Synthesis of N-linked pentasaccharides with isomeric glycosidic linkage

As part of a program to explore the structural requirement of N-glycans in the carbohydrate-mediated biological interactions, N-linked pentasaccharide core structure was stereochemically modified in terms of glycosidic linkage. Three isomers, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, and -D-Man-(13)-[-D-man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, were synthesized. Synthesis of the pentasaccharide with natural linkage is also described.     

Ultrastructure of four types of striated muscle fibers in the atlantic hagfish (Myxine glutinosa,L.)

Summary Four types of striated muscle fibers with distinctive ultrastructure were defined in the Atlantic hagfish (Myxine glutinosa, L.): white, intermediate, and red fibers of m. parietalis, and red fibers of m. craniovelaris.White fibers are thick, contain very few mitochondria and fat vacuoles, and possess distinct and separate myofibrils with thin Z-disks and distinct M-lines. Intermediate fibers are thinner, possess largely similar myofibrils that often are even better separated due to a higher content of fat vacuoles and especially mitochondria and glycogen granules. Red fibers of m. parietalis contain large amounts of mitochondria, fat vacuoles, and glycogen granules. Their myofibrils possess M-lines, and although branching more, the myofibrils of red fibers conform with a Fibrillenstruktur pattern like those of white and intermediate fibers. Red fibers of m. craniovelaris are very thin, possess many smaller fat vacuoles, and large amounts of mitochondria and glycogen granules. The myofibrils are significantly thinner than in m. parietalis fibers, run as quite independent well separated units, possess thicker Z-disks, and lack M-lines. Large amounts of myosatellite cells are associated with these red fibers.Triads are located near A/I-junctions in all four fiber types and occur irregularly, the density of triads being different in the various fiber types.We are indebted to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supply of hagfishes, and we also wish to thank Dr. Jan K. S. Jansen, Institute of Physiology, University of Oslo, for valuable suggestions during this study.     

Evolutionary origin of the class A and class C β-lactamases

Summary The protein sequences of 18 class A -lactamases and 2 class C -lactamases were analyzed to produce a rooted phylogenetic tree using the DD peptidase of Streptomyces R61 as an outgroup. This tree supports the penicillin-binding proteins as the most likely candidate for the ancestoral origin of the class A and class C -lactamases, these proteins diverging from a common evolutionary origin close to the DD peptidase. The actinomycetes are clearly shown as the origin of the class A -lactamases found in other non-actinomycete species. The tree also divides the -lactamases from the Streptomyces into two subgroups. One subgroup is closer to the DD peptidase root. The other Streptomyces subgroup shares a common branch point with the rest of the class A -lactamases, showing this subgroup as the origin of the non-actinomycete class A -lactamases. The non-actinomycete class A -lactamase phylogenetic tree suggests a spread of these -lactamases by horizontal transfer from the Streptomyces into the non-actinomycete gram-positive bacteria and thence into the gram-negative bacteria. The phylogenetic tree of the Streptomyces class A -lactamases supports the possibility that horizontal transfer of class A -lactamases occurred within the Streptomyces.     

Localization of blood-group-related linear poly-N-acetyllactosamine structure in different human tissues by Griffonia simplicifolia agglutinin-II staining following endo-β-galactosidase digestion

