 (MCP-1) was expressed at high levels in Pichia pastoris with the alcohol oxidase promoter. It was secreted from the yeast when either its natural signal sequence or the Saccharomyces cerevisiae alpha-factor signal peptide was used. SDS-PAGE and Western blot revealed two immunoreactive MCP-1 species at 15 and 8.5 kDa designated MCP-1H and MCP-1L, respectively; both were purified by cation-exchange chromatography. MCP-1H could be converted to MCP-1L by treatment with peptide N-glycosidase F, showing that the former is an N-glycosylated form of the latter. Laser desorption mass spectrometry showed that MCP-1L actually consisted of a mixture of three polypeptides of 8449, 8614, and 8780 Da and MCP-1H showed a broad peak at 11,134 Da. N-terminal peptide sequencing indicated that nearly half of MCP-1L lacked the two N-terminal amino acids found in the native protein. Both MCP-1H and MCP-1L could induce monocyte migration and calcium influx in THP-1 monocytic leukemia cells, although these activities were about 10- to 100-fold lower than those of MCP-1 produced in insect cells.

Inhibition of Aspergillus serine proteinase by Streptomyces subtilisin inhibitor and high-level expression of this inhibitor in Pichia pastoris.

Aspergillus fumigatus encodes an extracellular serine proteinase of the subtilisin family that is thought to be involved in invasive aspergillus infection of immunocompromised patients. When the structure of proteinase K was used to model this Aspergillus serine proteinase, Streptomyces subtilisin inhibitor (SSI) was predicted to be capable of binding to the serine proteinase. SSI purified from S. albogriseolus inhibited the serine proteinase with Ki of 1 x 10(-9)M. This value is higher than that for subtilisin probably because the serine proteinase lacks the S(4-6) site that interacts with the P(4-6) site of SSI. A high level expression for SSI, established in Pichia pastoris, yielded 0.5g of SSI per liter of culture medium. The secreted product was easily purified to homogeneity and biochemically characterized. The recombinant SSI showed a Ki of 1.1 x 10(-9) and 1 x 10(-8) for the serine proteinases from A. fumigatus and A. flavus, respectively.

Site-directed mutagenesis of monocyte chemoattractant protein-1 identifies two regions of the polypeptide essential for biological activity.

Monocyte chemoattractant protein-1 (MCP-1) mediates monocyte migration into tissues in inflammatory diseases and atherosclerosis. We have investigated structure-activity relationships for human MCP-1. Mutations were introduced based upon differences between MCP-1 and the structurally related but functionally distinct molecule interleukin-8 (IL-8). Mutant proteins produced using the baculovirus/insect cell expression system were purified and their ability to stimulate monocyte chemotaxis and elevation of intracellular calcium in THP-1 monocytic leukaemia cells was measured. Two regions in MCP-1 were identified as important for its biological activity. One region consists of the sequence Thr-Cys-Cys-Tyr (amino acids 10-13). Point mutations of Thr-10 to Arg and Tyr-13 to Ile greatly lowered MCP-1 activity. The second functionally important region is formed by Ser-34 and Lys-35. Insertion of a Pro between these two residues, or their substitution by the sequence Gly-Pro-His, caused nearly complete loss of MCP-1 activity. Competition binding experiments showed that the mutations that affected activity also lowered the ability to compete with wild-type MCP-1 for receptors on THP-1 cells. Point mutations at positions 8, 15, 30, 37, 38 and 68 had little effect on MCP-1 activity. The important regions that we have identified in MCP-1 correspond with previously identified functionally important regions of IL-8, suggesting that the two molecules bind to their respective receptors by similar contacts.

The cytomegalovirus US28 protein binds multiple CC chemokines with high affinity.

