Pattern of dietary supplement use and its psychosocial predictors among females visiting fitness centers in Saudi Arabia: Findings from a cross-sectional study.

Background: Establishing a balanced diet is necessary for obtaining the required daily micronutrients and macronutrients. However, the importance of using dietary supplements to complement one's diet is a significant aspect to consider. While studies have reported the prevalence and patterns of dietary supplements in Saudi Arabia, most focused on university students, women in their reproductive life-particularly during pregnancy-or male athletes. Hence, the objective of this study is to estimate the prevalence and patterns of dietary supplement usage, along with the associated sociodemographic and psychiatric predictors among women attending fitness centers in Saudi Arabia. Methods: A cross-sectional design was utilized using a structured questionnaire to collect data on dietary intakes such as consumption, nutrition supplements, type of nutrition supplements, and reason for consuming dietary supplements. Data collection occurred in female fitness centers, with participants accessing the questionnaires through QR codes linked to online Google forms. The dietary usage was assessed using a structured questionnaire developed by the study authors, and depressive and anxiety symptoms were assessed using the PHQ-9 and the GAD-7 scales, respectively. SPSS 23.0 was utilized to analyze the data, and a multivariable logistic regression analysis was performed to determine the association between sociodemographic and psychiatric predictors and patterns of dietary supplement usage. The results were presented by reporting an unadjusted and adjusted odds ratio (AOR) with 95 % Confidence Intervals (CIs). Results: The total number of participants in this study was 453 women. Half of the 453 women (46.1 %) consumed dietary supplements, and (84.0 %) reported taking proteins, caffeine, creatinine, mineral salts, fish oil, and/or amino acids or a combination of these dietary supplements. A considerable number of women (41.0 %) reported that a doctor, dietitian, or trainer motivated them to take the dietary supplement, and (38.0 %) of women self-prescribed dietary supplements. More than half (51 %) of the women consumed dietary supplements to compensate for nutrient deficiency, and 48 % used them to improve their health. Women who did not work to earn were 53 % less likely to consume dietary supplements (AOR = 0.47; [95 % CI: 0.30-0.72]) than women who reported working to earn either in the private or government sector. Depressed women were (42 %) less likely to consume dietary supplements than non-depressed women (AOR = 0.58; [95 % CI: 0.33-0.99]) after adjusting for sociodemographic variables and other medical conditions. Conclusion: It has been evident that a large proportion of women enrolled in the fitness center report consuming dietary supplements, mostly to compensate for energy and to improve overall health. The findings highlight that non-working and depressed women were less likely to use dietary supplements than their counterparts. By understanding these patterns, the community can tailor health interventions and policies to ensure equitable access to proper dietary support, especially for those facing challenges like depression and unemployment. Future research on a larger sample of women outside fitness centers is encouraged to explore further dietary supplement consumption and patterns among diverse population groups, and to ultimately develop health guidelines for the appropriate use of supplements.

REGECEL (an Oxidized Regenerated Cellulose) Provides Superior Bioactivity Effect on Microorganisms.

Introduction: Patients who need to be readmitted to the hospital because of complications from infections or require long-term care and rehabilitation face substantial financial hardships. To ensure the safety of patients undergoing surgery, it is crucial to implement measures that prevent wound infections before and after the procedure. Antibacterial wound dressings are essential to prevent infections during surgical procedures. There are various types of antibacterial wound dressings available on the market, such as silver-based dressings, hydrocolloid dressings, polyhexamethylene biguanide, alginate dressings, collagen-based dressings, and iodine-based dressings. Methods: We used each type (standard, knit, fibril, and non-woven) of a commercial brand of oxidized regenerated cellulose (ORC) called Regecel to test bacterial growth. The choice of antibacterial wound dressing depends on the type of wound being treated. Different bacterial strains require specific culture conditions to thrive and grow in laboratory settings. To obtain accurate and reliable results, it is vital to follow the precise culture conditions required for each bacterial strain. Results: The evaluation of ORC highlighted its potential to inhibit bacterial growth, showing promising results against various bacterial strains and Candida albicans. Different variants of ORC, such as Regecel, have demonstrated impressive capacity to hinder the growth of 32 distinct bacterial strains, with inhibition rates ranging from 40-100%. These bacteria include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and penicillin-resistant Streptococcus pneumoniae. Conclusion: This study supports the usage and development of ORC (Regecel) as an innovative approach to treating bacterial infections.

