bluuebunny/crossref_metadata_2025_split huggingface dataset:
Corneal epithelial-specific cell surface antigen recognized by a monoclonal antibody
Authors: Langer Marian G. , Sundarraj C. V. , Sundarraj Nirmala 
Published: April 2021

ABSTRACT
Monoclonal antibodies, specific against cell surface differentiation antigens of human corneal epithelial cells, were developed using epithelial cells resected from human corneas as the immunogens. One of these antibodies reacted specifically with corneal epithelial cells and not with epithelial cells of other tissues when tested by an indirect immunoperoxidase technique. Nonidet P-40 extracts of different subcellular fractions of human corneal epithelial cells were tested for their reactivity against this antibody using an enzyme-linked immunosorbent assay. The results indicated that the antigen recognized by this antibody is associated with the plasma membrane. This was further verified by immuno-electron-microscopic analysis using ferritin-conjugated anti-mouse IgG antibody. This antigen was not detectable in the corneal epithelial cells in primary cultures nor in the epithelial cells from early stages of developing cornea (12 to 18 weeks in utero) but was present in the epithelial cells in the corneas of an 8-month-old infant. Therefore, this surface-associated antigen identified in the present study is a developmentally regulated marker of human corneal epithelium.
The nature of developmental restrictions in Xenopus laevis embryos
Authors: Heasman Janet , Snape Alison , Smith J. C. , Wylie C. C. 
Published: April 2021

ABSTRACT
Fate maps of the late blastula stage of the Xenopus laevis embryo indicate that the cells of the vegetal pole area are destined to become part of the endoderm germ layer (Keller, 1975; Heasman, Wylie, Hausen & Smith, 1984). By labelling single cells from this region and transplanting them into the blastocoel cavity of host embryos, we have shown that the determinative process that restricts blastomeres to this their normal fate occurs between the early blastula and early gastrula stages (Heasman et al. 1984). To progress towards an understanding of this process, we need to establish some fundamental points. In particular, the following issues are discussed here. (1) Is cell interaction required for determination to proceed? (2) What is the cellular nature of determination?
We have used the labelling and transplantation technique described previously (Heasman, Snape, Smith & Wylie, 1985; Heasman, Snape, Smith, Holwill & Wylie, 1985) to study these questions in relation to the mechanism of determination of vegetal pole cells in Xenopus laevis.
Regeneration in the anterior—posterior axis of the insect thoracic segment
Authors: French Vernon , Rowlands Tamara F. 
Published: April 2021

ABSTRACT
After removal of a transverse strip of ventral thorax from the beetle, Tenebrio molitor, interaction occurred between epidermis posterior to the mesothoracic leg and that anterior to the metathoracic leg. Depending on the size and position of the excision, this interaction resulted in either the regeneration of the extirpated tissue or its replacement by an A/P reversed pattern of sclerites and supernumerary leg. By either route, local pattern continuity was restored between the normal meso- and metathoracic legs. Similarly, when a leg plus adjacent tissue was extirpated, continuity was restored by leg regeneration or by formation of an A/P reversed duplication of sclerites.
The results of these strip excisions can be understood in terms of two current models of the ventral thorax (the Boundary Model and the Polar Coordinate Model), each of which postulates a distinct compartment or region intervening between the epidermis surrounding the bases of successive legs. However, the models do not explain the large differences in the frequency of formation of the duplication/deletion pattern after excisions of different widths. The results are also compatible with a different model, involving an A-P sequence of positional values similar to that proposed for the abdominal segment. Regeneration would restore continuity within the sequence by the shortest route, forming either the midsegment (including the leg) or the intersegmental region.
The meso- and metathorax differ in the structure of the ventral sclerites and in the segmentation of the tarsus of the leg. The structures regenerated after the various excisions show that the segment border is not crossed during regeneration and indicate that an A/P compartment border running through the leg is usually also respected. There is no sign, however, of a third line of lineage restriction that would indicate a subdivision of the segment into three compartments (as proposed in the Boundary Model).
Stimulatory effects of insulin-like growth factors on DNA synthesis in the human embryonic cornea
Authors: Hyldahl Louise , Engström Wilhelm , Schofield Paul N. 
Published: April 2021

