A study of GDP binding to purified thermogenin protein from brown adipose tissue.

1. The binding of GDP to purified thermogenin protein was studied by using fluorescence spectroscopy and equilibrium dialysis. 2. GDP binding to thermogenin diminished fluorescence emission in a concentration-dependent manner that exhibited saturation. 3. Kd values for binding of nucleoside di- and tri-phosphates were lower than those for nucleoside monophosphates. 4. The GDP-induced fluorescence quenching was decreased by increasing pH, but the apparent Kd was unaltered by pH changes. 5. Equilibrium dialysis showed a Kd change from 3 to 6 microM when the pH was increased from 6.6 to 8.5. 6. The apparent pK of the fluorescence changes induced by pH (8.3) was identical with the apparent pK of the GDP-binding response. 7. The data are consistent with the existence of protonated and non-protonated forms of thermogenin protein that both bind GDP.     

Effects of nutritional status and acute variation in substrate supply on cardiac and skeletal-muscle fructose 2,6-bisphosphate concentrations.

Vasopressin was found to be an effective inhibitor of protein labelling in isolated liver cells. Its effect shows the following distinct characteristics: (1) in contrast with alpha-adrenergic agonists, its effect is observable under a wide range of cellular Ca2+-loading conditions; (2) it is not influenced by the nutritional state of the animal. The lack of vasopressin effect on valine production, and its ability to decrease protein labelling from near-saturation concentrations of [3H]valine, indicate that the observed variations in protein labelling reflect actual changes in the rate of protein synthesis. The action of vasopressin is primarily exerted on the initiation step of protein synthesis and this effect is accompanied by a decreased activity of eukaryotic initiation factor 2. Activators of protein kinase C showed similar but not additive effects on protein synthesis, as did vasopressin. It seems plausible to conclude that protein kinase C activation may play an important regulatory role in hepatic protein synthesis as a transducer of hormonal and perhaps other type of signals.     

Evidence that the functional beta-actin gene is single copy in most mice and is associated with 5'' sequences capable of conferring serum- and cycloheximide-dependent regulation.

Hybridization to synthetic oligonucleotides representing conserved regions in the promoter and first intron of several vertebrate beta-actin genes was used to discriminate between what appears to be a single functional beta-actin gene and numerous pseudogenes in the mouse genome. Sequences derived from the 5' end of this gene were shown to confer serum-inducible expression upon a heterologous reporter gene when transfected into mouse fibroblasts. Moreover, these sequences rendered reporter gene expression superinducible by a combination of serum and cycloheximide. These experiments indicate that the 5' end of the mouse beta-actin gene contains sequence elements which mediate the stimulatory effects of serum growth factors and which are responsive to both positive and negative regulators of gene expression.     

Antibodies to steroid receptor deoxyribonucleic acid binding domains and their reactivity with the human glucocorticoid receptor

Functional properties of the DNA-binding domain of the human glucocorticoid receptor were investigated using high titer polyclonal antibodies produced against single synthetic peptides or a mixture of peptides whose sequences were derived from the DNA-binding domain of steroid receptor proteins. Three of seven antisera recognized both native and denatured forms of the glucocorticoid receptor, although considerably lower antisera dilutions were required for antibody binding to native receptor. Activation of the glucocorticoid receptor to its DNA-binding form was required for antibody recognition of the native receptor. Antisera to the second finger region of the DNA-binding domain caused a portion of the activated 4S glucocorticoid receptor to sediment as 7 or 9S in sucrose gradients containing 0.4 M KCl, but did not alter the sedimentation of the nontransformed 8S receptor. Specificity of the glucocorticoid receptor-antibody interaction was demonstrated by loss of reactivity after preabsorption with peptide antigens. Antisera that interacted specifically with the glucocorticoid receptor inhibited DNA binding of the activated receptor by as much as 80%. Thus, antibody probes directed against DNA-binding domain sequences provide immunological evidence that glucocorticoid receptor activation exposes the DNA-binding region of the receptor.     

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     

Active immunization of intact mares against gonadotropin-releasing hormone: differential effects on secretion of luteinizing hormone and follicle-stimulating hormone

Five lighthorse mares were actively immunized against gonadotropin releasing hormone (GnRH) to determine the relative importance of this hypothalamic hormone in the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Five mares immunized against the conjugation protein served as controls. Mares were initially immunized in November and received secondary immunizations 4 wk later, and then at 6-wk intervals until ovariectomy in June. All mares immunized against GnRH exhibited an increase (p less than 0.01) in the binding of tritiated GnRH by plasma, an indication that antibodies against this hormone had been elicited. Concentrations of LH, FSH and progesterone in weekly blood samples were lower (p less than 0.05) in GnRH-immunized mares than in controls after approximately 4 mo of immunization. However, the LH concentrations were affected to a greater degree than were FSH concentrations. All five control mares exhibited normal cycles of estrus and diestrus in spring, whereas no GnRH-immunized mare exhibited cyclic displays of estrus up to ovariectomy. All mares were injected intravenously with a GnRH analog (which cross-reacted less than 0.1% with the anti-GnRH antibodies) in May, after all control mares had displayed normal estrous cycles, to characterize the response of LH and FSH in these mares; two days later, the mares were injected with GnRH. The LH response to the analog, which was assessed by net area under the curve, was lower (p less than 0.01) by approximately 99% in mares immunized against GnRH than in control mares. In contrast, the FSH response to the analog was similar for both groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence and distribution of halophilic vibrios in subtropical coastal waters of Hong Kong.

The summer occurrence and distribution of halophilic vibrios in the subtropical coastal waters of Hong Kong were investigated. The density of vibrios in six sample sites ranged from 90 to 6,700 per ml, which made up 0.41 to 40% of the total bacterial populations of these sample sites. The sucrose-positive vibrios were found to be much more common (88% of total vibrios) than the sucrose-negative ones. A total of 48 strains belonging to six Vibrio species were fully characterized. Among these, Vibrio alginolyticus was the most frequently isolated, followed by V. parahaemolyticus, V. harveyi, V. vulnificus, V. campbellii, and V. fluvialis. The finding that eight of the nine strains of V. harveyi showed a positive Kanagawa reaction warrants further study.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.