Effects of either glucose or fructose and metabolic regulators on bovine embryo development and lipid accumulation in vitro.

Our objective was to determine if replacing glucose with fructose would decrease cytoplasmic lipid accumulation during culture of embryos with or without regulators of metabolism. In vitro-produced bovine zygotes were cultured 60 hr in chemically defined medium-1 (CDM-1) plus 0.5% BSA and 0.5 mM fructose or glucose in Experiment 1, and glucose in Experiment 2. In both experiments, 8-cell embryos were next cultured 135 hr in CDM-2 plus 2 mM fructose or glucose in factorial combination with five treatments: (Experiment 1: control, 10% fetal calf serum (FCS), 0.3 microM phenazine ethosulfate (PES), 30 microM dinitrophenol (DNP), and PES + DNP), and (Experiment 2: control, PES, PES + DNP, and 1 and 3 microg/ml cerulenin (C1 and C3)). Day 7.5 blastocysts were stained with Sudan Black B to quantify cytoplasmic lipid droplets as small (SD, <2 microm), medium (MD, 2-6 microm), or large (LD, >6 microm). Blastocyst rates per oocyte were 22% (Experiment 1) and 15% (Experiment 2) higher (P < 0.05) for fructose than glucose. For Experiment 1, numbers of MD were lower for PES, DNP, and PES + DNP than control and FCS (P < 0.05). LD were lower for PES and DNP than control, and higher for FCS than all other treatments (P < 0.05). For Experiment 2, MD were lower (P < 0.05) for PES, and PES + DNP than C1, C3, and control. For LD, PES was lower (P < 0.05) than control, C1, and C3, but not different from PES + DNP. The only effect of hexose on lipids was that fructose resulted in fewer MD (P < 0.01) in Experiment 2. In conclusion, fructose produced more blastocysts than glucose, and PES reduced lipid accumulation.

Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer.

Protein labeling with green fluorescent protein derivatives has become an invaluable tool in cell biology. Protein quantification, however, is difficult when cells express constructs with overlapping fluorescent emissions. Under these conditions, signal separation using emission filters is inherently inefficient. Spectral imaging solves this problem by recording emission spectra directly. Unfortunately, linear unmixing, the algorithm used for quantifying individual fluorophores from emission spectra, fails when resonance energy transfer (RET) is present. We therefore sought to develop an unmixing algorithm that incorporates RET. An equation for spectral emission incorporating RET was derived and an assay based on this formalism, spectral RET (sRET), was developed. Standards with defined RET efficiencies and with known Cerulean/Venus ratios were constructed and used to test sRET. We demonstrate that sRET analysis is a comprehensive, photon-efficient method for imaging RET efficiencies and accurately determines donor and acceptor concentrations in living cells.

First record of host defence peptides in tadpoles. The magnificent tree frog Litoria splendida.

Tadpoles of the Magnificent Tree Frog Litoria splendida produce host defence peptides early in their development and well before metamorphosis. Peptides were identified and characterized using high performance liquid chromatography and electrospray mass spectrometry. No host defence peptides were identified in the eggs. The neuropeptide caerulein was detected 10 d after egg deposition, and the antibiotic peptides caerin 1.1, caerin 1.6 and caerin 3.1 first appeared at 14 d. The concentration of peptides increases with the onset of metamorphosis at 84 d, when the host-defence peptide profile is the same as that of the adult.

Ectopic expression of bioactive peptides and serotonin in the sacciform gland cells of teleost skin.

Immunohistochemical methods identified serotonin, and the peptides bombesin and caerulein, in the skin of the teleosts Lepadogaster candollei and Mastacembelus erytrotaenia. In both species, the secretory content of epidermal sacciform cells reacted positively for all three substances. These results are compared with studies on the skin glands of amphibians, which also contain multiple active compounds, and on various neuroendocrine cells of fish. The precise functions of the secretions are not known.

Immunocytochemical evidence for the colocalization of neurotensin/xenopsin- and gastrin/caerulein-immunoreactive substances in Xenopus laevis gastrointestinal tract.

