Flunitrazepam binding sites in rat diaphragm. Receptors for direct neuromuscular effects of benzodiazepines?

The evidence for direct muscle relaxant effects of benzodiazepines is controversial. We now show that a crude membrane preparation of rat diaphragm possesses binding sites for [3H]flunitrazepam (FNZ). Scatchard analysis gave a binding site density of 1689 +/- 143 fmol/mg protein (Kd = 25.6 +/- 2.6 nM). These sites are of the "peripheral" type since clonazepam fails to displace [3H]FNZ as effectively as R05-4864 (IC50 values: 7.5 x 10(-6) M and 8 x 10(-9) M, respectively). Diazepam is almost as effective as R05-4864 and potently displaces [3H]FNZ binding (IC50 = 3 x 10(-8) M). We propose that the previously described effects of diazepam on rat diaphragm are mediated through high-affinity binding sites.     

Inducible expression of murine IP-10 mRNA varies with the state of macrophage inflammatory activity

We have examined the expression of inducible inflammatory genes in murine macrophages from different tissues and at different stages of inflammatory activity. Although i.v. administration of IFN-gamma (10,000 U/mouse) strongly induced expression of IP-10 mRNA in the adherent cell population of the spleen, thioglycollate-elicited peritoneal macrophages were essentially unresponsive at the same dose. In contrast, D3 mRNA was expressed in both cell populations. This differential sensitivity of IP-10 mRNA expression was not restricted to stimulation by IFN-gamma as it was also seen when LPS (25 micrograms/mouse) was administered i.v. Expression of JE and KC mRNA, which encode cytokines related to IP-10, were also differentially expressed in elicited peritoneal macrophages from mice injected with LPS. Differential sensitivity was at least partially related to the state of macrophage activation because IP-10 mRNA was highly inducible in resident but not thioglycollate-elicited peritoneal macrophages. The eliciting agent was also an important determinant because proteose-peptone-elicited peritoneal macrophages were nearly as sensitive as splenic macrophages with respect to expression of IP-10 mRNA. IFN-gamma treatment induced IP-10 and D3 mRNA rapidly and transiently with the same time course in the spleen. IP-10 mRNA was not induced by IFN-gamma in TG-elicited macrophages regardless of the time after treatment. This differential expression of IP-10 was a consequence of different concentration requirements for IFN-gamma in the two cell types; thioglycollate-elicited macrophages required five- to 10-fold more IFN-gamma than did resident cells to achieve comparable IP-10 mRNA levels whether the agent was provided in vitro or in vivo. Thus variable sensitivity for induction of IP-10 mRNA was a characteristic of the macrophage itself and was not mediated by other cellular or molecular elements present in the inflammatory peritoneal cavity. The reduced sensitivity to IFN-gamma or LPS for expression of IP-10, JE, and KC mRNA as compared with TNF-alpha or D3 mRNA suggests that this distinct pattern of regulation may be restricted to members of these two related cytokine gene families that exhibit cell-type specific chemoattractant activity.     

An analytical and numerical study of the stability of bone remodelling theories: dependence on microstructural stimulus.

The origin of unstable bone remodelling simulations using strain-energy-based remodelling rules was studied mathematically in order to assess whether the unstable behavior was due to the mathematical rules proposed to characterize the processes, or to the numerical approximations used to exercise the mathematical predictions. A condition which is necessary for the stability of a strain-energy-based remodelling theory was derived analytically using the calculus of variation. The analytical result was derived using a simple elastic model which consists of a long beam loaded by an axial force and a bending moment. This loading situation mimics the coupling between local density and global density distributions seen in vivo. A condition necessary for a stable remodelling scheme is arrived at, but the conditions necessary to guarantee a stable remodelling scheme are not. In this remodelling scheme, the elastic modulus is proportional to volumetric density raised to an exponent n, and the microstructural stimulus is taken as the strain energy density divided by volumetric density raised to an exponent m. In order for a remodelling scheme to be stable in this loading situation, m must be greater than n. Finite-difference time-stepping is used to verify the predictions of the analytical study. These numerical studies appear to confirm the analytical studies. Physiologic interpretation of the behavior found with n greater than m indicates that this type of unstable behavior is unlikely to be observed in vivo. Since numerical approximations are not made in deriving this stability condition, we conclude that the mathematical rules proposed to characterize bone remodelling based on strain energy density should meet this condition to be relevant to physiologic bone remodelling.     

