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Gap Junction Formation in Heart Valves in Response to Mechanical LoadingO'Malley, Karen L. 28 June 2013 (has links)
Valvular interstitial cells (VICs) are responsible for the maintenance of heart
valve leaflet structure, however their responses to mechanical loading are not fully
understood. Further characterization of VIC responses with regards to phenotype
(quiescent or activated via ?-smooth muscle actin [?-SMA]) and communication (through
gap junction proteins connexins 43 and 26) were studied. Tissue strips from porcine
aortic, pulmonary, and mitral valves were cyclically stretched in the circumferential
direction at normal and above normal membrane tensions for 48 hours at 1 Hz, 37°C, and
5% CO2. Unloaded tissues were statically incubated concurrently with loaded tissues, and
fresh tissue controls were collected immediately. VIC phenotype was identified by ?-
SMA via immunohistochemical staining and cell enumeration, as well as by gene
expression via RT-PCR. Gap junction protein Cx43 was also evaluated via
immunohistochemical staining and cell enumeration and by gene expression via RT-PCR,
whereas Cx26 was evaluated using immunohistochemical staining and cell enumeration
only. Within the range tested, it was found that mechanical loading did not affect ?-SMA
or gap junction protein levels, nor were any differences in responses noted between valve
types. However, the ?-SMA gene expression level was significantly lower in the mitral
valve compared to the aortic and pulmonary valves. This may indicate a difference in the
genetic response pathways among the valves, but not in the functional outcomes. This
difference may be explained by embryological origins, since the mitral valve, unlike the
aortic and pulmonary valves, contains only VICs and no neural crest cells.
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Investigation of biological macromolecules using atomic force microscope-based techniquesBippes, Christian Alexander 19 August 2009 (has links) (PDF)
The atomic force microscope (AFM) provides a powerful instrument for investigating and manipulating biological samples down to the subnanometer scale. In contrast to other microscopy methods, AFM does not require labeling, staining, nor fixation of samples and allows the specimen to be fully hydrated in buffer solution during the experiments. Moreover, AFM clearly compares in resolution to other techniques.
In general, the AFM can be operated in an imaging or a force spectroscopy mode. In the present work, advantage was taken of this versatility to investigate single biomolecules and biomolecular assemblies.
A novel approach to investigate the visco-elastic behavior of biomolecules under force was established, using dextran as an example. While a molecule tethered between a solid support and the cantilever tip was stretched at a constant velocity, the thermally driven oscillation of the cantilever was recorded. Analysis of the cantilever Brownian noise provided information about the visco-elastic properties of dextran that corresponded well to parameters obtained by alternative methods. However, the approach presented here was easier to implement and less time-consuming than previously used methods.
A computer controlled force-clamp system was set up, circumventing the need for custom built analogue electronics. A commercial PicoForce AFM was extended by two computers which hosted data acquisition hardware. While the first computer recorded data, the second computer drove the AFM bypassing the manufacturer's microscope control software. To do so, a software-based proportional-integral-differential (PID) controller was implemented on the second computer. It allowed the force applied to a molecule to be held constant over time. After tuning of the PID controller, response times obtained using that force-clamp setup were comparable to those of the recently reported analogue systems. The performance of the setup was demonstrated by force-clamp unfolding of a pentameric Ig25 construct and the membrane protein NhaA. In the latter case, short-lived unfolding intermediates that were populated for less than 10 ms, could be revealed.
Conventional single-molecule dynamic force spectroscopy was used to unfold the serine:threonine antiporter SteT from Bacillus subtilis, an integral membrane protein. Unfolding force patterns revealed the unfolding barriers stabilizing structural segments of SteT. Ligand binding did not induce new unfolding barriers suggesting that weak interactions with multiple structural segments were involved. In contrast, ligand binding caused changes in the energy landscape of all structural segments, thus turning the protein from a brittle, rigid into a more stable, structurally flexible conformation. Functionally, rigidity in the ligand-free state was thought to facilitate specific ligand binding, while flexibility and increased stability were required for conformational changes associated with substrate translocation. These results support the working model for transmembrane transport proteins that provide alternate access of the binding site to either face of the membrane.
