Effect of experimentally induced hypertension on cerebellum of postmenopausal rat

Cerebellum seems to be a specific target for both the decrease of estrogen and hypertension in menopause. The aim of this study was to investigate the hypertension and menopause-induced changes in rat’s cerebellar cortex and the possible mechanisms of these changes. Rats were divided into four groups: the sham-operated control (SC-group), the ovariectomized (OVX-group), the hypertensive (H-group), and the ovariectomized-hypertensive (OVX-H-group) group. The mean arterial pressure (MAP), serum nitric oxide (NO), lipid peroxides and antioxidant catalase enzyme levels were assayed. Cerebellar tissue homogenization for analysis of lipid peroxides, antioxidant catalase enzyme, tumor necrosis factor-α (TNF-α), and estradiol was done. Quantification of adrenomedullin (AM) and interleukin-10 (IL-10) mRNA was also done. Cerebella were processed for histological, immunohistochemical and transmission electron microscopic examination. In the OVX-group, insignificant structural and biochemical changes were observed compared with the SC-group apart from the significantly increased lipid peroxides and decreased NO and catalase levels in serum. The H-group showed an elevated lipid peroxides and TNF-α levels, reduced catalase level, numerous degenerated Purkinje cells, vacuolations of the neuropil, some axonal degeneration, and few ghosts in the granular cell layer (GL). However, in OVX-H-group, oxidative stress, inflammation, and cerebellar damage were exacerbated and cerebellar estrogen was reduced associated with reduction in GL thickness and decreased Purkinje cells number. Most axoplasms had degenerated neurofilaments with abnormal myelination. The immunoexpression of glial fibrillary acidic protein were significantly increased in both OVX-group and H-group and significantly decreased in OVX-H group. Gene expression of AM and IL-10 were increased in cerebellar tissues of H-group compared with the SC-group but it was significantly decreased in OVX-H-group compared with H-group. Taken together, postmenopausal rats with hypertension suffered from structural cerebellar changes than rats with only hypertension or estrogen deficiency separately due to augmentation of the increased oxidative stress markers and the proinflammatory cytokines (TNF-α) with down regulation of the anti-inflammatory cytokine (IL-10) and the blood pressure regulator, AM. These suggested that high blood pressure is a critical factor for postmenopausal cerebellum.     

