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Biomedical subjects

S M King

Publications and source records attributed to S M King.

At least 37 records · Page 2Linked to original sources
PubMed2001

Update on perinatal HIV.

QUESTION: Many patients in my practice come from countries where HIV is endemic. Are there current figures on their risk of being infected during pregnancy? ANSWER: In Ottawa and Toronto, Ont, about half the pregnant women identified as HIV-positive were born in countries where HIV is endemic. If you suspect a woman has HIV or have other questions, call the Motherisk HIV-Healthline at 1-888-246-5840. Human immunovirus infection among pregnant women continues to be a serious public health concern. Most fetuses can be protected from contracting HIV if appropriate antiretroviral (ARV) therapy is instituted.

Anti-HIV Agents↗
PubMed2000

The dynein microtubule motor.

Dyneins are large multi-component microtubule-based molecular motors involved in many fundamental cellular processes including vesicular transport, mitosis and ciliary/flagellar beating. In order to achieve useful work, these enzymes must contain motor, cargo-binding and regulatory components. The ATPase and microtubule motor domains are located within the very large dynein heavy chains that form the globular heads and stems of the complex. Cargo-binding activity involves the intermediate chains and several classes of light chain that associate in a subcomplex at the base of the soluble dynein particle. Regulatory control of dynein motor function is thought to involve the phosphorylation of various components as well as a series of light chain proteins that are directly associated with the heavy chains. These latter polypeptides have a variety of intriguing attributes, including redox-sensitive vicinal dithiols and Ca(2+)-binding, suggesting that the activity of individual dyneins may be subject to multiple regulatory inputs. Recent molecular, genetic and structural studies have revealed insight into the roles played by these various components and the mechanisms of dynein-based motility.

Animals↗
PubMed2000

The light chain composition of chicken brain myosin-Va: calmodulin, myosin-II essential light chains, and 8-kDa dynein light chain/PIN.

Class V myosins are a ubiquitously expressed family of actin-based molecular motors. Biochemical studies on myosin-Va from chick brain indicate that this myosin is a two-headed motor with multiple calmodulin light chains associated with the regulatory or neck domain of each heavy chain, a feature consistent with the regulatory effects of Ca(2+) on this myosin. In this study, the identity of three additional low molecular weight proteins of 23-,17-, and 10 kDa associated with myosin-Va is established. The 23- and 17-kDa subunits are both members of the myosin-II essential light chain gene family, encoded by the chicken L23 and L17 light chain genes, respectively. The 10-kDa subunit is a protein originally identified as a light chain (DLC8) of flagellar and axonemal dynein. The 10-kDa subunit is associated with the tail domain of myosin-Va.

Amino Acid Sequence↗
PubMed2000

Investigation of protein-protein interactions within flagellar dynein using homobifunctional and zero-length crosslinking reagents.

The dynein molecular motor is a highly complex enzyme containing up to 15 different protein components and consists of several distinct domains identifiable by electron microscopy. One of the current challenges is to understand the supramolecular organization of this motor and to determine the location and function of the various components. Recently, we have used covalent crosslinking by amine-selective reagents and a carbodiimide, which results in zero-length crosslink, to investigate protein-protein associations within Chlamydomonas flagellar dynein. This approach also has enabled us to identify previously undescribed interactions between the dynein arms and other components of the flagellar axoneme. In this report, we detail methods we have developed to probe intradynein and intraaxonemal interactions and discuss the variety of factors that need be addressed to perform a successful crosslinking experiment.

Animals↗
PubMed2000

An axonemal dynein at the Hybrid Sterility 6 locus: implications for t haplotype-specific male sterility and the evolution of species barriers.

