Cardioactive neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) and novel related peptides are encoded in multiple copies by a single gene in the snail Lymnaea stagnalis

The neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) is a potent cardioactive neuropeptide in Lymnaea stagnalis. Isolation and sequencing of 2 cDNAs and a genomic clone shows that a single gene encodes a precursor protein which contains 9 copies of the FMRFamide peptide, 2 copies of the related peptide Phe-Leu-Arg-Phe-NH2 (FLRFamide), and single copies of the putative pentapeptides Gln-Phe-Tyr-Arg-lle-NH2 (posttranslationally modified to pQFYRlamide) and Glu-Phe-Leu-Arg-lle-NH2 (EFLRlamide). The gene is transcribed in the CNS and gives rise to a single RNA of 1.7 kb in size. The organization of the Lymnaea gene is significant with respect to the evolution of FMRFamide and related peptides in other organisms.     

Evidence for an enkephalinergic system in the nervous system of the pond snail, Lymnaea stagnalis

Evidence for the presence of an enkephalinergic system in the ganglia of the pond snail, Lymnaea stagnalis, has been obtained with 3 experimental approaches. Scatchard analysis with [3H]etorphine reveals a monophasic high-affinity opiate binding site (Kd 2.3 nM) which is naloxone-sensitive. Immunocytochemical localization of Met- and Leu-enkephalin-like substances as well as alpha-MSH- and ACTH-like materials was demonstrated within specific populations of neurons. Substances with Met- and Leu-enkephalin and Met-enkephalin sulfoxide RIA reactivities were detected also in HPLC fractions corresponding to the retention times of authentic enkephalin standards. Together, the results provide strong evidence for the presence of enkephalinergic mechanisms in the nervous system of Lymnaea stagnalis. Additionally, the report provides indirect evidence for the existence of a macromolecular opioid precursor. This enkephalinergic system shows striking similarities to opioid mechanisms found in vertebrates and bespeaks a common evolutionary origin.     

Distribution of the peptide Ala-Pro-Gly-Trp-NH2 (APGWamide) in the nervous system and periphery of the snail Lymnaea stagnalis as revealed by immunocytochemistry and in situ hybridization.

Immunocytochemistry and in situ hybridization were used to identify 200-250 central neurons that synthesize and contain the peptide APGWamide in the central nervous system of Lymnaea. The majority of these cells reside within the right anterior lobe of the cerebral ganglion and most appear to have projections to the ventral lobe of the ganglion. The neurites then branch to innervate the lobe and to send further projections into the penial nerve and to the PeIb cluster of the right pedal ganglion. The right ventral lobe also contains a cluster of about 30-40 somata, which apparently synthesize and contain APGWamide. Other populations of cells found in both the right and left anterior lobes project ipsilaterally to the pleural, parietal, and visceral ganglia. Prominent somata are also located in clusters in the visceral and right parietal ganglia. These cells project ipsilaterally into caudal neuropilar regions of the cerebral ganglia. Peripheral projections of central neurons form a dense plexus of varicose, APGWamide-containing fibres in superficial layers of the penis and preputium. Other peripheral projections were noted in the prostate and vas deferens. No peripherally located cell bodies appeared to contain or synthesize the peptide. The results show that APGWamide is widely present in the central nervous system and male reproductive organs and suggest that it plays a major role in control of reproduction.     

Behavioural and neural deficits induced by rotenone in the pond snail Lymnaea stagnalis. A possible model for Parkinson's disease in an invertebrate

Parkinson's disease is a neurodegenerative disorder, related to the loss of dopamine (DA)-containing neurons in the substantia nigra. In experimental animals, both vertebrates and invertebrates, rotenone, a commercially available organic pesticide, induces symptoms of Parkinson's disease. We found that that rotenone is toxic to the pond snail Lymnaea stagnalis (4-day LC50 0.8 microM). Rotenone, at concentrations from 0.1 to 5 microM, caused progressive and irreversible behavioural deficits in both acute and chronic exposure. Chronic exposure to 0.5 microM rotenone led to a progressive decrease in spontaneous locomotion and in feeding, reaching almost 100% inhibition of both behaviours by the 7th day of rotenone treatment. In the central nervous system preparation made on the 7th day of treatment the postsynaptic potentials evoked by the identified dopaminergic RPeD1 neuron disappeared whereas the synaptic inputs received by the RPeD1 from a peptidergic neuron (VD4) were still functional. Immunostaining revealed that the tyrosine hydroxylase immunoreactivity decreased below the detectable level in both the RPeD1 cell body and its axonal processes. Finally, HPLC assay showed a significant (25%) decrease in DA level in the CNS by the 7th day of rotenone treatment. We conclude that, as in vertebrates, rotenone disrupts feeding and locomotion of the model mollusc Lymnaea stagnalis. One possible target of rotenone is the dopaminergic neurons in the CNS. We therefore suggest that Lymnaea stagnalis is a suitable invertebrate model for the study of Parkinson's disease, allowing direct analysis of the response of dopaminergic systems to rotenone at behavioural, cellular and neuronal levels.     

