Increased natriuretic peptides in fetal hearts of diabetic rats.

The main purpose of these studies was to determine whether diabetic pregnancy altered maternal and fetal atrial natriuretic peptide (ANP). Diabetes was induced in rats by intravenous injection of 40 mg streptozotocin/kg on day 2 of gestation. Immunoreactive ANP in plasma, amniotic fluid and hearts on day 20 of gestation was measured by radioimmunoassay; fetal cardiac natriuretic peptides (ANP, proANP and BNP) were separated by reverse-phase high pressure liquid chromatography. Diabetes caused an increase in fetal plasma insulin, placental weight, amniotic fluid volume, the ratio of the fetal heart to body weight, maternal and fetal plasma ANP, fetal cardiac ANP and fetal cardiac BNP. It is suggested that the maternal diabetes-induced increase in fetal ANP might be related to fetal myocardial hypertrophy and could contribute to hydramnios.

Cloning of a novel rat placental prolactin-like protein C-related cDNA.

Prolactin-like protein C (PLP-C) is a major rat placental protein which is expressed during the second half of pregnancy and belongs to the growth hormone-prolactin family. Here we report on the isolation of overlapping rat placental cDNAs which specify a transcript of 915 base pairs and predict a 205-amino acid translated product. The full-length cDNA shares 93% homology with the nucleotide sequence reported for PLP-C, and the putative protein, which we designate PCRP (prolactin-like protein C-related protein), exhibits 88% homology with the PLP-C precursor protein. PCRP lacks the signal sequence and the first 2 N-terminal cysteine residues present in PLP-C. Northern blot analysis indicated the basal zone-specific expression of PCRP mRNA, with no detectable expression in decidua and labyrinth. Southern blot analysis of rat genomic DNA using PCRP cDNA as a probe demonstrated multiple hybridization bands, suggestive of a family of genes encoding prolactin-like proteins. Western immunoblot analysis of basal zone culture media using a PCRP antipeptide antiserum revealed at least 5 immunoreactive proteins. The existence of a PLP-C family of proteins in rat placenta after midpregnancy suggests their functional significance in the maintenance of pregnancy and fetal development.

Cloning and expression of a rat placental cDNA encoding a novel cathepsin L-related protein.

Cathepsin L is a major lysosomal cysteine protease produced by mouse placenta and fibroblasts. This study characterizes a novel cathepsin L-related mRNA expressed in rat placenta. Immunological and nucleotide screening of a rat placenta library identified six positive clones, the largest, pCLRP-9, being 924 base pairs in length. The combined sequences of all the clones contain an open reading frame of 711 nucleotides, a termination codon, a polyadenylation site, and 197 nucleotides of 3' untranslated region, but lack the 5' translation initiation codon. The pCLRP nucleotide sequence showed 60-64% identity to those of mouse, rat, and human cathepsin L. The deduced amino acid sequence of pCLRP codes for 237 amino acids, which align with the carboxy-terminal sequence of cathepsin L and has the active site residues characteristic of the cysteine protease family. Northern blot analysis showed hybridization of pCLRP with a major mRNA transcript of 1.3 kilobases expressed in placenta, but not kidney or liver. In contrast, a cDNA for mouse pro-cathepsin L hybridized with a transcript of 1.7 kilobases expressed in rat kidney, as well as placenta. During late gestation, steady-state levels of rat placental pCLRP mRNA were highest on day 18, whereas those of mouse procathepsin L were greatest on day 20 of gestation. Antiserum to mouse cathepsin L cross-reacted with four proteins of molecular weights 36,000 to 42,000 in rat placental culture medium, of which two were absent in the kidney. These data indicate that rat placenta expresses several species of cathepsin L-type proteins, which may be involved in placental function and nutrient supply.

Expression and characterization of recombinant rat placental prolactin-like protein C.

