Ultrastructural similarity of endocrine-like cells of the human lung and some related cells of the gut

Summary The ultrastructure of endocrine-like cells of the human lung was compared to the ultrastructure of endocrine-like cells of the stomach and pancreas in both adult and foetal material.Three types of endocrine-like cells were found in the human foetal lung. Type 1 or P₁ cells contained very small granules (about 110 nm) of two varieties, cored and vesicular; type 2 or P₂ cells with cored granules measuring about 130 nm; and type 3 cells with cored granules of about 180–190 nm. In the adult lung only one type P_a cells with cored granules could be found.Cells resembling foetal P₁ cells were not found in foetal or adult gastric mucosa, or in the pancreas. In the gastric mucosa cells resembling pulmonary P_a or P₂ cells were moderately represented and often difficult to distinguish from each other. Thus, they were grouped together as gastric P cells. Cells with granules resembling those of pulmonary type 3 cells were found most numerous in the adult oxyntic mucosa. Cells resembling gastric P cells (and pulmonary P₂ cells) were rather numerous in foetal pancreas, but very rare in adult pancreas. Few cells containing granules somewhat resembling those of pulmonary type 3 cells were present in both foetal and adult pancreas.The results were discussed in respect to 1) the similarities between some gastric or pancreatic carcinoids and lung carcinoids, 2) the gastro-pancreatic P cells as a separate cell population, 3) the possible secretion by the lung endocrine-like cells of active substances, either amines or peptides, 4) the similarity between the secretory granules of P_a and P₁ cells and neurosecretory granules of the hypothalamus and between P₂ cells and some endocrine cells of the pituitary.Supported in part by the Italian National Research Council (Grants N. 75.00630.04 and N. 76.01558.04)     

Mixed function oxidase activity in the harbour seal (Phoca vitulina) from Sable Is., N.S

One adult male, eight pups (including two full term foetuses) and nine adult female harbour seals (Phoca vitulina) were analysed for indices of mixed function oxidase (MFO) activity. MFO activity was present in liver samples, but was at or below detection limits in samples of kidney, lung and pancreas. Hepatic ethoxyresorufin O-de-ethylase and benzo[a]pyrene hydroxylase activities were similar to those reported in other seals and in other mammals. Cytochromes P-450 and b5 concentrations were slightly lower than those observed in other mammals. MFO activities in newborn pups and foetuses were significantly lower than those in adult females. No qualitative differences in cytochrome P-450 isozyme distribution between foetal and adult samples could be discerned by electrophoresis.     

Age-related changes in the chemical composition of bovine articular cartilage. The structure of high-density proteoglycans

Proteoglycans were extracted from the articular cartilage of foetal, calf and adult bovine metacarpal–phalangeal joints with 4m-guanidinium chloride. After extraction, the high-density proteoglycans (PG-I fractions) were prepared by sedimentation in two sequential CsCl-density-gradient procedures [Swann, Powell & Sotman (1979) J. Biol. Chem. 254, 945–954]. The PG-I fractions from foetal, calf and adult tissues accounted for 75%, 52% and 46% respectively of the extracted components. The glucosamine, galactose, N-acetylneuraminic acid and protein contents increased with age. The overall amino acid compositions of PG-I fractions were similar. Fractionation of PG-I-fraction samples on a Bio-Gel A-50m column indicated that the molecular weight decreased with age. The PG-I fractions were specifically ³H-labelled by treatment with galactose oxidase followed by reduction with NaB³H₄. The ³H radioactivity was incorporated into both galactose and galactosamine residues of different carbohydrate side chains. The elution profiles of alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples on a Sepharose 6B column showed that the molecular weights of chondroitin sulphate chains were 13500, 12000 and 10500 in foetal, calf and adult tissues respectively. Fractionation of the alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples and ³H-labelled calf and adult PG-I-fraction samples on a Bio-Gel P-10 column showed that there was an inverse relationship between the low-molecular-weight O-linked oligosaccharides and the higher-molecular-weight sialic acid-containing constituents at different ages. The oligosaccharide components of foetal, calf and adult PG-I-fraction samples represented 79%, 69% and 36% respectively of the total sialic acid content of the proteoglycans. Similarly in the ³H-labelled calf and adult samples 75% and 30% of the total radioactivity were present in the oligosaccharide components respectively. Digestion with chondroitinase AC-II and infrared analyses showed that the PG-I-fraction F and C samples contained primarily chondroitin 4-sulphate chains whereas PG-I-fraction sample A was 6-sulphated. These studies show that the major proteoglycans (PG-I fractions) in the articular cartilage of foetal, calf and adult animals differ in the content, types and structure of the chondroitin sulphate, keratan sulphate and oligosaccharide constituents. These changes in proteoglycan structure reflect the gross age-related changes in the chemical composition of the tissue.     

