Uterine blood flow during the development and regression of the decidual cell reaction in ovariectomized, steroid-treated mice

Uterine blood flow was assessed in mice by measuring organ uptake of intravenously injected [14C]butanol. In ovariectomized mice, injection of 100 ng oestradiol-17 beta increased blood flow 5-fold over that of untreated controls. The injection of oestradiol-17 beta in progesterone-treated mice also increased uterine blood flow at the time of maximal sensitivity to a decidual stimulus, but not 4 days later. Absolute values of blood flow increased during development of the decidual cell reaction in proportion to the increase in uterine weight, reaching maximal values 96 h after decidual induction. When progesterone injections were stopped 72 h after decidual induction, a rapid decrease in absolute and relative blood flow values preceded the decrease in uterine weight. This decrease in uterine blood flow occurred within 12 h of removing a subcutaneous implant containing progesterone. These results are consistent with the view that increased uterine blood flow during decidual development may be necessary to support the rapid increase in uterine weight at implantation and the subsequent decrease in both relative and absolute uterine blood flow on withdrawal of progesterone may promote decidual regression in the mouse.     

Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy

We have calculated three-dimensional maps from images of myosin subfragment-1 (S1)-decorated thin filaments and S1-decorated actin filaments preserved in frozen solution. By averaging many data sets we obtained highly reproducible maps that can be interpreted simply to provide a model for the native structure of decorated filaments. From our results we have made the following conclusions. The bulk of the actin monomer is approximately 65 X 40 X 40 A and is composed of two domains. In the filaments the monomers are strongly connected along the genetic helix with weaker connections following the long pitch helix. The long axis of the monomer lies roughly perpendicular to the filament axis. The myosin head (S1) approaches the actin filament tangentially and binds to a single actin, the major interaction being with the outermost domain of actin. In the map the longest chord of S1 is approximately 130 A. The region of S1 closest to actin is of high density, whereas the part furthest away is poorly defined and may be disordered. By comparing maps from decorated thin filaments with those from decorated actin, we demonstrate that tropomyosin is bound to the inner domain of actin just in front of the myosin binding site at a radius of approximately 40 A. A small change in the azimuthal position of tropomyosin, as has been suggested by others to occur during Ca2+-mediated regulation in vertebrate striated muscle, appears to be insufficient to eclipse totally the major site of interaction between actin and myosin.     

Adipocyte plasma-membrane Gi and Gs in insulinopenic diabetic patients.

The matrix method for calculating the overall sensitivities (including control coefficients) of a metabolic system, described by Crabtree & Newsholme [Biochem. J. 247, 113-129 (1987)], is simplified by a preliminary partitioning of the initial matrix equation. This reduces the size of the matrix to be inverted and thereby removes a major drawback with the original method. The resulting procedure is simpler and more systematic than the alternative methods currently available, especially when the system is extensively branched.     

Persistent activation of the alpha subunit of Gs promotes its removal from the plasma membrane.

As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5'-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.     

Elevated levels of the guanine nucleotide binding protein, Go, are associated with differentiation of neuroblastoma x glioma hybrid cells

Each of a range of pharmacological agents which function to increase intracellular levels of cAMP caused a morphological 'differentiation' of neuroblastoma x glioma hybrid, NG108-15, cells grown in tissue culture. Associated with this differentiation, increased incorporation of [32P]ADP-ribose catalysed by pertussis toxin was noted into a band of some 39-40 kDa in membranes derived from these cells. Immunoblotting using two antipeptide antisera which identify different regions of Go alpha demonstrated marked increases in the levels of this polypeptide in membranes of the differentiated cells. However, levels of the beta-subunit did not increase appreciably with differentiation.     

Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates.

We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.     

Opioid peptides promote cholera-toxin-catalysed ADP-ribosylation of the inhibitory guanine-nucleotide-binding protein (Gi) in membranes of neuroblastoma x glioma hybrid cells.

A novel assay for 5'-nucleotidase is described in which 1,N6-etheno-AMP is converted into ethenoadenosine. The product ethenoadenosine is neither a substrate for nor an inhibitor of adenosine deaminase. Ethenoadenosine appears to have little effect at adenosine receptors on adipose-tissue cells.     

Sequence analysis and comparison of avocado fruit and bean abscission cellulases

A 1700 nucleotide cDNA clone for a bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase (endo-(1,4)-β-d-glucanase) has been identified and sequenced. This cDNA clone contains a 1485 nucleotide open reading frame which includes coding sequences for a putative signal peptide and mature protein. The nucleotide and deduced amino acid sequences for the bean abscission cellulase are compared to the previously reported sequences of an avocado fruit ripening cellulase. Optimal alignment of these sequences shows 64% and 50% identically matched nucleotides and amino acids, respectively. Analysis of the deduced amino acid sequences for the mature bean and avocado cellulases indicates that these two proteins share similar molecular weights, position of cysteine residues, and hydropathic character, but have very different isoelectric points and glycosylation. Genomic blot data suggest that the avocado fruit cellulase belongs to a small gene family, whereas the bean abscission cellulase appears to be encoded by a single gene or a few very closely related genes.     

The GTP-binding regulatory proteins of neuroblastoma x glioma, NG108-15, and glioma, C6, cells. Immunochemical evidence of a pertussis toxin substrate that is neither Ni nor No

Amounts of the guanine nucleotide binding regulatory proteins which are also pertussis toxin substrates (such as Ni and No) were measured in rat glioma, C6BU-1, cells and in neuroblastoma X glioma, NG108-15, hybrid cells. Measurements were performed both by quantitating pertussis toxin catalyzed ADP-ribosylation and by quantitative immunoblotting with affinity purified antibodies specific for Ni or No. The amounts of pertussis toxin substrate in C6 and NG108-15 cells are 7.5 and 0.6 pmol/mg membrane protein, respectively. These levels are minimum values and higher estimates of the total amounts of N proteins in the two cells are obtained by quantitative immunoblot analysis of the beta-subunit common to all N proteins. Immunoblots with specific antibodies show that NG108-15 cells contain 3.8 pmol/mg of No and detectable but small (less than 0.1 pmol/mg) amounts of Ni. In contrast, C6 cell membranes contain no detectable No and only 0.14 pmol/mg Ni. Thus, C6 cells contain large amounts of a pertussis toxin substrate which is neither Ni nor No.     

Extracellular and intracellular acid-base status following strenuous activity in the sea raven (Hemitripterus americanus)

Summary Exhausting activity in the sea raven resulted in a pronounced extracellular acidosis, which consisted of a large, short-lived respiratory component and a small, longer-lived metabolic component. Thi disturbance had been corrected by 12 h. White muscle experienced a pronounced intracellular acidosis of chiefly metabolic origin, with pH_i dropping from a resting value of 7.51 to a low of 7.10 immediately post-activity. The recovery of pH_i was associated with a reduction in muscle lactate. Despite the large increase in , cardiac muscle pH_i remained constant postactivity, actually showing an alkalosis at 30 min into recovery. Maintenance of cardiac muscle pH_i was achieved by an accumulation of HCO ₃ ^– intracellularly.