Incorporation of human foamy virus (HFV) envelope proteins into murine leukemia virus (MuLV) particles was studied in a transient transfection packaging cell system. We report here that wild-type HFV envelope protein can pseudotype MuLV particles, albeit at low efficiency. Complete or partial removal of the HFV cytoplasmic tail resulted in an abolishment or reduction of HFV-mediated infectivity, implicating a role of the HFV envelope cytoplasmic tail in the pseudotyping of MuLV particles. Mutation of the endoplasmic reticulum retention signal present in the HFV envelope cytoplasmic tail did not result in a higher relative infectivity of pseudotyped retroviral vectors. However, a chimeric envelope protein, containing an unprocessed MuLV envelope cytoplasmic domain fused to a truncated HFV envelope protein, showed an enhanced HFV specific infectivity as a result of an increased incorporation of chimeric envelope proteins into MuLV particles.

Amino Acid Sequence↗

PubMed1997

Simian foamy virus isolated from an accidentally infected human individual.

Evidence for natural foamy virus (FV) infections in humans is still lacking. However, accidental infections of humans with simian FV have been demonstrated by serology and PCR, but all previous attempts to recover infectious virus in such cases have failed. Here we describe the isolation of a simian FV from peripheral blood mononuclear cells (PBMC) of a healthy animal caretaker, who acquired the virus 20 years ago from an African green monkey (AGM) bite. Properties of the human isolate such as host range in cell cultures including human PBMC and ability to induce neutralizing antibodies in the primate host proved to be similar to those of FV obtained from AGM. The genomic sequence of the isolate was found to be virtually identical to the proviral sequence present in the host lymphocytes and related to AGM isolates but distinct from those of all FV isolates handled in the laboratory. For successful virus isolation, it was essential to stimulate the host lymphocytes by phytohemagglutinin and interleukin-2 for 2 weeks prior to cocultivation with permissive cells. In contrast to the situation found in FV-infected monkeys, virus isolation from the saliva of the animal caretaker was not possible, and no evidence for FV transmission to family contacts was obtained. We conclude that, in contrast to active infection in monkeys, FV persists in a state of latency following accidental infection of humans.

Accidents↗

PubMed1997

Baroreflex sensitivity and heart rate variability in conscious rats with myocardial infarction.

The baroreflex sensitivity (BRS) and the heart rate variability (HRV) were studied in conscious rats after myocardial infarction (MI; induced by coronary artery ligation) and after sham operation (SH). BRS was determined by linear regression of R-R interval vs. arterial pressure changes induced by nitroprusside or methoxamine (intravenous bolus). HRV was calculated from 3-min electrocardiogram recordings. Left ventricular end-diastolic pressure and plasma atrial natriuretic peptide were increased after MI; plasma norepinephrine and basal heart rate (HR) remained unchanged. At 3 and 28 days after MI, BRS was reduced as indicated by decreased reflex bradycardia (RB) (MI, 0.66 +/- 0.13 and 0.78 +/- 0.07 ms/mmHg; SH, 1.27 +/- 0.16 and 1.48 +/- 0.14 ms/mmHg, respectively; P < 0.05 MI vs. SH). At 56 days after MI, BRS was normalized. RB was unaffected by atropine 3 and 28 days after MI but reduced in all other groups. The increase of basal HR by atropine 3 and 28 days after MI was less than in all other groups. HRV (SD of mean N-N interval, coefficient of variance, low- and high-frequency power; studied at 28 and 56 days) was similar in all groups. It is concluded that BRS is transiently depressed in rats with left ventricular dysfunction after MI probably due to a reduced reflex vagal activity. Even though basal HR and HRV are unchanged after MI, a temporary attenuation of tonic vagal activity is unmasked after autonomic blockade.

Analysis of Variance↗

PubMed1996

Stimulation of Ca2+ release from rat liver mitochondria by the dithiol reagent alpha-lipoic acid.

Rat liver mitochondria contain a Ca(2+)-specific release pathway stimulated by Ca(2+)-dependent hydrolysis of oxidized intramitochondrial pyridine nucleotides to ADP ribose and nicotinamide. We have previously shown that NAD+ hydrolysis and subsequent Ca2+ release are inhibited by cyclosporine A and that they are only possible when some critical thiols are cross-linked or oxidized, e.g. by phenylarsine oxide, gliotoxin, or peroxynitrite. We now report that the antioxidant alpha-lipoic acid stimulates Ca2+ release from intact mitochondria, i.e. with preservation of the mitochondrial membrane potential and without large-amplitude swelling. The release stimulated by alpha-lipoic acid is inhibited by cyclosporine A and is more effective when the pyridine nucleotides are oxidized. The results strongly suggest that alpha-lipoic acid stimulates the Ca(2+)-specific release pathway from intact mitochondria by oxidizing some vicinal thiols, thereby stimulating hydrolysis of oxidized pyridine nucleotides. These observations further corroborate that intact rat liver mitochondria contain a specific Ca2+ release pathway stimulated by modification of vicinal thiols. Prolonged stimulation of Ca2+ release by lipoic acid followed by its re-uptake (Ca2+ "cycling") may contribute to the detrimental, prooxidant-like effects seen with higher concentrations of lipoic acid.

