Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extration was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25–30 mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level ofN-acetylglucosaminlytransferase IV activity in CHO cell extracts.Abbreviations PA 2-aminopyridine - SDS sodium dodecyl sulfate - GlcNAc N-acetylglucosamine - GnT N-acetylglucosaminyltransferase - Gn,Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-tri-PA GlcNAc1-2(GlcNAc1-4)Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-trí-PA GlcNAc1-2Man1-3({GlcNAc1-2(GlcNAc1-6)Man1-6})Man1-4GlcNac1-4GlcNAc-2-aminopyridine - Gn,(Gn),Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-4)(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine     

Partial trisomy 4 resulting from a complex maternal rearrangement of chromosomes 2, 4, and 18 with interstitial translocation

Summary A woman presented a complex chromosome rearrangement with translocation between chromosome 2 and 4 in addition to an insertion of the band 4q12q13 in the long arm of chromosome 18. The authors present a case study of the daughter who displayed the abnormal chromosome 18 and trisomy of band 4q12q13.     

Full trisomy 7 and Potter syndrome

Summary A patient with typical Potter's syndrome and full trisomy 7 is described. All previous reports on chromosome 7 abnormalities, whether monosomic or trisomic, p or q, are reviewed and discussed, establishing two 7q trisomy snydromes: 7q227q31 and 7q22, q317qter. Some implications of the finding of full trisomy 7 in a case of Potter's syndrome are discussed.     

Partial trisomy 18q in a newborn with typical 18 trisomy phenotype

Summary This paper describes a case of partial trisomy of almost the entire long arm of chromosome 18 in a newborn with classic trisomy-18 phenotype, resulting from a de novo unbalanced 18q/21p translocation: karyotype: 46,XX,-21,t(18;21)(18qter18q11::21p1221qter). A review of the other reported cases of partial trisomy 18 suggests that a critical segment in chromosome 18, corresponding to bands q11-q12, might be responsible for most of the signs of trisomy 18, including failure to thrive and early death.     

Assignment of human phosphoribosylglycinamide synthetase locus to region 21q221

Summary The enzymatic activity of phosphoribosylglycinamide synthetase (GARS) has been studied in several cases of partial monosomies and full and partial trisomies 21. An excess of GARS activity was found in regular trisomy 21 with a trisomy 21/normal ratio equal to 1.55. A 0.99 ratio was found in 21q2121pter monosomy; a 0.54 ratio was found in 21qter21q22 monosomy; a 0.88 ratio, in 21q2121pter trisomy, and a 1.46 ratio, in 21q22.1 trisomy. Consequently, the GARS gene locus, assigned to chromosome 21, could be localized in subband 21q22.1.     

SOD-A and chromosome 21

Summary A balanced maternal chromosome translocation (9p24;21q214) resulted in two offspring with unbalanced karyotypes. One of these, a girl trisomic for both segment 9pter9p24 and segment 21pter21q214, was found to have a SOD-A activity not significantly different from those found in a group of five cases with trisomy 21. However, clinical evaluation of this girl revealed no symptoms of the Down syndrome. These findings suggest that, providing the gene dosage theory is correct, the gene for SOD-A is probably localized on chromosome 21 proximal to, or in, band q21.     

Disomic homozygosity in 21-trisomic cells: a mechanism responsible for transient myeloproliferative syndrome

Summary Nine patients with transient myeloproliferative syndrome (TMS) with or without Down syndrome (DS) phenotype were studied cytogenetically, particularly with regard to the origin of trisomy 21. Of six DS patients, five had standard trisomy 21 and one a mosaic consisting of 21-tetrasomic, trisomic and disomic cell lines. The other three non-DS patients were mosaics with both 21-trisomic and-disomic cell lines. In all nine patients, the leukemoid cells in TMS stage were largely or exclusively composed of trisomy or tetrasomy 21, an indiction that the additional chromosome(s) 21 plays an important role in the occurrence of TMS. Sequential Q-and R-banding analysis of heteromorphisms demonstrated that all these patients had a duplication of a chromosome 21, as revealed by an aab pattern, regardless of DS or normal phenotype or parental origin of the extra chromosome 21. Irrespective of the possibility of recombination, the aa chromosomes are homozygous, i.e. show disomic homozygosity: this may in turn result in the duplication of a gene that controls the proliferation of the myelogenous cells, thereby leading to TMS.     