Molecular Profiling of Inflammatory Mediators in Human Respiratory Syncytial Virus and Human Bocavirus Infection.

Infections due to human respiratory syncytial virus (HRSV) and human bocavirus (HBoV) can mediate the release of several pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α, which are usually associated with disease severity in children. In this study, the change in the expression profile of cytokines and chemokines were determined during HRSV, HBoV, and HRSV coinfection with HBoV in 75 nasopharyngeal aspirates (NPAs) samples, positive real-time reverse transcriptase PCR Assay (rRT-PCR) for HRSV (n = 36), HBoV (n = 23) infection alone or HRSV coinfection with HBoV (n = 16). The samples were collected from hospitalized children. qPCR-based detection revealed that the levels of IL-6, IL-8, IL-10, IL-13, IL-33, and G-CSF were significantly (p < 0.05) greater in patients than in controls. IL-4, IL-17, GM-CSF, and CCL-5 were significantly elevated in children with HRSV coinfection with HBoV than in other groups (p < 0.05). TNF-α, IL-6, IL-8, IL-10, IL-13, and IL-33 in children with HRSV were significantly increased in severe infections compared to mild infections. Whereas, IL-10, IL-13, and IL-33 were significantly increased in severe infection in compared a mild infection in children with HBoV. Further large-scale investigations involving isolates are needed to enhance our knowledge of the association between viral infections and cytokine expression patterns during the different stages of HRSV and HBoV infection.

A Physiologically Based Pharmacokinetic Model to Predict Systemic Ondansetron Concentration in Liver Cirrhosis Patients.

INTRODUCTION: Ondansetron is a drug that is routinely prescribed for the management of nausea and vomiting associated with cancer, radiation therapy, and surgical operations. It is mainly metabolized in the liver, and it might accumulate in patients with hepatic impairment and lead to unwanted adverse events. METHODS: A physiologically based pharmacokinetic (PBPK) model was developed to predict the exposure of ondansetron in healthy and liver cirrhosis populations. The population-based PBPK simulator PK-Sim was utilized for simulating ondansetron exposure in healthy and liver cirrhosis populations. RESULTS: The developed model successfully described the pharmacokinetics of ondansetron in healthy and liver cirrhosis populations. The predicted area under the curve, maximum systemic concentration, and clearance were within the allowed twofold range. The exposure of ondansetron in the population of Child-Pugh class C has doubled in comparison to Child-Pugh class A. The dose has to be adjusted for liver cirrhosis patients to ensure comparable exposure to a healthy population. CONCLUSION: In this study, the developed PBPK model has described the pharmacokinetics of ondansetron successfully. The PBPK model has been successfully evaluated to be used as a tool for dose adjustments in liver cirrhosis patients.

