Localization of Pyrophosphatase and V-ATPase in Chlamydomonas reinhardtii

Microsomal membranes of Chlamydomonas reinhardtii possess PPase and V-ATPase activities. By immunogold labelling we have shown that H⁺-pyrophosphatase (PPase) is localized to membranes of lytic and contractile vacuoles of Chlamydomonas, in which the density of antigen in the latter is much higher. In addition, PPase is conspicuously present in trans cisternae and transpole elements of the Colgi apparatus. Such a distribution for PPase has hitherto not been reported. A positive in situ identification for PPase at the plasma membrane, including the flagellar membrane, was also made, and has also been confirmed by Western blotting and activity measurements on isolated plasma membranes. V-ATPase antisera which cross react with polypeptides of this transport complex from maize roots failed to recognize anything in Western blots of Chlamydomonas microsomal membranes. Thus immunogold labelling for V-ATPase was not possible with Chlamydomonas. On the other hand, surfaces of contractile vacuole membranes as revealed by deepetching were covered by conspicuous 9 ? 11.5 nm diameter smooth particles which had a central hole. These were very similar to those previously identified by Heuser et al., (1993) as the V,-head of V-ATPase in Dictyostelium contractile vacuoles. Another type of membrane image, designated “intermediate-sized vesicle”, was found associated with the contractile vacuole. It was characterized by densely-packed 6 ? 7.5nm diameter polygonal particles, which upon rotation analysis showed both 5- and 6-fold symmetries, also with a central hole. These particles are interpreted as representing either PPase complexes or the V₀ body of the V-ATPase in etched fractured membrane surfaces. We have incorporated these findings into a model of contractile vacuole function.     

PVA based nanofiber containing cellulose modified with graphitic carbon nitride/nettles/trachyspermum accelerates wound healing

Today, bacterial cellulose has received a great deal of attention for its medical applications due to its unique structural properties such as high porosity, good fluid uptake, good strength, and biocompatibility. This study aimed to fabricate and study bacterial cellulose/graphitic carbon nitride/nettles/trachyspermum nanocomposite by immersion and PVA/BC/g-C₃N₄/nettles/trachyspermum nanofiber by electrospinning method as a wound dressing. The g-C₃N₄ and g-C₃N₄ solution were synthesized and then were characterized using Fourier transform infrared, X-ray diffraction, Zeta Potential, and scanning electronic microscope analyzes. Also, the antibacterial properties of the synthesized materials were proved by gram-positive and gram-negative bacteria using the minimum inhibitory concentration method. Besides, the toxicity, migration, and cell proliferation results of the synthesized materials on NIH 3T3 fibroblasts were evaluated using MTT and scratch assays and showed that the BC/PVA/g-C₃N₄/nettles/trachyspermum composite not only had no toxic effect on cells but also contributed to cell survival, cell migration, and proliferation has done. To evaluate the mechanical properties, a tensile strength test was performed on PVA/BC/g-C₃N₄/nettles/trachyspermum nanofibers, and the results showed good strength of the nanocomposite. In addition, in vivo assay, the produced nanofibers were used to evaluate wound healing, and the results showed that these nanofibers were able to accelerate the wound healing process so that after 14 days, the wound healing percentage showed 95%. Therefore, this study shows that PVA/BC/g-C₃N₄/nettles/trachyspermum nanofibers effectively inhibit bacterial growth and accelerate wound healing.     

Detection of K-ras and p53 mutations in sputum samples of lung cancer patients using laser capture microdissection microscope and mutation analysis

Mutations in the p53 tumor suppressor gene and the K-ras oncogene have been frequently found in sputum and bronchoalveolar lavage (BAL) samples of lung cancer patients and other patients prior to presenting clinical symptoms of lung cancer, suggesting that they may provide useful biomarkers for early lung cancer diagnosis. However, the detection of these gene mutations in sputum and BAL samples has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells and, in the case of p53 gene, at many codons. In this study, sputum cells were collected on a filter membrane by sputum cytocentrifugation and morphologically analyzed. Epithelial cells were selectively taken by using a laser capture microdissection microscope and analyzed by polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP) for p53 mutations and by PCR and denaturing gradient gel electrophoresis (DGGE) for K-ras mutations. This method was used to analyze sputum of 15 Chinese women with lung cancer from Xuan Wei County, China and detected mutations in sputum of 7 (46.7%) patients, including 5 patients with p53 mutations, 1 patient with a K-ras mutation, and 1 patient with K-ras and p53 mutations. For comparison, only two of the mutations were detected by conventional methods. Therefore, the laser capture/mutation analysis method is sensitive and facilitates the detection of low-fraction mutations occurring throughout the p53 and K-ras genes in sputum of lung cancer patients. This method may be applicable to the analysis of epithelial cells from clinically normal sputum or BAL samples from individuals with a high risk for developing lung cancer.     

