Pharmacodynamics and proteomic analysis of acalabrutinib therapy: similarity of on-target effects to ibrutinib and rationale for combination therapy.

Acalabrutinib, a highly selective Bruton's tyrosine kinase inhibitor, is associated with high overall response rates and durable remission in previously treated chronic lymphocytic leukemia (CLL); however, complete remissions were limited. To elucidate on-target and pharmacodynamic effects of acalabrutinib, we evaluated several laboratory endpoints, including proteomic changes, chemokine modulation and impact on cell migration. Pharmacological profiling of samples from acalabrutinib-treated CLL patients was used to identify strategies for achieving deeper responses, and to identify additive/synergistic combination regimens. Peripheral blood samples from 21 patients with relapsed/refractory CLL in acalabrutinib phase I (100-400 mg/day) and II (100 mg BID) clinical trials were collected prior to and on days 8 and 28 after treatment initiation and evaluated for plasma chemokines, reverse phase protein array, immunoblotting and pseudoemperipolesis. The on-target pharmacodynamic profile of acalabrutinib in CLL lymphocytes was comparable to ibrutinib in measures of acalabrutinib-mediated changes in CCL3/CCL4 chemokine production, migration assays and changes in B-cell receptor signaling pathway proteins and other downstream survival proteins. Among several CLL-targeted agents, venetoclax, when combined with acalabrutinib, showed optimal complementary activity in vitro, ex vivo and in vivo in TCL-1 adoptive transfer mouse model system of CLL. These findings support selective targeting and combinatorial potential of acalabrutinib.

Longitudinal Multilevel Modeling of Facial Pain, Muscle Tension, and Stress.

The role of masticatory muscle activation on pain in temporomandibular muscle and joint disorders (TMJD) is controversial. This single-group, prospective panel study examined the relationships among masticatory muscle tension, emotional distress, and TMJD pain in a sample of 7,023 observations obtained from 171 individuals using longitudinal multilevel modeling. Three main hypotheses were tested. The first posited that emotional distress and muscle tension directly influenced pain (hypothesis 1a: Distress → TMJD Pain; hypothesis 1b: Muscle Tension → TMJD Pain). The second posited that emotional distress directly influenced muscle tension (Distress → Muscle Tension), and the third posited that the effect of emotional distress on pain was mediated by muscle tension (Distress → Muscle Tension → TMJD pain). We also examined the fit of the data to possible alternative models. All the data used in this study were collected via an experience sampling methodology. The fit of the preferred models was better than that of the alternative models, with the preferred models explaining large proportions of the data, especially for level 2 variance (hypothesis 1a = 41% variance; hypothesis 1b = 69% variance; hypothesis 2 = 48% variance). In the mediation model, the addition of muscle tension to the model reduced the impact of emotional distress. The findings support a causal role for masticatory muscle tension in TMJD pain. Clinically, the results suggest that addressing tension and other oral parafunctions in those diagnosed with TMJDs should be an important part of the conservative, noninvasive care of individuals diagnosed with the myofascial pain or arthralgia of TMJD.

