Identification of procalcitonin in a rat medullary thyroid carcinoma cell line

As a first step in studying the biosynthesis of the peptide hormone calcitonin, we have identified procalcitonin species in CA-77 cells, a newly developed rat medullary thyroid carcinoma cell line. mRNA extracted from the cells directed the synthesis of a putative procalcitonin in a reticulocyte lysate translation system containing microsomal membranes. Both this species and a radiolabeled form of immunoreactive calcitonin from intact cells had the same retention time during reverse phase high performance liquid chromatography. The putative cellular procalcitonin was also immunoprecipitated by antiserum to a synthetic peptide whose sequence constitutes the COOH-terminal 16 residues of preprocalcitonin. The polypeptide had a Mr = 13,400, as estimated by gel filtration chromatography under denaturing conditions. Microsequencing of the [35S]methionine-labeled polypeptide indicated that residues 13, 32, and 34 of procalcitonin were methionine. Similar analysis of the peptide labeled with [3H]proline indicated that residues 2 and 11 of the precursor were proline. The positions of methionine and proline could be aligned in a unique manner with the NH2-terminal half of the preprocalcitonin sequence inferred from cDNA analyses. These results indicate that procalcitonin consists of 111 amino acids and suggest that a 25-residue signal sequence is cotranslationally cleaved from preprocalcitonin. From the procalcitonin sequence we can now predict the sequence of likely biosynthetic intermediates and mature secretory products derived from the NH2-terminal as well as COOH-terminal regions of the precursor.     

Influence of malnutrition and alterations in dietary protein on murine rotaviral disease

The possible correlation between malnutrition and degree of severity of rotavirus-associated infantile diarrhea which appears to occur in human populations was studied using a mouse model. To determine the effects of general malnutrition or altered levels of dietary protein, female mice were fed throughout pregnancy and infection periods with diets diluted with 0, 300, or 600 g glucose/kg, designated as normal nutrient to calorie ratio (N/C) diet, 70% N/C diet, or 40% N/C diet or with diets containing 75, 150, or 300 g casein/kg, as low-, normal-, or high-protein diets. Murine rotavirus was given by gavage to the 2-day-old offspring of these dams, and the extent of infection determined. Marked increases in severity of diarrheal disease were seen in the infants from dams receiving the 40 and 70% N/C diets and the low-protein diet. Severity of infection was seen as increased deaths, reduced weight gain, and increased passage of diarrheic feces. Intestinal viral levels and intestinal diarrhea scores did not vary appreciably. Serum interferon remained below detectable limits throughout the studies, but serum antibody was determined in dams 30 days post-virus exposure. The latter titers were lower in the infected mice from dams fed the 40 and 70% N/C diets, but were essentially the same in all the protein diet groups. Cross-fostering was done using the 40 and 100% N/C diets, wherein mice from dams fed either diet were placed on mothers fed the opposite diet. Increased severity of infection was again seen when the virus was given 2 days after the exchange, although the greatest infection occurred in animals from dams fed 40% N/C diet which were then fostered by other similarly fed dams. The increased host sensitivity to the rotaviral infection appeared to be a result of both pre- and postnatal dietary effects.     

Fine Structure of Thiobacillus thiooxidans

Mahoney, Robert P. (Skidmore College, Saratoga Springs, N.Y.), and Mercedes R. Edwards. Fine structure of Thiobacillus thiooxidans. J. Bacteriol. 92: 487-495. 1966.-Thin section analysis of the chemosynthetic autotroph Thiobacillus thiooxidans revealed structures comparable to gram-negative heterotrophic bacteria. Although this species is unique in that it oxidizes elemental sulfur for energy, uses carbon dioxide as its sole source of carbon, and can withstand a pH of less than 1, thin sections revealed a profile of the cell envelope (cell wall and plasmalemma) similar to other gram-negative species which have more common physiological traits. The cell wall is composed of five layers with an overall width of approximately 200 A, and the plasmalemma appears as a conventional "unit membrane" with a width of about 85 A. Volutin granules and less-dense bodies of similar shape and size were frequently observed in close association with the nucleoplasm. The nature and function of these bodies are unknown at this time.     

The impenetrability of 5-(N-hexadecanoyl)aminofluoroscein through endothelial cell monolayers is dependent upon its solution properties, not the presence of tight junctions.

