Genetic comparison between Spirometra erinacei and S. mansonoides using PCR-RFLP analysis.

The only observed morphological difference between Spirometra erinacei and S. mansonoides is the uterine shape of the mature proglottid. Two species of worms are thought to be evolutionarily closely related. Biomolecular comparison of the two worms by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was conducted to observe the genetic distance. The 28s rDNA, mitochondrial cytochrome c oxidase subunit I (mCOI), and ribosomal internal transcribed spacer 1 (ITS1) fragments were obtained from the worms by PCR. The PCR products were cleaved by 5 four-base pair restriction enzyme combinations (Msp I, Hae III, Alu I, Cfo I, Rsa I), electrophoresed and analyzed with PAUP 3.1.1. The fragment patterns of 28S rDNA and ITS1 demonstrated that two worms were in identical systematic tree with bootstrap number 94 and 100, respectively. As for mCOI, bootstrap number was 74 in a different tree. Above results are indicative of recent common ancestry between S. erinacei and S. mansonoides.

Comparison of isozyme patterns between Spirometra erinacei and Spirometra mansonoides by isoelectric focusing.

No differences were observed in the isozyme patterns of 4 enzymes examined between fresh samples stored at -80 C and samples stored at room temperature for 10 days after lyophilization, which supports the validity of comparing lyophilized samples to fresh frozen tissue. Mature proglottids as well as plerocercoids of Spirometra erinacei from Japan and Australia were indistinguishable by comparison of isozyme patterns after isoelectric focusing. The isozyme patterns of acid phosphatase, glucosephosphate isomerase (GPI), and mannosephosphate isomerase from plerocercoids of Spirometra mansonoides were distinctly different from those of plerocercoids of S. erinacei. The adenylate kinase isozyme patterns of the mature proglottids of S. mansonoides were also distinctly different from those of the mature proglottids and the plerocercoids of S. erinacei. The GPI isozyme pattern of the mature proglottids of S. mansonoides was also distinguishable from the GPI patterns of those of S. erinacei. These electrophoretic data suggest that the S. erinacei from Japan and Australia are closely related, if not identical, but that S. mansonoides is genetically distinct from S. erinacei.

Molecular characterization of the Spirometra mansonoides genome: renaturation kinetics, methylation, and hybridization to human cDNA probes.

High molecular weight DNA from pleroceroid larvae of the tapeworm Spirometra mansonoides was purified from isolated nuclei by conventional techniques. The DNA so isolated has a melting temperature (Tm) of 87 degrees C and a guanine plus cytosine (G/C) content of 44%. 5-Methyl cytosine could not be detected in plerocercoid DNA by HPLC analysis of DNA hydrolysates, by radiolabeling 5'-termini of MspI digests with polynucleotide kinase, or by comparing restriction patterns generated by MspI and HpaII. Renaturation kinetics demonstrated that the genome of S. mansonoides contains repetitive as well as single copy sequences and has a genome size estimated at approx. 1.6 X 10(9) bp. Hybridization was carried out between plerocercoid DNA and cDNAs for human beta-actin, alpha-tubulin and growth hormone (hGH). Rationale for this analysis was based on known homologies among actin and tubulin genes in numerous species and on apparent similarities between hGH and a plerocercoid growth factor that may be reflected in similar DNA sequence. Scanning densitometry of dot blots demonstrated that the hGH probe annealed to the same extent at low stringency (1 M NaCl, 55 degrees C) to DNA from plerocercoids, rat liver and chicken erythrocytes; but this interaction was less than to DNA from human lymphocytes, calf thymus and mouse skin. Similar results were obtained when restriction endonuclease digests of these DNAs were analyzed by Southern transfer. Little or no hybridization of the growth hormone probe to plerocercoid DNA was evident at higher stringency (1 M NaCl, 65 degrees C). In contrast, human tubulin and actin probes showed extensive hybridization to pleroceroid restriction fragments under the high stringency conditions.

The growth factor from plerocercoid larvae of the tapeworm, Spirometra mansonoides, stimulates growth but is not diabetogenic.