Production of dodecaploid plants of Japanese persimmon (Diospyros kaki L.) by colchicine treatment of protoplasts

Summary Dodecaploid plants of Japanese persimmon (Diospyros kaki L.) were obtained by colchicine treatment of protoplasts. Callus protoplasts of Jiro (2n=90, x=15) were cultured in modified KM8p medium with 0.1% colchicine for 3–9 days. After colchicine treatment, they were cultured using agarose bead culture. Microcalli were recovered from the protoplasts after 3 months. Flow cytometric measurement showed that nine of 31 callus lines obtained from 6 days of colchicine treatment had twice the nuclear DNA content as non-treated controls. Plantlets were regenerated from the calli with twice the nuclear DNA content. Microscopic observation of root tip cells showed that their somatic chromosome number was 2n=180 (x=15). Compared with Jiro, dodecaploid plants had longer stomatal guard cells and lower stomatal densities, consistent with increased ploidy.     

A deletion in chromosome 22 can cause digeorge syndrome

Summary An association between DiGeorge's syndrome and an unbalanced chromosomal rearrangement leading to trisomy 20pter20q11 and monosomy 22pter22q11 was found in four individuals belonging to one family. These and other data from the literature are interpreted to suggest that DiGeorge's syndrome can be caused by deletion of a gene located in chromosome 22, probably in band 22q11.     

Die Bedeutung der “feedback”-Hemmung der Threonin-Desaminase für die Synthese von Valin und Leucin

Zusammenfassung In Arthrobacter Stamm 23 führte die durch Mutation verursachte allosterische Unempfindlichkeit der Threonin-Desaminase zur dereprimierten Bildung der Enzyme im Isoleucin-Valin-Leucin-Biosyntheseweg. Derepression erfolgte auch, wenn Wildtypzellen in Gegenwart von -Ketobuttersäure inkubiert wurden. In beiden Fällen wurde Isoleucin überproduziert und ins Kulturmedium ausgeschieden. Wie aus Wachstumsexperimenten hervorging, verursachte der Überschuß an -Ketobuttersäure im Medium primär einen Valin- und Leucin-Mangel, der zu einer vorübergehenden Wachstumshemmung führte. Durch die dereprimierte Bildung der Enzyme im Isoleucin-Valin-Biosyntheseweg konnte die Wachstumshemmung überwunden werden.Der vorübergehende Hemmeffekt der -Ketobuttersäure ließ sich auf eine Konkurrenz der Substrate am ersten gemeinsamen Enzym im Isoleucin-Valin-Biosyntheseweg, der Acetohydroxysäure-Synthase, zurückführen. Wegen des niedrigen K _m-Wertes für -Ketobuttersäure wird dieses Substrat vom Enzym bevorzugt umgesetzt. Durch gaschromatographische Bestimmungen der Acetoin- und Acetyläthylcarbinol-Bildung in Enzymtests mit variierten Substrat-Konzentrationen konnte nachgewiesen werden, daß relativ geringe Konzentrationen an -Ketobuttersäure genügen, um die -Acetolacetat-Bildung vollständig zu unterdrücken. Diese Ergebnisse erklären die durch -Ketobuttersäure verursachte vorübergehende Wachstumshemmung bei Bakterien.

