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.     

ααααanti-3.7 type II: a new α-globin gene rearrangement suggesting that the α-globin gene duplication could be caused by intrachromosomal recombination

Summary We report here a new human -globin gene rearrangement carrying the two normal, 2 and 1, and two hybrid, 1/2, globin genes in the order 5-2-1/2-1/2-1-3. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the -thalassaemia IVS 1 nt 1 GA mutation caused thalassaemia intermedia. We also report a case of an -globin gene rearrangement in the twin of one of the -globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an -anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the -globin gene rearrangements could be of the intra-chromosomal rather than the interchromosomal type.     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

The sensitivity of head and neck squamous cell carcinomas to tumor necrosis factor-α

Sensitivities to recombinant human tumor necrosis factor- (TNF-) and chemotherapeutic agents (cisplatin, peplomycin, methotrexate) were evaluated in 20 tumor cells of head and neck squamous cell carcinomas, using a dye uptake method. Also, numbers of TNF receptors of these tumor cells were measured by Scatchard plot analysis. There was no relationship between the number of TNF- receptors and the sensitivity to TNF-. Furthermore, there was no correlation between the sensitivity to TNF- and that to chemotherapeutic drugs, nor between the sensitivity to TNF- and the clinical response to chemotherapy including of cisplatin and peplomycin. The sensitivity to TNF- was higher in poorly differentiated carcinomas than in well differentiated ones.Abbreviations BSA Bovine serum albumin - CDDP Cisplatin - 5-Fu 5-fluorouracil - IC50 Inhibition concentration 50 - MTX Methotrexate - PLM Peplomycin - TNF- Tumor necrosis factor-     

Transformation of lithocholic acid to a new bile acid, 3α, 15β-dihydroxy-5β-cholanic acid by Cunninghamella blakesleeana ST-22

Summary A fungus identified as Cunninghamella blakesleeana (Lendner) can carry out 15-hydroxylation of lithocholic acid to a new bile acid (3,15-dihydroxy-5-cholanic acid). By optimizing the fermentation conditions, the amount of the product increased from 0.17 g/l to 1.2 g/l. Hydrophilicity measurements and in vitro cholesterol solubilization tests showed that 3, 15-dihydroxy-5-cholanic acid was as effective as ursodeoxycholic acid in cholesterol solubilization.Abbreviations LCA lithocholic acid (3-hydroxy-5-cholanic acid) - 3, 15-DHC (3, 15-dihydroxy-5-cholanic acid) - DMSO dimethyl sulfoxide - CHES 2-[N-cyclohexylamino]ethanesulfonic acid     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

The effect of culture and membrane potential on Goα expression in neonatal rat cardiac myocytes

The effects of culture and membrane potential on G_o39 expression were examined in neonatal rat cardiac myocytes. During six days of culture, the amount of G_o39 in myocytes increased six-fold. The increase in G_o39 appeared to be programmed, since G_o39 of rat hearts also increased in vivo within three days after birth before declining by six days after birth. Furthermore, the age of the rat from which cardiac myocytes were isolated determined the amount of G_o39 that accumulated in cultured cells with myocytes from two day-old rats producing more G_o39 than myocytes from six day-old rats. In addition, agents which alter membrane potential (KCl and bupivacaine) inhibited the accumulation of G_o39 in cultured myocytes. In an attempt to identify the signaling pathway in which cardiac G_o39 is involved, muscarinic receptor-stimulated inositol phosphate production was examined, but was found to be comparable in myocytes that had six-fold differences in G_o39 content. Thus G_o39 does not appear to couple muscarinic receptors to phospholipase C in rat cardiac myocytes.     

Glycosylated human interleukin-1α, neoglyco IL-1α, coupled with N-acetylneuraminic acid exhibits selective activities in vivo and altered tissue distribution

In order to study the effect of glycosylation on its biological activities and to develop IL-1 with less deleterious effects, N-acetylneuraminic acid (NeuAc) with C9 spacer was chemically coupled to human recombinant IL-1. NeuAc-coupled IL-1 (NeuAc-IL-1) exhibited reduced activities in vitro and receptor-binding affinities by about ten times compared to IL-1. In this study, we examined a variety of IL-1 activities in vivo. NeuAc-IL-1 exhibited a marked reduction in the activity to up-regulate serum IL-6, moderate reduction in the activities to up-regulate serum amyloid A and NOx. However, it exhibited comparable activities as IL-1 to down-regulate serum glucose and to improve the recovery of peripheral white blood cells from myelosuppression in 5-fluorouracil-treated mice. In addition, tissue level of NeuAc-IL-1 was high compared to IL-1. These results indicate that coupling with NeuAc enabled us to develop neo-IL-1 with selective activities in vivo and enhanced tissue level.     