A factor produced by plerocercoids of the tapeworm Spirometra mansonoides is similar to human growth hormone (hGH) in that it stimulates body growth, binds to hGH receptors, cross-reacts with anti-hGH antibodies, and has lactogenic and insulin-like activities. The purpose of this study was to determine whether plerocercoid growth factor (PGF) is similar to hGH in expressing diabetogenic activity in the genetically obese (ob/ob) mouse. To determine an effective dose for use in the obese mice, the ability of daily injections of PGF to stimulate growth of phenotypically normal mice of the same strain was assessed in a 10-day weight gain assay. Injections of PGF stimulated a dose-dependent weight gain (r = 0.83) and 25 ng eq/day of PGF stimulated a response not significantly different from that produced by 100 micrograms of bovine growth hormone/day. Diabetogenicity was assessed using fasting blood glucose and glucose tolerance tests in obese mice that had been injected for 3 days with saline, hGH, or PGF. Human growth hormone caused a significant increase (P less than 0.005) in fasting blood glucose and glucose tolerance of the obese mice was impaired (P less than 0.01). All of the doses of PGF used to test diabetogenicity in the obese mice were at least twice that required to stimulate a maximal growth response in normal mice, yet none of the doses of PGF increased fasting blood glucose or decreased glucose tolerance. These results show that PGF was a potent growth stimulant but was not diabetogenic.

In vitro insulin-like actions of the growth factor from the tapeworm, Spirometra mansonoides.

In vitro actions of purified plerocercoid growth factor (PGF) were compared with those of insulin and human growth hormone (hGH) in adipose tissue from normal male rats. Insulin-like effects were measured by the ability of PGF, insulin, or hGH to stimulate oxidation of [U-14C]glucose to 14CO2, to stimulate lipogenesis, and to inhibit epinephrine-induced lipolysis. PGF and insulin stimulated significant increases in glucose oxidation and lipogenesis in adipose tissue that had not been preincubated as well as in tissue that had been preincubated. hGH stimulated insulin-like effects only in tissue that had been preincubated for 3 hr. Insulin, hGH, and PGF inhibited epinephrine-induced lipolysis of preincubated (3 hr) adipose tissue. hGH produced a dramatic lipolytic response in tissue freshly removed from normal rats but no dose of PGF was lipolytic. PGF did not displace 125I-insulin from its receptors on adipocytes but did competitively inhibit 125I-hGH binding to adipocytes. These results suggest that PGF has direct insulin-like actions which are initiated by binding a GH receptor, but PGF had no anti-insulin action and the insulin-like activity of PGF was unaffected by refractoriness of adipose tissue to GH.

The growth hormone-like factor produced by the tapeworm Spirometra mansonoides specifically binds receptors on cultured human lymphocytes.

Plerocercoid larvae of the tapeworm Spirometra mansonoides produce a factor with activities similar to those of growth hormone (GH). Highly selective receptors for GH have been described on cultured human lymphocytes (IM-9 cells) and these cells have been used as a model of binding essentially restricted to human GH (hGH). We compared the displacement of [125I]hGH by hGH and partially purified plerocercoid growth factor (PGF) in assays using rabbit hepatic membranes and IM-9 cells. PGF displaced [125I]hGH from both rabbit hepatic membranes and IM-9 cells in a dose-dependent manner (r greater than 0.98). These results show that PGF specifically binds to hGH receptors on human IM-9 cells and suggest the possibility that PGF will have somatotropic activity in humans.

Use of receptor affinity chromatography in purification of the growth hormone-like factor produced by plerocercoids of the tapeworm Spirometra mansonoides.

The plerocercoid stage of the tapeworm Spirometra mansonoides produces a functional analog of human growth hormone (hGH). Among the similarities between plerocercoid growth factor (PGF) and hGH is competition for the same receptors on rabbit liver membranes. To take advantage of this characteristic in a purification scheme for PGF, rabbit liver microsomes were solubilized in Triton X-100 and the hGH receptors were purified over an hGH affinity column. The purified receptors from six rabbit livers were coupled to Affi-Gel-10 to create a receptor affinity column which was used to purify PGF. Chromatography of crude PGF over the receptor column resulted in a 1044 fold increase in specific activity. SDS-PAGE in the presence of 2-mercaptoethanol showed that the affinity-purified PGF contained three protein bands with apparent Mrs of 27.5 K, 22 K, and 16.7 K. Injections of the partially-purified PGF into hypophysectomized rats produced a dose-dependent growth response and 400 ng eq of PGF each day for 10 days stimulated a growth response not significantly different from that produced by 250 micrograms of bovine GH each day. Receptor affinity chromatography was an effective method to purify small amounts of PGF in a single step with negligible loss of biological activity.

