Extent and high frequency of a short conversion between the human Aγ and Gγ fetal globin genes

Summary Southern blotting and DNA sequencing after polymerase chain reaction (PCR) amplification provide evidence for the frequent occurrence (in 7 out of 24 chromosomes) of a short conversion GA in the 3 end of the human fetal A globin gene. This short conversion is characterized by the presence, 3 nucleotides downstream from the termination codon of the A gene, of the TCAC sequence that is normally present at the equivalent position at the 3 end of the G gene; it is therefore identical to a conversion already described. Interestingly, we have found that this conversion is associated with the presence of theHindIII polymorphic restriction site in the A IVS2, occuppying an equivalent position in both the G and A genes. Our observations strengthen the hypothesis that the presence of the HindIII polymorphic restriction site in A IVS2 and the presence of the sequence TCAC at the 3 end of the A gene might be the result of a single conversion event.     

Recombinant interferon γ up-regulates in vivo and down-regulates in vitro monocyte CD14 antigen expression in cancer patients

Summary The expression of the monocyte membrane glycoprotein CD14 was measured and related to the serum interferon (IFN) concentration in thirteen patients with disseminated cancer during treatment with human recombinant interferon (rIFN). The drug was administered by continuous subcutaneous infusion using an escalating dose schedule, starting at 50 µg/day or 100 µg/day and increasing weekly up to 600 µg/day, if tolerated. Treatment was continued at a mean maximal tolerated dose of 200 µg/day for a median duration of 43 days. Serum IFN concentration and monocyte CD14 antigen expression (immunofluorescence with the monoclonal antibody LeuM3 and fluorescence-activated cell sorting analysis) were determined weekly. The serum IFN concentration was positively correlated with the rIFN dose (P <0.05). Therapy induced a dose-dependant enhancement of CD14 antigen expression. The increase in mean CD14 fluorescence intensity was on average 60% after 3 weeks of treatment at a mean dose of 220 µg rIFN/day and was reversed after withdrawal of therapy. Patients with a rapidly rising serum IFN concentration (starting dose 100 µg/day) showed a smaller increment in CD14 fluorescence intensity than those with slowly rising serum IFN levels (starting dose 50 µg/day). Since rIFN is known to down-regulate CD14 antigen expression in vitro, monocytes from patients off therapy and from healthy volunteers were cultured with this cytokine. A similar decrease of CD14 fluorescence was observed in both groups. In patients several factors, such as IFN concentration, duration of drug effect and type of serum, were evaluated and could not explain the discrepant in vivo and in vitro findings. In conclusion, the monocyte marker CD14 was found to be differentially regulated by rIFN in vivo and in vitro. In vivo, secondary mediators, induced by rIFN and acting on a constantly renewed cell population, may contribute to the enhanced CD14 expression.     

Carvedilol prevents cardiac hypertrophy and overexpression of hypoxia-inducible factor-1α and vascular endothelial growth factor in pressure-overloaded rat heart

Summary The use of -blockers has emerged as a beneficial treatment for cardiac hypertrophy. Hypoxia-inducible factor-1 (HIF-1) is tightly regulated in the ventricular myocardium. However, the expression of HIF-1 in cardiac hypertrophy due to pressure overload and after treatment with -blocker is little known. To evaluate the effect of carvedilol on both myocardial HIF-1 expression and cardiac hypertrophy, infra-renal aortic banding was performed for 4 weeks in adult Sprague-Dawley rats to induce cardiac hypertrophy. Carvedilol at 50 mg/kg body weight per day after surgery was given. Heart weight and the ratio of heart weight and body weight increased significantly after aortic banding for 4 weeks in the absence of drug treatment. Mean arterial pressure increased from 80 ± 9 mmHg in the sham group to 94 ±5 mmHg (p < 0.001) in the banding group. Echocardiography showed concentric hypertrophy after aortic banding. Mean arterial pressure decreased after treatment with carvedilol. The increased wall thickness and heart weight was reversed to normal by carvedilol. Western blot showed that HIF-1, vascular endothelial growth factor (VEGF) and brain natriuretic peptide (BNP) proteins were up-regulated and nerve growth factor- (NGF-) down-regulated in the banding group. Treatment with valsartan, doxazosin, or N-acetylcysteine did not significantly affect HIF-1 and VEGF proteins expression in the banding groups. Real-time polymerase chain reaction showed that mRNA of HIF-1, VEGF and BNP increased and mRNA of NGF- decreased in the banding group. Treatment with carvedilol reversed both protein and mRNA of HIF-1, VEGF, BNP, and NGF- to the baseline values. Increased immunohistochemical labeling of HIF-1, VEGF, and BNP in the ventricular myocardium was observed in the banding group and carvedilol again normalized the labeling. In conclusion, HIF-1, VEGF, and BNP mRNA and protein expression were up-regulated, while NGF- mRNA and protein was downregulated in the rat model of pressure-overloaded cardiac hypertrophy. Treatment with carvedilol is associated with a reversal of abnormal regulation of HIF-1,VEGF, BNP, and NGF- in the hypertrophic myocardium.     

