Characterization of hexamerin proteins and their mRNAs in the adult lubber grasshopper: The effects of nutrition and juvenile hormone on their levels.

The lubber grasshopper (Romalea microptera) has three major hemolymph proteins with apparent sizes on native PAGE of 90, 270, and 500kDa and subunit sizes (79, 81 and 82kDa respectively) determined by SDS-PAGE . Trypsin fragments from each protein band were sequenced, used to design degenerate primers to amplify core cDNA fragments, which were extended by 5' and 3' RACE. All three cDNAs were closely related to insect hexamerins, had an N-terminal signal sequence, and their transcripts were found solely in the fat body. Adult females fed an ad libitum diet had their highest hexamerin levels on day 18 when oocytes begin rapid growth. Hexamerin levels fell as oocytes reached their maximum length on day 30. Animals fed a restricted diet had their highest hexamerin levels on day 30 which then fell as oocytes reached their maximum length on day 36. Hexamerin mRNA levels were only modestly different for animals on the two diets, indicating that nutrition affected translation of the hexamerin mRNA. Allatectomized animals treated with juvenile hormone III (JH) or methoprene caused the appearance of vitellogenin in the hemolymph, but had no effect on hexamerin levels. Thus, JH does not appear to directly regulate hexamerin production.

Donor HLA-C genotype has a profound impact on the clinical outcome following liver transplantation.

Late allograft dysfunction is a significant problem following liver transplantation and its pathogenesis is uncertain. HLA-C is the major inhibitory ligand for killer immunoglobulin-like receptors (KIRs) that regulate the cytotoxic activity of natural killer (NK) cells. HLA-C alleles can be allocated into two groups, termed HLA-C1 and HLA-C2, based on their KIR specificity. HLA-C2 interactions are more inhibiting to NK cell activation. We studied the clinical importance of HLA-C genotype in a large liver transplant cohort and found that possession of at least one HLA-C2 allele by the donor allograft was associated with less histological evidence of chronic rejection and graft cirrhosis, a 16.2% reduction in graft loss (p = 0.003) (hazard ratio: 2.7, 95% CI 1.4-5.3) and a 13.6% improvement in patient survival (p = 0.01) (hazard ratio: 1.9, 95% CI 1.1-3.3) at 10 years. Transplantation of an HLA-C2 homozygous allograft led to a particularly striking 26.5% reduction in graft loss (p < 0.001) (hazard ratio: 7.2, 95% CI 2.2-23.0) at 10 years when compared to HLA-C1 homozygous allografts. Donor HLA-C genotype is therefore a major determinant of clinical outcome after liver transplantation and reveals the importance of NK cells in chronic rejection and graft cirrhosis. Modulation of HLA-C and KIR interactions represents an important novel approach to promote long-term graft and patient survival.

Roles of IGF-I and the estrogen, androgen and IGF-I receptors in estradiol-17beta- and trenbolone acetate-stimulated proliferation of cultured bovine satellite cells.

Although numerous studies have shown that both androgenic and estrogenic steroids increase rate and efficiency of muscle growth in steers, there is little consensus as to their mechanism of action. A combined estradiol 17beta (E2)/trenbolone acetate (TBA) implant causes a significant increase in muscle IGF-I mRNA and both E2 and TBA stimulate a significant increase in IGF-I mRNA level in bovine satellite cell (BSC) cultures in media containing 10% fetal bovine serum (FBS). Consequently, increased IGF-I expression may play a role in anabolic-steroid-enhanced muscle growth. However, even though treatment of cultured BSC with E2 or TBA in media containing 1% IGFBP-3-free swine serum (SS) results in increased proliferation there is no effect on IGF-I mRNA expression, suggesting that increased IGF-I expression may not be responsible for anabolic-steroid-enhanced BSC proliferation. To further examine the role of estrogen, androgen and IGF-I receptors and their respective ligands in E2- and TBA-stimulated BSC proliferation, we assessed the effects of specific inhibitors on E2- or TBA-stimulated proliferation of BSC. Both ICI 182 780 (an estrogen receptor blocker) and flutamide (an inhibitor of androgen receptor) suppressed (p<0.05) E2- and TBA-stimulated BSC proliferation, respectively. JB1 (a competitive inhibitor of IGF-I binding to type I IGF receptor) reduced (p<0.05) both E2- and TBA-stimulated proliferation in BSC cultures. Both the Raf-1/MAPK kinase (MEK)1/2/ERK1/2, and the phosphatidylinositol 3-kinase (PI3K)/Akt pathways play significant roles in the actions of IGF-I on proliferation and differentiation of myogenic cells. PD98059, an inhibitor of the MAPK pathway, and wortmannin, an inhibitor of the PI3K pathway, both suppressed (p<0.05) E2- and TBA-stimulated proliferation of cultured BSC. Our data suggest that IGF-I plays a role in E2- and TBA-stimulated proliferation of cultured BSC even in the absence of increased IGF-I expression.

