Biomedical subjects

P Swart

Publications and source records attributed to P Swart.

At least 19 recordsLinked to original sources

PubMed2005

A robust approach to studying the adsorption of Pluronic F108 on nonporous membranes.

A method for poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) desorption from synthetic nonporous polymeric membranes, using hexane:isopropanol treatment and subsequent colorimetric quantification, is described. The polymers polysulfone, poly(vinyldiene fluoride), and poly(ether imide) were used to fabricate solid adsorption matrices. The desorbed Pluronic F108 forms a color complex with ammonium ferrothiocyanate (NH4FeSCN) and is based on partitioning of a chromophore present in NH4FeSCN from an aqueous phase to a chloroform phase in the presence of Pluronic. The protocols for Pluronic desorption and detection are simple, sensitive, inexpensive, rapid, and reproducible over a wide range of Pluronic coating concentrations and membrane surface chemistries. A linear response over the concentration range from 3 to 130 microg ml(-1) is obtained. The adsorption isotherms for flat sheet membranes are also described and the Langmuir equation provides the best fit for the adsorption data obtained within the concentration range studied. The absence of any significant interference from certain proteins, vitamins, carbohydrates, plasma, and halogenated derivatives makes the assay equally suitable for the estimation of Pluronic F108 in the attendant Pluronic conjugates or in biomedical applications. Using nonporous hollow fine fibers and capillary membranes as model curved substrates we were also able to correlate an increase in the radius of curvature with a corresponding increase in the surface interfacial adsorption of Pluronic F108.

Adsorption↗

PubMed2004

Ex vivo modulation of chemical-induced mutagenesis by subcellular liver fractions of rats treated with rooibos (Aspalathus linearis) tea, honeybush (Cyclopia intermedia) tea, as well as green and black (Camellia sinensis) teas.

Male Fischer rats were given unprocessed (not oxidized) and processed (oxidized) rooibos and honeybush teas as well as green and black teas as a sole source of drinking fluid for 10 weeks, and sub cellular liver fractions were prepared. Cytosolic fractions of rats consuming the unprocessed herbal teas, green and black teas significantly (P < 0.05) protected against 2-acetylaminofluorene (2-AAF)-induced mutagenesis in the Salmonella mutagenicity test with strain TA 98, using Aroclor 1254-induced microsomes. A marginal or no protection was obtained with the processed herbal teas. The mutagenic response of aflatoxin B1 (AFB1) against Salmonella strain TA 100 was significantly (P < 0.05) inhibited by cytosolic fractions from rats treated with processed and unprocessed herbal teas, while no effect was obtained with the green and black teas. Microsomal fractions prepared from livers of rats treated with both the processed and unprocessed rooibos teas and the unprocessed honeybush tea, significantly (P < 0.05) reduced the activation of AFB1 while no protection was observed against 2-AAF-induced mutagenesis. In contrast, microsomal fractions from rats treated with the green, black and unprocessed honeybush teas significantly (P < 0.05) enhanced the mutagenic response of 2-AAF. None of the tea treatments significantly affected the concentration of the microsomal liver cytochrome P450.

Animals↗

PubMed2004

The effect of Sutherlandia frutescens on steroidogenesis: confirming indigenous wisdom.

Sutherlandia frutescens (Cancer bush), a Southern African indigenous plant, is traditionally used to treat stress related maladies linked to the endocrine system. Extracts of the shrub were used to investigate the claimed stress-relieving properties of the shrub. Dysregulation of the stress response is associated with elevated glucocorticoid levels. A model of chronic intermittent immobilization stress was investigated in 40 adult male Wistar rats to determine the effect of Sutherlandia. Immobilization stress resulted in increased corticosterone levels in the control group while rats receiving Sutherlandia extract showed significantly decreased corticosterone levels (P < 0.005). Since the biosynthesis of glucocorticoids in the adrenals is catalyzed by the cytochrome P450-dependent enzymes, the influence of Sutherlandia extracts on adrenal steroidogenesis was determined in ovine adrenocortical microsomes and mitochondria, using spectral binding and enzyme conversion assays. Water extracts showed inhibition of substrate binding to cytochrome P450 21-hydroxylase (CYP21) by 38% and cytochrome P450 11beta-hydroxylase (CYP11B1) by 60%. The conversion of progesterone and pregnenolone was inhibited by 34% and 30%, respectively. Subsequent extractions with chloroform and methanol showed inhibition of substrate binding and conversion with hydrophobic compounds exhibiting a greater inhibitory effect on deoxycorticosterone binding to CYP11B1 (30%) and on progesterone binding to CYP21 (50%). The inhibition of binding of pregnenolone to CYP17 by the chloroform extract was 62%, with negligible inhibition by the methanol extract. The chloroform extract showed a greater inhibitory effect than the methanol extract on progesterone and pregnenolone metabolism (20%-50%).