ABSTRACT
10- to 12-week-old human embryonic eye globes were microdissected so that a passage was opened between the outer environment and the anterior chamber which rendered free access of tissue culture medium to the endothelial cell monolayer. The dissected eye globes were maintained in organ culture for 24 h in the continuous presence of tritiated thymidine. Cross sections were cut through the whole eye globes and subjected to autoradiographic analysis in order to estimate the mitogenic response of human embryonic corneal endothelial cells to externally supplied growth factors and hormones.
It was found that the corneal endothelial cells could be stimulated to initiate DNA synthesis by exposure to insulin-like growth factor I (IGF-I). The thymidine-labelling index doubled after IGF-I supplementation.
Northern blot analysis revealed the abundant presence of IGF-II transcripts in the posterior eye. In contrast, the anterior portion of the eye, including the cornea, contains barely detectable levels of IGF-II transcripts. IGF-I transcripts were detected in both parts of the eye at much lower concentrations than those for IGF-II. No insulin transcripts were found. These results demonstrate that mRNA for both IGF-I and IGF-II is present in the late first trimester eye. The observed stimulatory effects of IGF-I in organ culture suggest that local production of IGF-I and IGF-II may stimulate cell proliferation in vivo.
The development of XO gynogenetic mouse embryos
Authors: Mann Jeff R. , Lovell-Badge Robin H. 
Published: April 2021

ABSTRACTDiploid gynogenetic embryos, which have two sets of maternal and no paternal chromosomes, die at or soon after implantation. Since normal female embryos preferentially inactivate the paternally derived X chromosome in certain extraembryonic membranes, the inviability of diploid gynogenetic embryos might be due to difficulties in achieving an equivalent inactivation of one of their two maternally derived X chromosomes. In order to investigate this possibility, we constructed XO gynogenetic embryos by nuclear transplantation at the 1-cell stage. These XO gynogenones showed the same mortality around the time of implantation as did their XX gynogenetic counterparts. This shows that the lack of a paternally derived autosome set is sufficient to cause gynogenetic inviability at this stage. Autosomal imprinting and its possible relation to X-chromosome imprinting is discussed.
Cell proliferation is not required for the initiation of early cleft formation in mouse embryonic submandibular epithelium in vitro
Authors: Nakanishi Yasuo , Morita Toshiteru , Nogawa Hiroyuki 
Published: April 2021

ABSTRACT
An X-ray irradiation method was employed to analyse the role of cell proliferation in vitro in the cleft formation of mouse embryonic submandibular epithelium at early stages. When the mid 12-day gland was exposed to 200 rad of X-rays, the growth was severely retarded. In contrast, late 12-day and early 13-day glands grew apparently in a normal fashion, as did the control gland, for up to 40 h. In either case, they formed shallow clefts within 10 h of culture. With 1000 rad irradiation, the mid 12-day gland did not grow at all, but formed clefts within 20 h of culture followed by a rapid degeneration. Under, the same conditions, the growth of the late 12-day gland, which was at the stage just before branching, was retarded until 10 h of culture, followed by a slight increase in epithelial size, but cleft formation was also observed within 6-10 h, as in the control gland. When exposed to a dose of 1000 rad of X-rays, the early 13-day and the late 12-day glands exhibited similar radiosensitivity; the initial narrow clefts in the epithelium deepened and new clefts began to form within 6-10 h of culture. [3H]thymidine incorporation studies revealed that a dose of 1000 rad reduced DNA synthesis of mid and late 12-day glands by 72 and 65 %, respectively. Histological examination of X-irradiated late 12-day gland showed that mitotic figures were rarely seen in the epithelium at 6 h of culture. Aphidicolin, a specific inhibitor of DNA synthesis, could not halt the cleft formation of the late 12-day gland. In this experiment 89 % of DNA synthesis was inhibited. Treatment of an X-ray irradiated late 12-day gland with aphidicolin blocked 92 % of the DNA synthesis, but did not prevent cleft formation taking place. These results indicate that neither cell division nor DNA synthesis, is required for the initiation process of the cleft formation of the mouse embryonic submandibular epithelium at early morphogenetic stages in vitro.
A monoclonal antibody detects a difference in the cellular composition of developing and regenerating limbs of newts
Authors: Fekete Donna M. , Brockes Jeremy P. 
Published: April 2021