Localization of insulin-like growth factor (IGFBP)-3 in cultured porcine embryonic myogenic cells before and after TGF-beta1 treatment.

Insulin-like growth factor binding protein (IGFBP)-3 binds IGFs with high affinity and affects their biological activity. IGFBP-3 that is not bound to IGF also affects cells via mechanisms involving binding to specific cell surface receptors and/or transport into the cell. IGFBP-3 is produced by porcine embryonic myogenic cell (PEMC) cultures. Additionally, IGFBP-3 facilitates the proliferation-suppressing actions of TGF-beta(1) and myostatin in PEMC cultures via mechanisms that do not involve IGF binding. Moreover, these mechanisms do not involve preventing myostatin or TGF-beta(1)-induced increases in phosphosmad2 or phosphosmad3 level. Consequently, the mechanism(s) by which IGFBP-3 facilitates the proliferation-suppressing actions of TGF-beta(1) and myostatin in PEMC is unclear. Since IGFBP-3 reportedly interacts with nuclear proteins that regulate transcription, TGF-beta(1) or myostatin-induced translocation of IGFBP-3 into the nucleus may facilitate the proliferation-suppressing actions of these cytokines. Here, we show that IGFBP-3 is localized in cells containing the muscle specific protein desmin, thus establishing the presence of this IGFBP in myogenic cells. IGFBP-3 is present in the cytoplasm of all myogenic cells and approximately 50% of the nuclei of proliferating PEMC. IGFBP-3 is also detectable in fused myotubes. IGFBP-3 suppresses IGF-I-stimulated differentiation of PEMC but has no affect on Long-R3-IGF-I-stimulated differentiation of PEMC. Treatment of PEMC for 24h with TGF-beta(1) (20 ng/ml) results in a 78% (p<0.01) increase in the number of nuclei that contain detectable IGFBP-3. These results suggest that translocation of IGFBP-3 into the nucleus of PEMC could play a role in mediating the proliferation-suppressing action of TGF-beta(1).

Effect of constitutive expression of porcine IGFBP-3 on proliferation and differentiation of L6 myogenic cells.

We have previously shown that exogenous recombinant porcine IGFBP-3 (rpIGFBP-3) suppresses proliferation and differentiation of L6 myogenic cells in an IGF-I-dependent manner and suppresses proliferation of L6 myogenic cells via an IGF-I-independent mechanism. In order to assess the effects of endogenously produced IGFBP-3, we have transfected L6 myogenic cells with a pEF6/V5 vector containing pIGFBP-3 cDNA under the control of the human elongation factor 1alpha (hEF-1alpha) promoter and with the empty vector. We have isolated a cell population that constitutively produces porcine IGFBP-3 (tL6 cells) and a stable mock transfected cell population containing the empty vector (mtL6 cells). Constitutive expression of IGFBP-3 slightly reduced the expression of IGFBP-5 but had no effect on IGFBP-4 production by L6 myogenic cells. Immunoneutralization of IGFBP-3 increased both IGF-I- and Long-R3-IGF-I-stimulated proliferation of tL6 cells (58 and 33%, respectively) (P<0.01). These data indicate endogenous pIGFBP-3, like exogenous rpIGFBP-3, suppresses the proliferation of L6 myogenic cells via both IGF-I-dependent and -independent pathways. Immunoneutralization of IGFBP-3 also increased IGF-I-stimulated differentiation (21%, P<0.05) but had no effect on Long-R3-IGF-I stimulated differentiation of tL6 myogenic cells. Results indicate that exogenous and endogenous IGFBP-3 affect proliferation and differentiation of L6 myogenic cells in a similar way. Immunohistochemical localization data reveal that pre-incubation with anti-pIGFBP-3 dramatically reduces the level of intracellular IGFBP-3 in tL6 myogenic cells indicating that endogenously produced IGFBP-3 must first be secreted before it is internalized and that anti-pIGFBP-3 prevents internalization of IGFBP-3. TL6 and mtL6 cells provide a good system to further investigate the mechanisms by which IGFBP-3 affects proliferation and differentiation of myogenic cells.