Transfer ribonucleic acid methylase activity in adult and foetal rat liver.

Foetal rat liver extracts were found to have higher tRNA methylene activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers. The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100%. The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 muM for Escherichia coli tRNA and 2.1 muM for S-adenosyl-L-methionine. After T1-T2 ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts. It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

Pancreatic cholesterol esterases. 1. Pancreatic cholesterol esterase induction during maturation

The activities of pancreatic cholesterol esterase from calf and cow pancreas were examined in detail. A 1300-fold enhancement of enzymatic activity was found after maturation, even though cholesterol esterase activity levels in other organs did not change from the juvenile to the adult species. Radioimmunoassays also showed that the calf pancreas contained at least 100-fold less cholesterol esterase protein. Decreased amounts of protein were not due to enhanced proteolysis, since cytosol from cow pancreas degrades exogenously added cholesterol esterase faster than that from calf pancreas. Rather, enhancement of pancreatic cholesterol esterase activity associated with bovine maturation was the result of specific, increased synthesis of a 72-kDa enzyme. This labile 72-kDa cholesterol esterase species was purified to homogeneity by a two-step process in 75% yield and is the major form of bovine pancreatic cholesterol esterase (99%). A much less abundant 67-kDa species, accounting for less than 1% of total pancreatic cholesterol esterase activity, was also purified to homogeneity in a similar two-step process. These results demonstrate that a specific form of pancreatic cholesterol esterase is induced during maturation, and they bear importantly on understanding juvenile cholesterol metabolism as related to dietary absorption of this sterol.     

SdPI, the first functionally characterized Kunitz-type trypsin inhibitor from scorpion venom

Background

Kunitz-type venom peptides have been isolated from a wide variety of venomous animals. They usually have protease inhibitory activity or potassium channel blocking activity, which by virtue of the effects on predator animals are essential for the survival of venomous animals. However, no Kunitz-type peptides from scorpion venom have been functionally characterized.

Principal Findings

A new Kunitz-type venom peptide gene precursor, SdPI, was cloned and characterized from a venom gland cDNA library of the scorpion Lychas mucronatus. It codes for a signal peptide of 21 residues and a mature peptide of 59 residues. The mature SdPI peptide possesses a unique cysteine framework reticulated by three disulfide bridges, different from all reported Kunitz-type proteins. The recombinant SdPI peptide was functionally expressed. It showed trypsin inhibitory activity with high potency (K_i = 1.6×10⁻⁷ M) and thermostability.

Conclusions

The results illustrated that SdPI is a potent and stable serine protease inhibitor. Further mutagenesis and molecular dynamics simulation revealed that SdPI possesses a serine protease inhibitory active site similar to other Kunitz-type venom peptides. To our knowledge, SdPI is the first functionally characterized Kunitz-type trypsin inhibitor derived from scorpion venom, and it represents a new class of Kunitz-type venom peptides.     