Assessment of Proinflammatory Cytokines Among Patients with Middle East Respiratory Syndrome Coronavirus Infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with significant morbidity and mortality. This study was performed to assess the proinflammatory cytokines profile among MERS-CoV patients. A total of 46 MERS-CoV-infected patients (27 symptomatic and 19 asymptomatic) were assessed and compared with 52 normal healthy controls for plasma levels of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-17, IL-7, IL-6, interferon (IFN)-α, and IL-15 using a customized luminex kit. Whereas asymptomatic MERS-CoV patients and controls were no different; the mean plasma levels among MERS-CoV symptomatic patients were significantly higher than the normal controls: IL-1ß (16.89 ± 1.23 vs. 12.80 ± 0.59 pg/mL; p < 0.001), TNF-α (14.04 ± 0.93 vs. 10.35 ± 0.29 pg/mL; p < 0.0001), IL-17 (14.3 ± 0.89 vs. 11.47 ± 0.61 pg/mL; p < 0.001), IL-7 (21.56 ± 1.00 vs. 16.31 ± 0.30 pg/mL; p < 0.0001), IL-6 (156.5 ± 37.90 vs. 18.60 ± 1.59 pg/mL; p < 0.0001), and IFN-α (68.73 ± 13.06 vs. 23.57 ± 1.05 pg/mL; p < 0.0001). The mean plasma levels of IL-7 (24.81 ± 1.63 vs. 19.79 ± 0.94 pg/mL; p < 0.01), IL-6 (312.7 ± 94.67 vs. 101.2 ± 25.67 pg/mL; p < 0.01), and IFN-α (89.00 ± 18.97 vs. 51.05 ± 8.68 pg/mL; p < 0.05) were significantly elevated among MERS-CoV symptomatic patients with fatal outcome compared with MERS-CoV symptomatic patients who survived. Only IL-7 was found to have a higher risk ratio of mortality (4.76, 95% confidence interval: 1.5-14.94; p < 0.01). No differences were observed in IL-15 levels among the groups. Significantly elevated proinflammatory cytokines among symptomatic MERS-CoV-infected patients may contribute to manifestations of cytokine storm frequently observed among critically ill MERS-CoV patients and IL-7 may serve as a marker for disease activity.

Can periodontal pockets and caries lesions act as reservoirs for coronavirus?

The periodontal pocket and likely caries lesions may act as a reservoir and source of dissemination and development of systemic infections. While periodontal pockets have been found to harbor several viral species, there is no information on its ability to serve as a reservoir for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have used a real-time polymerase chain reaction (RT-PCR) approach to evaluate SARS-CoV-2 in periodontal pockets and cavitated caries lesions in a cross-sectional study of 72 participants who were divided into six groups: symptomatic positive COVID-19 cases with periodontal pockets, symptomatic positive with cavitated caries lesions, asymptomatic positive with periodontal pockets, asymptomatic positive with cavitated caries lesions, positive control, and negative control. A total of 180 samples were interrogated by RT-PCR to amplify the SARS-CoV-2 E and S genes. SARS-CoV-2 was present in 41.7% of symptomatic positive COVID-19 cases with periodontal pockets and 16.7% of symptomatic positive with cavitated caries lesions. The mean Ct value of E and S genes in periodontal pockets patients were 36.06±0.46 and 30.06±6.73, respectively, and the mean Ct value for both genes in caries lesions patients were 35.73±4.14, and 34.78±1.93, respectively. The sensitivity, specificity, and accuracy to detect SARS-CoV-2 among periodontal pockets were 20.8% (95% CI 7.13-42.15), 100% (95% CI 73.54-100.0), and 47.2% (95% CI 30.22-64.51), respectively. Among cavitated caries lesions patients, they were 8.3% (95% CI 1.03-27.0), 100% (95% CI 73.54-100.0), and 38.9% (95% CI 23.14-56.54), respectively. SARS-CoV-2 can be detected in periodontal pockets and caries lesions, and these sites may act as reservoirs for the virus. However, the sensitivity of SARS-CoV-2 detection is low compared with other methods. To our knowledge, this report is the first to investigate the relationship between SARS-CoV-2 and periodontal pockets and caries.

The short-term effect of different chlorhexidine forms versus povidone iodine mouth rinse in minimizing the oral SARS-CoV-2 viral load: An open label randomized controlled clinical trial study.