Finally, high-resolution imaging was exploited to visualize the extracellular surface of Cx26 gap junction hemichannels (connexons). AFM topographs reveal pH-dependent structural changes of the extracellular connexon surface in presence of HEPES, an aminosulfonate compound. At low pH (< 6.5), connexons showed a narrow and shallow channel entrance, which represented the closed pore. Increasing pH values resulted in a gradual opening of the pore, which was reflected by increasing channel entrance widths and depths. At pH > 7.6 the pore was fully opened and the pore diameter and depth did not increase further. Importantly, coinciding with pore gating a slight rotation of the subunits was observed. In the absence of aminosulfonate compounds, such as HEPES, acidification did not affect pore diameters and depths, retaining the open state. Thus, the intracellular concentration of taurine, a naturally abundant aminosulfonate compound, might be used to tune gap junction sensitivity at low pH.
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Investigation of biological macromolecules using atomic force microscope-based techniquesBippes, Christian Alexander 18 August 2009 (has links)
The atomic force microscope (AFM) provides a powerful instrument for investigating and manipulating biological samples down to the subnanometer scale. In contrast to other microscopy methods, AFM does not require labeling, staining, nor fixation of samples and allows the specimen to be fully hydrated in buffer solution during the experiments. Moreover, AFM clearly compares in resolution to other techniques.
In general, the AFM can be operated in an imaging or a force spectroscopy mode. In the present work, advantage was taken of this versatility to investigate single biomolecules and biomolecular assemblies.
A novel approach to investigate the visco-elastic behavior of biomolecules under force was established, using dextran as an example. While a molecule tethered between a solid support and the cantilever tip was stretched at a constant velocity, the thermally driven oscillation of the cantilever was recorded. Analysis of the cantilever Brownian noise provided information about the visco-elastic properties of dextran that corresponded well to parameters obtained by alternative methods. However, the approach presented here was easier to implement and less time-consuming than previously used methods.
A computer controlled force-clamp system was set up, circumventing the need for custom built analogue electronics. A commercial PicoForce AFM was extended by two computers which hosted data acquisition hardware. While the first computer recorded data, the second computer drove the AFM bypassing the manufacturer's microscope control software. To do so, a software-based proportional-integral-differential (PID) controller was implemented on the second computer. It allowed the force applied to a molecule to be held constant over time. After tuning of the PID controller, response times obtained using that force-clamp setup were comparable to those of the recently reported analogue systems. The performance of the setup was demonstrated by force-clamp unfolding of a pentameric Ig25 construct and the membrane protein NhaA. In the latter case, short-lived unfolding intermediates that were populated for less than 10 ms, could be revealed.
Conventional single-molecule dynamic force spectroscopy was used to unfold the serine:threonine antiporter SteT from Bacillus subtilis, an integral membrane protein. Unfolding force patterns revealed the unfolding barriers stabilizing structural segments of SteT. Ligand binding did not induce new unfolding barriers suggesting that weak interactions with multiple structural segments were involved. In contrast, ligand binding caused changes in the energy landscape of all structural segments, thus turning the protein from a brittle, rigid into a more stable, structurally flexible conformation. Functionally, rigidity in the ligand-free state was thought to facilitate specific ligand binding, while flexibility and increased stability were required for conformational changes associated with substrate translocation. These results support the working model for transmembrane transport proteins that provide alternate access of the binding site to either face of the membrane.