Adrenal Cortex↗

PubMed2004

The influence of the amino acid substitution I98K on the catalytic activity of baboon cytochrome P450 side-chain cleavage (CYP11A1).

Cytochrome P450 side-chain cleavage (CYP11A1) catalyzes the first and "rate-limiting" step in steroidogenesis, the conversion of cholesterol to pregnenolone. In an effort to gain further insight into the structure/function relationship of this key enzyme, CYP11A1 was characterized in the Cape baboon (Papio ursinus), a species closely related to humans. Baboon cDNA was isolated from adrenal tissue and direct sequence analysis showed mature baboon and human CYP11A1 share 98% deduced amino acid homology. The cDNA was subsequently amplified and two recombinant constructs, CYP11A1a and CYP11A1b, were cloned. Sequence analyses of the constructs revealed four amino acid substitutions. The constructs were expressed in nonsteroidogenic mammalian COS-1 cells with 25-hydroxycholesterol as substrate. Apparent Km values of 1.62 and 4.53 microM were determined for CYP11A1a and CYP11A1b, respectively. Homology modeling revealed that the lower substrate affinity of CYP11B1b could be attributed to an I98K substitution, which lies between the B and C helices, providing further evidence for the importance of this domain in the catalytic activity of CYP11A1.

Amino Acid Substitution↗

PubMed2002

Baboon CYP11B1: the localization and catalytic activity in baboon adrenal tissue.

A third gene encoding baboon CYP11B1 was isolated and was shown to catalyze only the metabolism of deoxycorticosterone (DOC) to corticosterone. The investigation into the localization of CYP11B1 in the baboon adrenal tissue, using in situ hybridization, showed that mRNA transcripts were predominantly present in the zona reticularis (ZR) and zona fasciculata (ZF). Signal was also observed in the zona glomerulosa (ZG) and scattered within the medulla. Immunohistochemical studies, using rabbit anti-sheep CYP11B1 IgG, indicated that CYP11B1 was expressed only in the zona fasciculata, zona reticularis and in the medulla. CYP11B1 was not detected in the zona glomerulosa. Subsequent Western Blot investigations into the presence of CYP11B1 in baboon adrenal cortex and medullary homogenates indicated CYP11B1 as a single band in the cortex and as two distinct bands in the medulla. CYP11A was present only in the baboon adrenal cortex. The metabolism of deoxycorticosterone and corticosterone was subsequently investigated in the baboon adrenal cortex and medulla. In cortex homogenates, deoxycorticosterone was converted to corticosterone, and neither 18-hydroxycorticosterone nor aldosterone was detected. In medulla homogenates, however, corticosterone was metabolized to aldosterone, as confirmed by APcI-MS.

Adrenal Glands↗

PubMed2001

Sequence specific stabilization of a linear analog of the antifungal lipopeptide iturin A2 by sodium during low energy electrospray ionization mass spectrometry conditions.

The structures and stability of sodiated species of 8-Beta, a linear lipopeptide analog (beta-aminotetradecanoyl-NYNQPNS) of the antifungal peptide iturin A2, were evaluated by electrospray ionization mass spectrometry (ESI-MS). Association of the lipopeptide, 8-Beta, with sodium afforded protection from fragmentation at high cone voltages and increasing collision energy conditions in the ESI-MS. The order of decreasing stability was found as 8-Beta 1Na > 8-Beta 2Na > 8-Beta 3Na > 8-Beta. Substantial differences were found between fragmentation patterns of the free and sodiated molecular species. Breakage of the N-terminal peptide bond of L-Pro generated the major product ions of the free 8-Beta parent ion. Impaired fragmentation of the sodium adducts of 8-Beta, indicated that this bond is protected by sodium complexation. Fragmentation patterns of the sodiated lipopeptide further revealed two specific binding sites for a nonsolvated sodium ion within the two type II beta-turn sequences (beta-aminotetradecanoyl-NYN and QPNS) of the natural iturin A2. It is proposed that specific interaction with sodium takes place with most of the peptide bond oxygens in these turns, and with the Gln sidechain. This interaction leads to stabilized structures in which the peptide backbone, specifically the peptide bonds in which L-Pro participates, is protected against low-energy fragmentation during ESI-MS.