Endocrine Measurements and Calving Performance of Swedish Red and White and Swedish Holstein Dairy Cattle with Special Respect to Stillbirth

During 3 consecutive calving seasons, calving performance, placental characteristics and endocrine profiles of total 98 pregnancies of late pregnant Swedish Red and White (SRB) and Swedish Holstein (SLB) dairy heifers and cows, were investigated. Ninety-four singleton pregnancies and 4 sets of twins were recorded. In animals with singleton pregnancy, 8 stillbirths, 7 weak calves, 3 premature parturitions and 1 abortion were registered. In the SLB heifers, 19% of stillbirth (5/26) were observed, while 5% (2/42) were noted for the SRB heifers. One stillborn calf derived from the SRB cows and none was found from the SLB cows. In the heifers and cows delivering a normal living calf with unassisted parturition, the placentome thickness monitored by ultrasonography was constant towards the end of pregnancy. The numbers of foetal cotyledons varied individually between animals but in total, fewer cotyledons were found in the foetal membranes of the SRB animals than in the SLB animals (69 ± 19) vs. (88 ± 29) (p < 0.05). No morphological and numerical differences of the placentome thickness in animals delivering a stillborn or weak calf, compared to animals delivering a normal living calf, could be observed. In animals with unassisted parturition and without birth complications, the levels of progesterone (P4), PGF_2α metabolite (PG-metabolite), cortisol, oestrone sulphate (E1SO4) and pregnancy associated glycoproteins (PAGs) were not different by breeds and parities. In animals carrying stillbirth, higher levels of E1SO4 were found in 3 SRB animals and 1 SLB heifer, whereas lower levels of E1SO4 were recorded in 3 SLB heifers during the last week of pregnancy, compared to the profiles found in animals with unassisted parturition. Additionally, the levels of PAGs remained low and constant in 1 SRB cow (delivering a stillborn calf), 1 SRB heifer (giving birth prematurely), 4 animals (carrying twins) and 1 aborting SRB cow. Our results show a very high rate of stillbirth in especially SLB heifers and deviating profiles of E1SO4 and PAGs in animals with impaired parturition were recorded.     

Stimulation of lung maturity: investigation of ambroxol in various animal models

The surfactant or phospholipid lining of the alveolar surface is essential for lung function and airway stability. In premature neonates, the idiopathic respiratory distress syndrome (IRDS) is usually due to a prenatal deficiency in pulmonary surfactant. Experimental and clinical investigations with corticosteroids and other drugs were conducted to study various aspects of lung maturation. The present study of Ambroxol, a new compound with surfactant stimulating properties, was carried out on adult and foetal animals. In adult experimental animals an increase in the activity of Type II pneumocytes was shown by measuring various biochemical parameters. In models of premature animals comparable to clinical IRDS conditions, the antenatal treatment of foetal lambs and rabbits with Ambroxol enhanced lung maturation.     

Characterization of acidic and basic fibroblast growth factors in brain, retina and vitreous chick embryo

We have purified acidic and basic fibroblast growth factors (c-aFGF, c-bFGF) from 11 day-old chick embryo brain, retina and vitreous by heparin-Sepharose chromatography and reverse phase HPLC. The analysis of their biological activity as well as their molecular weight indicates that they were analogous to basic or acidic human and bovine FGF. The ratio of c-aFGF to c-bFGF activity depended of the tissue. In brain c-aFGF represented 66% of the total mitogenic activity retained on the heparin-sepharose column and c-bFGF 34% while retina contained 16% of c-aFGF and 84% of c-bFGF; vitreous 78% of c-aFGF and 22% of c-bFGF. Like human aFGF, Heparin stimulated purified c-aFGF mitogenic activity in the absence of serum but inhibited the activity of the retina acid soluble extract, in the presence of foetal calf serum (FCS). Thus, chick embryo and adult human acidic and basic FGF respectively share the same biochemical properties. Since there are no blood vessels in chick retina or vitreous, their presence in these tissues suggests that angiogenesis is not the only role of these growth factors.     

