Differential effects of two anti-apo-cytochrome c-specific monoclonal antibodies on the function of apo-cytochrome c-specific murine T cell clones.

A murine monoclonal antibody (SJL 2-4) specific for the antigen apo-cytochrome c was shown to inhibit both antigen-induced proliferation and lymphokine secretion by an apo-cytochrome c-specific BALB/c helper T cell clone. The inhibition was specific because additional apo-cytochrome c-specific T cell clones were not inhibited by the same monoclonal antibody. Time course studies of the inhibition indicated that the initial 8 hr of contact between T cell clones and antigen-presenting cells were critical for activation of the T cell clones. Inhibition of T cell functions by antigen-specific antibodies appeared to correlate with the antibody-antigen binding constant because a second monoclonal antibody (Cyt-1-59), with identical specificity but with a lower affinity constant for apo-cytochrome c, had very little inhibitory effect on the proliferation or lymphokine secretion of apo-cytochrome c-specific T cell clones.

Fine specificity of a BALB/c T cell clone directed against beef apo cytochrome c.

A BALB/c cloned T cell line directed against beef apo cytochrome c was shown to exhibit the Lyt-1+2- cell surface phenotype. The fine specificity of antigen recognition exhibited by the T cell clone was assessed by using a variety of peptide preparations obtained from cytochrome c of different sources. The peptide segment recognized by this T cell clone, in conjunction with I-A region gene products, appeared similar to that bound by a monoclonal antibody specific for beef apo cytochrome c derived from the same strain of mice.

CYP3A5 and ABCB1 genes influence blood pressure and response to treatment, and their effect is modified by salt.

The permeability-glycoprotein efflux-transporter encoded by the multidrug resistance 1 (ABCB1) gene and the cytochromes P450 3A4/5 encoded by the CYP3A4/5 genes are known to interact in the transport and metabolism of many drugs. Recent data have shown that the CYP3A5 genotypes influence blood pressure and that permeability-glycoprotein activity might influence the activity of the renin-angiotensin system. Hence, these 2 genes may contribute to blood pressure regulation in humans. We analyzed the association of variants of the ABCB1 and CYP3A5 genes with ambulatory blood pressure, plasma renin activity, plasma aldosterone, endogenous lithium clearance, and blood pressure response to treatment in 72 families (373 individuals; 55% women; mean age: 46 years) of East African descent. The ABCB1 and CYP3A5 genes interact with urinary sodium excretion in their effect on ambulatory blood pressure (daytime systolic: P=0.05; nighttime systolic and diastolic: P<0.01), suggesting a gene-gene-environment interaction. The combined action of these genes is also associated with postproximal tubular sodium reabsorption, plasma renin activity, plasma aldosterone, and with an altered blood pressure response to the angiotensin-converting enzyme inhibitor lisinopril (P<0.05). This is the first reported association of the ABCB1 gene with blood pressure in humans and demonstration that genes encoding for proteins metabolizing and transporting drugs and endogenous substrates contribute to blood pressure regulation.

Identification of snails within the Bulinus africanus group from East Africa by multiplex SNaPshotTManalysis of single nucleotide polymorphisms within the cytochrome oxidase subunit I

Identification of populations of Bulinus nasutus and B. globosus from East Africa is unreliable using characters of the shell. In this paper, a molecular method of identification is presented for each species based on DNA sequence variation within the mitochondrial cytochrome oxidase subunit I (COI) as detected by a novel multiplexed SNaPshotTM assay. In total, snails from 7 localities from coastal Kenya were typed using this assay and variation within shell morphology was compared to reference material from Zanzibar. Four locations were found to contain B. nasutus and 2 locations were found to contain B. globosus. A mixed population containing both B. nasutus and B. globosus was found at Kinango. Morphometric variation between samples was considerable and UPGMA cluster analysis failed to differentiate species. The multiplex SNaPshotTM assay is an important development for more precise methods of identification of B. africanus group snails. The assay could be further broadened for identification of other snail intermediate host species.