Production of recombinant porcine IGF-binding protein-5 and its effect on proliferation of porcine embryonic myoblast cultures in the presence and absence of IGF-I and Long-R3-IGF-I.

IGF-binding protein-5 (IGFBP-5) is produced by porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. IGFBP-5 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-5 reportedly may either suppress or stimulate proliferation or differentiation of cultured cells depending on cell type and culture conditions. Additionally, IGFBP-5 has been shown to possess both IGF-dependent and IGF-independent actions in some cell types. The goal of this study was to produce recombinant porcine IGFBP-5 (rpIGFBP-5) and assess its IGF-I-dependent and IGF-I-independent actions on the proliferation of PEMCs. To accomplish this, we have expressed porcine IGFBP-5 in the baculovirus system, purified and characterized the expressed rpIGFBP-5 and produced an anti-porcine IGFBP-5 antibody that neutralizes the biological activity of porcine IGFBP-5. rpIGFBP-5, purified to 98% homogeneity using nickel affinity chromatography and IGF-I affinity chromatography, suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner (P>0.05). rpIGFBP-5 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMCs (P>0.05), even in the presence of significant molar excess of Long-R3-IGF-I compared with rpIGFBP-5, demonstrating the IGF-independent activity that rpIGFBP-5 possesses in PEMCs, since Long-R3-IGF-I is an IGF analog that has very low affinity for the IGFBPs but retains its ability to bind to the type I IGF receptor and thereby can stimulate proliferation. The anti-rpIGFBP-5 IgY produced against rpIGFBP-5 specifically recognized native porcine IGFBP-5 in PEMC media that also contained porcine IGFBP-2, -3, and -4. This antibody is capable of neutralizing the effects of both rpIGFBP-5 and endogenously produced porcine IGFBP-5 on PEMCs as well as detecting IGFBP-5 in Western blots. The production of rpIGFBP-5 and a neutralizing antibody to porcine IGFBP-5 provides a powerful tool to investigate the role of IGFBP-5 in porcine myogenic cell proliferation and differentiation. The data provided here demonstrated that IGFBP-5 has the potential to affect proliferation of PEMCs during critical periods of in vitro muscle cell development and therefore may impact the capacity for ultimate postnatal muscle mass development in vivo.

The effect of a limit-fed diet and slow-feed hay nets on morphometric measurements and postprandial metabolite and hormone patterns in adult horses.