Regional Development of Glutamate Dehydrogenase in the at Brain

The development of glutamate dehydrogenase enzyme activity in rat brain regions has been followed from the late foetal stage to the adult and through to the aged (greater than 2 years) adult. In the adult brain the enzyme activity was greatest in the medulla oblongata and pons greater than midbrain = hypothalamus greater than cerebellum = striatum = cortex. In the aged adult brain, glutamate dehydrogenase activity was significantly lower in the medulla oblongata and pons when compared to the 90-day-old adult value, but not in other regions. The enzyme-specific activity of nonsynaptic (free) mitochondria purified from the medulla oblongata and pons of 90-day-old animals was about twice that of mitochondria purified from the striatum and the cortex. The specific activity of the enzyme in synaptic mitochondria purified from the above three brain regions, however, remained almost constant.     

Hepatotropin mRNA expression in human foetal liver development and in liver regeneration

A 569 bp probe against the β-chain of hepatotropin was used to examine expression of RNA for this growth factor in human adult and foetal liver, foetal kidney and pancreas, and rat liver after partial hepatectomy. Low level expression of a 6kb RNA occurred in human adult and normal rat liver. 70% hepatectomy increased expression, peaking at 10 h and returning to near normal levels 24 h after resection. The 6 kb band was strongly expressed in human foetal liver, as compared with adult, but not in foetal kidney or pancreas, suggesting a major role for hepatotropin in both foetal development and regeneration of the liver.     

Thymidine kinase in rat liver during development

1. The activity of thymidine kinase in rat liver supernatant decreased with development to a value in the adult that was 1% of that in the 17-day foetus. 2. The foetal enzyme was more stable than the adult to gel filtration on Sephadex G-25 at 0 degrees . 3. The greater stability of the foetal enzyme to incubation at 45 degrees was attributable to the presence of higher concentrations of nucleotides in foetal liver supernatant. 4. The K(m) values for foetal and adult enzymes were approx. 2.5mum- and 2.1mum-thymidine respectively. 5. The foetal enzyme was more sensitive to inhibition by thymidine triphosphate. 6. The decline in enzyme activity during the neonatal period was correlated with a shift in the enzyme properties from the foetal to the adult type, and may reflect the decrease in the proportion of haemopoietic tissue in the liver.     

Uridine kinase activities in developing, adult and neoplastic rat tissues.

Uridine kinase activities were found chiefly in the soluble fractions of rat tissues. In normal adults the activities ranged from 13 munits/g in skeletal muscle to 178 munits/g in colon. Enzyme activities in several rat neoplasms were significantly higher (e.g. in a fibrosarcoma, mammary carcinoma, renal carcinoma, pancreatic carcinoma and lymphocytic lymphoma, but not in a fast-growing Morris hepatoma). The activities were not related to tumour growth rates or sizes. In normal foetal liver, lung, brain, heart and kidney, uridine kinase concentrations equalled or exceeded those in the adult homologous tissue, but maximal activities in liver were reached 3--5 days post partum. In suckling rats the intestinal activity decreased substantially immediately after birth and normally did not rise again until late in the third postnatal week. Premature upsurges could be evoked by an injection of cortisol or by starvation of the pups overnight. Pancreatic activity was absent from 1-day-old rats, and only about 5% of the adult activity was reached by day 20; adult activities were attained rapidly after weaning. In pancreas, precocious formation or uridine kinase was elicited by overnight starvation of 2-week-old rats.     

