Renin: structural features of active enzyme and inactive precursor

To determine the structural basis for the unique catalytic mechanism of renin and the mechanism of activation of inactive renin, renin and inactive renin were isolated in pure form. The active site of renin consists of two aspartyl residues, two tyrosyl residues, and one arginyl residue, analogous to pepsin and other acid proteases. The complete amino acid sequence of mouse submaxillary gland renin was determined. Of the amino acids, 43% were identical to those in porcine pepsin. Combination of various chromatographic techniques permitted the separation of inactive renin from active renin in human plasma and kidney. Inactive renin of hog kidney was completely purified. Inactive renin consists of a single polypeptide chain and is activated by proteolysis but not by dissociative reagents such as 4 M NaCl or detergent. Thus it was concluded that the inactive renin in these tissues is renin zymogen rather than a renin-inhibitor complex.     

Localisation of malignant germ-cell tumours by external scanning after injection of radiolabelled anti-alpha-fetoprotein.

Sheep IgG antibody to alpha-fetoprotein was labelled with 131I and used to identify human germ-cell tumours by emission scanning. Eleven patients were studied after resection of their primary tumours. Ten had malignant teratoma and one an endodermal sinus tumour. All eight patients with raised serum alpha-fetoprotein concentrations had metastases apparent in the antibody scans. Of the remaining three patients with normal serum alpha-fetoprotein concentrations, two had positive scans. Three of the patients with positive results were scanned twice; the second scans were negative after treatment, when the alpha-fetoprotein concentrations had returned to normal. These result suggest that antibody scans are useful in the clinical management of patients with germ-cell tumours.     

Correlation of the conformation of a modified ribonuclease octapeptide, homologous to peptide T, with its ability to induce CD4-dependent monocyte chemotaxis

Peptide T, from the human immunodeficiency virus (HIV), whose sequence is Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, has been shown to inhibit attachment of this virus to T cells and neural cells bearing the CD4 receptor. This peptide shares extensive homology with the 19–26 segment of ribonuclease A (RNase A), whose sequence is Ala-Ala-Ser-Ser-Ser-Asn-Tyr-Cys. Based on comparison of the structures of peptides occurring in proteins of known structure that are homologous to peptide T,viz, RNase A and endothiapepsin and on conformational energy calculations, we predicted that peptide T adopts a structure much like that for residues 19–26 in RNase A. A critical feature is a bend involving residues Thr 4-Asn 7 in peptide T corresponding to Ser 22-Tyr 25 in the RNase A peptide. Our proposed structure for peptide T has recently been confirmed by Cotelleet al. (Biochem. Biophys. Res. Commun. 171, 596–602). We now show directly that the RNase A peptide, with Met replacing Cys 26 to prevent disulfide exchange reactions, strongly induces monocyte-chemotaxis that is blocked by anti-CD4 monoclonal antibody. Both peptide T and RNase A fail to induce chemotaxis, however, in neutrophils which do not express surface CD4 receptors. These results suggest that both peptides interact with the CD4 receptor in inducing monocyte chemotaxis. We have also prepared cyclo-RNase A peptide with Met 26. Using molecular dynamics and conformational energy calculations, we find that the cyclic peptide cannot form a bend structure involving Ser 22-Tyr 25 that is superimposable on the RNase A bend. Indeed, we find that this peptide is inactive in inducing monocyte chemotaxis despite the fact that its amino acid sequence is identical to that of the open chain form. This result suggests that a correlation between the -bend structure of the RNase A peptide and peptide T and their abilities to bind to the CD4 receptor.     

Population distribution in North and Central America of PGM1 and Gc subtypes as determined by isoelectric focusing (IEF)

Gc subtypes of 20 North and Central American populations and PCM₁ subtypes in 11 populations were analyzed to identify interpopulation variation of the respective gene frequencies for common alleles. A total of 23,304 phenotypings were done. Absolute heterozygosity levels (D) generally increased twofold when phenotyping by isoelectric focusing was compared with conventional electrophoresis. Graphic representation of the Gc subtypes and multivariate analysis to identify genetic affinities of the populations under study reveal genetic clusters consistent with major historical and geographical groupings of man.     