Prolactin-like protein C (PLP-C) is a member of the rat placental family of proteins which are structurally related to pituitary prolactin (PRL). In an effort to characterize the receptor specificity and biological activity of PLP-C, we used a PLP cDNA to express the recombinant protein in a bacterial system. The PLP-C cDNA was modified by oligonucleotide mutagenesis and ligated into a human carbonic anhydrase II (hCAII) expression vector. Following a single step affinity purification, the hCAII-PLP-C fusion protein was digested with enterokinase to release a 25 kDa protein. N-Terminal sequence analysis of the 25 kDa band demonstrated identity with PLP-C. A polyclonal antiserum to the fusion protein cross reacted with seven major proteins in rat placental culture media of which two were the native forms of PLP-C. Recombinant PLP-C was not mitogenic in the Nb2 lymphoma bioassay and did not exhibit high affinity binding to rat PRL receptor. The choice of hCA-II fusion allows for rapid purification of rPLP-C which will aid in further investigation of the biological role of PLP-C.

Isolation and characterization of a cytostatic histone H2B-like protein from fetal lungs of non-diabetic and diabetic rats.

It was observed in the course of other studies that rat fetal lung extracts inhibited proliferation of fetal lung cells in culture. The purpose of the present study was to isolate and characterize this cytostatic factor. It was found that fetal lungs contained a 16 kDa cytostatic factor and its concentration was twofold greater in fetal lungs of diabetic rats compared with control rats. This fetal lung cytostatic protein (FLCP) was purified by reversed-phase, heparin-affinity and gel filtration high-performance liquid chromatography and SDS-PAGE. The purified protein was electroblotted onto polyvinylidene difluoride membrane and subjected to sequence analysis. The amino-terminal sequence of this fetal lung cytostatic protein was P E P A K S A P A P X K G I G K Q X X K A X X K A ... and showed significant homology with histone H2B; however, the amino acid composition of FLCP suggested that it may be structurally distinct from histone H2B. Ion-spray mass spectrometry suggested that FLCP was made up of at least two species of the protein with molecular weights of 13,776.1 and 14,007.3 and was different from the molecular weight of rat histone H2B predicted by its cDNA sequence. The concentration of FLCP, based on amino acid compositions, was 0.32 nmol/g and 0.83 nmol/g wet fetal lung from non-diabetic and diabetic rats respectively. These findings suggest that the fetal rat lung produces a regulatory factor bearing considerable homology with but possibly different from histone H2B and that fetal lung immaturity during diabetic pregnancy might be contributed to by an increase in this factor.

Cytotoxic factor in the serum of diabetic rats and its increase during pregnancy.

Diabetic pregnancy is associated with a high incidence of fetal growth abnormalities which cannot be solely ascribed to fetal hyperglycaemia and hyperinsulinaemia. We therefore examined the possibility of other contributing factors using rats made diabetic with streptozotocin as the experimental model. Blood serum from virgin diabetic rats and, to a much greater extent, from pregnant diabetic rats inhibited [3H]thymidine incorporation by fetal lung cells in culture in a concentration- and time-dependent manner; cell number was also decreased. The cytotoxic activity of the serum was decreased by treatment of pregnant diabetic rats with insulin. Sera from non-diabetic rats and from rats at 6 h and 24 h after the injection of streptozotocin were not cytotoxic. The cytotoxic activity of diabetic rat serum was retained after dialysis and was not destroyed by heating it for 60 min at 60 degrees C. Diabetic rat serum antagonized the stimulatory effects of fetal bovine serum, insulin and insulin-like growth factors on thymidine incorporation by lung cells and inhibited corticosterone production by adrenal cells. Ultrafiltration of diabetic rat serum and high-performance gel permeation chromatography in phosphate buffer suggested that the molecular weight of the cytotoxic factor was approximately 40 kDa. However, gel permeation chromatography in 40% acetonitrile of the cytotoxic eluate from reversed-phase high-performance liquid chromatography using a C18 and C4 column revealed that the cytotoxic factor was a low molecular weight substance, which contained no amino acids. The apparent discrepancy in molecular weights using different separation procedures suggests that the cytotoxic factor is bound to serum proteins. The u.v. spectrum of this factor was different from those of ketone bodies but its exact chemical identity could not be established because of the scarcity of the material. It is suggested that the sera of pregnant diabetic rats and their fetuses contain a cytotoxic factor, which may contribute to fetal developmental abnormalities.