The solution properties of two fluorescent lipophilic analogues were examined in conjunction with their ability to penetrate the tight junctions of bovine aortic endothelial cell monolayers. 5-(N-dodecanoyl)aminofluoroscein was shown to label both the apical and basolateral plasma membrane domains of confluent monolayers at 4 degrees C and pH 7.3, but 5-(N-hexadecanoyl)aminofluoroscein was shown to label only the apical membrane domain. When used under more soluble conditions at 20 degrees C and pH 8.5, both probes labeled apical and basolateral plasma membrane domains more equally. This indicates that solubility conditions, and not tight junctions, dictate the penetration of 5-(N-hexadecanoyl)aminofluoroscein from the apical to the basolateral plasma membrane domain.     

Production and composition of phenylpyrrole metabolites prodcued by Pseudomonas cepacia

Summary In shaken cultures, a strain of Pseudomonas cepacia isolated from apple leaves produced pyrrolnitrin and four other phenylpyrrole antibiotics. The concentrations of these metabolites were determined at intervals for 7 days in three different media at two initial pH levels. Optical density measurements revealed maximum cell concentrations after 24 h in nutrient broth, after 48 h in King's B medium, and after 96 h in minimum salts solution. The effects caused by initiating fermentations at pH 5.8 rather than 7.0 were in most cases not dramatic, although in some instances, especially in minimum salts broth, higher concentrations of metabolites were produced with the lower initial pH. Concentrations of the phenylpyrrole antibiotics were greatly affected by choice of culture medium and incubation time. Concentrations of the two nitrophenyl metabolites, pyrrolnitrin and 2-chloropyrrolnitrin, rose throughout the 7-day incubation and were more than 20 times greater in minimum salts medium than in either King's B medium or nutrient broth. The maximum concentrations of each of the three aminophenyl metabolites (dichloroamino, trichloroamino and monochloroamino) occurred in different media, the monochloro compound in nutrient broth, the dichloro compound in Kings B medium and the trichloro compound in minimum salts medium. The time dependence of the concentrations of the five metabolites supports the proposed biosynthesis of these pyrroles from tryptophan by successive chlorinations followed by oxidation of the amino group at the end of the pathway.     

Aflatoxin variability in pistachios.

Pistachio fruit components, including hulls (mesocarps and epicarps), seed coats (testas), and kernels (seeds), all contribute to variable aflatoxin content in pistachios. Fresh pistachio kernels were individually inoculated with Aspergillus flavus and incubated 7 or 10 days. Hulled, shelled kernels were either left intact or wounded prior to inoculation. Wounded kernels, with or without the seed coat, were readily colonized by A. flavus and after 10 days of incubation contained 37 times more aflatoxin than similarly treated unwounded kernels. The aflatoxin levels in the individual wounded pistachios were highly variable. Neither fungal colonization nor aflatoxin was detected in intact kernels without seed coats. Intact kernels with seed coats had limited fungal colonization and low aflatoxin concentrations compared with their wounded counterparts. Despite substantial fungal colonization of wounded hulls, aflatoxin was not detected in hulls. Aflatoxin levels were significantly lower in wounded kernels with hulls than in kernels of hulled pistachios. Both the seed coat and a water-soluble extract of hulls suppressed aflatoxin production by A. flavus.     

Inhibition of Aflatoxin Production by Surfactants

The effect of 12 surfactants on aflatoxin production, growth, and conidial germination by the fungus Aspergillus flavus is reported. Five nonionic surfactants, Triton X-100, Tergitol NP-7, Tergitol NP-10, polyoxyethylene (POE) 10 lauryl ether, and Latron AG-98, reduced aflatoxin production by 96 to 99% at 1% (wt/vol). Colony growth was restricted by the five nonionic surfactants at this concentration. Aflatoxin production was inhibited 31 to 53% by lower concentrations of Triton X-100 (0.001 to 0.0001%) at which colony growth was not affected. Triton X-301, a POE-derived anionic surfactant, had an effect on colony growth and aflatoxin production similar to that of the five POE-derived nonionic surfactants. Sodium dodecyl sulfate (SDS), an anionic surfactant, and dodecyltrimethylammonium bromide, a cationic surfactant, suppressed conidial germination at 1% (wt/vol). SDS had no effect on aflatoxin production or colony growth at 0.001%. The degree of aflatoxin inhibition by a surfactant appears to be a function of the length of the hydrophobic and hydrophilic chains of POE-derived surfactants.     

Restriction enzymes in cells, not eppendorfs

Restriction enzymes are essential reagents to molecular biologists, but their relevance to bacterial populations is less obvious. Most bacteria encode restriction and modification systems and these are commonly considered to be a barrier to phage infection. Current evidence also supports a more general role for them in genetic recombination.