Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Concluding remarks

On the basis of symposium contributions onChlorella, Hibbertia, Eucalyptus, Ambrosia and on numerical approaches some fundamental problems of (bio)systematics, evolution, and taxonomic categories are discussed: Methods available for analysing affinities; conflicting evidence from phenetic, biochemical, cytogenetic and other analyses; further classification problems in cases of intermediacy, etc. While sibs of various levels and their natural hierarchy often can be objectively defined, this appears impossible for particular taxonomic levels itself (e. g. species). A single objective taxonomic system of organisms is unrealistic. Certain guiding lines for relative and practicable concepts of species and genus are proposed.Presented at the symposium Speciation and the Species Concept during the XIIth International Botanical Congress, Leningrad, July 8, 1975.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

No linkage to the 3β-HSD gene cluster in a kindred affected with 3β-hydroxy-Δ5-C27steroid dehydrogenase deficiency and early onset hepatic failure

We studied the segregation of the genes for 3-hydroxy-C_19/21-steroid dehydrogenase types I and II (3-HSD I and II) in a consanguineous family affected with 3-hydroxy-⁵-C₂₇steroid dehydrogenase (3-OH-C₂₇-SD) deficiency. The results show that the C₂₇ and C_19/21 steroid dehydrogenase activities are encoded by distinct genes that are not in genetic linkage. Further kindreds would assist in screening for linkage of 3-OH-C₂₇-SD to other members of the 3-hydroxysteroid dehydrogenase gene family.     

Stimulation by hexose esters of lactate production by rat erythrocytes,insensitivity to 3-O-methyl-D-glucose and inhibition by 2-deoxy-D-glucose and its tetraacetic ester

Selected esters of D-glucose were recently proposed as tools to provide the sugar to cells, whilst bypassing the carrier system for hexose transport across the plasma membrane. In the present study, -D-glucose pentaacetate, -D-glucose pentaacetate, -D-mannose pentaacetate and, to a lesser extent, 6-O-acetyl-D-glucose, all tested at a 1.7 mM concentration, were found to increase lactate production above basal value in rat erythrocytes. Over 90 min incubation, the increment in lactate production ranged from about 1.2 (-D-glucose pentaacetate) to 0.6 (6-O-acetyl-D-glucose) mol/l of erythrocytes. Little or no change in lactate production was observed in cells exposed to -L-glucose pentaacetate, -D-glucose pentaethylsuccinate, -D-galactose pentaacetate or -D-galactose pentaacetate. The metabolic response to -D-glucose pentaacetate was resistant to 3-O-methyl-D-glucose (10-80 mM) which suppressed, however, that evoked by D-glucose. D-mannoheptulose (10 mM) virtually failed to affect the response to D-glucose and its pentaacetate ester. On the contrary, 2-deoxy-D-glucose (10.6 mM) inhibited to the same relative extent (55% decrease) lactate production in erythrocytes exposed to either unesterified D-glucose or -D-glucose pentaacetate. The tetraacetic ester of 2-deoxy-D-glucose was more efficient than unesterified 2-deoxy-D-glucose in inhibiting lactate production from -D-glucose pentaacetate. It is proposed that selected esters of saccharides represent useful tools to bypass defects in hexose transport, and to increase their nutritional or therapeutic efficiency.     

The NGF complex from the African ratMastomys natalensis

The 7S NGF complex from the male mouse submaxillary gland consists of the , and subunits in the ratio ₂₂. The (NGF) subunit contains all the known biolocial activity of 7S NGF. The and subunits are both members of glandular kallikrein gene family, yet only subunit has protease activity. The subunit plays a role in the processing of preproNGF to its mature form, while the role of the subunit is not yet understood. Despite the fact that 7S NGF has been extensively characterized, no other NGF complex has been characterized, nor have the or subunits been observed in tissues which express NGF. We have therefore purified and characterized the NGF complex from the submaxillary glands of the ratMastomys natalensis in order to more fully understand the roles of the and subunits. The NGF complex from M. natalensis contains subunits similar to those found in mouse 7S NGF. Although similar, there are significant differences between mouse and M. natalensis NGF complexes, especially in the degree of post-translational modification of the and NGF subunits, the expression of esterase activity and the ease with which the complexes dissociate. Evidence is presented that suggests that the NGF complex from M. natalensis may consist of subunits in the ratio ₂. The amino acid sequence of the M. natalensis NGF suggests some, but not all, ways in which these differences arise.Special issue dedicated to Dr. Lawrence Austin     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

Lipid profile of rat myelin subfractions

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of light, heavy and membrane fraction lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the membrane fraction resembles in its lipid and fatty acid composition other cell membranes (particulary oligodentrocytes); ii) light and heavy myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nohhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the heavy subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.     

