Establishment of a galactocerebrosidase-deficient twitcher mouse cell line that expresses galactocerebrosidase activity in hybrids with control human fibroblasts

Summary Primary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation of skin obtained from 3-d-old littermates of atwi+/twi×twi+/twi mating. Galactocerebrosidase activity remained deficient for two twitcher cell lines, TM-1 and TM-2, and both lines demonstrated an initial period of growth decline, followed by accelerated growth. The TM-2 line has been subcultured for more than 3.5 yr, has a modal chromosome number of 63, a doubling time of approximately 16 h, and has remained galactocerebrosidase deficient throughout its life span. These data indicate this to be an established twitcher cell line that can be continuously maintained in culture as a transformed galactocerebrosidase-deficient mouse cell line. This established line was rendered 6-thioguanine resistant so that the cells could be fused with control human fibroblasts and selected for hybrid lines in hypoxanthine-aminopterin-thymidine medium. Also, the established twitcher cells were crossed with neomycin-resistant control human fibroblasts and selected in G418 medium. Several of the hybrid lines from both crosses had higher than deficient levels of galactocerebrosidase activity initially, followed by a decrease to twitcher levels during subculture, whereas other lines retained high levels of activity. These results indicate that twitcher-human somatic cell hybrids will express galactocerebrosidase activity and thus may be useful for determining the human chromosome or chromosomes associated with this expression. Partial support for these studies was provided by a National Institutes of Health AREA grant (HD21222-01) and a NIH subcontract to Clark University from the Shriver Center for Mental Retardation This research forms a portion of studies performed to fulfill the requirements for the Ph.D. degree in biology at Clark University for J. T. K.     

The action of theI(1)npr-1 + locus on theDrosophila glue geneSgs-3 is cell-autonomous

The 2B5 chromosomal locus inDrosophila contains a gene,I(1)npr-1 ⁺, whose product required intrans for the expression of the larval salivary gland-specific geneSgs-3. We have addressed the question as to whether this factor acts in a cell-autonomous manner or not. This was made possible by the use of a transformant strain that makes anSgs-3-E. coli β-galactosidase (lacZ) fusion protein, under the control of anSgs-3 promoter, allowing the cellular examination of gene expression by a histochemical assay for enzyme activity. Using genetic methods, larvae that were mosaic for the loss of function mutationl(l)npr-1 were generated. The expression of theSgs-3-lacZ fusion gene was assayed histochemically in such larvae. Our results strongly indicate a cell-autonomous requirement for the product ofl(1)npr-1+. This is in contrast to another factor, the hormone ecdysterone, which is also required forSgs-3 expression, but acts in a non-autonomous manner     

Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis

Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here, we introduce morphogen‐age measurements as a novel approach to test models of morphogen gradient formation. Using a tandem fluorescent timer as a protein age sensor, we find a gradient of increasing age of Bicoid along the anterior–posterior axis in the early Drosophila embryo. Quantitative analysis of the protein age distribution across the embryo reveals that the synthesis–diffusion–degradation model is the most likely model underlying Bicoid gradient formation, and rules out other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bicoid gradient formation and demonstrate how morphogen‐age information can complement knowledge about movement, abundance, and distribution, which should be widely applicable to other systems.     

Potency Analysis of Mesenchymal Stromal Cells Using a Combinatorial Assay Matrix Approach

1. Download high-res image (159KB)
2. Download full-size image

     

Transition metal-saccharide chemistry: d-glucose complexes of Mn(II), Co(II), Ni(II), Cu(II) and Zn(II)

Metal complexes of d-glucose (d-Glc) from large cation containing dibromo-dichloro salts of dipositive metals [NEt₄]₂[MBr₂Cl₂] (M = Mn, Co, Ni, Cu and Zn) and the disodium salt of glucose were synthesized from a MeOH:MeCN mixture. The complexes were characterized by UV-vis absorption, circular dichroism, IR and proton magnetic resonance spectroscopies, and by elemental analysis, and were found to be Na[M(d-Glc)(OMe)Cl]. Cyclic voltammetric studies of these complexes, in the acidic to neutral pH range, indicated no dissociation, even in highly acidic conditions.This paper is dedicated to Professor Richard H. Holm (Harvard University) on the occasion of his 60th birthday.     

Transplanted wing and leg imaginal discs in Drosophila melanogaster demonstrate interactions between epidermis and myoblasts in muscle formation

 Adult muscle development in Drosophila is intimately associated with the development of the nervous system and epidermis. During metamorphosis, myoblasts from the wing imaginal disc reach target sites on the developing pupal epidermis and begin the formation of multinucleate myofibres of the dorsal thorax. The paths taken by pupal myoblasts could be specified by the nervous system and/or the epidermis. Using genetically marked donor pupal wing and leg discs transplanted onto pupal hosts, we have generated animals that have ectopic wings or legs and have examined the formation of adult muscle types. We show that thoracic myoblasts migrate over both host and donor epidermis when the transplant site on the host is thoracic. However, when the transplant site is on the abdomen, thoracic myoblasts do not migrate over abdominal epidermis. Our results show that the epidermis plays an important role in determining the migration pattern of myoblasts. Since muscles are multinucleate cells that form by the fusion of myoblasts, one way in which their molecular characteristics could be achieved is by some myoblasts acting as ”founders”. These myoblasts could influence the pattern of gene expression of those nuclei that fuse with them. We have examined, again using disc transplant experiments, if myoblasts on discs have the capacity to express fibre-specific genes as distinct from this property being conferred by other extra-discs myoblasts. Our results demonstrate that disc-associated myoblasts can indeed fuse with each other to express fibre-specific genes. We synthesize the results presented here with those from earlier experiments to suggest a mechanism for muscle patterning in the adult thorax. Received: 8 January 1995 / Accepted in revised form: 22 January 1996     