Finally, high-resolution imaging was exploited to visualize the extracellular surface of Cx26 gap junction hemichannels (connexons). AFM topographs reveal pH-dependent structural changes of the extracellular connexon surface in presence of HEPES, an aminosulfonate compound. At low pH (< 6.5), connexons showed a narrow and shallow channel entrance, which represented the closed pore. Increasing pH values resulted in a gradual opening of the pore, which was reflected by increasing channel entrance widths and depths. At pH > 7.6 the pore was fully opened and the pore diameter and depth did not increase further. Importantly, coinciding with pore gating a slight rotation of the subunits was observed. In the absence of aminosulfonate compounds, such as HEPES, acidification did not affect pore diameters and depths, retaining the open state. Thus, the intracellular concentration of taurine, a naturally abundant aminosulfonate compound, might be used to tune gap junction sensitivity at low pH.
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Integration von Connexonen in Lipidmembranen auf porösen Oberflächen / Integration of connexons in lipid bilayers on porous substratesGaßmann, Helmut Albin Oliver 15 July 2010 (has links)
No description available.
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Genetic aspects of hearing loss in the Limpopo Province of South Africa.Kabahuma, Rosemary I. 27 August 2010 (has links)
The aetiological diagnosis of recessive non-syndromic hearing loss poses a challenge owing
to marked heterogeneity and the lack of identifying clinical features. The finding that up to
50% of recessive non-syndromal genetic hearing loss among Caucasians was due to
mutations in GJB2, the gene encoding Connexin 26 (Cx26) was a breakthrough, whose value
as a diagnostic tool has been limited by the significant variation in the prevalence of deafness
genes and loci among population groups. The significant association of the GJB6-D13S1830
deletion among individuals with one mutant GJB2 allele highlighted the need to explore
population specific genetic mutations for NSHL. Although data from Sub-Saharan Africa is
limited, reported studies found a high prevalence of R143W GJB2 mutation among
Ghanaian, the 35delG mutation in 5 out of 139 Sudanese and a low prevalence of GJB2
variations among 385 Kenyan deaf children. The mutation spectrum of Waardenburg
Syndrome (WS) in Africans has not been documented.
During a visit to a School for the Deaf in the Limpopo Province of South Africa in 1997, it
was noted that a high number of students came from Nzhelele sub-district. All had childhood
onset hearing loss with no associated anomalies or disorders. The question arose as to
whether there was a high-risk area for deafness in the Limpopo Province and what the
aetiology of this hearing loss was.The main aim of this study was to investigate the role of
GJB2, the GJB6-D13S1830 deletion, and the four common mitochondrial mutations,
A1555G, A3243G, A7511C and A7445G, in the African hearing-impaired population of
Limpopo province in South Africa, and to identify the mutation spectrum of the deafness
genes found. The type and degree of hearing loss in this hearing impaired population would
also be assessed. Secondly, this study sought to identify the mutations in a sibling pair with
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clinical WS and to use the findings in a future study to establish the mutation spectrum of WS
in the African population of the Limpopo province and of South Africa in general.
The study was designed as a two phase study, in which phase 1 was used for hypothesis
formulation and phase 2 was for hypothesis testing. While phase 1 was a descriptive
retrospective case study, phase 2 was a combination of sample survey and prospective
descriptive case study. In phase 1, demographic data of 361 students in two schools of the
deaf in the Limpopo province was analyzed for evidence of areas of high risk populations for
deafness in the province. In phase 2, a group of 182 individuals with genetic non-syndromic
hearing loss (NSHL) and two siblings with clinical WS from two schools for the Deaf in the
Limpopo Province of South Africa were investigated. A thorough clinical examination,
audiological evaluation and urinalysis were done. Mutational screening was carried out in all
184 subjects using genomic DNA using single-strand conformation polymorphism (SSCP),
multiplex polymerase chain reaction (PCR), and direct sequencing for GJB2, and Restriction
Fragment-Length Polymorphism (PCR–RFLP) analysis for GJB6, and SSCP, hetero-duplex
analysis, and direct sequencing of the first 8 exons of PAX3 and all of MITF for Waarenburg
syndrome. Data analysis was by geographical mapping, frequency tables, tests of association
with calculation of odds ratios, and binary logistic regression analysis using STATA and GIS
mapping systems.
