Inhibition of the hemorrhagic and proteolytic activities of Lansberg's hognose pit viper (Porthidium lansbergii hutmanni) venom by opossum (Didelphis marsupialis) serum: isolation of Didelphis marsupialis 0.15Dm fraction on DEAE-cellulose chromatography.

Earlier studies have revealed the ability of sera from several mammals to neutralize the toxic effects of snake venom. The Venezuelan opossum (Didelphis marsupialis) is one that has been found to inhibit hemorrhagic and proteolytic activities of venoms from many species of snakes. In this article it is shown that the opossum sera and its 0.15DM fraction were able to completely neutralize both hemorrhagic and hydrolysis (proteolysis) of casein effects induced by venom of the Lansberg's hognose pit viper (Porthidium lansbergii hutmanni). We have used DEAE-cellulose ion exchange chromatography to collect protein fractions from D. marsupialis sera which were able to defend mice from the lethal effects of P.l. hutmanni venom. The fractions separated were homogeneous by conventional electrophoresis using SDS-PAGE. The protein bands obtained contained molecular weights of approximately 6 to 220 kDa. These results revealed the presence of proteases inhibitors in the opossum sera fractions and the inhibition of venom activity by opossum sera suggesting a reciprocal adaptation at the molecular level.

Effects of deslorelin implants on reproduction in the common brushtail possum (Trichosurus vulpecula).

The present study investigated the effects of slow-release implants containing the gonadotrophin-releasing hormone (GnRH) agonist deslorelin on reproduction in the common brushtail possum (Trichosurus vulpecula). Captive female brushtail possums were assigned to control (placebo implant), low dose (4.7 mg deslorelin) or high dose (9.4 mg deslorelin) groups; males were assigned to control or high dose (9.4 mg deslorelin) groups. The acute effects of deslorelin treatment at the level of the pituitary gland were similar between the two sexes, where a transient rise in luteinising hormone concentration was induced over the first 24 h. In females, this was associated with the disruption of the normal oestrous cycle and mating within 2-10 days in some treated individuals, but no young were subsequently detected. By 3 weeks after treatment, treated females became anoestrus and remained infertile for at least one breeding season. The effects of treatment were reversible in a subset of females that had their implants removed, although the time taken to produce offspring was variable. Paradoxically, male brushtail possums remained fertile during chronic deslorelin exposure. Despite significant declines in basal follicle-stimulating hormone and testosterone concentrations, as well as an inability to respond to a GnRH challenge, treated males sired as many offspring as control males and there was no evidence of testicular regression. In conclusion, there is potential to control reproduction in female brushtail possums by using chronic GnRH agonist treatment.

[Trypanosomatidae of public health importance occurring in wild and synanthropic animals of rural Venezuela]. / Trypanosomatidae de importancia en salud pública en animales silvestres y sinantrópicos en un area rural del municipio Tovar del estado Mérida, Venezuela.

INTRODUCTION: Chagas disease and leishmaniasis are important public health problems due to their high frequency and broad distribution in Latin America. Understanding of the roles of reservoir animals is crucial for a global assessment of the epidemiology of these diseases. OBJECTIVE: To identify parasites classed as Trypanosomatidae as they occurred in sylvatic animals, and to establish rates of coinfection. MATERIALS AND METHODS: Sylvatic animals were systematically captured in the rural area of El Carrizal, Merida State, Venezuela, betweenJuly, 1998 and February, 2000. The captures were made in Tomahawk type homemade traps, placed 15 nights per month throughout the study period. Blood was extracted from each captured and anesthetized animal by means of cardiac puncture. The search for trypanosomatids was undertaken by fresh blood examination, Giemsa stained blood smears and by means of blood-agar culture. Occasional xenodiagnoses were made to check diagnostic accuracy. The isolates obtained in culture media were identified by restriction fragment analysis and hybridization with specific probes. RESULTS: Three species of sylvatic animals (n = 215) were captured: Rattus spp. (135), Sigmodon hispidus (73) and Didelphis marsupialis (7). From them, three species of Trypanosomatidae were identified: Leishmania (Viannia) guyanensis, Trypanosoma cruzi and Trypanosoma lewisi. Trypanosoma. cruzi was identified in D. marsupialis (4/7), S. hispidus (1/73) and Rattus spp. (1/ 135), whereas L. (V.) guyanensis and T. lewisi were identified only in Rattus spp., 1/135 and 12/ 135, respectively. CONCLUSIONS: The coexistence of these genetically related hemoflagellates in sylvatic hosts was important for understanding the immunological interactions that may be established in reservoir animals, and the possible implications that this may have for the susceptible host. Finally, the identification of L. (V.) guyanensis in Rattus spp and T. cruzi in S. hispidus constituted the first reports of this relationship in Venezuela.