Modern horse management systems tend to limit a horse's opportunity to forage, rely on meal feeding, and may contribute to the increase in equine obesity. The use of slow-feed hay nets represents an opportunity to extend foraging time while feeding a restricted diet. The objectives of this study were to determine if limit feeding combined with a slow-feed hay net would affect morphometric measurements and postprandial metabolite and hormone patterns in overweight adult horses. Eight adult Quarter horses (BW 563 kg ± 4.6 kg; BCS 7.2 ± 0.3) were used in a randomized complete block design, with 4 horses assigned to feeding hay off the stall floor (FLOOR) and 4 horses assigned to feeding from a slow-feed hay net (NET). Horses were fed in individual stalls at 1% BW each day, split evenly between 2 meals at 0700 and 1600 h. Body weight, BCS, neck and girth circumference, cresty neck score, and ultrasound measurements of average rump fat, longissimus dorsi (LD) depth, and LD thickness were taken on d 0, 14, and 28. Three 24-h blood samplings were conducted on d 0, 14, and 28 and were analyzed for glucose, insulin, cortisol, and leptin concentrations. Samplings occurred every 30 min for 3 h postfeeding, with hourly samples occurring between feedings. Horses feeding from the FLOOR took less time to consume their hay meal compared with horses feeding from the NET ( < 0.001). All horses lost weight over the 28-d period ( < 0.0001); however, no difference was observed between treatments. There was no difference in BCS, neck and girth circumference, cresty neck score, rump fat, or LD depth between days or treatments ( ≥ 0.25). There was an effect of day on LD thickness in horses feeding from the NET. Longissimus dorsi thickness was lower on d 28 compared with that on d 0 ( = 0.0257). Only time to peak insulin and peak cortisol were affected by treatment ( ≤ 0.037), with horses feeding from the NET having lower values than horses feeding from the FLOOR. Average glucose, insulin, cortisol, and leptin were affected by day ( ≤ 0.0102). Glucose and insulin values increased, whereas cortisol and leptin levels decreased throughout the 28-d study. The use of a slow-feed hay net coupled with a limit-fed diet appears to be an effective method for decreasing BW and maintaining more homeostatic levels of postprandial metabolites and hormones when feeding overweight adult horses.

Identification of Eimeria bovis merozoite cDNAs using differential mRNA display.

Differences in gene expression between Eimeria bovis sporozoites and first-generation merozoites were analyzed using the technique of differential mRNA display. Approx. 5% of the sequences detected in first-generation merozoites appear to be unique relative to sporozoites. Several of the bands corresponding to merozoite-specific gene expression were isolated and cloned. Northern blot analysis revealed that the cDNA fragments DMZ-7, DMZ-8 and NMZ-6 hybridized to mRNAs expressed at > 50-fold higher levels in merozoites relative to sporozoites. A fourth cDNA fragment, NMZ-4, hybridized to a mRNA expressed at 3-fold higher levels in merozoites. Further characterization demonstrated that expression of DMZ-8 in E. bovis-infected bovine cells begins as early as 12 h after sporozoite invasion and continues throughout the entire 14 days of first-generation schizogony. Sequence analysis of each of the four merozoite cDNAs failed to identify any significant similarity to any entries in the GenBank database, suggesting that these developmentally regulated genes may be unique to coccidian parasites.

Recurrence of autoimmune hepatitis after liver transplantation.

BACKGROUND: The literature data on the recurrence of autoimmune hepatitis (AIH) after orthotopic liver transplantation (OLTX) is scanty. METHODS: We analyzed the frequency of recurrent AIH in 47 patients who had been transplanted for AIH and survived at least 1 year after surgery. The following criteria were applied to diagnose recurrence: (1) positive autoantibodies in the titer> or =1:40; (2) hypertransaminasemia; (3) histological features of chronic hepatitis; (4) need of reintroduction or significant increase of steroids; and (5) lack of serum markers of viral hepatitis. RESULTS: A total of 13 patients (1 male/12 females) developed recurrent AIH after an interval of 6-63 months after OLTX (mean 29 months). Mean AST level at recurrence was 542+/-129 U/L. Three patients from this group needed regrafting. Mismatch of DR3+ recipient and DR3- donor was not more common in the recurrent disease group (37%) compared to the nonrecurrence group (31%) (P=NS). CONCLUSIONS: Recurrence of AIH after OLTX was diagnosed in a high proportion of patients and some of them required regrafting. DR3+ patients are not particularly prone to develop recurrence.

A positive crossmatch in liver transplantation--no effect or inappropriate analysis? A prospective study.