Human cytomegalovirus encodes several proteins with high similarity to seven transmembrane domain receptors. We investigated the ability of one of these proteins, the product of the US28 open reading frame, to bind various chemoattractant ligands. When transfected into COS-7 cells, the US28 product conferred high affinity binding to the labeled chemokines monocyte chemoattractant protein-1 (MCP-1) (Kd = 6.0 x 10(-10) M) and RANTES (Kd = 2.7 x 10(-10) M). Binding of these labeled ligands could be competed by the unlabeled macrophage inflammatory proteins MIP-1 alpha and MIP-1 beta, with Kd values in the range 1.2 x 10(-9) to 7.5 x 10(-9) M. Comparisons of the sequences of US28 and other receptors that bind chemokines should help to define regions responsible for receptor-ligand interactions.

Tissue-specific alternative splicing of the Drosophila dopa decarboxylase gene is affected by heat shock.

The Drosophila dopa decarboxylase gene, Ddc, is expressed in the hypoderm and in a small number of cells in the central nervous system (CNS). The unique Ddc primary transcript is alternatively spliced in these two tissues. We investigated whether Ddc splicing in the CNS is a general property of the CNS or a unique property of the cells that normally express Ddc by expressing the Ddc primary transcript ubiquitously under the control of an Hsp70 heat shock promoter. Under basal expression conditions, Ddc splicing shows normal tissue specificity, indicating that the regulation of Ddc splicing in the CNS is tissue specific rather than cell specific. Previous studies have shown that severe heat shock blocks mRNA splicing in cultured Drosophila melanogaster cells. Our results show that splicing of the heat shock-inducible Hsp83 transcript is very resistant to heat shock. In contrast, under either mild or severe heat shock, the splicing specificity of the heat shock-induced Ddc primary transcript is affected, leading to the accumulation of inappropriately high levels of the CNS splice form in non-CNS tissues. The chromosomal Ddc transcript is similarly affected. These results show unexpected heterogeneity in the splicing of individual mRNAs as a response to heat shock and suggest that the Ddc CNS-specific splicing pathway is the default.

Conversion of monocyte chemoattractant protein-1 into a neutrophil attractant by substitution of two amino acids.

The small cytokine monocyte chemoattractant protein-1 has structural similarity to the neutrophil chemoattractant interleukin-8, but each protein is specific in attracting its own target cell. To investigate the structural basis of this cell type specificity, we have developed an Escherichia coli expression system for the monocyte chemoattractant and mutagenized selected amino acid residues to ones found at the corresponding positions of interleukin-8. We find that a double mutation of tyrosine 28 and arginine 30 to leucine and valine, respectively, causes a drastic decrease in chemotactic activity toward monocytes with the appearance of a novel (interleukin-8-like) neutrophil chemotactic activity. Computer graphic analysis predicts that, with the double substitution, a putative receptor binding groove of the monocyte chemoattractant protein would become topographically similar to that of interleukin-8. We therefore postulate that one or both of these amino acid residues are part of the binding contact of these small cytokines and their receptors.

Muscle abnormalities in Drosophila melanogaster heldup mutants are caused by missing or aberrant troponin-I isoforms.

We have investigated the molecular bases of muscle abnormalities in four Drosophila melanogaster heldup mutants. We find that the heldup gene encodes troponin-I, one of the principal regulatory proteins associated with skeletal muscle thin filaments. heldup3, heldup4, and heldup5 mutants, all of which have grossly abnormal flight muscle myofibrils, lack mRNAs encoding one or more troponin-I isoforms. In contrast, heldup2, an especially interesting mutant wherein flight muscles are atrophic, synthesizes the complete mRNA complement. By sequencing mutant troponin-I cDNAs we demonstrate that the molecular basis for muscle degeneration in heldup2 is conversion of an invariant alanine residue to valine. We finally show that degeneration of heldup2 thin filament/Z-disc networks can be prevented by eliminating thick filaments from flight muscles using a null allele of the sarcomeric myosin heavy chain gene. This latter observation suggests that actomyosin interactions exacerbate the structural or functional defect resulting from the troponin-I mutation.