Rickettsial Infections among Ctenocephalides felis and Host Animals during a Flea-Borne Rickettsioses Outbreak in Orange County, California.

Due to a resurgence of flea-borne rickettsioses in Orange County, California, we investigated the etiologies of rickettsial infections of Ctenocephalides felis, the predominant fleas species obtained from opossums (Didelphis virginiana) and domestic cats (Felis catus), collected from case exposure sites and other areas in Orange County. In addition, we assessed the prevalence of IgG antibodies against spotted fever group (SFGR) and typhus group (TGR) rickettsiae in opossum sera. Of the 597 flea specimens collected from opossums and cats, 37.2% tested positive for Rickettsia. PCR and sequencing of rickettsial genes obtained from C. felis flea DNA preparations revealed the presence of R. typhi (1.3%), R. felis (28.0%) and R. felis-like organisms (7.5%). Sera from opossums contained TGR-specific (40.84%), but not SFGR-specific antibodies. The detection of R. felis and R. typhi in the C. felis fleas in Orange County highlights the potential risk for human infection with either of these pathogens, and underscores the need for further investigations incorporating specimens from humans, animal hosts, and invertebrate vectors in endemic areas. Such studies will be essential for establishing a link in the ongoing flea-borne rickettsioses outbreaks.

Isolation and characterization of DM40 and DM43, two snake venom metalloproteinase inhibitors from Didelphis marsupialis serum.

From Didelphis marsupialis serum, two antihemorrhagic proteins were isolated by DEAE-Sephacel, Phenyl-Sepharose and Superdex 200 and characterized. Their masses by mass spectrometry were 40318 AMU for DM40 and 42373 and 43010 AMU for DM43, indicating the presence of isoforms for the last. Molecular masses of 44.8 and 47.3 were obtained by SDS-PAGE, respectively for DM40 and DM43. Both inhibitors showed isoelectric points lower than 3.5 and glycosylation percentages varying from 20.5 to 29.0%, as estimated by chemical deglycosylation and amino acid analysis. N-terminal sequences of the first 17 residues of DM40 and DM43 were identical except for the exchange of R9 for P9. Both were homologous to oprin, a similar inhibitor from Didelphis virginiana serum. No evidence of complex formation between DM40 and DM43 was observed either by native PAGE or gel filtration chromatography. In addition to the antihemorrhagic activity, DM40 and DM43 inhibited the hydrolysis of casein, fibrinogen and fibronectin by Bothrops jararaca venom. DM43 also showed antilethal, antiedematogenic and antihyperalgesic activities. None of the inhibitors showed enzymatic activity on casein. Both proteins formed stable complexes with jararhagin and inhibited its hemorrhagic effect as well as the enzymatic activity of this toxin on fluorogenic substrate.

X-linked gene expression in the Virginia opossum: differences between the paternally derived Gpd and Pgk-A loci.

Expression of X-linked glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase-A (PGK-A) in the Virginia opossum (Didelphis virginiana) was studied electrophoretically in animals from natural populations and those produced through controlled laboratory crosses. Blood from most of the wild animals exhibited a common single-banded phenotype for both enzymes. Rare variant animals, regardless of sex, exhibited single-banded phenotypes different in mobility from the common mobility class of the respective enzyme. The laboratory crosses confirmed the allelic basis for the common and rare phenotypes. Transmission of PGK-A phenotypes followed the pattern of determinate (nonrandom) inactivation of the paternally derived Pgk-A allele, and transmission of G6PD also was consistent with this pattern. A survey of tissue-specific expression of G6PD phenotypes of heterozygous females revealed, in almost all tissues, three-banded patterns skewed in favor of the allele that was expressed in blood cells. Three-banded patterns were never observed in males or in putatively homozygous females. These patterns suggest simultaneous, but unequal, expression of the maternally and paternally derived Gpd alleles within individual cells (i.e., partial paternal allele expression). The absence of such partial expression was noted in a parallel survey of females heterozygous at the Pgk-A locus. Thus, it appears that Gpd and Pgk-A are X-linked in D. virginiana and subject to preferential paternal allele inactivation, but that dosage compensation may not be complete for all paternally derived X-linked genes. The data establish the similarity between the American and Australian marsupial patterns of X-linked gene regulation and, thus, support the hypothesis that this form of dosage compensation was present in the early marsupial lineage that gave rise to these modern marsupial divisions. In addition, the data provide the first documentation of the differential expression of two X-linked genes in a single marsupial species. Because of its combination of X-linked variation, high fecundity, and short generation time, D. virginiana is a unique model for pursuing questions about marsupial gene regulation that have been difficult to approach through studies of Australian species.

Evolution of transthyretin gene expression in the liver of Didelphis virginiana and other American marsupials.

The occurrence of the thyroid hormone-binding plasma protein transthyretin in the bloodstream was investigated for four American marsupial species. Serum samples were analyzed by incubation with radioactive T4, followed by electrophoresis, then autoradiography, and Western blotting. Transthyretin was found in serum from Monodelphis domestica, Didelphis virginiana, Caluromys lanatus, and Dromiciops australis. For unambiguous identification, transthyretin from D, virginiana was purified from serum and its N-terminal amino acid sequence was determined. The obtained results suggest that the initiation of transthyretin gene expression in the liver of marsupials occurred independently in several lineages of American marsupials, all of which are at the ends of phylogenetic branches. The expression of the transthyretin gene in the liver of the American polyprotodont marsupials contrasts with the lack of transthyretin gene expression in the liver of all 22 previously investigated Australian Polyprotodonta.

Plasma growth hormone and growth hormone-binding protein during development in the marsupial brushtail possum (Trichosurus vulpecula).

Plasma concentrations of growth hormone (GH) were measured in the brushtail possum (Trichosurus vulpecula) pouch young from 25 through to 198 days post-partum (n=71). GH concentrations were highest early in pouch life (around 100 ng/ml), and thereafter declined in an exponential fashion to reach adult concentrations (10.8+/-1.8 ng/ml; n=21) by approximately 121-145 days post-partum, one to two months before the young is weaned. Growth hormone-binding protein (GHBP), which has been shown to modify the cellular actions of GH in eutherian mammals, was identified for the first time in a marsupial. Based on size exclusion gel filtration, possum GHBP had an estimated molecular mass of approximately 65 kDa, similar to that identified in other mammalian species, and binding of (125)I-labelled human GH (hGH) was displaced by excess hGH (20 microg). An immunoprecipitation method, in which plasma GHBP was rendered polyethylene glycol precipitable with a monoclonal antibody to the rabbit GHBP/GH receptor (MAb 43) and labelled with (125)I-hGH, was used to quantitate plasma GHBP by Scatchard analysis in the developing (pooled plasma samples) and adult (individual animals) possums. Binding affinity (K(a)) values in pouch young aged between 45 and 54 and 144 and 153 days post-partum varied between 1.0 and 2.4 x 10(9)/M, which was slightly higher than that in adult plasma (0.96+/-0.2 x 10(9)/M, n=6). Binding capacity (B(max)) values increased from non-detectable levels in animals aged 25-38 days post-partum to reach concentrations around half that seen in the adult (1.4+/-0.2 x 10(-9) M) by about 117 days post-partum and remained at this level until 153 days post-partum. Therefore, in early pouch life when plasma GH concentrations are highest, the very low concentrations of GHBP are unlikely to be important in terms of competing with GH-receptor for ligand or altering the half-life of circulating GH.

Inhibitory properties of the anti-bothropic complex from Didelphis albiventris serum on toxic and pharmacological actions of metalloproteases and myotoxins from Bothrops asper venom.

