Corrosion casts as a method for investigation of the insect tracheal system

Summary The tracheal systems of five insect species (two species of ants, worker bee, housefly and the cabbage butterfly) have been studied by scanning electron microscopy of corrosion casts. This technique, which is commonly used for the investigation of vertebrate vasculature, is adapted to demonstrate the ultrastructure of the insect respiratory organ. The problem of filling a blind ending system was solved by injecting the resin Mercox into the evacuated tracheae through a thoracal spiracle. After polymerization of the resin, the tissue was digested enzymatically and chemically. The three-dimensional structure of the tracheal system was investigated by scanning electron microscopy. The technique used here displays for the first time the complex morphology of the entire tracheal system in fine detail, especially the structure of spiracles, airsacs, tracheae and tracheoles. Smooth-walled terminal tracheoles show up in flight muscles. The finest tracheoles that could be identified have diameters of approximately 70 nm. This approaches the finest tracheoles portrayed by transmission electron micrographs.     

Mutant proteins of human interleukin 2. Renaturation yield, proliferative activity and receptor binding

Muteins, i.e. proteins altered by mutation of their genes, of interleukin 2 (Il2) were generated by oligonucleotide-directed mutagenesis in vitro. All acidic and basic residues conserved between man and mouse were exchanged as well as four lipophilic residues contained within four hydrophobic segments of the protein. The muteins were produced in Escherichia coli and submitted to a renaturation and purification protocol, before bioactivity and receptor binding of each of them was determined. All muteins besides two (K44/T125 and Q110/T125) could be renatured and purified. One mutein (K94/T125) exhibited a more than tenfold-improved renaturation yield. One amino exchange (Asp-20 to Asn) resulted in an about 20-fold reduction in proliferative activity and high-affinity receptor binding. The binding to the low-affinity Il2-binding protein (Tac antigen) was unimpaired. A second exchange (Arg-38 to Gln) had no effect on proliferative activity. The binding to both the high- and the low-affinity receptor, however, was reduced about 20-fold. Preliminary trials on the stability of these muteins by guanidinium hydrochloride denaturation studies detected no differences between wild-type interleukin 2 and muteins. It is suggested that Asp-20 forms part of the binding site for the large receptor subunit whereas Arg-38 is involved in the contact site to the small subunit.     

Mutation in phenol-type herbicide resistance maps within the psbA gene in Synechocystis 6714

A Synechocytis 6714 mutant resistant to the phenol-type herbicide ioxynil was isolated and characterized. Sensitivity to DCMU and atrazine was tf measured in whole cells and isolated thylakoids. The mutant presents the same sensitivity to atrazine as the wild type and a slightly increased sensitivity to DCMU. A point mutation has been found at codon 266 in the psbAI coding locus (AAC to ACC) resulting in an amino acid change from asparagine to threonine in the D1 protein.     

Evidence for two closely related isozymes of arylamine N-acetyltransferase in human liver

Acetyl CoA-dependent arylamine N-acetyltransferase (EC 2.3.1.5) is the target of a genetic polymorphism in the metabolism of drugs and carcinogens. N-Acetyltransferase was purified 1000-fold from cytosol of human liver and its identity was verified by amino acid sequence homology of two of its tryptic peptides with published rabbit and chicken N-acetyltransferase sequences. Enzyme activity correlated with the presence of two proteins, NAT-1 and NAT-2, with indistinguishable molecular masses (31 kDa). NAT-1 and NAT-2 could be separated by anion-exchange chromatography and were functionally distinguished by their different apparent affinities for the acceptor amine sulfamethazine (SMZ). Antibodies raised against NAT-1 were able to recognize both isozymes on Western blots.     

Enkephalin regulation of L-valine transport in rabbit ileum

We studied the influence on ionic basal transport (Na+ and Cl-) and L-valine transport of two enkephalins which are not metabolized and act in delta and mu receptors respectively. Transports have been indirectly determined measuring the transepithelial electric potential and the short circuit current. DADLE does not significantly influence ion and amino-acid transport, while DAGO alters both of them in the presence of the myenteric plexus (muscle layers present) or inhibits only L-valine transport in the absence of the plexus (muscle layers removed).     

Expression of a cytochrome c2 isozyme restores photosynthetic growth of Rhodobacter sphaeroides mutants lacking the wild-type cytochrome c2 gene

Deletion of the cytochrome c2 gene in the purple bacterium Rhodobacter sphaeroides renders it incapable of phototrophic growth (strain cycA65). However, suppressor mutants which restore the ability to grow phototrophically are obtained at relatively high frequency (1-10 in 10(7)). We examined two such suppressors (strains cycA65R5 and cycA65R7) and found the expected complement of electron transfer proteins minus cytochrome c2: SHP, c', c551.5, and c554. Instead of cytochrome c2 which elutes from DEAE-cellulose between SHP and cytochrome c', at about 50 mM ionic strength in wild-type extracts, we found a new high redox potential cytochrome c in the mutants which elutes with cytochrome c551.5 at about 150 mM ionic strength. The new cytochrome is more acidic than cytochrome c2, but is about the same size or slightly smaller (13,500 Da). The redox potential of the new cytochrome from strain cycA65R7 (294 mV) is about 70 mV lower than that of cytochrome c2. The 280 nm absorbance of the new cytochrome is smaller than that of cytochrome c2, which suggests that there is less tryptophan (the latter has two residues). In vitro kinetics of reduction by lumiflavin and FMN semiquinones show that the reactivity of the new cytochrome is similar to that of cytochrome c2, and that there is a relatively large positive charge (+2.6) at the site of reduction, despite the overall negative charge of the protein. This behavior is characteristic of cytochromes c2 and unlike the majority of bacterial cytochromes examined. Fourteen out of twenty-four of the N-terminal amino acids of the new cytochrome are identical to the sequence of cytochrome c2. The N-termini of the cycA65R5 and cycA65R7 cytochromes were the same. The kinetics and sequence data indicate that the new protein may be a cytochrome c2 isozyme, which is not detectable in wild-type cells under photosynthetic growth conditions. We propose the name iso-2 cytochrome c2 for the new cytochrome produced in the suppressor strains.     

