Photosensitivity and heat resistance conferred by BrdU incorporation upon a thymidine kinase-deficient mouse cell line with persistent mitochondrial enzyme activity

The selective incorporation of 5-bromodeoxyuridine (BrdU) into mitochondrial DNA (mit-DNA) in the LM(TK-) ClID cell line, a thymidine kinase-deficient derivative of L fibroblasts with persistent mitochondrial enzyme activity, has been utilized to specifically damage mit-DNA by 'visible' light irradiation. ('Visible light' indicates the source of light used, although the components most active photochemically on BrdU-substituted DNA are in the near-visible range, 300-340 nm.) (Szybalski & Opara-Kubinski, 1965). LM(TK-) Cl ID cells, which had been grown in the presence of 30 mug/ml BrdU, were irradiated with 'visible' light. Analysis of the pre-existing mit-DNA in these cells, which had been long-term labelled with [5-3H]deoxycytidine, showed a progressive decrease, with increasing duration of irradiation, in the proportion of the closed-circular form and an increase in that of the open-circular form of mit-DNA, with the subsequent appearance of fragments of this DNA. Furthermore, there was a decrease during irradiation in the total amount of mit-DNA, which became about 35% of the non-irradiated control after 65 h irradiation. On the other hand, irradiation with 'visible' light failed to cause any quantitative or qualitative change, with respect to the non-irradiated control, in mit-DNA from cells grown in the absence of BrdU and long-term labelled with [Me-3h]thymidine. An analysis of the incorporation of [5-3H]deoxycytidine into mit-DNA of BrdU-grown cells, during a 3-h exposure of the cells to the precursor following irradiation, showed a fairly rapid decline of mit-DNA labelling; this became about 50% of the non-irradiated control after 12 h irradiation, decreasing to about 25% in the next 48 h. By contrast, no effect of irradiation was observed on the subsequent pulse-labelling of mit-DNA with [Me-3H]thymidine in cells grown in the absence of BrdU. Furthermore, no change in the size of the extracted nuclear DNA was found in irradiated BrdU-grown cells. The progressive and selective damage and destruction of mit-DNA during irradiation with 'visible' light of Cl ID cells correlate fairly well with the kinetics of loss of cell viability occurring under the same conditions, as described in the accompanying paper, strongly suggesting a link between the two phenomena

Construction of a recombinant plasmid containing xynb gene from Bacillus subtilis subsp. spizizeniiW23

This study aimed to clone thexynB gene from Bacillussubtilissubsp. spizizeniiW23,encodinga xylan l,4-beta-xylosidase to pMMB67EH plasmid which then be used to transformed EscherichiacoliDH-5a and Origami host cells. The xynBgenewas amplifiedby polymerasechain reaction (PCR) technique using a pair of primers flanking the gene sequence,and chromosomal DNA of the W23 strain as a template. Analyses of the recombinant plasmid were done by restriction analyses, and PCR detection. The result showed that the xynB has been cloned on pMMB67EH vector. The recombinant plasmid contained the xynB gene which was confirmed by restriction analyses and by PCR detection using primers pair's specific for the xynB gene and for the vector. The xylanaseactivity of xynB gene in E.coliDH-5a and Origami host cells was assayedon luria-Bertani-xylan plate qualitatively with addition of isopropyl B-D-thio-galactoside (IPTG) as an inducer.Uponsprayingwith Congored, the cells bearing the pMMB-xynB recombinant plasmid showed a xylan-degrading activity by the appearance ofclear zanearound the colonies whilethe transformant bearing an empty plasmid showed no clear zone. It could be concluded that the cloning Arocess was succeeded

