bluuebunny/crossref_metadata_2025 huggingface dataset:
Size-controlled human adipose-derived stem cell spheroids hybridized with single-segmented nanofibers and their effect on viability and stem cell differentiation
Authors: Lee Jinkyu , Lee Sangmin , Kim Sung Min , Shin Heungsoo
Published: April 2021

Background

Fabrication of three-dimensional stem cell spheroids have been studied to improve stem cell function, but the hypoxic core and limited penetration of nutrients and signaling cues to the interior of the spheroid were challenges. The incorporation of polymers such as silica and gelatin in spheroids resulted in relatively relaxed assembly of composite spheroids, and enhancing transport of nutrient and biological gas. However, because of the low surface area between cells and since the polymers were heterogeneously distributed throughout the spheroid, these polymers cannot increase the cell to extracellular matrix interactions needed to support differentiation.

Methods

We developed the stem cell spheroids that incorporate poly(ι-lactic acid) single-segmented fibers synthesized by electrospinning and physical and chemical fragmentation. The proper mixing ratio was 2000 cells/μg fibers (average length of the fibers was 50 μm - 100 μm). The SFs were coated with polydopamine to increase cell binding affinity and to synthesize various-sized spheroids. The function of spheroids was investigated by in vitro analysis depending on their sizes. For statistical analysis, Graphpad Prism 5 software (San Diego, CA, USA) was used to perform one-way analysis of variance ANOVA with Tukey’s honest significant difference test and a Student’s
t
-test (for two variables) (
P
 < 0.05).

Results

Spheroids of different sizes were created by modulating the amount of cells and fibers (0.063 mm
2
–0.322 mm
2
). The fibers in the spheroid were homogenously distributed and increased cell viability, while cell-only spheroids showed a loss of DNA contents, internal degradation, and many apoptotic signals. Furthermore, we investigated stemness and various functions of various-sized fiber-incorporated spheroids. In conclusion, the spheroid with the largest size showed the greatest release of angiogenic factors (released VEGF: 0.111 ± 0.004 pg/ng DNA), while the smallest size showed greater effects of osteogenic differentiation (mineralized calcium: 18.099 ± 0.271 ng/ng DNA).

Conclusion

The spheroids incorporating polydopamine coated single-segmented fibers showed enhanced viability regardless of sizes and increased their functionality by regulating the size of spheroids which may be used for various tissue reconstruction and therapeutic applications.
Patient oriented research in mental health: matching laboratory to life and beyond in Canada
Authors: Johnston Jenessa N. , Ridgway Lisa , Cary-Barnard Sarah , Allen Josh , Sanchez-Lafuente Carla L. , Reive Brady , Kalynchuk Lisa E. , Caruncho Hector J.
Published: April 2021

As patient-oriented research gains popularity in clinical research, the lack of patient input in foundational science grows more evident. Research has shown great utility in active partnerships between patient partners and scientists, yet many researchers are still hesitant about listening to the voices of those with lived experience guide and shape their experiments. Mental health has been a leading area for patient movements such as survivor-led research, however the stigma experienced by these patients creates difficulties not present in other health disciplines. The emergence of COVID-19 has also created unique circumstances that need to be addressed. Through this lens, we have taken experiences from our patient partners, students, and primary investigator to create recommendations for the better facilitation of patient-oriented research in foundational science in Canada. With these guidelines, from initial recruitment and leading to sustaining meaningful partnerships, we hope to encourage other researchers that patient-oriented research is necessary for the future of mental health research and foundational science.
A case of left ventricular free wall rupture after insertion of an IMPELLA® left ventricular assist device diagnosed by transesophageal echocardiography
Authors: Mizuno Akito , Kawamoto Shuji , Uda Shuji , Tatsumi Kenichiro , Takeda Chikashi , Tanaka Tomoharu , Fukuda Kazuhiko
Published: April 2021

Background

The IMPELLA® is a minimally invasive left ventricular assist device. We report a case in which transesophageal echocardiography (TEE) was useful in diagnosis of left ventricular rupture after IMPELLA® insertion.

Case presentation

A 75-year-old man presented to the emergency room with chest pain and underwent percutaneous coronary intervention for 100% stenosis of the left anterior descending branch #7. An IMPELLA® was inserted to stabilize the circulation, but hypotension persisted. Transthoracic echocardiography revealed increased pericardial effusion and suspicion of free wall left ventricular rupture, leading to emergency surgery. TEE revealed the IMPELLA® straying into the left ventricle apical wall and cardiac tamponade. Hemorrhage was observed from the thinning free wall and the tip of the IMPELLA® was palpable. The IMPELLA® was removed and the left ventricular wall was repaired.