The amino acid sequence of low-potential cytochrome c550 from the cyanobacterium Microcystis aeruginosa

The low-potential cytochrome c550 has been purified from the cyanobacterium Microcystis aeruginosa and its amino acid sequence has been determined. The protein contains 135 amino acid residues with the Cys-X-X-Cys-His heme binding site at residues 37 to 41. The sequence from residue 28 to 45 shows similarity to cytochrome c553 residues 1 to 18 when the heme binding sites are aligned. Another region of similarity is in the carboxyl-terminal regions of these two proteins. The two aligning regions of cytochrome c553 correspond to helical segments in other related cytochromes. A partial sequence of cytochrome c550 from Aphanizomenon flos-aquae was obtained and showed a 48% identity to the sequence of the M. aeruginosa cytochrome. The single methionine residue in cytochrome c550 of M. aeruginosa occurs at position 119 but there is no methionine in this region in the A. flos-aquae cytochrome, indicating that methionine is not the sixth ligand to the heme iron atom. Histidine 92 is a possible sixth ligand in M. aeruginosa cytochrome c550. The far-uv circular dichroism spectrum indicates that this protein is approximately 17% alpha helix, 42% beta-pleated sheet, and 41% random coil.     

The CYP2D gene subfamily: analysis of the molecular basis of the debrisoquine 4-hydroxylase deficiency in DA rats

The DA rat has been proposed as an animal model for the human debrisoquine 4-hydroxylase/bufuralol 1'-hydroxylase genetic deficiency. To determine the mechanism of this deficiency, we isolated and sequenced five cDNAs in the CYP2D gene subfamily including a new IID1 allele and two cDNAs of novel P450s, designated IID3 and IID5. IID3 and IID5 cDNA-deduced amino acid sequences contained 500 and 504 residues with calculated molecular weights of 56,683 and 57,081, respectively. IID5 displayed 20 amino acid differences with the IID1, yet bore only 72% and 76% similarity to IID2 and IID3. Despite an overall nucleotide similarity of 80-98% between the 4 cDNAs, a region of 134 nucleotides of sequence exists that contains only 1 base difference. This region is probably the result of gene conversion events between the P450 IID genes. Although all IID cDNAs were expressed into immunodetectable proteins using the COS cell SV40-based expression system, only IID1 could effectively catalyze the oxidation of the prototype substrate bufuralol. Expression of a cDNA isolated in an earlier study [Gonzalez, F. J., Matsunaga, T., Nagata, K., Meyer, U. A., Nebert, D. W., Pastewka, J., Kozak, C. A., Gillette, J., Gelboin, H. V., & Hardwick, J. P. (1987) DNA 6, 149-161], previously called db1 and now designated IID1v, produced a protein with a drastically reduced activity as compared to cDNA-expressed IID1 despite only four amino acid differences between the two cDNA-deduced protein sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperosmotic relaxation lysis of chromaffin granules is caused by interactions between the granular membrane and intragranular vesicles

Bovine chromaffin granules undergo irreversible structural changes during osmotic shrinkage in hypertonic sucrose and salt solutions, such that, on reexposure to isoosmotic conditions they do not regain their original morphology, but undergo lysis ('hyperosmotic relaxation lysis'). Irreversible alterations of granules were induced by hypertonic incubations lasting for as little as 1 min. Fluorescence and EPR membrane labelling experiments showed that hypertonicity did not induce membrane loss for instance by inwardly or outwardly directed pinching off of membrane material. The mean sizes of chromaffin granules as a function of increasing and subsequently decreasing osmotic pressure were measured by photon correlation spectroscopy; there was no significant difference in sizes of hyperosmotically pretreated granules as compared with controls. Freeze-fracture electron micrographs showed the formation of 'twins' and 'triplets' under hypertonic conditions. They also revealed intragranular vesicles of 50-200 nm in diameter in both hypertonically and isotonically suspended granules. 'Twin' and 'triplet' granules were formed by the attachment of intragranular vesicles to the granule membranes. We suggest that hyperosmotic relaxation lysis is caused by the fact that this adhesion partly prevents the granule membrane from reexpanding, thus, leading to its rupture.     

A second nonrandom translocation, der(16)t(1;16)(q21;q13), in Ewing sarcoma and peripheral neuroectodermal tumor

The majority of Ewing sarcomas and peripheral neuroectodermal tumors (PNET) that have been karyotyped contain a specific translocation, t(11;22)(q23;q11). We report here a second nonrandom translocation, der(16)t(1;16)(q21;q13), in 2 of 20 cases of Ewing sarcoma (seven previously unreported) and 2 of 7 cases of PNET (all previously unreported). All cases with this translocation also contained the t(11;22). Comparison of C-banding patterns in tumor and peripheral lymphocyte karyotypes in one case indicated that the likely breakpoints were 1q21 and 16q13. The presence of this translocation in cell lines will enable further investigation of the molecular events important in the pathogenesis of Ewing sarcoma and PNET.