Reactivation of proliferin gene expression is associated with increased angiogenesis in a cell culture model of fibrosarcoma tumor progression.

Proliferin (PLF) is an angiogenic placental hormone. We now report that PLF gene expression can also occur in a progressive fibrosarcoma mouse tumor cell model. PLF mRNA and protein are detectable at very low levels in cell lines derived from the mild noninvasive stage of tumor development. Expression is greatly augmented in cell lines from the aggressively invasive stage of development, a stage at which the tumor becomes highly angiogenic, and PLF expression remains high in cell lines from the end stage of fibrosarcoma. Activator protein 1 factors present at high levels in the more invasive stages of the tumor may in part allow for increased PLF expression, as cells from the mild stage in which c-jun and junB are stably expressed secrete levels of PLF comparable to that of the advanced stages. Secreted PLF protein is functionally important in tumor cell angiogenic activity, as demonstrated by the reduction of angiogenic activity in fibrosarcoma cell culture medium by immunodepletion of PLF. These results suggest that an extraembryonic genetic program, which has evolved to support fetal growth, may be reactivated in certain tumors and contribute to tumor growth.

Placental prolactins and the physiology of pregnancy.

Mammalian pregnancy is characterized by a concerted and widespread series of changes in maternal physiology, many of which are direct responses to the binding of placental hormones to maternal targets. Among these placental hormones are proteins closely related to prolactin. In rodents, a large number of these placental prolactin-related hormones are expressed that have a broad spectrum of activities, including activities on endothelial cells and blood cells.

Identification of three prolactin-related hormones as markers of invasive trophoblasts in the rat.

An expressed-sequence tag database search has identified three rat cDNA clones in the prolactin/growth hormone family, including a homologue of mouse proliferin-related protein (PRP). The encoded proteins of the two novel clones, designated prolactin-like proteins L (PLP-L) and M (PLP-M), are predicted to be synthesized as precursors of 229 and 227 amino acids, modified by N-linked glycosylation, and secreted as mature glycoproteins of 199 and 200 residues, respectively. Murine homologues to PLP-L and PLP-M were also identified. The open reading frame of rat PRP encodes a precursor protein of 245 amino acids and predicts a secreted 215-amino acid glycoprotein with 81% identity to mouse PRP. All three rat mRNAs are expressed in the placenta, and expression is not detected in other tissues. PLP-L mRNA expression is observed from Days 11-20, with highest levels at Day 13; highest levels of PLP-M are observed from Day 11 until parturition, with peak levels also on Day 13; and highest levels of PRP are also observed from Day 11 until term, with maximal expression on Day 17. All three genes are most highly expressed in invasive trophoblast cells lining the central placental vessel. The identification of molecular markers for endovascular trophoblasts serves to highlight the invasive nature of rodent placentation and may prove useful for future studies of placental function.

Prolactin (PRL)-like protein J, a novel member of the PRL/growth hormone family, is exclusively expressed in maternal decidua.

A search of a nonmouse, nonhuman, expressed sequence tag database for messenger RNAs in the PRL/GH family has identified a novel rat complementary DNA clone. The encoded protein, designated PRL-like protein J (PLP-J), is predicted to be synthesized as a precursor of 211 amino acids, modified by N-linked glycosylation, and secreted as a mature glycoprotein of 182 residues. PLP-J messenger RNA synthesis is limited to early pregnancy with abundant expression on day 7, slightly declining expression on day 9, and no detectable expression by day 11. Unlike most other PRL family members, PLP-J does not appear to be synthesized by placental trophoblasts but, rather, by decidual cells surrounding the implantation site. By sequence similarity to rat PLP-J, a murine clone was identified in a mouse expressed sequence tag database. Mouse PLP-J was used to map the gene to a 700-kb region of mouse chromosome 13 that includes other members of the PRL/GH family.

Path and extent of cross-bridge rotation during muscle contraction.

