A conjugate of 5-fluorouridine-poly(L-lysine) and an antibody reactive with human colon carcinoma

The ribose moiety of 5-fluorouridine (FUR) was oxidized with periodate and the product was bound through a poly(L-lysine) bridge to monoclonal antibodies, denoted SF25MAb, reactive with a human colon carcinoma LS180. The antibody was linked via its polysaccharide (previously oxidized with periodate) to the poly(L-lysine)-drug conjugate. The linking of FUR-poly(L-lysine) to the antibody markedly increased the latter's binding to the tumor cells. A relatively lower increase was also observed with conjugates of nonrelated antibodies, such as anti-hepatitis B surface antigen and anti-epidermal growth factor receptor antibodies. The pharmacological activity of the specific conjugate FUR-poly(L-lysine) -SF25MAb was higher than that of the drug-substituted polymer alone. The poly(L-lysine) bridge caused toxic effects in vivo, even though substituted both by FUR and by antibody. Therefore, the additional unreacted lysyl residues were blocked by succinylation. Partial blocking of free amino groups on the conjugate rendered it nontoxic but decreased its cell-binding capacity, though to a level still higher than that of the original unmodified antibody. The pharmacological activity of the specific conjugate after blocking was also reduced and necessitated prolonged incubation periods or higher concentrations. Following periodate oxidation and reduction, FUR was as effective as the clinically preferred compound 5-fluoro-2'-deoxyuridine in vitro and in vivo, against the LS180 colon carcinoma. Experiments in nude mice, with LS180 tumor subcutaneous xenotransplants, showed that FUR-poly(L-lysine)-SF25MAb (blocked by succinylation) was not toxic and was effective in the retardation of tumor growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

A polypeptide conjugate of bilirubin from human bile.

An alkali-stable bilirubin conjugate has been obtained from human T-tube bile as its phenylazo derivative. The conjugate consists of a polypeptide probably of molecular weight 7000 to which the azo pigment of bilirubin is linked covalently through its carboxyl group. It thus constitutes the first biliprotein found in mammals. It is not known whether both carboxyl groups of native bilirubin participate in the binding of the conjugating protein, nor has it been possible to determine the number of pigment moieties occurring on a single polypeptide chain. The isolation makes use of the tendency of the conjugate to form large aggregates and involves the following steps: azo coupling of the native bile, (NH4)2S04 precipitation of macromolecules and aggregates, removal of low molecular weight contaminants by dialysis and gel filtration (first on Sepharose 6B IN 6 M guanidine, then on Sephadex LH-20 in 50% acqueous 2-chloroethanol) and a concluding purification by chromatography on p-aminobenzyl cellulose using a PH gradient. The final preparation appeared to be homogeneous on polyacrylamide electrophoresis.     

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.     

Temperature control of biotin binding and release with A streptavidin-poly(N-isopropylacrylamide) site-specific conjugate.

The many laboratory and diagnostic applications utilizing streptavidin as a molecular adaptor rely on its high affinity and essentially irreversible interaction with biotin. However, there are many situations where recovery of the biotinylated molecules is desirable. We have previously shown that poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, can reversibly block biotin association as the polymer's conformation changes at its lower critical solution temperature (LCST). Here, we have constructed a streptavidin-PNIPAAm conjugate which is able to bind biotin at room temperature or lower and release bound biotin at 37 degrees C. The conjugate can repeatedly bind and release biotin as temperature is cycled through the LCST. A genetically engineered streptavidin mutant, E116C, which has only one cysteine residue, was conjugated site specifically via the sulfhydryl groups with a PNIPAAm that has pendent sulfhydryl-reactive vinyl sulfone groups. The conjugation site is near the tryptophan 120 residue, which forms a van der Waals contact with biotin that is important in generating the large binding free energy. The temperature-induced conformational change of the polymer at position 116 may lead to structural changes in the region of tryptophan 120 that are responsible for the reversible binding between biotin and the conjugated streptavidin.     

A morphological and experimental study of the mesencephalic neural crest cells in the mouse embryo using wheat germ agglutinin-gold conjugate as the cell marker

The distribution of the mesencephalic neural crest cells in the mouse embryo was studied by mapping the colonization pattern of WGA-gold labelled cells following specific labelling of the neuroectoderm and grafting of presumptive neural crest cells to orthotopic and heterotopic sites. The result showed that (1) there were concomitant changes in the morphology of the neural crest epithelium during the formation of neural crest cells, in the 4- to 7-somite-stage embryos, (2) the neural crest cells were initially confined to the lateral subectodermal region of the cranial mesenchyme and there was minimal mixing with the paraxial mesoderm underneath the neural plate, (3) labelled cells from the presumptive crest region colonized the lateral cranio-facial mesenchyme, the developing trigeminal ganglion and the pharyngeal arch, (4) the formation of neural crest cells was facilitated by the focal disruption of the basal lamina and the cell-cell interaction specific to the neural crest site and (5) the trigeminal ganglion was colonized not only by neural crest cells but also by cells from the ectodermal placode.     