Complement receptor 3-like immunoreactivity in the light green cells and the canopy cells of the pond snail, Lymnaea stagnalis

We observed CR3-like immunoreactivity in the central nervous system (CNS) and its surrounding peripheral nerves of the pond snail, Lymnaea stagnalis. In the CNS of L. stagnalis, the immunoreactivity presenting meshwork-like structure was detected in some neurosecretory cells, which are the light green cells (LGCs) and the canopy cells (CCs), both controlling the body growth. The immunoreactivity was also observed along the edges of median lip nerves. The immunoreactive regions in the median lip nerves appeared to form the axonal plates, from which the LGCs and the CCs release molluscan insulin-related peptides (MIPs) into the blood. By contrast, no immunoreactivity was detected in other neurosecretory cells or their release sites, for example the caudodorsal cells and the cerebral commissure, which release ovulation hormones. The present findings, therefore, suggested that CR3 expresses only in the neurosecretory cells releasing MIPs in L. stagnalis.     

Photoinactivation of neurones axonally filled with the fluorescent dye 5(6)-carboxyfluorescein in the pond snail, Lymnaea stagnalis.

We describe a new, simple and reliable technique to fill molluscan neurones from their cut axons with sufficient fluorescent dye for photoinactivation experiments. The fluorescent dye 5(6)-carboxyfluorescein (5-CF) travels quickly up the nerves of the gastropod mollusc, Lymnaea stagnalis into the buccal ganglia and fills the cell bodies in 1-3 h. 5-CF filled neurones can be located in the intact ganglia with low intensity blue light. Impalement shows that they are alive and show normal resting, action and synaptic potentials. Intense laser light (wavelength 442 nm, intensity 0.5 MW.m-2) kills all the 5-CF filled cells in less than 5 min in laboratory reared snails. Unstained neurones are not killed. 5-CF fills neurones quicker than Lucifer yellow (LY) when the dye is applied axonally. Neurones stained with Lucifer yellow do not contain sufficient dye to be killed with 5 min laser illumination, but this irradiation reduces the membrane resistance to less than 25%.     

Processing of the FMRFamide Precursor Protein in the Snail Lymnaea stagnalis: Characterization and Neuronal Localization of a Novel Peptide, 'SEEPLY'

In the pulmonate snail Lymnaea stagnalis , FMRFamide-like neuropeptides are encoded by a multi-exon genomic locus which is subject to regulation at the level of mRNA splicing. We aim to understand the post-translational processing of one resulting protein precursor encoding the tetrapeptide FMRFamide and a number of other putative peptides, and determine the distribution of the final peptide products in the central nervous system (CNS) and periphery of Lymnaea. We focused on two previously unknown peptide sequences predicted by molecular cloning to be encoded in the tetrapeptide protein precursor consecutively, separated by the tetrabasic cleavage site RKRR. Here we report the isolation and structural characterization of a novel non-FMRFamide-like peptide, the 22 amino acid peptide SEQPDVDDYLRDWLQSEEPLY. The novel peptide is colocalized with FMRFamide in the CNS in a number of identified neuronal systems and their peripheral motor targets, as determined by in situ hybridization and immunocytochemistry. Its detection in heart excitatory motoneurons and in nerve fibres of the heart indicated that the novel peptide may play a role, together with FMRFamide, in heart regulation in the snail. The second predicted peptide, STEAGGQSEEMTHRTA (16 amino acids), was at very low abundance in the CNS and was only occasionally detected. Our current findings, suggestive of a distinct pattern of post-translational processing, allowed the reassessment of a previously proposed hypothesis that the two equivalent sequences in the Aplysia FMRFamide gene constitute a molluscan homologue of vertebrate corticotrophin releasing factor-like peptides.     

