Use of sialylated or sulfated derivatives and acrylamide copolymers of Galβ1,3GalNAcα- and GalNAcα- to determine the specificities of blood group T- and Tn-specific lectins and the copolymers to measure anti-T and anti-Tn antibody levels in cancer patients

Sialylated or sulfated derivatives and acrylamide copolymers of blood group T-(Gal1,3GalNAc-) and Tn-(GalNAc) haptens were studied for their interaction with the lectins of peanut (PNA),Agaricus bisporus-(ABA),Helix pomatia-(HPA) andVicia villosa B₄-(VVA), using asialo Cowper's gland mucin (ACGM), which contains both T and Tn epitopes, as the coating substrate in enzyme linked lectin assay. Both T and Tn copolymers (40 haptens) showed high affinity and strict specificity; although the T-copolymer at 0.05–0.07 µm concentration caused 50% inhibition of interaction of either PNA or ABA with ACGM, there was little inhibition of the HPA and VVA interactions at over 100 times that concentration. The Tn-copolymer at 0.02–0.05 µm inhibited HPA or VVA interaction with ACGM by 50% but gave virtually no inhibition of PNA and ABA binding. Sialyl, sulfate or methyl group substitution on C-6 of GalNAc of the T-haptene did not prevent interaction with PNA but almost abolished interaction with ABA. In contrast, sialyl or sulfate group on C-6 and sulfate on C-3 of Gal in Gal1,3GalNAc- inhibited almost completely the interaction of PNA with ACGM but had only a slight effect on the interaction of ABA; C-6 substitution with either sialic acid or sulfate on GalNAc- almost abolished the interaction of both HPA and VVA with ACGM. Preliminary studies revealed a significant depression in the serum level of anti-T (two to three-fold decrease) and anti-Tn ( two-fold decrease) antibodies in breast cancer compared with normal control subjects when the acrylamide T- and Tn-copolymers were used as coating substrates in enzyme linked immunoassays.Abbreviations PNA peanut agglutinin - ABA agaricus bisporus agglutinin - HPA helix pomatia agglutinin - VVA (B4),vicia villosa agglutinin - ACGM Asialo Copwer's gland mucin - CA carcinoma - BSA bovine serum albumin - HRP Horseradish peroxidase - ABTS 2,2-azino-di (3-ethyl-benzthiazoline sulfonate) - ELISA enzyme-linked immunosorbent assay - Al allyl - Bn benzyl - AA acrylamide - CP copolymer     

Dihydropteridine reductase variation in man and the characid fish “Cheirodon axelrodi”: Evidence for a dimeric enzyme structure

Summary Genetic evidence for a dimeric structure of dihydropteridine reductase in man and in the fish species Cheirodon axelrodi and Salmo irideus is presented. A single locus in man and two loci in the fishes examined encode this enzyme. Zymograms revealed two alleles for the locus in man and two alleles for each locus in the fish Cheirodon axelrodi. The liver homogenate of a patient with dihydropteridine reductase deficiency showed no detectable activity in the gel, while his parents showed the normal electrophoretic phenotype.     

Einige theoretische Ergebnisse zur pflanzenzüchterischen positiven Massenauslese bei Berücksichtigung von Konkurrenzeffekten

