Abnormal functional development of sympathetic nerve terminal-smooth muscle complex in neonatal spontaneously hypertensive rats

In neonatal and young adult SH and WK rats, maturation of a functional unit consisting of sympathetic nerve terminals and smooth muscle (levator palpebrae) was assessed $\text{in vivo}$ by measuring the contractile response to tyramine, an agent which releases endogenous norepinephrine from sympathetic nerve terminals. Responses in SH are comparable to WK at 5–6 days postnatally, smaller from the 8th through 19th day and larger in young adults (41–46 days old). Results indicate that functional maturation of the nerve terminal-smooth muscle complex is retarded in SH relative to WK during the 2nd and 3rd postnatal weeks. It is suggested that retardation in the neonatal SH rat is an expression of a genetic defect in the growth of the complex and that the enhanced response in young adult SH is a consequence of the neonatal abnormality.     

A gene between polA and glnA retards growth of Escherichia coli when present in multiple copies: physiological effects of the gene for spot 42 RNA.

We have isolated the single gene for spot 42 RNA of Escherichia coli on a 20-kilobase DNA fragment. Physical characterization of this cloned DNA fragment showed that it is homologous to a region at 86 min on the genetic map and extends from the 23S to 5S rRNA coding region of rrnA to the coding region of glnA, the gene for glutamine synthetase. Other genes included on this cloned DNA fragment are polA, ntrC (glnG), and ntrB (glnL). E coli cells transformed with a multicopy plasmid clone of the gene for spot 42 RNA had about a 10-fold increase in the amount of spot 42 RNA they contained. The amount of 6S RNA in these cells was increased about twofold, although the gene for 6S RNA was not located on this plasmid or on the larger 20-kilobase fragment. Presence of this multicopy plasmid also affected the growth of cells. The generation time was increased under a variety of growth conditions, especially when cells were grown in medium with succinate as the carbon source. In addition, some strains of E. coli which have multicopy plasmids carrying the gene for spot 42 RNA were unable to respond normally to a shift into richer medium: upon upshift from minimal glucose to LB broth or minimal glucose plus 1% Casamino Acids, there was a 3- to 4-h lag before the culture adapted to the new medium. More than 90% of the cells in such cultures stopped dividing, although they remained viable. The plating efficiency of minimal-glucose-grown cells was 100-fold less on rich media than on minimal glucose medium. One revertant was isolated which regained the phenotype of pBR322-transformed cells. Analysis of this strain showed that the plasmid it contained had an insertion of an IS1 element into the 5' end of the coding region for the gene for spot 42 RNA.     

Ionic charge on phospholipids and their interaction with the mitochondrial adenosine triphosphatase

The activity of the lipid-depleted, oligomycin-sensitive mitochondrial ATPase has been measured in the presence of liposomes prepared from mixtures of phosphatidylglycerol and phosphatidylglycerol lysine. Enzyme activity increased linearly with an increase in the negative charge of liposomes prepared from the phosphatidylglycerol-phosphatidylglycerol lysine mixtures. The electrophoretic mobility and activating capacity of liposomes of several other phospholipids were determined. A linear relationship between electrophoretic mobility of the liposomes and oligomycin-sensitive activity was again apparent. These observations demonstrate that the activity of the ATPase is directly proportional to the ionic charge on phospholipid activators if the acyl chain composition of the phosphoglycerides is relatively constant.     

The immune response of inbred mouse strains to DNP-BGG

The immune response of six inbred mouse strains (SJL, A, C57BL/6, CBA, BALB/c, and DBA/1) to DNP₅₆BGG was tested under three separate immunization schedules: 1 Μg DNP-BGG in 1 mg Al(OH)₃ adjuvant, 50 Μg DNP-BGG in 1 mg A1(OH)₃ adjuvant, and 1 Μg DNP-BGG in complete Freund's adjuvant. Individual serum samples were titered using a modified Farr assay. It was found that the first schedule allowed classification of the mice into responder (SJL, A) and nonresponder (C57BL/6, CBA, BALB/ c, DBA/1) strains. The second schedule produced quantitative as well as qualitative differences among the strains and allowed classification of the mice into higher-responder (SJL, A), intermediate-responder (C57BL/6, CBA, BALB/c), and low-responder (DBA/1) categories. When complete Freund's adjuvant was used in the third schedule, the differences among strains became insignificant. The sera from each strain were pooled and assayed for relative antibody affinity and IgM content. Both of these parameters were dependent largely on the dose of antigen and type of adjuvant used, rather than on the particular mouse strain being studied. The mechanism of adjuvant action, and possible cell interactions in the genetic control of the immune response, are discussed.     

Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluoral Yellow 088 in Polyethylene Glycol-Glycerol

Polyethylene glycol (400) with 90% glycerol (aqueous) is introduced as an efficient solvent system for lipid stains. Various lipid-soluble dyes were dissolved in this solvent system and tested for their intensity, contrast, and specificity of staining of suberin lamellae in plant tissue. The stability (i.e., lack of precipitation) of the various staining solutions in the presence of fresh tissue was also tested. When dissolved in polyethylene glycol-glycerol, Sudan red 7B (fat red) was the best nonfluorescent stain and fluorol yellow 088 (solvent green 4) was an excellent fluorochrome. These two dyes formed stable staining solutions which efficiently stained lipids in fresh sections without forming precipitates. Estimations of the solubilities of these dyes in the solvent compared with their solubilities in lipids of various chemical types indicated that they should both be effective stains for lipids in general.     

Murine p53 inhibits the function but not the formation of SV40 T antigen hexamers and stimulates T antigen RNA helicase activity

We have characterized the effects of p53 on several biochemical activities of simian virus 40 (SV40) large tumor (T) antigen. While p53 induced a strong inhibition of the T antigen DNA helicase activity, surprisingly, its RNA helicase activity was stimulated. This supports the liklihood that the DNA and RNA helicase activities of T antigen reflect discrete functions. p53 did not significantly affect the ATP-dependent conversion of T antigen monomers to hexamers. However, the ability of these hexamers to assemble on a DNA fragment containing the viral origin was impaired by p53. Thus, these results suggest that p53 inhibits the function but not the formation of T antigen multimers. This conclusion was further supported by the observation that the addition of a purified p53:T antigen complex was as inhihitory as free p53 to the DNA helicase activity of free T antigen. Thus our data indicates that the targets of p53 inhibition are the functional units of T antigen, namely the hexamers.     

Use of a Plasmid DNA Probe To Monitor Populations of Bacillus pumilus Inoculant Strains in Hay

We are evaluating naturally occurring isolates of Bacillus pumilus for use as microbial hay preservatives. Seven isolates of B. pumilus from hay contained a 42-kb cryptic plasmid (pMGD296). We wished to determine whether pMGD296 could be used as a molecular marker to follow populations of these isolates in hay over time. Southern blots and colony blots of 69 isolates of B. pumilus and other Bacillus spp. were probed with ³²P-labeled pMGD296. Twenty-nine probe-positive isolates were identified; of these, 28 contained a plasmid with a restriction profile identical to that of pMGD296. One isolate from untreated hay contained a 40-kb plasmid (pMGD150) that was homologous to pMGD296 but had a different restriction fragment pattern. Regions of homology between the two plasmids were identified by Southern blotting, and a 1.9-kb HindIII-PstI fragment of pMGD296 lacking strong homology to pMGD150 was cloned in pUC18. The cloned fragment hybridized only with isolates containing pMGD296 and was used to estimate populations of these isolates in treated and untreated hay.     

Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor.

Plasminogen binding to cell surfaces results in enhanced plasminogen activation, localization of the proteolytic activity of plasmin on cell surfaces, and protection of plasmin from alpha 2-antiplasmin. We sought to characterize candidate plasminogen binding sites on nucleated cells, using the U937 monocytoid cell as a model, specifically focusing on the role of cell-surface proteins with appropriately placed lysine residues as candidate plasminogen receptors. Lysine derivatives with free alpha-carboxyl groups and peptides with carboxy-terminal lysyl residues were effective inhibitors of plasminogen binding to the cells. One of the peptides, representing the carboxy-terminal 19 amino acids of alpha 2-antiplasmin, was approximately 5-fold more effective than others with carboxy-terminal lysines. Thus, in addition to a carboxy-terminal lysyl residue, other structural features of the cell-surface proteins may influence their affinity for plasminogen. Affinity chromatography has been used to isolate candidate plasminogen receptors from U937 cells. A major protein of Mr 54,000 was recovered and identified as alpha-enolase by immunochemical and functional criteria. alpha-Enolase was present on the cell surface and was capable of binding plasminogen in ligand blotting analyses. Plasminogen binding activity of a molecular weight similar to alpha-enolase also was present in a variety of other cell types. Carboxypeptidase B treatment of alpha-enolase abolished its ability to bind plasminogen, consistent with the presence of a C-terminal lysyl residue. Thus, cell-surface proteins with carboxy-terminal lysyl residues appear to function as plasminogen binding sites, and alpha-enolase has been identified as a prominent representative of this class of receptors.