Distribution and developmental changes in GABA-like immunoreactive neurons in the central nervous system of pond snail, Lymnaea stagnalis

We examined three-dimensionally the arrangement of gamma-aminobutyric acid (GABA)-like immunoreactive neurons in the central nervous system (CNS) of the pond snail, Lymnaea stagnalis, by a combination of immunohistochemistry and confocal laser scanning microscopy on whole-mount preparations. GABA-like immunoreactivity was detected in all ganglia of the adult CNS. The following distribution of immunoreactive cell bodies was noted in the adult snail. Buccal ganglia: one cell body and five pairs of cell bodies, cerebral ganglia: one pair of cell bodies, pedal ganglia: two single cell bodies, two pairs of cell bodies, and three pairs of cell clusters, and pleural ganglia: one pair of cell bodies. In the asymmetrical parietal ganglia, three cell bodies were located in the left parietal ganglion; three cell bodies and three cell clusters were located in the right parietal ganglion. In the single visceral ganglion, a few scattered individual cell bodies and a cell cluster were GABA-like immunoreactive. Our results showed that the occurrence of GABA is widely spread in the CNS of adult L. stagnalis. GABA-like immunoreactivity in the CNS was not detected in the embryo but was observed after hatching, although the number of stained cells was less than in the adult, with the exception of those in the cerebral ganglia where their number decreased with maturation. Our results provide detailed maps of the central GABA-like immunoreactive neurons in juveniles, immatures, and adults of L. stagnalis.     

Identification, Distribution and Physiological Activity of Three Novel Neuropeptides of Lymnaea: FLRlamide and pQFYRlamide Encoded by the FMRFamide Gene, and a Related Peptide

We are interested in analysing the detailed modulation of defined neuronal systems by multiple neuropeptides encoded in the FMRFamide locus of the snail Lymnaea. Cloning of the FMRFamide gene has predicted the existence of two novel peptides previously unknown from biochemical analysis, the pentapeptides EFLRlamideand QFYRlamide. These peptides may form part of a new family of peptides sharing the sequence motif –FXRlamide. In this paper we adopt a novel approach to first identify and characterize –FXRlamide-like peptides in extracts from the central nervous system of Lymnaea. By a combination of high-performance liquid chromatography (HPLC) and continuous-flow fast atom bombardment mass spectrometry, we identify three novel peptides: EFLRlamide, pQFYRlamide and pQFLRlamide. The first two are those predicted in exon II of the FMRFamide locus whereas the last is, interestingly, a product which cannot be derived from post-translational modification of the predicted peptides but must be encoded by as yet unidentified nucleotide sequences. A specific antibody raised to EFLRlamide, and immuno reactive to all three peptides, revealed EFLRlamide-like expression throughout the central nervous system in the same cells where exon II is transcribed and the peptide SEEPLY (a post-translational product of exon II) was localized. Additional cells, however, were also identified. Immunoreactivity was mapped in a number of identified neurons in the central nervous system, including two heart cardio excitatory motoneurons, the E_he cells (E heart excitors of the visceral ganglion) and penialmotoneurons in the right cerebral ganglion. The peripheral tissues (heart and penial complex) that the serespective classes of neurons innervate also exhibited EFLRlamide immunoreactivity. The central and peripheral localization of EFLRlamide-like immunoreactivity suggested that EFLRlamide/pQFYRlamide may have an important physiological role in both these peripheral systems as well as in the central nervous system. This was confirmed by physiological experiments that showed that EFLRlamide and pQFYRlamide inhibited many centralneurons and in particular the Bgp neurons in the right parietal ganglion. EFLRlamide had complex biphasic effects on the frequency of heart-beat: an initial inhibitory response was followed by a long-lasting increase in the rate of beating. Taken together with earlier work, this study now completes the analysis and localization of the full set of post-translational products of the FMRFamide precursor in Lymnaea and supplies further evidence towards the characterization of the physiological systems which such peptides may modulate in concert.     