Conclusions

The IMPELLA® requires implantation of the tip in the left ventricle, but it should be noted that a fragile ventricular wall can be easily perforated.
Synthesis, characterization, and biological evaluation of some 4-((thiophen-2-yl-methylene)amino)benzenesulfonamide metal complexes
Authors: Sekhar E. Vijaya , Karki Subhas S. , Rangaswamy Javarappa , Bhat Mahesh , Kumar Sujeet
Published: April 2021

Background

Sulfonamides (sulfa drugs) and the metals like mercury, copper, and silver bear antimicrobial properties. The discovery of broad-spectrum antibiotics such as penicillins, cephalosporins, and fluoroquinolones has reduced their use. However, in some instances these drugs are the first-line treatment. The metal-based sulfonamide (e.g., silver sulfadiazine) is considered as first choice treatment in post-burn therapy while the use of silver nanoparticle-cephalexin conjugate to cure Escherichia coli infection explains the synergistic effect of sulfa drugs and their metal conjugates. With growing interest in metal-based sulfonamides and the Schiff base chemistry, it was decided to synthesize sulfonamide Schiff base metal complexes as antioxidant and antimicrobial agent.

Results

The Fe (III), Ru (III), Co (II), Ni (II), Cu (II), Pd (II), Zn (II), Cd (II), and Hg (II) metal complexes of 4-((thiophen-2-ylmethylene)-amino)-benzenesulfonamide (TMABS) were prepared and studied for thermal stability, geometry, and other electronic properties. The ligand TMABS (Schiff base) and its metal complexes were screened in-vitro for 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and antimicrobial properties against Gram-positive (+ve) Bacillus subtilis (MTCC-441), Staphylococcus aureus (MTCC 7443), Gram-negative (-ve) Escherichia coli (MTCC 40), Salmonella typhi (MTCC 3231), and fungal strains Aspergillus niger (MTCC-1344) and Penicillium rubrum by agar well diffusion method. Results summarized in Tables 3, 4, and 5 represent the inhibitory concentration (IC50) in micromole (μM). The zone of inhibition (ZI) in millimeter (mm) represents antimicrobial properties of TMABS and its metal complexes.

Conclusions

The synthesized sulfanilamide Schiff base (TMABS) behaved as a neutral and bidentate ligand coordinating with metal ions through its azomethine nitrogen and thiophene sulfur to give complexes with coordination number of 4 and 6 (Fig. 3). The nucleophilic addition of sulfanilamide amino group (–NH2) group to carbonyl carbon (>C=O) of benzaldehyde gave sulfanilamide Schiff base (imine) (Fig. 2). All the metal complexes were colored and stable at room temperature. With IC50 of 9.5 ± 0.1 and 10.0 ± 0.7 μM, the Co, Cu, and Pd complexes appeared better antioxidant than the ligand TMABS (155.3±0.1 μM). The zone of inhibition (ZI) of Hg (28 mm) and Ru complexes (20 mm) were similar to the ligand TMABS (20 mm) against Aspergillus niger (MTCC-1344) as in Figs. 4, 5, and 6. None of the synthesized derivatives had shown better antimicrobial properties than the standard streptomycin sulfate and fluconazole.
Design and optimization of nano invasomal gel of Glibenclamide and Atenolol combination: in vitro and in vivo evaluation
Authors: Anitha P. , Satyanarayana S. V.
Published: April 2021

Background

There are many circumstances where chronic disease is associated with other disorders, especially in diseases such as diabetes with noncommunicable disease risk factors, such as hypertension. The current therapies for treating such chronic comorbid diseases are limited and challenging due to the difficulties in overcoming the side effects from complex therapeutic treatment regimen. The present study is aimed to develop and optimize the combinational nano invasomal gel of Glibenclamide (GLB) and Atenolol (ATN) as a novel combination therapy for comorbid treatment of diabetic hypertensive patients. The developed formulations were characterized for various parameters, including in-vitro skin permeation, skin irritation, in-vivo antidiabetic, and antihypertensive activities.

Results

OCNIG showed that the % entrapment efficiency of GLB is 96.67 ± 0.65% and % entrapment efficiency of ATN is 93.76 ± 0.89%, flux of GLB (240.43 ± 1.76 μg/cm2/h), and flux of ATN (475.2 ± 1.54 μg/cm2/h) which was found to conform to the expected value. The results indicated desired release and permeation profiles. Optimized formulation showed significant pharmacokinetic properties, which shows improvement in bioavailability by 134.30% and 180.32% respectively for two drugs, when compared to marketed oral preparation. Pharmacodynamic studies showed improved and prolonged management of diabetes and hypertension in Wistar rats, compared to oral and drug-loaded nano invasomes formulations.

Conclusion

Overall, the results showed that nano invasomal gel was found to be a useful and promising transdermal delivery system for the treatment of concurrent diseases.
Stress: a global multidimensional common denominator
Authors: Halbreich Uriel
Published: April 2021

The manifestations of stress are diverse and culturally and individually determined, but it is present in every culture, every socioeconomic level and – it is global. Introducing a special BJPsych Advances issue on stress, this editorial outlines research questions to be examined and first actions to be taken in the multidimensional sphere of stress, highlighting the need for a collaborative interdisciplinary approach in both research and practice.
A Cell in Waiting
Authors: McGrath Brian
Published: April 2021