Summary Endo--galactosidase from Escherichia freundii cleaves polylactosaminyl structures as follows: R-GlcNAc1-3Gal1-4GlcNAc1-R + H₂O R-GlcNAc1–3Gal + GlcNAc1-R. By staining with Griffonia simplicifolia agglutinin-II following the enzyme digestion, the distribution of R-GlcNAc1–3Gal1–4GlcNAc can be demonstrated in tissue sections. This carbohydrate chain is one of the backbone structures carrying the blood-group-related antigens and, thus, localization of this structure may provide detailed information about the distribution of variants with different backbone structures. Various formalin-fixed, paraffin-embedded tissue sections were stained by Griffonia simplicifolia agglutinin-II with or without prior enzyme digestion and the reactivity of the agglutinin imparted by enzyme digestion was studied in the following tissues and cells: pancreatic acinar cells, gastric surface mucosae, duct cells and mucous cells of salivary glands and tracheal glands, surface epithelium of trachea, goblet cells of large intestine, columnar epithelium of uterine cervical glands, distal and collecting tubules of kidney, certain cells of anterior lobe and colloid of middle lobe of pituitary glands, epithelial reticular cells and Hassall's corpuscles of thymus and Kupffer cells of liver. In gastric surface mucosae, the reactivity of the agglutinin appeared in non-secretor individuals but not in the secretor individuals, and in mucous cells of salivary and tracheal glands the reactivity appeared in Le(a - b -) non-secretor individuals but not in Le(a + b -) non-secretor or secretor individuals. In pancreatic acinar cells and duct cells of salivary glands from fetuses and newborn infants, prior fucosidase digestion markedly enhanced the Griffonia simplicifolia agglutinin-II reactivity elicited by endo--galactosidase digestion. Prior fucosidase digestion was also a prerequisite for revealing the reactivity of this agglutinin by endo--galactosidase digestion in gastric surface mucosae from secretor individuals. -Galactosidase digestion disclosed reactivity of this agglutinin in pancreatic acinar cells and duct cells of salivary glands even after the removal of endo--galactosidase-labile lactosamine structures by sequential digestion with endo--galactosidase and -N-acetylhexosaminidase. These results demonstrate that the procedures developed in this study provide a useful means for detecting different types of lactosamine structures which carry blood-group antigens in humans tissues.     

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.     

Interactions of Aβ(1–40) with Glycerophosphocholine and Intact Erythrocyte Membranes: Fluorescence and Circular Dichroism Studies

Deposition of amyloid peptide in human brain in the form of senile plaques is a neuropathological hallmark of Alzheimers disease (AD). Levels of a phospholipid breakdown product, glycerophosphocholine (GPC), also increase in AD brain. The effect of GPC on amyloid (1–40) peptide (A) aggregation in PBS buffer was investigated by circular dichroism and fluoresence spectroscopy; interactions of A and GPC with the intact erythrocyte membrane was examined by fluoresence spectroscopy. Fluorescamine labeled A studies indicate GPC enhances A aggregation. CD spectroscopy reveals that A in the presence of GPC adopts 14% more -sheet structure than does A alone. Fluorescamine anisotropy measurements show that GPC and A interact in the phospholipid head-group region of the erythrocyte membrane. In summary, both soluble A and GPC insert into the phospholipid head-group region of the membrane where they interact leading to -sheet formation in soluble A which enhances A aggregation.     

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (_m), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of _m may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of _m may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of _m has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of _m early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of _m may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect _m. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in _m.     

Endocrine cells in the human fetal small intestine

Summary In this report we describe the time of appearance and ultrastructural features of enteroendocrine cells (EECs) in the human fetal small intestine (SB) between 9 and 22 weeks gestation. Thirteen distinctive EECs were identified in fetal SB. Two of these, not found in normal adult SB, appeared within the stratified epithelium of the proximal SB at 9–10 weeks. They were arbitrarily termed primitive and precursor cells. As in all fetal EECs, the pale cytoplasm of the primitive cell contains a distinctive population of secretory granules (SGs). Primitive cell SGs average 200–330 nm; some have dense cores with lucent halos while others are filled with a homogeneous dense or flocculent material. The SGs of the precursor cells are larger, averaging up to 1 m in diameter and their contents vary in electron density. A third group of cells not described in normal adult SB was arbitrarily termed transitional cells. These have two populations of SGs; one resembles the SGs of the precursor cells, and the other resembles the SGs of some of the specific adult type EECs. Transitional EC, S, I and G cells are seen. In addition, mature appearing EC, S, G, I, L, D, and D₁ cells were identified by 12 weeks of gestation. The primitive, precursor, and transitional cells may represent sequential developmental precursors of adult type EECs.Supported by Research Grant AM-17537 from the National Institutes of Health, Besthesda, MarylandThe authors would like to thank Ms. Linda Barstein for her excellent technical assistance     

Consequences of harvesting for genetic diversity in American ginseng (<Emphasis Type="Italic">Panax quinquefolius</Emphasis> L.): a simulation study

