85 research outputs found
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Internal iamin structures within G1 nuclei of human dermal fibroblasts
The nuclear lamina is a mesh-like network of fibres subjacent
to the inner nuclear membrane that is believed
to be involved in the specific spatial reorganisation of
chromatin after mitosis. To determine how the lamina
might be involved in chromatin reorganisation, we have
performed indirect immunofluorescence studies on quiescent
and proliferating human dermal fibroblasts
(HDF). Two monoclonal antibodies recognising human
lamins A and C and three different fixation methods
were employed. In indirect immunofluorescence studies,
cultures of quiescent cells displayed a uniform perinuclear
distribution of the antibodies. In proliferating cultures
two distinct populations of cells were observed:
one population displayed a typical perinuclear antibody
distribution, while the second population displayed an
unusual pattern consisting of a series of spots and fibres
within the nucleus. By inducing cell-cycle synchrony in
cultures we were able to determine that the unusual
internal distribution of the lamin antibodies was
restricted to cells in G1. Optical sectioning and 3-D
reconstruction of the lamina structures in G1 nuclei was
performed with a confocal laser scanning microscope
(CLSM). This revealed that the internal lamin structures
consisted of small foci and fibres proliferating
throughout the nucleus. These structures were shown to
be closely associated with areas of condensed chromatin
but not nuclear membrane. As cells progress towards S
phase the internal lamin foci disappear
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Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication
The major residual structure that remains associated with
the nuclear envelope following extraction of isolated nuclei
or oocyte germinal vesicles with non-ionic detergents,
nucleases and high salt is the lamina (Fawcett, 1966;
Aaronson and Blobel, 1975; Dwyer and Blobel, 1976). The
nuclear lamina is composed of intermediate filament
proteins, termed lamins (Gerace and Blobel, 1980; Shelton
et al., 1980), which polymerise to form a basket-weave
lattice of fibrils, which covers the entire inner surface of the
nuclear envelope and interlinks nuclear pores (Aebi et al.,
1986; Stewart and Whytock, 1988; Goldberg and Allen,
1992). At mitosis, the nuclear envelope and the lamina both
break down to allow chromosome segregation. As a consequence,
each structure has to be rebuilt during anaphase
and telophase, allowing cells an opportunity to reposition
chromosomes (Heslop-Harrison and Bennett, 1990) and to
reorganise looped chromatin domains (Franke, 1974;
Franke et al., 1981; Hochstrasser et al., 1986), which may
in turn control the use of subsets of genes. Because of the
position that it occupies, its dynamics during mitosis and
the fact that it is an essential component of proliferating
cells, the lamina has been assigned a number of putative
roles both in nuclear metabolism and in nuclear envelope
assembly (Burke and Gerace, 1986; Nigg, 1989). However,
to date there is little clear cut evidence that satisfactorily
explains the function of the lamina in relation to its
structure. In this Commentary we will describe some of the
recent work that addresses this problem and attempt to
provide a unified model for the role of lamins in nuclear
envelope assembly and for the lamina in the initiation of
DNA replication
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The timing of the formation and usage of replicase clusters in S-phase nuclei of human diploid fibroblasts
The sites of nascent DNA synthesis were compared
with the distribution of the proliferating cell nuclear antigen (PCNA) in S-phase nuclei of human diploid fibroblasts (HDF) by two in vitro techniques. Firstly, proliferating fibroblasts growing in culture that had been synchronised at S-phase were microinjected with the thymidine analogue biotin-11-dUTP. The sites of incorporation of biotin into injected cells
were compared with the distribution of PCNA by
indirect immunofluorescence microscopy and laser
scanning confocal microscopy (LSCM). In common
with other studies, a progression of patterns for both biotin incorporation and PCNA localisation was observed. However, we did not always observe
coincidence in these patterns, the pattern of biotin incorporation often resembling the expected, preceding distribution of PCNA. In nuclei in which the pattern of biotin incorporation appeared to be identical to the distribution of PCNA, LSCM revealed that not all of the sites of PCNA immunofluorescence were incorporating biotin at the same time. Secondly,
nuclei which had been isolated from quiescent
cultures of HDF were innoculated into cell-free
extracts of Xenopus eggs which support DNA replication in vitro. Following innoculation into these extracts DNA replication was initiated in each nucleus. The sites of DNA synthesis were detected by biotin-11-dUTP incorporation and compared with the distribution of PCNA by indirect immunofluorescence. Only a single pattern of biotin incorporation and PCNA distribution was observed. PCNA accumulated
at multiple discrete spots some 15min before any biotin incorporation was observed. When biotin incorporation did occur, LSCM revealed almost complete coincidence between the sites of DNA synthesis and the sites at which PCNA was localised.Brunel Open Access Publishing Fun
Influence of the initial chemical conditions on the rational design of silica particles
The influence of the water content in the initial composition on the size of silica particles produced using the Stöber process is well known. We have shown that there are three morphological regimes defined by compositional boundaries. At low water levels (below stoichiometric ratio of water:tetraethoxysilane), very high surface area and aggregated structures are formed; at high water content (>40 wt%) similar structures are also seen. Between these two boundary conditions, discrete particles are formed whose size are dictated by the water content. Within the compositional regime that enables the classical Stöber silica, the structural evolution shows a more rapid attainment of final particle size than the rate of formation of silica supporting the monomer addition hypothesis. The clearer understanding of the role of the initial composition on the output of this synthesis method will be of considerable use for the establishment of reliable reproducible silica production for future industrial adoption
External tagging does not affect the feeding behavior of a coral reef fish, Chaetodon vagabundus (Pisces: Chaetodontidae)
Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of for personal use, not for redistribution. The definitive version was published in Environmental Biology of Fishes 86 (2009): 447-450, doi:10.1007/s10641-009-9545-9.Increasingly, the ability to recognize individual fishes is important for studies of population
dynamics, ecology, and behavior. Although a variety of methods exist, external tags remain one
of the most widely applied because they are both effective and cost efficient. However, a key
assumption is that neither the tagging procedure nor the presence of a tag negatively affects the
individual. While this has been demonstrated for relatively coarse metrics such as growth and
survival, few studies have examined the impact of tags and tagging on more subtle aspects of
behavior. We tagged adult vagabond butterflyfish (Chaetodon vagabundus) occupying a 30-ha
insular reef in Kimbe Bay, Papua New Guinea, using a commonly-utilized t-bar anchor tag. We
quantified and compared feeding behavior (bite rate), which is sensitive to stress, of tagged and
untagged individuals over four separate sampling periods spanning four months post-tagging.