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The effect of the feedback inhibition of threonine deaminase on valine-leucine biosynthesis

In Arthrobacter strain 23 the allosteric insensitivity of threonin deaminase caused by mutation resulted in derepressed formation of the enzymes of the isoleucine-valine-leucine pathway. Derepression was also observed, when wild type cells were incubated in the presence of -oxobutyrate. In both cases isoleucine was overproduced and excreted. As growth experiments indicated the excess of -oxobutyrate in the medium caused endogenous valine and leucine deficiency and a transient inhibition of growth. Derepressed formation of the isoleucinevaline biosynthetic enzymes resulted in relief of growth inhibition.The transient inhibitory effect of -oxobutyrate has been traced back to substrate competition at the first enzyme common to the isoleucine and valine pathway, acetohydroxy acid synthase. Due to the low K _m of the enzyme for -oxobutyrate this substrate is preferentially converted. As proven by gaschromatographical measurements of acetoin and acetylethyl carbinol produced in enzyme (acetohydroxy acid synthase) assays with varied substrate concentrations, relatively low concentrations of -oxobutyrate are able to suppress the formation of -acetolactate completely. These results explain the transient inhibitory effect of -oxobutyrate on the growth of bacteria.

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Abkürzungen -KBS -Ketobuttersäure - FAD Flavin-adenin-dinucleotid - AHS Acetohydroxysäure - IPM Isopropylmalat - TPP Thiaminpyrophosphat     

Trisomy 16q23→qter arising from a maternal t(13;16)(p12;q23): case report and evidence of the reciprocal balanced maternal rearrangement by the Ag-NOR technique

Summary We describe a female new-born with partial trisomy of the long arm of chromosome 16. The chromosome anomaly was the result of an unbalanced segregation of a maternal translocation t(13;16)(p12;q23). Dynamic (RBG, GBG) banding and the Ag-NOR technique ascertained the reciprocal balanced maternal translocation between the 16q23qter and 13q12pter segments because nucleolar organizers were present on the tip of long arms of the derivative 16 maternal chromosome. As monosomy 13p has little or no deleterious effect we consider our case as exhibiting the phenotype of trisomy 16q23qter free from any monosomic feature. Clinical effects are of less consequence as compared with previously published cases of partial trisomy 16q.     

Linear differentiation of cereal chromosomes

Summary The BSG test was used in a comparative study of the linear chromosome differentiation and the idiograms of T. Macha ssp. tubalicum v. letschchumicum Dek. et Men., T. georgicum Dek., T. timopheevi. Zhuk., T. carthlicum Nevski, T. dicoccum Schrank, v. rufum, T. durum Desf. v. Arnautka were compiled.The karyotype of each polyploid wheat species consists of two groups of chromosomes. The first is formed by ten pairs of constant chromosomes occurring almost in all species and the second by all the rest of the variable chromosomes that are either fully specific for the species in question or occur only in a few species. T. timopheevi largely differs from other species of polyploid wheats in the high level and specific localization of structural heterochromatin on chromosomes. The rols of introgression in wheat evolution and the necessity of establishing a General Cytological Nomenclature of Cereal Chromosomes are discussed.     

Adjacent 2 meiotic disjunction. Report of a case resulting from a familial 13q;15q balanced reciprocal translocation and review of the literature

Summary An abnormal short-lived female infant with almost complete trisomy 13 (pterq32 or 33) and partial monosomy 15 (pterq14 or 15) resulting from an adjacent 2 meiotic disjunction of a paternal reciprocal translocation is described. Cases with monosomy of chromosome 15 material are reviewed. It appears likely that monosomy of an interstitial long arm segment, approximating to 15q2124, imparts the lethality associated with the full monosomic condition. Adjacent 2 disjunction in man has been further characterised by reviewing the literature.     

