ACTH determination in a petrosal sinus venous specimen after corticotrophin releasing factor provides the best clue on the laterality of microadenoma in Cushing's disease

A 42-year-old woman with Cushing's disease was endocrinologically cured after transsphenoidal selective excision of a left-sided microadenoma. Simultaneous samplings from the bilateral inferior petrosal sinuses with ovine corticotrophin-releasing factor (CRF) stimulation were performed preoperatively to evaluate some relations between the localization of a possible microadenoma and the levels of ACTH in inferior petrosal sinuses. The data for the venous samplings were as follows:-(1) The basal levels of ACTH obtained simultaneously from both inferior petrosal sinuses and peripheral vein were about the same. (2) A significant difference, with a high level of ACTH from the ipsilateral side of the microadenoma, was demonstrated after CRF stimulation. (3) The magnitude of increase in ACTH on the contralateral side after CRF was similar to that of the peripheral response. It is recommended that all patients with ACTH dependent Cushing's syndrome and negative radiological findings should have bilateral simultaneous inferior petrosal venous sampling with CRF stimulation.     

Single amino acid replacements affecting the thermostability of kanamycin nucleotidyltransferase

Summary Amino acid residues of the carboxyl-terminal region of kanamycin nucleotidyltransferase were modified using segment-directed mutagenesis. Six different mutant enzymes with single amino acid replacements were selected out of 59 clones by DNA sequence analyses. The mutant enzymes were purified and it was found that the thermostability of one mutant enzyme was identical to the wild type, whereas the other five were less thermostable at varying degrees. The data suggested that changes in the enzyme thermostability depend not only on the position but also on the species of amino acid residue replaced.     

Nucleotide sequence of the penicillinase repressor gene penI of Bacillus licheniformis and regulation of penP and penI by the repressor.

Bacillus licheniformis penicillinase genes, penP and penI, are coded on a 4.2-kilobase EcoRI fragment of pTTE21 (T. Imanaka, T. Tanaka, H. Tsunekawa, and S. Aiba, J. Bacteriol. 147:776-186, 1981). The EcoRI fragment was subcloned in a low-copy-number plasmid pTB522 in Bacillus subtilis. B. subtilis carrying the recombinant plasmid pPTB60 (Tcr penP+ penI+) was chemically mutagenized. Of about 150,000 colonies, two penI(Ts) mutant plasmids, pPTB60D13 and pPTB60E24, were screened by the plate assay at 30 and 48 degrees C for penicillinase. By constructing recombinant plasmids between wild-type and mutant plasmids, the mutation points were shown to be located in a 1.7-kilobase EcoRI-PstI fragment. The EcoRI-PstI fragments of the wild-type plasmid and two mutant plasmids were sequenced. A large open reading frame, composed of 384 bases and 128 amino acid residues (molecular weight, 14,983), was found. Since the mutation points were located at different positions in the protein coding region (Ala to Val for pPTB60D13 and Pro to Leu for pPTB60E24), the coding region was concluded to be the penI gene. A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site (ATG). A probable promoter sequence which is very similar to the consensus sequence was also found upstream of the penP promoter, but in the opposite direction. A consensus twofold symmetric sequence (AAAGTATTA CATATGTAAGNTTT) which might have been used as a repressor binding region was found downstream and in the midst of the penP promoter and also downstream of the penI promoter. The regulation of penP and penI by the repressor is discussed.     

Enhancement of tryptophan production by Escherichia coli as an application of genetic engineering

Summary A feedback resistant trp operon plasmid that transformed a multiple mutant (trpR tnaA) of Escherichia coli was found to enhance remarkably the production of tryptophan in a bench-scale fermentor. 5.5 g of tryptophan was accumulated per litre of culture medium at 24th hr in batch. The productivity was 0.229 g/l/hr. This productivity is the highest among those ever reported by other workers. The recombinant plasmid (Tc^r Trp⁺ I^-) used was completely stable in each run when tetracycline was added by 10 g/l into the medium.     