Transfer ribonucleic acid methylase activity in adult and fœtal rat liver

Foetal rat liver extracts were found to have higher tRNA methylase activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers.The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100 per cent.The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 μM for Escherichia coli tRNA and 2.1 μM for S-adenosyl-l-methionine.After T₁-T₂ ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts.It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

The effects of newborn calf serum and foetal bovine serum on the Na+ + K+-activated adenosine triphosphatase activity in 3T3 and SV3T3 cells grown to confluency

The effects of cell density and growth in 10% foetal bovine serum and 10% newborn calf serum on the activity of the enzyme (Na+ + K+)-ATPase were studied in 3T3 and SV3T3 cells. The enzyme activity decreases in 3T3 cells grown in foetal bovine serum as the cells approach confluency while in those grown in newborn calf serum the enzyme activity increases. The (Na+ + K+)-ATPase activity does not change with increase in cell density in SV3T3 cells grown in foetal bovine serum while the enzyme activity in those grown in newborn calf serum increases with increase in cells density up to about 1.35 x 10(5) cells/sq. cm. and then decreases with further increase in cell number. At confluency it was found that the enzyme activity is higher in the SV3T3 as compared to the 3T3 cells when the cells were grown in 10% foetal bovine serum, whereas in those grown in 10% newborn calf serum the enzyme activity is higher in the 3T3 as compared to the SV3T3 cells.     

hDlk-1: a cell surface marker common to normal hepatic stem/progenitor cells and carcinomas

Advances in stem cell biology have clarified that a tumour is a collection of heterogeneous cell populations, and that only a small fraction of tumour cells possesses the potential to self-renew. Delta-like 1 protein (Dlk-1) is a surface antigen present on foetal hepatic stem/progenitor cells but absent from mature hepatocytes in neonatal and adult rodent liver. Using a monoclonal antibody (mAb) against hDlk-1, Yanai et al. (Dlk-1, a cell surface antigen on foetal hepatic stem/progenitor cells, is expressed in hepatocellular, colon, pancreas and breast carcinomas at a high frequency. J. Biochem. 2010;148:85-92) have shown that human (h) Dlk-1 is expressed in human foetal, but not adult, liver and that 20% of all hepatocellular carcinomas (HCCs) are hDlk-1(+). Importantly, an even higher percentage of HCCs in younger patients are hDLK-1(+). These authors also found that hDlk-1 is present at high frequency in colon adenocarcinomas, pancreatic islet carcinomas and small cell lung carcinomas. Here, I discuss the implications of the expression of foetal hepatic stem/progenitor cell antigens on carcinoma cells.     

Phosphoenolpyruvate carboxykinase and pyruvate carboxylase in developing rat liver

1. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase were measured in foetal, newborn and adult rat liver extracts by a radiochemical assay involving the fixation of [¹⁴C]bicarbonate. 2. Pyruvate-carboxylase activity in both foetal and adult liver occurs mainly in mitochondrial and nuclear fractions, with about 10% of the activity in the cytoplasm. 3. Similar studies of the intracellular distribution of phosphoenolpyruvate carboxykinase show that more than 90% of the activity is in the cytoplasm. However, in the 17-day foetal liver about 90% of the activity is in mitochondria and nuclei. 4. Pyruvate-carboxylase activity in both particulate and soluble fractions is very low in the 17-day foetal liver and increases to near adult levels before birth. 5. Phosphoenolpyruvate-carboxykinase activity in the soluble cell fraction increases 25-fold in the first 2 days after birth. This same enzyme in the mitochondria has considerable activity in the foetal and adult liver and is lower in the newborn. 6. Kinetic and other studies on the properties of phosphoenolpyruvate carboxykinase have shown no differences between the soluble and mitochondrial enzymes. 7. It is suggested that the appearance of the soluble phosphoenolpyruvate carboxykinase at birth initiates the rapid increase in overall gluconeogenesis at this stage.     

Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage.

