Nuclear matrix proteins in well and poorly differentiated human breast cancer cell lines

The nuclear matrix, besides providing the structural support of the nucleus, is involved in various cellular functions of the nucleus. Nuclear matrix proteins (NMPs), which are both tissue- and cell type–specific, are altered with transformation and state of differentiation. Furthermore, NMPs have been identified as informative markers of disease states. Here, the NMP profiles from human breast cancer cell lines and breast tumours were analyzed using two-dimension gel electrophoresis. We identified NMPs that are associated with well and poorly differentiated human breast cancer cells in vitro and in vivo. Five NMPs (NMBC 1–5) were found to be exclusive for well-differentiated human breast cancer cells, while one NMP (NMBC-6) was found to be present only in poorly differentiated human breast cancer cells. The identification of these proteins suggests the potential use of nuclear matrix proteins as prognostic indicators. J. Cell. Biochem. 66:9–15, 1997. © 1997 Wiley-Liss, Inc.     

研究程序化细胞死亡的科学方法

运用科学认识论和方法论,围绕2002年诺贝尔生理医学奖,讨论了程序化细胞死亡的研究过程及方法特点,得出了几点重要结论,对于未来生物学新现象的探索具有积极作用。     

Sustaining an efficient and effective CHO cell line development platform by incorporation of 24‐deep well plate screening and multivariate analysis

Efficient and effective cell line screening is paramount toward a successful biomanufacturing program. Here we describe the implementation of 24‐deep well plate (24‐DWP) screening of CHO lines as part of the cell line development platform at AbbVie. Incorporation of this approach accelerated the identification of the best candidate lines for process development. In an effort to quantify and predict process performance comparability, we compared cell culture performance in and in shake flasks, for a panel of Chinese Hamster Ovary cell lines expressing a monoclonal antibody. The results in 24‐DWP screening showed reduced growth profiles, but comparable viability profiles. Slow growers in 24‐DWP achieved the highest productivity improvement upon scaling‐up to shake flasks. Product quality of the protein purified from shake flasks and 24‐DWP were also compared. The 24‐DWP culture conditions were found to influence the levels of acidic species, reduce the G0 N‐glycan species, and increase the high‐mannose N‐glycan species. Nevertheless, the identification of undesirable profiles is executed consistently with the scaled‐up culture. We further employed multivariate data analysis to capture differences depending on the two scales and we could demonstrate that cell line profiles were adequately clustered, regardless of the vessel used for the development. In conclusion, the 24‐DWP platform was reasonably predictive of the parameters crucial for upstream process development activities, and has been adapted as part of the AbbVie cell line development platform. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:175–186, 2018     

Directed Evolution of the DnaK Chaperone: Mutations in the Lid Domain Result in Enhanced Chaperone Activity

We improved the DnaK molecular chaperone system for increased folding efficiency towards two target proteins, by using a multi-parameter screening procedure. First, we used a folding-deficient C-terminal truncated chloramphenicol acetyl transferase (CAT_Cd9) to obtain tunable selective pressure for enhanced DnaK chaperon function in vivo. Second, we screened selected clones in vitro for CAT_Cd9 activity after growth under selective pressure. We then analyzed how these variants performed as compared to wild type DnaK towards folding assistance of a second target protein; namely, chemically denatured firefly luciferase. A total of 11 single point DnaK mutants and 1 truncated variant were identified using CAT_Cd9 as the protein target, while 4 of the 12 selected variants showed improved luciferase refolding in vitro. This shows that improving the DnaK chaperone by using a certain target substrate protein, does not necessarily result in a loss or reduction in its ability to assist other proteins. Of the 12 identified mutations, half were clustered in the nucleotide binding domain, and half in the lid domain (LD) of DnaK. The truncated variant is characterized by a 35-residue C-terminal truncation (Cd35) and exhibited the highest improvement for luciferase refolding. Cd35 showed a 7-fold increase in initial refolding rate for denatured luciferase and resulted in a 5-fold increase in maximal luminescence as compared to wild type DnaK. Given that the best in vitro performing mutants contained LD substitutions, and that the LD is not involved in ATP binding, ATP hydrolysis or client protein association, but is involved in allosteric regulation of the chaperone cycle, we propose that improved DnaK variants result in changes to allosteric domain communication, ultimately retuning the ATP-dependent chaperone cycle.     