Poor sperm motility characterized by a distinct aberration in flagellar waveform known as "curlicue" is a hallmark of t haplotype (t) homozygous male sterility. Previous studies have localized "curlicue" and a flagellar developmental defect, "whipless", to the Hybrid Sterility 6 locus (Hst6), between the markers Pim1 and Crya1. More recent heterospecific breeding experiments between Mus spretus (Spretus) and Mus musculus domesticus (Domesticus) have mapped the primary source(s) of both "curlicue" and "whipless" to a small sub-locus of Hst6, Curlicue a (Ccua). Here we report the complete physical isolation of the Ccua locus and the identification of a candidate gene for expression of both "whipless" and "curlicue" at its proximal end, an axonemal dynein heavy chain gene, Dnahc8, formerly mapped by interspecific backcross analysis near Pim1. Dnahc8 mRNA expression commences in the Domesticus wild-type testis just prior to flagellar assembly and is testis-specific in the adult male. However, expression of Dnahc8 is not readily evident in the testis of either Spretus or "whipless" animals (Domesticus males homozygous for the Spretus allele of Dnahc8). Our results argue that Dnahc8 is fundamental to flagellar organization and function in Domesticus, but not Spretus, and suggest that Dnahc8 is integral to both Hst6- and t-specific male infertility.

Animals↗
PubMed2000

Solution structure of a dynein motor domain associated light chain.

Dyneins are molecular motors that translocate towards the minus ends of microtubules. In Chlamydomonas flagellar outer arm dynein, light chain 1 (LC1) associates with the nucleotide binding region within the gamma heavy chain motor domain and consists of a central leucine-rich repeat section that folds as a cylindrical right handed spiral formed from six beta-beta-alpha motifs. This central cylinder is flanked by terminal helical subdomains. The C-terminal helical domain juts out from the cylinder and is adjacent to a hydrophobic surface within the repeat region that is proposed to interact with the dynein heavy chain. The position of the C-terminal domain on LC1 and the unexpected structural similarity between LC1 and U2A' from the human spliceosome suggest that this domain interacts with the dynein motor domain.

Amino Acid Motifs↗
PubMed2000

The molecular anatomy of dynein.

Recent molecular, genetic and functional studies have led to an unparalleled growth in our understanding of dynein and the roles played by the various polypeptides of these massive macromolecular assemblies. Dyneins are highly complex 1-2MDa complexes that function as molecular motor and move the cargo to which they are attached towards the minus-end of a microtubule. Dynein motor function is a property of the heavy chains, whereas the intermediate chains are involved in attachment to the appropriate cargo. In order for useful work to be obtained, motor and cargo-binding activities must be tightly controlled. Current data suggest that this is the role played by certain accessory light-chain proteins. The LC8 is highly conserved and found in many enzyme systems. This protein is essential in multicellular organisms. The dynein light chains Tctex1 and Tctex2 have been implicated in the non-Mendelian transmission of variant forms of mouse chromosome 17.

Animals↗
PubMed2000

Dynein light chain binding to a 3'-untranslated sequence mediates parathyroid hormone mRNA association with microtubules.

The 3'-untranslated region (UTR) of mRNAs binds proteins that determine mRNA stability and localization. The 3'-UTR of parathyroid hormone (PTH) mRNA specifically binds cytoplasmic proteins. We screened an expression library for proteins that bind the PTH mRNA 3'-UTR, and the sequence of 1 clone was identical to that of the dynein light chain LC8, a component of the dynein complexes that translocate cytoplasmic components along microtubules. Recombinant LC8 binds PTH mRNA 3'-UTR, as shown by RNA electrophoretic mobility shift assay. We showed that PTH mRNA colocalizes with microtubules in the parathyroid gland, as well as with a purified microtubule preparation from calf brain, and that this association was mediated by LC8. To our knowledge, this is the first report of a dynein complex protein binding an mRNA. The dynein complex may be the motor that is responsible for transporting mRNAs to specific locations in the cytoplasm and for the consequent is asymmetric distribution of translated proteins in the cell.

3' Untranslated Regions↗
PubMed2000

AAA domains and organization of the dynein motor unit.