Diphthongization of /u/ in Midwestern American English

Some researchers have noted that /u/ in modern Midwestern American English (MAE) seems to deviate from past documentations of the vowel, but none have conducted acoustic analyses that specifically target /u/ in MAE. I conducted a survey to examine how /u/ surfaces when produced by native Midwestern speakers. I used recordings that I obtained from native Midwestern speakers reading a word list that I designed to test /u/ in a variety of phonetic environments. Using Praat and acoustic analysis techniques, I compared the F2 values produced for various test words to expected F2 values for monophthong /u/. I found widespread surface realizations that were diphthongal rather than monophthongal. Factors such as age, gender, and individual language history are taken into account to see if any of these influence how the vowel surfaces. This project considers two possible theories to account for this phenomenon: historical processes beginning in Middle English, or a relatively new vowel shift. I examine the data collected from my speakers from both of these possible theoretical perspectives, and discuss the potential consequences and questions that positing either theory as the motivator of the diphthongization creates

An investigation of the link between the typical geometry errors and the Van Hiele levels of geometric thought of grade 9 learners

South African learners perform poorly in the geometry sections of both national and international assessments. Numerous assessment reports mention multiple errors that keep re-occurring and play a big role in the learners’ poor performance. For this research, the link between the grade 9 learners Van Hiele levels of thought and the typical errors that they made were investigated. In this mixed method study, 194 grade 9 learners in two schools in Port Elizabeth, South Africa were tested using a Van Hiele based test. A test was set up containing multiple-choice and open-ended questions and was used to determine firstly, the predominant level of geometric reasoning of the learners and secondly, to determine their typical errors. Semi-structured interviews were held with six learners to gain more insight into some of the typical errors uncovered in the tests. The quantitative data revealed that the learners’ predominant levels of geometric thought were low. Furthermore, the qualitative data revealed typical error patterns concerning angles and sides, parallel lines, hierarchy of quadrilaterals and incorrect reasons in the proofs. The quantitative and qualitative data was merged to determine if the errors could be linked to the Van Hiele levels. From the findings, it was concluded that most of their typical errors could be linked to the Van Hiele levels of the learners

Pengaruh Konflik Peran, Ketidakjelasan Peran, dan Komitmen Organisasi terhadap Kinerja Auditor

The purpose of this study was to collect evidence of three fraud factors which influenced auditor performance. They were conflict role, ambiguity role, and organization commitment at public accountant in Surabaya.Data were obtained by distributing questionnaire and by using multiple regression analysis. The results from this study showed that there were positive significant effect of organization commitment in auditor performance, and negative significant effect of conflict role, ambiguity role in auditor performance

Prevalence and Determinants of Overweight and Obesity in Preschoolers in Miami-Dade County

Background: The prevalence of childhood overweight in the United States continues to be a national public health problem. The child care and household environments play an important role in the nutrition, physical activity, and screen-time behaviors of preschoolers. Aims: This dissertation aimed to (1) estimate the prevalence of overweight and obesity in 3-5 year old children enrolled in child care centers in Miami-Dade County; (2) identify early life, dietary, and screen-time risk factors of overweight, and (3) assess child care centers\u27 level of adherence to nutrition, physical activity, and screen-time regulations by center socioeconomic location (SEP). Methods: A total of 366 children ages 3-5 years old from 34 child care centers participated in the study. Caregivers completed a questionnaire on socioeconomic, early life, dietary, and screen-time factors. We measured children\u27s heights and weights. Chi-square and logistic regression analyses were used to examine the association between child overweight and determinants. Results: the prevalence of overweight was 29.8% in this sample. The middle-SEP group had the highest prevalence of overweight (35.3%) when compared to low and high-SEP groups. Being Hispanic was associated with a three-fold risk of overweight/ obesity (AOR: 2.91, 95% CI 1.36, 6.21). Lack of daily fruit consumption increased the risk of overweight in Hispanic children. Middle-SEP children reported lowest consumption of fruits and vegetables. There were significant differences in breastfeeding practices by ethnicity and SEP. Assessment of child care practices resulted in all child care centers adhering to two-hour screen-time regulation for children older than 2-years old. Low and middle SEP centers fared better in serving of fruits , vegetables, and low fat/ fat free milk. The centers had incorporated quite and active play in their routines. Conclusions: This is the first study to examine prevalence by SEP in Miami-Dade County. Findings highlight opportunities for improvement in early life nutrition, as well as dietary and screen-time practices in the household and child care environments