In the Death Penalty seminar, Derrida is on the side of abolition. The seminar has in mind an end: the end of the death penalty. But this end is not simple or straightforward, for as Derrida explores, the death penalty is nothing other than an attempt to control the end by making death (the end of all ends) calculable. The death penalty aims to put an end to the surprise of death. Though Derrida announces an aim for the Death Penalty seminar, at several key moments he is at pains to distinguish the work of reading, the work of the seminar—the work of a seminar that reads, that develops a reading—from what he calls the “militant activist cell.” The militant activist cell aims for direct intervention; it aims, for instance, to abolish the death penalty. Derrida’s Death Penalty seminar teaches us much about the death penalty, but the death penalty, as read through the seminar, also teaches us about the seminar. How does the aim of the seminar—a seminar devoted to reading—differ from that of the militant activist cell (with its imagined telos, aim, or end)?
Response
Authors: Rapaport Herman
Published: April 2021

This article contextualizes Derrida’s seminars within the European seminar tradition familiar to French and German academic cultures as a prerequisite to discussing individual essays in a special issue of Poetics Today devoted to Derrida. These contributions engage with both published and unpublished seminars that Derrida delivered chiefly in Europe and North America. Highlights of the various essays are reviewed and at various points some few issues of a critical nature are raised for consideration. The seminars Life Death (1975–76), Hospitality (1995–96) and Death Penalty (1998–2001) are of main concern, though significant mention is made of numerous other seminars that Derrida gave over a forty year span. Discussants have been very careful to consider the specific characteristics of Derrida’s seminars from a textual point of view informed by Derrida’s well-known conceptions of écriture and dissemination. Major themes concern issues of education, modeling, production-reproduction, language, the foreign, philosophical nationalism, and borders.
How to Go Mad without Losing Your Mind
Authors: Bruce La Marr Jurelle
Published: April 2021

“Hold tight. The way to go mad without losing your mind is sometimes unruly.” So begins La Marr Jurelle Bruce's urgent provocation and poignant meditation on madness in black radical art. Bruce theorizes four overlapping meanings of madness: the lived experience of an unruly mind, the psychiatric category of serious mental illness, the emotional state also known as “rage,” and any drastic deviation from psychosocial norms. With care and verve, he explores the mad in the literature of Amiri Baraka, Gayl Jones, and Ntozake Shange; in the jazz repertoires of Buddy Bolden, Sun Ra, and Charles Mingus; in the comedic performances of Richard Pryor and Dave Chappelle; in the protest music of Nina Simone, Lauryn Hill, and Kendrick Lamar, and beyond. These artists activate madness as content, form, aesthetic, strategy, philosophy, and energy in an enduring black radical tradition. Joining this tradition, Bruce mobilizes a set of interpretive practices, affective dispositions, political principles, and existential orientations that he calls “mad methodology.” Ultimately, How to Go Mad without Losing Your Mind is both a study and an act of critical, ethical, radical madness.
Determination of Cell Function in an Insect
Authors: Wigglesworth V. B.
Published: April 2021

I Propose to consider two kinds of determination and differentiation which have been studied in the hemipteron Rhodnius prolixus. (i) The determination of the cell or group of cells, with their subsequent differentiation to produce a given part of the body, (ii) The determination or control of the characters of that part—whether these are to be juvenile (larval) or adult (imaginal). Discussion of this second type of determination will require consideration of the role of hormones in controlling differentiation in insects.
The Effect of Excess Vitamin A on the Differentiation of Chick Ectoderm in Culture1
Authors: Fell Honor B.
Published: April 2021

This investigation was made in collaboration with Sir Edward Mellanby, F.R.S. (National Institute for Medical Research, Mill Hill). It has long been known that in animals suffering from vitamin A deficiency, mucous epithelia are replaced by keratinizing epithelium of the epidermoid type. Since keratinization is promoted by lack of vitamin A, it seemed possible that it might be inhibited by an excess of the vitamin. Series of cultures were therefore made in which explants of the ectoderm and underlying tissue from the trunk and limbs of 6–7-day chick embryos were grown by the watchglass or hanging-drop methods; the explants from one side of each embryo were grown in normal medium, and corresponding fragments from the opposite side in medium to which about 1,800 i.u. per 100 c.c. of vitamin A had been added ( + A medium).
Interference of Amino-acid Analogues with Normal Embryonic Development1
Authors: Herrmann Heinz
Published: April 2021

The study of the reactions of the embryonic organism to chemical substances is one of the main approaches to the analysis of embryonic development. Well known is the use of the lithium ion which produces in embryos of diverse species a variety of developmental abnormalities. However, in the case of a substance like lithium an analysis of the particular sequence of chemical reactions which leads eventually to the manifestation of a morphological abnormality encounters a peculiar difficulty. Since lithium reacts with numerous proteins, a wide range of reactions has to be considered as the initial step in the teratogenic mechanism.
Basic Mechanisms in the Differentiation of Pigment Cells
Authors: Willier B. H.
Published: April 2021

