Further evidence for non-pancreatic insulin immunoreactivity in guinea pig brain

The existence of large amounts of insulin in rat brain and of a porcine- or rat-like insulin in guinea pig brain have been disputed on the basis of differing results from standard (Method I) and hydrophobic adsorption techniques (Method II) for concentrating insulin from acid ethanol extracts. To try to resolve these differences, acid ethanol extracts of rat and guinea pig brains were divided into equal aliquots and concentrated for insulin radioimmunoassay (RIA) by both techniques. The RIA used guinea pig anti-porcine insulin serum, with 50% B0 for purified pancreatic porcine, rat and guinea pig insulin standards being 1.35, 2.38 and greater than 1,000 ng/ml, respectively. Oral glucose (4 g/kg) produced plasma glucose of 377 mg/dl in a guinea pig by 20 min but was not associated with any porcine- or rat-like immunoreactive insulin. Dilutions of guinea pig and rat brain extracts had parallel cross-reactivity with insulin standard curves. Insulin contents of rat brain (uncorrected for recovery) against porcine and rat insulin standards, respectively, were 1.33 and 1.93 ng/g (Method I) and 5.93 and 11.67 ng/g (Method II). Rat plasma was 0.85 and 1.42 ng/ml, respectively. Guinea pig contained 1.35 and 1.89 ng/g (uncorrected), respectively (Method I), and 2.99 and 5.62 ng/g, respectively (Method II). Guinea pig plasma was below the sensitivity of the RIA (less than 0.15 ng/ml). These results suggest that a porcine- or rat-like insulin may exist in guinea pig brain.     

Age-related changes in chemical composition and physical properties of mucus glycoproteins from rat small intestine.

Mucus glycoproteins from newborn and adult rat small intestine were radiolabelled in vivo with Na2 35SO4 and isolated from mucosal homogenates by using Sepharose 4B column chromatography followed by CsCl-density-gradient centrifugation. Non-covalently bound proteins, lipids and nucleic acids were not detected in the purified glycoproteins. Amino acid, carbohydrate and sulphate compositions were similar to chemical compositions reported for other intestinal mucus glycoproteins, as were sedimentation properties. There were, however, important differences in the chemical and physical characteristics of the mucus glycoproteins from newborn and adult animals. The buoyant density in CsCl was higher for the glycoproteins from newborn rats (1.55 g/ml versus 1.47 g/ml). On sodium dodecyl sulphate/polyacrylamide/agarose-gel electrophoresis, the glycoprotein from newborn rats had a greater mobility than the adult-rat sample. Although both preparations had similar general amino acid compositions, variations were observed for individual amino acids. The total protein content was greater in the glycoprotein from newborn animals (27%, w/w, versus 18%, w/w). The molar ratio of carbohydrate to protein was less in the newborn, primarily owing to a decreased fucose and N-acetylgalactosamine content. Comparison of the molar ratio of fucose and sialic acid to galactose for both glycoproteins demonstrated a reciprocal relationship similar to that described by Dische [(1963) Ann. N.Y. Acad. Sci. 106, 259-270]. The sulphate content was greater in the glycoprotein from newborn rats (5.5%, w/w, versus 0.9%, w/w). Both had similar sedimentation coefficients in a dissociative solvent. These results suggest an age-related difference in the types of mucus glycoproteins synthesized by small intestine.     

The origins, fate, and ecological significance of free amino compounds released by freshwater pulmonate snails