American ginseng, Panax quinquefolius L., is one of the most heavily traded medicinal plants in North America. The effect of harvest on genetic diversity in ginseng was measured with a single generation culling simulation program. Culling scenarios included random harvest at varying levels, legal limit random harvest and legal limit mature plant harvest. The legal limit was determined by the proportion of legally harvestable plants per population (% mature plants per population). Random harvest at varying levels resulted in significant loss of genetic diversity, especially allelic richness. Relative to initial levels, average within-population genetic diversity (H _e) was significantly lower when plants were culled randomly at the legal limit (Mann–Whitney U=430, p<0.001) or when only mature plants were culled (Mann–Whitney U=394, p<0.01). Within-population genetic diversity was significantly higher with legal limit mature plant harvest (H _e=0.068) than when plants were culled randomly at the legal limit (H _e=0.064; U=202, p<0.01). Based on these simulations of harvest over one generation, we recommend that harvesting fewer than the proportion of mature plants could reduce the negative genetic effects of harvest on ginseng populations.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Opioid and Cannabinoid Receptors Share a Common Pool of GTP-Binding Proteins in Cotransfected Cells, But Not in Cells Which Endogenously Coexpress the Receptors

1. Opioid (, , ) and cannabinoid (CB1, CB2) receptors are coupled mainly toG_i/G_o GTP-binding proteins. The goal of the present study was to determine whether different subtypes of opioid and cannabinoid receptors, when coexpressed in the same cell, share a common reservoir, or utilize different pools, of G proteins.2. The stimulation of [³⁵S]GTPS binding by selective opioid and cannabinoid agonists was tested in transiently transfected COS-7 cells, as well as in neuroblastoma cell lines. In COS-7 cells, cotransfection of - and -opioid receptors led to stimulation of [³⁵S]GTPS binding by either -selective (DAMGO) or -selective (DPDPE) agonists. The combined effect of the two agonists was similar to the effect of either DAMGO or DPDPE alone, suggesting the activation of a common G-protein reservoir by the two receptor subtypes.3. The same phenomenon was observed when COS-7 cells were cotransfected with CB1 cannabinoid receptors and either - or -opioid receptors.4. On the other hand, in N18TG2 neuroblastoma cells, which endogenously coexpress CB1 and -opioid receptors, as well as in SK-N-SH neuroblastoma cells, which coexpress - and -opioid receptors, the combined effects of the various agonists (the selective cannabinoid DALN and the selective opioids DPDPE and DAMGO) were additive, implying the activation of different pools of G proteins by each receptorsubtype.5. These results suggest a fundamental difference between native and artificially transfected cells regarding the compartmentalization of receptors and GTP-binding proteins.     

The effects of collembola grazing on microbial activity in decomposing leaf litter

Summary Ground leaf litter was inoculated with the fungus Coriolus versicolor and incubated in respirometers for 6 days (fresh cultures) or 33 days (senescent cultures) before different number of Folsomia candida were added. Grazing by 5 animals stimulated O₂ consumption in both series of cultures but 10, 15 or 20 animals inhibited microbial respiration. The stimulatory effect was less marked in the senescent cultures. Bacterial and fungal standing crops increased in fresh cultures during the course of the experiment but grazing by collembola increased bacterial and reduced fungal standing, crops in proportion to the grazing intensity. Microbial standing crops were not determined for senescent cultures. Microarthropod feeding activities can therefore exert a strong differential effect on fungal and bacterial populations which has not been previously recognised.     

Peptide des β-Alanins als Ersatz für die freie Aminosäure bei pantothensäurebedürftigen Hefezellen und ihr Verhalten gegenüber dem β-Alanin-Antagonisten Asparagin

Zusammenfassung Pantothensäurebedürftige Hefezellen können ihren Bedarf an diesem Vitamin nicht allein aus -Alanin decken, sondern auch aus Benzoyl--Alanin, -Alanyl-d,l-Norleucin und -Alanyl-l-Histidin. Der Antagonist Asparagin hemmt die Verwertung dieser Peptide genauso wie diejenige der freien Aminosäure. Durch höhere Konzentrationen an -Alanin oder -Alanyl-d,l-Norleucin läßt sich die Hemmwirkung nicht allein kompensieren, es kommt sogar zu einer Förderung des Hefewachstums. Der Antagonist wird dann zum Synergisten.