Some of the basic mechanisms of cellular differentiation have been elucidated by an experimental analysis of the underlying factors and conditions which control the processes of differentiation of the pigment cell in the chick embryo. The pigment cell, owing to its singular capacity to synthesize melanin pigment granules, is an ideal cell for locating its site of origin and tracing its subsequent pathways to the skin and derivatives, the feather papillae. It has the further advantage that its specific behaviour is recorded as colour or colour pattern in the mature feather. In other words, by using the pigment-forming cell as a ‘marker’ many of its properties in time and space may be ascertained from the moment that it is first set aside as a specialized strain of cell until it reaches its ultimate position and visible expression in the growing feather papilla.
Formation of primary and secondary myotubes in rat lumbrical muscles
Authors: Ross J. J. , Duxson M. J. , Harris A. J.
Published: April 2021

Numbers of myoblasts, primary myotubes and secondary myotubes in developing rat embryo hindlimb IVth lumbrical muscles were counted at daily intervals up until the time of birth, using electron mi-croscopy. Motoneurone death at the spinal cord level supplying the lumbricals was assessed by counting axons in the 4th lumbar ventral root. Death of the motoneurones that supply the intrinsic muscles of the hindfoot was monitored by comparing the timecourse of development of total muscle choline acetyltransferase activity in control embryos with that in embryos where motoneurone death was inhibited by chronic paralysis with TTX, and by counting axons in the mixed nerve trunks at the level of the ankle at daily intervals. Condensations of undifferentiated cells marking the site of formation of the muscle were seen on embryonic day 15 (E15). Primary myotubes began to appear on E16 and reached a stable number (102 ± 4) by E17. Secondary myotubes first appeared two days later, on E19, and numbered 280 at the time of birth (E22). The adult total of about 1000 muscle fibres, derived from both primary and secondary myotubes, was reached at postnatal day 7 (PN7) so considerable generation of secondary myotubes occurred after birth. There was a linear correlation between the number of undifferentiated mononucleate cells in a muscle and the rate of formation of secondary myotubes. The major period of motoneurone death in lumbar spinal cord was during E16-E17, when axon numbers in the L4 ventral root fell from 12000 to 4000, but a discontinuity in the curve of muscle ChAT activity versus time indicated that death in the lumbrical motor pool occurred during E17-E19, after all primary myotubes had formed and before generation of secondary myotubes began. We suggest that motoneurone death, by regulating the final size of the motoneurone pool, regulates the ratio of secondary to primary myotube numbers in a muscle.
Immunochemical demonstration of the clonal organization of chimaeric mouse epidermis
Authors: Schmidt Günter H. , Bloun Margaret A. , Ponder Bruce A. J.
Published: April 2021

The clonal organization of chimaeric mouse epidermis was demonstrated by in situ staining of whole-mount preparations using monoclonal antibodies directed against H-2k and H-2b antigens. A striking pattern of transversely oriented stripes or patches was found which extended from mid-dorsum to the flank region. The orientation of these patches indicates a preferred directional expansion of clones during the development of dorsal/lateral epidermis. The clonal pattern of the belly region differed in that stripes were not found, but a marked ventral midline boundary was observed. This demarcation line may be due to a physical effect, i.e. isolation of the left and right ventral halves of the epidermis during early embryogenesis with relatively little cell mingling following closure of the abdominal wall. The obvious nonhomogenous distribution of chimaeric components in dorsal/lateral and ventral epidermis contradicts assumptions of homogenous, fine-grained patchiness derived from electrophoretic analysis of tissue samples and used in studies of skin carcinogenesis.

The observation that hair follicles may contain cells of both parental genotypes implies a polyclonal origin. Epidermal proliferative units as described by Potten (1974) were not revealed by the pattern of mosaicism at the cellular level in these chimaeric tissue sheets. This indicates that the proliferative compartment of each putative epidermal unit is polyclonal.
Do trophectoderm and inner cell mass cells in the mouse blastocyst maintain discrete lineages?
Authors: Dyce Jonathan , George Martin , Goodall Harry , Fleming Tom P.
Published: April 2021

The extent to which trophectoderm (TE) and inner cell mass (ICM) lineages in the mouse blastocyst remain distinct during the period from the commencement of cavitation up until 48 h later in culture was investigated. Fluorescent latex microparticles were used to label exclusively all TE cells in nascent blastocysts and the position of labelled progeny in cultured blastocysts was examined by disaggregation, by serial sectioning and by whole-mount analyses. The results indicate that, in most blastocysts (80–90 %), TE and ICM lineages are entirely separate during this period while in the remainder lineage crossing is limited usually to only one or two cells of either tissue.
Cytokeratin polypeptide expression during the histogenesis of guinea pig submandibular salivary gland
Authors: Marshak Gabriel , Leitner Orith , Geiger Benjamin
Published: April 2021