Zusammenfassung Zur Untersuchung der Auswirkung der Parzellenränder-Konkurrenzwirkungen zwischen benachbarten Parzellen in parzellenweise angelegten Feldversuchen wurde eine umfangreiche Feldversuchsserie mit Fichtenkreuzungen verschiedenen Auswertungen unterzogen: 1) Eine Auswertung mit weitgehender Ausschaltung der Konkurrenzeffekte an den Parzellenrändern sowie 2) eine Auswertung ohne explizite Berücksichtigung und Eliminierung der Parzellenränder-Konkurrenzwirkungen (siehe Hühn 1974). Diese Untersuchungen zur quantitativen Einschätzung von Konkurrenzeffekten in Pflanzenbeständen führten schließlich — unter anderem — zu Größenbeziehungen (Ungleichungen) zwischen den phänotypisehen Gesamtvarianzen dieser beiden Auswertungen mit (V*) und ohne (V) Berücksichtigung der Parzellenränder-Konkurrenzwirkungen: V*V sowie zu Größenbeziehungen zwischen verschiedenen Varianzkomponenten, die aus den varianzanalytischen Verrechnungen dieser beiden Auswertungen erhalten wurden.In der vorliegenden Arbeit werden nun unter ausschließlicher Verwendung der Ungleichung V*V interessante theoretische Folgerungen über 1) die Kovarianz zwischen Versuchsglied- und Konkurrenzeffekten und 2) über die Bestimmtheitsmaße bzw. Korrelationskoeffizienten zwischen phänotypischem Wert und Versuchsgliedeffekt sowie zwischen phänotypischem Wert und Konkurrenzeffekt abgeleitet.Zur quantitativen Beschreibung dieser Zusammenhänge erweist sich das Verhältnis f der Konkurrenzvarianz zur Varianz der Versuchsgliedeffekte als besonders geeignet.Im Sonderfall V*=V, der jedoch von einer außerordentlichen praktischen Bedeutung ist, ergibt sich dabei für einen großen Teil des in Frage kommenden Bereiches (0f2) für den Anteil H der Varianz der Versuchsgliedeffekte an der phänotypischen Gesamtvarianz (unter bestimmten Voraussetzungen ist dies also die Heritabilität im weiteren Sinn) eine interessante Deutung als multiple Bestimmtheit R (phänotypischer Wert in Abhängigkeit von Versuchsgliedeffekt und Konkurrenzeffekt), wobei R für diesen Bereich explizit völlig unabhängig von der Größe der Konkurrenzvarianz ist.Im Hauptteil der Arbeit werden dann für diesen praktisch äußerst bedeutsamen Fall V*=V züchterische Anwendungen (positive Massenauslese) diskutiert. Ausgehend von einer einfachen Formel für den Korrelationskoeffizienten zwischen phänotypischem Wert und Versuchsgliedeffekt (wobei die Forderung eines bestimmten zu überschreitenden Mindestwertes c des entsprechenden Bestimmtheitsmaßes als Selektionsbedingung benutzt wird) lassen sich Bedingungen für eine sinnvolle züchterische Anwendung der positiven Massenauslese ableiten und durch quantitative Beziehungen präzisieren.Bei der Formulierung dieser Bedingungen kommt man zu Aussagen über: Mindestwerte für H (in Abhängigkeit von der Anzahl N der zu selektierenden Individuen und c), Angabe von für die positive Massenauslese nicht zulässigen f-Intervallen (in Abhängigkeit von N, H und c, wobei sich ein interessanter Sonderfall für sehr hohe Individuenanzahlen (N ) ergibt) und schließlich die Berechnung der mindestens notwendigen Anzahlen der zu selektierenden Individuen (in Abhängigkeit von f, H und c), wobei hier besonders der unter praktischen züchterischen Gesichtspunkten interessierende Fall: Signifikant von Null verschiedene Korrelation zwischen phänotypischem Wert und Versuchsgliedeffekt diskutiert wird.