Augmentation of postresection mucosal hyperplasia by plerocercoid growth factor (PGF). Analog of human growth hormone.

Postresection villus hyperplasia is a major compensatory mechanism in the short-bowel patient. Substances capable of augmenting postresection mucosal hyperplasia could have therapeutic implications. Human growth hormone (hGH) and human growth hormone releasing factor (hGHRF) stimulate growth of the gastrointestinal tract; however, the diabetogenic actions of growth hormone limit its usefulness in clinical practice. Plerocercoid larvae of the tapeworm Spirometra mansonoides produce an analog of hGH void of diabetogenic side effects. We assessed effects of plerocercoid growth factor (PGF) on mucosal adaptation following 70% proximal jejunoileal resection in young rats. Mucosal weight, DNA, protein, and total sucrase activity per centimeter of bowel were increased in resected PGF-treated animals compared to resected controls. We conclude PGF augments intrinsic postresection mucosal hyperplasia following extensive intestinal resection.

Antihuman growth hormone (GH) antibodies cross-react with the GH-like factor from plerocercoids of the tapeworm Spirometra mansonoides.

A factor produced by the plerocercoid stage of S. mansonoides mimics some, but not all, of the actions reported for hGH. The biological actions of plerocercoid growth factor (PGF) suggest structural similarity to human GH (hGH). Plerocercoid membranes were solubilized, and PGF was purified more than 1000-fold by hGH receptor affinity chromatography. The ability of purified PGF to displace [125I]hGH from monoclonal antibodies specific for four distinct nonoverlapping antigenic determinants of hGH and from an anti-hGH polyclonal antibody was tested in liquid phase RIA. All of the hGH antibodies cross-reacted with PGF, with potencies ranging from more than 60% to less than 1% that of the hGH standard. Of the four major epitopes of hGH defined by the monoclonal antibodies used in this study, only one is not represented to a significant extent in PGF. The epitope of hGH that is only marginally present in PGF is highly conformationally dependent, and a minor difference in the structure of PGF (compared to hGH) could result in a significant conformational change. The dramatic cross-reactivity between anti-hGH antibodies and PGF suggests that the similarities in biological activities between these two substances are based in significant molecular homology.

Plerocercoid growth factor: a homologue of human growth hormone.

Plerocercoids of the tapeworm, Spirometra mansonoides, produce a factor with characteristics similar to those of mammalian growth hormone (GH). Plerocercoid growth factor (PGF) stimulates growth and mimics other actions of GH but does not possess the anti-insulin/diabetogenic activities intrinsic to mammalian growth hormones. Duplication of activities unique to human GH, chemical and physical similarities, plus crossreactivity with strictly specific anti-hGH monoclonal antibodies, underlie the hypothesis that S. mansonoides has obtained and expresses a human gene for GH. In this article, Kirk Phares discusses the similarities between the two hormones.

Insulin-like effects in the rat of the purified growth factor from Spirometra mansonoides plerocercoids.

The acute effects of injections of the human growth hormone-like factor purified from plerocercoids of the tapeworm Spirometra mansonoides on carbohydrate, lipid, and protein metabolisms were determined in intact rats. Male rats were injected ip with saline, insulin, or various doses of partially purified PGF. The rats injected with insulin had significantly reduced serum glucose concentrations but no dose of PGF caused a change in serum glucose levels. Insulin and PGF stimulated [14C]glucose and [14C]leucine oxidation to 14CO2 in adipose tissue and muscle and increased incorporation of both [14C]glucose carbons into lipids and [14C]leucine into protein in fat and muscle. The responses to PGF were dose-dependent and persisted after 3 hr of incubation in vitro. Injections of naloxone prior to injecting PGF to block the stress response did not prevent the stimulation of insulin-like responses by PGF. Therefore, PGF has intrinsic insulin-like activities in normal male rats.

Some biochemical effects of the growth hormone analogue produced by plerocercoids of the tapeworm Spirometra mansonoides on carbohydrate metabolism of adipose tissue from normal, diabetic, and hypophysectomized rats.