Distribution and association of neurotensin (NT)- and xenopsin (XP)-like peptides were investigated using immunocytochemical techniques in the amphibian gut. Antisera against both groups of peptides showed an identical distribution pattern of NT- and XP-positive cells in Xenopus laevis gastrointestinal tract. Immunolabeling of consecutive semithin sections revealed the coexistence of NT- and XP-like substances within cells of the stomach and small intestine. Recent reports of the colocalization of XP-like material with gastrin in mammalian G cells led us to study the association of NT/XP-like peptides with members of the gastrin/cholecystokinin (CCK)/caerulein (G/C) family in amphibians. The data obtained from immunolabeling serial sections with NT/XP-specific and G/C-specific antisera show that in some intestine NT/XP- and G/C-like peptides do exist in the same cells. In the stomach, however, G/C-like material is confined to endocrine cells of the antral region, while NT/XP-like substances occur in distinct cells accumulating in cardial glands but absent in the pyloric glands. Our findings thus indicate that in amphibian gastrointestinal tract there is some association between the regulatory peptide families NT/XP and G/C, similar to mammals. The regional distribution of both hormone families, however, is different from that in mammals.

Novel peptide fragments originating from PGLa and the caerulein and xenopsin precursors from Xenopus laevis.

Skin secretions from the South African frog Xenopus laevis have been chromatographed by high performance liquid chromatography (HPLC), fractionated, and analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The HPLC chromatograms showed the secretion to be a complex mixture with over 30 components at similar levels to the four peptides previously isolated from X. laevis skin, i.e. xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa. FAB-MS analysis of the HPLC fractions gave numerous protonated molecular ions ranging from m/z 491 to 2662. Preliminary assignments of these components were made by comparing these experimental molecular weights to those predicted for regions within the xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa precursors. These results suggested that many of these skin secretions were peptides originating from additional processing of the xenopsin, caerulein, and PGLa precursors, primarily involving cleavage at single arginine residues, and a novel cleavage at the NH2-terminal side of single lysines. These assignments were subsequently confirmed by Edman degradation, FAB-MS peptide sequencing, and amino acid analysis. All of these peptides contain one or more lysines and would be expected to have amphiphilic structures. As yet, nothing is known about their activity, although they resemble in composition the mast cell degranulating peptides melittin and the bombolitins. These precursor fragments were also found to have limited sequence homology to bombinin, a hemolytic amphibian peptide isolated from the European Bombina toad.

Sequence of preprocaerulein cDNAs cloned from skin of Xenopus laevis. A small family of precursors containing one, three, or four copies of the final product.

From skin of Xenopus laevis, cDNA libraries were constructed and clones coding for the precursors of caerulein were isolated and sequenced. Using restriction endonuclease digestions, three different types of preprocaerulein cDNAs could be discerned. These were termed types I, III, and IV in accordance with the number of caerulein copies present in the sequence, the type III being the most abundant one. An incomplete copy of a fourth variant, termed type I', was also found. Besides deletions/insertions encompassing one or two caerulein sequences, these types also differ from each other by several point mutations. In the homologous precursor polypeptides deduced from the nucleotide sequence of these cloned cDNAs, the caerulein copies are flanked by complex processing sequences. These are Arg-Arg-Phe-Ala-Asp-Gly or Arg-Arg-Asp-Gly at the amino-terminal side and Gly-Arg-Arg at the carboxyl end. Between caerulein copies, highly homologous segments are present both at the polypeptide and cDNA level. This homology is evident both within a given precursor, where up to three such segments are present, and between the different types of precursors. We conclude that preprocaerulein cDNAs in the skin of X. laevis represent a small family, at least part of which is derived from different genes rather than being formed by alternative splicing of pre-mRNAs.

Active peptides in the skins of two hundred and thirty American amphibian species.

Extracts prepared from dried or fresh skins of more than 200 American amphibian species were subjected to biological screening in order to determine occurrence and contents of peptides active on smooth muscle preparations, systemic blood pressure and, subordinately, external secretions, anterior pituitary and the central nervous system. The peptide families identified in skin extracts were as follows: caruleins (caerulein, phyllocaerulein), tachykinins (physalaemin, phyllomedusin), bombesins (phyllolitorin, [Leu8]phyllolitorin, rohdeilitorin), bradykinins (phyllokinin and others), sauvagine, dermorphins (dermorphin, [Hyp6]dermorphin), tryptophyllins (numerous peptides) and, finally, miscellaneous peptides. None of the above peptide families showed a widespread distribution, but all were restricted to particular amphibian genera or stocks. The hylid frogs of the Phyllomedusinae family occupy a unique position, as their skin displayed the greatest variety and abundance of active peptides ever found in any amphibian is destined to increase because numerous other peptide molecules await isolation, elucidation of structure and definition of possible biological activities.

