Leptin is a four-helix bundle: secondary structure by NMR.

Leptin is a signaling protein that in its mutant forms has been associated with obesity and Type II diabetes. The lack of sequence similarity has precluded analogies based on structural resemblance to known systems. Backbone NMR signals for mouse leptin (13C/15N -labeled) have been assigned and its secondary structure reveals it to be a four-helix bundle cytokine. Helix lengths and disulfide pattern are in agreement with leptin as a member of the short-helix cytokine family. A three-dimensional model was built verifying the mechanical consistency of the identified elements with a short-helix cytokine core.

Enantioselective reduction of 3,4-methylene-dioxyphenylacetone using Candida famata and Zygosaccharomyces rouxii.

In an effort to prepare 3,4-methylene-dioxyphenyl-(S)-isopropanol from 3,4-methylene-dioxyphenylacetone, an initial screen of microbes indicated that Candida famata could catalyze this reaction efficiently at low substrate concentration. A dilute, large-scale process was developed to provide experimental material for the chemical synthesis to be explored. However, the productivity number of this process [0.134 g product (g wet weight cells)-1 day-1 was too low to be practical. C. famata was also extremely sensitive to concentrations of both the ketone and the alcohol greater than 2 g/l. A more extensive screen of yeast and fungi revealed that Zygosaccharomyces rouxii was more tolerant to higher substrate concentrations and had a higher productivity number [0.8 g (g wet weight cells)-1 day-1]. These characteristics suggested that Z. rouxii could be used in a large-scale process at high substrate concentrations.

Escherichia coli K12 does not colonize the gastrointestinal tract of Fischer-344 rats.

The colonizing potential of Escherichia coli K12 containing a vector coding for somidobove (bovine somatotropin) was determined. Treated male and female Fischer-344 rats were given a single oral gavage inoculum of sucrose with/without tetracycline (15 micrograms/ml). Untreated control animals received similar drinking water regimes. All animals survived until termination. There were no clinical signs of toxicity observed and no treatment-related effect upon body weight, food consumption, or efficiency of food utilization. Fresh fecal samples were collected from each rat every 24 h following inoculation and the population of the marked strain was quantitated until no bacterial colonies were observed for two consecutive days. While all inoculated rats were positive at 24 h, by 72 and 96 h all had become negative for the test (marked) strain, as were the corresponding control group throughout the test. The frozen stock of the marked strain used as the positive control demonstrated that the agar plates were selective for the test strain. Fourteen days following inoculation, all groups of rats were killed and the gastrointestinal tracts removed and treated to recover the marked strain. There was no evidence of the marked strain in the gastrointestinal tract of any from any group. Thus, the E. coli K12 host/vector system used in this experiment does not colonize the gastrointestinal tract of Fischer-344 rats.

Measurement of the change in aerosol size distribution with bacterial growth in a pilot scale fermentor.

A number of industrial processes require the addition of materials to the fermentation broth that are hazardous to health and environment. Agitation of broths inoculated with microorganisms can potentially release aerosols large enough to carry the microorganisms. The influence of agitation, air flow, and bacterial growth on aerosol size distribution, air flow, and bacterial growth on aerosol size distribution was investigated in an industrial pilot scale fermentor. A decrease in particle concentration was observed with increase in bacterial growth; this change was more pronounced in the size range above 2 microm. The aerosol size distribution was found to be practically independent of air flow rate and agitation rate for sizes less than 2 microm. However, for particles larger than 2 microm, the concentration was found to increase with an increase in air flow rate and agitation rate.

Immunocytochemical demonstration of human proinsulin chimeric polypeptide within cytoplasmic inclusion bodies of Escherichia coli.

Immunocytochemical techniques were used to identify human proinsulin chimeric protein in cytoplasmic inclusion bodies of genetically modified Escherichia coli. Antibodies to proinsulin chimeric protein (human proinsulin coupled at its amino-terminus to a portion of the E. coli tryptophan E gene product) were localized in E. coli using post-embedding staining with protein A-peroxidase labelling for transmission electron microscopy. The observable distribution of the labelled antibody was limited to that portion of the E. coli cytoplasm occupied by inclusion bodies. The localization of human peptides as insoluble masses within the bacterial cytoplasm has important implications in relation to the synthesis, recovery and purification of pharmacologically useful substances produced through the application of recombinant DNA technology.

Cytoplasmic inclusion bodies in Escherichia coli producing biosynthetic human insulin proteins.

Escherichia coli that has been genetically manipulated by recombinant DNA technology to synthesize human insulin polypeptides (A chain, B chain, or proinsulin) contains prominent cytoplasmic inclusion bodies. The amount of inclusion product within the cells corresponds to the quantity of chimeric protein formed by the bacteria. At peak production, the inclusion bodies may occupy as much as 20 percent of the Escherichia coli cellular volume.

Primary malignant teratoma of the thyroid: case report and literature review of cervical teratomas in adults.

A case of primary malignant teratoma of the thyroid in a gravid 37-year-old female is reported and the literature on cervical teratomas in adults reviewed. The patient was treated by total thyroidectomy and left radical neck dissection followed by cervical and mediastinal radiotherapy and chemotherapy. She fied of recurrent tumor and related complications 9 months after discovery of the lesion. This is the 6th case of primary teratoma of the thyroid and the 9th case of cervical teratoma reported in a adult. These tumors are characteristically large, rapidly growing and highly malignant. The longest survival reported is 10 months. The differentiation of this lesion from carcinosarcomas of the thyroid is discussed.

Biosynthesis of mycophenolic acid: purification and characterization of S-adenosyl-L-methionine: demethylmycophenolic acid O-methyltransferase.

The final step in the biosynthesis of mycophenolic acid involves the transfer of a methyl group from S-adenosylmethionine to demethylmycophenolic acid. The enzyme, S-adenosylmethionine:demethylmycophenolic acid O-methyltransferase, was isolated from Penicillium stoloniferum and purified 2,700-fold by ammonium sulfate fractionation and diethylaminoethyl-cellulose and Sephadex G-200 chromatography. Maximum enzyme activity was achieved at pH 7.5 and a temperature of 27 to 28 C. The apparent K(m) for demethylmycophenolic acid was 3.1 x 10(-6) M. The enzyme preparation was 50% inactivated when exposed to 33 C for 15 min. Mycophenolic acid, homocystine, S-adenosyl-homocysteine, ethanol, and Mg(2+) inhibited the methyltransferase. This enzyme appears to be subject to end product inhibition which may regulate the synthesis of mycophenolic acid. The methyltransferase activity was highest during the early phases of the fermentation.