Bite rates did not differ between tagged and untagged individuals at each sampling period and,
combined with additional anecdotal observations of normal pairing behavior and successful
reproduction, suggest that tagging did not adversely affect individuals.The authors gratefully
acknowledge funding from the Fulbright Program, National Science Foundation and the
Australian Research Council
Dramatic effect of pop-up satellite tags on eel swimming
The journey of the European eel to the spawning area in the Sargasso Sea is still a mystery. Several trials have been carried out to follow migrating eels with pop-up satellite tags (PSATs), without much success. As eels are very efficient swimmers, tags likely interfere with their high swimming efficiency. Here we report a more than twofold increase in swimming cost caused by a regular small satellite tag. The impact was determined at a range of swimming speeds with and without tag in a 2-m swimming tunnel. These results help to explain why the previous use of PSATs to identify spawning sites in the Sargasso Sea was thus far unsuccessful
Employing Relative Entropy Techniques for Assessing Modifications in Animal Behavior
In order to make quantitative statements regarding behavior patterns in animals, it is important to establish whether new observations are statistically consistent with the animal's equilibrium behavior. For example, traumatic stress from the presence of a telemetry transmitter may modify the baseline behavior of an animal, which in turn can lead to a bias in results. From the perspective of information theory such a bias can be interpreted as the amount of information gained from a new measurement, relative to an existing equilibrium distribution. One important concept in information theory is the relative entropy, from which we develop a framework for quantifying time-dependent differences between new observations and equilibrium. We demonstrate the utility of the relative entropy by analyzing observed speed distributions of Pacific bluefin tuna, recorded within a 48-hour time span after capture and release. When the observed and equilibrium distributions are Gaussian, we show that the tuna's behavior is modified by traumatic stress, and that the resulting modification is dominated by the difference in central tendencies of the two distributions. Within a 95% confidence level, we find that the tuna's behavior is significantly altered for approximately 5 hours after release. Our analysis reveals a periodic fluctuation in speed corresponding to the moment just before sunrise on each day, a phenomenon related to the tuna's daily diving pattern that occurs in response to changes in ambient light
The Mixed-Lineage Kinase DLK Is a Key Regulator of 3T3-L1 Adipocyte Differentiation
The mixed-lineage kinase (MLK) family member DLK has been proposed to serve as a regulator of differentiation in various cell types; however, its role in adipogenesis has not been investigated. In this study, we used the 3T3-L1 preadipocyte cell line as a model to examine the function of DLK in adipocyte differentiation.Immunoblot analyses and kinase assays performed on 3T3-L1 cells showed that the expression and activity of DLK substantially increase as differentiation occurs. Interestingly, DLK appears crucial for differentiation since its depletion by RNA interference impairs lipid accumulation as well as expression of the master regulators of adipogenesis C/EBPalpha and PPARgamma2 at both the mRNA and protein levels. In contrast, neither the expression nor the DNA binding activity of C/EBPbeta, an activator for C/EBPalpha and PPARgamma, is affected by DLK loss.Taken together, these results suggest that DLK is important for expression of mature adipocyte markers and that its action most likely takes place via regulation of C/EBPbeta transcriptional activity and/or initiation of C/EBPalpha and PPARgamma2 gene transcription
Occurrence of Ancylostoma in dogs, cats and public places from Andradina city, São Paulo state, Brazil
The Mutant Form of Lamin A that Causes Hutchinson-Gilford Progeria Is a Biomarker of Cellular Aging in Human Skin
Hutchinson-Gilford progeria syndrome (HGPS, OMIM 176670) is a rare disorder characterized by accelerated aging and early death, frequently from stroke or coronary artery disease. 90% of HGPS cases carry the LMNA G608G (GGC>GGT) mutation within exon 11 of LMNA, activating a splice donor site that results in production of a dominant negative form of lamin A protein, denoted progerin. Screening 150 skin biopsies from unaffected individuals (newborn to 97 years) showed that a similar splicing event occurs in vivo at a low level in the skin at all ages. While progerin mRNA remains low, the protein accumulates in the skin with age in a subset of dermal fibroblasts and in a few terminally differentiated keratinocytes. Progerin-positive fibroblasts localize near the basement membrane and in the papillary dermis of young adult skin; however, their numbers increase and their distribution reaches the deep reticular dermis in elderly skin. Our findings demonstrate that progerin expression is a biomarker of normal cellular aging and may potentially be linked to terminal differentiation and senescence in elderly individuals
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