Histochemische und biochemische Untersuchung der α-glucosidasen mit 2-naphthyl-α-D-glucosid

Summary Histochemical and biochemical studies yield the following method of choice for the in situ detection of neutral (microvillous) and acid (lysosomal) -glucosidases: 12 mg 2-naphthyl--D-glucoside (dissolved in 0.5 ml N,N-dimethylformamide) and 0.6–0.8 ml hexazonium-p-rosaniline in 10 ml 0.1 M citric acid phosphate buffer for aqueous or 5 ml buffer mixed with equal parts of 2% agar for incubation with semipermeable membranes, pH 5 or 6.5.With this method neutral -glucosidases can be exactly demonstrated in the brush border of the small intestine (glycoamylase, sucrase-isomaltase) and kidney of mammals, birds, fishes, amphibia and reptiles; localization of acid -glucosidases is achieved at the cellular level in many organs and tissues.Fluorometric and photometric measurements prove that 2-naphthyl--D-glucoside is superior to 6-brom-2-naphthyl--D-glucoside for the demonstration of -glucosidases in situ due to the lower Michaelis constant and higher maximal reaction velocity of the naphthol derivative. — Among the coupling reagents tested neutral -glucosidases can be localized correctly with hexazotized p-rosaniline (with and without semipermeable membranes) for simultaneous coupling. Fast Blue B delivers false positive results in the suczedaneous and simultaneous coupling procedure using aqueous incubation media; in combination with the membrane technique azo dye can not be observed in the sections. Hexazonium-p-rosaniline inhibits neutral and acid -glucosidases to nearly the same extent as Fast Blue B.Fixation of blocks of tissue in formaldehyde and glutaraldehyde suppresses -glucosidases in the intestine and epididymis. The inhibition rates amount to 50 and 70% respectively. Washing in sugar solution rises enzyme activity to 65 and 50%.Species and organ dependent activity differences of neutral and acid -glucosidases and changes of enzyme activity in the intestine and kidney after castration as well as in the course of pregnancy can be detected by means of biochemistry but not with the histochemical assay including minimal incubation. In comparison with p-nitrophenyl--D-glucoside the 2-naphthyl derivative is also the substrate of choice for the biochemical determination of -glucosidases. — Agar gel electrophoresis reveals one band in the neutral and acid pH range.

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Histochemische und biochemische Untersuchung der -glucosidasen mit 2-naphthyl--D-glucosid

Zusammenfassung Vergleichend histochemisch-biochemische Untersuchungen ergeben folgende Methode der Wahl zum in situ-Nachweis der neutralen (mikrovillären) und sauren (lysosomalen) -Glucosidasen: 12 mg 2-Naphthyl--D-glucosid (gelöst in 0,5 ml N,N-Dimethylformamid) und 0,6–0,8 ml Hexazonium-p-rosanilin in 10 ml 0,1 M Citronensäure-Phosphat-Puffer zur wäßrigen oder in 5 ml Pufferlösung 1:1 gemischt zur Inkubation mit semipermeablen Membranen, pH 5 und 6,5.Mit diesem Verfahren können die neutralen -Glucosidasen in Bürstensaum von Dünndarm (Glucoamylase, Saccharase-Isomaltase) und Niere bei Säugern, Fischen, Vögeln, Reptilien und Amphibien exakt dargestellt werden; die Lokalisation der sauren -Glucosidase gelingt auf Zellebene in zahlreichen Organen und Geweben.Fluorometrische und photometrische Messungen zeigen, daß 2-Naphthyl--D-glucosid dem Alternativsubstrat 6-Br-2-Naphthyl--glucosid zur histochemischen Untersuchung wegen seiner kleineren Michaelis-Konstante und höheren maximalen Reaktionsgeschwindigkeit überlegen ist. — Unter den geprüften Kupplungssubstanzen kann nur Hexaonium-p-rosanilin als Simultankuppler mit und ohne Membrantechnik vor allem die neutralen -Glucosidasen korrekt erfassen; Fast Blue B in wäßrigen Medien lokalisiert bei Post- und Simultankupplung falsch-positiv und liefert in Verbindung mit semipermeablen Membranen negative Resultate. Die Hemmung der -Glucosidasen durch hexazotiertes p-Rosanilin entspricht etwa der durch Fast Blue B.Stückfixation in Form- und Glutaraldehyd inhibiert die neutralen und sauren -Glucosidasen in Dünndarm und Nebenhoden zu ca. 50 bzw. 70%; nach Auswaschen liegen die Aktivitäten bei 65 bzw. 50%.Art- und organspezifische Aktivitätsdifferenzen der sauren und neutralen -Glucosidasen und Änderungen der Enzymaktivität nach Kastration sowie während der Gravidität in Dünndarm und Niere deckt zuverlässig nur die biochemische Untersuchung auf; der histochemische -Glucosidasen-Nachweis versagt hier selbst bei Minimalinkubation weitgehend. Verglichen mit p-Nitrophenyl--glucosid ist das 2-Naphthylderivat auch das biochemische Substrat der Wahl. — Mittels Agargelelektrophorese kann im alkalischen und sauren pH-Bereich 1 Bande nachgewiesen werden.