Calmodulin binding to Arabidopsis cyclic nucleotide gated ion channels

Recently we have reported that the αC-helix in the cyclic nucleotide binding domain (CNBD) is required for channel regulation and function of cyclic nucleotide gated ion channels (CNGCs) in Arabidopsis. A mutation at arginine 557 to cysteine (R557C) in the αC-helix of the CNBD caused an alteration in channel regulation. Protein sequence alignments revealed that R557 is located in a region that is important for calmodulin (CaM) binding. It has been hypothesized that CaM negatively regulates plant CNGCs similar to their counter parts in animals. However, only a handful of studies has been published so far and we still do not have much information about the regulation of CNGCs by CaM. Here, we conducted in silico binding prediction of CaM and Arabidopsis CNGC12 (AtCNGC12) to further study the role of R557. Our analysis revealed that R557 forms salt bridges with both D79 and E83 in AtCaM1. Interestingly, a mutation of R557 to C causes the loss of these salt bridges. Our data further suggests that this alteration in CaM binding causes the observed altered channel regulation and that R557 plays an important role in CaM binding.Key words: calmodulin, CaM, cyclic nucleotide gated ion channels, CNGC, environmental effect, defense responses, temperatureWe recently reported about the role of the αC-helix in the cyclic nucleotide binding domain (CNBD) for channel regulation and function of cyclic nucleotide gated ion channels (CNGCs) in Arabidopsis.¹ CNGCs were first discovered in vertebrate retinal photoreceptors and olfactory sensory neurons.^2,3 They are composed of a cytoplasmic N-terminus, six membrane spanning regions (S1–S6), a pore domain located between S5 and S6 and a cytoplasmic C-terminus and share similarities with the voltage-gated outward rectifying K⁺-selective ion channel (Shaker) proteins.² However, CNGCs are ligand gated and opened by the direct binding of cyclic nucleotides monophosphates (cNMPs), such as cAMP and cGMP (cNMPs).⁴ The cytoplasmic C-terminus contains a CNBD that is connected to the pore domain by a C-linker region.⁵ The function and regulation of CNGCs has been extensively studied in retinal photoreceptor and olfactory sensory neurons and it has been reported that their channel activity is moderated by Ca²⁺/calmodulin (CaM).⁶ A variety of Ca²⁺ permeable channels are known to be regulated by Ca²⁺/CaM. In many cases, CaM downregulates their activity, thereby creating negative feedback regulation for Ca²⁺ entry.⁷ CNGCs are also considered to follow this mode of regulation. Some animal CNGCs possess a CaM binding domain in the cytoplasmic N-terminus. For example, it has been reported that CaM binds to a short segment in the N-terminal region of the A2 subunit of CNGCs of olfactory sensory neurons in a Ca²⁺ dependent manner.^8,9The first plant CNGC, HvCBT1 (Hordeum vulgare calmodulin (CaM)-binding transporter), was identified as a CaM-binding protein in barley.¹⁰ Interestingly, in contrast to animal CNGCs, the CaM binding domain in HvCBT1 was shown to be located at the cytoplasmic C-terminal region.¹⁰ Subsequently, several CNGCs were identified from Arabidopsis and Nicotiana tabacum^11,12 and some of these CNGCs have also been shown to possess the CaM binding domain in the C-terminal region that partially overlaps with the CNBD.^11–14 This difference in the location of the CaM binding domain between animal and plant CNGCs implies that different mechanisms for CNGC regulation may have evolved in animals and plants.The Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22), which contains a novel chimeric CNGC, AtCNGC11/12, shows environmentally sensitive constitutive defense responses, such as heightened salicylic acid accumulation and hypersensitive response-like spontaneous programmed cell death.^15,16 It has been reported that the expression of AtCNGC11/12 and its channel activity is attributable for the cpr22 phenotype.^17,18 A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12.^1,18 These intragenic mutants are excellent tools to study the structure-function relationship of plant CNGCs. One of these mutants, suppressor S58, possesses a single amino acid substitution, arginine 557 to cysteine (R557C), in the αC-helix of the CNBD. The suppressor S58 lost all cpr22 related phenotypes, such as spontaneous cell death formation, under ambient temperature conditions. However, these phenotypes were recovered at 16°C, suggesting that the stability of channel function is affected by temperature.¹ Interestingly, this temperature sensitivity was also observed in the original mutant, cpr22.¹⁹ All salicylic acid-dependent phenotypes of cpr22 are enhanced under low temperature and low humidity conditions. Furthermore, this type of environmental sensitivity has been reported not only for cpr22, but also for various other pathogen resistance mutants as well as for defense responses in wild type plants.