Nephelometry and Turbidimetry
Engrailed negatively regulates the expression of cell adhesion molecules connectin and neuroglian in embryonic Drosophila nervous system. (1/616)Engrailed is expressed in subsets of interneurons that do not express Connectin or appreciable Neuroglian, whereas other neurons that are Engrailed negative strongly express these adhesion molecules. Connectin and Neuroglian expression are virtually eliminated in interneurons when engrailed expression is driven ubiquitously in neurons, and greatly increased when engrailed genes are lacking in mutant embryos. The data suggest that Engrailed is normally a negative regulator of Connectin and neuroglian. These are the first two "effector" genes identified in the nervous system of Drosophila as regulatory targets for Engrailed. We argue that differential Engrailed expression is crucial in determining the pattern of expression of cell adhesion molecules and thus constitutes an important determinant of neuronal shape and perhaps connectivity. (+info)
Mechanical and chemical unfolding of a single protein: a comparison. (2/616)Is the mechanical unraveling of protein domains by atomic force microscopy (AFM) just a technological feat or a true measurement of their unfolding? By engineering a protein made of tandem repeats of identical Ig modules, we were able to get explicit AFM data on the unfolding rate of a single protein domain that can be accurately extrapolated to zero force. We compare this with chemical unfolding rates for untethered modules extrapolated to 0 M denaturant. The unfolding rates obtained by the two methods are the same. Furthermore, the transition state for unfolding appears at the same position on the folding pathway when assessed by either method. These results indicate that mechanical unfolding of a single protein by AFM does indeed reflect the same event that is observed in traditional unfolding experiments. The way is now open for the extensive use of AFM to measure folding reactions at the single-molecule level. Single-molecule AFM recordings have the added advantage that they define the reaction coordinate and expose rare unfolding events that cannot be observed in the absence of chemical denaturants. (+info)
Proteinuria is associated with persistence of antibody to streptococcal M protein in Aboriginal Australians. (3/616)Aboriginal communities in Northern Australia with high rates of group A streptococcal (GAS) skin infection in childhood also have high rates of renal failure in adult life. In a cross-sectional study of one such high risk community, albuminuria was used as a marker of renal disease. The prevalence of albuminuria increased from 0/52 in subjects aged 10-19 years to 10/29 (32.9%) in those aged 50 or more (P < 0.001). Antibodies to streptococcal M protein, markers of past GAS infection, were present in 48/52 (92%) at ages 10-19 years, 16/32 (50%) at ages 30-39, and 20/29 (69%) in those aged 50 or more. After allowing for the age-dependencies of albuminuria and of M protein antibodies (P < 0.001) albuminuria was significantly associated with M protein antibodies (P < 0.01). Thus, 72% of adults aged 30 or more with M protein antibodies also had albuminuria, compared with only 21% of those who were seronegative. More detailed modelling suggested that although most Aboriginal people in this community developed M protein antibodies following GAS infection in childhood, the development of proteinuria was associated with the persistence of such seropositivity into adult life. The models predicted that proteinuria developed at a mean age of 30 years in seropositive persons, at 45 years in seronegative persons who were overweight, and at 62 years in seronegative persons of normal weight. We demonstrated a clear association between evidence of childhood GAS infection and individual risk of proteinuria in adult life. This study provided a strong rationale for prevention of renal disease through the more effective control of GAS skin infections in childhood and through the prevention of obesity in adult life. (+info)
Strength of a weak bond connecting flexible polymer chains. (4/616)Bond dissociation under steadily rising force occurs most frequently at a time governed by the rate of loading (Evans and Ritchie, 1997 Biophys. J. 72:1541-1555). Multiplied by the loading rate, the breakage time specifies the force for most frequent failure (called bond strength) that obeys the same dependence on loading rate. The spectrum of bond strength versus log(loading rate) provides an image of the energy landscape traversed in the course of unbonding. However, when a weak bond is connected to very compliant elements like long polymers, the load applied to the bond does not rise steadily under constant pulling speed. Because of nonsteady loading, the most frequent breakage force can differ significantly from that of a bond loaded at constant rate through stiff linkages. Using generic models for wormlike and freely jointed chains, we have analyzed the kinetic process of failure for a bond loaded by pulling the polymer linkages at constant speed. We find that when linked by either type of polymer chain, a bond is likely to fail at lower force under steady separation than through stiff linkages. Quite unexpectedly, a discontinuous jump can occur in bond strength at slow separation speed in the case of long polymer linkages. We demonstrate that the predictions of strength versus log(loading rate) can rationalize conflicting results obtained recently for unfolding Ig domains along muscle titin with different force techniques. (+info)