Morbidity profile of admissions to GF Jooste Hospital; Manenberg; Cape Town

BackgroundSecondary hospitals play an important; yet overlooked; role in reflecting public health status; both locally and nationally. Relatively few reports analysing the causes of secondary hospital admissions exist; which is especially unfortunate in the case of developing countries; considering the huge numbers of admissions and people at risk. In developing countries like South Africa; the quality of records varies among institutions. Some hospitals have computerised data; while others may keep no records whatsoever. A major problem facing the quality of hospital records is the constant shortage of staff in rural and urban hospitals. Thorough documentation is essential in providing an invaluable database for researchers; but morbidity statistics are unfortunately scarce.GF Jooste Hospital in Manenberg is the busiest hospital in Cape Town - serving 1.1 million people; with 224 beds and over 12 000 admissions annually. Budgetary constraints in the South African public health sector means that providing healthcare services at higher levels than necessary is too costly. Because hospitals consume the largest share of the public healthcare budget; they have been the focus in cost cutting. In particular; the budgets of referral (tertiary or teaching) hospitals have been trimmed in order to promote primary and secondary care. It is imperative to identify those services that are required most at secondary hospitals in order to improve budgeting and; more appropriately; train doctors and medical students for the job at hand. Identifying the morbidity profile of the population for which the hospital caters can aid the optimal utilisation of the available resources; as well as focusing the continuing medical education of hospital physicians. We determined disease patterns of admissions over a three-year period (2001-2003); primarily as insight towards optimal hospital resource management.MethodsA retrospective study examined ward records; totalling 36 657 admissions; from which a random sample (N=608) was selected. A stratified sample (N=462) was constructed; considering the relative proportions admitted to the wards. The International Statistical Classification of Diseases (ICD) directed diagnosis sorting. Disease prevalence was expressed as the percentage of patients allocated to each ICD category among those admitted to the hospital and respective wards and; additionally; the percentage of diagnoses for each ICD subcategory among patients assigned to each major category.ResultsTrauma (represented by ICD categories S/T 23and V/X/Y 16); specifically assault-related; was most prevalent. This was followed by circulatory diseases (22) and infectious diseases (19); dominated by HIV (61) and associated diseases like TB (57). The age of the patients ranged from 13 to 87 (mean: 40 years); with the 20 to 30-year-olds predominating. Surgical patients were younger (mean: 35 years) than medical (mean: 45 years). In the medical wards; infectious (39in men; 38in women) and circulatory aetiologies (39and 41in men and women respectively) dominated. In the surgical wards; the trend varied according to sex: assault (43) and other injuries (61) for males; pregnancy-related (42) for females. ConclusionThe morbidity distribution reflects the ills affecting South African urban society; with young trauma admissions predominating. The hospital's budget is insufficient; considering its population's demands

A specialized mitochondrial molecular chaperone system: a role in formation of Fe/S centers.

Mitochondria contain a specialized system of molecular chaperones that plays a critical role in the biogenesis of Fe/S centers. This Hsp70:J-protein system shows many similarities to the system found in bacteria, but the precise role of neither chaperone system has been defined. However, evidence to date suggests an interaction with the scaffold protein on which a transient Fe/S center is assembled, and thus implies a role in either assembly of the center or its transfer to recipient proteins.

Jac1, a mitochondrial J-type chaperone, is involved in the biogenesis of Fe/S clusters in Saccharomyces cerevisiae.

A minor Hsp70 chaperone of the mitochondrial matrix of Saccharomyces cerevisiae, Ssq1, is involved in the formation or repair of Fe/S clusters and/or mitochondrial iron metabolism. Here, we report evidence that Jac1, a J-type chaperone of the mitochondrial matrix, is the partner of Ssq1 in this process. Reduced activity of Jac1 results in a decrease in activity of Fe/S containing mitochondrial proteins and an accumulation of iron in mitochondria. Fe/S enzyme activities remain low in both jac1 and ssq1 mutant mitochondria even if normal mitochondrial iron levels are maintained. Therefore, the low activities observed are not solely due to oxidative damage caused by excess iron. Rather, these molecular chaperones likely play a direct role in the normal assembly process of Fe/S clusters.

Genetic evidence for selective transport of opsin and arrestin by kinesin-II in mammalian photoreceptors.

To test whether kinesin-II is important for transport in the mammalian photoreceptor cilium, and to identify its potential cargoes, we used Cre-loxP mutagenesis to remove the kinesin-II subunit, KIF3A, specifically from photoreceptors. Complete loss of KIF3A caused large accumulations of opsin, arrestin, and membranes within the photoreceptor inner segment, while the localization of alpha-transducin was unaffected. Other membrane, organelle, and transport markers, as well as opsin processing appeared normal. Loss of KIF3A ultimately caused apoptotic photoreceptor cell death similar to a known opsin transport mutant. The data suggest that kinesin-II is required to transport opsin and arrestin from the inner to the outer segment and that blocks in this transport pathway lead to photoreceptor cell death as found in retinitis pigmentosa.

CENP-meta, an essential kinetochore kinesin required for the maintenance of metaphase chromosome alignment in Drosophila.