Isolation of cobamides from Methanothrix soehngenii: 5-methylbenzimidazole as the α-ligand of the predominant cobamide

Methanothrix soehngenii was found to contain five different cobamides when grown on vitamin B₁₂ supplemented as well as vitamin B₁₂ free media. In both cases, it was shown by HPLC-chromatography and UV/VIS spectroscopy, that -5-methylbenzimidazolyl--cyanocobamide was the predominant cobamide, accounting for 27% and 23%, respectively, of the total corrinoid content. Vitamin B₁₂ and -5-hydroxybenzimidazolyl--cyanocobamide could also be detected in both cell batches in varying amounts. Cells grown on vitamin B₁₂ free medium contained significantly more baseless cobamides, indicating biosynthesis of cobamides.Abbreviations (5-MeBza)CNCba -5-methylbenzimidazolyl--cyanocobamide - (5-HOBza)CNCBa -5-hydroxybenzimidazolyl--cyanocobamide (factor III) - (5-MeOBza)CNCba -5-methoxybenzimidazolyl--cyanocobamide (factor III_m) - (Bza)CNCba -benzimidazolyl--cyanocobamide     

A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Summary Cholic acid (3,7,12-trihydroxy-5-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7- and 12-hydroxysteroid dehydrogenase and, the second, by 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with -ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.     

Die Feinstruktur der Zirbeldrüse normaler,trächtiger und experimentell beeinflußter Meerschweinchen

Zusammenfassung In der Meerschweinchenzirbeldrüse lassen sich elektronenmikroskopisch helle und dunkle Pinealzellen sowie einzelne Gliazellen nachweisen. In den bei weitem überwiegenden hellen Pinealzellen zeichnet sich ein Teil der vesicle-crowned rodlets (VCR) durch lokale Auftreibungen aus. Von VCR deutlich abzugrenzen sind die vesicle-crowned balls (VCB). Erstmalig beschrieben wird das Vorkommen von sog. Zylindern, die als Vorstufen von VCB aufgefaßt werden. In den relativ seltenen dunklen Pinealzellen, die sich durch chromatinreiche Kerne und elektronendichtes Zytoplasma auszeichnen, sind Vesikel, VCR, VCB und Zylinder seltener als in hellen Pinealzellen. Die reichlich vorhandenen marklosen Nervenfasern finden sich vor allem in perivasculären Räumen, seltener im Parenchym. Synapsen zwischen Nerven und Pinealzellen wurden nicht beobachtet. In den Zirbeldrüsen trächtiger Meerschweinchen zeichnen sich in der 2. Hälfte der Tragzeit die hellen Pinealzellen durch stärkere Lappung der Kerne, gehäuftes Auftreten von laktiven Zonen, Vermehrung von Mitochondrien, glattem ER, agranulären Vesikeln, VCR, VCB und Zylindern aus. Die dunklen Pinealzellen nehmen während der Tragzeit an Zahl zu. Post partum bilden sich diese Veränderungen innerhalb einer Woche zurück. Längerer Aufenthalt der Tiere in Dunkelheit führt zu einer Aktivierung der hellen Pinealzellen mit auffallender Vermehrung der VCR und zu einer Zunahme der dunklen Zellen. Unter Dauerbelichtung kommt es in den hellen Zellen zu einer Abnahme fast aller Zellorganellen und zu einer starken Vermehrung der VCR, die nach 70 Tagen auch Formveränderungen aufweisen. Nach Reserpinbehandlung beobachtet man eine Verminderung und degenerative Veränderungen der VCR. Es wird diskutiert, daß die VCR als prae- bzw. postsynaptische Strukturen der Erregungsübertragung von Nerven zu Pinealzellen bzw. von Pinealzellen untereinander dienen könnten.