Genetic diversity of Triatoma infestans (Hemiptera: Reduviidae) in Chuquisaca, Bolivia based on the mitochondrial cytochrome b gene

Partial cytochrome b DNA sequences for 62 Triatoma infestans were analyzed to determine the degree of genetic variation present in populations of this insect in the northwest region of Chuquisaca, Bolivia. A total of seven haplotypes were detected in the localities sampled. The phylogenetic relationship and population genetic structure of the haplotypes found in this region, indicate that there is greater variation in this relatively small region of Bolivia than what has been previously reported by studies using the same gene fragment, for more distant geographic areas of this country. In addition, a comparison of rural and peri-urban localities, indicate that there is no difference in the genetic variation of T. infestans between these two environments.

Molecular characterization of Echinococcus granulosusfrom Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene

Echinococcus granulosus, the etiologic agent of cystic echinococcosis (CE) in humans and other animal species, is distributed worldwide. Ten intra-specific variants, or genotypes (G1-G10), have been defined based on genetic diversity. To determine the genotypes present in endemic areas of Peru, samples were collected from cattle (44), sheep (41) and humans (14) from Junín, Puno Huancavelica, Cusco, Arequipa and Ayacucho. DNA was extracted from protoscolex and/or germinal layers derived from 99 E. granulosus isolates and used as templates to amplify the mitochondrial cytochrome C oxidase subunit 1 gene. The resulting polymerase chain reaction products were sequenced and further examined by sequence analysis. All isolates, independent of the host, exhibited the G1 genotype. Phylogenetic analysis showed that three isolates from Ayacucho shared the same cluster with microvariant G1(4). The G1 genotype is considered the most widespread and infectious form of E. granulosusworldwide and our results confirm that the same patterns apply to this country. Therefore, these findings should be taken into consideration in developing prevention strategies and control programs for CE in Peru.

False phylogenies on wood mice due to cryptic cytochrome-b pseudogene.

The phylogeny and phylogeography of the Old World wood mice (subgenus Sylvaemus, genus Apodemus, Muridae) are well-documented. Nevertheless, the distributions of species, such as A. fulvipectus and A. ponticus remain dubious, as well as their phylogenetic relationships with A. sylvaticus. We analysed samples of Apodemus spp. across Europe using the mitochondrial cytochrome-b gene (cyt-b) and compared the DNA and amino-acid compositions of previously published sequences. The main result stemming from this study is the presence of a well-differentiated lineage of Sylvaemus including samples of various species (A. sylvaticus, A. fulvipectus, A. ponticus) from distant locations, which were revealed to be nuclear copies of the mitochondrial cyt-b. The presence of this cryptic pseudogene in published sequences is supported by different pathways. This has led to important errors in previous molecular trees and hence to partial misinterpretations in the phylogeny of Apodemus.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Concentrations of the enantiomers of fluoxetine and norfluoxetine after multiple doses of fluoxetine in cytochrome P4502D6 poor and extensive metabolizers.

Plasma concentrations of the enantiomers of fluoxetine (FLX) and norfluoxetine (NFLX) were measured at days 7, 14, and 23 of oral administration of 20 mg of racemic fluoxetine in 11 patients who were comedicated with risperidone. Eight patients were genotyped as being cytochrome P4502D6 extensive metabolizers (EMs) and three as cytochrome P4502D6 poor metabolizers (PMs). No statistically significant differences were calculated between EMs and PMs in the concentrations of (R)-FLX and (R)-NFLX for all days examined (day 23, mean +/- SD for (R)-FLX and (R)-NFLX in EMs, 16 +/- 5 and 29 +/- 20 ng/mL, respectively; in PMs, 16 +/- 1 and 20 +/- 2 ng/mL, respectively). However, concentrations of (S)-FLX and (S)-NFLX were higher and lower, respectively, in PMs as compared with EMs (day 7, p = 0.037 and p = 0.036; day 14, p = 0.014 and p = 0.014; day 23, p = 0.068 and p = 0.038). On day 23, mean (S)-FLX and (S)-NFLX in EMs were (mean +/- SD) 39 +/- 26 and 63 +/- 26 ng/mL, and in PMs they were 88 +/- 7 and 19 +/- 2 ng/mL. This study confirms the results of the single-dose studies showing that CYP2D6 is involved in the demethylation of FLX to NFLX, with a stereoselectivity toward the (S)-enantiomer. The data also clearly show that the CYP2D6 genotype has an important influence on the concentrations of the (S)- but not of the (R)-enantiomer of FLX and NFLX after multiple doses.