CENP-meta has been identified as an essential, kinesin-like motor protein in Drosophila. The 257-kD CENP-meta protein is most similar to the vertebrate kinetochore-associated kinesin-like protein CENP-E, and like CENP-E, is shown to be a component of centromeric/kinetochore regions of Drosophila chromosomes. However, unlike CENP-E, which leaves the centromere/kinetochore region at the end of anaphase A, the CENP-meta protein remains associated with the centromeric/kinetochore region of the chromosome during all stages of the Drosophila cell cycle. P-element-mediated disruption of the CENP-meta gene leads to late larval/pupal stage lethality with incomplete chromosome alignment at metaphase. Complete removal of CENP-meta from the female germline leads to lethality in early embryos resulting from defects in metaphase chromosome alignment. Real-time imaging of these mutants with GFP-labeled chromosomes demonstrates that CENP-meta is required for the maintenance of chromosomes at the metaphase plate, demonstrating that the functions required to establish and maintain chromosome congression have distinguishable requirements.

Role of the mitochondrial Hsp70s, Ssc1 and Ssq1, in the maturation of Yfh1.

The mitochondrial matrix of the yeast Saccharomyces cerevisiae contains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported into ssc1-3 mutant mitochondria, remaining in a protease-sensitive precursor form. As reported earlier, the Yfh1 intermediate form was only slowly processed to the mature form in Deltassq1 mitochondria (S. A. B. Knight, N. B. V. Sepuri, D. Pain, and A. Dancis, J. Biol. Chem. 273:18389-18393, 1998). However, the intermediate form in both wild-type and Deltassq1 mitochondria was entirely within the inner membrane, as it was resistant to digestion with protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold excess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature form in isolated mitochondria. However, the steady-state level of mature Yfh1 in Deltassq1 mitochondria is approximately 75% of that found in wild-type mitochondria, indicating that this retardation in processing does not dramatically affect cellular concentrations. Therefore, Ssq1 likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of Deltassq1 cells, as well as the accumulation of mitochondrial iron and the defects in Fe/S enzyme activities normally found in Deltassq1 mitochondria. Deltassq1 mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests that Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.

Understanding the functions of kinesin-II.

Species ranging from Chlamydomonas to humans possess the heterotrimeric kinesin-II holoenzyme composed of two different motor subunits and one non-motor accessory subunit. An important function of kinesin-II is that it transports the components needed for the construction and maintenance of cilia and flagella from the site of synthesis in the cell body to the site of growth at the distal tip. Recent work suggests that kinesin-II does not directly interact with these components, but rather via a large protein complex, which has been termed a raft (intraflagellar transport (IFT)). While ciliary transport is the best-established function for kinesin-II, evidence has been reported for possible roles in neuronal transport, melanosome transport, the secretory pathway and during mitosis.

Dual role of the mitochondrial chaperone Mdj1p in inheritance of mitochondrial DNA in yeast.

Mdj1p, a homolog of the bacterial DnaJ chaperone protein, plays an essential role in the biogenesis of functional mitochondria in the yeast Saccharomyces cerevisiae. We analyzed the role of Mdj1p in the inheritance of mitochondrial DNA (mtDNA). Mitochondrial genomes were rapidly lost in a temperature-sensitive mdj1 mutant under nonpermissive conditions. The activity of mtDNA polymerase was severely reduced in the absence of functional Mdj1p at a nonpermissive temperature, demonstrating the dependence of the enzyme on Mdj1p. At a permissive temperature, the activity of mtDNA polymerase was not affected by the absence of Mdj1p. However, under these conditions, intact [rho(+)] genomes were rapidly converted to nonfunctional [rho(-)] genomes which were stably propagated in an mdj1 deletion strain. We propose that mtDNA polymerase depends on Mdj1p as a chaperone in order to acquire and/or maintain an active conformation at an elevated temperature. In addition, Mdj1p is required for the inheritance of intact mitochondrial genomes at a temperature supporting optimal growth; this second function appears to be unrelated to the function of Mdj1p in maintaining mtDNA polymerase activity.