The present study was directed towards the characterization of cell-specific histogenetic markers for the various epithelial elements of the adult and the developing guinea pig submandibular salivary gland. We have employed immunofluorescent labelling using three cytokeratin monoclonal antibodies, for which the polypeptide specificities towards guinea pig cyto-keratins were determined. All the epithelial elements of the adult gland were positively labelled with two monoclonal antibodies, namely KG 8.13 (‘broad spectrum’ anti-cytokeratin) and antibody KsB.18 (reactive with a simple cytokeratin-specific polypeptide of 49× 103Mr). Antibody KS8.58 (reactive with a guinea pig cytokeratin polypeptide of 50×103Mr) labelled the basal cells of the large ducts, as well as the myoepithelium. During development of the gland, the submandibular anlage and its primary and secondary branches with their terminal buds, were uniformly labelled with the three antibodies; however, the cytokeratin polypeptides reactive with antibody KS8.58, which were apparently expressed in all cells of the developing ducts, gradually disappear from most of the ductal cells, starting at about 6 weeks of gestation, and remain only in the basal or reserve cells of the large ducts and the myoepithelium. These observations support the notion that the basal cells retain at least some of the properties of the embryonic glandular epithelium and could be considered as pluripotent reserve cells which may function as progenitors for other epithelial elements in the salivary glands epithelia.
Axis and germ line deficiencies caused by u.v. irradiation of Xenopus oocytes cultured in vitro
Authors: Holwill S. , Heasman J. , Crawley C. R. , Wylie C. C.
Published: April 2021

An intriguing aspect of developmental biology is the extent to which early development is controlled by the spatial arrangement of molecules in the oocyte. Ultraviolet (u.v.) irradiation of the vegetal pole of the fertilized egg of Xenopus laevis affects both the development of the embryonic dorsal axis and also the formation of primordial germ cells (PGCs). However, the importance of cytoplasmic localization in the oocyte has been difficult to assess because, until recently, it has proved impossible to mature and fertilize cultured oocytes routinely.

In this report, we describe a method for routinely maturing and fertilizing cultured oocytes of Xenopus. We find that the u.v.-sensitive period for PGC and dorsal axis formation extends back into stage-VI oocytes, thus demonstrating a true oocyte contribution to these processes. This method also allows greater time for experimental intervention and should facilitate the eventual isolation of the molecules concerned.
The development of animal cap cells in Xenopus: the effects of environment on the differentiation and the migration of grafted ectodermal cells
Authors: Jones E. A. , Woodland H. R.
Published: April 2021

We have used blastocoel and vegetal pole grafts to investigate the effect of environment on differentiation and movement of animal pole cells of Xenopus. In the blastocoel of embryos earlier than stage 10, fragments of animal pole primarily form mesoderm. The cells are either integrated into normal host tissues or they organize a secondary posterior dorsal axis. If either host or graft is later than stage 9 the graft forms ectoderm and its cells all migrate into the host ectoderm. Inner layer animal cells form sensorial layer; outer cells move to the epidermis. Thus considerable powers of appropriate movement are seen. In the vegetal pole no movement occurs. If the graft is stage 9 or earlier, or the host is stage 10| or earlier, the graft forms mesoderm, including striated muscle in the gut. This shows that muscle can develop in wholly the wrong environment, it suggests that the dorsal inductive signal from mesoderm is rather general in the vegetal mass and suggests that dorsal mesoderm development involves little subsequent adjustability. If the host is stage 11 or later, or the graft later than stage 9, the graft forms epidermis in the gut. This shows that the epidermal pathway of development is also insensitive to environment.
Distribution of yolk polypeptides in the follicle cells during the differentiation of the follicular epithelium in Sarcophaga bullata egg follicles
Authors: Geysen Johan , Cardoen Johan , Loof Arnold De
Published: April 2021

In S. bullata, the ovaries contribute to the synthesis of yolk polypeptides. A specific antiserum for yolk polypeptides was used to visualize the presence of yolk polypeptides in the follicle cells during their differentiation. After vitellogenesis has started, all follicle cells contain yolk polypeptides. The squamous follicle cells covering the nurse cells and the border cells lose yolk polypeptides before mid-vitellogenesis, whereas the follicle cells over the oocyte contain yolk polypeptides until after late vitellogenesis. All follicle cells are immunonegative afterwards. In vitro translation of poly(A)+ RNA demonstrated that the presence of yolk polypeptide mRNA correlates well with follicle cell immunopositivity for yolk polypeptides. This suggests that the follicle cells synthesize the ovarian yolk polypeptides. Differences in cellular and nuclear morphology, total and poly(A)+ RNA synthesis and the rate of yolk polypeptide synthesis were shown to be correlated with the presence or absence of yolk polypeptides in the differentiating follicular epithelium. The possible relationship between these different aspects of follicle cell differentiation, follicle cell polyploidy and the extracellular current pattern around follicles are discussed.
Ultrabithorax gene expression in Drosophila imaginal discs and larval nervous system
Authors: Brower Danny L.
Published: April 2021