BACKGROUND: Controversy over the relationship of preformed lymphocytotoxic antibodies and liver graft outcome remains. Because graft loss associated with preformed lymphocytotoxic antibodies probably occurs early after transplant, analysis of long-term survival is of questionable value. We therefore prospectively analyzed the effect on short- and long-term graft survival of the presence of lymphocytotoxic alloantibody in 207 primary adult liver allograft recipients. METHODS: Pretransplant serum was tested for donor-specific lymphocytotoxic antibodies and panel-reactive antibodies (PRA) using donor splenic lymphocytes and lymphocytes obtained for routine tissue typing. RESULTS: A positive crossmatch was detected in 24 recipients (11.5%): T-cell positive in 11 recipients and B-cell positive in 13 recipients. PRA were detected in 68 of 179 recipients tested (37.4%). High T-cell PRA (>55%) was detected in 17 recipients, and high B-cell PRA was detected in 20 recipients. Low PRA (<15%) against T cells was detected in 19 recipients and against B cells in 24 recipients. Graft failures occurred in 5 of 24 (21%) crossmatch-positive recipients and in 7 of 172 (4%) crossmatch-negative recipients. Graft survival was significantly lower in crossmatch-positive recipients at 1 month after transplant (chi-square=10.3, P=0.00133) but not at 3 months or 1 year. Causes of early graft loss were associated with immunological mechanisms, whereas later losses were due to nonimmunological mechanisms. CONCLUSIONS: Early graft loss may be increased in those recipients who are crossmatch positive. However, the logistical problems and consequences associated with allocation probably outweigh the benefits of prospective crossmatching.

Increased incidence of chronic rejection in adult patients transplanted for autoimmune hepatitis: assessment of risk factors.

BACKGROUND/AIM: It remains uncertain whether autoimmune hepatitis (AIH), as an original indication for orthotopic liver transplantation (OLTX), predisposes to the development of chronic rejection (CR) after surgery and published reports on heterogeneous groups of patients provided conflicting data. In this work we analyzed the incidence and risk factors for CR in a large cohort of adult patients transplanted for AIH in our unit. RESULTS: A total of 1190 adult patients received OLTX in our center between 1982 and 1998. A total of 77 patients (6.5%) were transplanted for AIH and 12 (15.6%) patients from this group developed clinical and histological features of CR within a median time of 3.5 months after OLTX. Patients with AIH who developed CR were younger than other AIH patients at OLTX (32 vs. 44.2 ys; P=0.015) and more often had histological features of moderate or severe acute rejection (83 vs. 34%; P=0.002) on early post-OLTX biopsies. The incidence of CR in AIH patients was significantly higher than in subjects transplanted for other indications such as primary biliary cirrhosis (8.2%; P<0.05), primary sclerosing cholangitis (5.2%; P<0.05) or alcoholic cirrhosis (2.0%; P<0.001). Also, we observed a tendency to decreased incidence of CR with time in all transplanted subjects. CONCLUSIONS: Apart from younger age at OLTX and higher incidence of severe acute rejection, patients with AIH who developed CR did not differ from other subjects transplanted for this indication. Unlike other studies, not stratified by diagnosis, recipient CMV negative status, young donor age, and HLA DR matching were not identified as risk factors for CR in AIH.

Direct visualization and quantitation of cytomegalovirus-specific CD8+ cytotoxic T-lymphocytes in liver transplant patients.

BACKGROUND: CMV infection remains a significant clinical problem in the context of LT. Changes in the magnitude of the CMV-specific CTL response after LT have not previously been assessed but may be important in determining the outcome of CMV infection. METHOD: We used a fluorescent HLA-B*0702-CMV peptide tetrameric complex to directly visualize and quantitate CMV-specific CD8+ CTL both in immunosuppressed patients after LT and in immunocompetent controls. RESULTS: CMV-specific CD8+ CTL, at a frequency ranging from 0.1 to 5.8% of CD8+, were detected in the peripheral blood of 22 of 25 B*0702, CMV immunoglobulin G seropositive individuals, with no difference observed between immunocompetent controls and patients >3 years after LT. In CMV seropositive LT recipients who did not have symptomatic CMV infection during the first 3 months after LT, CMV-specific CD8+ CTL magnitude initially decreased, then increased up to 5 times higher than pre-LT levels within 3 months. Two CMV seronegative recipients of seropositive donors had symptomatic CMV infection in association with high viral load. In both patients, no CD8+ CTL response was detected before the onset of symptoms, and a reduction in viral load was observed during antiviral therapy. However, polymerase chain reaction negativity was achieved only when a demonstrable CMV-specific CD8+ CTL response was generated. Responses were never observed in asymptomatic CMV seronegative patients. CONCLUSIONS: We suggest that the generation of CMV-specific CD8+ CTL may be driven by, and seems to coincide with the suppression of, viral reactivation. Direct monitoring of CMV-specific CD8+ CTL using an HLA-peptide tetramer may prove to be of value in the management of patients after LT.

