Genetic controls of T cell-independent Thy-1 alloantibody responses

Early and late primary IgM antibody responses of mice to Thy-1.1 antigens showed different antigenic and cellular requirements. We studied genetic controls of the early primary responses, which could be induced by subcellular thymocyte antigens independently of host T-cell activity. All Thy-1.2 mouse strains of Igh ^a(BALB/c and BC8), Igh-V ^aC^b(BAB14), Igh ^d(AKR/Cum), Igh ^j(CBA/J, C3H/HeN, C3H.SW, and C3H.JK), and Igh ⁿ(NZB) definitely responded early to Thy-1.1 antigens from AKR/J (Igh ^d), A.Thy-1.1 (Igh ^e), or B10.Thy-1.1 (Igh ^b) mice or SD rats, whereas all strains of Igh ^b(C57BL/6, C57BL/10, B10.D2, B10.BR, B10.A, CB20 and CWB), Igh ^c(DBA/2), Igh ^e(A/J), and Igh ^o(C.AL20) responded poorly to the same antigens. This contrasts with the observation that both strains of Igh ^j(C3H/HeN) and Igh ^b(B10.BR) responded well at later times. As was the case for late responses, the matching of H-2 between donor and recipient resulted in early responses of exceptional quality in high-responder strains. It was concluded that under the influence of H-2, whose incompatibility between donor and recipient partially interferes with responses, early but not late primary Thy-1.1-specific antibody responses are selectively controlled by Igh-V or closely linked Ir gene(s) as a new V _Hmarker.Abbreviations used in this paper Tl T cell-independent - TD T cell-dependent - PFC plaque-forming cell(s) - Igh immunoglobulin heavy chain - V _H variable region of heavy chain - C _H constant region of heavy chain     

Regulation of low-density lipoprotein receptors in cultured bovine adrenocortical cells

Bovine adrenocortical cells in monolayer culture produce cortisol and respond to corticotropin (ACTH) by an increase in cortisol secretion. Several lines of evidence are indicative that much of the cholesterol that serves as precursor for steroid hormone biosynthesis by these cells is derived from low-density lipoprotein (LDL) cholesterol that is taken up endocytotically by means of specific receptors localized in bovine adrenocortical plasma membranes. ACTH stimulated this process concomitant with an increase in steroid production. In the absence of LDL, ACTH had no effect on steroid biosynthesis. ACTH action in bovine adrenocortical cells resulted in an increase in the number of LDL receptor sites in the membrane fractions, whereas the dissociation constant for LDL binding was not changed. Chloroquine and NH₄Cl, considered to be inhibitors of lysosomal degradative activity, caused an increase in the number of [¹²⁵I]iodoLDL binding sites in the plasma membrane but the effect of ACTH was still apparent in the presence of these agents. These results are suggestive that the lifetime of the LDL receptor is increased when lysosomal activity is inhibited. When aminoglutethimide was added to block cholesterol side-chain cleavage activity and inhibit steroid production, the number of [¹²⁵I]iodoLDL binding sites in the membrane fractions prepared from bovine adrenocortical cells cultured in the presence of ACTH was reduced to 50% of that in cells maintained in aminoglutethimide-free medium. However, under these conditions the number of binding sites was still significantly greater than in cells maintained in the absence of ACTH. The effects of aminoglutethimide on uptake and degradation of [¹²⁵I]iodoLDL were similar to the effects on the number of [¹²⁵I]iodoLDL binding sites. Based on these results, we conclude that the action of ACTH to stimulate LDL metabolism in bovine adrenocortical cells results from an increase in the number of LDL binding sites in the plasma membranes. This action of ACTH appears to be, at least in part, independent of cholesterol utilization for cortisol biosynthesis. However, the effect of aminoglutethimide is indicative that changes in the intracellular cholesterol concentration might modulate the action of ACTH to increase the number of LDL binding sites and therefore to stimulate LDL degradation.     

Synthesis of human big endothelin-1 by sequence-specific proteolysis of a fusion protein in Escherichia coli.

Three DNA constructs, pETB-40, 41, and 42, encoding human big endothelin-1 (ET-1) preceded by the specific recognition sequence (Ile-Glu-Gly-Arg) for the activated blood coagulation factor Xa (FXa), fused in frame to the N-terminal portion of beta Gal, were expressed in Escherichia coli. The fusion proteins, pETB-40P, 41P, or 42P, consisted of the 55-, 51-, or 42-aa N-terminal peptide of beta Gal and the 38-aa of big ET-1, and had 1, 0, or 0 Cys residues and 5, 5, or 1 Arg residues in the N-terminal peptide of beta Gal, respectively. Enzymatic cleavage of the purified fusion proteins by FXa or trypsin allowed the recovery of authentic human big ET-1. The rates of conversion of pETB-40P, 41P, and 42P to big ET-1 by FXa digestion were 5.6, 11.2, and 30.0%, respectively. pETB-40P with a deletion of one Cys residue and four Arg residues in the N-terminal part was a better substrate than the other two for FXa or trypsin in the production of big ET-1.     

