Recent studies suggest that synthesis of the Colony-stimulating factor (CSF) is a well regulated process. However, the molecular mechanisms of the signal transduction of the various inducers of CSF such as monokines and lymphokines are not well understood. Using Interleukin 1 (IL-1) stimulation of CSF-1 in the MIA PaCa-2 cell line as a model system, the involvement of G-protein has been studied. The IL-1 induction of CSF-1 synthesis can be inhibited by both Pertussis toxin and Cholera toxin, which are known to modify the Gᵢ and Gₛ proteins respectively, thus activating adenylate cyclase to release more cAMP. The toxin inactivation can be prevented by inhibitors of the ADP-ribosylation such as, benzamide and MBAMG. Addition of dibutyryl-cAMP inhibits the IL-1 induced CSF production. Both Theophylline and Forskolin which increase cAMP by inhibiting phosphodiesterase and stimulating adenylate cyclase respectively, also inhibit CSF-1 production. Results from these studies have shown that cAMP level inversely regulates the biosynthesis of CSF-1. Preincubation of MIA PaCa-2 cells with IL-1 and 5'- guanylylimidodiphosphate (GppNHp) prevents the inhibitory effect of pertussis toxin on CSF-1 production. These data are consistent with the hypothesis that IL-1 binds to its receptor and couples to Gᵢ∝ resulting in the inhibition of adenylate …

A highly specific steroid regulated transcription system system in Saccharomyces cerevisae was used to screen for phytoestrogens indioecious plants. Yeast cells were co-transformed with a human estrogen receptor expression plasmid and a reporter plasmid containing the E. coli β-galactosidase gene.

TOL plasmids encode enzymes responsible for utilization of toluene and related aromatic compounds by Pseudomonas putida, ultimately converting them to central metabolic intermediates. The nucleotide sequence for the 5.6 kb xylGFJQK region of the pDK1 TOL meta operon was determined. DNA sequence analysis revealed the presence of five open reading frames corresponding to xylG (1458 bp), xylF (846 bp), xylJ (783 bp), xylQ (936 bp) and xylK (1047 bp), encoding predicted protein products of 51.6, 31.3, 27.8, 32.8, and 36.6 kDa in size, respectively. The average G+C content of the xylLTEGFJQK region was 65.7%, somewhat higher than the 58.9% seen in the immediately upstream xylXYZ region and substantially more than the 50% G+C content reported for the upper TOL operon of this plasmid. Homology comparisons were made with genes and proteins of related catabolic plasmids. The dmpCDEFG and pWWO xylGFJQK regions exhibit consistently high levels of nucleotide and amino acid homology to pDK1 xylGFJQK throughout the entire region. In contrast, although the nucleotide sequence homology of the Acinetobacter atdCDE region to xylGFJ is high, the homology of atdFG to xylQK is markedly less. Such radical changes in homology between corresponding regions of different operons, combined with variable base and codon …

Rhizobia are Gram-negative, rod-shaped, soil bacteria with the ability to fix atmospheric nitrogen into ammonia as symbiont bacteroids within nodules of leguminous plant roots. Here, resident Rhizobium plasmids were studied as possible sources of components for the construction of a cloning vector for Rhizobium species.

TOL plasmids of Pseudomonas putida encode enzymes for the degradation of toluene and related aromatics. These genes are organized into two operons regulated by the Xy1R and Xy1S transcriptional activators. Previous analysis of the TOL pDK1 catechol-2,3-dioxygenase gene (xy1E) and a comparison of this gene to xy1E from the related TOL plasmid pWW0, revealed the existance of a substantial level of sequence homology (82%).

The complete nucleotide sequence of the region encoding the DHCDH function of the pDK1 lower operon was determined. DNA analysis has shown the presence of two open reading frames, one gene consisting of 777 nucleotides encoding a polypeptide of 27.85 kDa and another gene of 303 nucleotides encoding a polypeptide of 11.13 kDa. The results of enzymatic expression studies suggest that DHCDH activity is associated only with xy1L. However although the addition of xy1T cell-free extracts to xy1L cell-free extracts does not produce an increase in DHCDH activity, subclones carrying both xy1L and xy1T exhibit 300- 400% more DHCDH activity than subclones carrying only xy1L.

Cell-free extracts from Pseudomonas fluorescens NCIMB 11764 catalyzed the degradation of cyanide into products that included C02, formic acid, formamide and ammonia. Cyanide-degrading activity was localized to cytosolic cell fractions and was observed at substrate concentrations as high as 100 mM. Two cyanide degrading activities were identified by: (i) the determination of reaction products stoichiometries, (ii) requirements for NADH and oxygen, and (iii) kinetic analysis. The first activity produced CO2 and NH3 as reaction products, was dependent on oxygen and NADH for activity, and displayed an apparent Km for cyanide of 1.2 mM. The second activity generated formic acid (and NH3) pfus formamide as reaction products, was oxygen independent, and had an apparent Km of 12 mM for cyanide. The first enzymatic activity was identified as cyanide oxygenase whereas the second activity consists of two enzymes, a cyanide nitrilase (dihydratase) and putative cyanide hydratase. In addition to these enzymes, cyanide-grown cells were also induced for formate dehydrogenase (FDH), providing a means of recycling NADH utilized by cyanide oxygenase.

The Pseudomonas aeruginosa ATCase was cloned and sequenced to determine the correct size, subunit composition and architecture of this pivotal enzyme in pyrimidine biosynthesis. During the course of this work, it was determined that the ATCase of Pseudomonas was not 360,000 Da but rather present in a complex of 484,000 Da consisting of two different polypeptides (36,000 Da and 44,000 Da) with an architecture similar to that of E. coli ATCase, 2(C3):3(r2). However, there was no regulatory polypeptide found in the Pseudomonas ATCase.

The regulation of short chain fatty acid metabolism has been examined. Metabolism of acetoacetate, and short chain fatty acids such as butyrate and valerate, is predicated upon the expression of genes of the ato operon. Acetoacetate induces expression of a CoA transferase (encoded by the atoDA genes) and expression of a thiolase (encoded by the atoB gene). Metabolism of saturated short chain fatty acids requires the activities of the transferase and thiolase and enzymes of 6-oxidation as well. Spontaneous mutant strains were isolated that were either constitutive or that were inducible by valerate or butyrate instead of acetoacetate.

Triosephosphate isomerase (EC 5.3.1.1., TPI) undergoes specific posttranslational modifications (deamidation and oxidation) which are believed to initiate protein turnover by destabilization of the dimer. The crystal structures, amino acid sequences, and aging related changes of TPI from various species have been independently characterized by several laboratories. TPI has thus become the prototype enzyme for examining the initial steps in protein turnover. The binding of substrate enhances the specific deamidation of the mammalian enzyme, and a general mechanism of 'molecular wear and tear' [Gracy, R. W., Yiiksel, K. 0., Chapman, M. L., and Dimitrijevich, S. D. (1990) in Isozymes-Structure, Function and Use in Biology and Medicine (Ogita, Z-I., and Markert, C. L., Eds) pp. 787-817, Wiley-Liss, New York] has been proposed to explain how enzymes may wear out.