Plerocercoids of Spirometra mansonoides produce a functional analogue of mammalian growth hormone (GH). Plerocercoid growth factor (PGF) mimics the growth-promoting actions of GH, but has not been shown to duplicate all of the actions reported for GH. The purpose of this study was to determine the effects of plerocercoid infection (chronic PGF treatment) on glucose metabolism of adipose tissue and to compare the effects to those elicited by insulin and GH in intact, diabetic, and hypophysectomized male rats. Groups of rats were constantly exposed to PGF (via plerocercoid infection) or injected twice daily with bovine GH, insulin, or saline for 10 days. Basal oxidation rates of [U-14C]glucose to 14CO2 in adipose tissue segments were measured in vitro immediately after tissue removal. Other aliquots of adipose tissue were preincubated in hormone-free medium for 3 hr prior to testing the ability of the tissue to respond to insulin or human GH (hGH) added in vitro. Adipose tissue from PGF-treated intact and hypophysectomized rats had significantly elevated basal glucose oxidation rates, and the tissue was sensitive to further stimulation by insulin or hGH. The results obtained with intact and hypophysectomized rats were essentially the same, indicating that the effects of PGF were not due to suppression of endogenous GH. The basal glucose oxidation rate in adipose tissue from diabetic rats was stimulated (P less than 0.01) by PGF, but the tissue was not sensitive to insulin added in vitro. Furthermore, PGF had no effect on body growth or blood glucose concentrations of diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Regression of rat mammary tumors associated with suppressed growth hormone.

Serum growth hormone (GH) was suppressed in female rats bearing mammary tumors induced by 7, 12, dimethylbenz(a)anthracene (DMBA) or N-nitrosomethylurea(NMU). Serum GH was suppressed due to treatment with a human GH analog produced by the plerocercoid stage of the tapeworm Spirometra mansonoides. Rats treated with plerocercoid growth factor (PGF) via plerocercoid infection had accelerated growth rates despite marked reductions in GH levels. Approximately two-thirds of the mammary tumors induced by either DMBA or NMU regressed during three weeks of exposure to PGF while most of the control tumors continued to grow. The data support an important regulatory role for GH in growth of mammary tumors in rats.

Reduction of lactogenic receptors in female hamster liver due to the human growth hormone analog produced by plerocercoids of the tapeworm, Spirometra mansonoides.

The inductive effect of GH on hepatic lactogenic receptors is suspected of being due to a direct somatogenic action. Plerocercoid larvae of the tapeworm, Spriometra mansonoides, produce a factor that stimulates body growth, suppresses endogenous GH, and specifically displaces [125I]human (h) GH from hepatic receptors. Plerocercoid growth factor (PGF) mimics the growth-promoting actions of GH, but it has not been shown to duplicate all of the activities reported for GH. An important function of GH is its role in the maintenance of liver receptors for lactogenic hormones. This study was undertaken to determine if treatment of female hamsters with PGF would increase, decrease, or have no effect on liver receptors that bind hGH. Since hGH binds to somatogenic as well as lactogenic receptors, it was necessary to demonstrate the specificity of PGF's effects on [125I]hGH binding. PGF-treated (15 pleocercoids sc) hamsters had accelerated body growth, suppressed serum GH, and a marked reduction in [125I]hGH and [125I]ovine PRL binding to hepatic microsomes. Specific binding of [125I] bGH was unaltered by PGF treatment. The difference in [125I] hGH binding was due to a reduction in receptor number and not to receptor occupancy or reduced affinity. Serum GH was normalized after 10 days of estradiol benzoate (25 micrograms/day) injections, but the binding capacity for [125I]hGH of the PGF-treated group was less than half that of the control group. The fact that estrogen injections normalized serum GH, but not hGH binding, indicates that down-regulation of these receptors by PGF cannot be entirely explained on the basis of reduced levels of serum GH. The lack of any effect of PGF treatment on [125I]bGH binding suggests that the hepatic somatogenic receptors were not involved and that the reduction in receptors for [125I]hGH was associated with the lactogenic component of hGH.

Suppression of receptors for prolactin and estrogen in rat liver due to treatment with the growth hormone analogue produced by the tapeworm Spirometra mansonoides.