Using a monoclonal antibody and image-processing procedures, the patterns of expression of the Ultrabithorax (Ubx) gene product have been characterized in Drosophila larvae. As reported previously, the metathoracic imaginal discs stain most intensely with anti-Ubx, with some mesothoracic and no prothoracic expression detectable. In the metathoracic discs, the greatest modulation in anti-Ubx staining is along the proximodistal axis. Ubx is generally expressed at higher levels in the posterior regions of metathoracic discs, although relatively high anterior expression is found in some areas. Expression in the mature wing disc is confined to the squamous peripodial membrane cells; in younger wings, Ubx expression fills the posterior half of the peripodial side of the disc. The mesothoracic leg stains with a pattern that is qualitatively similar (but not identical) to that of the metathoracic leg; Ubx is expressed in some anterior regions of the mesothoracic leg, in parasegment 4. Double staining with anti-Ubx and anti-engrailed reveals that discontinuities in Ubx expression that have been suggested to correspond to compartment borders do not coincide with the compartment boundaries in some cases. In the larval ventral ganglion, Ubx expression is greatest in parasegments 5 and 6, as in the embryonic nervous system.
Nerve-dependent accumulation of myosin light chain 3 in developing limb musculature
Authors: Merrifield Peter A. , Konigsberg Irwin R.
Published: April 2021

Myosin alkali light chain accumulation in developing quail limb musculature has been analysed on immunoblots using a monoclonal antibody which recognizes an epitope common to fast myosin light chain 1 (MLCif) and fast myosin light chain 3 (MLC3f). The limb muscle of early embryos (i.e. up to day 10 in ovo) has a MLC profile similar to that observed in myotubes cultured in vitro-, although MLC)r is abundant, MLC3r cannot be detected. MLC3f is first detected in 11-day embryos. To determine whether this alteration in MLC3f accumulation is nerve or hormone dependent, limb buds with and without neural tube were cultured as grafts on the chorioallantoic membrane of chick hosts. Although differentiated muscle develops in both aneural and innervated grafts, innervated grafts contain approximately three times as much myosin as aneural grafts. More significantly, although aneural grafts reproducibly accumulate normal levels of MLCif, they fail to accumulate detectable levels of MLC3f. In contrast, innervated grafts accumulate both MLCif and MLC3f, suggesting that the presence of neural tube in the graft promotes the maturation, as well as the growth, of muscle tissue. This is the first positive demonstration that innervation is necessary for the accumulation of MLC3f that occurs during normal limb development in vivo.
Immunolocalization of three oocyte nuclear proteins during oogenesis and embryogenesis inPleurodeles
Authors: Abbadie C. , Boucher D. , Charlemagne J. , Lacroix J. C.
Published: April 2021

The location of three proteins of the oocyte nucleus of Pleurodeles was studied during oogenesis and embryogenesis using monoclonal antibodies A33/22, C3/1 and C36/1. Immunoblotting of two-dimensional gel electophoregrams of oocyte nuclear proteins showed that these antibodies recognized proteins whose relative molecular masses and isoelectric points were 80X103 and 6 4, 175×103 and 5 and 270×103 and 7, respectively. In the oocyte, all three proteins were nucleoplasmic; those revealed by antibodies A33/22 and C36/1 were detected on lampbrush chromosomes: the first one on the RNP matrix of the loops, and the second one on both the loops and the chromomeres. Protein A33/22 was observed in most nuclei during embryonic, larval and adult development, except for the young embryo, before the midblastula transition. The distribution of this protein in the oocyte and its behaviour during development suggest that it might be involved in the packaging of RNAs during transcription. Antibody C3/1 recognized an oocyte nucleoplasmic protein with biochemical and biophysical properties similar to those of protein N1-N2, After oocyte maturation, the protein moved into the cytoplasm of the animal hemisphere and, from fertilization to the midblastula stage, it shifted from the cytoplasm into the nuclei as cell division proceeded. Starting from the gastrula stage, this protein became specific to the endoderm nuclei. After hatching, it was no longer detectable. This behaviour seems to correspond to that of a nuclear protein issued from the maternal stock pile. Protein C36/1 behaved similarly during early development, but remained in most nuclei after neurulation until the adult age, with a pattern similar to that of protein A33/22. In addition, it was present on the mitotic chromosomes. Its association with mitotic as well as lampbrush chromosomes connects it with the DNP fibre proteins.
Avoidance of posterior tectal membranes by temporal retinal axons
Authors: Walter Jochen , Henke-Fahle Sigrid , Bonhoeffer Friedrich
Published: April 2021

Membrane carpets consisting of alternating membrane stripes were prepared from plasma membranes of anterior and posterior chick optic tectum. Axons from retinal explants extend neurites on these carpets. Axons of the nasal retina do not distinguish between the stripes. Axons of the temporal retina prefer to extend neurites on anterior tectal membranes.

Treatment of the membrane fragments with high temperature interferes with the pattern of neurite outgrowth from temporal axons. When growing on carpets consisting of treated anterior and posterior tectal membranes, temporal retinal axons no longer distinguish between the stripes. Treatment of posterior membranes alone is sufficient to abolish the preference of temporal axons to extend neurites on anterior tectal membranes. Treatment of the anterior membranes alone has no effect.