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Summary To quantitatively investigate the competitive effects at the borders of neighbouring plots (using field experiments arranged in plots) an extensive series of field experiments, where the treatments were certain crosses of spruce, had been analysed by two different methods: 1) An analysis with almost complete elimination of the competitive effects at the borders of the plots; and 2) an analysis without explicit consideration and elimination of these competition-border-effects (see: Hühn 1974).These studies on estimating competitive effects in plant stands quantitatively finally resulted in relations (inequalities) between the phenotypic total variances of these two methods of analysis, with (V*) and without (V) eliminating the competition-border-effects: V* V. Furthermore, relations between different variance components were obtained from the analysis of variance computations of these two methods. The main purpose of the present paper is to draw some interesting theoretical conclusions, using only the inequality V* V, about 1) the covariance between treatment(genetic)-effects and competitive effects and 2) about the coefficients of determination and correlation-coefficients between the phenotypic values and the treatment-effects and between the phenotypic values and the competitive effects. To describe these relations quantitatively the ratio f of the competitional variance to the variance of the treatment-effects is especially suitable.In the special case V*=V, which, however, has exceptional practical relevance, one obtains — for a large part of the possible interval (0f2) — for the ratio H of the variance of the treatment-effects to the total phenotypic variance (under certain assumptions this is the broad sense heritability), an interesting interpretation as coefficient of multiple determination R (phenotypic value dependent on treatment-effects and competitive-effects). For 0f2 this R is explicitly totally independent of the magnitude of the competitional variance.In the main part of this publication, applications for breeding (positive mass selection) are discussed for the case V*=V, which is of special practical relevance. Starting with a simple formula for the correlation-coefficient between the phenotypic values and the treatment effects, quantitative conditions for a possible application of positive mass selection in breeding are derived; with this the demand of a certain minimal value c of the corresponding coefficient of determination, which must be exceeded, is used as a condition for selection.In the formulation of these conditions one obtains results about: 1) minimal values for H (dependent on c and on the number N of individuals, which must be selected); 2) derivation of f-intervals, where positive mass selection should not be applied (dependent on N, H and c, where an interesting special case arises for very large numbers of individuals (N )); 3) the computation of the necessary minimal number of individuals which must be selected (dependent on f, H and c), where the case: correlation between phenotypic values and treatment-effects significantly different from zero, which is of special interest from the point of view of practical breeding, is discussed in detail.

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Angenommen durch H. Stubbe     

Gonadal structure and P450scc and 3β-HSD immunoreactivity in the gobiid fish Trimma okinawae during bidirectional sex change

The process of sex change in the gobiid fish Trimma okinawae was investigated by gonad histology and immunohistochemistry of two steroidogenic enzymes, P450 cholesterol-side-chain-cleavage (P450scc) and 3-hydroxysteroid dehydrogenase (3-HSD). Irrespective of sexual phase, gonads comprised both ovarian and testicular tissues. Females changed sex to male within 7 days, reverting again to female over an 11-day period. In each sexual phase of the females, the 2nd (2DF-M) and 4th (4DF-M) day after the initiation of sex change to male, the males, and 2nd (2DM-F), 4th (4DM-F), and 6th (6DM-F) days after the initiation of reversion from male to female, histological observations were made. In the ovary during the female, 2DF-M, 4DF-M, and 6DM-F phases, both vitellogenic and previtellogenic oocytes were present, but only previtellogenic oocytes were found in the other phases. The testis contained sperm in all phases, but sperm ducts were not visible in the female phase. In the ovary, P450scc immunoreactivity of interstitial cells was strongly or moderately detected, although weak in the male phase. In contrast, P450scc immunoreactivity in thecal cells was found in all but the male and 2DM-F phases. 3-HSD immunoreactive interstitial cells were detected in all phases, but only weakly so in the male and 2DM-F phases. 3-HSD immunoreactive thecal cells were observed in all stages without the male and 2DM-F and 4DM-F phases. In the testis, moderate P450scc and 3-HSD immunoreactivity was regularly found in the Leydig cells in all the phases. These results suggest that functional steroids including testosterone are produced in any sexual phases.     

Ecological study of fungi in the tidal mud-flats of Kuwait

In the present study, fungal flora of the uncolonized mud flats of Kuwait is investigated. Known isolation techniques, based on different concepts, are used in a trial to distinguish between species present in the active vegetative phase and others existing in the dormant spore phase.According to the frequency of isolation, the fungi reported are classified into four groups showing different existence patterns. Group A fungi; comprised species that most probably occur in the active vegetative phase. Species assigned to this group are: Fusarium oxysporum, Acremonium strictum, Penicillium frequentans A. terricola, Mucor cricinelloides, Rhizopus arrhizus, Stachybotrys atra and Trichoderma koningii. Fungi of group B occur partly in the active vegetative phase and partly in the dormant spore phase whilst members of groups C & D most likely exist only in the spore phase. Reasons for these trends are suggested.     