Biological activities of peptides and peptide analogues derived from common sequences present in thrombospondin, properdin, and malarial proteins

Thrombospondin (TSP), a major platelet-secreted protein, has recently been shown to have activity in tumor cell metastasis, cell adhesion, and platelet aggregation. The type 1 repeats of TSP contain two copies of CSVTCG and one copy of CSTSCG, per each of the three polypeptide chains of TSP and show homology with peptide sequences found in a number of other proteins including properdin, malarial circumsporozoite, and a blood-stage antigen of Plasmodium falciparum. To investigate whether these common sequences functioned as a cell adhesive domain in TSP, we assessed the effect of peptides corresponding to these sequences and an antibody raised against one of these sequences, CSTSCG, in three biological assays which depend, in part, on the cell adhesive activity of TSP. These assays were TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis. We found that a number of peptides homologous to CSVTCG promoted the adhesion of a variety of cells including mouse B16-F10 melanoma cells, inhibited platelet aggregation and tumor cell metastasis, whereas control peptides had no effect. Anti-CSTSCG, which specifically recognized TSP, inhibited TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis, whereas control IgG had no effect. These results suggest that CSVTCG and CSTSCG present in the type I repeats function in the adhesive interactions of TSP that mediate cell adhesion, platelet aggregation, and tumor cell metastasis. Peptides, based on the structure of these repeats, may find wide application in the treatment of thrombosis and in the prevention of cancer spread.     

Inhibition of S-phase progression in macrophages is linked to G1/S-phase suppression of DNA synthesis genes.

Some of the important controlling events regulating eukaryotic S-phase progression are considered to occur late in the G1 stage of the cell cycle. We show here that stimulation of DNA synthesis in bone marrow-derived macrophages (BMM) by macrophage CSF-1 is preceded by G1 expression of three genes which encode proteins associated with the DNA synthesis machinery--the M1 and M2 subunits of ribonucleotide reductase and proliferating cell nuclear Ag (PCNA). Increased expression for these genes correlated well with the mitogenic response and sustained expression required de novo RNA and protein synthesis and also the presence of CSF-1 for at least most of G1. Inhibitors of BMM proliferation (LPS, TNF-alpha, IFN-gamma, and cAMP elevating agents) suppressed CSF-1-induced expression of M1, M2, and PCNA mRNA measured at 22 h. This suppression occurred even when added up to 12 h after the CSF-1, a period coinciding with the G1/S-phase boundary. The delayed kinetics of this effect parallels the ability of these agents to maximally inhibit CSF-1-induced BMM DNA synthesis when added at similar times. Decreased expression of M1, M2, and PCNA was not merely a consequence of DNA synthesis inhibition because the S-phase inhibitor, hydroxyurea, did not suppress CSF-1-induced gene expression. These results suggest that inhibition of DNA synthesis by antiproliferative agents involves inhibition of expression of several genes associated with the DNA synthesis machinery.     

More covert sex: The elusive females of myrmecolacidae

Among evolutionary biologists, considerable interest currently surrounds organisms that seem to be represented entirely by females. Here we discuss a less familiar problem - that of organisms in which females appear to be lacking. Our main example is the Myrmecolacidae (Strepsiptera), an unusual group of entomophagous parasites with dual hosts. Males parasitize ants and females parasitize orthopterans. Although the free-living adult males are frequently collected, the permanently endoparasitic neotenic females are elusive and are seldom encountered.     

The superovulation of synchronous adult rats using follicle-stimulating hormone delivered by continuous infusion

The estrous cycles of adult female rats were synchronized with an LHRH agonist on the morning of Day -4 (Day 0 = day of mating). On Day -2, animals received s.c. implants of continuous-infusion osmotic minipumps containing different doses of an FSH preparation (Folltropin) in combination with hCG at various ratios of hCG:FSH or were given single injections of eCG in doses ranging from 15 IU to 60 IU. Rats infused with the optimal dose (3.4 U/day) of FSH ovulated 44.1 +/- 5.4 oocytes/rat while rats treated with the most effective dose (60 IU) of eCG ovulated only 20.5 +/- 4.3 oocytes/rat on the morning of Day 1. The inclusion of hCG in pumps at ratios from 0.188:1 to 0.75:1 (hCG:FSH) had no significant effect on ovulation rate. The importance of synchronization of estrus in successful superovulation was demonstrated by the finding that only 70% of the unsynchronized animals ovulated (29.1 +/- 4.8 oocytes/rat) whereas 95% of the synchronized animals ovulated (51.0 +/- 3.6 oocytes/rat). Oocyte viabilities were assessed by determining fertilization rates and embryonic development in vivo following mating with fertile males. In rats superovulated by use of the FSH regimen, 92% (39.0 +/- 4.1) of the recovered embryos were 1-cell zygotes on Day 1, 89% (36.3 +/- 5.6) were at the 2-cell embryo stage of development on Day 2, and 88% (28.8 +/- 2.2) were at the morula and blastocyst stages on Day 5 following mating on Day 0. The high ovulation rates and oocyte viability in rats receiving infusions of Folltropin following estrus synchronization offer a reliable method for superovulation of adult rats.