AbstractWe have previously described a monoclonal antibody (called 22/18) that reacts with the early blastemal cells of the regenerating limb of the newt (Notophthal-mus viridescens). In embryos of two newt species the antibody reacts with the epidermis, glial cells in the neural tube, the lens and cells in a restricted region of the aorta. In the developing limb bud less than 1 % of the mesenchymal cells were reactive with 22/18, although most cells stained brightly with an antibody to another cytoskeletal component. When limbs were amputated prior to the arrival of nerves (axons and Schwann cells) at the amputation plane there was no extra reactivity with 22/18 as compared to the contra-lateral unamputated control, even though the amputated buds regenerated satisfactorily. Limbs amputated after nerves are present at the plane of amputation respond by forming a 22/ 18-positive blastema. The appearance of the 22/18 response is a function of the stage of limb development as shown by amputation of forelimb and hindlimb buds at a larval stage where development of the forelimb is greatly advanced relative to the hindlimb. The distribution of the 22/18-positive cells in larval blastemas showed them to be closely associated with axons as detected by double staining with an antiserum to a neurofilament subunit. The clear antigenic difference between development and regeneration may be related to the relationship between embryonic regulation and epimorphic regeneration, and also to the acquisition of nerve-dependent proliferation of blastemal cells.
Recycling of the yeast V-SNARE Sec22p involves COPI-proteins and the ER transmembrane proteins Ufe1p and Sec2Op
Authors: Ballensiefen Wolfgang , Ossipov Dmitri , Schmitt Hans Dieter 
Published: April 2021

ABSTRACT
Vesicle-specific SNAP receptors (v-SNAREs) are believed to cycle between consecutive membrane compartments. The v-SNARE Sec22(Sly2)p mediates the targeting of vesicles between endoplasmic reticulum (ER) and early Golgi of Saccharomyces cerevisiae. To analyze factors involved in targeting of Sec22(Sly2)p, an α-factor-tagged Sec22 protein (Sec22-α) was employed. Only on reaching the late Golgi, can α-factor be cleaved from this hybrid protein by Kex2p, a protease localized in this compartment. In wild-type cells Kex2p-cleavage is observed only when Sec22-α is greatly overproduced. Immunofluorescence microscopy and subcellular fractionation studies showed that Sec22-α is returned to the ER from the late Golgi (Kex2p) compartment. When Sec22-α is expressed in wild-type cells at levels comparable to the quantities of endogenous Sec22p, very little of this protein is cleaved by Kex2p. Efficient cleavage, however, occurs in mutants defective in the retrograde transport of different ER-resident proteins indicating that Sec22-α rapidly reaches the late Golgi of these cells. These mutants (sec20-1, sec21-1, sec27-1 and ufe1-1) reveal Golgi structures when stained for Sec22-α and do not show the ER-immunofluorescence observed in wild-type cells. These results show consistently that Sec22p recycles from the Golgi back to the ER and that this recycling involves retrograde COPI vesicles.
Novel features of intermediate filament dynamics revealed by green fluorescent protein chimeras
Authors: Ho Chung-Liang , Martys Jayme L. , Mikhailov Alexei , Gundersen Gregg G. , Liem Ronald K. H. 
Published: April 2021