The angular distribution of myosin cross-bridges in muscle fibers was investigated in four physiological states using a multiple probe analysis of varied extrinsic probes of the cross-bridge [Burghardt & Ajtai (1994) Biochemistry (preceding paper in this issue)]. The analysis combines data of complementary techniques from different probes giving the highest possible angular resolution. Four extrinsic probes of the fast reactive sulfhydryl (SH1) on myosin subfragment 1 (S1) were employed. Electron paramagnetic resonance (EPR) spectra from paramagnetic probes, deuterium- and 15N-substituted for greater sensitivity to orientation, on S1 were measured when the protein was freely tumbling in solution and when it was decorating muscle fibers. The EPR spectra from labeled S1 tumbling in solution were measured at X- and Q-band microwave frequencies to uniquely specify the orientation of the probe relative to the S1 principal hydrodynamic frame. The EPR spectra from labeled S1 decorating muscle fibers in rigor and in the presence of MgADP were measured at X-band and used in the multiple probe analysis of cross-bridge orientation. The time-resolved fluorescence anisotropy decay (TRFAD) of fluorescent probes on S1 was measured when the protein was freely tumbling in solution, and fluorescence polarization (FP) intensities from fluorescent probes modifying SH1 in intact muscle fibers were measured for fibers in rigor, in the presence of MgADP, in isometric contraction, and in relaxation at low ionic strength. The TRFAD measurements limit the range of possible orientations of the probe relative to the S1 principal hydrodynamic frame. The FP intensity measurements were used in the multiple probe analysis of cross-bridge orientation. The combination of the EPR and FP data determined a highly resolved cross-bridge angular distribution in rigor, in the presence of MgADP, in isometric contraction, and in relaxation at low ionic strength. These findings confirm earlier observations of a rigid body rotation of the SH1 region in the myosin head group upon physiological state changes and indicate the path and extent of cross-bridge rotation during contraction. The rotation of the cross-bridge is visualized with computer-generated space-filling models of actomysin in six states of the contraction cycle.

Stereospecific reaction of muscle fiber proteins with the 5' or 6' isomer of (iodoacetamido)tetramethylrhodamine.

The labeling of muscle fiber proteins with iodoacetamido)tetramethylrhodamine (IATR) was reinvestigated with the purified 5' or 6' isomers of IATR. Both isomers modify the myosin heavy chain within the 20-kDa fragment of myosin subfragment 1 (S1) but with different rates, and only the 5'-IATR alters K(+)-EDTA- and Ca(2+)-activated ATPases. Absorption spectroscopic and ATPase studies of probe stoichiometry indicate that for 5'-IATR there are two probes per myosin sulfhydryl 1 (SH1). Quantitative fluorograms of the SDS-PAGE gels confirm that there are one covalent and one noncovalent probe per SH1 when S1 is labeled with 5'-IATR (5'-IATR-S1) and that there are one covalent and two noncovalent probes per S1 when S1 is labeled with 6'-IATR (6'-IATR-S1). The 5'- and 6'-IATR probes have similar fluorescent lifetimes when bound to S1, but quenching studies with potassium iodide show that 5'-IATR-S1 has a single class of strongly bound chromophores while 6'-IATR-S1 has two or more classes of chromophores. It is possible that 5'-IATR labels SH1 as a dimer. The polarization anisotropies of 5'- and 6'-IATR-S1 indicate that 5'-IATR is immobilized, while 6'-IATR is moving independently, on the surface of S1. The emission spectrum from 5'-IATR-S1 is unaffected by the addition of MgATP, while 6'-IATR-S1 shows a spectral shift and total intensity change. When labeling muscle fibers, 5'-IATR labels myosin SH1 and differentiates between the fiber physiological states by indicating cross-bridge rotation in quantitative agreement with previous results [Burghardt et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7515]. 6'-IATR reacts preferentially with actin in muscle fibers and does not differentiate between fiber physiological states as expected for an actin probe. The stereospecificity of the rhodamine isomers for SH1 indicates features of the local protein structure. The experimental results are used with theoretical methods for determining molecular structure to suggest a qualitative scheme for the specific interaction of 5'-IATR with its binding pocket on the surface of S1.