Novel dendritic kinesin sorting identified by different process targeting of two related kinesins: KIF21A and KIF21B.

Neurons use kinesin and dynein microtubule-dependent motor proteins to transport essential cellular components along axonal and dendritic microtubules. In a search for new kinesin-like proteins, we identified two neuronally enriched mouse kinesins that provide insight into a unique intracellular kinesin targeting mechanism in neurons. KIF21A and KIF21B share colinear amino acid similarity to each other, but not to any previously identified kinesins outside of the motor domain. Each protein also contains a domain of seven WD-40 repeats, which may be involved in binding to cargoes. Despite the amino acid sequence similarity between KIF21A and KIF21B, these proteins localize differently to dendrites and axons. KIF21A protein is localized throughout neurons, while KIF21B protein is highly enriched in dendrites. The plus end-directed motor activity of KIF21B and its enrichment in dendrites indicate that models suggesting that minus end-directed motor activity is sufficient for dendrite specific motor localization are inadequate. We suggest that a novel kinesin sorting mechanism is used by neurons to localize KIF21B protein to dendrites since its mRNA is restricted to the cell body.

Situs inversus and embryonic ciliary morphogenesis defects in mouse mutants lacking the KIF3A subunit of kinesin-II.

The embryonic cellular events that set the asymmetry of the genetic control circuit controlling left-right (L-R) axis determination in mammals are poorly understood. New insight into this problem was obtained by analyzing mouse mutants lacking the KIF3A motor subunit of the kinesin-II motor complex. Embryos lacking KIF3A die at 10 days postcoitum, exhibit randomized establishment of L-R asymmetry, and display numerous structural abnormalities. The earliest detectable abnormality in KIF3A mutant embryos is found at day 7.5, where scanning electron microscopy reveals loss of cilia ordinarily present on cells of the wild-type embryonic node, which is thought to play an important role in setting the initial L-R asymmetry. This cellular phenotype is observed before the earliest reported time of asymmetric expression of markers of the L-R signaling pathway. These observations demonstrate that the kinesin-based transport pathway needed for flagellar and ciliary morphogenesis is conserved from Chlamydomonas to mammals and support the view that embryonic cilia play a role in the earliest cellular determinative events establishing L-R asymmetry.

Interaction of the Escherichia coli DnaA protein with bacteriophage lambda DNA.

Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage lambda DNA demonstrated differential binding of DnaA along the whole lambda DNA. Interaction of DnaA with the lambda replication region (from the promoter pR to the origin of replication, orilambda) demonstrated a strong binding of DnaA to the region around the p(o) promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the lambda DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.

Role of adenine nucleotides, molecular chaperones and chaperonins in stabilization of DnaA initiator protein of Escherichia coli.

DnaA protein of Escherichia coli is a sequence-specific DNA binding protein required for the initiation of DNA replication from the chromosomal origin, oriC, and of several E. coli plasmids. At a moderate ionic strength, purified DnaA protein has a strong tendency to aggregate; the self-aggregate form is inactive in DNA replication. Binding of ATP or ADP to DnaA protein protected it from aggregation to maintain its replication activity. AMP or cyclic AMP had no protective effect. The molecular chaperone DnaK protected DnaA protein from aggregation with or without ATP. DnaJ and GrpE were not stimulatory. Chaperonins GroEL and GroES were also able to prevent aggregation but only in the presence of ATP. The studies presented here show that for DnaA protein to be active in the initiation of DNA replication, it must be prevented from forming a self-aggregate by the binding of adenine nucleotides, and/or by the action of molecular chaperones.

Identification, partial characterization, and genetic mapping of kinesin-like protein genes in mouse.