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Summary The -alanine containing peptides benzoyl--alanine, -alanyl-d,l-norleucine and -alanyl-l-histidine can substitute for the amino acid -alanine in a pantothenic acid requiring yeast. Asparagine, an antagonist of -alanine, affects these peptides in a similar manner. In combination with an overdose of -alanine or -alanyl-d,l-norleucine, asparagine is no longer an antagonist but becomes a synergist.

     

The inheritance of radiation induced semi-sterility in Rhodnius prolixus

Eggs from crosses of 40 adult male R. prolixus irradiated with 6K rad -rays with normal females had a mean fertility of 23.9%, only 2 crosses being completely sterile. The 86 F₁ progeny of both sexes, when outcrossed with normal mates, had a mean egg fertility of 12.6%, and 43 of these matings were completely sterile. Twenty-eight F₂ bugs reared from F₁ x normal crosses were mated with normal partners and had a mean fertility of 44.6%, 6 of them being fully fertile, a reversal towards normal fertility. Cytogenetic examination of F₁ F₂ and F₃ males showed that these changes in fertility correlated well with the degree of chromosomal abnormality found. The very high recovery rate of translocations in F₁ generation males can be related to the holocentric chromosomes of these bugs which precludes the formation of dicentric chromosomes which are inviable in monocentric species. In F₁ and F₂ males the majority of translocations were associated as chains of III+I or as chains of IV. Only one bug was found with a ring of IV chromosome association and it is suggested that chromosome morphology, combined with a low chiasma frequency, favours chain association. Most chain multivalents showed linear orientation which may lead to duplication deficiencies and zygotic death. However parallel, indifferent and the more stable convergent modes of chain orientation were also all observed indicating that survival of some translocations in this species may be possible. The survival to the F₂ generation of chromosomal fragments confirmed the holocentric nature of triatomine chromosomes. It is suggested that semi-sterile males would prove more effective than releases of completely sterile males for reducing wild populations of R. prolixus, because of the delayed effects of sterilizing radiation consequent upon the holocentric structure of triatomine chromosomes.     

The cytology of phytophthora infestans

Summary Phytophthora infestans has three kinds of somatic nuclei: an oval shaped nucleus (approx. 3.1×2.7 ) which stains diffusely except for a crescent shaped Feulgen positive cap which stains intensely; a granular nucleus whose contents are organized into a fairly constant number of stained bodies, and, a deeply staining condensed nucleus. The capped nucleus is thought to be metabolic or resting and the granular nucleus is thought to be dividing as it is most commonly found in hyphal tips. Attenuated forms of all three kinds of nuclei are found.Nuclear division is mitotic and intranuclear. Eight—ten chromosomes are seen at metaphase.Sporangia have a mean of 6.3 nuclei which is constant for age and strain of culture. Sporangia become multinucleate as a result of nuclear migration and not by division in the developing sporangium. Zoospores are usually uninucleate.The nuclear cap is persistent throughout nuclear division when it also divides. It is associated with flagella production and nuclear migration and has some of the properties of a blepharoplast.     

MHC class II sequences of an HLA-DR2 narcoleptic

Narcolepsy has a 98% association with the DR2-Dw2/DQw1 haplotype. To establish if a disease-specific allele is present in narcolepsy, a cDNA library was made from a B-cell line from a DR2,4/DQw1,3 narcoleptic. Clones encoding the two expressed DR2 chains, along with DQw1 and chains, were isolated and completely sequenced. The coding regions of these four genes were similar to published nucleotide and protein sequences from corresponding healthy controls, with some minor exceptions. The 3 untranslated region of one of the DR2 genes in the narcoleptic was extended by 42 bp. Complete sequences were not available for DQw1.2 or from healthy individuals, but first domain nucleotide sequences showed only a single nonproductive difference in DQ. Partial protein sequences of both DQ and from published data were identical. Although the effects of minor differences cannot be ruled out completely, it is concluded that there are probably no narcolepsy-specific DR or DQ / sequences, and that the alleles found in narcolepsy are representative of those found in the healthy population.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Unique sequences for two HLA-DRB1 genes expressed on distinct DRw6 haplotypes