Several investigations evaluated the possibility of different types of mouth wash rinse in minimizing the SARS-CoV-2 load. However, results still controversial. The study aim is to assess the short-term efficiency of several over-the-counter mouth rinses and lozenges in minimizing the salivary viral load for SARS-CoV-2 in patients with confirmed COVID-19 in comparison to saline. This is a randomized controlled clinical trial with 4 arms. The recruited cases were randomized using a simple randomization technique and were assigned to chlorhexidine digluconate mouth rinse (CHX mouth rinse), 2 mg of chlorhexidine digluconate lozenges (CHX lozenges), povidone iodine mouth rinse (PVP-I mouth rinse) or saline as a control group. Saliva were collected from all study subjects by passive drool technique at two time points. First, prior to intervention with mouth rinse or the lozenges, the baseline saliva sample was collected. Second saliva samples were collected immediately after the mouth rinse. Real time PCR was conducted and the value threshold cycle (Ct) for each sample was recorded. Majority of the participants had an education level of high school or less (60%), were married (68.3), males (58.3%), and non-smokers (58.5%). No statistically significant differences between groups at the two times test (P > .05). However, a significant decrease of salivary viral load in all four groups combined (P-value for E genes = .027, and for S genes = .006), and in PVP-I mouth rinse specifically (P = .003 and P = .045, respectively). Povidone iodine mouth rinse showed a potential influence on the reduction of the viral load on a short-term basis. However, longer-term studies of the effect of these products should be conducted.

Possible vertical transmission of corona virus disease 19 (COVID-19) from infected pregnant mothers to neonates: a multicenter study.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious with various possible routes of transmission, resulting in high mortality globally. Controversy exists regarding the vertical transmission of the SARS-CoV-2 infection to fetuses of COVID-19-infected women. The aim of this study was to investigate the possibility of the vertical transmission of SARS-CoV-2 from COVID-19-infected mothers to their neonates. MATERIALS AND METHODS: We prospectively collected demographical and clinical characteristics of 31 COVID-19 positive pregnant women and their neonates. All mothers and neonates were tested for SARS-CoV-2 infection using the real-time polymerase chain reaction on nasopharyngeal swabs and breast milk samples. Antenatal and placental abnormalities were ultrasonically and histopathologically examined. In cord blood samples, the immunoglobins (Ig) M and IgG were estimated qualitatively. RESULTS: The women's mean age and gestational age were 31 years and 38 weeks, respectively, with 58% undergoing an elective cesarean section. Gestational diabetes was reported in 29% of cases, 64.5% of women were medically free and only 16.12% were symptomatic. A normal antenatal ultrasound was observed in 77.42% of cases. Nine cord blood samples were positive for IgG. Villous infarction (24%), villous agglutination, and chorangiosis (51%), accelerated villous maturation (21%) and reduced and hypercoiling were reported for 6.97% of the umbilical cords. Three newborns had possible vertical transmission of SARS-CoV-2 infection, of which, two were preterm and IUFD. The third neonate was born full-term, admitted to NICU and later discharged in good health. CONCLUSION: Our findings support the possibility of the direct vertical transmission of the SARS-CoV-2 infection to neonates from infected mothers. Further studies with a larger sample size are required to validate the current findings.

Correction to: Influenza vaccine coverage, awareness, and beliefs regarding seasonal influenza vaccination among people aged 65 years and older in Central Saudi Arabia.

The original version of this article unfortunately contained a mistake in affiliation of the second author.

Influenza vaccine coverage, awareness, and beliefs regarding seasonal influenza vaccination among people aged 65 years and older in Central Saudi Arabia.