Microtubule-dependent motors of the kinesin superfamily have undergone structural and functional diversification during evolution and play crucial roles in cell division and intracellular transport. Degenerate oligonucleotides homologous to highly conserved regions of sequence within the motor domain were used in a polymerase chain reaction to isolate five new members (KIF3C, KIFC2, KIFC3, KIFC4, and KIF22) of the kinesin superfamily from a mouse brain cDNA library. Northern analysis showed that KIF3C and KIFC2 are expressed mainly in neural tissues, that KIFC4 and KIF22 are expressed primarily in proliferative tissues and cell lines, and that KIFC3 is apparently ubiquitous. To elucidate the organization of genes encoding kinesin-like motors in the mouse genome and to explore the potential associations of these genes with classical mouse mutations or human genetic diseases, these new genes as well as genes encoding the previously reported KIF3A and KIF3B motors were mapped to mouse chromosomes by using an interspecific backcross panel of DNAs from The Jackson Laboratory. The data indicate that the gene KIFC4 is present in three copies in the mouse genome on chromosomes 13 (KIFC4A), 10 (KIFC4B), and 17 (KIFC4C). The gene KIF22 is present in two copies on chromosomes 7 (KIF22A) and 1 (KIF22B). The genes KIF3A, KIF3B, KIF3C, KIFC2, and KIFC3 are each single loci and map to chromosomes 11, 2, 12, 15, and 8, respectively.

Neurofilament subunit NF-H modulates axonal diameter by selectively slowing neurofilament transport.

To examine the mechanism through which neurofilaments regulate the caliber of myelinated axons and to test how aberrant accumulations of neurofilaments cause motor neuron disease, mice have been constructed that express wild-type mouse NF-H up to 4.5 times the normal level. Small increases in NF-H expression lead to increased total neurofilament content and larger myelinated axons, whereas larger increases in NF-H decrease total neurofilament content and strongly inhibit radial growth. Increasing NF-H expression selectively slow neurofilament transport into and along axons, resulting in severe perikaryal accumulation of neurofilaments and proximal axonal swellings in motor neurons. Unlike the situation in transgenic mice expressing modest levels of human NF-H (Cote, F., J.F. Collard, and J.P. Julien. 1993. Cell. 73:35-46), even 4.5 times the normal level of wild-type mouse NF-H does not result in any overt phenotype or enhanced motor neuron degeneration or loss. Rather, motor neurons are extraordinarily tolerant of wild-type murine NF-H, whereas wild-type human NF-H, which differs from the mouse homolog at > 160 residue positions, mediates motor neuron disease in mice by acting as an aberrant, mutant subunit.

Mechanisms of selective motor neuron death in transgenic mouse models of motor neuron disease.

To examine the mechanism(s) of disease underlying ALS, transgenic mouse models have been constructed that express aberrant neurofilaments or mutations in the abundant, cytoplasmic enzyme superoxide dismutase 1 (SOD1). In addition to progressive weakness arising from selective motor neuron death, mice expressing a modest level of a point mutant in neurofilament subunit NF-L show most of the pathologic hallmarks observed in familial and sporadic ALS, including perikaryal proximal axonal swellings, axonal degeneration, and severe skeletal muscle atrophy. Additional mice expressing familial ALS-linked mutations in the cytoplasmic enzyme SOD1, the only proven cause of ALS and which accounts for approximately 20% of familial disease, have demonstrated that at least one mutation causes disease through acquisition of an adverse property by the mutant enzyme, rather than elevation or loss of SOD1 activity. These animals not only provide a detailed look at the pathogenic progression of disease, but also represent a tool for testing hypotheses concerning the specific mechanism(s) of neuronal death and for testing therapeutic strategies.

Domains of DnaA protein involved in interaction with DnaB protein, and in unwinding the Escherichia coli chromosomal origin.

DnaA protein of Escherichia coli is a sequence-specific DNA-binding protein required for the initiation of DNA replication from the chromosomal origin, oriC. It is also required for replication of several plasmids including pSC101, F, P-1, and R6K. A collection of monoclonal antibodies to DnaA protein has been produced and the primary epitopes recognized by them have been determined. These antibodies have also been examined for the ability to inhibit activities of DNA binding, ATP binding, unwinding of oriC, and replication of both an oriC plasmid, and an M13 single-stranded DNA with a proposed hairpin structure containing a DnaA protein-binding site. Replication of the latter DNA is dependent on DnaA protein by a mechanism termed ABC priming. These studies suggest regions of DnaA protein involved in interaction with DnaB protein, and in unwinding of oriC, or low-affinity binding of ATP.