Survey of environmental enhancement programs for laboratory primates

Animal welfare regulations in the United States require that nonhuman primate environmental enhancement plans be made in accordance with currently accepted professional standards; however, little information is available for quantifying common practice. Here we report the results of a 2003 survey that was sent to individuals overseeing enrichment programs at a variety of primate research institutions. The surveys requested information on program administration and management, implementation standards, procedures, and constraints pertaining to major categories of environmental enrichment, as well as intervention plans for animals exhibiting behavioral pathologies. Data were obtained on the management of 35,863 primates in 22 facilities. Behavioral scientists performed program oversight at the majority of facilities. Most programs reported recent changes, most commonly due to external site visits, and least commonly resulting from internal review. Most facilities' institutional animal care and use committees (IACUCs) included of individuals with behavioral expertise, and about two-thirds reported that enrichment issues could influence research protocol design. While most primates were reported to be housed socially (73%), social housing for indoor-housed primates appears to have changed little over the past 10 years. Research protocol issues and social incompatibility were commonly cited constraints. Implementation of feeding, manipulanda, and structural enrichment was relatively unconstrained, and contributions to these aspects of behavioral management generally included individuals in a wide variety of positions within a facility. In contrast, enrichment devices were used on a less widespread basis within facilities, and positive reinforcement programs that involved dedicated trainers were rare. We suggest that altering the role of the IACUC would be a productive avenue for increasing the implementation of social housing, and that an emphasis on prevention rather than intervention against behavioral pathology is warranted. The data from this survey may be useful for anticipating future program evaluations, establishing more effective internal evaluations, and assessing program progress and resource allocation.     

Prevalence of Weight Cycling and its Relation to Health Indicators in Finland

Objective: This study was performed to identify weight cyclers and to assess the prevalence of weight cycling and its relation to health indicators in Finnish adults. Research Methods and Procedures: Data for the study consisted of 3320 men and 3540 women (25 to 64 years of age). The subjects went through a health examination, and data on intentional weight losses and regains during the last 10 years were collected by a questionnaire. The subjects were divided into five groups: severe weight cyclers (weight loss ≥ 5 kg at least three times with regain), mild weight cyclers (weight loss ≥ 5 kg one to two times and regain), successful dieters (weight loss ≥ 5 kg with no regain), nonobese nondieters, and obese nondieters. Results: Approximately 7% of men and 10% of women were defined as severe weight cyclers, and an additional 11% and 19% were defined as mild weight cyclers, respectively. In men, 20% of severe weight cyclers and 15% of obese nondieters perceived their health as poor, whereas in other groups, these proportions were <10%. In both sexes, severe weight cyclers seemed to have visited a doctor more frequently than nonobese nondieters. Severe weight cyclers were also more likely to use some medication compared with other groups. Discussion: Weight cycling is more common in women than in men, and it seems to be associated with more regular visits to a doctor and poor self‐perceived health. However, because of the cross‐sectional design of the study, causal conclusions cannot be drawn.     

Inducible nitric oxide synthase haplotype associated with hypertension and responsiveness to antihypertensive drug therapy