Comparison of the effectiveness of various procedures for reducing or eliminating insulin-like growth factor-binding protein interference with insulin-like growth factor-I radioimmunoassays on porcine sera.

We have examined the efficacy of various methods for reducing the interference of insulin-like growth factor-binding proteins (IGFBPs) with insulin-like growth factor-I (IGF-I) radioimmunoassays (RIAs) run on porcine sera. Acid-ethanol (AE) extraction, AE extraction followed by cryoprecipitation, glycyl-glycine (GG) extraction, GG extraction followed by Sephadex G-50 chromatography in 1 mol acetic acid/l (GG/G-50), and Sep-Pak chromatography were analysed. To provide a range of IGF-I and IGFBP levels, sera obtained from control, hypophysectomized, diabetic and somatotrophin-treated pigs were used. Recoveries of IGF-I added to sera prior to treatments other than Sep-Pak chromatography ranged from 85 to 105% and were not significantly different. In contrast, Sep-Pak chromatography gave extremely variable recoveries. 125I-Labelled IGF-I ligand blotting showed that GG extraction followed by acid G-50 chromatography was by far the most effective method of removing or inactivating IGFBPs in porcine sera. Consequently, this procedure was used as a standard against which to compare other extraction procedures. GG extraction alone removed or inactivated low molecular weight binding proteins but appeared to have little effect on IGFBP-3. AE extraction reduced the level of IGFBP-3 but had little effect on lower molecular weight binding proteins. Even though none of the tested procedures completely removed or inactivated the binding proteins, all samples yielded IGF-I displacement curves that were parallel to that obtained for IGF-I standard. Despite yielding parallel displacement curves, sera extracted by various methods gave dramatically different apparent IGF-I levels when subjected to IGF-I RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of recombinant porcine IGF-binding protein-3 on proliferation of embryonic porcine myogenic cell cultures in the presence and absence of IGF-I.

IGF-binding protein (IGFBP)-3 is produced by cultured porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. Levels of secreted IGFBP-3 and IGFBP-3 mRNA are significantly reduced during differentiation and increase after differentiation is complete, suggesting that IGFBP-3 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-3 reportedly may either suppress or stimulate proliferation of cultured cells depending on cell type. Additionally, IGFBP-3 has been shown to affect proliferation via both IGF-dependent and IGF-independent mechanisms in some cell types but not all. Currently, the effect, if any, of IGFBP-3 on myogenic cell proliferation is not known. Consequently, the goal of this study was to assess the IGF-I-dependent and IGF-I-independent actions of recombinant porcine IGFBP-3 on proliferation of cultured porcine myogenic cells. To facilitate these investigations, we have expressed porcine IGFBP-3 in the baculovirus system, purified and characterized the expressed recombinant porcine IGFBP-3 (rpIGFBP-3), and produced and characterized an anti-porcine IGFBP-3 antibody that neutralizes the biological activity of porcine IGFBP-3. rpIGFBP-3 suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner with equimolar concentrations of IGF-I and rpIGFBP-3, resulting in complete suppression of IGF-I-stimulated proliferation. rpIGFBP-3 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMC, indicating that rpIGFBP-3 possesses IGF-independent activity in this cell system. These data have established that IGFBP-3 has the potential to affect proliferation of PEMCs during critical periods of muscle development that may impact ultimate muscle mass achievable postnatally.

Spermatogenesis in Octomacrum lanceatum (Monogenoidea, oligonchoinea, mazocraeidea).