The addition of foetal calf serum to explant cultures of adult bovine articular cartilage is known to stimulate proteoglycan synthesis in a dose-dependent manner. We have now shown the activity in serum responsible for this effect to be heat- and acid-stable, to be associated with a high-Mr complex in normal serum but converted to a low-Mr form under acid conditions. The activity has an apparent Mr approximately 10,000 and isoelectric points similar to those reported for insulin-like growth factors (IGFs). Addition of a monoclonal antibody against insulin-like growth factor-I (IGF-I) prevented foetal calf serum from stimulating proteoglycan synthesis. Physiological concentrations of recombinant IGF-I or pharmacological levels of insulin when added to cartilage cultures mimicked the proteoglycan-stimulatory activity of serum. IGF-I appeared to act by increasing the rate of proteoglycan synthesis and did not change the nature of the proteoglycan synthesized nor the rate of proteoglycan catabolism by the tissue, suggesting that IGF-I may be important in the regulation of proteoglycan metabolism in adult articular cartilage. Furthermore, IGF-I can replace foetal calf serum in the culture medium, thereby allowing the use of a fully-defined medium which will maintain the synthesis and tissue levels of proteoglycan in adult articular cartilage explants for up to 5 days.     

myo-Inositol homeostasis in foetal rabbit lung

In several species, lung maturation is accompanied by a decline in the phosphatidylinositol content of lung surfactant and a concomitant increase in its phosphatidylglycerol content. To examine the possibility that this developmental change is influenced by the availability of myo-inositol, potential sources of myo-inositol for the developing rabbit lung were investigated. On day 28 of gestation the myo-inositol content of foetal rabbit lung tissue (2.3±0.5μmol/g of tissue) was not significantly different from that of adult lung tissue but the activity of d-glucose 6-phosphate:1l-myo-inositol 1-phosphate cyclase (cyclase) in foetal lung tissue (81.0±9.0nmol·h⁻¹·g of tissue⁻¹) was higher than that found in adult lung tissue (23.2±1.0nmol·h⁻¹·g of tissue⁻¹). Day 28 foetal rabbit lung tissue was found also to take up myo-inositol by a specific, energy-dependent, Na⁺-requiring mechanism. Half-maximal uptake of myo-inositol by foetal rabbit lung slices was observed when the concentration of myo-inositol in the incubation medium was 85μm. When the myo-inositol concentration was 1mm (but not 100μm) the addition of glucose (5.5mm) stimulated myo-inositol uptake. myo-Inositol uptake was observed also in adult rabbit lung and was found to be sub-maximal at the concentration of myo-inositol found in adult rabbit serum. The concentration of myo-inositol in the serum of pregnant adult rabbits (47.5±5.5μm) was significantly lower than that of non-pregnant adult female rabbits (77.9±9.2μm). On day 28 of gestation the concentration of myo-inositol in foetal serum (175.1±12.0μm) was much less than on day 25, but more than that found on day 30. A transient post-partum increase in the concentration of myo-inositol in serum was followed by a rapid decline. Much of the myo-inositol in foetal rabbit serum probably originates from the placenta, where on day 28 of gestation a high cyclase activity (527±64nmol·h⁻¹·g of tissue⁻¹) was measured. The gestational decline in serum myo-inositol concentration, together with the decreasing cyclase activity of the lungs, is consistent with the view that maturation of the lungs is accompanied by decreased availability of myo-inositol to this tissue.     

Amino acid sequence and disulfide bridges of affinity purified Kunitz-type chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L.) DC)

The primary sequence of the affinity purified chymotrypsin inhibitor, WBCI, isolated from the albumin fraction of Psophocarpus tetragonolobus (L.) DC cv. UPS-122 seed was determined. The inhibitor consisted of a single polypeptide chain of 183 amino acids (Mr 20285) and the four half-cystine residues in the molecule formed two intramolecular disulfide bridges equivalent to those in other Kunitz-type seed inhibitors. The sequence of this chymotrypsin inhibitor was identical to that of chymotrypsin inhibitor-3 from cultivar UPS-31 and it showed about 50% sequence similarity to the winged bean acidic (WBTI-2, pI 5.1) and basic (WBTI-1, pI 8.9) trypsin inhibitors. Sequence similarities to other Kunitz-type seed inhibitors are discussed.