Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

Variations in problem conceptualizations and intended solutions among Hong Kong students

Cognitive schema were used to explain health and illness behaviors among Chinese students. University students in Hong Kong were asked to attribute causes and suggest solutions to five health/mental health problems: Weakness/Fatigue, Tension/ Anxiety, Sleep Difficulty, Hollow/Emptiness, and Headache. The patterns of endorsement on the causal and solution categories used for the five problems were compared using a new asymptotic chi-squared test. The response patterns were found to be significantly different across the five problems. Each problem was attributed to multiple causes including psychological, social, situational, somatic, and existential factors. The intended solutions were often related to the nature of the causal attributions especially when the problems were mild. In lay help-seeking, the Hong Kong students would attempt a variety of self-help measures. However, for professional consultation, the medical doctor would be the primary care professional the students would turn to for most of the problems except in the case of Hollow/Emptiness.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.     

Longitudinal,genome-scale analysis of DNA methylation in twins from birth to 18 months of age reveals rapid epigenetic change in early life and pair-specific effects of discordance

Background

The extent to which development- and age-associated epigenetic changes are influenced by genetic, environmental and stochastic factors remains to be discovered. Twins provide an ideal model with which to investigate these influences but previous cross-sectional twin studies provide contradictory evidence of within-pair epigenetic drift over time. Longitudinal twin studies can potentially address this discrepancy.

Results

In a pilot, genome-scale study of DNA from buccal epithelium, a relatively homogeneous tissue, we show that one-third of the CpGs assayed show dynamic methylation between birth and 18 months. Although all classes of annotated genomic regions assessed show an increase in DNA methylation over time, probes located in intragenic regions, enhancers and low-density CpG promoters are significantly over-represented, while CpG islands and high-CpG density promoters are depleted among the most dynamic probes. Comparison of co-twins demonstrated that within-pair drift in DNA methylation in our cohort is specific to a subset of pairs, who show more differences at 18 months. The rest of the pairs show either minimal change in methylation discordance, or more similar, converging methylation profiles at 18 months. As with age-associated regions, sites that change in their level of within-pair discordance between birth and 18 months are enriched in genes involved in development, but the average magnitude of change is smaller than for longitudinal change.

Conclusions

Our findings suggest that DNA methylation in buccal epithelium is influenced by non-shared stochastic and environmental factors that could reflect a degree of epigenetic plasticity within an otherwise constrained developmental program.     

Noise Exposure and Hearing Impairment among Chinese Restaurant Workers and Entertainment Employees in Hong Kong

Background

Noise-induced hearing loss (NIHL) is a major concern in the non-manufacturing industries. This study aimed to investigate the occupational noise exposure and the NIHL among Chinese restaurant workers and entertainment employees working in the service industry in Hong Kong.

Methods

This cross-sectional survey involved a total of 1,670 participants. Among them, 937 were randomly selected from the workers of Chinese restaurants and 733 were selected from workers in three entertainment sectors: radio and television stations; cultural performance halls or auditoria of the Leisure and Cultural Services Department (LCSD); and karaoke bars. Noise exposure levels were measured in the sampled restaurants and entertainment sectors. Each participant received an audiometric screening test. Those who were found to have abnormalities were required to take another diagnostic test in the health center. The “Klockhoff digit” method was used to classify NIHL in the present study.

Results

The main source of noise inside restaurants was the stoves. The mean hearing thresholds showed a typical dip at 3 to 6 KHz and a substantial proportion (23.7%) of the workers fulfilled the criteria for presumptive NIHL. For entertainment sectors, employees in radio and television stations generally had higher exposure levels than those in the halls or auditoria of the LCSD and karaoke bars. The mean hearing thresholds showed a typical dip at 6 KHz and a substantial proportion of the employees fulfilled the criteria for presumptive NIHL (38.6%, 95%CI: 35.1–42.1%). Being male, older, and having longer service and daily alcohol consumption were associated with noise-induced hearing impairment both in restaurant workers and entertainment employees.

Conclusion

Excessive noise exposure is common in the Chinese restaurant and entertainment industries and a substantial proportion of restaurant workers and entertainment employees suffer from NIHL. Comprehensive hearing conservation programs should be introduced to the service industry in Hong Kong.     

Dynamin is functionally coupled to insulin granule exocytosis

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 beta-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of beta-cell insulin secretion.     

Development and evaluation of a protein microarray chip for diagnosis of hepatitis C virus

A protein chip diagnostic kit was developed for the diagnosis of hepatitis C virus (HCV) based on the protein chip technique and the immuno-concentration method. This kit was designed for low-density protein chips and also for the availability of multiple sample screening. Applicability of the chip was evaluated using 96 blood specimens and the results were compared to results of an anti-HCV enzyme immunoassay (EIA) test. With further development, the technology associated with the development of this chip could be applied to the simultaneous detection of multiple protein-protein, protein-ligand interactions.