Ultrastructural aspects of spermatozoon development and morphology in Octomacrum lanceatum are presented. Spermiogenesis involves development of zones of differentiation from the surface of cytophores followed by formation of a middle cytoplasmic process and 2 free flagella whose axonemes form from basal bodies located within each zone of differentiation. The mitochondrion and nucleus penetrate through the zone of differentiation into the middle cytoplasmic process. An intercentriolar body is present. Initially the axes of the basal bodies are perpendicular to that of the intercentriolar body but subsequently rotate about 90 degrees to parallel positions as the flagella develop. Flagella are initially free, but eventually fuse with the middle cytoplasmic process from the proximal to distal end (proximodistal fusion). Subsurface microtubules occur within the zone of differentiation but are lacking from the lateral regions of the middle cytoplasmic process. The mature sperm possesses two axonemes, one mitochondrion, and a complete ring of cortical microtubules (sperm pattern 1). The finding of sperm pattern 1 in the Octomacridae suggests that many features of the spermatozoon of the Diplozoidae are autapomorphic.

First in-vivo trials of a fiber Bragg grating based temperature profiling system.

We describe the results of in-vivo trials of a portable fiber Bragg grating based temperature profile monitoring system. The probe incorporates five Bragg gratings along a single fiber and prevents the gratings from being strained. Illumination is provided by a superluminescent diode, and a miniature CCD based spectrometer is used for demultiplexing. The CCD signal is read into a portable computer through a small A/D interface; the computer then calculates the positions of the center wavelengths of the Bragg gratings, providing a resolution of 0.2 degree C. Tests were carried out on rabbits undergoing hyperthermia treatment of the kidney and liver via inductive heating of metallic implants and comparison was made with a commercial Fluoroptic thermometry system.

Identification and characteristics of a 37,000 M(r) insulin-like growth factor binding protein in canine serum.

Insulin-like growth factors (IGFs) are potent stimulators of cellular growth and their half-life and biological activity are regulated by specific IGF binding proteins (IGFBPs). Western ligand blots of non-reduced human, bovine, ovine and porcine sera reveal an IGFBP-2 band at approximately 34,000 M(r). However, canine sera appear to contain a unique 37,000 M(r) IGFBP and lack the 34,000 M(r) IGFBP-2 band. In order to identify and characterize the 37,000 M(r) IGFBP, adult canine serum was subjected to non-reducing SDS polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose paper, followed by [125I]-IGF-1 ligand blotting or immunoblotting with commercially available IGFBP antibodies. The 37,000 M(r) canine IGFBP reacted with an anti-IGFBP-2 antibody indicating that it is a canine analogue of IGFBP-2. However, the large difference in apparent molecular size indicates that this is a unique molecular form of IGFBP-2. N- or O-glycanase treatment of canine sera did not alter the molecular size of canine IGFBP-2 indicating that it is not a glycosylated variant of the IGFBP. Subjecting canine sera to reducing SDS-PAGE followed by anti-IGFBP-2 western immunoblotting revealed that the actual molecular weight of the canine IGFBP-2 is similar to that of reduced IGFBP-2 from other species indicating similar peptide lengths. Thus, the increased non-reduced size of the canine 37,000 M(r) IGFBP-2 is possibly due to a unique secondary structure.

Effect of differentiation on levels of insulin-like growth factor binding protein mRNAs in cultured porcine embryonic myogenic cells.

Insulin-like growth factor binding proteins (IGFBPs) have been shown to affect proliferation of several cell types via insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms. The goal of this study was to determine if levels of IGFBP-2, -3, -4 and -5 mRNA changed during differentiation of cultured porcine embryonic myogenic cells. Total RNA was isolated from muscle cultures at various stages of differentiation and Northern blots of this RNA were probed with 32P-labeled cDNA probes specific for individual IGFBPs. Fusion, myogenin mRNA, and creatine phosphokinase activity were used as markers of differentiation. The level of IGFBP-3 mRNA in differentiating cultures (120 h in culture) was only one-third of the level in myogenin negative, nonfused cultures (72 h in culture) (P < 0.05, n = 4). In contrast, the level of IGFBP-3 mRNA in extensively fused cultures (144 h in culture) was increased by three-fold as compared to the level in myogenin negative, nonfused cultures (P < 0.05, n = 4) and approximately seven-fold as compared to the 120-h cultures (P < 0.05, n = 4). No significant change in the level of IGFBP-5 mRNA was observed during differentiation of myogenic cultures. IGFBP-2 mRNA levels were not significantly different at 72, 96 and 120 h, but at 144 h IGFBP-2 mRNA level was increased three-fold as compared to nonfused cultures (72 h) (P < 0.05, n = 4). IGFBP-4 mRNA was not detectable on Northern blots of total RNA from porcine myogenic cultures at any stage of differentiation. Changes in IGFBP-3 and IGFBP-2 mRNA levels are associated with differentiation of embryonic porcine myogenic cells in culture and this may indicate that these IGFBPs play a role in differentiation of these cells.