The mass-specific accumulation rates (MSAR) of both total (TFAC) and individual free amino compounds (FAC) in conditioned media were measured by HPLC, using the orthophthaldialdehyde (OPA) methods, in the following cases: (a) laboratory-reared freshwater snails (B. glabrata) with chemosterilized shells; (b) Biomphalaria glabrata with non-chemosterilized shells; (c) B. glabrata faeces; (d) isolated shells of B. glabrata; and (e) 10 other species of freshwater gastropods from the Lewes Brooks, East Sussex, U.K. The MSAR values for B. glabrata show that 95% of the TFAC's (predominantly ethanolamine, phosphoserine, and the amino acids leucine, isoleucine, valine, aspartic acid, and glycine/threonine) originated from the snails themselves as the faeces and shells contributed only 5.0 and 0%, respectively. In contrast, epizootic organisms on the shells of all 10 snail species from the Lewes Brooks released significant amounts of FAC with the two smallest species (Planorbis vortex and Planorbis contortus) having the highest MSAR values. The MSAR for isolated B. glabrata mucus was 42.45 micromol x g(-1)h(-1). As 500 mg snails can release 16.67 mg of mucus daily, this could potentially result in the daily loss of 707.5 micromol of FAC. The cost/benefits of mucus secretion and the various anatomical, physiological, biochemical, and ecological mechanisms which allow freshwater snails to recover FAC's lost as a result of a high rate of urine production in their hypotonic environment, are discussed.     

Only trace amounts of fatty acids are found in pure mucus glycoproteins

The presence of noncovalently associated lipids and covalently bound fatty acids was investigated in preparations of mucus glycoproteins obtained by using density-gradient centrifugation in CsCl/guanidinium chloride. No phospholipids, glycolipids, cholesterol, or triglycerides could be detected. However, small amounts of extractable fatty acids were consistently found, the sum of which ranged from 0.3 to 0.9 micrograms/mg of glycoprotein. The amount of fatty acid released after subsequent treatment with KOH ranged from 0 to 27 ng/mg of glycoprotein. We conclude that density-gradient centrifugation in CsCl/guanidinium chloride is very efficient in removing noncovalently associated lipids from mucus glycoproteins and that covalently bound fatty acids are probably not present in the macromolecules.     

Studies on extraction of fumonisins from rice, corn-based foods and beans

Different solvent mixtures were examined for extraction of fumonisins from various naturally contaminated and spiked foods and foodstuffs: rough rice, retail rice, rice flour, white corn flour, corn meal, corn starch, corn flakes, tortilla/corn chips, white bean flour, white beans, mung beans, adzuki beans and infant cereals. Most of the naturally contaminated samples were analyzed using the extraction solvent mixtures methanol-acetonitrile-water (25:25:50) (solvent A) and methanol-water (75:25 or 80:20) (solvents B, BB); some were extracted with 0.1 M sodium hydrogen phosphate-acetonitrile (1:1, adjusted to pH 3.0 with o-phosphoric acid) (solvent C) and methanol-0.025 M borate buffer (3:1, adjusted to pH 9.2 with 1 N sodium hydroxide) (solvent D). A 1-ml SAX solid phase extraction column was used for the cleanup in all cases except for infant cereals, for which immunoaffinity chromatography was used; fumonisin concentrations were determined by liquid chromatography. Solvent A gave slightly better extraction of fumonisins from one of two samples of naturally contaminated rough rice than solvent B (fumonisin B₁: 4080 ng/g versus 3150 ng/g; fumonisin B₂:1100 ng/ g versus 922 ng/g) and much better extraction than solvent C (1210 ng/g fumonisin B₁ and 315 ng/g fumonisin B₂) or solvent D (372 ng/ g fumonisin B1 and 191 ng/g fumonisin B2). However, spike recoveries on a similar rice naturally contaminated at a lower level were only in the 43–53% range (solvent A). Recovery of fumonisins was very poor from spiked white rice flour but satisfactory from other rice foods. Solvent A similarly gave slightly better extraction of fumonisins from a sample of naturally contaminated white corn flour than solvent B (fumonisin B₁ 1260 ng/g versus 931 ng/g; fumonisin B₂: 511 ng/g versus 447 ng/g ) and better extraction than solvents C and D. Solvent A was also a better solvent for extraction of fumonisins from naturally contaminated tortilla chips and infant cereals. Study of naturally contaminated corn starch was confounded by instability of fumonisins in this food. Recovery of fumonisins from spiked corn meal, tortilla chips, corn flakes, various types of beans and infant cereals with solvent A and/or solvent B (or BB) was satisfactory.     