Anti-bothropic complex (ABC) was isolated from the serum of the South American opossum (Didelphis albiventris) by single-step affinity chromatography using a Sepharose-immobilized metalloprotease (BaP1) from Bothrops asper as the binding protein. Biochemical characterization of ABC showed the presence of two glycosylated subunits of 43 and 45 kDa, respectively, with an isoelectric point < 4. The two subunits were separated by ion-exchange HPLC. The N-terminal sequences of both subunits (LKAMDPTPXLWIETESP, where X is Arg-9 and Pro-9, respectively) showed a high degree of identity with other serum inhibitors isolated from different marsupials. Functional studies pointed out that ABC inhibits the hemorrhagic and proteolytic activities on fibrin, fibrinogen, and casein induced by the metalloproteases BaP1 and BaH4 isolated from B. asper venom. In addition to the anti-hemorrhagic and anti-proteolytic activities, ABC also showed anti-myotoxic, anti-lethal, and anti-edematogenic effects against myotoxic phospholipases A(2) isolated from the same venom. Moreover, it had inhibitory effects on the phospholipase A(2) activity of the crude venom as well as the isolated venom phospholipases A(2).

Mesotocin in the brain and plasma of an Australian marsupial, the brushtail possum (Trichosurus vulpecula).

Oxytocic peptides extracted from the brain and plasma of the brushtail possum, Trichosurus vulpecula, were separated by reverse-phase high pressure lipid chromatography (HPLC) and quantified by specific radioimmunoassays for oxytocin (OT) and mesotocin (MT). The pituitary, hypothalamus and cerebral cortex were found to contain MT only in quantities of 3.9 +/- 0.2 (SE) ug, 17.6 +/- 0.6 ng and 21.0 +/- 2.6 ng respectively. The plasma concentration of MT varied according to the degree of stress of the possum. In anaesthetized animals values of 39.7 +/- 9.7 pg/ml (11 males) and 31.5 +/- 12.9 pg/ml (6 females) were obtained; in four conscious catheterized animals, 9.4 +/- 6.3 pg/ml. Samples taken from three anaesthetized animals during exsanguination contained 271 +/- 102 (SD) pg MT/ml. It was concluded that hypothalamic and extra-hypothalamic MT is present in the marsupial brain and that as in placental mammals, stress stimulates the secretion of mesotocin.

Antivenom activity of opossum (Didelphis marsupialis) serum fraction.

We have found an opossum serum fraction of approximately 97,000 mol. wt to be highly proficient in inactivating the haemorrhagic and proteolytic fractions of Bothrops lanceolatus venom. This antivenom substance, isolated from opossum serum or a synthetic peptide based on the aforementioned protein, would probably be useful in the medical management of Bothrops accidents.

Isolation of antihemorrhagic factors in opossum (Didelphis virginiana) serum using a monoclonal antibody immunoadsorbent.

The antihemorrhagic factor in opossum (Didelphis virginiana) serum isolated by Sephadex G-200 gel filtration and DEAE A-50 ion exchange chromatography was used as antigen to immunize BALB/c mice. Hybrid cell lines secreting monoclonal antibodies against antihemorrhagic factor were produced by fusion of Sp2/0 myeloma cells with spleen cells of the immunized mice. The ascites fluid was produced in BALB/c mice. The monoclonal antibody in the ascites fluid was partially purified by DEAE A-50 ion exchange and coupled to CNBr-activated isolation of isolation of antihemorrhagic factor. The neutralization capacity of the conventionally isolated antihemorrhagic factor was 14.6 times and the affinity isolated antihemorrhagic factor was 16.8 times that of crude opossum serum. Both antihemorrhagic factors were homogeneous, with one fast migrating band in the area of albumin shown by polyacrylamide gel electrophoresis. However, the antihemorrhagic factor showed one heavy band and one faint band in SDS-polyacrylamide electrophoresis, as well as in isoelectric focusing. The molecular weight of the heavy band was estimated to be 65,000 with a value of p1 4.8 and the faint band was 57,000 with a value of pI 4.1.

Inhibitory properties of the antibothropic complex from the South American opossum (Didelphis marsupialis) serum.

The South American opossum Didelphis marsupialis is known to be highly resistant to snake envenomation. In this paper it is shown that the opossum serum inhibits haemorrhage induced by both Crotalinae and Viperinae venoms. Tested against Bothrops jararaca (jararaca) venom, the antibothropic complex (ABC) isolated from the opossum serum was at least six times more antihaemorrhagic than the commercial antivenom. ABC showed no proteolytic activity by itself and was not hydrolysed by the venom. It inhibited the hydrolysis of casein by B. jararaca venom, but did not inhibit its hydrolytic activities upon N alpha-benzoyl-L-arginine ethyl ester (BAEE) and N alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA). The inhibitor did not interfere with trypsin and bacterial collagenase activities on BAPNA and N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA), respectively. It reduced chymotrypsin hydrolysis of N-acetyl-L-tyrosine ethyl ester (ATEE) because ABC is also a substrate for this enzyme. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, B. jararaca venom preferentially degraded fibrinogen A alpha-chain and fibrin alpha-chain. Tested on extracellular matrix proteins, the venom hydrolysed collagen IV, gelatins I and V, laminin and fibronectin, besides depolimerizing collagen I alpha-chain dimers. Fibrillar collagen V was not digested. These hydrolyses were inhibited by ABC and by EDTA. Our results show that the antibothropic complex is a venom metalloproteinase inhibitor, which could, at least partially, account for its antihaemorrhagic activity. Electrophoretic evidence indicated non-covalent complex formation between the antihaemorrhagic factor and component(s) of B. jararaca venom.

Isolation of protein factors from opossum (Didelphis albiventris) serum which protect against Bothrops jararaca venom.

The fractionation of Didelphis albiventris serum by DEAE-Sephadex A50 yields a fraction (DA2) which protects the opossum against Bothrops venom. One polypeptide (DA2-II) responsible for this protection was isolated from fraction DA2 by ion exchange chromatography and biochemically characterized. DA2-II is a 43,000 mol. wt glycoprotein with the following N-terminal sequence: LKAMDTTPPLKIKKEPVK. Pairwise comparison of the amino acid sequence with four anti-hemorrhagic factors isolated from other opossum species indicated that DA2-II possesses high similarity (60-80%) with these proteins.

Isolation and partial characterization of an anti-bothropic complex from the serum of South American Didelphidae.

An anti-bothropic fraction (ABF) with anti-Bothrops jararaca venom activity tested in mice was isolated from the serum of some South American Didelphidae (Didelphis marsupialis, Philander opossum and Lutreolina crassicaudata) by DEAE-Sephacel chromatography. ABF from D. marsupialis was shown to be 12 times more active in protection assays on a weight basis than the serum proteins. A similar fraction obtained from Metachirus nudicaudatum serum was shown to be inactive. An anti-bothropic complex (ABC) was isolated from D. marsupialis ABF. HPLC gel permeation chromatography of ABC from D. marsupialis indicated the presence of a main peak with mol. wt of 84,000. SDS-PAGE of this ABC showed the presence of two subunits of 48,000 and 43,000. The active ABF isolated from P. opossum and L. crassicaudata also showed the presence of these subunits by SDS-PAGE. Isolation of the 48,000 mol. wt D. marsupialis subunit by HPLC-hydrophobic interaction chromatography demonstrated that the 43,000 subunit was essential for the protective action of the complex. Both subunits from D. marsupialis, P. opossum and L. crassicaudata were Western-blotted and N-terminal sequenced. No N-terminal amino acid was found for the 43,000 subunit, whereas for the 48,000 subunit a high degree of homology was found: D. marsupialis: H2N-L K A M D P T P P L W I K T E X P . ; L. crassicaudata: H2N-L K A M D P T P P L W I Q T E . . . ; P. opossum: H2N-L K A M D T T P E . . . No significant homology with known proteins was detected.

Release of sarcoplasmic enzymes from skeletal muscle by Bothrops jararacussu venom: antagonism by heparin and by the serum of South American marsupials.

The venom of B. jararacussu induced a time- and dose-dependent (2-100 micrograms/ml) increase in the rate of release of sarcoplasmic enzymes (CK and LDH) from isolated rat and frog muscles. This effect, which we attribute to sarcolemmal damage by the venom, persisted in a Ca2+-free media, suggesting that phospholipase A activity was not required. The venom-induced enzyme release from the isolated muscles was reversibly inhibited by the sera (1-10 microliters/ml) of the marsupials Didelphis marsupialis aurita and Philander opossum, by an acidic glycoprotein fraction isolated from the opossum serum (50 micrograms/ml), and by heparin (50 micrograms/ml). Incubation of the B. jararacussu venom with opossum sera or heparin prevented the increase in plasma CK level observed in mice in which the snake venom (2.5-5.0 micrograms/g) was injected i.m. It is suggested that formation of acid-base complexes between basic myotoxins of B. jararacussu venom and either acidic components in the marsupial sera or the polyanionic heparin could account for the inhibition of the in vitro and in vivo effects of the venom on the release of sarcoplasmic enzymes from skeletal muscles.

Inhibition of the hyperalgesic activity of Bothrops jararaca venom by an antibothropic fraction isolated from opossum (Didelphis marsupialis) serum.

The antibothropic fraction (ABF) already isolated from Didelphis marsupialis serum, inhibits the haemorrhagic, oedematogenic, myonecrotic and lethal activities of Bothrops jararaca venom (Bjv). The aim of this work was to verify the capability of ABF to inhibit the hyperalgesic activity of Bjv. Intraplantar injection of Bjv induced hyperalgesia in a time- and dose-dependent manner and ABF administered in situ concomitantly with Bjv or i.v. 30 min before venom injection reduced the induced hyperalgesia. This same effect was observed when ABF was intravenously injected at 5 and 15 min after Bjv. Our results show that ABF inhibits also the hyperalgesia induced by Bjv.

PO41, a snake venom metalloproteinase inhibitor isolated from Philander opossum serum.

PO41 was isolated from Philander opossum serum by DEAE-Sephacel, Phenyl Superose and Superdex 200 chromatographies and showed a molecular mass of 41,330 Da by MALDI-TOF MS. Molecular masses of 81.5 and 84.5 kDa were obtained by size exclusion chromatography and dynamic laser light scattering, respectively, suggesting that PO41 is dimeric. Its isoelectric point was estimated to be lower than 3.5. PO41 presented similar amino terminal sequence to those of DM40 and DM43, two antihaemorrhagins previously isolated from Didelphis marsupialis serum and was recognized by polyclonal antibodies raised against D. marsupialis antibothropic fraction. To study the inhibitory properties of this protein, the metalloproteinases bothrolysin and jararhagin were isolated from Bothrops jararaca venom by chromatographies on Superdex 200 and Phenyl Superose. Jararhagin was further submitted to a Mono Q column. The proteolytic and haemorrhagic effects of these haemorrhagins were neutralized by PO41. Both snake venom metalloproteinases formed stable complexes with PO41. The stoichiometry of the complex PO41-jararhagin was one inhibitor subunit to one molecule of the enzyme. These results show that PO41 has physicochemical, structural, immunoreactive and biological properties similar to other metalloproteinase inhibitors belonging to the supergene family of immunoglobulins.

Studies on the energy metabolism of opossum Didelphis virginiana erythrocytes--IV. Red cells have low adenosine deaminase activity and high levels of deoxyadenosine nucleotides.

Alkaline extracts of adult opossum red cells were used to determine triphosphates of adenosine, deoxyadenosine and guanosine by anion exchange HPLC. Mean (nm/g Hg) ATP content of erythrocytes was 3713 and that of dATP 1913 (n = 12). Sonicates of red cells deaminated adenosine (ADO) at a rate of 1.55 nm/mg Hg/h and deoxyadenosine (dADO) at 1.82 nm/mg Hg/h. dATP synthesis from provided dADO was one order of magnitude greater in opossum than in human erythrocytes at both low and high dADO and Pi concentrations.

The acute phase response of plasma proteins in the polyprotodont marsupial Monodelphis domestica.

In eutherians, patterns of plasma protein levels in blood change during the acute phase response to trauma and inflammation. Until now, such an acute phase response has not been characterised in a noneutherian species. Here we describe the acute phase response in a marsupial species, the South American polyprotodont marsupial Monodelphis domestica, after brain surgery or injection of lipopolysaccharide. Several days after brain surgery, transthyretin was not detected in plasma. For 48 hr following injection of lipopolysaccharide, the concentration of haptoglobin in plasma increased, that of transthyretin decreased, and the concentration of albumin in plasma did not change significantly. The American polyprotodont marsupials are probably more closely related to the common ancestor marsupial than the Australian marsupials are. It is most likely that the transthyretin gene was not expressed in the liver of this common ancestor. As the transthyretin gene is expressed in the liver of M. domestica, it seems that as soon as transthyretin is synthesised by the liver, it is under negative acute phase control.