ABSTRACT
In order to study the dynamic behavior of intermediate filament networks in living cells, we have prepared constructs fusing green fluorescent protein to intermediate filament proteins. Vimentin fused to green fluorescent protein labeled the endogenous intermediate filament network. We generated stable SW13 and NIH3T3 cell lines that express an enhanced green fluorescent protein fused to the N-terminus of full-length vimentin. We were able to observe the dynamic behavior of the intermediate filament network in these cells for periods as long as 4 hours (images acquired every 2 minutes). In both cell lines, the vimentin network constantly moves in a wavy manner. In the NIH3T3 cells, we observed extension of individual vimentin filaments at the edge of the cell. This movement is dependent on microtubules, since the addition of nocodazole stopped the extension of the intermediate filaments. Injection of anti-IFA causes the redistribution or ‘collapse’ of intermediate filaments. We injected anti-IFA antibodies into NIH3T3 cells stably expressing green fluorescent protein fused to vimentin and found that individual intermediate filaments move slowly towards the perinuclear area without obvious disassembly. These results demonstrate that individual intermediate filaments are translocated during the collapse, rather than undergoing disassembly-induced redistribution. Injections of tubulin antibodies disrupt the interactions between intermediate filaments and stable microtubules and cause the collapse of the vimentin network showing that these interactions play an important role in keeping the intermediate filament network extended. The nocodazole inhibition of intermediate filament extension and the anti-IFA microinjection experiments are consistent with a model in which intermediate filaments exhibit an extended distribution when tethered to microtubules, but are translocated to the perinuclear area when these connections are severed.
Actomyosin motor in the merozoite of the malaria parasite, Plasmodium falciparum: implications for red cell invasion
Authors: Pinder J. C. , Fowler R. E. , Dluzewski A. R. , Bannister L. H. , Lavin F. M. , Mitchell G. H. , Wilson R. J. M. , Gratzer W. B. 
Published: April 2021

ABSTRACT
The genome of the malaria parasite, Plasmodium falciparum, contains a myosin gene sequence, which bears a close homology to one of the myosin genes found in another apicomplexan parasite, Toxoplasma gondii. A polyclonal antibody was generated against an expressed polypeptide of molecular mass 27,000, based on part of the deduced sequence of this myosin. The antibody reacted with the cognate antigen and with a component of the total parasite protein on immunoblots, but not with vertebrate striated or smooth muscle myosins. It did, however, recognise two components in the cellular protein of Toxoplasma gondii. The antibody was used to investigate stage-specificity of expression of the myosin (here designated Pf-myo1) in P. falciparum. The results showed that the protein is synthesised in mature schizonts and is present in merozoites, but vanishes after the parasite enters the red cell. Pf-myo1 was found to be largely, though not entirely, associated with the particulate parasite cell fraction and is thus presumably mainly membrane bound.
It was not solubilised by media that would be expected to dissociate actomyosin or myosin filaments, or by non-ionic detergent. Immunofluorescence revealed that in the merozoite and mature schizont Pf-myo1 is predominantly located around the periphery of the cell. Immuno-gold electron microscopy also showed the presence of the myosin around almost the entire parasite periphery, and especially in the region surrounding the apical prominence. Labelling was concentrated under the plasma membrane but was not seen in the apical prominence itself. This suggests that Pf-myo1 is associated with the plasma membrane or with the outer membrane of the subplasmalemmal cisterna, which forms a lining to the plasma membrane, with a gap at the apical prominence. The results lead to a conjectural model of the invasion mechanism.
Expression and characterization of splice variants of PYK2, a focal adhesion kinase-related protein
Authors: Xiong Wen-Cheng , Macklem Marlene , Parsons J. Thomas 
Published: April 2021

ABSTRACT
Focal adhesion kinase and the recently identified proline-rich tyrosine kinase 2 (PYK2), also known as cell adhesion kinase β, related adhesion focal tyrosine kinase or calcium-dependent protein tyrosine kinase, define a new family of non-receptor protein tyrosine kinases. Activation of PYK2 has been implicated in multiple signaling events, including modulation of ion channels, T- and B-cell receptor signaling and cell death. Mechanisms underlying the functional diversity of PYK2 are unclear. Here, we provide evidence for two novel alternatively expressed isoforms of PYK2. One isoform, designated PYK2s (PYK2 splice form), appears to be a splice variant of PYK2 lacking 42 amino acids within the C-terminal domain. A second isoform, referred to as PRNK (PYK2-related non-kinase), appears to be specified by mRNAs that encode only part of the C-terminal domain of PYK2. Northern blot analysis indicates that the unspliced PYK2 is expressed at high levels in the brain and poorly expressed in the spleen, whereas PYK2s and PRNK are expressed in the spleen. In situ hybridization studies of rat brain demonstrate that the unspliced PYK2 is selectively expressed at high levels in hippocampus, cerebral cortex and olfactory bulb, whereas PYK2s and PRNK are expressed at low levels in all regions of rat brain examined. Immunofluorescence analysis of ectopically expressed PRNK protein shows that PRNK, in contrast to full-length PYK2, is localized to focal adhesions by sequences within the focal adhesion targeting domain. In addition, PYK2, but not PRNK, interacts with p130casand Graf. These results imply that PRNK may selectively regulate PYK2 function in certain cells by binding to some but not all PYK2 binding partners, and the functional diversity mediated by PYK2 may be due in part to complex alternative splicing.
Vesicle-associated brain myosin-V can be activated to catalyze actin-based transport
Authors: Evans L. L. , Lee A. J. , Bridgman P. C. , Mooseker M. S. 
Published: April 2021