Urinary 1-hydroxypyrene and mutagenicity in bus drivers and mail carriers exposed to urban air pollution in Denmark

BACKGROUND: Previous studies in Denmark have shown that bus drivers and tramway employees were at an increased risk for developing several types of cancer and that bus drives from central Copenhagen have high levels of biomarkers of DNA damage.AIMS: The present study evaluates 1-hydroxypyrene concentrations and mutagenic activity in urine as biomarkers of exposure in non-smoking bus drivers in city and rural areas on a work day and a day off and in non-smoking mail carriers working outdoors (in the streets) and indoors (in the office). METHODS: Twenty-four hour urine samples were collected on a working day and a day off from 60 non-smoking bus drivers in city and rural areas and from 88 non-smoking mail carriers working outdoors (in the streets) and indoors (in the office). The concentration of 1-hydroxypyrene was measured by means of HPLC and the mutagenic activity was assessed by the Ames assay with Salmonella tester strain YG1021 and S9 mix. The N-acetyltransferase (NAT2) phenotype was used as a biomarker for susceptibility to mutagenic/carcinogenic compounds. RESULTS: Bus drivers excreted more 1-hydroxypyrene in urine than did mail carriers. The differences were slightly smaller when NAT2 phenotype, cooking at home, exposure to vehicle exhaust, and performing physical exercise after work were included. The NAT2 slow acetylators had 29% (1.29 [CI: 1.15-1.98]) higher 1-hydroxypyrene concentrations in urine than the fast acetylators. Male bus drivers had 0.92 revertants/mol creatinine [CI: 0.37-1.47] and female bus drivers 1.90 revertants/mol creatinine [CI: 1.01-2.79] higher mutagenic activity in urine than mail carriers. CONCLUSION: The present study indicates that bus drivers are more exposed to polycyclic aromatic hydrocarbons (PAH) and mutagens than mail carriers. Mail carriers who worked outdoors had higher urinary concentration of 1-hydroxypyrene, a marker of exposure to PAH, than those working indoors. The individual levels of urinary mutagenic activity were not correlated to excretion of 1-hydroxypyrene. This might be due to the fact that the most potent mutagenic compounds in diesel exhaust are not PAH but dinitro-pyrenes. Among bus drivers, fast NAT2 acetylators had higher mutagenic activity in urine than slow NAT2 acetylators and female bus drivers had higher mutagenic activity than male bus drivers.     

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.     

Effects of different gelling agents on the different stages of rice regeneration in two rice cultivars

Plant tissue culture technology offers a solution for meeting the increasing commercial demand on economically important plants such as rice, a widespread dietary staple. However, significant genotype-specific morphogenetic responses constitute a considerable on rice regeneration in plant biotechnology contexts. Aside from genotype dependency, the components of the nutrient media including gelling agents have an important impact on regeneration efficiency. The current study explores the effect of different gelling agents on various stages of rice regeneration in two Egyptian rice cultivars-Sakha104 and Giza178. Media solidified with varying concentrations of a variety of gelling agents (agar, bacto agar, gelrite and phytagel) were tested for their impact on the frequency of callus induction, shoot regeneration and rooting. The results indicated gellan gum (gelrite and phytagel) was superior to agar products (agar and bacto agar) for callus induction. By contrast, no significant differences were found between different gelling agents for shoot regeneration. Gellan gum and media solidified with bacto agar were found to lead to significantly higher root regeneration than agar. The Sakha104 cultivar showed better responses than Giza 178 for callus induction and similar performance to the Giza 178 cultivar for root regeneration irrespective of the gelling agent. This work provides insights into the impact of different gelling agents on the morphogenetic response of two rice cultivars and can be used to help maximize the frequency of rice regeneration.     