Different domains of the M-band protein myomesin are involved in myosin binding and M-band targeting. (5/616)Myomesin is a 185-kDa protein located in the M-band of striated muscle where it interacts with myosin and titin, possibly connecting thick filaments with the third filament system. By using expression of epitope-tagged myomesin fragments in cultured cardiomyocytes and biochemical binding assays, we could demonstrate that the M-band targeting activity and the myosin-binding site are located in different domains of the molecule. An N-terminal immunoglobulin-like domain is sufficient for targeting to the M-band, but solid-phase overlay assays between individual N-terminal domains and the thick filament protein myosin revealed that the unique head domain contains the myosin-binding site. When expressed in cardiomyocytes, the head domains of rat and chicken myomesin showed species-specific differences in their incorporation pattern. The head domain of rat myomesin localized to a central area within the A-band, whereas the head domain of chicken myomesin was diffusely distributed in the cytoplasm. We therefore conclude that the head domain of myomesin binds to myosin but that this affinity is not sufficient for the restriction of the domain to the M-band in vivo. Instead, the neighboring immunoglobulin-like domain is essential for the precise incorporation of myomesin into the M-band, possibly because of interaction with a yet unknown protein of the sarcomere. (+info)
Expression and functional characteristics of calpain 3 isoforms generated through tissue-specific transcriptional and posttranscriptional events. (6/616)Calpain 3 is a nonlysosomal cysteine protease whose biological functions remain unknown. We previously demonstrated that this protease is altered in limb girdle muscular dystrophy type 2A patients. Preliminary observations suggested that its gene is subjected to alternative splicing. In this paper, we characterize transcriptional and posttranscriptional events leading to alterations involving the NS, IS1, and IS2 regions and/or the calcium binding domains of the mouse calpain 3 gene (capn3). These events can be divided into three groups: (i) splicing of exons that preserve the translation frame, (ii) inclusion of two distinct intronic sequences between exons 16 and 17 that disrupt the frame and would lead, if translated, to a truncated protein lacking domain IV, and (iii) use of an alternative first exon specific to lens tissue. In addition, expression of these isoforms seems to be regulated. Investigation of the proteolytic activities and titin binding abilities of the translation products of some of these isoforms clearly indicated that removal of these different protein segments affects differentially the biochemical properties examined. In particular, removal of exon 6 impaired the autolytic but not fodrinolytic activity and loss of exon 16 led to an increased titin binding and a loss of fodrinolytic activity. These results are likely to impact our understanding of the pathophysiology of calpainopathies and the development of therapeutic strategies. (+info)
Mechanically driven contour-length adjustment in rat cardiac titin's unique N2B sequence: titin is an adjustable spring. (7/616)The giant elastic protein titin is largely responsible for passive forces in cardiac myocytes. A number of different titin isoforms with distinctly different structural elements within their central I-band region are expressed in human myocardium. Their coexpression has so far prevented an understanding of the respective contributions of the isoforms to myocardial elasticity. Using isoform-specific antibodies, we find in the present study that rat myocardium expresses predominantly the small N2B titin isoform, which allows us to characterize the elastic behavior of this isoform. The extensibility and force response of N2B titin were studied by using immunoelectron microscopy and by measuring the passive force-sarcomere length (SL) relation of single rat cardiac myocytes under a variety of mechanical conditions. Experimental results were compared with the predictions of a mechanical model in which the elastic titin segment behaves as two wormlike chains, the tandem immunoglobulin (Ig) segments and the PEVK segment (rich in proline [P], glutamate [E], valine [V], and lysine [K] residues), connected