OBJECTIVES: To investigate the level of awareness, sources of knowledge, and beliefs about the influenza vaccine in people ≥65 years in central Saudi Arabia. METHODS: A cross-sectional study that used self-administered questionnaires conducted in 3 primary healthcare centers in Riyadh, Saudi Arabia between May 2018 and August 2018. RESULTS: There were 496 Saudis participants (70.2% male) were included, of whom, 47.8% had been vaccinated against influenza at least once. Of unvaccinated individuals, 46% believed that the vaccine was unnecessary. The majority of participants (70.2%) preferred to receive medical information on vaccination from doctors and health workers. Participants with higher levels of education were significantly more likely to have been vaccinated (p less than 0.05). Compared to the vaccinated participants, unvaccinated individuals were significantly less likely to be aware of the Ministry of Health campaign against influenza, believe that influenza vaccine does not weaken the immune system, know that elderly people and people with long-standing health problems should be regularly vaccinated against influenza, and believe that the influenza vaccine was the best way of prevention. Approximately 40% of the participants considered the influenza vaccine to be very effective and safe. CONCLUSIONS: There is a need for further programs in order to increase the awareness level of influenza vaccination. Also, health workers need to make more effort to educate patients because they are the preferred source of information.

Screening for Q fever. A tertiary care hospital-based experience in central Saudi Arabia.

OBJECTIVES: To evaluate the presence of Coxiella burnetii  (C. brunetii) infection among patients presenting with fever of unknown origin (FUO). METHODS: A cross-sectional study of 100 patients (54 men and 46 women; mean age: 34.3 ± 19.2 years) with FUO was conducted at King Khalid University Hospital, Riyadh, Saudi Arabia between March 2015 and June 2016. Phase 1 and phase 2 C. burnetii-specific antibodies in serum samples were detected by enzyme-linked immunosorbent assay. RESULTS: Coxiella burnetii phase 1 and phase 2 antibodies were detected in 16% of the patients. Phase 2 IgM was present in 2% of the patients, whereas phase 2 IgG antibodies were detected in 11%  of the patients. Coxiella burnetii-specific phase 1 IgG was found in 2% of the patients, and 8% of the patients harbored phase 1 IgA antibodies in their serum. CONCLUSION: The presence of C. burnetii-specific antibodies in many patients suffering from FUO highlights the importance of Q fever screening among patients presenting with febrile illness.

Clinical validation of 3 commercial real-time reverse transcriptase polymerase chain reaction assays for the detection of Middle East respiratory syndrome coronavirus from upper respiratory tract specimens.

Since discovery of Middle East respiratory syndrome coronavirus (MERS-CoV), a novel betacoronavirus first isolated and characterized in 2012, MERS-CoV real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays represent one of the most rapidly expanding commercial tests. However, in the absence of extensive evaluations of these assays on positive clinical material of different sources, evaluating their diagnostic effectiveness remains challenging. We describe the diagnostic performance evaluation of 3 common commercial MERS-CoV rRT-PCR assays on a large panel (n = 234) of upper respiratory tract specimens collected during an outbreak episode in Saudi Arabia. Assays were compared to the RealStar® MERS-CoV RT-PCR (Alton Diagnostics, Hamburg, Germany) assay as the gold standard. Results showed i) the TIB MolBiol® LightMix UpE and Orf1a assays (TIB MolBiol, Berlin, Germany) to be the most sensitive, followed by ii) the Anyplex™ Seegene MERS-CoV assay (Seegene, Seoul, Korea), and finally iii) the PrimerDesign™ Genesig® HCoV_2012 assay (PrimerDesign, England, United Kingdom). We also evaluate a modified protocol for the PrimerDesign™ Genesig® HCoV_2012 assay.

Prevalence and characterization of occult hepatitis B infection among blood donors in central Saudi Arabia.