Angiostrongylus cantonensis: Development following pulmonary arterial transfers into permissive and nonpermissive hosts

The survival, growth, and egg-laying capacity of young adult Angiostrongylus cantonensis, surgically transferred from intracranial sites into pulmonary arteries, were studied. A variety of experimental animals (rats, guinea pigs, mice, and mastomys) were chosen as donor animals and as recipient hosts (rats, guinea pigs, and rabbits). These species were specifically chosen to span the spectrum of host permissiveness relative to worm development in an attempt to understand the mechanisms which underlie species-dependent resistance. Recipient animals were monitored not only for the development of parasites per se but also for antibody production and histopathologic changes. The results indicated that these procedures were technically feasible, with good worm development following intra-rat transfers, as early as 15 days after initial exposure. Studies were performed to analyze the constraints of development both on initial, i.e., prelung and subsequent i.e., postlung development. When worms were obtained from permissive species such as rat or mastomys, transfer into rats resulted in good growth and development; however, worms which developed initially in exposed mice or guinea pigs developed less well in the rat. Conversely, worms which developed initially in permissive host such as the rat, when transferred into a variety of less permissive hosts such as the guinea pig and rabbit, apparently did not survive and caused significant morbidity and mortality within the nonpermissive host. Histopathologic evaluation revealed a strong eosinophilic perivascular and peribronchiolar infiltration as well as granulomatous reactions surrounding the worms in the lungs of recipient guinea pigs and rabbits, changes not observed in the lungs of permissive rat recipients. As reaginic antibody responses were also more prominent in nonpermissive than in permissive animals, it is possible that IgE responses may be more directly related to the phenomenon of morbidity and/or permissiveness than are other aspects of immune response. In support of this contention was the finding of nearly equivalent hemagglutinating antibody production between permissive rats and nonpermissive guinea pigs and rabbits.     

Calcium sensitivity of contractile proteins from chicken gizzard muscle.

Actin, myosin, and "native" tropomyosin (NTM) were separately isolated from chicken gizzard muscle and rabbit skeletal muscle. With various combinations of the isolated contractile proteins, Mg-ATPase activity and superprecipitation activity were measured. It was thus found that gizzard myosin and gizzard NTM behaved differently from skeletal myosin and skeletal NTM, whereas gizzard actin functioned in the same wasy as skeletal actin. It was also found that gizzard myosin preparations were often Ca-sensitive, that is, that the two activities of gizzard myosin plus actin without NTM were activated by low concentrations of Ca2+. The Mg-ATPase activity of a Ca-insensitive preparation of gizzard myosin was not activated by actin even in the presence of Ca2+. When Ca-sensitive gizzard myosin was incubated with ATP (and Mg2+) in the presence of Ca2+, a light-chain component of gizzard myosin was phosphorylated. The light-chain phosphorylation also occurred when Ca-insensitive myosin was incubated with gizzard NTM and ATP (plus Mg2+) in the presence of Ca2+. In either case, the light-chain phosphorylation required Ca2+. Phosphorylated gizzard myosin in combination with actin was able to exhibit superprecipitation, and Mg-ATPase of the phosphorylated gizzard myosin was activated by actin; the actin activation and superprecipitation were found to occur even in the absence of Ca2+ and NTM or tropomyosin. The phosphorylated light-chain component was found to be dephosphorylated by a partially purified preparation of gizzard myosin light-chain phosphatase. Gizzard myosin thus dephosphorylated behaved exactly like untreated Ca-insensitive gizzard myosin; in combination with actin, it did not superprecipitate either in the presence of Ca2+ or in its absence, but did superprecipitated in the presence of NTM and Ca2+. Ca-activated hydrolysis of ATP catalyzed by gizzard myosin B proceeded at a reduced rate after removal of Ca2+ (by adding EGTA), whereas that catalyzed by a combination of actin, gizzard myosin, and gizzard NTM proceeded at the same rate even after removal of Ca2+. However, addition of a partially purified preparation of gizzard myosin light-chain phosphatase was found to make the recombined system behave like myosin B. Based on these findings, it appears that myosin light-chain kinase and myosin light-chain phosphatase can function as regulatory proteins for contraction and relaxation, respectively, of gizzard muscle.