Characterization of trophoblast cell isolations by a modified flow cytometry assay.

The purity of isolated trophoblast cells is an important checkpoint in in vitro studies on the human placenta. Maintaining viability of primary cells for a prolonged period, or achieving cell proliferation in primary cell cultures, is often a matter of concern. In this paper we present a method for characterizing the purity of isolated trophoblast cells based on the expression of cytoskeletal proteins, as well as for assessing their cell cycle status, by flow cytometry. We show that after a simple permeabilization and fixation step in 70 per cent methanol, staining for cytokeratin 7 and vimentin could discriminate between trophoblast cells and contaminating populations. The method was applicable to trophoblast cells both from villous and extravillous origin. By staining the proliferation-related antigen Ki-67 and DNA, information was gained about the cell cycle status and viability of freshly isolated and cultured villous trophoblast cells. This method may help to quickly and quantitatively characterize preparations of isolated trophoblast, as well as to search for culture conditions favouring long-term survival and proliferation.

Cytogenetic and DNA-fingerprint characterization of choriocarcinoma cell lines and a trophoblast/choriocarcinoma cell hybrid.

We report the successful fusion of human choriocarcinoma cells with normal human trophoblast cells to a choriocarcinoma/trophoblast hybrid. The hybrid cells ACH1P were derived from fusion of primary male trophoblast cells with the HGPRT-defective choriocarcinoma cell line AC1-1. The karyotypes of the parental choriocarcinoma cell line JEG-3, its HGPRT-defective mutant clones AC1-1, AC1-5, and AC1-9, and the choriocarcinoma/trophoblast hybrid ACH1P are presented, together with a detailed characterization of the AC1-specific chromosomal marker add(X)(q26) using conventional cytogenetic banding techniques and multiplex-fluorescence in situ hybridization (M-FISH). To our knowledge, this is the first report of a stably proliferating human cell hybrid of trophoblastic origin, providing a unique cell culture model to study trophoblast-related invasion and its underlying genetic mechanisms.

The effect of neuropeptides from Limulus on its circadian rhythm in retinal sensitivity.

Retinal responses of the Limulus lateral eyes to light are greater at night than during the day. A circadian clock in the brain of the horseshoe crab controls these rhythmic changes of light sensitivity. The increase in sensitivity (as measured by the amplitude of the electroretinogram) is mediated at least in part by octopamine that is released from efferent axons terminating in the visual cells. Earlier studies indicate that certain factors in Limulus hemolymph can act in conjunction with octopamine. More recently, five neuropeptides (LP1-LP4 and Lip-HP) had been isolated from acetone extracts of the Limulus central nervous system using HPLC fractionation and radioimmunoassay with antisera against FMRFamide-like peptides for detection. Presently, we have injected into the Limulus lateral eye these five peptides and observed changes in retinal sensitivity. Injection during daytime had no immediate effect on that daytime electroretinogram but decreased the electroretinogram amplitude for the entire subsequent night (12 h). However, upon injection at night, we observed an immediate but only transitory decrease in electroretinogram amplitude for about 1 h without effect on the subsequent daytime electroretinogram. We suggest that the peptides act antagonistically to octopamine and are highly dependent upon the activity state of the efferent nerve terminals.

Extracellular matrix components of the placental extravillous trophoblast: immunocytochemistry and ultrastructural distribution.

Invasive extravillous trophoblast cells of the human placenta are embedded in a self-secreted extracellular matrix, the matrix-type fibrinoid. The ultrastructure and molecular composition of the matrix-type fibrinoid of the term human placenta were studied by transmission electron microscopy and immunogold labelling. We used antibodies directed against different matrix proteins such as collagen type IV, laminin, vitronectin, heparan sulfate, various fibronectin isoforms, and against the oncofetal blood group antigen, "i". Immunogold labelling patterns of matrix proteins are the basis for the subdivision of the trophoblast-derived matrix-type fibrinoid into mosaic-like patches of structurally and immunocytochemically different compartments. Firstly, fine granular patches with structural similarities to basal lamina material are composed solely of collagen type IV and laminin. Secondly, an ultrastructurally amorphous glossy substance shows reactivity with antibodies against heparan sulfate and vitronectin. A third type of patches, fine fibrillar networks embedded in the above-mentioned glossy matrix, are reactive with antibodies against normal fibronectin isoforms (IST-4, IST-6, IST-9) and oncofetal isoforms (BC-1, FDC-6). The blood group precursor antigen "i" was not only expressed on the surfaces of the extravillous trophoblast cells but was associated with the fibronectin-positive fibrils. In conclusion, within this extracellular matrix, clear compartments of different composition can be distinguished from each other. Glycosylation with "i" in this matrix may be involved in immunological masking, thus preventing rejection of placenta and fetus.