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The fine structure of the pineal gland of normal, pregnant and experimentally affected guinea-pigs

Summary By means of electron microscopy light and dark pinealocytes can be distinguished in the guinea-pig pineal gland. Glial cells are rare. In the light pinealocyte. the most frequent cell type, some vesicle-crowned rodlets (VCR) show circumscribed thickenings. From these structures vesicle-crowned balls (VCB) have to be clearly distinguished. Furthermore cylinders occur, which, it is suggested, are precursors of VCB. Dark pinealocytes characterized by chromatin-rich nuclei and electron-dense cytoplasm are rare and contain fewer vesicles, VCR, VCB and cylinders than light pinealocytes. Numerous non-myelinated nerve fibres are situated within perivascular spaces, a few also in the parenchyma. Synapses between nerve fibres and pinealocytes were not observed. In the pineal gland of pregnant guinea-pigs the following changes can be observed in the second half of gestation. The light cells show many nuclear indentations and an increase of active zones, mitochondria, smooth ER, agranular vesicles, VCR, VCB, and cylinders respectively. The dark cells increase in number. After birth these changes reverse to normal within one week. Constant darkness leads to an activation of the light cells accompanied by an increase of the VCR and to an increase in number of the dark cells. Under constant illumination the light cells show a decrease of their organelles and a strong increase of the VCR. After 70 days the VCR also show a change in shape. Following reserpine treatment the VCR decrease in number and show signs of degeneration. It is discussed that the VCR function as pre- or postsynaptic structures and that they are involved either in transmitting impulses from nerve fibres to pinealocytes or from one pinealocyte to the other.

Untersuchung unter Leitung von Univ.-Doz. Dr. L. Vollrath.     

Fever and feeding: Differential actions of macrophage inflammatory protein-1 (MIP-1), MIP-1α and MIP-1β on rat hypothalamus

Changes in body temperature (T_b) and feeding were characterized in unrestrained rats following the micro-injection into the anterior hypothalamic preoptic area (AH/POA) of macrophage inflammatory protein-1 (MIP-1), MIP-1 or MIP-1. After the rats recovered from the stereotaxic implantation of a single guide tube placed in the AH/POA, either one of the MIP-1 compounds or control CSF was micro-injected in a volume of 1.0 l into this area. Changes in body temperature (T_b) and food and water intakes were monitored throughout each experiment. When micro-injected into the AH/POA in a dose of 28 or 280 pg, doublet MIP-1 and MIP-1 evoked a monophasic fever which increased above baseline to a mean maximum of 2.17±0.14°C and 2.1±0.24°C, respectively. MIP-1 micro-injected similarly evoked a biphasic fever, with the T_b declining transiently at the 30 min point 0.4°C lower than the congruent rises in T_b evoked by doublet MIP-1 or MIP-1. The secondary rise in T_b induced by MIP-1 had a latency of 1.5–2.0 hrs and reached a maximum of 1.56±0.16°C. Although all three cytokines significantly attentuated the rats' mean intake of food during the 24 hr interval after their micro-injection into the AH/POA, doublet MIP-1 exerted the most potent anorexic effect in comparison to that of the saline control rats. However, neither body weight nor intake of water was altered significantly by the three cytokines. These results demonstrate a functional distinction between the febrile actions of MIP-1 and MIP-1 on cells of the AH/POA. It is envisaged that MIP-1 underlies the initial phase of a pyrogen-induced fever, whereas MIP-1 could mediate the secondary phase of a biphasic fever. Clearly, the localized elevation of either MIP-1 or MIP-1 in the AH/POA perturbs the neuronal mechanism underlying the set-point for body temperature. Thus, both MIP-1 and MIP-1 could play a functional role in the pathological events occurring in neurons of the AH/POA in response to an endotoxin or other pyrogen challenge.     

Slowly adapting stretch-receptor organs: Periodic stimulation with and without perturbations