Single-strand conformational polymorphism and direct sequencing applied to carrier testing in families with ornithine transcarbamylase deficiency

Single-strand conformational polymorphism (SSCP) and direct sequencing were used to confirm or deny carrier status in three families with ornithine transcarbamylase (OTC) enzyme deficiency. Two male probands with late onset OTC deficiency, whose private mutations were previously characterized, inherited the mutations form their heterozygous mothers. One of the heterozygous mothers had a false negative allopurinol test. Three female siblings of the two male probands were tested, one proved to be a carrier of the respective mutation while the other two were found to have normal alleles. In the third family, the proband was a female with late onset presentation of OTC deficiency. We found a new point mutation in this girl consisting of a guanine-tocytosine transversion at nucleotide 520 resulting in a substitution of proline for alanine at amino acid 142 of the mature OTC protein. We confirmed that this mutation occurred spontaneously and that neither of the two parents carries this mutation. We conclude that SSCP, in conjunction with direct sequencing, is a useful technique that can be practically applied for carrier testing in families with OTC deficiency.     

Untersuchungen über die terminale Strombahn im Bereiche der Pars subcapsularis der menschlichen Milz

Zusammenfassung Obwohl die alte Streitfrage der offenen oder geschlossenen Milzblutbahn sehr an Schärfe verloren hat, stehen auch heute noch die arteriellen Endigungen in der Milz im Brennpunkt des Interesses. Mit Injektionsversuchen allein ist dem Problem ebensowenig beizukommen wie mit der üblichen Schnittuntersuchung gespülter Milzen, da in beiden Fällen Artefakte zu befürchten sind. Einen Ausweg aus dieser Situation hat neuerdings die Perjodsäure-Schiff-Reaktion eröffnet: Sie liefert auch ohne Spülung, d. h. bei unverändertem Zellbestand der roten Pulpa, ein angioarchitektonisches Bild der Milz, das an Klarheit dem mit einer Durchspülung erzielten nicht nachsteht (Tischendorf 1956). Auf der Basis dieser methodischen Vorarbeiten untersuchte Verfasser an einem umfangreichen, operativ gewonnenen (Milzruptur, Magen-Karzinom usw.) und lebendfrisch fixierten (Bouin) Material die terminale Strombahn im Bereiche der Pars subcapsularis der menschlichen Milz. Zur Untersuchung (Paraffinschnittserien von 5 , PJS-Reaktion) gelangten nur Organpartien, die keinerlei pathologische Veränderungen aufwiesen. Um Täuschungen über den Gefäßverlauf auszuschließen, wurden die Einzelbefunde jeweils mit Hilfe photographischer Reihenaufnahmen nach dem Vorbild der graphischen Rekonstruktion zu einem Gesamtbefund vereinigt.Nach einleitenden Bemerkungen über die mikroskopische Anatomie der menschlichen Milz im Bilde der PJS-Reaktion beschreibt Verfasser an Hand von Mikrophotogrammen das Verhalten der arteriellen Kapillaren zu den Milzsinus in der Pars subcapsularis, die er (nach der Sinusdichte und Anordnung der Hülsen) in eine Innen-, Zwischen- und Außenzone unterteilt. Die arteriellen Kapillaren verzweigen sich zum Teil schon innerhalb der Hülse und machen auch danach noch bis zu vier Teilungen durch. Das Schema von Weidenreich (1901) verzeichnet nur die erste davon, und es läßt sich nachrechnen, daß Weidenreich die arteriellen Kapillaren nicht in ganzer Länge zu Gesicht bekommen hat. Da die letzten Kapillargabeln in Höhe der Endigungen von Herrlingers Rekonstruktion (1949) liegen, ist die Gesamtzahl der Äste eines Penicillus erheblich größer als bisher angenommen. Bei den in der Literatur als Ampullen, Endkämmerchen oder -kölbchen bezeichneten blinden Kapillarendigungen handelt es sich um Durchspülungsartefakte. Sie treten in der ungespülten Milz nicht auf, sind aber durch eine Spülung willkürlich hervorzurufen. Auch die trichterförmigen freien Kapillarendigungen sind auf die Milzspülung bzw. auf spontane postmortale Veränderungen zurückzuführen. Das Problem der offenen oder geschlossenen Milzblutbahn ist, wie auch die Vitalbeobachtung sinusreicher Nagermilzen (Knisely 1934, 1936 u. a.) zeigt, nicht zuletzt eine Fixierungsfrage.Im