Somatogenic hormones play an important role in regulation of receptors for prolactin (PRL) and estrogen. Plerocercoids of the tapeworm, S. mansonoides produce a factor which mimics some, but not all of the actions reported for GH. Intact female rats were subjected to a constant infusion of plerocercoid growth factor (PGF) via a subcutaneous infection for two weeks to determine if PGF influences receptors for PRL, GH or estradiol. The rate of weight gain in the PGF-treated rats was accelerated in spite of a marked reduction in serum GH. Partially-purified PGF specifically displaced [125I]hGH from rat liver receptors but microsomes prepared from rats treated with PGF specifically bound significantly less [125I]hGH than microsomes from control rats. The reduction in [125I]hGH binding was not due to occupancy or to a change in affinity but to a suppression in receptor concentration. Scatchard analysis of [3H]estradiol binding in rat liver cytosols shows a 50% reduction in receptor concentration in the PGF-treated group. Specific binding of [3H]estradiol in anterior pituitary was also suppressed by PGF treatment.

Insulin-like effects of fatty acid synthesis in liver of hamsters infected with plerocercoids of the tapeworm, Spirometra mansonoides.

Elevated serum lipids are associated with infections of laboratory rodents with plerocercoids of Spirometra mansonoides. The effect of infection with these larval tapeworms on triglyceride degradation and hepatic de novo fatty acid synthesis was investigated in Syrian hamsters. Serum lipoprotein electrophoresis revealed a consistent elevation in very low density lipoproteins in the infected animals. Lipoprotein lipase activity was enhanced in the infected animals. After seven days of plerocercoid infection the activity of acetyl-CoA carboxylase (E.C. 6.4.1.2) was significantly elevated after 6, 12 and 18 hours of fasting. Fatty acid synthetase was significantly increased after 0, 6, 12, 18 and 24 hours of fasting. Therefore, a chronic insulin-like activity on lipid metabolism of hamsters is associated with plerocercoid infection.

Isolation and quantitation of immune complexes in diabetic syrian hamsters: a chronological study.

Immune complexes (IC) were isolated from the serum of streptozotocin (Sz)-induced diabetic hamsters by coprecipitation with an equine rheumatoid-C1q factor (RhC). The IC, comprised of an unknown antigen(s) and IgG, occurred at 2 distinct time intervals during the 12-week chronological study. Although all diabetic hamsters with hyperglycemia of 300 mg/dl had IC, there was no correlation between the occurrence or concentration of IC and the degree of hyperglycemia. The cycling nature of IC in the serum of diabetic hamsters suggests that the antigen component either fluctuates chronologically or that the IC contain different antigens that do not occur simultaneously.

Delay of puberty onset in males due to suppression of growth hormone.

Infection with Spirometra mansonoides at 22 days of age was associated with delayed sexual maturation in the male rat. Balanopreputial separation occurred at 46.7 +/- 0.8 days in controls and at 50.6 +/- 0.9 days in worm-treated rats (p less than 0.02). This was accompanied by a delay in the normal prepubertal testicular and seminal vesicle weight increases. LH remained normal but serum FSH was depressed as early as 25 days of age. The castration response in worm-treated rats was comparable to that of controls, when the FSH levels were normalized as percentages of resting level. The LH response to castration was diminished. The acute FSH and LH response to GnRH was normal and pituitary stores of gonadotrophin, as estimated by radioimmunoassayable concentrations of FSH and LH, were normal. It can be concluded that puberty onset in males is not linked to body weight gain pe se, and that the absence of endogenous GH secretion affects both sexual maturation and gonadotrophin secretion in the prepubertal period.

Spirometra mansonoides: effects of plerocercoid infection on glycogen deposition in rats.

The effects of infection with plerocercoids of Spirometra mansonoides on tissue glycogen deposition of rats was determined. Hypophysectomized rats infected for two days had higher liver glycogen concentrations than controls and this effect was greatest after one week. Elevated liver glycogen associated with plerocercoid infection was observed in fed animals both at the beginning and at the end of the light period as well as after an overnight fast. Glycogen phosphorylase (1,4 alpha D glucan: orthophosphate alpha glucosyltransferase EC 2.4.1.1.) was inhibited but glucose-6-phosphatase (EC 3.1.3.9) was unaffected in the livers of infected hypophysectomized rats. While this effect is similar to actions of both growth hormone and insulin, plerocercoid infection had no influence on glycogen of cardiac or skeletal muscle at any time. Plerocercoid infection had no effect on the glycogen concentration of any tissue of intact rats.