Detection of sugar residues in rabbit embryo teeth with lectin-horseradish peroxidase conjugate: II. A light microscopal study

The cellular distribution and changes of sugar residues during tooth development in embryos of the rabbit Oryctolagus cuniculus were investigated by using horseradish peroxidase-conjugated lectins (lectin-HRP). The lectins SBA, ECA, and LTA show no binding to any region of the dental cap and bell stages, whereas BS-1 and UEA-1 bind to dental cells at both stages. Appropriate control studies confirmed the specificity of the binding of the lectins. At cap stage, the lectins BS-1 and UEA-1 show moderate binding to the (pre)-ameloblast and (pre)-odontoblast cells. These results suggest that the acetylgalactosamine and α-L-fucose residues present in (pre)-ameloblasts and (pre)-odontoblasts, respectively, are common to determined but relatively undifferentiated cells capable of forming matrices of hard tissues. Since the odontoblast and ameloblast express dentin and enamel, respectively, it can be speculated that the abundance of these residues in these cells might be associated with the maintenance of the capacity of the cells to produce such matrices. At the bell stage, the odontoblasts display considerable amounts of α-L-fucose, whereas α-L-fucose is poorly localized in ameloblasts. However, ameloblasts contain significant quantities of N-acetylgalactosamine, whereas only a diffuse positivity for this carbohydrate is apparent in odontoblasts. The marked changes of the glycosylation pattern of these glycoconjugates might indicate that they play a role during the cell-to-cell interaction and might also be involved in the odontoblastic and ameloblastic functional activity. Such a possibility is entirely speculative until specific in vitro experiments are conducted. J. Morphol. 231:175–184, 1997. © 1997 Wiley-Liss, Inc.     

Human embryo (12 mm) with mesohydronephrosis and ureterohydronephrosis.

This report describes a human embryo with a length of 12 mm (horizon XVII Streeterm 35 +/- 1 day old), in which the following malformations appear: Dilation on both sides of the Wolffian ducts, and the more caudal mesonephritic glomerules (mesohydronephrosis). Dilatation of both ureters and renal pelvis (uretero-hydronephrosis). These malformations are the result of the accumulation of liquid secreted by the mesonephros in a cloaca which is dilated, unwalled and blocked by an epithelial plug which is continuous with the cloacal membrane.     

Expression of DNA methyltransferase (Dnmt1) in testicular germ cells during development of mouse embryo

The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.     

Glucose responsive insulin production from human embryonic germ (EG) cell derivatives

Type 1 diabetes mellitus subjects millions to a daily burden of disease management, life threatening hypoglycemia and long-term complications such as retinopathy, nephropathy, heart disease, and stroke. Cell transplantation therapies providing a glucose-regulated supply of insulin have been implemented clinically, but are limited by safety, efficacy and supply considerations. Stem cells promise a plentiful and flexible source of cells for transplantation therapies. Here, we show that cells derived from human embryonic germ (EG) cells express markers of definitive endoderm, pancreatic and beta-cell development, glucose sensing, and production of mature insulin. These cells integrate functions necessary for glucose responsive regulation of preproinsulin mRNA and expression of insulin C-peptide in vitro. Following transplantation into mice, cells become insulin and C-peptide immunoreactive and produce plasma C-peptide in response to glucose. These findings suggest that EG cell derivatives may eventually serve as a source of insulin producing cells for the treatment of diabetes.     

Cryptic causation of human disease: reading between the (germ) lines

Most cases of complex human diseases arise sporadically. However, usually there is a significant level of familial aggregation of risk and genetic mapping has identified the responsible gene in a few mendelian cases. Although a disease can be causally genetic, intensified mapping efforts have so far been unable to identify genes that account for more than a small fraction of the familial risk, perhaps because the responsible variation arises by somatic mutation (SM). SM explains the kind of epidemiological pattern seen in cancer, and might have a comparable role in many other diseases. For example, in epilepsy, which has largely defied mapping analysis, the underlying disease pathology, undamped neuronal signaling, is closely connected to gene function. Better technologies to detect and characterize SM are becoming available. However, until it is studied directly, SM will remain a cryptic etiological force, even for diseases that are essentially "genetic".     

Concanavalin A and wheat germ agglutinin binding to sea urchin embryo basal laminae

Summary The basal laminae and inner extracellular matrices of Lytechinus pictus and Arbacia punctulata embryos were characterized on the basis of lectin binding. Developmental stage specific patterns of lectin binding were observed after microinjection of Con A-FITC and WGA-FITC. Lectin-specific patterns differed between control, sulfate free sea water (SFSW) and tunicamycin treated embryos. Con A injection resulted in the rounding-up of cells in the epithelium and was most pronounced in embryos cultured in the presence of tunicamycin. Basal laminae were isolated by Triton X-100 extraction of whole embryos. Proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose and incubated in biotinylated lectins. Lectin-binding glycoproteins were detected with avidin peroxidase. The electrophoretic pattern of Con A-binding proteins in early developmental stages of Arbacia was similar with several low molecular weight species appearing at gastrulation in control and SFSW embryos. WGA-binding in Arbacia and Lytechinus control embryos was limited to a 125,000 Mr glycoprotein (gp125). In addition to gp125, several high molecular weight WGA-binding glycoproteins were also detected in SFSW embryos. The evidence suggests that mesenchyme migration and gastrulation are correlated with changes in the molecular composition of the ECM.