     

Spatiotemporal expression patterns of sialoglycoconjugates during nephron morphogenesis and their regional and cell type-specific distribution in adult rat kidney

The expression of 2,6- and 2,3-linked sialic acids on N-glycans was studied in embryonic, postnatal, and adult rat kidney. Histochemistry and blotting using Polyporus squamosus and Sambucus nigra lectins for 2,6-linked sialic acids and the Maackia amurensis lectin for 2,3-linked sialic acids were performed and sialyltransferase activity was assayed. N-glycans with 2,6- and 2,3-linked sialic acid were differently expressed in the two embryonic anlagen and early stages of nephron. Metanephrogenic mesenchyme was positive for 2,3-linked sialic acid but not for the 2,6-linked one, which became detectable initially in the proximal part of S-shaped bodies. Collecting ducts were positive for 2,6-linked sialic acid, whereas 2,3-linked sialic acid was restricted to their ampullae. Although positive in embryonic kidney, S1 and S2 of proximal tubules became unreactive for 2,3-linked sialic acid in postnatal and adult kidneys. In adult kidney, intercalated but not principal cells of collecting ducts were reactive for 2,3-linked sialic acid. In contrast, 2,6-linked sialic acids were detected in all cells of adult kidney nephron. Blot analysis revealed a different but steady pattern of bands reactive for 2,6- and 2,3-linked sialic acid in embryonic, postnatal, and adult kidney. Activity of 2,6 and 2,3 sialyltransferases was highest in embryonic kidney and decreased over postnatal to adult kidney with the activity of 2,6 sialyltransferase always being three to fourfold that of 2,3 sialyltransferase. Thus, 2,6- and 2,3-linked sialic acids are differently expressed in embryonic anlagen and mesenchyme-derived early stages of nephron and show regional and cell type-specific differences in adult kidney.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The -isopropylmalate synthase (EC 4.1.3.12) from Alcaligenes eutrophus H 16 was inhibited by l-leucine and -ketoisocaproate. The extent of inhibition was influenced by substrate- and inhibitor concentrations as well as by the pH. Intermediary plateaus, which always appeared in the inhibition curves, suggested cooperative effects. The maximal Hill coefficient was found to be two. At low concentrations of leucine the inhibition mechanism was of the competitive type with respect to substrate acetyl coenzyme A and of the noncompetitive type with respect to substrate -ketoisovalerate. The inhibition was specifically relieved by the addition of valine or isoleucine. The anomalous effect of temperature on enzyme activity was diminished by leucine. The Arrhenius energy of the reaction increased from about 11 kcal/mole in the absence of leucine to about 18 kcal/mole in the presence of leucine. The further addition of valine reversed this effect. The physiological relevance of the -ketoisocaproate-mediated inhibition is discussed.Abbreviations IPM -isopropylmalate - KIC -ketoisocaproate - KIV -ketoisovalerate - DTNB 5:5 Dithiobis-(2-nitrobenzoe acid)     

Synthesis of methyl α-isomalto-oligosaccharides specifically deoxygenated at position 2 of the terminal glycopyranosyl unit

Methyl -isomaltoside and methyl -isomaltotrioside specifically deoxygenated at position C-2 of the terminal glucopyranosyl unit were synthesized by trimethylsilyltriflate-mediated condensation of 3,4,6-tri-O-benzoyl-1-O-tert-butyl(dimethyl)silyl-2-deoxy--d-arabino-hexopyranose with suitably blocked derivatives of methyl -d-glucopyranoside and methyl -isomaltoside, respectively.To whom correspondence should be addressed.     