ABSTRACT
Myosin-V has been linked to actin-based organelle transport by a variety of genetic, biochemical and localization studies. However, it has yet to be determined whether myosin-V functions as an organelle motor. To further investigate this possibility, we conducted a biochemical and functional analysis of organelle-associated brain myosin-V. Using the initial fractionation steps of an established protocol for the purification of brain myosin-V, we isolated a population of brain microsomes that is approx. fivefold enriched for myosin-V, and is similarly enriched for synaptic vesicle proteins. As demonstrated by immunoelectron microscopy, myosin-V associates with 30-40% of the vesicles in this population. Although a majority of myosin-V-associated vesicles also label with the synaptic vesicle marker protein, SV2, less than half of the total SV2-positive vesicles label with myosin-V. The average size of myosin-V/SV2 double-labeled vesicles (90±45 nm) is larger than vesicles that label only with SV2 antibodies (60±30 nm). To determine if these vesicles are capable of actin-based transport, we used an in vitro actin filament motility assay in which vesicles were adsorbed to motility assay substrates. As isolated, the myosin-V-associated vesicle fraction was nonmotile. However, vesicles pre-treated with ice-cold 0.1% Triton X-100 supported actin filament motility at rates comparable to those on purified myosin-V. This dilute detergent treatment did not disrupt vesicle integrity. Furthermore, while this treatment removed over 80% of the total vesicle proteins, myosin-V remained tightly vesicle-associated. Finally, function-blocking antibodies against the myosin-V motor domain completely inhibited motility on these substrates. These studies provide direct evidence that vesicle-associated myosin-V is capable of actin transport, and suggest that the activity of myosin-V may be regulated by proteins or lipids on the vesicle surface.
Compensation of Hypercapnic Acidosis in the Aquatic Blue Crab, Callinectes Sapidus: The Predominance of External Sea Water Over Carapace Carbonate as the Proton Sink
Authors: Cameron James N. 
Published: April 2021

ABSTRACT
Radioactive labelling of the CaCO3 in the crab’s carapace was employed as a tool to study the contribution of the carapace carbonates to acute buffering of acid-base disturbances. Since Ca2+ uptake is extremely rapid during the post-moult period, crabs that moulted in the laboratory were incubated with 45Ca for 5 days immediately following the moult in order thoroughly to load the carapace carbonate pool with radiolabel. After a subsequent 2-week interval for feeding and completion of the post-moult carapace mineralization phase, these 45Ca-loaded crabs were subjected to a 24 h control period and a 24 h hypercapnic period (water equilibrated with 2% CO2 in air) to induce an internal acidosis. Compared with the control period, a compensatory increase in [HCO3−] of 17 mequivl−1 was observed in the blood, along with an apparent H+ excretion to the external sea water of 10-8 mequivkg−1. A statistically significant increase in circulating [Ca2+] and in the specific radioactivity of the blood Ca2+ reached a maximum during the first 3 h of the compensatory phase. By measuring the radioactivity appearing in the water and the blood, and the specific radioactivity of the carapace carbonates, the contribution of the carapace carbonates was calculated to be 7·5% of the total compensatory H+ disposal. The rapid exchange of Ca2+ with the external medium, coupled with physiological regulation of blood [Ca2+] minimized changes in blood [Ca2+]. The total dissolution of carapace CaCO3 was approximately 0·9 mequivkg−1, less than 0·1% of the quantity contained in the shell. The carapace carbonates, therefore, do contribute to acute buffering of hypercapnic acidosis, but their quantitative importance is small, with the gills serving to conduct most of the compensatory exchanges with the environment.