PJS-Präparat der ungespülten, lebendfrisch fixierten menschlichen Milz münden die letzten arteriellen Kapillaren unmittelbar ins Sinusnetz. Die Vereinigung mit den Sinus erfolgt meist End zu End, seltener schräg-seitlich. Die Kapillarwand geht im Bereiche des perisinuösen Maschenmantels kontinuierlich und allmählich in die Sinuswand über. Es ist nicht ausgeschlossen, daß in der äußeren Subcapsularis ein Teil der schräg-seitlich in einen Sinus mündenden Kapillaren im Zustand erhöhter Permeabilität vorübergehend auch mit dem Maschengangsystem kommuniziert. Das Pulparetikulum zerfällt anatomisch und funktionell in einen intersinuösen und einen perisinuösen Anteil. Der intersinuöse wird erst agonal oder postmortal durch Auflösung der Kapillarwand zur roten Pulpa, der perisinuöse steht schon intravital zeitweise mit der Sinuslichtung — in Kapselnähe möglicherweise auch mit der Kapillarlichtung — in Verbindung. Zu einer wirklichen freien Endigung arterieller Kapillaren im intersinuösen Pulparetikulum kommt es niemals, auch nicht vorübergehend. Die These, der Milzkreislauf sei strukturell stets offen (funktionell bald offen, bald geschlossen), ist also für die menschliche Milz — und den Sinustyp schlechthin (vgl. Knisely; Peck und Hoerr) — nicht länger aufrechtzuerhalten. Der Normalzustand der menschlichen Milz ist vielmehr die strukturell geschlossene Blutbahn. Die Sinus stellen demgemäß auch nicht den Beginn des Venensystems, sondern das neutrale Bindeglied (vgl. Weidenreich, Herrlinger) zwischen arteriellem und venösem System dar.Ein besonderer Regulationsapparat steuert zugleich mit dem Sinusrhythmus (v. Herrath, Knisely) die Blutverteilung innerhalb der roten Pulpa. Als alternierende Zuflußsperren füngierende Engpässe finden sich vor den Teilungsstellen der Kapillaren, auf dem Wege zum Sinus und gelegentlich auch am Übergang in den Sinus. Die ihnen zugrunde liegende zeitweilige Kapillarverengerung beruht offensichtlich auf Endothelschwellung. Eine direkte Verbindung arterieller Kapillaren mit Pulpavenen im Sinne der capillary shunts von Knisely, Peck und Hoerr konnte Verfasser nicht nachweisen; auch sind die Kapillarhülsen nicht als arteriovenöse Anastomosen aufzufassen. Einen Umgehungskreislauf, durch den Pulpaarterien und -venen vorübergehend kurz geschlossen werden, bringen jedoch die Sinus von Zeit zu Zeit durch den Übergang von der Speicherzur Stromphase zustande. — Die abschließend in einem Schema zusammengefaßten Untersuchungsergebnisse beziehen sich zunächst nur auf die Pars subcapsularis der menschlichen Milz, gelten mit gewissen Abweichungen indessen auch für die Pars interfollicularis. Das Verhalten der Pars perifollicularis bleibt abzuwarten, sehr wahrscheinlich findet sich aber auch hier das Prinzip der strukturell geschlossenen Blutbahn verwirklicht.Herrn Prof. Dr. O. Veit zum 75. Geburtstag gewidmet.Durchgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Production and characterization of antibodies to the β-(1→6)-galactotetraosyl group and their interaction with arabinogalactan-proteins

Rabbit antisera were raised against -(16)-galactotetraose coupled to bovine serum albumin (Gal₄-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of -l-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, -(14)-galactan, and -(13)-galactan, indicating their high specificity toward the consecutive -(16)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immobilized -(16)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (16)-linked -galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal₄-BSA as antigen, which revealed that -(16)-galactotriose and-tetraose were potent inhibitors, while -(13)-or -(14)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or -(16)-galactotetraose.Abbreviations AGP arabinogalactan-protein - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - Gal₃-BSA -(16)-galactotriose coupled to BSA - Gal₄-BSA -(16)-galactotetraose coupled to BSA - Ig immunoglobulin - 4-Me-GlcpA 4-O-methyl-d-glucopyranosyluronic acid - M_r relative molecular mass The authors wish to thank Dr. J. Ohnishi of Department of Biochemistry, Saitama University, for his help in preparing protoplasts.     