Production of biologically active hirudin in plant seeds using oleosin partitioning

A plant oleosin was used as a carrier for the production of the leech anticoagulant protein, hirudin (variant 2). The oleosin-hirudin fusion protein was expressed and accumulated in seeds. Seed-specific expression of the oleosin-hirudin fusion mRNA was directed via an Arabidopsis oleosin promoter. The fusion protein was correctly targeted to the oil body membrane and separated from the majority of other seed proteins by flotation centrifugation. Recombinant hirudin was localized to the surface of oil bodies as determined by immunofluorescent techniques. The oleosin-hirudin fusion protein accumulated to ca. 1% of the total seed protein. Hirudin was released from the surface of the oil bodies using endoprotease treatment. Recombinant hirudin was partially purified through anion exchange chromatography and reverse-phase chromatography. Hirudin activity, measured in anti-thrombin units (ATU), was observed in seed oil body extracts, but only after the proteolytic release of hirudin from its oleosin carrier. About 0.55 ATU per milligram of oil body protein was detected in cleaved oil body preparations. This activity demonstrated linear dose dependence. The oleosin fusion protein system provides a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for purification of the desired polypeptide.     

Analysis of the effect of rye chromosomes 4R, 6R and telomeric heterochromatin on 7R on homologous chromosome pairing in pentaploid hybrids (AABBR,AABBD)

Summary Meiotic pairing in Triticum turgidum cv. Ma (4x) with a mean chiasmata frequency of 27.16 per cell was compared with chiasmata frequencies in its hybrids with several triticale strains, Chinese Spring wheat and its addition lines for Imperial rye chromosomes 4R and 6R. In hybrids between Ma and x Triticosecale cv. Rosner the chiasmata frequency was marginally reduced by an average of 1.25%, by 8.8% in hybrids with x Triticosecale cv. DRIRA HH and by 6.7% with DRIRA EE (lacking 90% telomeric heterochromatin from chromosome arm 7RL). In pentaploid hybrids between Ma and T. aestivum cv. Chinese Spring the reduction was an average of 10.30%, while addition lines with rye chromosome 6R reduced chiasmata frequencies by an average of 7.4% and rye addition line for 4R showed the greatest depression in chiasmata frequency in hybrids by a 25.04% reduction. An interchange difference involving long chromosome segments was observed between Ma and Rosner.Contribution No. 819 Ottawa Research Station     

Psychological changes associated with self-regulatory treatments of irritable bowel syndrome

The psychological side effects of self-regulatory treatment (a combination of relaxation, thermal biofeedback, and cognitive therapy) for irritable bowel syndrome (IBS) were compared among 20 successfully treated patients, 12 unsuccessfully treated patients, and 9 patients who merely monitored symptoms for 12 weeks. Pretreatment and posttreatment scores on the Beck Depression Inventory, State-Trait Anxiety Inventory, and Psychosomatic Symptom Checklist were examined. Successfully treated patients had significant (p<.01) reductions on all measures and significantly greater reductions on depression and state anxiety than the symptom monitoring group. Interestingly, the failures also showed a significant (p=.027) reduction in trait anxiety and no significant increases on other measures.     

On age morphological changes of males of Chydoridae (Cladocera)

Summary Young and adult males of 11 species of Chydoridae are studied, their figures being published here (fig. 1–15). The necessity is stressed to distinguish young forms of males and gynandromorphic individuals.Pleuroxus balatonicus is considered to be described from the population ofPleuroxus unicatus having under Balaton Lake conditions retarded transformation of young males into adult form, and accordingly having unusually numerous young males. \qO\qs\qn\qo\qv\qn\qy\ye \qr\ye\qz\qu\ql\Qj\qt\qa\qt\qy 11 (. 1–15). . , Pleuroxus uncinatus , Pleuroxus balatonicus.     

Identification of a 4A/7R and a 7B/4R wheat-rye chromosome translocation

Summary By producing chromosome substitutions with Imperial rye chromosomes 4R (C) and 7R (D) in the wheat cultivar Chinese Spring two spontaneous translocation lines were obtained. One involves segments of wheat chromosome 4A and rye chromosome 7R, the other involves portions of wheat chromosome 7B and rye chromosome 4R     

Possible intrachromosomal duplication in a case of trisomy 9p

Summary A 5-year-old boy with multiple minor anomalies and mental retardation was found to have chromosomal condition of 46,XY,inv dup(9p)(pterp13::p21p24::p13qter). The clinical features of the propositus fit well with those of trisomy 9p which have been established to be a clinical entity.