Dyneins contain one-three microtubule motor units that are each derived from the C-terminal globular head of a heavy chain. The N-terminal regions of the heavy chains form stems that are required for intra-dynein associations. The microtubule-binding sites are located at the terminus of a short stalk that emanates from each globular head. Recent electron microscopic analysis indicates that the dynein head has a heptameric toroidal organization. This finding is echoed by the identification of six AAA (ATPases associated with cellular activities) domains and a seventh unrelated unit within this heavy chain region. At least two of these AAA domains can bind nucleotide, although only one appears able to hydrolyze ATP. Several other AAA domain proteins exhibit a similar annular organization of six AAA units. Detailed structural information is available for several AAA proteins, including N-ethylmaleimide-sensitive vesicle-fusion protein and the RuvB motor involved in DNA migration and resolution of Holliday junctions. The resulting structural parallels allow intriguing predictions to be made concerning dynein organization and motor function.

Adenosine Triphosphatases↗
PubMed1999

Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis.

Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement. To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants. One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis. These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation. The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here. Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae. Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein. We propose that roadblock/LC7 family members may modulate specific dynein functions.

Amino Acid Sequence↗
PubMed1999

Light chain 1 from the Chlamydomonas outer dynein arm is a leucine-rich repeat protein associated with the motor domain of the gamma heavy chain.

The LC1 light chain from Chlamydomonas outer arm dynein is tightly bound to the gamma heavy chain. Molecular cloning revealed that LC1 is a member of the SDS22+ subclass of the leucine-rich repeat protein family and as such is likely involved in mediating interactions between dynein and the components of a signal transduction pathway. Through the combination of covalent cross-linking and vanadate-mediated photolysis, LC1 was found to associate with that portion of the gamma HC that is C-terminal to the P1 loop. This region comprises most of the globular head domain of the heavy chain and includes the stalk-like structure that is involved in microtubule binding. Attachment of LC1 to this region represents the only known example of an accessory polypeptide directly associated with a dynein motor domain. Additional cross-linking experiments revealed that LC1 also interacts directly in situ with an approximately 45 kDa axonemal component; this interaction is disrupted by the standard high salt treatment used to remove the outer arm from the axoneme. These data suggest that LC1 acts to mediate the association between this 45 kDa axonemal polypeptide and the motor unit of the gamma HC.

Amino Acid Sequence↗
PubMed1999

Assigning secondary structure from protein coordinate data.

We have developed a program to convert the three dimensional coordinates describing protein structure in the Brookhaven Data Bank into an assignment of secondary structure. The program assigns secondary structure in the same way a person assigns structure visually. It uses two angles and three distances to assign alpha-helix, 3(10)-helix, beta-strand, hydrogen-bonded beta-turn, non-hydrogen-bonded beta-turn, and poly (L-proline) II type 3(1)-helix. The program is concerned with amide-amide interactions and should be particularly useful to spectroscopists.

Amino Acid Sequence↗
PubMed1999

Escape-related behaviours in an unstable, elevated and exposed environment. II. Long-term sensitization after repetitive electrical stimulation of the rodent midbrain defence system.

The behavioural indices of anxiety/fear/panic range from freezing/inhibition of ongoing behaviour to active defence strategies i.e. fight/flight, depending on the proximity or intensity of the aversive stimulus. However, evidence suggests that when the initial stressor is sufficiently intense, the neural defence circuitry may undergo a long-term increase in sensitivity. Such sensitization has been evoked to explain the chronically hyper-aroused fight/flight state of patients suffering from extreme anxiety states. The purpose of this study was to establish whether direct activation of the rodent midbrain defence system could result in a similar long-term alteration of the animal's responses to subsequent stressful events. This was achieved by studying the behaviour of animals in a threatening environment after repetitive electrical stimulation of the superior colliculus. Animals were then placed on an unstable, elevated and exposed plus maze and their behaviour recorded. Testing was carried out regularly over 3 months. Stimulated animals reliably exhibited significantly increased levels of behaviours designed to escape the aversive conditions of the unstable plus maze. These included visual scanning, end-reaching, preparing to jump, and jumping off the apparatus. Unstimulated control animals, on the other hand, exhibited decreased levels of these behaviours post-stimulation. In contrast, the experimental animals' performance on standard anxiety tests did not differ from controls. These results demonstrate that repetitive tactile stimulation can produce a long-term change in reactions to threat, and is proposed as a functional model of extreme anxiety.