We have used restriction fragment length polymorphism (RFLP) analysis and DNA sequencing to characterize two distinct DRB1 alleles expressed on DRw52 and DQw7-associated haplotypes but not readily defined by conventional DR serology. These two haplotypes, designated HLA-D HAG and PEV, react variably with DRw13(w6), DRw14(w6), and the more broad DR 3+6 antisera. Analysis of RFLP revealed that HLA-D HAG and PEV are associated with different DRw52 variants, and that HAG is indistinguishable from DRw18(3) haplotypes. Sequencing of the HAG and PEV DRB1 genes showed each to represent novel alleles. Nevertheless, these sequences show similarities with the other alleles of the DR5, w6, and w8 family. HAG (DRB1*1303) appears to have arisen either from two recombinational events involving at least three DRB1 sequences (DRB1*1101, DRB1*0803, DRB1*0401) or from a single recombinational event together with multiple point mutational events. PEV appears to represent a DRB1*1301-1302/DRB1*1101 recombinant allele, with recombination having occured in the region of bases 175 – 198. The results of this study suggest that the DRw52 family haplotypes is derived from a relatively restricted number of ancestral sequences, with diversity among DRB1 alleles within this family arising through gene conversion or recombination events.     

Trophic Assemblages of the Rocky Intertidal Zone in Vostok Bay,Sea of Japan

The ratios between the indices of relative abundance for different trophic life forms have been used to characterize bionomical types of a rocky intertidal zone. It has been shown that the distribution of life forms is determined by the geomorphological peculiarities of the surveyed intertidal areas. A brief critical historical review has been provided of the terms bionomy and bionomical type.     

Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin a: Preferential inhibition of glycation by nucleophiles at sites of low isomerization potential

The relative roles of the two structural aspects of nonenzymic glycation sites of hemoglobin A, namely the ease with which the amino groups could form the aldimine adducts and the propensity of the microenvironments of the respective aldimines to facilitate the Amadori rearrangement, in dictating the site selectivity of nonenzymic glycation with aldotriose has been investigated. The chemical reactivity of the amino groups of hemoglobin A forin vitro reductive glycation with aldotriose is distinct from that in the nonreductive mode. The reactivity of amino groups of hemoglobin A toward reductive glycation (i.e., propensity for aldimine formation) decreases in the order Val-1(), Val-1(), Lys-66(), Lys-61(), and Lys-16(). The overall reactivity of hemoglobin A toward nonreductive glycation decreased in the order Lys-16(), Val-1(), Lys-66(), Lys-82(), Lys-61(), and Val-1(). Since the aldimine is the common intermediate for both the reductive and nonreductive modification, the differential selectivity of protein for the two modes of glycation is clearly a reflection of the propensity of the microenvironments of nonenzymic glycation sites to facilitate the isomerization reaction (i.e., Amadori rearrangement). A semiquantitative estimate of this propensity of the microenvironment of the nonenzymic glycation sites has been obtained by comparing the nonreductive (nonenzymic) and reductive modification at individual glycation sites. The microenvironment of Lys-16() is very efficient in facilitating the rearrangement and the relative efficiency decreases in the order Lys-16(), Lys-82(), Lys-66(), Lys-61(), Val-1(), and Val-1(). The propensity of the microenvironment of Lys-16() to facilitate the Amadori rearrangement of the aldimine is about three orders of magnitude higher than that of Val-1() and is about 50 times higher than that of Val-1(). The extent of nonenzymic glycation at the individual sites is modulated by various factors, such as thepH, concentration of aldotriose, and the concentration of the protein. The nucleophiles—such as tris, glycine ethyl ester, and amino guanidine—inhibit the glycation by trapping the aldotriose. The nonenzymic glycation inhibitory power of nucleophile is directly related to its propensity to form aldimine. Thus, the extent of inhibition of nonenzymic glycation at a given site by a nucleophile directly reflects the relative role ofpK _a of the site in dictating the glycation at that site. The nonenzymic glycation of an amino group of a protein is an additive/synergestic consequence of the propensity of the site to form aldimine adducts on one hand, and the propensity of its microenvironment to facilitate the isomerization of the aldimines to ketoamines on the other. The isomerization potential of microenvironment plays the dominant role in dictating the site specificity of the nonenzymic glycation of proteins.