¹⁶ Therefore, it is possible that the basis of the temperature sensitivity observed in S58 may be related to a general environmental sensitivity in defense responses.Characterization of S58 and functional complementation using heterologous expression analyses suggested that R557 in the αC-helix of the CNBD is important for channel regulation, but not for basic channel function.¹ To further investigate this, we aimed to elucidate the molecular mechanism by which R557C (S58 mutation) alters channel regulation to determine the functional role of R557. Since R557 is located in the CNBD, it is possible that R557C alters cNMP binding resulting in disruption of channel opening. In animal systems, it has been reported that cNMPs bind within the pocket formed by the αC-helix and the β-barrel that is composed of the eight β sheets in the CNBD.^20,21 The αC-helix was suggested to function as the lid of this pocket that stabilizes cNMP binding by forming hydrophobic interactions with the bound cNMP.²¹ However, it is unlikely that R557 interacts directly with the bound cNMP because of its hydrophilic nature.Interestingly, a 19–20 amino acid sequence of the αC-helix was suggested to function as CaM binding domain in AtCNGC1 and AtCNGC2 by Köhler and Neuhaus using yeast two hybrid analysis.¹⁴ Arazi et al.¹³ biochemically demonstrated that a 23 amino acid sequence that overlaps with this 19–20 amino acids is the CaM binding domain in the tobacco CNGC, NtCBP4. Furthermore, they reported that the 4 additional amino acids (W R T/S W) which are located just outside of the 19–20 amino acid sequence are crucial for efficient binding to CaM. Sequence alignment revealed that R557 is located in this crucial sequence (W R T/S W).¹ Thus, it can be hypothesized that the R557C mutation causes a modification in the binding affinity to CaM resulting in an alteration in channel regulation. Therefore, we generated in silico models of CaM binding with the αC-helix of the CNBD in AtCNGC12 (identical to AtCNGC11/12) and AtCNGC12:R557C (S58). We first modeled Arabidopsis CaM1 (AtCaM1) based on the crystal structure of a potato CaM, PCM6 (PDB# 1RFJ).²² There are seven different CaM genes in Arabidopsis that encode two sets of identical isoforms (CaM1 and CaM4; CaM2, CaM3 and CaM5) and two additional distinct isoforms (CaM6 and CaM7).^23,24 All of them share high similarity with PCM6. Using the AtCaM1 model, we modeled possible interactions between AtCaM1 and the αC-helix of the CNBD in AtCNGC12 or AtCNGC12:R557C. As shown in Figure 1 (center part), a hydrophobic pocket of CaM that is necessary for binding with target proteins²⁵ was seen in AtCaM1 creating a binding pocket for the αC-helix of the CNBD in AtCNGC12. In this model, R557 creates salt bridges with both D79 and E83 of AtCaM1 (indicated by a box) and these salt bridges appear to play a role for binding to CaM. This type of salt bridges have been reported to be crucial for CaM binding with several different target proteins.²⁶ Interestingly, these salt bridges are no longer seen in AtCNGC12:R557C (Fig. 1, right part, indicated by a box); indicating that the mutation may cause an affinity change in CaM binding. Such an affinity change will likely cause an alteration in channel regulation. Since CaM is likely a negative regulator of CNGCs, this alteration in CaM affinity does not provide a simple mechanism for the R557C mutation. However, the change in CaM binding affinity may cause complex regulatory changes in CNGCs. Thus, we are currently conducting various biochemical analyses to validate this hypothesis.Open in a separate windowFigure 1Computational structural modeling of CaM binding with AtCNGC12 and AtCNGC12:R557C. Modeling of the tertiary structure of AtCaM1, and the αC-helix of AtCNGC12 and AtCNGC12:R557 was conducted using the crystallized structures of the potato CaM, PCM 6 (PDB# 1RFJ)²² and the cytoplasmic C-terminus of the invertebrate CNGC, SpIH (Flynn et al. 2007, PDB# 2PTM),²⁷ respectively, as templates. The protein fold recognition server (Phyre)²⁸ was used to model these proteins. The binding modeling was performed using an algorithm for molecular docking (PatchDock).²⁹ All the images were generated using PyMOL.³⁰ CaM is colored in cyan and the αC-helix is shown in magenta. Left part: overall binding model between AtCaM1 and AtCNGC12, Center part: close up of the boxed area of the left part in AtCNGC12, Right part: the same area in AtCNGC12:R557C. M73, M52 and M37 of AtCaM1 create a hydrophobic pocket together with F562 and I564 of AtCNGC12, which is a typical binding configuration between CaM and target proteins. R557 creates salt bridges with both D79 and E83 (center part). These salt bridges are no longer seen between AtCaM1 and AtCNGC12:R557C (right part).Although plants CNGCs have only recently been revealed to mediate multiple stress responses and also play important roles in some developmental pathways, studies that aim to elucidate their structural and regulatory properties are still very much in their infancy. Our current study will certainly contribute to a better understanding of the structure-function relationship and regulation of plant CNGCs.     

Injection Drug Use Is a Risk Factor for HCV Infection in Urban Egypt

Objective

To identify current risk factors for hepatitis C virus (HCV) transmission in Greater Cairo.

Design and Setting

A 1∶1 matched case-control study was conducted comparing incident acute symptomatic hepatitis C patients in two “fever” hospitals of Greater Cairo with two control groups: household members of the cases and acute hepatitis A patients diagnosed at the same hospitals. Controls were matched on the same age and sex to cases and were all anti-HCV antibody negative. Iatrogenic, community and household exposures to HCV in the one to six months before symptoms onset for cases, and date of interview for controls, were exhaustively assessed.

Results

From 2002 to 2007, 94 definite acute symptomatic HCV cases and 188 controls were enrolled in the study. In multivariate analysis, intravenous injections (OR = 5.0; 95% CI = 1.2–20.2), medical stitches (OR = 4.2; 95% CI = 1.6–11.3), injection drug use (IDU) (OR = 7.9; 95% CI = 1.4–43.5), recent marriage (OR = 3.3; 95% CI = 1.1–9.9) and illiteracy (OR = 3.9; 95% CI = 1.8–8.5) were independently associated with an increased HCV risk.

Conclusion

In urban Cairo, invasive health care procedures remain a source of HCV transmission and IDU is an emerging risk factor. Strict application of standard precautions during health care is a priority. Implementation of comprehensive infection prevention programs for IDU should be considered.     

Ovarian hydrobursitis in female camels (Camelus dromedaries): biochemical, bacterial and protozoal evaluation

The aim of this study was to evaluate female camels affected with ovarian hydrobursitis (n = 31) for hematological and biochemical findings and for bacterial and protozoal infections. Blood samples were obtained and surgical ablation of the affected bursa was performed. Bursal fluid, follicular fluid, and serum were subjected to hormonal and biochemical analyses. Bursal fluids were cultured and colonies were identified using BioMérieux Vitek two compact system. Passive haemagglutination test was used for detection of Trypanosoma evansi. Indirect ELISA technique was carried out for detection of anti-Hydatid cysts anti-bodies. Neutrophilia was found in the affected animals (P = 0.01) with tendencies for monocytosis (P = 0.06) and eosinophelia (P = 0.05). Bursal fluid had a tendency for high estradiol-17β concentration compared to blood serum (P = 0.07). Progesterone and cholesterol concentrations were similar in bursal fluid, follicular fluid and serum. Total protein, phosphorus, and magnesium concentrations were greater (P < 0.05) in the bursal fluid than in serum. Oligella urethralis, Alloiococcus otitis, Granulicatella adicens, Escherichia coli, Sphingobacterium thalpophilum, Streptococcus sanguinis, Aeromonas salmonicida, Pseudomonas stutzeri, Staphylococcus warneri, Staphylococcus hominis, and Rhizobium radiobacter were isolated from 46.7% of bursal fluids. T. evansi was positive in 9.7% of cases. None were positive for hydatid cyst. Accordingly, we suggest that the ovarian hydrobursitis syndrome is initially an inflammatory process and the accumulated bursal fluid is partially originated from follicular fluid.     

C-peptide corrects hepatocellular dysfunction in a rat model of type 1 diabetes

Journal of Physiology and Biochemistry - C-peptide is gaining much interest recently due to its well-documented beneficial effects on multiple organ dysfunction induced by diabetes. Our study was...     

Fibrinolytic activity of some fungi isolated from self-heated composted fertilizer

Mesophilic fungi isolated from organic fertilizer compost samples accounted for 70.94% of the total fungal count, while thermophilic and thermotolerant fungi constituted 29.05% of that count. Eight mesophilic fungal species, namelyAspergillus niger, Monilia sitophila, Paecilomyces divaricata, Penicillium chrysogenum, P. fellutanum, Scopulariopsis brevicaulis, S. brumptii andZygorhynchus japonicus; two thermophilic fungiHumicola grisea andOidiodendron flavum and three thermotolerant speciesAspergillus fumigatus, Thermomyces lanuginosus andZygorhynchus vuilleminii were isolated during the study. Most of the tested fungi showed a proteolytic activity and liquified gelatin in the test tube method and in cup plates. The thermophilic fungusO. flavum was the most potent proteolytic fungus. The comparative fibrinolytic assay revealed the following sequence in the ability of the tested fungi to hydrolyse fibrin:O. fiavum>S. brevicaulis>H. grisea>A. fumigatus>T. lanuginosus.     

Population structure and ecological role of Moringa peregrina (Forssk.) Fiori. at its northwestern range edge in the Hajar Mountains

Moringa peregrina (Moringaceae), an economically important tree that occurs in arid desert mountains, is currently threatened in large part of its distribution range in the Middle East and the Arabian Peninsula. We assessed the impact of topographic factors on population structure and performance of the species at its northwest range edge on the Hafit Mountain, UAE, where it faces harsh climatic conditions and human disturbances. Size structure, fruit production, and vitality were assessed on all 165 individual plants encountered in 12 gorges. The size distribution indicates that the population of M. peregrina is young; the proportion of small and medium-size individuals is high, but with few recorded juveniles. The frequency of plant occurrence and their performance were highest at middle altitudes. Large trees produced significantly more fruits than small and medium plants, with the larger trees of the population at middle altitude producing most fruits. M. peregrina facilitated the growth of grasses and small shrubs, but limited the presence of other big shrubs and trees beneath their canopies, compared to areas beyond. The study indicates that gorges play an important role in maintaining the M. peregrina population at its hyper-arid northwest range edge.     

Effects of sub-lethal concentrations of synthetic insecticides and Callitris glaucophylla extracts on the development of Aedes aegypti.

Synthetic and botanical insecticides can have a profound effect on the developmental period, growth, adult emergence, fecundity, fertility, and egg hatch, resulting in effective control at sub-lethal concentrations. This paper investigated sub-lethal concentrations of fenitrothion, lambda-cyhalothrin, and Callitris glaucophylla Joy Thomps. & L.P. Johnson (Cupressaceae) extract to characterize their effects on the development of Aedes aegypti L. (Diptera: Culicidae) mosquito larvae. The LC25, LC50, and LC75 (four replicates) were used for each synthetic insecticide and the LC25 and LC75 (four replicates) were used for C. glaucophylla. Observations of larval mortality, duration of larval stage, pupal mortality, duration of pupal stage, adult emergence, sex ratio, and malformations were recorded over 14 days. A dose-response effect was observed for all insecticides. Although C. glaucophylla extract doses were higher than synthetic insecticide doses, the LC75 treatment outperformed synthetics by completely prohibiting adult emergence. Consequently, this botanical is recommended for field application either in combination with synthetic or natural insecticides or alone.     

Impact of mycogenic zinc nanoparticles on performance,behavior, immune response,and microbial load in Oreochromis niloticus

This work aims to evaluate the antibacterial activity of biological zinc nanoparticles (BIO-ZnONPs) against pathogenic fish bacteria and assess the effect of BIO-ZnONPs on the performance, behavior, and immune response in Nile tilapia (Oreochromis niloticus) as compared to chemical zinc nanoparticles (CH- ZnONPs). Aspergillus niger TS16 fabricated the BIO-ZnONPs were spherical shape with the average size of 45 nm and net charge of ?27.23 mV. Generally, the results indicate that BIO-ZnONPs were more effective than CH- ZnONPs in enhancing the performance properties of Nile tilapia. Five experimental groups of Nile tilapia (initial body weight of 20.2 g) were treated with two concentrations of 0.5 and 1 mg L^?1 from biological and chemical ZnONPs, while the fifth group was served as a control. After ten weeks of treated water with ZnONPs, the performance, feed efficiency parameters, feeding, and swimming behaviors significantly improved in BIO-ZnONPs treated groups (P < 0.05). The liver function, LYZ activity, and NBT values were significantly enhanced in the 0.5 mg L^?1 BIO-ZnONPS group compared to CH- ZnONPs group and control (P < 0.05). Furthermore, the lowest cortisol and the highest testosterone and growth hormone levels were recorded in 1 mg L^?1 BIO-ZnONPs group. Regarding the antibacterial effects, BIO-ZnONPs displayed the lower total bacterial loads in water and fish tissues (intestine, gills, skin, and muscle) and the maximum antibacterial properties against pathogenic bacteria (Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Aeromonas hydrophila). Our study exemplifies novel findings of BIO-ZnONPs in the promotion of fish health and production and its antibacterial properties in Nile tilapia.     

Evidence for a dominant major gene conferring predisposition to hepatitis C virus infection in endemic conditions

Hepatitis C virus (HCV), infecting 170 million people worldwide, is a major public health problem. In developing countries, unsafe injections and blood transfusions are thought to be the major routes of transmission. However, our previous work in a population from Egypt, endemic for HCV, revealed highly significant familial correlations, strongly suggesting the existence of both familial transmission of the virus and genetic predisposition to HCV infection. We investigated the hypothesis of genetic predisposition by carrying out a segregation analysis of HCV infection in the same population. We used a logistic regression model simultaneously taking into account a major gene effect, familial correlations and relevant risk factors. We analyzed 312 pedigrees (3,703 subjects). Overall HCV seroprevalence was 11.8% and increased with age. The main associated risk factors were previous parenteral treatment for schistosomiasis and blood transfusions. We found strong evidence for a dominant major gene conferring a predisposition to HCV infection. The frequency of the predisposing allele was 0.013, reflecting a strong predisposition to HCV infection in 2.6% of the subjects, particularly those under the age of 20. This study provides evidence for the involvement of host genetic factors in susceptibility/resistance to HCV infection in endemic conditions.