OBJECTIVES: To evaluate the prevalence of occult hepatitis B viral infections (OBIs)  among blood donors considering the clinical and epidemiological importance of identifying OBIs. METHODS: A cross-sectional study conducted at King Khalid University Hospital, Riyadh, Saudi Arabia between January 2011 and January 2012. Blood donors (n=8501) were screened for Hepatitis B virus surface antigen (HBsAg) and hepatitis B core antibodies (HBcAb). All HBsAg-negative and HBcAb-positive samples were tested further for hepatitis B surface antibodies (HBsAb), hepatitis B virus (HBV)-DNA, and HBV genotyping.   RESULTS: Of the 8501 serum samples tested, 56 (0.7%) were positive and 8445 (99.3%) were negative for HBsAg. Among the HBsAg-negative samples, 198 (2.3%) were positive for HBcAb and these patients were suspected to have OBIs. Among the HBcAb-positive samples, 119 (60.1%) were positive while 79 (39.9%) were negative for HBsAb. Analysis of HBV-DNA for the suspected OBIs showed that 17 out of 198 samples (8.6%) yielded positive results, and all of them were HBsAb-negative. The viral load was low (less than 20-186 IU/mL) in all OBIs. Hepatitis B virus genotyping showed that 15 out of 17 samples (88.2%) were genotype D, and the other 2 samples (11.8%) were genotype E.  CONCLUSION: The prevalence of OBIs among blood donors in Riyadh was 0.2%. Therefore, it is recommended that HBV molecular testing should be incorporated with serological assays for screening of blood donors.

Homologous recombination between the tuf genes of Salmonella typhimurium.

The genes coding for the translation factor EF-Tu, tufA and tufB are separated by over 700 kb on the circular chromosome of Salmonella typhimurium. The coding regions of these genes have 99% identity at the nucleotide level in spite of the presumed ancient origin of the gene duplication. Sequence comparisons between S. typhimurium and Escherichia coli suggest that within each species the two tuf genes are evolving in concert. Here we show that each of the S. typhimurium tuf genes can transfer genetic information to the other. In our genetic system the transfers are seen as non-reciprocal, i.e. as gene conversion events. However, the mechanism of recombination could be reciprocal, with sister chromosome segregation and selection leading to the isolation of a particular class of recombinant. The amount of sequence information transferred in individual recombination events varies, but can be close to the entire length of the gene. The recombination is RecABCD-dependent, and is opposed by MutSHLU mismatch repair. In the wild-type, this type of recombination occurs at a rate that is two or three orders of magnitude greater than the nucleotide substitution rate. The rate of recombination differs by six orders of magnitude between a recA and a mutS strain. Mismatch repair reduces the rate of this recombination 1000-fold. The rate of recombination also differs by one order of magnitude depending on which tuf gene is donating the sequence selected for. We discuss three classes of model that could, in principle, account for the sequence transfers: (1) tuf mRNA mediated recombination; (2) non-allelic reciprocal recombination involving sister chromosomes; (3) non-allelic gene conversion involving sister chromosomes, initiated by a double-strand break close to one tuf gene. Although the mechanism remains to be determined, the effect on the bacterial cells is tuf gene sequence homogenisation. This recombination phenomenon can account for the concerted evolution of the tuf genes.

Mutations to kirromycin resistance occur in the interface of domains I and III of EF-Tu.GTP.

The antibiotic kirromycin inhibits protein synthesis by binding to EF-Tu and preventing its release from the ribosome after GTP hydrolysis. We have isolated and sequenced a collection of kirromycin resistant tuf mutations and identified thirteen single amino acid substitutions at seven different sites in EF-Tu. These have been mapped onto the 3D structures of EF-Tu.GTP and EF-Tu.GDP. In the active GTP form of EF-Tu the mutations cluster on each side of the interface between domains I and III. We propose that this domain interface is the binding site for kirromycin.

Mutants of EF-Tu defective in binding aminoacyl-tRNA.

Five single amino acid substitution variants of EF-Tu from Salmonella typhimurium were tested for their ability to promote poly(U)-translation in vitro. The substitutions are Leu120 Gln, Gln124 Arg and Tyr160 (Asp or Asn or Cys). They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III of the EF-Tu.GTP configuration. The different EF-Tu variants exhibit a spectrum of phenotypes. First, k cat/K(M) for the interaction between ternary complex and the programmed ribosome is apparently reduced by the substitutions Leu120 Gln, Gln124 Arg and Tyr160 Cys. Second, this reduction is caused by a defect in the interaction between these EF-Tu variants and aminoacyl-tRNA during translation. Third, in four cases out of five the affinity of the complex between EF-Tu.GTP and aminoacyl-tRNA is significantly decreased. The most drastic reduction is observed for the Gln124 Arg change, where the association constant is 30-fold lower than in the wild-type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant (kd) for the release of GDP from EF-Tu is increased 6-fold by the Tyr160 Cys substitution, but remains unchanged in the four other cases. These results show that the formation of ternary complex is sensitive to many different alterations in the domain I-III interface of EF-Tu.