Laminins, tenascin and type VII collagen in colorectal mucosa

Summary The distribution of different laminin polypeptides, type VII collagen and tenascin has been studied in adult and foetal colorectal mucosa by using the indirect immunofluorescence technique. Immunoreactivity for laminin 1 chain was located to basement membranes of epithelia, muscularis mucosae, and blood vessels, respectively in different segments of adult colon and rectum. Laminin 1 and 1 chains were additionally expressed in lamina propria. Laminin 2 chain was also found in lamina propria around the pericyptal fibrollasts. Immunoreactivity for laminin 2 chain was restricted to basement membranes in the muscularis mucosae and arteries. Laminin 3 and 3 chains, suggestive for laminin-5, were confined especially to surface epithelial basement membranes. Immunoreactivity for type VII collagen was confined to basement membrane of surface epithelium in a punctate manner, while that for tenascin was seen slightly more broadly in the basement membrane zone and also in the muscular layer. The distribution of laminin chains in 16-week-foetal colon mostly resembled that of corresponding adult tissue, although immunoreactivities for laminin 2 and 2 chains were lacking. Type VII collagen and the high molecular weight isoform of tenascin also absent from the foetal colon. The results show that the basement membrane of the surface epithelium of colon and rectum express the components of epithelial adhesion complex, laminin-5 (3-3-2) and type VII collagen, resembling in this respect small intestine and stomach while laminin-2 (2-1-1) appears to be associated with pericryptal fibroblasts, and laminin-1 (1-1-1) widely in most basement membranes.     

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.     

Patterned disjunction in Drosophila melanogaster. I. Assortment of SPA pol /+ and X,Y in the stock N1

N1 (= Nijmegen 1) D. melanogaster heterozygous for sparkling poliert (4) (= pol, here) were backcrossed as single pairs. When were not selected for departure from 1/1, pol/pol ⁺, many exceptional ratios were observed even though the net for all 67 pairs was approximately one-to-one; in the same experiment a net excess of was observed. In a second experiment were selected for departure from 1/1, pol/pol ⁺ratios. The net pol/pol ⁺ratios became significantly different from the 1/1 expected but the sex ratio approached normal. Lineage of the males in the second experiment were recorded and displayed as pedigrees. These together with tabulated data suggest that in some pairs, one of the four categories pol , pol , pol ⁺, pol ⁺ may be significantly greater or less than 1/4 of the total offspring recovered.     

Ontogenesis of cells producing polypeptide hormones (ACTH,MSH, LPH,GH, Prolactin) in the fetal hypophysis of the rat: Influence of the hypothalamus

Summary The ontogenesis of cells containing polypeptide hormones (ACTH, MSH, LPH, GH and Prolactin) was investigated in the fetal rat hypophysis by immunohistochemistry using the peroxidase-antiperoxidase complex.Corticotrophs, melanotrophs and lipotropic cells were revealed earlier in the pars distalis than in the pars intermedia. In the pars distalis, cells producing LPH were found in the morning of day 15 of gestation using anti-- or anti--LPH sera, and in the afternoon using anti-- or -endorphin sera. Cells containing -MSH were observed from the afternoon of day 15. The cells stainable with the anti--MSH, anti--(17-39)ACTH and anti--(l-24)ACTH sera appeared on day 16. In the pars intermedia, the cells producing -MSH, MSH, - and -endorphin, and -LPH were observed in the morning of day 17, while cells containing ACTH were only revealed in the afternoon of the same day of gestation. Based on the treatment of serial paraffin sections with various antisera, it was clearly shown that MSH, ACTH, and LPH occur in the same cells located in the pars distalis as in the pars intermedia.The development of the corticotrophs, melanotrophs and lipotropic cells does not require the presence of the fetal hypothalamus or other central nervous structures. The pituitary glands of 21 day-old fetuses encephalectomized on day 16 showed as many reactive cells as those of the littermate controls.The somatotrophs were first revealed in the pars distalis in the afternoon of day 19. The cells producing prolactin were not observed before day 21 of gestation. On some cases GH and prolactin were found together in one cell. The cytodifferentiation of GH and prolactin cells is apparently not under hypothalamic control.     