Anti-Bacterial Agents↗

PubMed2000

Membrane Pretreatment: A Method for Reducing Fouling by Natural Organic Matter.

The prevention of fouling of polysulphone ultrafiltration membranes, used for the purification of natural brown water, was investigated by pretreating the membranes prior to filtration. Polysulfone membranes were pretreated by commercial nonionic surfactants Triton X-100 and Pluronic F108. Specific characterisation techniques, developed by Maartens et al. (1998) and Jucker and Clarke, (1994), were used to determine and compare the effects induced by the adsorption of natural organic matter on the permeability of untreated as well as surfactant treated capillary ultrafiltration membranes. The extent of foulant adsorption and the quality of the resultant permeate solutions were determined by ultraviolet visible-light spectroscopy. The findings of this investigation provides information of importance for the operation of future natural brown water ultrafiltration plants. Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

Influence of an aziridine precursor on the in vitro binding parameters of rat and ovine corticosteroid-binding globulin (CBG).

Aziridines are highly reactive alkylating compounds used in cancer treatment. Salsola tuberculatiformis Botsch., which causes prolonged gestation in sheep and contraception in rats, contains a very labile hydroxy-phenylaziridine or its precursor. A less labile analogue, 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (Compound I), was synthesized and has been shown to be contraceptive in rats and to be stabilized by corticosteroid-binding globulin (CBG). The current study compared the binding parameters of rat and ovine CBG and evaluated the effect of the aziridine precursor, Compound I, on these parameters. Kd and Bmax values of 0.646 and 578 nM for corticosterone binding to rat CBG and 0.577 and 19.8 nM for cortisol binding to sheep CBG, respectively, were measured. In competitive binding studies with rat plasma, Ki values of 3.48 nM, 0.856 nM, 22.2 nM, 722 microM, and > 1,000,000 microM for cortisol, corticosterone, progesterone, Compound I, and synephrine (Compound II), respectively, were found, while in sheep plasma the values were 0.409 nM, 1.78 nM, 5.28 nM, 594 microM, and > 1,000,000 microM, respectively. Concentrations of Compound I equivalent to an effective pharmacological dose resulted in a significant (P < 0.01) decrease in CBG bound corticosterone and a significant (P < 0.01) increase in free corticosterone in rat plasma. In sheep, a similar effect was observed with cortisol. Progesterone binding, however, did not appear to be affected significantly by Compound I in either rat or sheep plasma. Compound I was found to be a competitive inhibitor of glucocorticoid binding to CBG. These results suggest that binding of Compound I to CBG with concomitant displacement of endogenous glucocorticoids, but not progesterone, may be part of the mechanism of action of these phenylaziridine compounds.

Acetates↗

PubMed2000

The interaction of analogues of the antimicrobial lipopeptide, iturin A2, with alkali metal ions.

Electrospray mass spectrometry was employed as a tool in this first study on the molecular interaction between the alkali metal ions and antifungal lipopeptide iturin A, and some analogues. Cationisation by sodium and signal intensity of lipopeptide species depended on sodium concentration, but was independent of sample solvent, carrier solvent polarity and sample pH between 4 and 11. 8-Beta, a linear analogue of iturin A2 (8-Beta; beta-aminotetradecanoyl-NYNQPNS), and its shorter linear lipopeptide analogues, associated either one or two alkali metal cations, while the N-->C cyclic peptides associated with only one cation. The chirality of the beta-NC14 residue had a limited influence on the cationisation. It was observed that 8-Beta contained at least four interaction sites for a cation of which two, the C-terminal carboxylate and the side-chain of tyrosine, can take part in ionic interaction with a cation. It is proposed that the remaining two interaction centres of alkali metal ions are within the two type II beta-turns found in conformation of natural iturin A. This was corroborated by the diminished capacity of the shorter peptides, in which one of the beta-turns was eliminated to bind a second larger cation. All the lipopeptides showed the same order of alkali metal ion selectivity: Na+ > K+ > Rb+. These results indicated a size limitation in the interaction cavity or cavities. The absence of, or observation of only low abundance, di-cationised complexes of cyclic peptides the indicated association of the cation in the interior of the peptide ring. It is thus hypothesised that alkali metal ions can bind in one of the two beta-turns in the natural iturin A molecule.