in series. The overall contour length was predicted from the sequence of N2B cardiac titin. According to mechanical measurements, above approximately 2.2 microm SL titin's elastic segment extends beyond its predicted contour length. Immunoelectron microscopy indicates that a prominent source of this contour-length gain is the extension of the unique N2B sequence (located between proximal tandem Ig segment and PEVK), and that Ig domain unfolding is negligible. Thus, the elastic region of N2B cardiac titin consists of three mechanically distinct extensible segments connected in series: the tandem Ig segment, the PEVK segment, and the unique N2B sequence. Rate-dependent and repetitive stretch-release experiments indicate that both the contour-length gain and the recovery from it involve kinetic processes, probably unfolding and refolding within the N2B segment. As a result, the contour length of titin's extensible segment depends on the rate and magnitude of the preceding mechanical perturbations. The rate of recovery from the length gain is slow, ensuring that the adjusted length is maintained through consecutive cardiac cycles and that hysteresis is minimal. Thus, as a result of the extensible properties of the unique N2B sequence, the I-band region of the N2B cardiac titin isoform functions as a molecular spring that is adjustable. (+info)
A response regulator that represses transcription of several virulence operons in the group A streptococcus. (8/616)A search for homologs of the Bacillus subtilis PhoP response regulator in the group A streptococcus (GAS) genome revealed three good candidates. Inactivation of one of these, recently identified as csrR (J. C. Levin and M. R. Wessels, Mol. Microbiol. 30:209-219, 1998), caused the strain to produce mucoid colonies and to increase transcription of hasA, the first gene in the operon for capsule synthesis. We report here that a nonpolar insertion in this gene also increased transcription of ska (encoding streptokinase), sagA (streptolysin S), and speMF (mitogenic factor) but did not affect transcription of slo (streptolysin O), mga (multiple gene regulator of GAS), emm (M protein), scpA (complement C5a peptidase), or speB or speC (pyrogenic exotoxins B and C). The amounts of streptokinase, streptolysin S, and capsule paralleled the levels of transcription of their genes in all cases. Because CsrR represses genes unrelated to those for capsule synthesis, and because CsrA-CsrB is a global regulatory system in Escherichia coli whose mechanism is unrelated to that of these genes in GAS, the locus has been renamed covR, for "control of virulence genes" in GAS. Transcription of the covR operon was also increased in the nonpolar insertion mutant, indicating that CovR represses its own synthesis as well. All phenotypes of the covR nonpolar insertion mutant were complemented by the covR gene on a plasmid. CovR acts on operons expressed both in exponential and in stationary phase, demonstrating that the CovR-CovS pathway is separate from growth phase-dependent regulation in GAS. Therefore, CovR is the first multiple-gene repressor of virulence factors described for this important human pathogen. (+info)
Early Postmortem Changes:
1. Cessation of metabolic processes: After death, the body's metabolic processes come to a standstill, leading to a decrease in body temperature, cellular respiration, and other physiological functions.
2. Decline in blood pressure: The heart stops pumping blood, causing a rapid decline in blood pressure.
3. Cardiac arrest: The heart stops beating, leading to a lack of oxygen supply to the body's tissues.
4. Brain death: The brain ceases to function, causing a loss of consciousness and reflexes.
5. Rigor mortis: The muscles become stiff and rigid due to the buildup of lactic acid and other metabolic byproducts.
6. Livor mortis: Blood settles in the dependent parts of the body, causing discoloration and swelling.
7. Algor mortis: The body's temperature cools, causing the skin to feel cool to the touch.
Late Postmortem Changes:
1. Decomposition: Bacteria and other microorganisms begin to break down the body's tissues, leading to putrefaction and decay.
2. Autolysis: Enzymes within the body's cells break down cellular components, causing self-digestion and softening of the tissues.
3. Lipid decomposition: Fats and oils in the body undergo oxidation, leading to the formation of offensive odors.
4. Coagulative necrosis: Blood pools in the body's tissues, causing damage to the cells and tissues.
5. Putrefaction: Bacteria in the gut and other parts of the body cause the breakdown of tissues, leading to the formation of gases and fluids.
It is important to note that postmortem changes can significantly impact the interpretation of autopsy findings and the determination of cause of death. Therefore, it is essential to consider these changes when performing an autopsy and interpreting the results.
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