A mass spectrometric method for the identification of novel peptides in Xenopus laevis skin secretions.

The peptides secreted by the South African frog Xenopus laevis were screened systematically using a strategy based on fabms. Hplc of crude and Sephadex G-10 chromatographed secretions showed that many more peptides were present in these secretions than those previously identified, i.e., xenopsin, caerulein, TRH, and PGLa. Fractions from the hplc were analyzed directly by fabms to determine the molecular weights of these novel peptides. Subsequent analyses, using a combination of fabms, manual Edman degradation, enzymatic digestions, and amino acid analyses identified the partial and sometimes complete sequences of these peptides which had molecular weights ranging from 700-2700. Many peptides with structural features that are often indicative of biological activity, e.g., C-terminal amides and pyroglutamic acid, were readily identified by fabms. In some cases, molecular weight data combined with partial sequence data was sufficient to identify peptides as originating from spacer regions in the precursors to xenopsin, caerulein, and PGLa.

A mass spectrometric assay for novel peptides: application to Xenopus laevis skin secretions.

The peptides secreted by the South African frog Xenopus laevis were screened systematically using a strategy based on fast atom bombardment mass spectrometry (FAB-MS). High performance liquid chromatography (HPLC) of crude and Sephadex G-10 chromatographed secretions showed that many more peptides were present in these secretions than those previously identified, i.e., xenopsin, caerulein, TRH and PGLa. Fractions from the HPLC were analyzed directly by FAB-MS to determine the molecular weights of these novel peptides. Subsequent analyses, using a combination of FAB-MS, manual Edman degradation, enzymatic digestions and amino acid analyses, identified the partial and sometimes complete sequences of these peptides which had molecular weights ranging from 700-2,700. Many peptides with structural features that are often indicative of biological activity, e.g., C-terminal amides and pyroglutamic acid, were readily identified by FAB-MS. In some cases, molecular weight data combined with partial sequence data was sufficient to identify peptides as originating from PGLa and the spacer regions in the precursors to xenopsin and caerulein.

Is caerulein amphibian CCK?

The amphibian skin decapeptide caerulein is structurally related to the mammalian peptides gastrin and CCK, suggesting that the peptides might share a common evolutionary history. It has been suggested that caerulein is the amphibian counterpart of gastrin and CCK, and that the Amphibia do not possess authentic gastric and CCK. High Performance Liquid Chromatography (HPLC) in conjunction with radioimmunoassay using a caerulein-specific antiserum and C-terminal CCK antisera, was used to characterize CCK-and caerulein-like peptides in amphibian brain and gut. In the brain of Xenopus laevis, two CCK-like peptides were present, one of which was indistinguishable by HPLC from mammalian CCK8. No decapeptide caerulein was detected in the brain of Xenopus laevis or Rana temporaria. In the stomach of Xenopus and in the intestine of both species studied, CCK-like and caerulein-like peptides were present. The results indicate therefore that the Amphibia possess CCK8-like rather than caerulein-like peptides in brain. In contrast, stomach and intestine contain both CCK-like and caerulein-like peptides, but the latter are however distinguishable from the decapeptide found in skin.

Biologically active gastrin/CCK-related peptides in the stomach of a reptile, Crocodylus niloticus; identified and characterized by immunochemical methods.

We have used immunochemical, chromatographic, and bioassay techniques to characterize peptides related to gastrin and CCK, from the stomach of the reptile Crocodylus niloticus. By immunocytochemistry gastrin/CCK-like peptides were localized in specific mucosal cells of the pylorus and in the duodenum. Boiling water extracts of pyloric antrum cross reacted with four antisera specific for the C-terminal region of gastrin or CCK, but estimates of concentration varied between antisera. Antisera specific for the N-terminus of heptadecapeptide gastrin (G17), intact G17, or the amphibian CCK-like peptide caerulein did not cross react with the crocodile extracts. Gel filtration of the extracts on Sephadex G50 resolved one major peak eluting significantly before G17 or CCK8, suggesting larger molecular size, whereas ion exchange on DE52 cellulose resolved two major immunoreactive peaks, both eluting before G17, indicating that they are less acidic. The more acidic of the two peptides stimulated gastric acid secretion in the rat, but had no CCK-like actions on the rat pancreas. Thus crocodile antrum contains gastrin-like peptides, which are however clearly distinguishable from any of the known mammalian forms of gastrin and CCK.