Amino Acid Sequence↗

PubMed2000

Investigation of the primary cause of hypoadrenocorticism in South African angora goats (Capra aegagrus): a comparison with Boer goats (Capra hircus) and Merino sheep (Ovis aries).

Our objective was to identify the primary site of the reduced adrenal function in South African Angora goats (Capra aegagrus) that causes a decrease in cortisol production and leads to severe losses of Angora goats during cold spells. Angora goats, Boer goats (Capra hircus), and Merino sheep (Ovis aries) were assigned to three intravenous treatments: 1) insulin, 2) corticotropin-releasing factor (CRF), and 3) ACTH. Blood cortisol concentrations were determined over a 90-min period to determine any differences in the response of the experimental animals to these treatments. For both the insulin and ACTH treatments, cortisol concentrations were less in Angora goats than in the other experimental animals. The adrenal gland was subsequently investigated as a possible cause for the observed hypoadrenocorticism. Primary adrenal cell cultures were prepared from these species, subjected to different treatments, and the cortisol production determined. Upon pregnenolone (PREG) addition, all the experimental animals' cortisol production increased significantly, with the production in Boer goats higher (P<.01) when compared with that in the other species. The stimulation of cortisol biosynthesis by ACTH was only obtained for Boer goats and Merino sheep. The stimulation of cortisol production by forskolin and cholera toxin were compared with ACTH, and, for Angora goats, only cholera toxin caused a significant increase in cortisol production. For Boer goats, no difference (P>.05) between the PREG, ACTH, forskolin, or cholera toxin treatments were observed. The Merino adrenal cells were increasingly stimulated in the following order: PREG, ACTH, forskolin, and cholera toxin (forskolin and cholera toxin stimulated cortisol production to the same extent). This investigation of the hypothalamic-pituitary-adrenocortical axis, therefore, identified the adrenal gland as the primary site of the Angora's hypoadrenocorticism.

Adrenal Insufficiency↗

PubMed2000

Adrenal function in Angora goats: a comparative study of adrenal steroidogenesis in Angora goats, Boer goats, and Merino sheep.

South African Angora goats (Capra aegagrus) are susceptible to stress conditions, possibly due to adrenal cortex malfunction. Selection for mohair production may reduce adrenal function and decrease cortisol production. Secretion of cortisol by the adrenal cortex is essential for the induction of several gluconeogenic enzymes that enable animals to survive stressful conditions, and adrenocortical insufficiency, therefore, precipitates a vulnerability to stress. In this study, Angora goats were compared with two breeds generally accepted as hardy, Boer goats (Capra hircus) and Merino sheep (Ovis aries). Adrenal steroidogenesis was studied using subcellular fractions prepared from the adrenal glands of freshly slaughtered animals. Adrenal microsomes and mitochondria were incubated with the relevant steroid substrates, and products were analyzed and quantified with TLC, HPLC, or RIA. Subsequently, the activity of individual enzymes involved in this pathway were further investigated. The cytochrome P450 content in the preparations was also compared. The results from these studies indicated that the activity of the cytochrome P450c17 enzyme in Angora goats differed (P < .01) from that of the other species investigated. This difference may contribute to the cause of the observed hypoadrenocorticism in Angora goats.

Adrenal Glands↗

PubMed2000

Inhibition of cytochrome P450c11 by biogenic amines and an aziridine precursor, 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride.