The length of isolated slowly adapting stretch-receptor organs (SAO) from crayfish was submitted to approximately sinusoidal modulations of 0.030–0.800 mm at frequencies of about 0.2, 1.0 or 3.0 cps. Sines were imposed either by themselves (clean) or mixed with (perturbed by) fast irregular length fluctuations or jitter. The amplitude of the latter remained within a specified fraction of the modulation, usually two-thirds (or x0.66), though from one twentieth to twice (or x0.05–2.00) were explored. The afferent impulse discharge was recorded: rate over bins of about 1/10 of the period was plotted as function of ongoing time. Within stationary epochs, average cycles were analyzed primarily in terms of lengths and discharge intensities. Each point in the displays corresponded to a particular bin along the average cycle, the abscissa and the ordinate being the length and the corresponding discharge rate, respectively; points were numbered in the order of their generation. Similar displays were constructed for rate vs velocity, rate vs acceleration, and rate-change vs velocity. Experiments without jitter. The non-perturbed state. With 0.2 and 1.0 cps, the rate vs length display had a clockwise loop with a flat extension to the left. The SAO at its shortest did not discharge and remained silent for some time. As length augmented, it eventually started firing, reaching maximum rate while being stretched (lead) or at maximum length. While length decayed, the organ fired less, slowing down, stopping at a length greater than where it had started, and then remaining silent until the end of the cycle. With 3.0 cps, the display had a counterclockwise loop with a flat extension on the left. The SAO did not discharge at its shortest, and fired more during relaxation than during stretching, peaking when relaxing (lag) and stopping at a length greater than where it had started. Artificial introduction of time separations between length and rate converted displays into acceptably straight lines only in a few, usually 0.2 cps, cases. The rate vs length relation exhibited consistent departures from monotonic increase and from linearity: departures were large fractions of the overall swings involving flat extensions, leads, lags, saturation effects, asymmetric rate sensitivities, and locking (which was frequent with 1.0 or 3.0 cps and could conceivably be more common with faster modulations). Other experimental paradigms have demonstrated multivaiued steady-state-rate-length relations. The variability from cycle to cycle of the rate in a particular bin was high or low for bins with low or high rates, respectively. Rate vs velocity plots were counter-clockwise loops with a base upon the abscissae, higher rates when velocity was decaying than when augmenting, and maxima at non-negative velocities. Rate-change vs velocity plots showed shortening associated with either discharge, decreases or small changes, and lengthenings associated with discharge increases, greater when augmenting if at 0.2 cps or when decaying if at 3.0 cps. Rate vs stimulus acceleration plots were, for the three tested frequencies, counterclockwise loops with a base upon the abscissae and higher rates when acceleration was decaying than when augmenting, suggesting a particular role in sensing accelerations. The sensitivity to length was evaluated by comparing the rate-swings at each frequency using identical bin-widths and depths. This procedure is considered to be most meaningful physiologically. Sensitivity was contingent upon the bin-width: with small bins (30 and 120 ms) it was highest for 3.0 cps, with intermediate bins (160 ms) it was highest for 1.0 cps, and with larger bins it was practically uniform. Differences involved factors of under x1.7. Experiments with jitter. The perturbed state. Jitter changed radically the rate vs length displays, converting all of them into clockwise folium-like loops, which implied a shift from lag to lead at 3.0 cps. Introduction of appropriate lags brought points acceptably close to straight, positively sloped segments: this occurred often at 0.2 cps, sometimes at 1.0 cps, and exceptionally at 3.0 cps. Jitter altered the variability profile along the cycle, decreasing peak variability and not affecting or increasing the minimum; variability along the cycle was thus reduced and uniformed and identification of stimuli on the basis of discharges was improved. Jitter amplitude was an important issue. The larger ones, about the modulation size, increased the lowest bin-rates, particularly at 0.2 and 1.0 cps, and increased the peaks at 0.2 cps, decreasing them at 1.0 and 3.0 cps. The smaller jitter, about 1/10 of the modulation, affected the lowest rates little, but increased the peak rate at 0.2 cps, decreasing it at 1.0 and 3.0 cps; display shapes changed little. A qualitative model of the SAO involves subsystems with linear features, as well as rectification, stiction-like and saturation-like properties. Depending upon back-ground length, modulation depth, jitter and several other issues, the organ can perform frankly nonlinearly, piece-wise linearly or linearly. Within each context, a particular stimulus is of interest and can be referred to as the signal; others are not, and can be referred to as noise, acting as perturbations. Small, erratic perturbations influence strongly transduction of large steps or sines, simplifying and making it more proportionate. Large regular perturbations act upon small signals too, but their influence has been explored less extensively.Supported by grants from NIH and NSF     

Complex sex-determining mechanisms in three species of South American grasshoppers (Orthoptera,Acridoidea)

The karyotype of three species of South American grasshoppers are studied in this paper. Leiotettix sanguineus has two chromosome races, one of them with 2n=23 and an XO sex mechanism and the other, as far as we know limited to the Cerro Chato population, with 2n=22 and an XY sex mechanism. Leiotettix politus has two kinds of individuals, one with 2n=14 and XY sex chromosomes and the other 2n=13 and an X₁X₂Y mechanism. Dichroplus dubius presents 2n=21 and an X₁X₂Y sex chromosomes. One of the three specimens studied shows aberrant behaviour in the meiotic process.     

UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO₂ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc N-acetylneuraminic acid - GalNAcol N-acetylgalactosaminitol - CGMG Cowper's gland mucin glycoprotein - GalNAc-CGMG Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine - Gal3GalNAc-CGMC Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains - MES 2-(N-morpholino) Ethane Sulfonic acid - PBS Phosphate Buffered Saline     

Genetic heterogeneity of propionic acidemia: Analysis of 15 Japanese patients

Summary Propionic acidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl CoA carboxylase (PCC) activity. We have analyzed the molecular heterogeneity of Japanese propionic acidemia patients using anti-human PCC antiserum and cDNA clones coding for the two protein subunits ( and ) of the enzyme. The steady state levels of both and subunits of PCC from 15 Japanese patients were determined by Western blot. Three patients had neither nor subunits, and the amounts of both and subunits were low in 3 other patients. According to our previous data, we classified these 6 patients as having subunit deficiency. In the remaining 8 patients, subunits were normal, but the subunits were aberrant. Two patients had low levels of normal-sized subunits and 6 had subunits smaller than normal in size and greatly reduced in quantity. These 8 patients were assigned to the subunit deficiency category. One patient had apparently normal and subunits. We could not determine this patient's primary defect. These data reveal the genetic heterogeneity of molecular defects causing propionic acidemia in the Japanese. Southern blot analysis did not reveal any gross alteration in gene structure when DNA was digested withHindIII,EcoRI andTaqI. However, DNA from 3 -subunit-deficient patients, when digested withMspI and probed with PCC cDNA, revealed a unique 2.7-kb band not observed in blots of DNA from any other patient or 15 normal controls. We conclude that this alteredMspI restriction map is the result of a mutation in the subunit gene of these patients.     

Variant forms ofα-2-l-fucosyltransferase in human submaxillary glands from blood group ABH “secretor” and “non-secretor” individuals

The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed.     

Polarities in Mammalian Evolution Seen Through Homologs of the Inner Cell Mass

Embryogenetic pathways differ markedly among monotremes, marsupials, and placentals, and their analysis provides information of fundamental importance to recognition of mammalian evolutionary directions. The cap of cuboidal cells of the marsupial late unilaminar blastocyst, generally known as the embryonic area, probably is induced to form (prior to origin of Hensen's node) by signals from earliest hypoblastic cells (anterior visceral endoderm). The thickened cap is a medullary plate of sauropsid terminology because it includes epiblastic cells presumptive to neurectoderm (including neural crest), Hensen's node, primitive streak, and gut endoderm. The remainder of the definitive embryo (i.e., parts of epidermal origin, including ectodermal placodes) derives from squamous ectoderm (surrounding the medullary plate) of the blastocyst's ill-named trophoblastic area. Amniotic ectoderm develops farther distally within the trophoblastic area. The autapomorphic inner cell mass (ICM) of placental mammals is homologous to medullary plate of the marsupial blastocyst plus morphologically undefined, proximal parts of surrounding ectoderm (of the trophoblastic area). Considerations of early cell lineages in marsupials are greatly affected by recognition that the boundary between future embryonic and extra-embryonic tissues does not match the margin of the medullary plate (i.e., embryonic area). Marsupials and monotremes largely conform to sauropsid early embryogenesis, but placentals express, at earliest developmental stages, innovations unique within Amniota that are linked to early establishment of the brain. Neonatal marsupials and hatchling monotremes are extremely altricial and closely comparable anatomically/physiologically; they share a temporal pattern in combining early morphogenesis of craniofacial features (related to suckling) with deferral of telencephalic completion into postnatal/posthatching life. Placentals contrast greatly in establishing the central nervous system prior to rudiments of the cranial skeleton and associated musculature, and they complete essentials of forebrain development before birth. Comparative evidence from transitory periderm suggests that primordial eutherians had extremely altricial hatchlings or newborns, whichever was the mode of early development. Details remain unknown about the origin of the unique specialization of ICM plus encapsulating trophoblast from the more generalized blastula of ancestral synapsids.     

Comparison of the β-lactamases ofBacteroides species

-Lactam antibiotic susceptibility and the presence of -lactamase were examined in clinical strains ofBacteroides species. All strains produced a noninducible, cell-associated cephalosporinase. Based on isoelectric focusing, molecular weight determinations, substrate profiles, and inhibition studies, it was concluded that allBacteroides strains examined produced a very similar, if not identical, -lactamase in terms of these enzymatic and physical characteristics.