Animals↗

PubMed1996

Refinement of vectors for use in the yeast two-hybrid system.

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Amino Acid Sequence↗

PubMed1996

Lymphocytes are the major reservoir for foamy viruses in peripheral blood.

Simian and human foamy virus (FV) DNA can be readily detected in peripheral blood leukocytes. However, it is unknown which leukocyte populations harbor the virus in vivo. We, therefore, analyzed blood samples from nine African green monkeys, four chimpanzees, and two humans for the presence of foamy virus proviral DNA in different FACS-purified leukocyte populations, using a highly sensitive nested polymerase chain reaction (PCR). The CD8+ lymphocytes were PCR positive in all 15 samples and the average viral burden was highest in this population. FV DNA was detected in 10 of 15 cell samples enriched for B lymphocytes, and 4 of 9 CD4+ lymphocyte, 3 of 13 CD14+ monocyte, and 4 of 13 polymorphonuclear leukocyte samples. A highly sensitive reverse transcriptase PCR was performed to detect viral transcripts in peripheral blood leukocytes. All samples were negative. In conclusion, lymphocytes, and especially CD8+ T lymphocytes, were found to be a major target for foamy virus in the peripheral blood, but viral gene expression was not detected.

Animals↗

PubMed1996

Peroxynitrite stimulates the pyridine nucleotide-linked Ca2+ release from intact rat liver mitochondria.

Rat liver mitochondria contain a specific Ca2+ release pathway which operates when oxidized mitochondrial pyridine nucleotides are hydrolyzed in a Ca2+-dependent manner to ADP-ribose and nicotinamide. We have previously shown that NAD+ hydrolysis is inhibited by cyclosporin A and is possible only when some vicinal thiols are cross-linked. Here we report that the thiol oxidant peroxynitrite (ONOO-), which can form from nitric oxide (nitrogen monoxide, NO.) and superoxide anion (O2-), at low concentrations stimulates the specific Ca2+ release pathway. Both peroxynitrite-induced pyridine nucleotide hydrolysis and Ca2+ release are inhibited by cyclosporin A, and peroxynitrite is ineffective when pyridine nucleotides are kept reduced. Ca2+ release induced by peroxynitrite occurs with maintenance of the mitochondrial membrane potential and is not accompanied by entry of sucrose into mitochondria. The results suggest that peroxynitrite stimulates the specific Ca2+ release from intact mitochondria by modifying critical mitochondrial thiols other than glutathione in such a way that hydrolysis of oxidized pyridine nucleotides is achieved. These findings provide further insight into the regulation of Ca2+ release from mitochondria by nitric oxide and its congeners.

Animals↗

PubMed1996

Control of apoptosis by the cellular ATP level.

Apoptosis is a physiological form of cell death. Its causes and execution mechanisms are not clearly understood. Oxidative stress, nitric oxide and its congeners, Ca2+, proteases, nucleases, and mitochondria are considered mediators of apoptosis. At present their importance and exact role are elusive but it is clear that mitochondria are both the target and the source of oxidative stress, nitric oxide, and Ca2+. The mitochondrial membrane potential (delta psi), which is the driving force for mitochondrial ATP synthesis, declines during apoptosis, and maintenance of delta psi prevents apoptosis. Since apoptosis is highly regulated and involves the activity of hydrolytic enzymes, chromatin condensation and vesicle formation apoptosis is likely to have a high energy demand. We propose that the cellular ATP level is an important determinant for cell death. This hypothesis is supported by circumstantial evidence, is consistent with the available data, has a corrolary in aging, and is amenable to direct experimental testing particularly with flow cytometry as a promising tool.

Adenosine Triphosphate↗

PubMed1996

Sequencing of a 35.71 kb DNA segment on the right arm of yeast chromosome XV reveals regions of similarity to chromosomes I and XIII.

In a shotgun approach we sequenced the cosmid pEOA284 containing a fragment derived from the right arm of chromosome XV of Saccharomyces cerevisiae. An analysis of the sequence revealed that it contained open reading frames (ORFs) corresponding to the known genes SLY41, SPS4, COT1, FAA1, PMT3, PRO2 and MYO2. Of the 18 unknown ORFs, five are contained totally within, and two, O6105 and O6163, partially overlap other ORFs. ORF O6116 and O6139 have putative introns. Regions of similarity with chromosomes I and XIII have been uncovered. Interestingly, most of the paired ORFs encode proteins of the same gene family. The relatedness of these ORFs suggests gene duplication.

Amino Acid Sequence↗

PubMed1996

Construction of the complete rat fatty acid synthase cDNA and its expression in Saccharomyces cerevisiae.

The 272 647-dalton polypeptide of fatty acid synthase (FAS) from Rattus norvegicus has been expressed in a proteinase-deficient strain of Saccharomyces cerevisiae. The seven overlapping cDNA clones for rat FAS spanning the entire coding region were the starting material for this undertaking. In a series of cloning steps an expression plasmid was constructed in which the cDNA was placed under the control of the yeast ADH1 promoter. Northern blotting of total RNA isolated from yeast transformed with this expression plasmid demonstrated a high rate of transcription of the 7.4-kb cDNA. However, a successful translation required further manipulation of the sequence immediately upstream of the rat FAS translational start codon. This was obtained when the 86 bp of the rat FAS cDNA immediately 5' to the start codon were replaced by a nonamer corresponding to the immediate 5'-vicinity of the translational start codon of the yeast ADH1 gene. Nevertheless, the translation product could be detected only by Western blotting. The FAS proteins of S. cerevisiae and rat are not functionally interchangeable. Using the purification protocol of rat FAS the heterologously expressed FAS could be enriched by at least one order of magnitude.

Animals↗

PubMed1996

Analysis of projections from the cochlear nucleus to the lateral paragigantocellular reticular nucleus in the rat.

Golgi-staining, retrograde and anterograde tract-tracing, and a two-color immunoperoxidase technique have been employed, at the light- and electron-microscopic levels, to analyze the auditory projections from the cochlear nucleus (CN) to the lateral paragigantocellular reticular nucleus (LPGi) in the rat. We have found that the auditory input originates predominantly in the posteroventral and cochlear root nuclei. The auditory axons terminate in the cell-poor, ventral portion of the LPGi, which is strongly invaded by beaded dendritic profiles, originating from parent cell bodies located in the dorsal half of the LPGi. Ultrastructural analysis has revealed that the anterogradely labeled auditory axons form functional synapses preferentially with dendritic shafts. These axo-dendritic contacts are apparently excitatory in nature. By means of a sequential two-color immunoperoxidase staining method, we have further characterized potential postsynaptic neurons in the LPGi. Black-stained auditory fibers intermingle with brown-stained serotonergic or adrenergic neurons. Varicose auditory axons are often closely apposed to immunoreactive dendritic profiles of serotonergic and adrenergic neurons, indicating the presence of possible synaptic contacts of auditory terminal fibers with these transmitter-classified cells. The monosynaptic auditory in- put from the CN may modulate the activity of B3 sero-tonergic and C1 adrenergic cells in the LPGi and may thus induce adaptive changes in response to acoustic stimuli.

Afferent Pathways↗

PubMed1996

Control of the pyridine nucleotide-linked Ca2+ release from mitochondria by respiratory substrates.

Oxidation of mitochondrial pyridine nucleotides followed by their hydrolysis promotes Ca2+ release from intact liver mitochondria. In most of the previous studies oxidation was achieved with pro-oxidants which were added to mitochondria respiring on succinate in the presence of rotenone, a site I-specific inhibitor of the respiratory chain. Here we investigate pro-oxidant dependent and independent Ca2+ release from mitochondria when respiration is supported either by the NAD(+)-linked substrate beta-hydroxybutyrate, or by succinate. In the presence, as well as in the absence, of the pro-oxidant t-butylhydroperoxide mitochondria retain Ca2+ much better with succinate than with beta-hydroxybutyrate as respiratory substrate. When Ca2+ release is induced by t-butylhydroperoxide succinate-supported Ca2+ retention is impeded by rotenone. Ca2+ release (pro-oxidant dependent or independent) is paralleled by oxidation and hydrolysis of intramitochondrial pyridine nucleotides, and Ca2+ retention is paralleled by reduction of pyridine nucleotides. It is concluded that the pyridine nucleotide-linked Ca2+ release from mitochondria can be controlled by respiratory substrates which regulate the intramitochondrial hydrolysis of oxidized pyridine nucleotides.

3-Hydroxybutyric Acid↗

PubMed1996

Regulation of the fatty acid synthase promoter by retinoic acid.

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Animals↗