Metabolism of Prostaglandin D2 by Human Cerebral Cortex into 9α, 11β-Prostaglandin F2 by an Active NADPH-Dependent 11-Ketoreductase

Abstract: In homogenates of rat cerebral neocortex prostaglandin D₂ (PGD₂) was found to be quantitatively the main PG biosynthesized by a cytosolic PGD synthetase from en-dogenously released arachidonic acid. Amounts of 628 ng/g wet weight were found after 30-min incubation periods compared with basal levels of 2.3 ng/g wet weight. In human cerebral cortex, whether obtained at biopsy or postmortem, only small amounts of PGD₂ (4.5–11.7 ng/g wet weight/30 min) were formed. Furthermore, PGD₂, added to homogenates of human biopsy temporal cortex, was converted efficiently into 9α,11β-PGF₂ by a NADPH-dependent 11-ke-toreductase as has been reported in other human tissues (liver and lung). PGF_2α was determined directly as the fl-butylbo-ronate derivative. It became clear that 9α,11β-PGF₂ was formed in considerably greater amounts than PGF_2α and that other metabolites are also formed. These results can account for the low amounts of PGD₂ found in incubations of human brain tissue. The rat brain does not contain 11-ketoreductase activity. The present results indicate that the 9α, 11β-PGF₂ must be considered along with other eicosanoids in pathophysiological situations in brain.     

Determination of the lipid classes and fatty acid profile of Niger (Guizotia abyssinica Cass.) seed oil

Niger seeds (Guizotia abyssinica Cass.), which are of interest as a new source of vegetable oils, were subjected to Soxhlet-extraction with n-hexane and the extract analysed using a combination of CC, GC, TLC and normal-phase HPLC. The total lipid content was ca. 300 mg/g seed material, and the fatty acid profile showed a high content of linoleic acid (up to 63%) together with palmitic acid (17%), oleic acid (ca. 11%), and stearic acid (ca. 7%). CC separation over silica gel eluted with solvents of increasing polarity yielded 291 mg/g of neutral lipids, 5.76 mg/g of glycolipids, and 0.84 mg/g of phospholipids. GC analysis showed that the major fatty acid present in all lipid classes was linoleic acid together with minor amounts of palmitic, oleic and stearic acids. Polar lipid fractions, however, were characterised by higher levels of palmitic acid and a lower content of linoleic acid. Phospholipid classes separated by normal-phase HPLC consisted of phosphatidylcholine (ca. 49%), phosphatidylethanolamine (22%), phosphatidylinositol (14%), phosphatidylserine (ca. 8%), and minor amounts (2-3%) of phosphatidylglycerol and lysophosphatidylcholine.     

Free amino acid content in the skin mucus of goldfish, Carassius auratus L.: influence of feeding

Free amino acid contents in skin extracts and influence of food and starvation on free amino acid content in skin mucus were analysed in sexually immature goldfish. Free amino acid concentration in skin mucus (91.1 mumol/g dry wt) was higher than in deep skin (54 mumol/g) or in whole skin (56.6 mumol/g) extracts. Free amino acid compositions were very similar in the latter extracts. They both differed from skin mucus extract in taurine, glutamic acid, glycine and histidine relative contents. Free amino acid composition in zooplankton used to feed goldfish was close to the composition found in corresponding skin mucus extracts, except in taurine content. Goldfish weighing 3 g (6 months old) and 17 g (1 year old) reared on zooplankton showed similar patterns of free amino acid composition in skin mucus. Comparison with free amino acid composition in skin mucus from goldfish fed on commercial food had big differences in glutamic acid, valine, methionine and lysine relative contents. During fasting, we observed an increase in the amount of mucus secreted and a concomitant decrease of the free amino acid concentration in the secretion. The origin of free amino acids found in skin mucus and their possible role in pheromonal and allelochemical communications of goldfish are discussed.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B2 (TXB2) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 microgram/g tissue) to both PGE2 (10.7 microgram/g tissue) and TXB2 (6.2 microgram/g tissue. Smaller amounts of PGF2alpha (0.9 microgram/g) and 6-oxoPGF1alpha were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE2, but formed very little TXB2, PGF2alpha or 6-oxoPGF1alpha. Homogenates from rabbit lungs converted arachidonic acid (100 microgram/g) mainly to PGE2, both before and after birth. The amount of PGE2 formed increased during gestation to a maximum of about 6 microgram/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 microgram/g) which was observed 8 days after birth, followed by an increase to about 4 microgram/g in older rabbits.     