Isolation and culture of fetal porcine myogenic cells and the effect of insulin, IGF-I, and sera on protein turnover in porcine myotube cultures.

Porcine myogenic cells isolated from 50 to 55-d porcine fetuses were frozen and stored in liquid nitrogen until they were needed to establish cultures. Approximately 75.8 +/- .59% of the clonal cultures established from these frozen stocks produced myotubes and 60.8 +/- 2.3% of the nuclei in differentiated mass cultures were in myotubes. Differentiated cultures contained higher levels of creatine phosphokinase activity than undifferentiated cultures. Additionally, differentiated cultures incorporated [35S]methionine into putative myosin heavy chain, alpha-actinin, and actin more rapidly than did undifferentiated cultures. Insulin, insulin-like growth factor I, and sera stimulated total protein synthesis rate and decreased total protein degradation rate in myotube cultures. Based on our initial characterization, we believe that we have developed an effective and practical procedure for isolating and culturing fetal porcine myogenic cells.

Effects of growth factors on insulin-like growth factor binding protein (IGFBP) secretion by primary porcine satellite cell cultures.

Insulin-like growth factor-binding proteins (IGFBP) regulate the biological functions of insulin-like growth factors (IGF) and may affect cell growth through IGF-independent actions. Growth factors and hormones have been shown to alter IGFBP production by target cells suggesting that the effects of these factors may be partially mediated by the local production of IGFBP. Growth factors, including IGF-I, transforming growth factor-beta1 (TGF-beta1), and basic fibroblast growth factor (bFGF) have potent effects on satellite cell proliferation and differentiation, and some of these factors have been shown to alter IGFBP production in various cell types. Consequently, some of their actions on muscle satellite cells may be mediated by the local production of IGFBP. In this study, we measured the effects of IGF-I, bFGF, and TGF-beta1 on IGFBP production by primary porcine satellite cell (PSC) cultures after first determining physiologically active concentrations of these growth factors to use according to [3H]thymidine incorporation dose responses. There is little information on the effects of these growth factors on IGFBP production in primary porcine myogenic cells due to the confounding affects of contaminating nonmuscle fibroblasts. Comparative studies show that primary porcine satellite cells produce IGFBP-3 and -5 whereas porcine muscle-derived nonfusing cells (FIB) produce IGFBP-2 and -4 but not IGFBP-3 or -5. Because of this, our investigations have focused on growth factor-induced production of IGFBP-3 and -5 in primary porcine satellite cells cultures. Both IGF-I and bFGF exhibited dose-dependent increases in [3H]thymidine incorporation with increasing concentration from 1 to 50 ng/mL (P < 0.05), whereas TGF-beta1 caused a dose-dependent decrease from 0.01 to 0.5 ng/mL (P < 0.05). When 20 ng/ mL of IGF-I was added to the media, IGFBP-3 was increased approximately 65% (P < 0.05) and IGFBP-5 was increased approximately twofold (P < 0.05). The addition of 0.5 ng/mL TGF-beta1 caused more than a two-fold increase in IGFBP-3 (P < 0.05) and approximately an 80% increase in IGFBP-5 (P < 0.05), whereas 50 ng/ mL of bFGF caused approximately 40% (P < 0.05) and 70% (P < 0.05) increases in IGFBP-3 and -5, respectively. Neither IGFBP-3 nor -5 was detectable in the conditioned media from fibroblasts whether or not IGF-I, TGF- beta1 or bFGF were present. These data suggest that the effects of IGF-I, TGF- beta1 and bFGF on porcine satellite cells may in part be through the autocrine/ paracrine production of IGFBP-3 and -5 by porcine satellite cells.