Missense substitutions lethal to essential functions of EF-Tu.

We have used a simple selection and screening method to isolate function defective mutants of EF-Tu. From 28 mutants tested, 12 different missense substitutions, individually lethal to some essential function of EF-Tu, were identified by sequencing. In addition we found a new non-lethal missense mutation. The frequency of isolation of unique mutations suggests that this method can be used to easily isolate many more. The lethal mutations occur in all three structural domains of EF-Tu, but most are in domain II. We aim to use these mutants to define functional domains on EF-Tu.

Origin-specific reduction of ColE1 plasmid copy number due to mutations in a distinct region of the Escherichia coli RNA polymerase.

Mutations affecting a region of the Escherichia coli RNA polymerase have been isolated that specifically reduce the copy number of ColE1-type plasmids. The mutations, which result in a single amino acid alteration (G1161R) or a 41-amino acid deletion (Delta1149-1190) are located near the 3'-terminal region in the rpoC gene, which encodes the largest subunit (beta ') of the RNA polymerase. The rpoC deletion and the point mutation cause over 20- and 10-fold reductions, respectively, in the copy number of ColE1. ColE1 plasmid numbers are regulated by two plasmid-encoded RNAs: RNA II, which acts as a preprimer for the DNA polymerase I to start initiation of replication, and RNA I, its antisense inhibitor. Altered expression from the RNA I and RNA II promoters in vivo was observed in the RNA polymerase mutants. The RNA I/RNA II ratio is higher in the mutants than in the wild-type strain and this is most probably the main reason for the reduction in the ColE1 copy number in the two rpoC mutants.

tRNA-ribosome interactions.

Direct measurements of the rates of dissociation of dipeptidyl-tRNA from the ribosome show that hyperaccurate SmP and SmD ribosomes have unstable A-site binding of peptidyl-tRNA, while P-site binding is extremely stable in relation to the wild type. Error-prone Ram ribosomes, on the other hand, have stable A-site and unstable P-site binding of peptidyl-tRNA. At least for these mutant ribosomes, we conclude that stabilization of peptidyl-tRNA in one site destabilizes binding in the other. Elongation factor Tu (EF-Tu) undergoes a dramatic structural transition from its GDP-bound form to its active GTP-bound form, in which it binds aa-tRNA (aminoacyl-tRNA) in ternary complex. The effects of substitution mutations at three sites in domain I of EF-Tu, Gln124, Leu120, and Tyr160, all of which point into the domain I-domain III interface in both the GTP and GDP conformations of EF-Tu, were examined. Mutations at each position cause large reductions in aa-tRNA binding. An attractive possibility is that the mutations alter the domain I-domain III interface such that the switching of EF-Tu between different conformations is altered, decreasing the probability of aa-tRNA binding. We have previously found that two GTPs are hydrolyzed per peptide bond on EF-Tu, the implication being that two molecules of EF-Tu may interact on the ribosome to catalyze the binding of a single aa-tRNA to the A-site. More recently we found that ribosomes programmed with mRNA constructs other than poly(U), including the sequence AUGUUUACG, invariably use two GTPs per peptide bond in EF-Tu function. Other experiments measuring the protection of aa-tRNA from deacylation or from RNAse A attack show that protection requires two molecules of EF-Tu, suggesting an extended ternary complex. To remove remaining ambiguities in the interpretion of these experiments, we are making direct molecular weight determinations with neutron scattering and sedimentation-diffusion techniques.