Organization of the serotonergic innervation of the feeding (buccal) musculature during the maturation of the pond snail Lymnaea stagnalis: a morphological and biochemical study

The serotonergic innervation of the buccal musculature responsible for feeding (radula protraction) was investigated during the maturation of the pond snail, Lymnaea stagnalis L., applying light and electron microscopic immunohistochemistry and biochemical approaches. According to epifluorescence and laser confocal microscopy, the first 5-HT-like-immunoreactive (5-HTLIR) processes appeared on the surface of the musculature at the postmetamorphic E80% embryonic stage. Until hatching, the innervation continued to increase in density, showing axon arborizations with projections into the deeper muscle levels. An adult-like pattern of 5-HTLIR innervation appeared at P2-P3 juvenile stages. At the ultrastructural level, close (16-20 nm) but mostly unspecialized neuromuscular contacts were formed by both unlabeled and 5-HTLIR axon profiles from the E80% embryonic stage. Labeled processes were also found located relatively far from the muscle cells. An HPLC assay showed a gradual increase of the 5-HT level in the buccal mass during development. The buccal mass was characterized by a single-component high-affinity 5-HT uptake system, and 5-HT release could be evoked by 100 mM K(+) and blocked in Ca(2+) -free medium. It is suggested that 5-HT plays a wide modulatory role in the peripheral feeding system and is also involved in the functional maturation of the muscle system.     

Development of catecholaminergic neurons in the pond snail,Lymnaea stagnalis: I. Embryonic development of dopamine-containing neurons and dopamine-dependent behaviors

The embryonic development of the catecholaminergic system of the pond snail, Lymnaea stagnalis, was investigated by using chromatographic and histochemical methods. High performance liquid chromatography suggested that dopamine was the only catecholamine present in significant concentrations throughout the embryonic development of Lymnaea. Dopamine first became detectable at about embryonic stage (E) 15 (15% of embryonic development) and then increased in amount during early development to reach about 120–140 fmol per animal by around E40. Dopamine content remained stable during mid-embryogenesis (E40–65), increased slowing for the next couple of days, and then increased rapidly to culminate at about 400 fmol per animal by hatching. The detection of aldehyde- and glyoxylate-induced fluorescence and of tyrosine hydroxylaselike immunoreactivity indicated that the first catecholaminergic cells appeared in the late trochophore or early veliger stage of embryonic development (E32–35). The paired perikarya of these transient apical catecholaminergic (TAC) neurons were located beneath the apical plate, remained outside of the central ganglia during embryogenesis, and no longer contained detectable catecholamines close to hatching. TAC neurons bore cilia on the ends of short processes that penetrated the overlying epithelium; their long processes branched repeatedly under the ciliated apical plate. Several smaller catecholaminergic cells first appeared in the anterior margin of the foot at a stage when the embryos began to metamorphose from the veliger form (E55). Similar bipolar cells later appeared in the tentacle and lips. The axons of all of these small peripheral cells projected centrally and terminated within the neuropil of different central ganglia. Central catecholaminergic neurons, including RPeD1, differentiated only after metamorphosis was complete (E75). Development of locomotor, respiratory, and feeding behaviors correlated with maturation of catecholaminergic neurons, as indicated by histology and chromatography. J. Comp. Neurol. 404:285–296, 1999. © 1999 Wiley-Liss, Inc.     

Distribution of catecholamines and of immunoreactivity to substances like vertebrate enzymes for the synthesis of catecholamines within the central nervous system of the snail, Lymnaea stagnalis

Catecholamines (CAs) were detected histochemically within over 185 cell bodies in the central nervous system (CNS) of juvenile and young adult Lymnaea. This distribution of CA-containing cells in all central ganglia except the pleural ganglia is more widespread than previously described but is consistent with other reports suggesting numerous roles for CAs within the nervous system. This study also describes the distribution of substances which are antigenically similar to four bovine enzymes for catecholamine synthesis, but the distribution patterns showed little or no overlap with each other or with CA. These results suggest the need for caution in the interpretation of such immunohistochemical studies.     

Isolation, characterization, and evolutionary aspects of a cDNA clone encoding multiple neuropeptides involved in the stereotyped egg-laying behavior of the freshwater snail Lymnaea stagnalis

The cerebral neurosecretory caudodorsal cells (CDCs) of the freshwater pulmonate snail Lymnaea stagnalis control egg laying, an event that involves a pattern of stereotyped behaviors. The CDCs synthesize and release multiple peptides, among which is the ovulation hormone (CDCH). It is thought that each peptide controls a specific aspect of the processes involved in egg laying. We isolated and characterized a CDC-specific cDNA clone that encodes the ovulation hormone (CDCH). RNA blot analysis and in situ hybridization experiments demonstrated that the CDCs are the major cell groups in the cerebral ganglia that transcribe the CDCH gene. In addition to CDCH, the 259-amino acid-long CDCH preprohormone contains 11 other predicted peptides. The overall homology of the CDCH preprohormone with the egg-laying hormone (ELH) preprohormones of the marine opisthobranch snails Aplysia californica and A. parvula is very low (29 and 26%, respectively). However, a more detailed comparison revealed a highly differential pattern of conservation of peptide regions. Significant homology was found between the regions containing (1) CDCH and ELH, (2) repeated pentapeptides, (3) alpha-caudodorsal cell peptide and alpha-bag cell peptide, and (4) 2 regions representing as yet unidentified peptides. Insignificant homology was found when comparing regions containing the other predicted peptides. The conserved peptides probably control similar aspects of the egg-laying fixed action patterns in these distantly related gastropod species. The pentapeptide region exhibits the highest level of homology (75%); in addition, an extra pentapeptide has been generated on the CDCH precursor. This indicates a vital function of these peptides in Aplysia, as well as in Lymnaea species.     

Axonal mapping of the giant peptidergic neurons VD1 and RPD2 located in the CNS of the pond snail Lymnaea stagnalis, with particular reference to the innervation of the auricle of the heart.

VD1 and RPD2 are two giant neuropeptidergic neurons located respectively in the visceral and right parietal ganglion of the central nervous system (CNS) of the pond snail Lymnaea stagnalis. They are the most prominent representatives of a system of neurons expressing a gene that is similar to the gene expressed in R15 of Aplysia californica. Both neuronal systems are involved in the regulation of cardio-respiratory phenomena. In the present study the axonal branches of VD1 and RPD2 were mapped using immunocytochemical and tracer studies. To this end the alpha 1-antiserum (directed to one of the VD1/RPD2 neuropeptides) was used in combination with Lucifer yellow (LY) and Ni-lys tracers. In whole mount preparations of the CNS, immunostained axons of VD1 and RPD2 were observed to run to the pleural, cerebral and pedal ganglia and in several nerves. Upon LY injection of VD1 thin axon branches were observed in the internal right parietal nerve. These run to the skin in the mantle area near the pneumostome and osphradium. The skin of the lips appeared to receive a similar innervation via the lip nerves. Thick LY filled axons of VD1 and RPD2 were observed in the intestinal nerve. They could be traced to the heart region. The pericardial branch of the intestinal nerve innervates the pericardium and heart (Ni-lys tracing). Immunocytochemically, using the alpha 1-antiserum, it was demonstrated that this nerve branch carries the axons of VD1 and RPD2 to the venous side of the auricle, where they enter the pericardial cavity and ramify in the auricle (but not in the ventricle).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transplantation and restoration of functional synapses between an identified neuron and its targets in the intact brain of Lymnaea stagnalis

Most information available to date regarding the cellular and synaptic mechanisms of target cell selection and specific synapse formation has primarily come from in vitro cell culture studies. Whether fundamental mechanisms of synapse formation revealed through in vitro studies are similar to those occurring in vivo has not yet been determined. Taking advantage of the regenerative capabilities of adult molluscan neurons, we demonstrate that when transplanted into the host ganglia an identified neuron reestablishes its synaptic connections with appropriate targets in vivo. This synaptogenesis, however, was possible only if the targets were denervated from the host cell. Specifically, the giant dopamine neuron right pedal dorsal 1 (RPeD1) located in the pedal ganglia was isolated from a donor brain and transplanted into the visceral ganglia of the recipient brain. We discovered that within 2-4 days the transplanted RPeD1 exhibited extensive regeneration. However, simultaneous intracellular recordings failed to reveal synapses between the transplanted cell and its targets in the visceral ganglia, despite physical overlap between the neurites. To test whether the failure of a transplanted cell to innervate its target was due to the fact that the targets continued to receive input from the native RPeD1, the latter soma was surgically removed prior to the transplantation of RPeD1. Even after the removal of host soma, the transplanted RPeD1 failed to innervate the targets such as visceral dorsal 4 (VD4)-despite extensive regeneration by the transplanted cell. However, when RPeD1 axon was allowed to degenerate completely, the transplanted RPeD1 successfully innervated all of its targets and these synapses were similar to those seen between host RPeD1 and its targets. Taken together, our data demonstrate that the transplanted cells will innervate their potential targets only if the targets were denervated from the host cell. These data also lend support to the idea that, irrespective of their physical location in the brain, the displaced neurons are able to regenerate, recognize their targets, and establish specific synapses in the nervous system.     