Purification and characterization of intestinal adenosine deaminase from mice

Adenosine deaminase (ADA) was isolated from small intestine of mice and purified to utmost homogeneity. SDS-PAGE of purified ADA gave a molecular weight of 41 kDa. Western blot analyses gave a single reactive band at 41 kDa and the other band was an associated ADA binding protein. The purified enzyme was more stable in the alkaline pH. The optimum pH and the pI values were about 7.0 and 4.96, respectively. Km values of the small intestinal ADA for adenosine and 2-deoxyadenosine were 23 and 16M, respectively. Purine riboside was a competitive inhibitor with Ki of 5 M, whereas 2-3-o-isopropylidene adenosine acted as an uncompetitive inhibitor (Ki 66 M). Activity of ADA was inhibited by the presence of theophylline (-40%), caffeine (-30%), and L-cysteine (-50%). Significantly, Hg²⁺ (100 M) inhibited 98% of the initial ADA activity. In addition, various purine analogs such as inosine, purine, -adenosine and adenine showed variable inhibitions on the activity of ADA. Relative ADA activity towards 3-deoxyadenosine and 6-chloropurine riboside was lower by 30% and 40%, respectively. However, the activity towards 2-o-methyl adenosine was higher (30%) compared to the activity obtained using adenosine.     

Use of methicillin and ampicillin mixture as a selective pressure in the cultivation of recombinant E. coli

Summary Ampicillin was rapidly degraded by an extracellular -lactamase, and was therefore ineffective to isolate plasmid-harboring cells in the cultivation of recombinant E. coli for the production of thermostable d-hydantoinase. An effective way of preventing the degradation of ampicillin, methicillin was employed as a -lactamase inhibitor. A mixture of methicillin and ampicillin was observed to effectively function as a selective pressure, and consequently plasmid stability and enzyme productivity of recombinant E. coli were highly maintained.     

Diallel analysis of submergence tolerance in rice,Oryza sativa L.

Summary The genetics of submergence tolerance in rice was studied in a 10 × 10 half-diallel cross set involving 10 lowland rice varieties, four of which were tolerant (FR13A, FR43B, Kurkaruppan, and Goda Heenati) and the remaining six were nontolerant (RD19, IR42, IR17494-32-1, IR19672-24-3, Jagannath, and CR1009). Estimates of genetic parameters following Hayman's method showed significant additive and nonadditive gene action and the latter appeared to be solely due to dominance. Narrow sense heritability (0.70) indicated that additive gene effects were more important in the inheritance of the trait. Tolerance was dominant over nontolerance and the average dominance was within the range of incomplete dominance. Dominant alleles were more concentrated in the three tolerant parents, FR13A, Kurkaruppan, and FR43B in that order. Wr/Vr graphic analysis suggested the involvement of both major and minor genes. Combining ability analysis by Griffing's method also indicated significance of both additive and nonadditive effects, and the former appeared to be more important than the latter. The hybrids involving FR13A with RD19, IR42, and IR17494-32-1, and those of Kurkaruppan with RD19 and CR1009 appeared to be promising for incorporating an adequate level of tolerance to submergence into lowland rice cultivars.     