Genetic dissection of Drosophila myofibril formation: effects of actin and myosin heavy chain null alleles.

We used null mutations of Drosophila actin and myosin genes to investigate two aspects of myofibril assembly. First, we eliminated all actin or myosin in flight muscles to evaluate contributions of thick and thin filaments to sarcomere formation. Results demonstrate that thick and thin filament arrays can assemble independently but that both are essential for sarcomeric order and periodicity. Second, we examined how filament stoichiometry affects myofibril assembly. We find that heterozygotes for actin (Act88F) or myosin heavy chain (Mhc36B) null alleles have complex myofibrillar defects, whereas Mhc36B-/+; Act88F-/+ double heterozygotes have nearly normal myofibrils. These results imply that most defects observed in single heterozygotes are due to filament imbalances, not deficits, and suggest that thick and thin filament interactions regulate myofibrillar growth and alignment.

Arthrin, a myofibrillar protein of insect flight muscle, is an actin-ubiquitin conjugate.

Flight muscles of some insects contain a myofibrillar protein termed arthrin, which is closely related to actin (mw 43,000). Here we demonstrate that arthrin (mw 55,000) is ubiquitinated actin. We show that in Act88FM342, a flightless Drosophila mutant wherein the Act88F actin gene specifies a glu93----lys replacement, isoelectric points of both actin III and arthrin are shifted, revealing that both are encoded by the same gene. Arthrin reacts with an anti-ubiquitin antibody, which demonstrates that its extra mass results from ubiquitin ligation. Approximately one-seventh of myofibrillar actin is stably ubiquitinated, suggesting that there may be one arthrin molecule per actin-tropomyosin-troponin cooperative unit. Arthrin formation lags several hours behind that of actin III, implying that ubiquitination coincides with some aspect of myofibril assembly.

Regulation of the Drosophila dopa decarboxylase gene in neuronal and glial cells.

A cis-regulatory element selectively required for the Drosophila melanogaster dopa decarboxylase gene (Ddc) in the central nervous system has been identified previously (Scholnick et al. 1986). Here, we show that at least one additional regulatory element is required for normal neuronal expression of Ddc. We find that Ddc is normally expressed in about 125 discrete neurons and in a diffused network comprising a subset of glial cells. The expression of in vitro-altered Ddc genes was studied by immunohistochemistry following germ line reintegration with P-element vectors. Normal neuron-specific Ddc gene expression requires both the initially identified element (element I) which is 60 bp upstream from the RNA start site, and an additional regulatory element located 800-2200 bp upstream. This latter element is required for neuronal expression but is not necessary for glial expression of Ddc. We provide a model to explain how interactions between multiple regulatory elements may serve to specify cell-specific gene expression.

High levels of intron-containing RNAs are associated with expression of the Drosophila DOPA decarboxylase gene.

We have examined the structure and expression during embryonic development of the Drosophila DOPA decarboxylase gene, Ddc. The Ddc gene is transcribed to make at least five different size classes of RNA. These RNA species first appear late in embryogenesis, coincident with induction of Ddc enzyme activity. The most abundant and smallest RNA appears to be Ddc mRNA. The sequences encoding this RNA are split by two intervening sequences. Each of the larger RNA species contains some or all of the intervening sequences. We have noted two unusual features of Ddc expression during embryogenesis. First, the intervening-sequence-containing RNAs are present as 20% or more of the polyadenylated Ddc RNA molecules, an exceptionally high proportion. Second, these RNAs do not disappear as rapidly as Ddc mRNA after Ddc enzyme activity reaches fully induced levels. These observations indicate slow rates of RNA processing relative to mRNA half-life and suggest that post-transcriptional steps participate in regulating Ddc expression. Although four of the five RNA species were detected at multiple developmental stages during which Ddc is expressed, one was found uniquely during embryogenesis. This RNA differs from Ddc mRNA in length and in time of expression during embryogenesis but is transcribed in the same orientation and from the same genomic sequences as the Ddc primary transcript.