This result is best explained by a repulsive component in the posterior tectal membranes. Temporal, but not nasal, axons specifically recognize and avoid that component, with the result that they do not extend neurites on posterior tectal membrane stripes. Once the repulsive component is destroyed, temporal axons are able to extend neurites on posterior tectal membranes.
Expression and segregation of nucleoplasmin during development in Xenopus
Authors: Litvin Judith , King Mary Lou
Published: April 2021

The spatial segregation of informational molecules in the unfertilized egg and embryo has been hypothesized to be a necessary phenomenon for the normal pro-gression of development leading to the determination of cellular phenotypes. This study describes the selec-tion of a monoclonal antibody (Mab:2G6) that ident-ifies an antigen (Ag:2G6) which is localized in the germinal vesicle of oocytes and has a discrete pattern of inheritance during embryogenesis. The antigen displayed biochemical and physical characteristics very similar to nucleoplasmin, which is the histone-binding and nucleosome-assembly protein previously described. Immunoblot analysis with purified oocyte nucleoplasmin confirmed this relationship.

Indirect immunofluorescence was used to study the temporal expression and spatial distribution of nucleo-plasmin. From early cleavage stages through gastru-lation, it is preferentially localized in nuclei of blasto-meres at the animal pole. By tadpole stages, it was detected only in nuclei of postmitotic cells of the central nervous system and in nuclei of striated muscle. It was not detected in adult tissues. Western blot analysis during embryogenesis revealed at least five immunologically related polypeptides that dis-played distinct patterns of expression during develop-ment. The different species observed most likely represent different levels of phosphorylation of nu-cleoplasmin. The more acidic forms, known to be more active in nucleosome assembly, were present during cleavage stages.

Analysis of labelled oocyte proteins by two-dimen-sional immunoblots and autoradiography revealed that synthesis of nucleoplasmin was first detected in stage-2 oocytes, reached 60 % maximum levels at stage 3, peaked at stage 4 and was undetectable in stage-6 oocytes. The amount of nucleoplasmin message pres-ent does not follow a similar pattern during oogenesis. These results suggest that the message undergoes pronounced changes in translational efficiency during oogenesis.

A comparative immunoblot analysis using proteins from a variety of adult tissues revealed that, whereas the polyclonal antisera against amphibian vitellogenic oocyte nucleoplasmin recognized several different, tissue-specific polypeptides, two different monoclonal antibodies (Mab:b7-1D1, Mab:2G6) failed to recog-nize any of the adult tissues tested. We conclude that nucleoplasmin is a family of closely related proteins with distinct embryonic and adult members.
Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA
Authors: Franks Roberta R. , Hough-Evans Barbara R. , Britten Roy J. , Davidson Eric H.
Published: April 2021

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32 % versus 8 %) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36 % of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12 % of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmeta-morphic juveniles was usually less than 0·1 (range 0·05–50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment.
Temporal and spatial pattern of differences in microtubule behaviour during Drosophila embryogenesis revealed by distribution of a tubulin isoform
Authors: Wolf Nurit , Regan Cathy L. , Fuller Margaret T.
Published: April 2021

Immunofluorescence staining of Drosophila embryos with a monoclonal antibody specific for acetylated α tubulin has revealed that acetylated and nonacetylated a-tubulin isoforms have different patterns of distribution during early development. Acetylated α-tubulin was not detected in either interphase or mitotic spindle microtubules during the rapid early cleavage or syncytial blastoderm divisions. Acetylated α-tubulin was first observed as interphase lengthened at the end of syncytial blastoderm, and at cycle 14 was localized to a ring of structures clustered around the interphase nuclei. These structures probably represent a set of stable microtubules involved in nuclear elongation. Absence of detectable acetylated α-tubulin prior to cellular blastoderm seems to be due to rapid turnover of microtubule arrays rather than to lack of the enzyme required for modification, since acetylated α-tubulin appeared in early embryos when microtubules were stabilized by taxol treatment or anoxia. Because acetylated α-tubulin seems to be characteristic of stable microtubule arrays, the appearance of the antigen at cycle 14 represents a fundamental change in microtubule behaviour in the somatic cells of the embryo. Acetylated α-tubulin was not detected in pole cells during the blastoderm or early gastrula stages, indicating that acetylation of α-tubulin is not merely a consequence of cellularization.

After the onset of gastrulation, interphase microtubule arrays in most cell types contain acetylated α-tubulin. However, cells in mitosis lack antibody staining. The resulting unstained patches reveal the stereotyped spatial pattern of cell division during gastrulation. Although the cells that give rise to the amnioserosa have acetylated α-tubulin in their interphase arrays at early gastrulation, by germ band elongation these large, plastic cells completely lack staining with anti-acetylated α-tubulin. In contrast, differentiated cell types such as neurones, which have arrays of stable axonal microtubules, stain brightly with the specific antibody. Although acetylated and nonacetylated α-tubulin are present in roughly equal amounts by the late stages of embryogenesis, acetylated α-tubulin is partitioned into the pellet during centrifugation of extracts of embryos homogenized at 4°C.
Regulation of neurotransmitter metabolic enzymes and tyrosine hydroxylase mRNA level by nerve growth factor in cultured sympathetic neurones
Authors: Raynaud Brigitte , Faucon-Biguet Nicole , Vidal Simone , Mallet Jacques , Weber Michel J.
Published: April 2021

The survival of new-born rat sympathetic neurones in culture was increased in a dose-dependent manner by 7S nerve growth factor (NGF). NGF also increased, in a parallel manner, the specific activities of tyrosine hydroxylase (TOH) and choline acetyltransferase (CAT). Total acetylcholinesterase (AcChE) activity increased with NGF concentration, although less distinctly than TOH and CAT. However, NGF caused a large induction of the asymmetric A12 form of AcChE, and to a lesser extent of the globular G, and G2 forms, whereas the globular G4 form was little affected. This suggests that NGF differentially regulates the syn-thesis and/or assembly of the various AcChE molecular forms.