<i>Trichoderma</i>-Induced Improvement in Growth,Photosynthetic Pigments,Proline, and Glutathione Levels in <i>Cucurbita pepo</i> Seedlings under Salt Stress

Salt stress is one of the major abiotic stress in plants. However, traditional approaches are not always efficient in conferring salt tolerance. Experiments were conducted to understand the role of Trichoderma spp. (T. harzianum and T. viride) in growth, chlorophyll (Chl) synthesis, and proline accumulation of C. pepo exposed to salinity stress. There were three salt stress (50, 100, and 150 mM NaCl) lavels and three different Trichoderma inoculation viz. T. harzianum, T. viride, and T. harzianum + T. viride. Salt stress significantly declined the growth in terms of the shoot and root lengths; however, it was improved by the inoculation of Trichoderma spp. C. pepo inoculated with Trichoderma exhibited increased synthesis of pigments like chl a, chl b, carotenoids, and anthocyanins under normal conditions. It was interesting to observe that such positive effects were maintained under salt-stressed conditions, as reflected by the amelioration of the salinity-mediated decline in growth, physiology and antioxidant defense. The inoculation of Trichoderma spp. enhanced the synthesis of proline, glutathione, proteins and increased the relative water content. In addition, Trichoderma inoculation increased membrane stability and reduced the generation of hydrogen peroxide. Therefore, Trichoderma spp. can be exploited either individually or in combination to enhance the growth and physiology of C. pepo under saline conditions.     

Effectiveness of naturally occurring Aphis gossypii on tomato plants as a bio-indicator for heavy metals in Riyadh and Hafar Al-Batin,Saudi Arabia

Although certain pollutants can be biologically degraded by microorganisms, rendering their impact short-term, others can not be impaired, such that their effect persists. The present study evaluates the effectiveness of using a field-collected aphid, Aphis gossypii, as a bio-indicator for heavy metals in tomato farms in Riyadh and Hafar Al-Batin, Saudi Arabia. Heavy metals were selected (Cd, Cu, Zn, and Pb) and measured for comparative screening in field-collected plants, soil, and aphids using inductively coupled plasma-mass spectrometry (ICP-MS). Field-collected aphids from both studied regions were identified as Aphis gossypii. In Riyadh, there was no significant difference observed for Cd, Cu, and Zn for all experimental samples, while, Pb was showed differences among samples especially tomato leaves None of the studied samples in Hafar Al-Batin were showed statistically significant differences in Cd, in reverse to significant differences in the other heavy metals. Comparing concentrations of selected heavy metals between the two studied regions was showed that neither region showed a significant difference in heavy metals except for Cu. This study demonstrates that tomato leaf samples showed the highest concentrations of most studied heavy metals, followed by soil, then aphids. Aphids were utilized as a bio-indicator of heavy metals in the studied regions.     

Two green and sensitive spectrofluorimetric approaches for determination of Ambroxol and guaifenesin in their single and combined pharmaceutical formulations

Ambroxol hydrochloride (AMX) and guaifenesin (GFN) are approved drugs utilized to treat coughs through their potent mucolytic and expectorant properties. Due to their massive, combined administration in many illnesses, there is a persistent need for their concurrent estimation in different pharmaceutical formulations. Two sensitive, environmentally friendly spectrofluorimetric methods were developed. AMX was determined using the first method (I) without interference from GFN. This method depends on the quenching of Erythrosine B (EB) native fluorescence at 552 nm after excitation at 527 nm due to the formation of a non-fluorescent AMX-EB ion-pair complex in Britton–Robinson buffer (BRB) solution pH (3.5). The concentration plot is linear over the 0.25–5.0 μg/mL range, with a mean percent found value of 99.74%. Method (II) depends on measuring the native fluorescence of aqueous GFN solution at two analytical wavelengths, either 300 or 600 nm, after excitation at 274 nm. Relative fluorescence intensity (RFI)–concentration plots are linear over the ranges of 0.02–0.5 and 0.1–2.0 μg/ml, with mean percent found at 99.96% and 99.91% at dual wavelengths, respectively. The proposed methods were successfully applied to assay both drugs in raw materials and different single and combined pharmaceutical formulations. These methods have been thoroughly validated following International Committee on Harmonisation (ICH) guidelines. National Environmental Methods Index, Analytical Eco-Scale, and Green Analytical Procedure Index were used to prove greenness, thereby enhancing their applicability. The proposed techniques provide straightforward, precise, and cost-effective solutions for routine formulation analysis in quality control laboratories.