Variation of glucose 6-phosphate dehydrogenase in the HeLa and Detroit 98 cell lines and their clonal derivatives

Contrary to other reports, we have found that the A type G6PD found in two permanent cell lines—HeLa (Gey), with its single cell clonal derivative HeLa S3, and Detroit 98, with its four clonal derivative lines—is not a single variant but rather at least three different isozymes. One is heat stable with normal specific activity and normal A type electrophoretic migration, another is heat labile with normal specific activity and normal A type electrophoretic migration, and the third is heat labile with reduced specific activity and slightly slow A type electrophoretic migration. We also found that in a mosaic cell population with respect to G6PD phenotype, the predominant G6PD phenotype varied randomly over a 5-month period, that the G6PD phenotype might be mutable in permanent cell lines, and that spontaneous human cell lines might not be HeLa cell contaminants as has been suggested.Aided by the National Institutes of Health General Research Support Grant # 5 S01 FR05507.     

Population studies of the HLA-linked SB antigens

Using allogeneic T-cell recognition we have previously defined five new histocompatibility antigens designated SB antigens. To standardize typing for these antigens, cryopreserved, primed lymphocytes are now used as standard reagents and a technique of cluster analysis has been modified to score typing results objectively. Two primed lymphocyte reagents are used to define each SB antigen; although derived from independent responder-stimulator combinations, the concordance between the reagents is good (r is greater than 0.86). The SB-antigen distribution in a population of 215 normal donors is consistent with Hardy-Weinberg equilibrium of alleles of a single locus. Estimated gene frequencies ranged between 3 percent (SB5) and 36 percent (SB4) with 31 percent blanks. Analysis of association between the SB antigens and A, B, DR antigens in 200 normal donors revealed that associations were generally weak with a few exceptions, in particular, the A1, B8, DR3, SB1 haplotype and also the B7, DR2, SB5 haplotype.Abbreviations MHC major histocompatibility complex - PLT primed lymphocyte typing - SB secondary B cell (antigen)     

In vitro effects of puromycin aminonucleoside on the ultrastructure of rat glomerular podocytes

Summary Puromycin aminonucleoside (PAN)-induced nephrosis in rats provides a model for studying the pathogenesis of severe proteinuric conditions, such as minimal change disease. The present study used scanning (SEM) and transmission (TEM) electron microscopy to investigate the in vitro effects of PAN on rat glomerular podocytes. Slices of rat kidney were incubated for up to 3 days in Medium 199 with Hanks' salts (control) or in medium with PAN. Semiquantitative SEM analysis of glomeruli on the upper surface of kidney slices indicated that incubation with PAN (100 g/ml and 500 g/ml) decreased the number of microvilli on podocyte cell bodies (days 1, 2 and 3), increased the number of glomeruli showing flattening of podocyte cell bodies and major processes (days 2 and 3), and increased the number of glomeruli showing surface membrane blebbing on podocyte foot processes (day 3) (p<0.001 in all cases). TEM morphometry revealed that incubation with 500 g/ml PAN retarded significantly (p<0.001 at days 2 and 3) the loss of podocyte foot processes observed in control cultures. Whilst the SEM changes to podocyte ultrastructure largely mimic those seen in PAN nephrosis in vivo, the retardation of foot process loss runs counter to the major TEM change observed in vivo.     

Hereditary triosephosphate isomerase (TPI) deficiency: two severely affected brothers one with and one without neurological symptoms

A 13-year-old Hungarian boy (B.J. Jr.) with congenital haemolytic anaemia (CHA) and hyperkinetic torsion dyskinesia was found to have severe triose-phosphate isomerase (TPI) deficiency. One of his two brothers (A.J.), a 23-year-old amateur wrestler, has CHA as well, but no neurological symptoms. Both have less than 10% TPI activity and a highly increased dihydroxyacetone phosphate (DHAP) level in their red blood cells. Their TPI had a slow electrophoretic mobility and was heat unstable. Both parents and a third brother are healthy heterozygous carriers of the defect. A.J. represents a unique phenotype from the point of view that all published homozygotes had severe neurological alterations from infancy or early childhood except one infant who died at 11 months, probably too young for neurological symptoms to be noted. In contrast to the two affected Hungarian brothers all but one homozygote has died before the age of 6 years. The striking difference in the clinical course of the defect between the two brothers with the same severe red blood cell enzyme deficiency may originate from unusual differences between two double heterozygous brothers resulting inter alia in different levels of TPI expression in various tissues. Significantly lower TPI activities were found in both the T- and B-cells of the propositus as compared to the respective cells of the neurologically symptom-free brother.     