Cytochrome P4503A4 metabolic activity, methadone blood concentrations, and methadone doses.

We examined in vivo the influence of cytochrome P4503A4 (CYP3A4) activity, measured by the 30 min plasma 1'OH-midazolam/midazolam ratio after oral administration of 7.5 mg midazolam, on the methadone steady-state trough plasma concentrations in a group of 32 patients in methadone maintenance treatment. Patients were grouped as receiving 'low' (up to 99 mg/day, n = 10), 'high' (100-199 mg/day, n = 11) and 'very high' (> or = 200 mg/day, n = 11) doses of methadone, and the CYP3A4 metabolic activity was compared between the three groups. (S)-methadone and (R,S)-methadone, but not (R)-methadone, concentrations to dose ratios significantly correlated with the midazolam ratios (r(2) = -0.17, P = 0.018; r(2) = -0.14, P = 0.032; r(2) = -0.10, P = 0.083, respectively), with a 76% higher CYP3A4 activity in the very high-dose group as compared with the low-dose group. Significant differences in the CYP3A4 activity were calculated between the three groups (P = 0.0036), and group-to-group comparisons, using the Bonferroni correction, showed a significant difference between the low-dose and the very high-dose group (P = 0.0039), between the high-dose and the very high-dose group (P = 0.0064), but not between the low-dose and the high-dose group (P = 0.070). The higher CYP3A4 activity measured in patients receiving very high methadone doses could contribute to the need for higher doses in some patients, due to an increased metabolic clearance. This, however, must be confirmed by a prospective study.

Tests pharmacogénétiques: bientôt avant chaque prescription [Pharmacogenetic testing: soon before every prescription?]

Genetic polymorphisms have currently been described in more than 200 systems affecting pharmacological responses (cytochromes P450, conjugation enzymes, transporters, receptors, effectors of response, protection mechanisms, determinants of immunity). Pharmacogenetic testing, i.e. the profiling of individual patients for such variations, is about to become largely available. Recent progress in the pharmacogenetics of tamoxifen, oral anticoagulants and anti-HIV agents is reviewed to discuss critically their potential impact on prescription and contribution/limits for improving rational and safe use of pharmaceuticals. Prospective controlled trials are required to evaluate large-scale pharmacogenetic testing in therapeutics. Ethical, social and psychological issues deserve particular attention.

Cytochrome b haplotype divergences in West European Apodemus

Sequencing of the cytochrome b mitochondrial gene (732 base pairs) in samples of Apodemus sylvaticus from Central Europe (eastern France, Switzerland, southern Germany and western Austria) revealed significant molecular variation not reflected in previous morphological and genetic studies of this species. A comparison with the sequences (150 bp) of 54 specimens available from GenBank (NCBI) showed that two problematic individuals originating from southern Germany have to be assigned to A. fulvipectus, a species not yet known in western Europe. A. sylvaticus specimens (n = 14) sampled north of the Alps exhibited a maximum intraspecific sequence divergence of about 5.1%, whereas the maximum divergence is much Lower in A. flavicollis (1%, n = 5) and in A. alpicola (0.5%, n = 4), although the samples originate from a similar geographic range of about 350 km. We also found a high rate of erroneously assigned specimens in GenBank, which indicates that the discrimination of Apodemus species remains a problem and requires further investigations.

Clonal analysis of the BALB/c T cell proliferative response to apo beef cytochrome c.

Murine T cell clones that proliferated specifically in response to the protein antigen apo cytochrome c were derived and maintained in continuous culture. Two distinct clonotypes were observed with respect to the proliferative responses observed when a variety of peptides prepared from several species of cytochrome c were tested. These 2 clonotypes appeared to recognize 2 different regions in the cytochrome c molecule. Only 1 of the 2 clonotypes tested demonstrated helper cell activity for antibody formation in vitro.