Perfluorinated Compounds Distribution and Source Identification in Sediments of Lake Victoria Gulf Basin

Perfluorooctanoic acid and perfluorooctane sulfonate were determined in the sediments from Winam Gulf, which is in the Kenyan side of Lake Victoria and in its source rivers. The sources of perfluorinated compounds within the Gulf of Lake Victoria have been identified and their levels determined for the first time, in this study, using SPE and HPLC-MS-MS analytical methodology. Variability in the concentrations of perfluorooctanoic acid and perfluorooctane sulfonate ranged from 1.4–99.1 and <1–57.5 ng/g in river sediments, respectively, which was higher than concentrations obtained from lake sediments (range perfluorooctanoic acid <1–24.1 ng/g and perfluorooctane sulfonate <1–4.0 ng/g). The results obtained suggested generalized point sources such as domestic and industrial waste indicated by significant correlation and regression of r² = 0.857. Sampling sites within and near sewage and water treatment facilities gave the highest concentrations of both analytes, and were observed to be the main source of perfluorinated compounds pollution. The lowest limit of quantification was 1 ng/g for both analytes and limits of detection were 0.1 and 0.2 ng/g for perfluorooctanoic acid and perfluorooctane sulfonate, respectively. Typical values for recovery obtained were higher than 78% from spiked amounts ranging from 1 to 150 ng. Quantifying perfluoro alkylated compounds in sediments have provided insights into their source, distribution, and mobility in the Lake Victoria Basin.     

Paternal mouthbrooding in the black-chinned tilapia, Sarotherodon melanotheron (Pisces: cichlidae): changes in gonadal steroids and potential for vitellogenin transfer to larvae

The black-chinned tilapia (Sarotherodon melanotheron) is a paternal mouthbrooder. Pairs of adult black-chinned tilapia were raised in freshwater and the males were sampled during the mouthbrooding cycle. Sampling also occurred 10 days after release of the free-swimming fry for comparison. During the first week of incubation of the eggs, total androgens and estradiol were low (<5 and <0.3 ng/ml, respectively). During the second week of brooding, when the eggs have hatched and they are called newly hatched embryos, plasma levels of gonadal steroids increased (13-38 ng androgen/ml and >0.6 ng estradiol/ml). The plasma concentrations of vitellogenin (VTG) in male parents changed during mouthbrooding, with decreases occurring between egg pickup and hatching of the embryo (Day 6 of mouthbrooding). The pattern of change in concentrations of VTG in surface mucus of male parents differed from the pattern in plasma, with peak concentrations occurring at the time of hatching. The amount of VTG in mucus was similar to that measured in the female Oreochromis mossambicus during mouthbrooding of embryos. The appearance of peak VTG levels in the mucus at the time of hatching when plasma levels have declined and the availability of comparable amounts of mucus VTG in both maternal and paternal mouthbrooding tilapia, despite unequivalent plasma levels, support the possibility that parental provisioning of the young occurs during mouthbrooding in tilapia.     

Requirement of heme for growth of Bacteroides fragilis.