Cardioinhibitory peptides from Limulus polyphemus: modulation of the neurogenic heart.

Four neuropeptides have been isolated and sequenced from acetone extracts of brains of the horseshoe crab Limulus polyphemus. They belong to a newly discovered peptide family in invertebrates. A possible role of the four peptides from Limulus as cardioregulatory neurotransmitters has been tested on the isolated Limulus heart. Three of the peptides (DEGHKMLYFamide, GHSLLHFamide, and PDHHMMYFamide) produce dose-dependent decreases in both amplitude and rate of the heart contractions, whereas DHGNMLYFamide reduces only the amplitude of the heartbeat. All four peptides differ in threshold, potency and duration of their effects.

The Sequences of Five Neuropeptides Isolated from Limulus using Antisera to FMRFamide.

Five neuropeptides were isolated from CNS extracts of the horseshoe crab Limulus polyphemus by high pressure liquid chromatography (HPLC). The peptides were identified by radioimmunoassays (RIAs) based on two antisera raised to FMRFamide-related peptides (FaRPs). The purified peptides were analyzed by automated sequencing and mass spectrometry, and the following sequences were obtained: DEGHKMLYFamide, GHSLLHFamide, PDHHMMYFamide, DHGNMLYFamide, and GGRSPSLRLRFamide. The first four peptides are members of a novel family with virtually no relationship to FMRFamide. GGRSPSLRLRFamide, on the basis of structural similarity, becomes the second member of a class of FaRPs known previously only from a peptide isolated from mosquito heads. At least one member of the novel family (GHSLLHFamide) inhibits the isolated heart of Limulus.

The effect of neuropeptides on the ERG of the crayfish Orconectes limosus.

CCAP (Crustacean Cardioactive Peptide), Proctolin, FMRFamide, Met- and Leu-enkephalin, Substance P, RPCH (red pigment concentrating hormone) and PDH (pigment dispersing hormone) were applied to the isolated retina of the crayfish Orconectes limosus. Changes in light sensitivity, measured as changes of the amplitude of the electroretinogram (ERG) were observed after application of RPCH, PDH and CCAP. RPCH caused an increase of the ERG amplitude to 133% of its reference value whereas PDH and CCAP decreased the amplitude to 78% and 30% respectively. A dose-response curve showed that 10(-9) mol/l CCAP produce a half-maximal effect.

Isolation and amino acid sequence of crustacean hyperglycemic hormone precursor-related peptides.

The crustacean hyperglycemic hormone (CHH) is synthesized as part of a larger preprohormone in which the sequence of CHH is N-terminally flanked by a peptide for which the name CPRP (CHH precursor-related peptide) is proposed. Both CHH and CPRP are present in the sinus gland, the neurohemal organ of neurosecretory cells located in the eyestalk of decapod crustaceans. This paper describes the isolation and sequence analysis of CPRPs isolated from sinus glands of the crab Carcinus maenas, the crayfish Orconectes limosus and the lobster Homarus americanus. The published sequence of "peptide H" isolated from the land crab, Cardisoma carnifex, has now been recognized as a CPRP in this species. Sequence comparison reveals a high level of identity for the N-terminal region (residues 1-13) between all four peptides, while identity in the C-terminal domain is high between lobster and crayfish CPRP on the one hand, and between both crab species on the other. Conserved N-terminal residues include a putative monobasic processing site at position 11, which suggests that CPRP may be a biosynthetic intermediate from which a potentially bioactive decapeptide can be derived.

Amino acid sequence of the crustacean hyperglycemic hormone (CHH) from the shore crab, Carcinus maenas.

Crustacean hyperglycemic hormone (CHH) was isolated from sinus glands of Carcinus maenas, and its primary structure was determined by manual microsequencing, using the DABITC-PITC double-coupling method. The neurohormone consists of 72 amino acid residues (8524 Da). Three disulfide bridges are present and both the N- and C-terminus are blocked. CHH does not show significant sequence homology to any known peptide hormone or protein.

