Insulin as a surface marker on isolated cells from rat pancreatic islets

Immunoreactive insulin was shown to exist as a surface molecule in the plasma membrane of dispersed rat pancreatic islet cells. The intact cells were stained by immunofluorescence with a guinea pig antisera specific for insulin. The hormone on the cell surface could not be accounted for by insulin bound to specific receptors or nonspecifically absorbed to cells. Thus, surface insulin was demonstrated to be a specific membrane antigen for islet cells. Furthermore, the proportion of islet cells with insulin on the cell surface was directly correlated with insulin secretion in several different settings. This correspondence was demonstrated by varying the glucose concentration in the medium, by withholding Ca2+, which inhibits secretion, and by adding theophylline, which potentiates secretion. Consequently, these results suggested that insulin as a membrane protein was a marker for cells that actively secreted the hormone and may have been derived in the fusion process of secretory granules with the plasma membrane.     

Immunohistochemical demonstration of keratins in human ovarian neoplasms. A comparison of methods

A comparison of five immunohistochemical methods for the demonstration of keratins in human ovarian neoplasms using affinity-purified polyclonal rabbit antibody was made. The use of indirect immunofluorescence on frozen sections briefly fixed in acetone was found to be the most sensitive method and demonstrated keratin in all 14 primary and 1 metastatic ovarian epithelial neoplasms studied. Protein A-peroxidase, peroxidase--antiperoxidase (PAP), indirect peroxidase, or the avidin--biotinylated peroxidase complex (ABC) methods applied to formalin-fixed tissues were less sensitive and led to false negative results in 9 of 15, 1 of 15, 8 of 15, and 6 of 15 cases, respectively. A single case of dysgerminoma failed to reveal keratin by any method.     

Correlation of Ca2+-and calmodulin-dependent protein kinase activity with secretion of insulin from islets of Langerhans.

A Ca2+-activated and calmodulin-dependent protein kinase activity which phosphorylates predominantly two endogenous proteins of 57kDa and 54kDa was found in a microsomal fraction from islet cells. Half-maximal activation of the protein kinase occurs at approx. 1.9 microM-Ca2+ and 4 micrograms of calmodulin/ml (250 nM) for phosphorylation of both protein substrates. Similar phosphoprotein bands (57kDa and 54kDa) were identified in intact islets that had been labelled with [32P]Pi. Islets prelabelled with [32P]Pi and incubated with 28 mM-glucose secreted significantly more insulin and had greater incorporation of radioactivity into the 54 kDa protein than did islets incubated under basal conditions in the presence of 5 mM-glucose. Thus the potential importance of the phosphorylation of these proteins in the regulation of insulin secretion is indicated both by activation of the protein kinase activity by physiological concentrations of free Ca2+ and by correlation of the phosphorylation of the substrates with insulin secretion in intact islets. Experiments undertaken to identify the endogenous substrates indicated that this calmodulin-dependent protein kinase may phosphorylate the alpha- and beta-subunits of tubulin. These findings suggest that Ca2+-stimulated phosphorylation of islet-cell tubulin via a membrane-bound calmodulin-dependent protein kinase may represent a critical step in the initiation of insulin secretion from the islets of Langerhans.     

Analysis of ENU-induced mutations at the Adh locus in Drosophila melanogaster

N-Ethyl-N-nitrosourea (ENU) was used to induce mutations in the Drosophila melanogaster, alcohol dehydrogenase (Adh) gene. Flies were treated with ENU and mated to homozygous intragenic Adh null mutants; Adh null mutations were selected by exposure of the F1 generation to 1-penten-3-ol. Fourteen Adh null mutations were recovered which included 11 from spermatozoa, 2 from oocytes and 1 from a premeiotic spermatocyte. 2 mutations from spermatozoa and 1 of the mutations from oocytes were multilocus deficiencies which included the Adh locus as determined by complementation tests. The remaining 11 intragenic Adh null mutations were sequenced using the Sanger dideoxy method. One Adh null mutation induced in an oocyte was an AT to TA transversion and the mutation induced in a premeiotic spermatocyte was a GC to AT transition, both of which resulted in a single amino acid substitution. The 11 null mutations induced in spermatozoa were a data set in which both the dose of ENU and the treated germ-cell stage were held constant; therefore, only these 11 mutations were used to calculate the mutation frequency and compare the mutations at the Adh locus with those recovered in other studies. The dose of ENU induced a sex-linked recessive lethal frequency approximately 300 times that of the spontaneous frequency; therefore, these mutations were assumed to have been induced by ENU. 2 of the 11 mutations induced in spermatozoa were multilocus deficiencies and 9 were intragenic mutations. 7 of the 9 intragenic mutations were GC to AT transitions which resulted in 5 single amino acid substitutions, 1 premature translation termination codon, and 1 splice site mutation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evolutionally guided enzyme design

A combination of "rational" and "irrational" strategies for the creation of enzymes with novel properties is proving to be a powerful concept in the field of enzyme engineering. Guided by principles of physical organic chemistry, rational design strategies are used to identify suitable target enzymes and to choose appropriate molecular biological methods for engineering purposes. In contrast, irrational (or random) strategies are centered around the biological paradigm of stochastic molecular evolution. As illustrated in this review, such a hybrid approach is particularly useful for the design of new modular enzymes. (c) 1996 John Wiley & Sons, Inc.     

Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins.

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases.     

Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: I. GRADIENT-PURIFIED MITOCHONDRIA

The mitochondrial fraction isolated from durum wheat seedlings by differential centrifugation demonstrated antimycin A- or cyanide-insensitive O₂ uptake. Further purification of this initial mitochondrial pellet using a linear Percoll (Pharmacia) density gradient separated the mitochondria into two bands of physiologically distinct activity. Based on the usual mitochondrial respiratory criteria of ADP/O and respiratory control values, these fractions were qualitatively similar to the crude pellet. However, we observed no antimycin A-insensitive O₂ uptake in either gradient band. Antimycin A-insensitive O₂ consumption could be restored to the upper gradient band of mitochondria by the addition of linoleic acid. This activity was inhibited either by salicylhydroxamic acid or propyl gallate, a known lipoxygenase inhibitor. Likewise, addition of linoleic acid to the crude mitochondrial pellet elicited a 4- to 5-fold increase in O₂ uptake. This O₂ consumption was insensitive to antimycin A and cyanide but was inhibited by either propyl gallate or salicylhydroxamic acid. Electron microscopic examination revealed that only the lower gradient band contained contamination-free mitochondria, which, in turn, lacked ability to oxidize linoleic acid. Antimycin A-insensitive O₂ consumption in the differential centrifugation fraction from germinating durum wheat seedlings decreased over 64 hours of development.     

Survival of recombination-deficient mutants of Escherichia coli during incubation with nalidixic acid.

The ability of several Escherichia coli strains deficient in recombination (rec) to survive in the presence of nalidixic acid was determined. Genetic blocks of the RecBC or the RecF pathways resulted in increased sensitivity to nalidixic acid when compared with the wild-type strain. Mutants lacking functional recA, recL, or recB recC recF genes showed the most rapid decrease in colony-forming ability when incubated with nalidixic acid. However, the uvrB gene also plays a role in maintaining cell viability.