Animals↗
PubMed1999

Escape-related behaviours in an unstable elevated and exposed environment. I. A new behavioural model of extreme anxiety.

The aim of this study was to validate a new behavioural test for extreme anxiety, designed to elicit unconditioned escape from animals on an unstable, elevated and exposed plus maze. The animals were also run on a standard elevated plus maze, so that well-validated anxiety measures could be compared with the animals' propensity to escape. The first experiment compared the behavioural repertoire of three rat strains; more escape behaviours were elicited from the Sprague Dawleys (SDs) and dark agouti (DAs) than from the hooded listers (HLs) on the unstable maze. Similarly, the SDs and DAs exhibited higher anxiety profiles than the HLs on the standard plus maze. There was a clear correlation, therefore, between the propensity to escape and well-validated anxiety measures. A further group of rats [HLs] was tested on both the unstable and standard plus mazes (Experiment 2) and principal components analyses carried out on all behavioural measures. The extracted factors identified separate and independent factors relating to escape-linked behaviours on the unstable plus maze and anxiety on the standard plus maze. The escape-linked behaviours exhibited on the unstable plus maze appear to reflect a different type of anxiety from that exhibited on the standard plus maze. The bias towards escape behaviours shown by some rats on the unstable plus maze may mimic the chronically hyper-aroused, persistent 'fight/flight' state exhibited by certain categories of anxiety patients, and hence provide a behavioural model of extreme anxiety.

Animals↗
PubMed1999

Toronto street youth and HIV/AIDS: prevalence, demographics, and risks.

PURPOSE: The purposes of this study were: (a) to identify human immunodeficiency virus (HIV) prevalence in Toronto street youth through paired blood and saliva specimens; (b) to identify the HIV risk and prevention behaviors of street involved youth; and (c) to identify demographic or other factors that may contribute to the risk of street youth becoming infected with HIV/acquired immunodeficiency syndrome (AIDS) in the future. METHODS: This was a cross-sectional convenience study of street-involved youth aged 14-25 years. The youth participated in interviews to identify HIV-related knowledge and personal risk and preventive behaviors. Following interviews, they were asked to provide a saliva sample, blood spot, or both. They could refuse one or both samples without jeopardizing their involvement or receiving an honorarium. Two males were the only participants who declined to provide a sample. RESULTS: Fifteen of 695 (2.2%) youth tested positive for HIV infection. All were male, ranging in age from 18 to 25 years. Same and opposite sex, intravenous (IV) drug use, prostitution, and incarceration were risk factors associated with positive HIV test results. The rate of HIV infection was seven times greater for the group 20 years of age and older (20-25) compared to the younger group aged 14-9 years. The proportion testing positive for HIV from small cities, towns, and rural communities in Ontario was 40%; yet, they represented 21% of the study population. Most (57%) youth had been on their own for no more than 3 years and had moved frequently. Nearly two thirds (60%) had stayed in hostels or homeless shelters in the previous 6 months. CONCLUSION: Street youth in Canada are at high risk of HIV infection with their risk increasing with age. Unprotected (same and opposite) sex, IV drug use, prostitution and incarceration were linked to their HIV infections. The high level of mobility identified by street youth challenges governments, communities, and public health officials to develop appropriate prevention strategies and to carefully monitor the spread of HIV infection in this vulnerable population.

Adolescent↗
PubMed1999

The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex.

Bcl-2 family members that have only a single Bcl-2 homology domain, BH3, are potent inducers of apoptosis, and some appear to play a critical role in developmentally programmed cell death. We examined the regulation of the proapoptotic activity of the BH3-only protein Bim. In healthy cells, most Bim molecules were bound to LC8 cytoplasmic dynein light chain and thereby sequestered to the microtubule-associated dynein motor complex. Certain apoptotic stimuli disrupted the interaction between LC8 and the dynein motor complex. This freed Bim to translocate together with LC8 to Bcl-2 and to neutralize its antiapoptotic activity. This process did not require caspase activity and therefore constitutes an initiating event in apoptosis signaling.

Amino Acid Sequence↗