In vitro culture of Cedrela fissilis Vellozo (Meliaceae)

An efficient micropropagation protocol was developed for Cedrela fissilis (Meliaceae) using nodal segments from juvenile origin for axillary shoot proliferation. Shoot proliferation was significantly affected by salt formulation, explant origin and 6-benzyladenine concentration. Maximum multiplication rates (6–7 new plants were produced in the second subculture cycle per single cotyledonary node cutting) were achieved on Murashige and Skoog media supplemented with 1.25–5.0 M 6-benzyladenine. Addition of -naphthaleneacetic acid to these media caused significant inhibition on shoot proliferation and growth and stimulated callus formation. High frequency callus initiation and synergistic effects on callus growth were achieved on Murashige and Skoog medium supplemented with 6-benzyladenine at either 1.25, 2.5 or 5.0 M combined, respectively, with 2.5, 1.25–5.0 or 5.0 M -naphthaleneacetic acid. Rooting was achieved, after 10–12 days, with 87–100% of the node cuttings on half strength Murashige and Skoog medium either without growth regulators or supplemented with 2.5 M indole-3-butyric acid. Regenerated plants were successfully acclimatized on sterilized sand, for 21 days, but for further plant development the sand:soil (1:1) mixture was the best substrate. The survival rate of plantlets under ex vitro conditions was 100% after 3 months. The optimized micropropagation and callus culture protocols offer the possibility to use the organ/cell culture techniques for vegetative propagation, cryopreservation and secondary metabolism studies.     

Sibling Cannibalism of Young Red Drum, Sciaenops ocellatus, in Relation to Size Disparity and Metabolic Rates

Cannibalism is a leading cause of young mortality in the red drum, Sciaenops ocellatus, culture, and size disparity is its primary causative factor. Cannibalism did not occur in young fish, graded by a difference of 0.5cm from 2.0 to 4.5cm in total length. There was a shift in the size distribution from unimodal (normal) when there was no cannibalism, to bimodal or trimodal when cannibalism occurred. The results suggested that the wider was the size disparity, the greater was the sibling cannibalism. Size disparity increased with growth and in turn with sibling cannibalism, which became insignificant when the young grew to a size of about 6cm. We present threshold levels of size disparity among siblings to prevent mutual cannibalism. We hypothesize that through sibling cannibalism a hierarchy may evolve in a red drum cohort, at which a stable population will form.     

On the role of enzyme kinetic parameters in determining the effectiveness with which channelling can decrease the size of a metabolite pool

Recently, it has been argued that the phenomenon of direct transfer of intermediate metabolites between adjacent enzymes, also known as metabolic channelling, would not decrease the concentration of those intermediates in the bulk solution. However, this conclusion has been drawn by extrapolation from the results of simulations with a rather restricted set of parameters. We show that, for a number of kinetic cases, the existence of metabolic channelling can decrease the size of the soluble pool of intermediates. When the enzyme(s) downstream of the channel have a catalytic capacity that is large relative to the enzymes upstream of the channel, the decrease of concentration can be substantial (3 orders of magnitude).     

One haplotype is associated with the Swiss type of hereditary persistence of fetal hemoglobin in the Yugoslavian population

Summary Blood samples from normal adults and from members of seven families with the Swiss type of hereditary persistence of fetal hemoglobin (HPFH) from Yugoslavia were analyzed for their fetal hemoglobin (Hb F) and ^G levels, while haplotyping defined the chromosomes at eight or nine polymorphic restriction sites. The data indicate that Swiss-HPFH, characterized by slightly elevated Hb F and ^G levels and no recognizable hematological abnormality, is associated with a chromosome whose restriction enzyme haplotype is identical to the no. 3 (Senegal) haplotype found in black sickle cell (SS) patients. Many adults with this chromosome have high ^G but normal Hb F levels. It is suggested that the Swiss-HPFH phenotype results from the action of more than one factor; one is linked to the -globin gene cluster and causes high ^G values, while others result in an increased Hb F production and are perhaps of different origins.     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography