Changes in whole body concentrations of thyroid hormones and cortisol in metamorphosing conger eel

Summary To clarify the hormonal regulation of metamorphosis of the conger eel (Conger myriaster), changes in whole body concentrations of thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), and cortisol during metamorphosis were examined, as well as the changes in the histological activity of the thyroid gland. In larvae before metamorphosis, T₄ and T₃ levels were less than 5 and 0.15 ng·g^-1 respectively. Levels of T₄ increased to about 30 ng·g^-1 during early metamorphosis, and decreased subsequently. Levels of T₃ increased gradually in early metamorphosis, and then increased abruptly to about 2.0 ng·g^-1 in late metamorphosis. Before metamorphosis, cortisol levels of the leptocephali less than 11 cm in total length were greater than 200 ng·g^-1. Cortisol levels decreased rapidly in larger premetamorphic leptocephali, and low levels were maintained throughout the metamorphic period. Histological observation revealed an activation of the thyroid gland in early metamorphosis; thyroid follicle epithelial cells became columnar and their nuclei larger. Active uptake of colloid by these cells and intensive vascularization of the gland were also observed. By the end of metamorphosis, follicle epithelial cells became squamous, indicating a low level of glandular activity. These results suggest that thyroid hormone plays an important role in regulation of conger eel metamorphosis.Abbreviations AL anal length - TL total length - T ₃ triiodothyronine - T ₄ thyroxine     

Retrovirus-mediated gene transfer into CD4+ and CD8+ human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells

Summary Studies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterialneo ^R gene. The presence of theneo ^R gene in the TIL population was demonstrated by Southern blot analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of theneo ^R gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4⁺ and CD8⁺ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4⁺ and CD8⁺ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4⁺ or CD8⁺ cells. In other experiments highly purified CD4⁺ and CD8⁺ T cell subpopulations from either TIL or PBMC could be successfully transduced with theneo ^R gene as demonstrated by Southern blot analysis and detection of the gene product neophosphotransferase activity. No such activity or vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4⁺ and CD8⁺ gene-modified TIL. Current address: Bone Marrow Transplantation, Hadassah University Hospital, 91120 Jerusalem, Israel     

Localization of the high affinity calcium-binding site on tubulin molecule

Tubulin is a calcium-binding protein. Two different modes of interaction of calcium with tubulin have been described: a high affinity interaction to one or two binding sites and lower affinity interactions to several other binding sites. In the present study, we have used limited proteolysis of tubulin with trypsin, chymotrypsin, and subtilisin to localize the high affinity calcium-binding sites. Our results indicate that two sites are located in the carboxyl-terminal region of both tubulin subunits, and that tubulin deprived of its carboxyl-terminal region is able to polymerize in the presence of 0.5 mM calcium.     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine     

Identification of lactaldehyde dehydrogenase and glycolaldehyde dehydrogenase as functions of the same protein in Escherichia coli

Lactaldehyde dehydrogenase is an enzyme involved in the aerobic metabolism of fucose in wild type Escherichia coli, and glycolaldehyde dehydrogenase is an enzyme involved in the metabolism of ethylene glycol in mutant cells able to utilize this glycol. Both enzyme sources display oxidative activity on either substrate with a constant ratio between these activities. We have found that both enzymatic activities present the same electrophoretic mobility when crude extracts were electrophoresed in polyacrylamide gels and the gels stained for enzyme activities. Furthermore, both enzymatic activities co-chromatograph in a DEAE-Sephadex column. If lactaldehyde dehydrogenase of wild type cells is purified near homogeneity and the purification procedure is screened for both aldehydes as substrates, only one enzyme is apparent, giving again a constant ratio between lactaldehyde and glycolaldehyde dehydrogenase activities. Genetic evidence of the fact that both activities are functions of the same protein is provided by the observation that mutation to thermosensitivity for the production of lactaldehyde dehydrogenase affected in the same way the production of glycolaldehyde dehydrogenase. Glycolaldehyde dehydrogenase from mutant cells is purified in a procedure coincident with the lactaldehyde dehydrogenase purification, yielding a single enzyme electrophoretically indistinguishable from the purified lactaldehyde dehydrogenase. Peptide mapping of the purified preparation after digestion with chymotrypsin or Staphylococcus aureus protease V8 gives an indistinguishable band pattern between both enzymes.     

Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death

Thirteen mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (M-MuLV) at a single locus (Mov locus) have been derived previously. Experiments were performed to investigate whether homozygosity at the Mov loci would be compatible with normal development. Animals heterozygous at an Mov locus were mated, and the genotype of the offspring was analyzed. From parents heterozygous at the loci Mov1 to Mov12, respectively, homozygous offspring were obtained with the expected Mendelian frequency. In contrast, no homozygous offspring or embryos older than day 15 of gestation were obtained from parents heterozygous at the Mov13 locus. When pregnant Mov13 females at day 13 and day 14 of gestation were analyzed, approximately 25% of the embryos were degenerated. Genotyping revealed that these degenerated embryos were invariably homozygous and the normal appearing embryos were either heterozygous or negative for M-MuLV. These results suggest that integration of M-MuLV at the Mov13 locus leads to insertion mutagenesis, resulting in embryonic arrest between day 12 and day 13 of gestation. It is possible that the Mov13 locus represents a gene or gene complex involved in the early embryonic development of the mouse.     

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

Fluorescein isothiocyanate-labeled beta-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed.