Mycoplasmosis in Poultry: An Evaluation of Diagnostic Schemes and Molecular Analysis of Egyptian Mycoplasma gallisepticum Strains.

Infections with Mycoplasma gallisepticum (MG) in poultry are associated with a wide range of disease conditions, including those affecting the respiratory and reproductive systems. The purpose of this study was to endorse the more sensitive diagnostic scheme for MG infection and identify the best molecular marker for MG phylogenetic analysis using six housekeeping genes: mgc2, mraW, atpG, ugpA, DUF31196, and lgT. For these purposes, 55 poultry flocks of different species were screened using either qRT-PCR or PCR techniques analogous to conventional culturing from non-cultured and cultured swabs on PPLO broth. The rate of MG positivity was the highest when using qRT-PCR from cultured broth (89.0%) and the lowest when using conventional culturing (34.5%). Compared to qRT-PCR from broth, statistical analysis using the Roc curve in MedCalc statistical software showed that the PCR schemes (qRT-PCR from swabs and PCR from swabs and broth) performed better than conventional culturing in terms of sensitivity, accuracy, and area under the curve (AUC), suggesting that they may be more reliable schemes. Further support was added by Cohen's kappa test, showing moderate agreement between the molecular approaches. Among the six screened genes, mgc2 and mraW had the highest detection rates (69% and 65.4%, respectively). The comparative phylogenetic analysis revealed that mgc2 or atpG gene sequences distinguished MG isolates into different clades with high discriminatory power.

Liposomal encapsulated oregano, cinnamon, and clove oils enhanced the performance, bacterial metabolites antioxidant potential, and intestinal microbiota of broiler chickens.

Encapsulated phytochemicals with augmented therapeutic and nutritional characteristics have become promising alternatives to antimicrobials in the poultry industry. Hence, our key target was to explore the efficacy of liposomal encapsulation, as a novel carrier, for essential oils (LEOs) on growth, digestibility, intestinal microbiota, and bacterial metabolites of broiler chickens. Moreover, the impact of encapsulated EOs on transcription mechanisms targeting the genes encoding digestive enzymes, gut barrier functions and antioxidant potential of broiler chickens was evidenced. Four equal broiler groups were fed 4 basal diets fortified with LEOs (oregano, cinnamon, and clove) at the levels of 0, 200, 300, and 400 mg/kg diet. Our findings revealed significant improvement in body weight gain and feed conversion ratio of birds fed higher levels of LEOs. These results came concurrently with increasing the activities of digestive enzymes at both serum and molecular levels and consequently nutrient digestibility (dry matter, ether extract, crude protein, and crude fiber) in these groups. Remarkably, the abundance of beneficial bacteria as well as the bacterial metabolites (valeric acid, butyric acid, propionic acid, acetic acid, and total short-chain fatty acids) was increased, while that of pathogenic ones was reduced following dietary inclusion of LEOs. Of note, the mRNA expression of genes encoding antioxidant stability [catalase (CAT), superoxide dismutase 1 (SOD-1), glutathione peroxidase 1 (GPX-1), nuclear factor erythroid 2-related factor 2 (NRF2), NAD(P)H dehydrogenase quinone 1 (NQO1), and heme oxygenase-1 (HO-1)] as well as barrier functions [mucin-2 (MUC-2)] and tight junction proteins, TJP [junctional adhesion molecule-2 (JAM-2) and occludin] were noticeably upregulated in broilers fortified with 400 mg/kg diet of LEOs. Overall, the present work recommended dietary inclusion of LEOs as beneficial additives for attaining targeted performance, gut health and antioxidant stability in poultry farming.

Different kinetics of chicken interferon-alpha signalling transduction responses following immunization of broiler chickens with different Newcastle disease virus vaccines and infection with virulent genotype VIId strain.

Newcastle disease virus (NDV) is a highly contagious and notifiable avian disease leading to grave economic losses in the poultry industry. Although the immune responses against NDV have been widely investigated, little is known regarding the virus interaction with the host innate immune responses. In this study, we tested the effect of different commercially applied Newcastle disease vaccines as well as virulent NDV genotype VIId on the expression pattern of the upstream regulator and downstream effector genes related to chicken interferon-alpha (chIFNα) signalling transduction pathway. Using quantitative real-time PCR analysis, mild transient induction of chIFNα-inducible genes was detected in bird spleen 72 h post-vaccination (hpv) with either live LaSota (respiratory) or VG/GA (enteric) strains. Vaccination with the enteric VG/GA strain led to stimulation of the investigated pathway as early as 24 hpv which continued up to 7 days in bird caecal tonsils. Subcutaneous injection with inactivated LaSota oil adjuvant-based vaccine led to continual stimulation of the investigated pathway up to 7 days post-vaccination (dpv). The recombinant herpesvirus of turkey (rHVT) - NDV vaccine led to remarkable stimulation of all the tested cytokines up to 17 dpv in comparison with LaSota and VG/GA NDV vaccines. Stronger but transient activation of all the tested cytokines was detected in spleens during the first 24 h post-challenge with virulent NDV (vNDV) which reduced gradually and diminished later due to the virus-induced lymphocytic depletion. This study will aid in the discovery of new approaches to control NDV.

Evolutionary Analysis of Infectious Bronchitis Virus Reveals Marked Genetic Diversity and Recombination Events.

In the last 5 years, frequent outbreaks of infectious bronchitis virus (IBV) are observed in both broiler and layer chicken flocks in the Kingdom of Saudi Arabia (KSA) in spite of extensive usage of vaccines. The IBV is a widespread avian coronavirus affecting both vaccinated and unvaccinated chicken flocks and is attributed to significant economic losses, around the globe. In the present study, 58 (n = 58) samples were collected from four different commercial poultry flocks from 8 KSA districts during 2019. A total of nine positive isolates (9/58; 15.5%), based on real-time reverse transcriptase PCR targeting nucleocapsid (N) gene, were used for further genetic characterization and evolutionary analysis. Genetic characterization of the partial spike (S1) gene revealed the clustering of the reported isolates into three different genotypes, whereas four additional isolates were grouped within 4/91 genotype, two isolates within IS/885 genotype, one isolate was closely related to IS/1494/06, and two isolates were grouped within classic serotype (vaccine-like strains). Phylodynamic revealed clustering of four isolated viruses within GI-13 lineage, three isolates within GI-23 lineage, and two isolates within GI-1 lineage. Results indicate that there are high evolutionary distances between the newly identified IBV strains in this study and the commercially used vaccines (GI-1), suggesting that IBV strains circulating in the KSA are under constant evolutionary pressures. Selective pressure biostatistics analyses consistently demonstrate the presence of a higher positive score which highlights the role of natural selection, a mechanism of virus evolution on sites located on the protein surface, within or nearby domains involved in viral attachment or related functions. Recombination analysis revealed emergence of two isolates through recombination events resulting in new recombinant viruses. Taken together, these finding demonstrate the genetic and evolutionary insights into the currently circulating IBV genotypes in KSA, which could help to better understand the origin, spread, and evolution of infectious bronchitis viruses, and to ascertain the importance of disease monitoring as well as re-evaluation for the currently used vaccines and vaccination programs.

A proposed soft pneumatic actuator control based on angle estimation from data-driven model.

This article proposes a bending angle controller for soft pneumatic actuators, which could be implemented in soft robotic rehabilitation gloves to assist patients with hand impairment, such as stroke survivors. A data-driven model is used to estimate the angle as pneumatic pressure is applied to the actuator. Furthermore, a finite element model was used to manually optimize the dimensions of the actuator. An embedded flex sensor, which together with a custom testing rig, was used to gather input data for the data-driven model. This rig contains a pneumatic pressure control circuit as well as a camera for image acquisition. Collected data were fed into a linear regression model to predict the data-driven model. Experiments were carried out to validate model's accuracy as well as modified proportional-integral-derivative controller angle controller performance. The latter controller is designed to mitigate the non-linear response of solenoid valves at different pressures of the actuator. The data-driven model along with the used controller allows more accurate estimation and quicker response.

Influence of Marek's disease virus vaccines on chicken melanoma differentiation-associated gene 5-dependent-type I interferon signal transduction pathway with a highlight on their secondary impact on the immune responses post Newcastle disease virus vaccination.

Marek's disease virus (MDV) leads to a lytic infection of B-lymphocytes in chickens, and also latently infects T-lymphocytes. Although Marek's disease vaccines have been widely in use, little is known about the innate immune response of this important livestock vaccine. In this study, we tested the effect of different commercially applied Marek's disease vaccines on the expression pattern of selected genes related to chicken interferon-alpha (chIFN-α) (melanoma differentiation associated gene 5 "MDA5″ dependent) signal transduction pathway. Both MDV serotype I (Rispens) and serotype III (Herpesvirus of turkey "HVT") vaccines could stimulate MDA5 dependent-type I interferon response as early as three days post vaccination in a dose-dependent manner. The stimulation continued up to 10 days in the instance of HVT vaccine and declined in the case of Rispens. Surprisingly, increasing the doses of the two vaccines led to dose-dependent down-regulation in the expression pattern of the investigated pathway, five and ten days post vaccination. Additionally, to shed the light on the consequent effect on the immune responses of the other viral vaccine, another experimental model based on Newcastle disease virus (NDV) vaccines was designed using HVT, HVT-VP2 and Rispens MDV vaccines. The three MDV vaccines were found to reduce chicken humoral immune response post NDV vaccination. However, only Rispens and HVT-VP2 had suppressive effects on the expression of MDA5-dependent-chIFN-α related cytokines. Consistent with this finding, the protection rate and NDV- humoral immune response post challenge with virulent NDV strain was lower in case of Rispens and HVT-VP2 vaccines.

Molecular survey and interaction of common respiratory pathogens in chicken flocks (field perspective).

AIM: The present study was designed for the detection of the most prevalent respiratory infections in chicken flocks and clarifying their interaction and impact on flock health. MATERIALS AND METHODS: A total of 359 serum samples were collected from 55 backyard chickens and tested using commercial enzyme-linked immunosorbent assay kits to determine the seroprevalence of Newcastle disease virus (NDV), infectious bronchitis virus (IBV), influenza type A, Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS). Molecular prevalence of NDV, IBV, low pathogenic avian influenza virus (LPAIV) H9N2, MG, and MS was carried out on swab, and tissue samples collected from 55 backyard flocks and 11 commercial broiler flocks suffered from respiratory infections using polymerase chain reaction (PCR) and reverse transcription-PCR. RESULTS: Seroprevalence of NDV, IBV, Influenza type A virus, MG, and MS in chicken backyard flocks was 56.4%, 50.9%, 12.7%, 14.5%, and 3.6%, respectively. Specific antibodies against one or more respiratory viruses and mycoplasma were detected in 36.4% of backyard flocks, indicating concurrent viral infections. The molecular survey showed that 90.9% of chicken backyard flocks were infected with common respiratory viruses (NDV, IBV, and LPAIV H9N2) while 81.8% of commercial broiler flocks were infected. The molecular prevalence rate of NDV, IBV, and LPAIV H9N2 was 46.97%, 56.1%, and 19.7% in backyard flocks, respectively. Combined viral and bacterial infection represented 40% and 63.6% of the respiratory infections, resulting in enhanced pathogenicity and increased mortalities of up to 87.5% and 27.8% in backyard and commercial flocks, respectively. Mixed infection of IBV, LPAIV H9N2, and/or Escherichia coli is the most prevalent mixed infection in broiler flocks, inducing severe clinical outcomes. Avian pathogenic E. coli was, respectively, isolated from 40% of backyard flocks and 81.82% of broiler flocks. Staphylococcus aureus was isolated from three backyard chicken flocks mixed with other respiratory pathogens with elevated mortality. Mixed infection of E. coli and MG reported in 9.1% of broiler flock. MG was detected in 14.5% of backyard flocks and 9.1% of broiler flocks while MS was detected only in 3.6% of backyard chickens mixed with E. coli, and other viruses. CONCLUSION: Our results confirm that mixed infections are more commonly prevalent and associated with dramatic exacerbation in clinical outcomes than a single infection. Bidirectional synergistic interaction between these concurrently interacted respiratory pathogens explains the severe clinical impact and high mortality rate. The high prevalence of IBV (either as a single or combined infection) with LPAIV H9N2 and/or E. coli, in spite of intensive use of commercial vaccines, increases the need for revising vaccination programs and the application of standard biosecurity measures. Backyard chickens impose a great risk and threaten commercial flocks due to the high prevalence of viral respiratory pathogens.

Molecular characterization of fowl aviadenoviruses species D and E associated with inclusion body hepatitis in chickens and falcons indicates possible cross-species transmission.

During the period from 2015 to 2017, frequent outbreaks of inclusion body hepatitis (IBH) were observed in broiler chickens and falcons in Saudi Arabia. Fifty samples were collected from both species. The histopathological examination and polymerase chain reaction confirmed the IBH infection in eight samples (five samples from chickens and three samples from falcons). The genomic sequence and phylogenetic analysis based on nucleotide and amino acid sequences of Saudi strains, reference fowl aviadenoviruses (FAdVs) and field viruses available in Genbank revealed that all investigated FAdVs clustered into FAdV-2 (species D) and FAdV-6 (species E). The host-dependent characterization revealed that falcon origin strains showed low identity (∼35%) with falcon adenoviruses isolated from USA, which clustered into a separate group. The identification of FAdV-D and FAdV-E in diseased falcons and chickens indicates cross-species transmission although falcons and chickens are phylogenetically different. The control of IBH infection in falcons and chickens should be based on the separation of carriers and susceptible chickens as well as falcons to prevent cross-species contact. Vaccination is an important method for prevention of IBH. The characterization of newly emerging FAdV strains provides valuable information for the development of an efficacious control strategy based on the molecular structure of current circulating FAdV strains in different species of birds.

Amelioration of panadol-induced nephrotoxicity via down-regulation of Bax/Bcl2 ratio with some antioxidants.

BACKGROUND: Overdoses of Panadol (APAP) result in hepatic and renal toxicity. Up till now, there is no effective drug for APAP-enhanced nephrotoxicity. This work aims to explore the protective effects of N-acetylcysteine, Thymoquinone (THQ), Curcumin (CUR) and α-Lipoic acid (LA) either alone or in combination against APAP nephrotoxicity, focused on modulation of Bax/Bcl2 pathway. METHODS: APAP was administrated at a single dose then treated with the fore mentioned antioxidants. RESULTS: APAP administration increased serum creatinine, urea, uric acid, tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) levels compared to control group. There is a marked depletion of reduced glutathione (GSH) levels and superoxide dismutase activity (SOD), Bax level was overexpressed, whereas Bcl2 was downregulated in renal tissue. Histopathological examination of the kidney tissue supported these biochemical findings. Treatment with the fore mentioned anti-oxidants ameliorated most of the previous evaluated parameters and returned the kidney nearly to its normal architecture. CONCLUSION: The expression of Bax and Bcl2 is considered one of the mechanisms underlying APAP-induced nephrotoxicity. The administration of THQ along with CUR could be a promising antidote for APAP renal damage through their antioxidant potential.

Emergence of new virulent rabbit hemorrhagic disease virus strains in Saudi Arabia.

Rabbit hemorrhagic disease is an acute fatal highly contagious viral infectious disease that causes high losses among rabbitries. The disease was first reported in China in 1984 and later on in Saudi Arabia in 1996. The aim of this study was to investigate the emergence and pathogenicity of new rabbit hemorrhagic disease virus (RHDV) strains in Saudi Arabia. The pathogenicity was confirmed by inoculation in susceptible rabbits. Three RHDV strains were detected by reverse transcriptase polymerase chain reaction (RT-PCR) using primers targeting VP60 capsid protein gene in infected rabbitries during 2012 and 2013. These strains clustered into two genetically distinct genogroups related to year of isolation (G2 and G3). All new Saudi Arabia viruses clustered with the European strains, while the old strains clustered with strains from China and America. Based on amino acids and nucleotide sequences, the Saudi Arabia strains (RHD/1/SA/2012, RHD/2/SA/2012, and RHD/3/SA /2013) had high identity with Mexico89, Ca11-ITA, and 00-13,FRA virus; on the other hand, there was a relatively high identity with Bahrain strain. The evolutionary relationship of Saudi RHDVs strains revealed significant nucleotides and amino acid substitutions in hypervariable region E, suggesting the emergence of new RHDVs circulating in Saudi Arabia rabbitries. These antigenic changes represented by the antigenic index might be a potential cause of vaccination failure and raises the need to review the vaccination strategies against RHD.

Diversity of Meq gene from clinical Marek's disease virus infection in Saudi Arabia.

AIM: The aim of this study was to demonstrate the genomic features of Meq gene of Marek's disease virus (MDV) recently circulating in Saudi Arabia (SA). MATERIALS AND METHODS: Two poultry flocks suffering from mortalities and visceral tumors were presented to the Veterinary Teaching Hospital, King Faisal University, SA. Subjected to different diagnostic procedures: Case history, clinical signs, and necropsy as well as polymerase chain reaction followed by Meq gene sequence analysis. RESULTS: Case history, clinical signs, and necropsy were suggestive of MDV infection. The Meq gene was successfully detected in liver and spleen of infected chickens. A 1062 bp band including the native Meq ORF in addition to a 939 bp of S-Meq (short isoform of Meq) were amplified from Saudi 01-13 and Saudi 02-13, respectively. The nucleotide and deduced amino acids sequences of the amplified Meq genes of both Saudi isolates showed distinct polymorphism when compared with the standard USA virulent isolates Md5 and GA. The sequence analysis of the S-Meq gene showed a 123 bp deletion representing 41 amino acids between two proline-rich areas without any frameshift. The Meq gene encoded four repeats of proline-rich repeats (PRRs sequences), whereas the S-Meq contains only two PRRs. Interestingly, the phylogenetic analysis revealed that both of SA MDV isolates are closely related to the MDV strains from Poland. CONCLUSION: The two MDV isolates contain several nucleotide polymorphisms resulting in distinct amino acid substitutions. It is suggested that migratory and wild birds, as well as world trading of poultry and its by-products, have a great contribution in the transmission of MDVs overseas.

Effect of phylogenetic diversity of velogenic Newcastle disease virus challenge on virus shedding post homologous and heterologous DNA vaccination in chickens.

Newcastle disease (ND) is a highly devastating disease for the poultry industry as it causes high economic losses. In this present study, a DNA vaccine containing the F and HN surface antigens of a highly virulent Newcastle disease virus (NDV), NDV/1/Chicken/2005 (FJ939313), was successfully generated. Cell transfection test indicated that the vaccine expressed the F and HN genes in Hep-2 cells. The main objective of this study was to compare the extent of protection induced by DNA vaccination after homologous and heterologous NDV-challenge as determined by the amount of NDV shedding after challenge. NDV-antibody-negative chickens were vaccinated either once, twice or thrice intramuscularly at 7, 14 and 21 days old and were challenged 14 days post vaccination with either homologous virus (vaccine-matched velogenic viscerotropic Newcastle disease virus (vvNDV) strain, FJ939313), phylogenetically related to group VII, or a phylogenetically divergent heterologous virus (unmatched vvNDV strain, AY968809), which belongs to genogroup VI and shows 84.1% nucleotide similarity to the NDV-sequences of the DNA vaccine. Our data indicate that birds, which received a single dose of the DNA vaccine were poorly protected, and only 30-40% of these birds survived after challenge with high virus shedding titre. Multiple administration of the DNA vaccine induced high protection rates of 70-90% with reduced virus shedding compared to the non-vaccinated and challenged birds. Generally, homologous challenge led to reduced tracheal and cloacal shedding compared to the heterologous vvNDV strain. This study provides a promising approach for the control of ND in chickens using DNA vaccines, which are phylogenetically closely related to the circulating field strains.

Sequence analysis of fusion protein gene of Newcastle disease virus isolated from outbreaks in Egypt during 2006.

BACKGROUND: Newcastle disease virus represents APMV-1 and is the most characterized among all APMV types. The F protein cleavage site sequence is a well-characterized determinant of NDV pathogenicity in chickens. In this study, the sequences of fusion protein (F) gene of three Newcastle disease virus (NDV) strains isolated from outbreak in chickens in the Al-Sharkia province of Egypt in 2006 were determined. FINDINGS: The viral genomic RNAs were extracted from the infective allantoic fluid and F gene is amplified using primer sets designed from the available sequences of NDV strains from GenBank. The pathogenicity of NDV strains was determined by three internationally recognized tests mean death time, intracerebral pathogenicity index, and intravenous pathogenicity index. The phylogenetic analysis showed that the Egypt isolates are closely related with the genotype II of class II NDV strains. CONCLUSIONS: The sequences of the F genes of the 2006 Egypt isolates are closely related to that of the 2005 Egypt isolate from the same province suggesting that these strains are probably circulating in the vaccinated bird population in Egypt until development of an outbreak.

Complete genome sequence of a virulent Newcastle disease virus isolated from an outbreak in chickens in Egypt.

The complete genome sequence of a virulent Newcastle disease virus (NDV) isolated from chickens in Egypt was determined and compared to the sequence of NDV strains isolated from different parts of the world. The genome is 15,186 nucleotides (nt) long and consists of 6 genes in the order of 3'-N-P-M-F-HN-L-5'. The genome contains a 55-nt leader region at the 3' end and a 114-nt trailer region at the 5' end. Interestingly, the phylogenetic analysis showed that strain Egypt is closely related with the NDV strains isolated in China. In addition, the sequence of the fusion protein cleavage site of strain Egypt was identical to that of the NDV strain recently isolated in Mali. Determination of complete genome sequences of additional NDV strains from Africa is necessary to understand the epidemiology of currently circulating viruses in Africa.

Fructose-amino acid conjugate and other constituents from Cyperus rotundus L.

Further phytochemical study on the aerial parts of Cyperus rotundus L. led to the isolation of a fructose-amino acid conjugate, N-(1-deoxy-alpha-D-fructos-1-yl)-L-tryptophan (16) and its tautomers, in addition to n-butyl-beta-D-fructopyranoside (1), ethyl-alpha-D-glucopyranoside (2), adenosine (3), (-)-(E)-caffeoylmalic acid (4), vitexin (5), isovitexin (6), orientin (7), epiorientin (8), myricetin 3-O-beta-D-galactopyranoside (9), luteolin 7-O-beta-D-glucuronopyranoside-6''-methyl ester (10), chlorogenic acid (11), luteolin 4'-O-beta-D-glucuronopyranoside (12), luteolin 7-O-beta-D-glucuronopyranoside (13), uridine (14) and ellagic acid (15). Their structures were elucidated on the basis of spectroscopic methods. Additionally, antioxidant and alpha-amylase inhibitory activities of some of the isolated phenolic compounds were carried out.

A new steroid glycoside and furochromones from Cyperus rotundus L.

Further phytochemical investigation of the aerial parts of Cyperus rotundus L. afforded a new steroid glycoside named sitosteryl (6'-hentriacontanoyl)-beta-D-galactopyranoside (4) in addition to three furochromones, khellin (2), visnagin (3) and ammiol (9). Furthermore, benzo-alpha-pyrone (coumarin) (1), salicylic acid (5), caffeic acid (6), protocatechuic acid (7), p-coumaric acid (8), tricin (10) and isorhamnetin (11) were isolated. The structures of these compounds were established by spectroscopic methods. The isolated furochromones were tested for insect antifeedant activity against larvae Spodoptera littoralis when incorporated in artificial diet and offered to larvae in a chronic feeding bioassay. Also, visnagin, khellin and sitosteryl (6'-hentriacontanoyl)-beta-D-galactopyranoside showed strong cytotoxic activity against L5178y mouse lymphoma cells and were also active in the brine shrimp lethality test.

The biochemical and morphological alterations following administration of melatonin, retinoic acid and Nigella sativa in mammary carcinoma: an animal model.

Worldwide, breast cancer is the second leading cause of cancer death among women and the third most common cancer. Although our understanding of the molecular basis of this fatal disease has improved, this malignancy remains elusive. Melatonin (Mel), retinoic acid (RA) and Nigella sativa (NS) are substances with anticancer effects. To date, our understanding of the mechanisms of therapeutic effects of these products in mammary cancer is still marginal. To look at the preventive and therapeutic values of these products, we carried out this investigation. An animal model formed of 80 rats was established. The animals were divided into eight groups of 10 animals each: (a) control group injected with the same vehicle used for treatments in the relevant dosages and routes; (b) carcinogen group injected with the known carcinogenic substance 7,12-di-methylbenz(a)anthracene (DMBA) that induces mammary carcinoma; (c) three prophylactic (Pro) groups (Mel-Pro, RA-Pro and NS-Pro) injected with test substances (Mel, RA and NS, respectively) 14 days before the intake of the carcinogenic substance DMBA and then continued until the end of the experiments; and (d) three treated (Tr) groups (Mel-Tr, RA-Tr and NS-Tr) injected with the vehicles after the intake of DMBA. In both the Pro and Tr groups, the drugs were daily administered for 3 months. The animals were killed, and their serum and tissues were evaluated for (a) markers of tumorigenicity [serum levels of total sialic acid (TSA) and lipid-bound sialic acid (LSA)], (b) markers of endocrine derangement (serum prolactin, estradiol and progesterone levels), (c) apoptotic changes [serum tumour necrosis factor (TNF)-alpha, tissue caspase-3 activity, percentage of DNA fragmentation and ultrastructural features of apoptosis] and (d) markers of oxidative stress (tissue levels of lipid peroxides and nitric oxide). Carcinoma was absent both in the control and in the NS-Pro groups. Mammary carcinoma occurred in DMBA and other Pro and Tr groups. The frequency of mammary carcinoma was high in the carcinogen DMBA group (60%), followed by the Tr (56%) and finally the Pro groups (33%). These tumours included papillary, comedo and cribriform carcinomas. As compared with the control group, the development of carcinoma in the carcinogen DMBA group was associated with increased levels of (a) markers of tumorigenicity (77.0 +/- 3.3 vs. 209.0 +/- 5.6 and P < 0.05 for TSA; 28.7 +/- 1.7 vs. 41.8 +/- 1.2 and P < 0.01 for LSA), (b) markers of endocrine derangement (2.5 +/- 0.1 vs. 3.6 +/- 0.3 and P < 0.05 for prolactin; 39.6 +/- 1.3 vs. 24.8 +/- 2.1 and P < 0.01 for progesterone and 31.0 +/- 0.7 vs. 51.1 +/- 3.4 and P < 0.01 for estradiol) and (c) markers of oxidative stress (2.3 +/- 0.2 vs. 5.2 +/- 0.7 and P < 0.01 for lipid peroxides and 4.4 +/- 0.2 vs. 7.6 +/- 0.8 and P < 0.01 for nitric oxide). Also, it was associated with decreased levels of markers of apoptotic activity (20.8 +/- 1.1 vs. 13.4 +/- 0.7 and P < 0.01 for caspase-3; 29.0 +/- 1.7 vs. 20.9 +/- 1.3 and P < 0.05 for percentage of DNA fragmentation; and 9.4 +/- 0.8 vs. 52.1 +/- 3.3 and P < 0.01 for TNF-alpha). When compared with the carcinogen DMBA group, the development of carcinoma in the Pro and Tr groups was associated with decreased levels of (a) markers of tumorigenicity, (b) markers of endocrine derangement and (c) markers of oxidative stress. Alternatively, carcinogenicity was associated with statistically significant (P < 0.01) increased levels of markers of apoptotic activity. To conclude, the administration of Mel, RA and NS reduced the carcinogenic effects of DMBA, suggesting a protective role. The possible underlying mechanisms of these effects await further investigations.

New luffariellolide derivatives from the Indonesian sponge Acanthodendrilla sp.

Investigation of the Indonesian sponge Acanthodendrilla sp. afforded five new luffariellolide-related sesterterpenes, acantholides A-E (1-5), in addition to luffariellolide and its 25-O-methyl and 25-O-ethyl derivatives. All structures were unambiguously established by 1D and 2D NMR and MS spectroscopy. Acantholide D and E are derivatives comprising the 1-acetylcyclopentan-5-ol moiety, which are new variants of the C(14)-C(20) segment for this type of linear sesterterpenes. Luffariellolide and its 25-O-methyl congener as well as acantholide E (5) were cytotoxic against the mouse lymphoma L5187Y cell line. Acantholide B (2), luffariellolide, and its 25-O-methyl congener were active against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, the Gram-negative bacterium Escherichia coli, the yeast Candida albicans, and the plant pathogenic fungus Cladosporium herbarum.

19F NMR spectrometric determination of the partition coefficients of some fluorinated psychotropic drugs between phosphatidylcholine bilayer vesicles and water.

A simple 19F NMR spectrometric method was proposed for the determination of the partition coefficients of fluorinated psychotropic drugs, trifluoperazine (TFPZ), flunitrazepam (FNZ) and flurazepam (FZ) between phosphatidylcholine (PC) bilayer of small unilamellar vesicles (SUVs) and water (buffer). Each 19F NMR spectrum of these drugs in the presence of PC SUV showed a single signal accompanying a PC concentration-depending shift change and broadening, which indicated a fast exchange of these drugs between the water phase and the PC bilayer of SUV. From the relationship between the 19F chemical shift change (Deltadelta) of each drug and the PC concentration, the molar partition coefficients (K(p)'s) were calculated and obtained with a good precision of RSD below 6%. The fractions of the partitioned drugs calculated by using the obtained K(p)-values were in a good agreement with the experimental values. The results demonstrate that the 19F NMR method can be usefully applied to the determination of partition coefficients of many drugs having fluorine atom(s) without any separation procedure, especially for drugs which do not have absorption in the ultraviolet or visible region, or those having absorption but show insignificant spectral changes according to their incorporation to PC bilayers (e.g. FNZ).