Conservation status of parrot populations in an Atlantic rainforest area of southeastern Brazil

A census of four species of syntopic parrots was carried out using distance sampling methods on São Sebastião island, SE Brazil. Most of the 33593 ha island is covered by mature and secondary Atlantic rainforest. Almost 80% of these forests are within the Ilhabela Park. Although the species counted have marked differences in size and weight, density (individuals/km²) and estimated population size in 23500 ha of well-preserved forests were similar: Amazona farinosa (13.82±5.94; 3247±1395), Pionus maximiliani (15.79±7.04; 3712±1654), Brotogeris tirica (15.05±4.87; 3537±1143) and Pyrrhura frontalis (13.06±5.53; 3068±1298). Encounter rates of Forpus crassirostris and Pionopsitta pileata were very low, which suggests that there is only a small population of these species on the island. The São Sebastião forests still support healthy populations of parrots. Although woodpecker population estimates on the island are large enough to provide nesting sites for parrots, competition for holes with other secondary cavity nesters such as toucans, flycatchers and tytiras, and the selective cutting of dead trees for canoe construction, which is a common practice on the island, may limit hole availability for parrots.     

Homocysteine stimulates the expression of monocyte chemoattractant protein-1 in endothelial cells leading to enhanced monocyte chemotaxis

The effect of intraperitoneal administration of tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the tocopherol treated group were not observed. The light emission was significantly higher in the control than in the tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbateFe²⁺ lipid peroxidation. The protector effect observed by tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria.     

Synaptonemal Complex Analysis in Oocytes and Spermatocytes of Threespine Stickleback Gasterosteus Aculeatus (Teleostei, Gasterosteidae)

A surface-spreading synaptonemal complex (SC) technique was employed to analyze spermatocytes and oocytes of stickleback, Gasterosteus aculeatus, in order to visualize the process of chromosome synapsis. The mean SC length was 150±18m in three males and 143±12m in one female analyzed. A representative SC karyotype with 21bivalents was also presented. Each SC had lateral elements of equal length. No bivalent displaying the atypical synaptic behaviour which is often associated with heteromorphic sex chromosomes was observed neither in males nor in the female analyzed.     

Reactivity pattern of 15 HLA-Dw1 homozygous typing cells in primary mixed lymphocyte culture

Summary The Dwl specificity was highly correlated with the serologically determined HLA-DR1 antigen in the Eighth International Histocompatibility Workshop 1980. By testing a large number of HLA-Dwl, DR1 defined homozygous typing cells (HTC) in a checkerboard primary mixed lymphocyte reaction, on a panel of about 30 HLA-DR1 heterozygous individuals, and in family segregation, three Dwl subtypes could be defined in association with certain HLA-A, -B, and -C-antigens. HTC TA, FRI, and FRA carrying the HLA haplotypes A11, B35, Cw4 or A3, B35, Cw4 in the homozygous state gave positive typing results with most HLA-DR1 positive panel members and stimulated only four other Dw1 HTCs (SRR35%). In contrast to this operationally broad specificity, Dw1-HTC-HEN (HLA-A2, B44, C-, homozygous) was non-stimulatory to all HTCs except one, but gave high responses against these, leading to the definition of a narrow specificity included in the broad one. Another such narrow specificity was represented by HTC FEE (HLA-A2, B27, Cw2 homozygous). Typing patterns with FEE were mostly different from those defined with other HTC. In family studies a specific typing pattern for this HTC could be shown to segregate with HLA. However, within some of these responses a contribution of the HLA haplotype in the trans position must be assumed.Supported in part by DFG grant Ri 164/14     

The Effect of Salicylic Acid on Peroxidase Activity in Wheat Calli Cocultured with the Bunt Pathogen Tilletia caries

The effect of salicylic acid (SA) on peroxidase activity in wheat (Triticum aestivum L.) calli cocultured with the bunt pathogen Tilletia caries was studied. Fungal infection was shown to activate cytoplasmic peroxidase. SA suppressed total peroxidase activity but did not inhibit the peroxidase with pI 9.8. A novel chitin-specific peroxidase with pI 3.5 appeared after the SA treatment. The infection of SA-treated cells with Tilletia caries activated the isoenzymes with pI 3.5, 4.8, and 7.5 and stimulated their secretion into the culture medium. The ability of SA to control wheat peroxidase activity during pathogenesis is discussed. The important role of this control in plant defense responses to the bunt pathogen is emphasized.     