Heme or protoporphyrin IX was required for growth of Bacteroides fragilis in a defined medium. The amount of heme necessary for half-maximal growth was 2 to 10 ng/ml (3.8 to 15 pmol/ml) among the Bacteroides species and strains tested. The growth rate, metabolic products from glucose fermentation, and cell yields were affected by the concentration of heme in the medium and by the length of time the culture was incubated. When heme was growth limiting (4 ng/ml), growth rates decreased by 50%, cultures started producing lactic and fumaric acids, and the cell yields declined. The cell yield for B. fragilis (ATCC 25285) at 24 h in medium containing 6.5 microgram of heme per ml was 69 g (dry weight) of cells per mol of glucose compared to 16 g (dry weight) of cells per mol of glucose with 4 ng of heme per ml. B. fragilis was unable to grow in defined medium when a porphyrin precursor, delta-aminolevulenic acid or porphobilinogen, was added in place of heme.     

Pregnenolone, dehydroepiandrosterone, and their sulfate and fatty acid esters in the rat brain

The rat brain contains large amounts of pregnenolone (P) and dehydroepiandrosterone (D) arising from local biosynthetic pathways. We have devised a procedure for the measurement of both "neurosteroids" either unconjugated or released from their sulfate (S) or fatty acid (L) esters. The measurements were performed at the acrophase of the circadian variation of neurosteroids, and confirmed the large accumulation of P (25 +/- 8 ng/g, mean +/- SD) and of PS (19 +/- 6 ng/g) and DS (2.1 +/- 0.5 ng/g) in the brain of adult male rats. We found that fatty acid esters constitute the major species of neurosteroids in brain (PL 46 +/- 14, and DL 36 +/- 7 ng/g, in adult males). The levels of P and DS were increased by daily injection of vehicle to intact males, whereas castration, without or with testosterone or estradiol supplementation (2 mg daily for 7 days), did not produce a significant change of neurosteroids concentrations. Measurements of neurosteroids had not been previously reported in cyclic females. The levels of P, PL, and DS were identical in proestrous females and in intact males, whereas PS (26 +/- 6 ng/g) and DL (50 +/- 16 ng/g) were increased in females. Compared to proestrous females, diestrous females had lower levels of PS (19 +/- 6 ng/g), DS (1.7 +/- 0.4 ng/g), and PL (43 +/- 19 ng/g). These differences suggested a modulatory role of ovarian secretions on the metabolism of neurosteroids.     

Salicylic acid accumulates in the roots and hypocotyl after inoculation of cucumber leaves with Colletotrichum lagenarium

Increased amounts of salicylic acid (SA) were detected in the roots and hypocotyl of cucumber plants (Cucumis sativus) using high-performance liquid chromatography following inoculation of the leaves with the anthracnose pathogen, Colletotrichum lagenarium. The concentrations of SA in the internodes immediately below the infected leaves increased to more than 1microg/g fresh weight. In contrast, the concentrations of SA in stems distant from, or above the infected leaves increased to 100-300ng/g. An increase in SA levels was observed in the upper stem 2d after inoculation, followed by the hypocotyl with an increase detected 4d after inoculation. An initial increase in the SA levels was detected in the stem, followed by an increase in SA levels in the root from a basal level of approximately 300ng/g to more than 1microg/g. The increased level of SA in the lower leaves was less than 100ng/g. These results indicate that the levels of SA in the hypocotyl and root increased significantly following inoculation of the leaves with a microorganism capable of inducing SAR.     

The leukotriene B4 paradox: neutrophils can, but will not, respond to ligand-receptor interactions by forming leukotriene B4 or its omega-metabolites.