The results indicate that there seem to be areas of genuine populations at risk for hearing loss
in the Limpopo province of South Africa, namely Mutale and parts of Makhado and
Thulamela municipalities. In Thulamela (NP343) wards 11-15, 26-30 and 31-35, and in
Mutale (NP 344) wards 6-10, together accounted for 67 (18%) of participants in phase 1, and
33 (18%) of the participants in phase 2 of the study. Mutale municipality in the Vhembe
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district gave with a projected prevalence of at least 13.14 deaf children per 100,000 African
population attending the local school for the deaf.
The observed hearing loss is a genetic, non-syndromic form, which is mainly severe and
severe to profound, although without any clear defining configuration or shape. It is a stable,
non-progressive and prelingual form of hearing loss, implying that this may be a recessive
form of deafness. No identifiable environmental confounding factors or associations were
identified. The deafness is not linked the common known auditory gene mutations in GJB2,
the GJB6-D13S1830 deletion, or the common mitochondrial mutations A1555G, A3243G,
A7511C and A7445G. Severe and profound levels of hearing loss were found in 22.8% and
75% of the cohort respectively, with the majority exhibiting flat (70.1%) or sloping (23.4%)
audiograms that were commonly symmetrical (81.5%). However, as indicated, there was no
clear pattern in the audiological findings overall.
None of the 184 hearing impaired individuals exhibited any of the reported disease causing
mutations of GJB2, including 35delG. There was, however, a high prevalence of two
variants, the C>T variant at position g.3318-15 and the C>T variant at position g.3318-34,
occurring in 21.4% and 46.2% of the deaf cohort respectively. The same variants were found
to occur in 35% and 42.6% of a normal hearing control group (n = 63) respectively,
indicating that these variations are polymorphisms. In three subjects (1.63% of the cohort), a
T>A homozygous variation at position g.3318-6 was detected. Its significance in the
causation of NSSNHL is yet to be determined. The GJB6-D13S1830 deletion was not
detected in any of the participants. None of the four mitochondrial mutations screened for
were found.
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These results indicate that GJB2 is not a significant deafness gene in the African population
of the Limpopo Province of South Africa and that significant genes for non-syndromic
recessive hearing loss in this population are yet to be found. The geographical clustering of
deafness found in this study, combined with the lack of identifiable common associated
clinical features among the subjects of this study (excluding the WS sibling pair), suggests
that these subjects have a genetic recessive non-syndromal type of hearing loss. In the context
of historical and cultural evidence of consanguinity in this population, a founder effect cannot
be ruled out.
A rare mutation, R223X, previously identified only once out of 470 WS patients, was
identified in the PAX3 gene among the WS sibling pair. A novel silent change GGG>GGT at
amino acid 293, was also identified. These identical findings document, for the first time, a
molecular defect in WS in an African sibling pair, and confirm WS Type I in this family,
which could be found in other WS type I South Africans in the Limpopo Province of South
Africa.
The current study demonstrated that parents of genetically hearing impaired children in these
areas are able to detect hearing loss at an early age, with over 60% suspecting their children’s
hearing loss below 6 months of age. A child-centered management model encompassing all
the areas relevant to childhood deafness/hearing impairment, which takes into consideration
the prevailing logistical and financial constraints of the available healthcare system, is
proposed. The implementation of this model requires a paradigm shift from the current
fragmented model of service delivery to a cohesive patient-centered approach, based on
concrete data from appropriate community based research, in which all the relevant parties
communicate and share resources.
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It would achieve the goals of early detection and intervention, as well as inclusive education
for all. The relevant health and education policies are already in place and the posts funded.
Equitable implementation of these policies would require appropriate community based
research, as well as improved communication and consultation between the various
stakeholders to ensure an efficient and affordable quality healthcare service for all hearing
impaired South Africans.
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