Nucleotide specificity of the enzymatic and motile activities of dynein, kinesin, and heavy meromyosin

The substrate specificities of dynein, kinesin, and myosin substrate turnover activity and cytoskeletal filament-driven translocation were examined using 15 ATP analogues. The dyneins were more selective in their substrate utilization than bovine brain kinesin or muscle heavy meromyosin, and even different types of dyneins, such as 14S and 22S dynein from Tetrahymena cilia and the beta-heavy chain-containing particle from the outer-arm dynein of sea urchin flagella, could be distinguished by their substrate specificities. Although bovine brain kinesin and muscle heavy meromyosin both exhibited broad substrate specificities, kinesin-induced microtubule translocation varied over a 50-fold range in speed among the various substrates, whereas heavy meromyosin-induced actin translocation varied only by fourfold. With both kinesin and heavy meromyosin, the relative velocities of filament translocation did not correlate well with the relative filament-activated substrate turnover rates. Furthermore, some ATP analogues that did not support the filament translocation exhibited filament-activated substrate turnover rates. Filament-activated substrate turnover and power production, therefore, appear to become uncoupled with certain substrates. In conclusion, the substrate specificities and coupling to motility are distinct for different types of molecular motor proteins. Such nucleotide "fingerprints" of enzymatic activities of motor proteins may prove useful as a tool for identifying what type of motor is involved in powering a motility-related event that can be reconstituted in vitro.     

Developmental arrest at early stages of Chinese hamster embryos homozygous for chromosomal rearrangements

Forty-three Chinese hamster stocks with autosomal rearrangements produced by X-irradiation were used. These rearrangements, 38 reciprocal translocations and 5 inversions, were chromosomally balanced. Heterozygotes for these rearrangements were all fertile and morphologically normal in both sexes except for one line with growth retardation. By crossing male and female heterozygotes for the same rearrangements, homozygotes were obtained in 37 lines. In the remaining 6 lines (5 with reciprocal translocations and 1 with an inversion), no homozygotes were viable. These 6 lines revealed arrested development of homozygous embryos at the two-cell stage, around the eight-cell stage, and after implantation, respectively. The bands of the breakpoints of rearrangements associated with lethality of homozygous embryos were different for each rearrangement. These results suggest that abnormal expression including embryonic lethality in homozygotes may be due to an influence of genes at the breakpoints.     

Plasma growth hormone, insulin-like growth factor-I, and milk production responses to exogenous human growth hormone-releasing factor analogs in dairy cows

Responses of plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I), and milk production to subcutaneous (sc) injection(s) of two synthetic human growth hormone-releasing factor (hGRF) analogs were studied in dairy cows. Two mg of each hGRF analog dissolved in 5 ml saline per cow were injected into the shoulder area of each experimental animal, and jugular venous blood samples were collected via an indwelling catheter or by venipuncture. Plasma GH and IGF-I concentrations were measured by radioimmunoassay methods. In dry cows, the mean concentration of plasma GH after a single sc injection of hGRF analogs rose to 22.0-28.3 ng/ml at about 5 h from 1.4-1.7 ng/ml at 0 h (just before injection), and returned to the level before injection after 10-12 h. On the other hand, the plasma IGF-I began to increase after a lag of 4-6 h following a single injection of hGRF analogs, and reached maximum values of 71.1-89.4 ng/ml at 20 h from 43.7-46.4 ng/ml at 0 h. The IGF-I concentration at 24 h after a single injection of hGRF analogs was still higher than the value for the dry cows given saline. In lactating cows, the plasma concentration of GH at 2 h after daily sc injections of hGRF analogs during 14 consecutive days (an injection period) was higher than those for the lactating cows which received saline. Also, during the injection period, the concentration of IGF-I was higher in the lactating cows which received hGRF analog injections than in the cows which received saline injections. During the last 7 days of the injection period, the administration of hGRF analogs increased the mean milk yield by 11-19% in comparison with those for the saline injected cows. A positive correlation was observed between the mean plasma IGF-I concentration and the mean milk yield in the lactating cows treated with hGRF analogs throughout the injection and a postinjection (11 consecutive days after cessation of hGRF analog injection) periods. The results demonstrate that a single sc injection of hGRF analogs stimulates both GH release and the circulating level of IGF-I in dry cows, and that daily sc injections of hGRF analogs over 14 days enhance milk production, and plasma GH and IGF-I levels in lactating cows.     

Regional distribution of endothelin receptor in porcine cardiovascular tissues

To clarify effecting sites of endothelin (ET) in a circulation system, we have identified specific receptors for porcine ET(ET-1) and investigated the distribution in the porcine cardiovascular tissues. Scatchard analysis of 125I-porcine ET binding indicated the presence of a single class of high-affinity binding sites. The binding was highly specific for ET-1, because (1) none of the other various peptides or Ca2+-channel antagonists affected the binding, (2) the scission of disulfide bonds, the digestion of the C-terminal 6-amino acid residues, or nitrophenylsulfenylization of the C-terminal Trp21 of ET-1 markedly reduced the binding ability and, (3) ET-1 showed the highest affinity for the vascular receptor among three ET isopeptides. Cardiac atria possessed the highest density (2.7 pmol/mg protein) of ET receptors of all the tissues examined, including thoracic aorta, cardiac atria and cardiac ventriculi, basilar, renal, coronary and pulmonary branch arteries, coronary, renal and jugular veins, and small vessels of pia mater encephali. Small vessels, renal and coronary arteries also showed relatively high density (0.8-1.4 pmol/mg protein). Various veins examined also showed considerable density (0.45-0.74 pmol/mg protein). The apparent Kd of cardiac ET receptors (0.76 nM) was significantly greater than that of the receptors of the other tissue (0.06-0.14 nM). The extensive distribution and the local enrichment of ET receptor in a cardiovascular system strongly suggests that ET is one of the essential endogenous substances to control the tone of the vasculature.