The levels of TOH mRNA in neurone cultures grown with increasing NGF concentrations were measured by Northern blot analysis with a rat cDNA probe. To correct for variations in the total mass of RNA per neurone, the filters were rehybridized with an 18S rRNA probe. The level of TOH mRNA, measured by the ratio (TOH: 18S) of the hybridization signals increased 3·4-fold between 92 and 740 ng ml-1 7S NGF. Increases of TOH specific activity of the same order of magnitude were observed in sister cultures. The deficit in the level of mature TOH mRNA at low NGF concentration was not accompanied by a compensatory accumulation in unprocessed TOH transcripts. As TOH induction is insensitive to RNA polymerase inhibitors, we suggest that NGF regulates the maturation of TOH pre-mRNAs, and that the unprocessed transcripts are rapidly degraded. The long-term regulation of TOH by NGF may thus constitute a case of process-versus-discard control, as defined by J. E. Darnell.
Colour pattern regulation after surgery on the wing disks of Precis coenia (Lepidoptera: Nymphalidae)
Authors: Nijhout H. Frederik , Grunert Laura W.
Published: April 2021

Partial ablations were done in situ on the imaginai disks of the hindwing in larvae of Precis coenia at ages between 2 and 9 days prior to pupation. While there was no regeneration of the wing lamina, the cut edge developed normal marginal scales and a marginal colour pattern if the ablation was done more than 3-5 days prior to pupation. The response of elements of the marginal colour pattern to partial ablation of the wing disk indicates that the wing margin has an important role in colour pattern determination and appears to act as a sink for a pattern-inducing signal. While the elements of the marginal colour pattern regulate to the shape and position of the new wing margin, the eyespots changed their shape and size but not their position upon partial ablation of the wing disk. When a cut was positioned near one of the dorsal eyespots, the outer rings of the eyspot opened up so that its central field became contiguous with the new margin. The behaviour of the dorsal eyespots of the hindwing in response to ablation of the wing disk, as well as to other developmental disturbances, appears to be the reverse of those on the forewing and ventral hindwing. We conclude that the central field of a dorsal eyespot and the wing margin share similar controlling properties with respect to pattern, and that both appear to act as sinks or as the inverse of the sources of pattern-inducing signal found in the eye-spots of the forewing.
Sequential activation of butyrylcholinesterase in rostral half somites and acetylcholinesterase in motoneurones and myotomes preceding growth of motor axons
Authors: Layer Paul G. , Alber Regina , Rathjen Fritz G.
Published: April 2021

By applying double-staining procedures that combine cholinesterase histochemistry (acetyl- and butyrylcholinesterase, respectively) as indicators of neuronal and myotomal tissue differentiation on longitudinal sections, together with detection of motor axons with antibodies to G4 antigen, we here describe the spatiotemporal expression of all components of the segmental motor units along the trunk of chicken embryos between stages 16–20.

In particular, BChE expression is spatially elevated on the rostral part of the differentiating somite. About 2–3 somites more rostrally (and thus developmentally later), AChE is expressed almost simultaneously in a nonsegmented fashion in neuronal cell bodies of the ventral horn and in the corresponding dermomyo-tomes. There it is first detectable in a rostromedial sector. With a delay (4—6 somites compared with AChE in motoneurones), motor axons begin to grow exclusively through the BChE-rich sclerotomal space towards the AChE-activated myotome anlage. On motor axons, AChE detection is significantly retarded.

We conclude that the rostrocaudal segmental asymmetry is not restricted to the sclerotomes (which other authors have described before by using different markers), but it extends into the dermomyotome, in which cholinesterases introduce an early subdivision. Hence, the entire process of first myotome differentiation, motor axon growth and establishment of first target contacts are taking place within the rostral half somite. We suggest that both cholinesterases might be involved in processes of motor unit differentiation and fibre guidance.

Abbreviations: AChE, acetylcholinesterase (EC 3.1.1.7); AChR, acetylcholine receptor; BChE, butyryl- or pseudo-, or serumcholinesterase (EC 3.1.1.8); BW 284C51, 1,5-bis(4-allyldimethylammoniumphenyl)pentane-3-one dibromide = specific inhibitor of AChE; ChE, cholinesterase; iso-OMPA, tetraisopropylpyrophosphor-amide = specific inhibitor of BChE; NCC, neural crest cells: PBS, phosphate-buffered saline; TBS, Tris-buffered saline; ATC, acetylthiocholine; BTC, butyrylthiocholine.