A “Bloom” in the Water of Amursky Bay (Sea of Japan) Caused by the Dinoflagellate Oxyrrhis marina Dujardin, 1841

A bloom in the water of Amursky Bay (Sea of Japan) caused by the dinoflagellate Oxyrrhis marina Dujardin, 1841 was recorded for the first time. The highest density of this species in the bloom area was 443.3 million cells/liter. The abundant development of microalga was observed from July to September 2002 at a water temperature of 17–24.5°C and a salinity of 7–18. Changes in the density of O. marina and other species of phytoplankton during the bloom period are analyzed. Possible reasons for the blooms of O. marina in Amursky Bay are discussed.     

Patterns of adventitious root formation in English ivy

Adventitious root formation by debladed petiole cuttings of English ivy (Hedera helix L.) proceeds via a direct rooting pattern for the easy-to-root juvenile phase, while the difficult-to-root mature phase roots through an indirect rooting pattern. Juvenile petiole cuttings treated with -naphthaleneacetic acid (NAA, 100 M) plus the polyamine biosynthesis inhibitor, difluoromethylarginine (DFMA, 1 mM), formed an increased number of roots per cutting initiated by the indirect rooting pattern. The increased root formation and change in rooting pattern were reversed by the addition of putrescine (1 mM). Delaying auxin application to petiole cuttings for 15 days also induced juvenile petioles to root by the indirect pattern. This could be reversed by rewounding the base of the cutting prior to auxin application after day 15. The data support the use of the terms competent root-forming cells and induced competent root-forming cells to describe the target cells for the initial events of root formation for the direct and indirect rooting patterns, respectively.Kentucky Experiment Station publication 90-10-122.     

Synthesis of Gal-β-(1→4)-GlcNAc-β-(1→6)-[Gal-β- (1→3)]-GalNAc-α-OBn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal: 3-O-sulfotransferases

Our recent studies have revealed the existence of two distinct Gal: 3-O-sulfotransferases capable of acting on the C-3 position of galactose in a Core 2 branched structure, e.g., Gal14GlcNAc16(Gal13)GalNac1OBenzyl as acceptor to give 3-O-sulfoGal14GlcNAc13(Gal13)GalNAc1OB 20 and Gal14GlcNAc16(3-O-sulfoGal13)GalNAc1OB 23. We herein report the synthesis of these two compounds and also that of other modified analogs that are highly specific acceptors for the two sulfotransferases. Appropriately protected 1-thio-glycosides 7, 8, and 10 were employed as glycosyl donors for the synthesis of our target compounds.     

Intrinsic fluorescence studies on saccharide binding toArtocarpus integrifolia lectin

The combining region ofArtocarpus integrifolia lectin has been studied by using the ligand-induced changes in the fluorescence of the lectin. The saccharide binding properties of the lectin show that C-l, C-2, C-4, and C-6 hydroxyl groups of D-galactose are important loci for sugar binding. The -anorner of galactose binds more strongly than its -counterpart. Inversion in the configuration at C-4 as in glucose results in a loss of binding to the lectin. The C-6 hydroxyl group is also presumably involved in binding as D-fucose does not bind to the lectin.The lectin binds to the Thomsen-Friedenreich antigen (Gal(13)GalNAc) more strongly than the other disaccharides studied, viz. Gal/ (14) Gal and Gal (13) GlcNAc, which are topographically similar to T-antigen. This observation suggests that the combining region ofArtocarpus lectin is complementary to that of T-antigen.Solvent accessibility of the protein fluorophores have been probed by the quenching of protein fluorescence by Iodide ion in the absence and presence of sugar. In the presence of sugar a slight inaccessibility of the fluorophores to the solvent has been observed.Abbreviations MeGal 1-O-methyl--glucopyranoside - MeGal 1-O-methyl--glucopyranoside - GalNAc 2-acetamido-2-deoxy-galactose - Gal Galactose     

Comparative study of the antitumor effect of two types of murine recombinant interferons, (β) and (γ), against B16-F10 melanoma