Leukotriene B4 (5S,12R-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid, LTB4) is released from neutrophils exposed to calcium ionophores. To determine whether LTB4 might be produced by ligand-receptor interactions at the plasmalemma, we treated human neutrophils with serum-treated zymosan (STZ), heat-aggregated IgG and fMet-Leu-Phe (fMLP), agonists at the C3b, Fc and fMLP receptors respectively. STZ (10 mg/ml) provoked the formation of barely detectable amounts of LTB4 (0.74 ng/10(7) cells); no omega-oxidized metabolites of LTB4 were found. Adding 10 microM-arachidonate did not significantly increase production of LTB4 or its metabolites. Addition of 50 microM-arachidonate (an amount which activates protein kinase C) before STZ caused a 40-fold increase in the quantity of LTB4 and its omega-oxidation products. Neither phorbol myristate acetate (PMA, 200 ng/ml) nor linoleic acid (50 microM), also activators of protein kinase C, augmented generation of LTB4 by cells stimulated with STZ. Neither fMLP (10(-6) M) nor aggregated IgG (0.3 mg/ml) induced LTB4 formation (less than 0.01 ng/10(7) cells). Moreover, cells exposed to STZ, fMLP, or IgG did not form all-trans-LTB4 or 5-hydroxyeicosatetraenoic acid; their failure to make LTB4 was therefore due to inactivity of neutrophil 5-lipoxygenase. However, adding 50 microM-arachidonate to neutrophil suspensions before fMLP or IgG triggered LTB4 production, the majority of which was metabolized to its omega-oxidized products (fMLP, 20.2 ng/10(7) cells; IgG, 17.1 ng/10(7) cells). The data show that neutrophils exposed to agonists at defined cell-surface receptors produce significant quantities of LTB4 only when treated with non-physiological concentrations of arachidonate.     

Formation of highly reactive A-ring and J-ring isoprostane-like compounds (A4/J4-neuroprostanes) in vivo from docosahexaenoic acid

Free radical-initiated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. Docosahexaenoic acid is the most abundant unsaturated fatty acid in the central nervous system. We have shown previously that this 22-carbon fatty acid can yield, upon oxidation, isoprostane-like compounds termed neuroprostanes, with E/D-type prostane rings (E(4)/D(4)-neuroprostanes). Eicosanoids with E/D-type prostane rings are unstable and dehydrate to cyclopentenone-containing compounds possessing A-type and J-type prostane rings, respectively. We thus explored whether cyclopentenone neuroprostanes (A(4)/J(4)-neuroprostanes) are formed from the dehydration of E(4)/D(4)-neuroprostanes. Indeed, oxidation of docosahexaenoic acid in vitro increased levels of putative A(4)/J(4)-neuroprostanes 64-fold from 88 +/- 43 to 5463 +/- 2579 ng/mg docosahexaenoic acid. Chemical approaches and liquid chromatography/electrospray ionization tandem mass spectrometry definitively identified them as A(4)/J(4)-neuroprostanes. We subsequently showed these compounds are formed in significant amounts from a biological source, rat brain synaptosomes. A(4)/J(4)-neuroprostanes increased 13-fold, from a basal level of 89 +/- 72 ng/mg protein to 1187 +/- 217 ng/mg (n = 4), upon oxidation. We also detected these compounds in very large amounts in fresh brain tissue from rats at levels of 97 +/- 25 ng/g brain tissue (n = 3) and from humans at levels of 98 +/- 26 ng/g brain tissue (n = 5), quantities that are nearly an order of magnitude higher than other classes of neuroprostanes. Because of the fact that A(4)/J(4)-neuroprostanes contain highly reactive cyclopentenone ring structures, it would be predicted that they readily undergo Michael addition with glutathione and adduct covalently to proteins. Indeed, incubation of A(4)/J(4)-neuroprostanes in vitro with excess glutathione resulted in the formation of large amounts of adducts. Thus, these studies have identified novel, highly reactive A/J-ring isoprostane-like compounds that are derived from docosahexaenoic acid in vivo.     