The interaction of several biogenic amines and Compound A (2-(4-acetoxyphenyl)-2-chloro- N-methyl-ethylammonium chloride), an analogue of the active substance in a HPLC fraction isolated from the shrub, Salsola tuberculatiformis Botsch., with cytochrome P450c11 was investigated. Noradrenaline, octopamine and Compound A inhibited the type I DOC induced difference spectrum of P450c11 and elicited a type II difference spectrum when added alone. The Ks-values for noradrenaline, octopamine, and Compound A were 0.8 mM, 0.16 mM and 0.36 mM, respectively. Dopamine, adrenaline and synephrine did not interact with, or inhibit, P450c11. Further investigation of Compound A indicated that it is a mixed inhibitor of sheep P450c11 with a stronger competitive (Kic = 106-110 microM) than uncompetitive (Kiu = 667-737 microM) element, and that it inhibits the conversion of deoxycorticosterone to corticosterone by human 11beta-hydroxylase and aldosterone synthase with EC50 values of 97 microM and 190 microM, respectively, in fetal calf serum.

Acetates↗

PubMed2000

Baboon cytochrome P450c11 is encoded by more than one gene.

Two P450c11 genes, sharing 99% homology, were cloned from Cape baboon adrenal tissue using RT-PCR. The cDNAs showed 96% and 94.6% sequence identity to human P450c11 and aldosterone synthase, respectively. One of the gene sequences contained a termination codon in exon one. The cloned cDNAs were expressed in COS 1 cells and the metabolism of deoxycorticosterone to corticosterone investigated. The expressed enzyme exhibited 1 beta hydroxylase activity, but no aldosterone synthase activity.

Adrenal Glands↗

PubMed1999

Salsola tuberculatiformis Botschantzev and an aziridine precursor analog mediate the in vivo increase in free corticosterone and decrease in corticosteroid-binding globulin in female Wistar rats.

Salsola tuberculatiformis Botschantzev causes prolonged gestation in sheep and contraception in rats. An active fraction isolated from the shrub, containing a highly labile hydoxyphenyl aziridine or precursor, and a more stable analog, compound A, inhibits sheep adrenal cytochrome P450c11. In addition, compound A has been shown to bind to and be stabilized by corticosteroid-binding globulin (CBG). Binding may result in concomitant displacement of endogenous steroids, which could contribute to the biological effects of these compounds. The present study was undertaken to establish which mechanism would predominate in female rats. Compound A significantly (P < 0.01) displaced glucocorticoids, but not progesterone, from rat CBG in vitro, whereas in vivo the percentage of free plasma corticosterone in both S. tuberculatiformis (P < 0.05)- and compound A (P < 0.01)-treated rats was also significantly higher due to displacement from CBG. In addition, both ACTH and CBG concentrations were significantly (P < 0.05) lower than control values. The levels of the gonadotropins were also reduced during treatment, but only LH values significantly (P < 0.05) so. These results suggest that binding of the test substances to CBG in female rat plasma and concomitant displacement of endogenous corticosterone could be part of the contraceptive mechanism of S. tuberculatiformis and the aziridine precursor, compound A.

Acetates↗

PubMed1999

Prognostic significance of diagnostic laparoscopy for spontaneous fertility.

OBJECTIVE: To determine the prognostic significance of laparoscopy results for fertility outcome. STUDY DESIGN: Consecutive patients undergoing hysterosalpingography and laparoscopy for subfertility in our department between May 1985 and November 1987 were identified from medical records. The impact of tubal occlusion, hydrosalpinx and adhesions as detected at laparoscopy was studied. Kaplan-Meier curves for the occurrence of spontaneous intrauterine pregnancy were constructed for patients without tubal pathology, with mild tubal pathology (unilateral pathology or adhesions) and with severe tubal pathology (bilateral pathology). Fecundity rate ratios (FRR) were calculated to express the association between findings at laparoscopy and the occurrence of spontaneous intrauterine pregnancy. RESULTS: Of the 200 cases that could be analyzed, 129 (65%) showed no tubal occlusion on laparoscopy, 40 (20%) had unilateral tubal occlusion, and 31 (15%) had bilateral tubal occlusion. Unilateral hydrosalpinx was present in 13 (7%) patients, whereas 19 (10%) patients had bilateral hydrosalpinx. Adjusted FRRs were 0.65 and 0.20 for unilateral and bilateral tubal occlusion, and 0.46 and 0.32 for unilateral and bilateral hydrosalpinx. Peritubal adhesions were detected in 43% of patients and seemed to have no prognostic significance. CONCLUSION: Severe tubal pathology detected at laparoscopy affects fertility prospects strongly. However, since spontaneous intrauterine pregnancies occurred even in patients with bilateral tubal occlusion at laparoscopy, this technique should not be considered the gold standard in the diagnosis of tubal infertility.

Adult↗

PubMed1997

Mechanism for the stabilization in vivo of the aziridine precursor --(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride by serum proteins.

Oral and intraperitoneal administration of 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (Compound A), an analogue of phenyl aziridine precursors that occur in the shrub Salsola tuberculatiformis Botsch, had a contraceptive effect on female Wistar rats with a concomitant decrease in total body, uterus, and every mass and an increase in abronal mass. Compound A elicited a Type II difference spectrum and inhibited the Type I deoxycorticosterone (DOC) induced difference spectrum of sheep adrenal cytochrome P450c11 in a manner similar to that of S2, a biologically active fraction isolated from S. tuberculatiformis. The effects of Compound A on the spectral properties of P450c11 were diminished with time in PBS. Electrospray mass spectrometry (ES-MS) indicated that the rate of cyclization of Compound A to the corresponding aziridine followed a time course similar to the attenuation of cytochrome P450c11 inhibition. It was concluded that the aziridine precursor. Compound A, rather than aziridine itself, was the inhibiting agent of sheep adrenal P450c11. Addition of sheep and rat plasma prevented the attenuation of the effect of Compound A on the spectral properties of cytochrome P450c11. Subsequent ES-MS analysis indicated that Compound A was stabilized in plasma by sex hormone binding globulin and corticosteroid binding globulin. These results suggest a mechanism whereby natural plant products, which are highly reactive and unstable in vitro, can be stabilized by binding to plasma proteins, and so remain biologically active in vivo.

Acetates↗

PubMed1997

Is hysterosalpingography an important tool in predicting fertility outcome?

OBJECTIVE: To determine the prognostic significance of hysterosalpingography (HSG) for fertility outcome. DESIGN: Retrospective cohort study. SETTING: Infertility department of an academic hospital. PATIENT(S): Consecutive patients undergoing HSG for subfertility between May 1985 and November 1987. INTERVENTION(S): Hysterosalpingography. MAIN OUTCOME MEASURE(S): Follow-up ended when pregnancy or tubal surgery occurred or at the day of last contact. Kaplan-Meier curves for the occurrence of spontaneous intrauterine pregnancy (IUP) were constructed for a normal HSG, a HSG with a one-sided abnormality, and a HSG with a two-sided abnormality. Fecundity rate ratios were calculated to express the association between HSG findings and the occurrence of spontaneous IUP. RESULT(S): Of 359 patients that were analyzed, 231 (64%) showed no tubal pathology on HSG, 67 (19%) had a one-sided tubal pathology, and 61 (17%) had a two-sided tubal pathology. The adjusted fecundity rate ratios were 0.81 (95% confidence interval 0.47 to 1.4) for a one-sided pathology and 0.30 (95% confidence interval 0.13 to 0.71) for a two-sided pathology. Correction for informative censoring and sensitivity analysis did not alter these results. CONCLUSION(S): One-sided tubal pathology detected on HSG has limited prognostic significance, whereas two-sided tubal pathology detected on HSG reduces fertility prospects considerably.

Adult↗

PubMed1996

Sequence of the 11 beta-hydroxylase gene from the Cape baboon (Papio ursinus).

We investigated the nucleotide sequence of the steroid 11 beta-hydroxylase gene (CYP11B1) from the Cape baboon (Papio ursinus). Six primers, previously used in studies on human CYP11B1, were utilised to amplify three overlapping fragments (A, B and C) of the baboon CYP11B1 by the polymerase chain reaction (PCR). Sequence analysis of the three fragments yielded the sequence of all the exons of baboon CYP11B1. The open reading frame of 1509 bases shows 57 nucleotide exchanges when compared to the human resulting in 18 amino acid substitutions. For eight of these exchanges we found the amino acid which is common for human aldosterone synthase at the corresponding position. Most of the remaining 10 amino acid substitutions were conservative. Eight of the substitutions were located in the first four exons with a cluster in the second half of exon 3. One substitution was in exon 5 (F280L) and the 10th was C494F at the end of the protein.

Animals↗