[Nutritive physiological effect of dietary fats in rations for growing swine. 2. Effect of an isocaloric exchange of carbohydrate energy versus fat energy in piglets on growth and various metabolic parameters in the subsequent fattening period]. / Untersuchungen über Ernährungsphysiologische Wirkungen von Futterfetten in Rationen für wachsende Schweine. 2. Mitteilung. Einfluss eines isokalorischen Austausches von Kohlenhydratenergie gegen Fettenergie im Ferkelstadium auf das Wachstum und einige Stoffwechsel-parameter in der anschliessenden Mastperiode.

The influence of different feeding regimes of piglets on fattening performance and blood composition of the subsequent fattening period (30-100 kg) was examined. Three groups of pigs fed on an isocaloric and isonitrogenous basis with rations containing either 5% fat (groups I), 18% fat (group II) or 35% fat (group III) until body weight of 30 kg, were fed ad libitum one diet from 30 kg onwards. At a body weight of 45 and 93 kg the blood concentrations of insulin, glucose, urea, free fatty acids, neutral lipids and cholesterol in response to feeding and of insulin and glucose in response to an oral glucose load was determined in pigs provided with a permanent jugular vein catheter. Moreover, the in-vitro fat synthesis from glucose was measured. At the end of the experiment the body composition was determined by chemical analysis of the carcass. Neither feed intake, daily body weight gain nor feed conversion differed significantly among the groups. Blood urea levels of the three groups did not suggest a different protein utilization. Neither feed consumption nor oral intake of glucose affected the insulin and glucose response of the three groups differently. The concentrations of free fatty acids, neutral lipids and cholesterol did not differ clearly among the groups although occasionally significance of difference was observed. In group I the in-vitro synthesis of fat was increased (p less than 0.05) at a body weight of 45 kg and appeared to be higher at a body weight of 93 kg as compared to the high fat group (group III). No clear differences between the groups were observed in the chemical composition of the carcasses. It is concluded, that isocaloric replacement of carbohydrates by fat in the diet of piglets does not affect protein and fat retention in the subsequent fattening period.

[Nutritional physiology effect of dietary fats in the diet of growing pigs. 1. Effect of an isocaloric exchange of carbohydrate energy compared with fat energy on protein retention, in vitro synthesis of fat from glucose and on several blood parameters in piglets]. / Untersuchungen über ernährungsphysiologische Wirkungen von Futterfetten in Rationen für wachsende Schweine. 1. Mitteilung. Einfluss eines isokalorischen Austausches von Kohlenhydratenenergie gegen Fettenergie auf den Proteinansatz, die in-vitro-Fettsynthese aus Glukose sowie auf einige Blutparameter bei Ferkeln.

Three groups of male, castrated piglets of the German Landrace breed, weight range 3-30 kg, were used to study the relation between fattening performance and blood parameters when feeding rations containing different amounts of fat. The fat content of the rations was either 5% (group I), 18% (group II) or 35% (group III). Concomitantly with the increased metabolizable energy (ME) content of the ration the content of digestible protein (DP) was increased and the amount of feed reduced in order to guarantee an equal intake of ME and DP in all three groups. The digestibility of the crude nutrients and protein retention of the subjects were determined in nine subsequent trials each lasting 7 days. At a body weight of 27 kg the blood concentrations of insulin, glucose, free fatty acids, neutral lipids and cholesterol were determined at different times after feeding. In addition, an oral glucose tolerance test was made and the in-vitro synthesis of fat from glucose was measured. The apparent digestibility of fat amounted, unexpectedly, to approximately 94% on the high rat rations II and III. In all groups a significant positive relation between body weight and digestibility of the fat was determined. Despite equal daily intakes of ME in all groups, in group III daily weight gain and protein retention were 7% (p less than 0.01) and 4% (p less than 0.01) higher than in group I, respectively. Blood urea levels of group III were 67% (p less than 0.01) lower than in group I. The mean daily nitrogen retention of the three groups rose gradually from 4 g at a body weight of 5 kg to 16-17 g at a body weight of 25 kg. The feed conversion of group I was lower by 22 and 36% as compared to groups II and III, respectively. Highly significant differences were observed among either groups (p less than 0.01). It has been calculated that in group III the consumption of ME/kg weight gain was about 8% (p less than 0.05) lower than in the two other groups. According to the increased fat percentage of the rations, body protein content diminished from 17,1% in group I to 16,5 and 16% in groups II and III, respectively. Only the difference between groups I and III proved to be significant (p less than 0.05). In group I feed intake resulted in an increase in the insulin level by 55% (p less than 0.05) whereas glucose did not change significantly.(ABSTRACT TRUNCATED AT 400 WORDS)