Hypertension is a multifactorial disorder associated with increased inducible nitric oxide synthase (iNOS) expression and activity. While genetic polymorphisms affect iNOS expression, it is not known whether iNOS gene polymorphisms affect the susceptibility to hypertension and the responses to antihypertensive therapy. This study aimed at assessing whether iNOS polymorphisms ((CCTTT)_n, g.-1026C > A, and g.2087G > A) and haplotypes are associated with hypertension and with responsiveness to drug therapy. We studied 115 well controlled hypertensive patients (HTN), 82 hypertensive patients resistant to optimized antihypertensive therapy (RHTN), and 113 normotensive healthy subjects (NT). Genotypings were carried out using real-time polymerase chain reaction (PCR) and PCR amplification followed by capillary electrophoresis. The software PHASE 2.1 was used to estimate the haplotype frequencies in each group. Variant genotypes (GA + AA) for the g.2087G > A polymorphism were more commonly found in hypertensive patients (HTN + RHTN) than in normotensives (P = 0.016; OR = 2.05). We found no associations between genotypes and responsiveness to therapy (P > 0.05). The S-C-A haplotype was more commonly found in hypertensive patients (HTN + RHTN) than in normotensives (P = 0.014; OR = 6.07). Interestingly, this haplotype was more commonly found in the HTN group than in the RHTN group (P = 0.012; OR = 0.14). Our findings indicate that the g.2087G > A polymorphism in the iNOS gene affects the susceptibility to hypertension. Moreover, while the S-C-A haplotype is associated with hypertension, it is also associated with responsiveness to antihypertensive therapy.     

Phylogenetic relationships among six species of Epistylis inferred from 18S-ITS1 sequences

Phylogenetic relationships among six species of Epistylis (i. e. E. plicatilis, E. urceolata, E. chrysemydis, E. hentscheli, E. wenrichi, and E. galea) were investigated using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA). Amplified rDNA fragment sequences consisted of 215 or 217 bases of the flanking 18S and 5.8S regions, and the entire ITS-1 region (from 145 to 155 bases). There were more than 33 variable bases between E. galea and the other five species in both the 18S region and the ITS-1 region. The affiliation of them was assessed using Neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) analyses. In all the NJ, MP and ML analyses E. galea, whose macronucleic position and shape are distinctly different from those of the other five species, was probably diverged from the ancestor of Epistylis earlier than the other five species. The topology in which E. plicatilis and E. hentscheli formed a strongly supported sister clade to E. urceol     

To hunt or not to hunt? A feeding enrichment experiment with captive large felids

It is often difficult to promote the successful performance of feeding behaviors in zoos, especially for carnivores. Feeding enrichment provides these opportunities and often improves behavioral indications of an animal's well‐being and the experience of the zoo visitor. The effectiveness of two different feeding enrichment techniques was evaluated on five subjects in two species of felids: African lions and Sumatran tigers. The activity budgets of each cat were compared before, during, and after enrichment, focusing on activity levels, frequency and variety of feeding behaviors, and occurrence of stereotypic behaviors. The presentation of live fish increased the variety and frequency of feeding behaviors, while presentation of horse leg bones increased the frequency of these behaviors. Fish reduced the tigers' stereotypic behavior from 60% of scans to 30% of scans on the day of presentation, and this change was maintained for 2 days following enrichment. Bone presentation also reduced stereotypic behavior and increased nonstereotypic activity in both species. Both of these techniques appear to have sustained effects on behavior lasting at least 2 days after presentation, which may indicate their ability to alter the animals' underlying activity patterns. Zoo Biol 22:189–198, 2003. © 2003 Wiley‐Liss, Inc.     

Environmental and Health Implications of Nanotechnology—Have Innovators Learned the Lessons from Past Experiences?

Nanotechnology (NT) is expected to bring about novel technological designs and materials resulting in a wide spectrum of applications. Experience gained from past innovations shows that new technologies are often accompanied by undesired side-effects. If such side-effects are neglected or underestimated, they may result in damage. In this article we examine whether innovators, the pioneers of technological advance in nanotechnology, are aware of the lessons that can be learned from adverse effects that have occurred in connection with several past innovations. Based on the results of a survey taken among innovators we discuss what consequences the innovators draw for the present innovation process and which priorities they set when dealing with environmental and health risks of nanotechnology. Results suggest that innovators may be not very sensitive to early scientific warnings regarding risks of nanotechnology. The innovators are confident that risks are assessable and manageable on a “business as usual” basis. They consider lacking public acceptance as a potential hurdle for innovation and many innovators are afraid of a backlash. Nevertheless, they seldom engage in risk communication or stakeholder dialogue. Picking up recommendations voiced by the innovators interviewed, we sketch some possible approaches as to how innovators could tackle the potential risks of nanotechnology in a proactive manner.