Possible role of 3'(2')-phosphoadenosine-5'-phosphate phosphatase in the etiology and therapy of bipolar disorder

Bipolar affective disorder (BPD) is a multifactorial, severe, chronic and disabling illness with 50% heritability that affects 1-2% of the population. Lithium ions (Li) are the drug of choice for BPD. Yet, 20-40% of patients fail to respond to Li. Although numerous biochemical and cellular effects have been attributed to Li, its therapeutic mechanism of action has not been elucidated. This review presents the possible involvement of 3'(2')-phosphoadenosine-5'-phosphate (PAP) phosphatase in the etiology of bipolar disorder and the mechanism of action of Li. Of the enzymes inhibited by Li, PAP phosphatase is inhibited with the lowest Ki (0.3 mM). At therapeutic concentrations of Li (0.5-1.5 mM), inhibition is greater than 80%. Therefore, PAP phosphatase is a strong candidate for Li's therapeutic mechanism of action. In yeast, a PAP phosphatase knockout mutation leads to the accumulation of PAP, which affects ribosomal-, transfer- and small nucleolar-RNA processing. PAP accumulation in the mammalian brain following Li inhibition of PAP phosphatase may very well account for the observed effects of Li on gene expression and behavior. Furthermore, we have reported significant changes in PAP phosphatase levels in postmortem frontal cortex of bipolar patients.     

T cell response to 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in multiple sclerosis patients

T cell responses targeting myelin antigens are possibly involved in the pathogenesis of demyelinating diseases, such as multiple sclerosis (MS). Little is known about human T cell responses to 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), the third most abundant myelin protein. We examined the primary peripheral T cell response to CNPase and characterized CNPase-specific CD4+ long-term T cell lines (TCL) from MS patients and healthy donors. The strongest primary responses were found in two MS patients with very active disease and were directed against CNP(343-373). We identified immunodominant epitope clusters in the regions CNP(343-373) and (356-388) that were recognized in the context of MS-associated HLA-DR2 and DR4 molecules. These data provide the immunological basis for further investigation of CNPase as a potential target self-antigen in MS.     

Oxidative stress and toxicity induced by the nucleoside reverse transcriptase inhibitor (NRTI)--2',3'-dideoxycytidine (ddC): relevance to HIV-dementia

Human immunodeficiency virus dementia (HIVD) is the most common form of dementia occurring among young adults. In HIVD, neuronal cell loss occurs in the absence of neuronal infection. With the advent of highly active anti-retroviral therapy (HAART), the incidence of HIVD has drastically reduced, though prevalence of milder forms of HIVD continues to rise. Though these agents have been used successfully in suppressing viral production, they have also been associated with a number of side effects. Here we examine the possible role of NRTIs, in particular 2',3'-dideoxycytidine (ddC), in the neuropathology of HIVD. Synaptosomes and isolated mitochondria treated and incubated for 6 h with CSF-achievable concentrations of ddC, i.e., 6-11 ng/ml, were found to show a significant increase in oxidative stress with 40 nM ddC as measured by protein carbonyls and 3-nitrotyrosine (3NT), effects that were not observed in the more tolerable NRTI, 3TC. Protection against protein oxidation induced by ddC was observed when brain mitochondria were isolated from gerbils 1 h after injection i.p. with the brain accessible antioxidant and glutathione mimetic, tricyclodecan-9-yl-xanthogenate (D609). In addition, there is a significant reduction in the levels of anti-apoptotic protein Bcl-2 and a significant increase in cytochrome c release and also a significant increase in the expression of pro-apoptotic protein caspase-3 after mitochondria were treated with 40 nM ddC. The results reported here show that ddC at 40 nM can induce oxidative stress, cause the release of cytochrome c, and in addition, reduce the levels of anti-apoptotic proteins, increase the levels of pro-apoptotic proteins, thereby increasing the possibility for induction of apoptosis. These findings are consistent with the notion of a possible role of the NRTIs, and in particular, ddC, in the mechanisms involved in HIVD.