Pethia sanjaymoluri,a new species of barb (Teleostei: Cyprinidae) from the northern Western Ghats,India

Pethia sanjaymoluri, a new cyprinid, is described from the Pavana and Nira tributaries of Bhima River, Krishna drainage, Maharashtra, India. It can be distinguished from congeners by a combination of characteristics that includes an incomplete lateral line, absence of barbels, upper lip thick and fleshy, 23–25 lateral series scales, 7–12 lateral‐line pored scales, 10 predorsal scales, 11–14 prepelvic scales, 17–20 pre‐anal scales, 4½ scales between dorsal‐fin origin and lateral line, four scales between lateral line and pelvic‐fin origin, 8–15 pairs of serrae on distal half of dorsal‐fin spine, 12–14 branched pectoral‐fin rays, 4 + 26 total vertebrae, 4 + 5 predorsal vertebrae, 4 + 13 abdominal vertebrae, 13 caudal vertebrae and a unique colour pattern comprising a humeral spot positioned below the lateral line and encompassing the third and fourth lateral‐line scales and one scale below, one caudal spot on 17th–21st lateral‐line scales with a yellow hue on its anterior side and apical half of dorsal fin studded with melanophores making the fin tip appear black. Genetic analysis based on the mitochondrial cytochrome b gene sequence suggests that the species is distinct from other known species of Pethia for which data are available.     

Randomised comparison of procedures for obtaining informed consent in clinical trials of treatment for cancer.

Methods of obtaining informed consent have evolved differently in Western countries without substantive information on the impact of these different practices on the patients. A randomised study was performed to compare two commonly adopted methods of seeking consent to randomised treatment: an individual approach at the discretion of each doctor and a uniform policy of total disclosure of all relevant information. The impact of both consent procedures on the patient''s understanding and anxiety levels and on the doctor-patient relationship was assessed by means of a questionnaire given soon after the consent interview. Fifty seven patients were assigned at random to two groups: to 29 patients an individual approach to seeking consent was adopted and to 28 patients all relevant information was given. Seven patients refused consent to randomised treatment, with slightly more refusals by patients in the total disclosure group (5 v 2, p = 0.25). The main effects of total disclosure of all information compared with an individual approach to seeking consent were: a better understanding of treatment and side effects and of research aspects of the treatments; less willingness to agree to randomised treatment; and increased anxiety. No significant differences were found in patients'' perceptions of the doctor-patient relationship. A repeat questionnaire given three to four weeks later no longer showed significant differences between the two groups.     

Enhancement of protein synthesis in isolated chloroplasts by irradiation of fern gametophytes with blue light

Irradiation of the gametophytes of Pteridium aquilinum with blue light led to a nearly 5-fold increase in the amino acid-incorporating activity of isolated chloroplasts. The blue light effect was not due to increased synthesis of ATP or other energy donors by the chloroplasts but was probably related to an increased production of chlorophyll and photosynthetic enzymes.     

STUDIES ON THE FLORAL HISTOGENESIS AND PHYSIOLOGY OF PERILLA. II. FLORAL INDUCTION IN CULTURED APICAL BUDS OF P. FRUTESCENS

Raghavan , V., and W. P. Jacobs . (Princeton U., Princeton, N. J.) Studies on the floral histogenesis and physiology of Perilla. II. Floral induction in cultured apical buds of P. frutescens. Amer. Jour. Bot. 48(9): 751–760. Illus. 1961.—The morphological and histological changes induced in apical buds and explants of P. frutescens (L.) Britt. var. ‘Tall Late’ in short days and long days when cultured in White's medium have been followed. When photoinduced in culture, apical buds showed visible signs of changes in 30.9 days, and produced normal flowers in 81.6 days. Apical buds in LD showed similar transformations at the apices, but with continued LD treatment, they elongated to form sterile structures, superficially resembling Selaginella cones. The normal flowers formed in culture were similar to those formed on the intact plant, while the individual florets of the LD-cone differentiated only the non-sporogenous tissues in them. A less pronounced sequence of changes resulted when apical buds stripped of their older pairs of apical leaves were LD- or SD-treated. When explants with the 1st pair of unfolded leaves or with the 1st and 2nd pairs of unfolded leaves were photoinduced in vitro, the responses were relatively fast. However, explants with the 1st and 2nd pairs of unfolded leaves in LD remained entirely vegetative. When unfolded leaves were implanted in the same medium separated from the buds and both were photoinduced or given LD, the buds formed the typical 1st signs, but did not differentiate into normal flowers. This inhibition of flowering resulted in the formation of sterile cones, consisting of florets with non-sporogenous tissues only. The results have suggested the possibility of the sterile cone-like structures being an intermediate stage in the flowering of Perilla. The role of a possible inhibitor produced by mature leaves of SD plants in LD is discussed.