Microbial transformation of α-pinene

Summary A bacterium capable of utilizing -pinene as a sole carbon and energy source was isolated from soil. This strain, named strain S201-1, which was identified as Pseudomonas maltophilia on the basis of its taxonomical properties, accumulated limonene, borneol, camphor, perillic acid, and 2-(4-methyl-3-cyclohexenylidene) propionic acid from -pinene in the culture broth. It was demonstrated that -pinene, -pinene, borneol, camphor, and a number of p-menthane derivatives were oxidized by this strain. Relations between the protonation of -pinene and the formation of the products by the microbe are discussed.     

The hemoglobins of Artemia salina. V. Genetic variation in hemoglobin 1, hemoglobin 2, and hemoglobin 3

Electrophoretic mobilities of three hemoglobins (Hb1, Hb2, and Hb3) were studied in 15 populations of brine shrimps. Genetic segregation data support the model that Hb2 contains n -polypeptides and n -polypeptides; Hb1 contains 2n -polypeptides. Hb3 contains neither - nor -polypeptides. There is no evidence of linkage of and loci with each other or with the locus (or loci) which governs Hb3 or with the nonhomologous portion of the sex chromosomes. Hemoglobins of different populations may be hybridized in vitro by incubation at high temperature. Reversible dissociation to subunits which contain only one ( or ) polypeptide occurs at 40 C (for Hb1) and at 50 C (for Hb2).Supported by Grant HD-11445 from the National Institutes of Health.     

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids

Summary Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C ^, -disubstituted amino acids.-Trifluoromethyl substituted amino acids, a subclass of C ^, -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containing-TFM amino acids towards proteolysis by-chymotrypsin. Furthermore, configurational effects of-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence of-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.     

A simple brassinolide analogue 2α, 3α-dihydroxy-17β-(3-methyl-butyryloxy)-7-oxa-B-homo-5α-androstan-6-one which induces bean second internode splitting

A new synthetic brassinolide analogue, 2,3-dihydroxy-17-(3-methylbutyryloxy)-7-oxa-B-homo-5-androstan-6-one (11), has been shown to exhibit typical brassinolide activity characterised by elongation, swelling, twisting and splitting of the bean second internode. It was prepared from the known lactone 2,3,17-trihydroxy-7-oxa-B-homo-5-androstan-6-one (4) which was transformed to an isopropylidenedioxy derivative. After protection of the 2- and 3-hydroxy groups it yielded the 2,3-isopropylidenedioxy-17-(3-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one (7) on treating with 3-methylbutyryl chloride in pyridine. The analogue with a 2-methylbutyric moiety (10, 2,3-dihydroxy-17-(2-methyl-butyryloxy)-7-oxa-B-homo-5-androstan-6-one) in position 17 stimulated only elongation and swelling of the bean second internode. However, in this bioassay 100 times more 10 or 11 compared to 24-epibrassinolide is required to obtain the same effects. Analogues with -oriented hydroxyl groups at C-2 and C-3 (14,15), a 6-ketone (17,18) or 6-oxa-7-oxo-lactone system (12,13) in ring B lack the typical brassinolide activity. In addition, the active brassinosteroids applied to the second internode stimulated a similar, but 30% lower elongation of the first internode. From data presented here we conclude that the presence of two hydroxy groups in the positions 22 and 23 of the brassinolide side chain, which are considered as a key structural requirement, is not absolutely necessary for a compound to exhibit typical brassinosteroid activity. Nevertheless, these compounds have generally 2–10 times lower activity than that having 22,23-vicinal diol in the side chain.     

Sheep α-globin gene sequences: Implications for their concerted evolution and for the down-regulation of the 3′ genes

In sheep as in man and most other mammals, there are two -globin genes (^I and ^II), which are expressed at different levels, the upstream gene being the most efficient. In -globin gene triplication and quadruplication, this trend is confirmed, i.e., the -chain output of the downstream genes progressively decreases. In this study, we have determined the complete sequence of the cDNAs and of both the introns in a triple- haplotype in which each gene could be recognized for the presence of distinct alleles. The sequence analysis reveals that the bodies of the three -globin genes are essentially identical (99.9% homology) and moreover indicates that the down-regulation of additional -globin genes in sheep is not the effect of sequence variation from the Cap to the Poly(A) addition sites. This striking similarity among -genes is higher than that seen in other mammals and is probably sustained by particularly efficient mechanisms of gene conversion and cross-over fixation. Correspondence to: Dr. M.S. Ristaldi