Summary A comparative study of the antitumor effect of murine recombinant interferon() Mu-rIFN() and murine recombinant interferon() Mu-rIFN() on B16-F10 melanoma was conducted. Administration of Mu-rIFN() i.p. into C57BL/6 mice on days 1 to 7 produced a higher suppressive effect than Mu-rIFN() both on the growth of s.c. implanted tumor and on the formation of artificial pulmonary metastasis. Pharmacokinetic study of Mu-rIFN() demonstrated that high plasma levels were retained for a long time. In clonogenic assay, Mu-rIFN() at 1000 units/ml showed about 80% inhibition of colonies of B16-F10 melanoma. However, Mu-rIFN() hardly inhibited the colonies, even at 1000 units/ml. Augmentation of natural killer (NK) cytotoxicity was much greater with Mu-rIFN() than Mu-rIFN(), whereas Mu-rIFN() enhanced the cytotoxicity of peritoneal macrophages more strongly than Mu-rIFN(). Injection of Mu-rIFN() i.p. 1 day before tumor challenge also inhibited the formation of pulmonary metastasis of B16-F10 melanoma. However, pretreatment of mice with carrageenan significantly suppressed the inhibitory effect of Mu-rIFN(). From these results, it is suggested that the inhibitory effect of Mu-rIFN() on the tumor growth and metastases of B16-F10 melanoma is mediated partly by direct antitumor effect and partly by the activation of macrophages, and that the augmentation of NK activity contributes mainly to the antitumor effect of Mu-rIFN().     

Defective remethylation of homocysteine is related to decreased synthesis of coenzymes B2 in thyroidectomized rats

Summary. We investigated the influence of hypothyroidism on homocysteine metabolism in rats, focusing on a hypothetical deficient synthesis of FAD by riboflavin kinases. Animals were allocated in control group (n=7), thyroidectomized rats (n=6), rats with diet deficient in vitamin B2, B9, B12, choline and methionine (n=7), thyroidectomized rats with deficient diet (n=9). Homocysteine was decreased in operated rats (2.6±1.01 vs. 4.05±1.0mol/L, P=0.02) and increased in deficient diet rats (29.56±4.52 vs. 4.05±1.0mol/L, P=0.001), when compared to control group. Erythrocyte-Glutathione-Reductase-Activation-Coefficient (index of FAD deficiency) was increased in thyroidectomized or deficient diet rats (P=0.004 for both). Methylenetetrahydrofolate-reductase and methionine-synthase activities were decreased in thyroidectomized rats but not in those subjected to deficient diet. Cystathionine--synthase was increased only in operated rats. Taken together, these results showed a defective re-methylation in surgical hypothyroidism, which was due in part to a defective synthesis of vitamin B2 coenzymes. This defective pathway was overcompensated by the increased Cystathionine--synthase activity.     

Preparation and purification of γγ enolase (neuron-specific enolase) using high performance anion exchange chromatography

A simple and rapid method, using only two chromatographic steps, is described for the purification and preparation of enolase isoenzymes from human and beef brain extracts. In the first step, a crude enolase was obtained by chromatography on Q-Sepharose Fast Flow column. The crude fraction was then purified by high performance anion exchange chromatography on a Mono-Q column. enolase obtained in this manner was shown to be homogeneous by two dimensional polyacrylamide gel electrophoresis and by high performance gel permeation chromatography. The yield of enolase by this method was 7–8 mg of pure enzyme per 100 g of brain.     

Die optischen Übertragungseigenschaften der Komplexaugen von Drosophila

Summary The transfer properties of the optical system in the arthropod compound eye are determined by the interommatidial angle , influencing the resolving power, and by the width of the visual fields of single ommatidia , influencing the response at high spatial frequencies of brightness distributions in the object space. The energy transfer/ receptor is proportional to ( )² and decreases with in-inreasing approximation of the perfect-imaging condition: gD 0; 0. However, a value > 0 has to be maintained in order to overcome the threshold of nervous excitation at a certain minimum-brightness level. Theoretical treatment yields /=0.62 to 0.88 as the corresponding optimum-imaging relation. The actual ratio can be derived from measurements of the optomotor reactions to the movement of periodic brightness patterns. The approximate value 0.76 is obtained from the fruitfly Drosophila with normal and mutant eye pigmentation. As a result, the parameters of this imaging system are found to be established in a way that enables optimum performance at sufficient illumination. An dieser Stelle möchte ich Dr. W. Reichardt für sein eingehendes Interesse und manche anregende Diskussion über die Sehvorgänge im Komplexauge meinen Dank sagen. Dr. K. Kirschfeld verdanke ich ebenfalls wertvolle Hinweise. Herrn E. Freiberg bin ich für die Anfertigung der Abbildungen sehr verbunden.