Lipoxygenase Metabolism of Arachidonic Acid in Brain

When blood-free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, gas chromatography/mass spectrometry confirmed that the major radioactive lipoxygenase enzyme product of arachidonic acid was 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), with lesser amounts of 5-hydroxy-5,6,8,11,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. When 12-[2H]HETE was used to measure endogenous 12-HETE in brain tissue frozen with liquid nitrogen, the level of 12-HETE was 41 +/- 6 ng/g of wet weight tissue. This frozen tissue level was not due to the presence of blood. When brain slices were incubated in vitro for 20 min, the 12-HETE level increased to 964 +/- 35 ng/g of wet weight tissue. Elimination of residual intravascular blood before tissue incubation reduced the brain slice 12-HETE concentration by one-half.     

Partial characterisation of high-molecular weight glycoconjugates in the trail mucus of the freshwater pond snail Lymnaea stagnalis

We have studied the glycoconjugates in trail mucus of the pond snail Lymnaea stagnalis. The mucus was dissolved with 6 M guanidinium hydrochloride (GuHCl) and the major component was comprised of very high-M_r glycoconjugates that were eluted in the void volume of a Sepharose CL-4B gel-filtration column. This high-M_r material was pooled and thereafter subjected to density gradient centrifugation first in 4 M GuHCl/CsCl and subsequently 0.2 M GuHCl/CsCl to further remove non-glycosylated proteins and DNA. The harvested glycoconjugate pool chromatographed in the void volume of Sepharose CL-2B. However, reduction of disulfide bonds lowered the molecular size of approximately 80% of the void material yielding a major fragment and some minor smaller fragments in gel chromatography. The reduced glycoconjugates were digested with papain and yielded high molecular weight, proteinase-resistant glycopeptides. This fragmentation pattern is similar to that found for oligomeric gel-forming mucins in mammals and the amino acid composition (60% Ser/Thr) and sugar analysis of the glycopeptides is consistent with mucin-like molecules, there being no significant amounts of xylose or uronic acids. The residual 20% of the preparation, which apparently resisted reduction and protease digestion, had a similar amino acid composition to the bulk, but was somewhat different in sugar composition, containing some xylose and a significant amount of glucuronic acid. The two groups of molecules had very different morphologies in the electron microscope. Taken together, these data suggest that trail mucus is a complex mixture of at least two families of protein-glycoconjugate molecules based upon the gel-forming mucin and proteoglycan families, though we cannot rule out that polysaccharides may also be present.     

Isolation and characterization of beta-D-galactosidase from gastric juice]

A method for beta-D-galactosidase isolation from cattle gastric juice has been developed. Gastric juice mucus was removed by and addition of equimolar amounts of Na2HPO4 and CaCl2. The removal of proteases and other proteins was achieved by the treatment with resins KB-51X2 and AN-22. The resulting preparation had specific activity of 0.14 units per mg of protein. Gel filtration on Sepharose 4B led to an increase of specific activity of the preparation up to 0,4 units per mg of protein. Some properties of the beta-D-galactosidase obtained were compared to relation of lactose and o-nitrophenyl-beta-D-galactoside (pH optima, temperature optimym and thermostability).     

Pharmacokinetics of free catecholestrogens and catecholestrogen benzoates

To provide a definite basis for studies on the biological effects of exogenously administered catecholestrogens, the time courses of the concentrations of these estrogens in serum, pituitary and CNS-tissues were studied in male rats after s.c. injection of either 150 μg of 4-hydroxyestradiol or 2-hydroxyestradiol (dissolved in 200 μl sesame oil/ethanol/ascorbic acid; 97.5/2.5/0.1; vol/vol/wt) or equimolar amounts of 4-hydroxyestradiol 3,4-dibenzoate or 2-hydroxyestradiol 2,3-dibenzoate (dissolved in 200 μl sesame oil). The injection of free catecholestrogens resulted in bolus-like elevations of the serum and tissue concentrations of the respective compound (max. values up to 9 ng/ml, half-life below 1 h) whereas the injection of catecholestrogen benzoates gave lower (max. values about 1 ng/ml) but prolonged elevations (half-life approx. 24 h and 32 h for 4-OHE₂ and 2-OHE₂) of the respective free catecholestrogen.