Physicochemical properties of Shiga toxigenic Escherichia coli

AIMS: To investigate the physicochemical surface properties, such as cellular surface charge, hydrophobicity and electron donor/acceptor potential of a selection of Shiga toxigenic Escherichia coli (STEC) isolates grown in broth and agar culture. METHODS AND RESULTS: Cellular surface charge was determined using zeta potential measurements. Hydrophobicity of the isolates was determined using bacterial adhesion to hydrocarbons assay, hydrophobic interaction chromatography and contact angle measurements. Microbial adhesion to solvents was used to determine the electron donor/acceptor characteristics. No differences of surface charge measurements were found between broth and agar grown cultures. Isolates belonging to serogroup O157 and serotypes O26:H11 and O111:H- were significantly (P < 0.05) less negatively charged than other STEC serotypes tested. All strains were hydrophilic with most methods and demonstrated a lower hydrophobicity in agar culture compared with broth culture. All strains demonstrated a strong microbial adhesion to chloroform indicating that STEC possess an electron donor and basic character. A relationship between serogroup O157 and other STEC serotypes was apparent using principal-component analysis (PCA). CONCLUSIONS: Combining the results for physicochemical properties using PCA differentiated between strains belonging to the O157 serogroup and other STEC/non-STEC strains. PCA found similar results for broth and agar grown cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Particular serotypes of STEC possess similar physicochemical properties which may play a role in their pathogenicity or potential attachment to various surfaces.     

Downregulation of calcineurin activity in cervical carcinoma

Background  

Calcineurin (CaN) is an important serine-threonine phosphatase (PP2B), which plays a crucial role in calcium-calmodulin mediated signal transduction events. Calcineurin has been implicated in pathogenesis of various diseases cardiac hypertrophy, diabetic neuropathy and Alzheimer's, however its role in neoplasia remains unclear.     

Enkephalin is a competitive antagonist of cholecystokinin in the gastrointestinal tract, as predicted from prior conformational analysis

Prior calculations based on ECEPP (Empirical Conformational Energies for Peptides Program) of the low energy minima for cholecystokinin (CCK) and Met-enkephalin have demonstrated that significant structural features of these two peptides are identical. This result suggested the possibility that Met-enkephalin, as well as other enkephalin analogues of similar structure, could associate with receptors for CCK. To test this theoretical result, we examined the ability of Met-enkephalin and its analogues to bind to peripheral CCK receptors in the rat gastrointestinal tract; in particular, we measured the ability of the opiate peptide to inhibit the effects of CCK in a physiological assay system which we have previously characterized: CCK-induced contraction of the isolated rat pyloric sphincter. We find that Met-enkephalin is an antagonist of the CCK-8-induced contraction, with a IC₅₀ of 110 nM. Furthermore, antibodies against CCK were found to cross-react with Met-enkephalin and its analogues in a manner which suggests a distinct structure-activity relationship. These experimental results strongly support the theoretical results of conformational analysis showing structural similarity between enkephalin and CCK. They further suggest that enkephalins could modulate the response of CCK systems under physiological conditions.     

Frequency of the ESD*5 allele in three ethnic groups in Minnesota

Summary Detection of the ESD*5 gene products was simplified by the use of isoelectric focusing (IEF). Gene frequencies for ESD*5 in whites, blacks and Amerindians from the State of Minnesota were 0.019, 0.002 and 0.000, respectively. The gene frequencies observed in the white data set were similar to those reported for European whites.     

The histone deacetylase inhibitor cambinol prevents acidic pHe-induced anterograde lysosome trafficking independently of sirtuin activity

Common features of the solid tumor microenvironment, such as acidic extracellular pH and growth factors, are known to induce the redistribution of lysosomes from a perinuclear region to a position near the plasma membrane. Lysosome/plasma membrane juxtaposition facilitates invasion by allowing for the release of lysosomal proteases, including cathepsin B, which contribute to matrix degradation. In this study we identified the sirtuin 1/sirtuin 2 (SIRT1/2) inhibitor cambinol acts as a drug that inhibits lysosome redistribution and tumor invasion. Treatment of cells with cambinol resulted in a juxtanuclear lysosome aggregation (JLA) similar to that seen upon treatment with the PPARγ agonist, troglitazone (Tro). Like Tro, cambinol required the activity of ERK1/2 in order to induce this lysosome clustering phenotype. However, cambinol did not require the activity of Rab7, suggesting that this drug causes JLA by a mechanism different from what is known for Tro. Additionally, cambinol-induced JLA was not a result of autophagy induction. Further investigation revealed that cambinol triggered JLA independently of its activity as a SIRT1/2 inhibitor, suggesting that this drug could have effects in addition to SIRT1/2 inhibition that could be developed into a novel anti-cancer therapy.