Cloning and gastrointestinal expression of rat hephaestin: relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.     

Genetic monogamy and biparental care in an externally fertilizing fish, the largemouth bass (Micropterus salmoides)

Breeding, male North American sunfish (Centrarchidae), are often brightly coloured and promiscuous. However, the largemouth bass (Micropterus salmoides) is sexually monomorphic in appearance and socially monogamous. Unlike some other nest-tending centrarchids in the genus Lepomis, largemouth bass have also been reported to provide biparental care to eggs and fry. Here we use microsatellite markers in order to test whether social monogamy predicts genetic monogamy in the largemouth bass. Offspring were collected from 26 nests each usually guarded by a pair of adults, many of which were also captured. Twenty-three of these progeny cohorts (88%) proved to be composed almost exclusively of full-sibs and were thus the product of monogamous matings. Cuckoldry by males was rare. The genetic data also revealed that some nests contain juveniles that were not the progeny of the guardian female, a finding that can be thought of as low-level 'female cuckoldry'. Overall, however, the data provide what may be the first genetic documentation of near-monogamy and biparental care in a vertebrate with external fertilization.     

Expression and in vitro properties of guinea pig IL-5: comparison to human and murine orthologs

Interleukin-5 (IL-5) is a key mediator of eosinophilic inflammation. The biological role of this cytokine in an allergic airway inflammatory response has been widely demonstrated in guinea pigs, yet the interaction of guinea pig IL-5 (gpIL-5) with its receptor has not been studied. Experiments were performed to quantitate the interaction of gpIL-5 with gpIL-5r and to compare this affinity with that of hIL-5 and mIL-5 and their cognate receptors. The cross-species affinity and agonist efficacy were evaluated to see if gpIL-5r had a restricted species reactivity (as is the case with mIL-5r) or did not distinguish between IL-5 orthologs (similar to hIL-5r). gpIL-5 was cloned using mRNA isolated from cells obtained by bronchoalveolar lavage. Recombinant gpIL-5 was expressed in T. ni insect cells and purified from spent media. Binding assays were performed using insect cells expressing hIL-5ralphabeta or gpIL-5ralphabeta1 as previously described (Cytokine, 12:858-866, 2000) or using B13 cells which express mIL-5r. The agonist potency and efficacy properties of each IL-5 ortholog were evaluated by quantitating the proliferative response of human TF-1 cells and murine B13 cells. gpIL-5 bound with high affinity to recombinant gpIL-5r as demonstrated by displacing [125I]hIL-5 (Ki = 160 pM). gpIL-5 also bound to hIL-5r with high affinity (Ki = 750 pM). hIL-5 and mIL-5 showed similar, high-affinity binding profiles to both gpIL-5r and hIL-5r. In contrast, gpIL-5 and hIL-5 did not bind to the mIL-5r as demonstrated by an inability to displace [125I]mIL-5, even at 1000-fold molar excess. These differences in affinity for IL-5r orthologs correlated with bioassay results: human TF-1 cells showed roughly comparable proliferative responses to guinea pig, human and murine IL-5 whereas murine B13 cells showed a strong preference for murine over guinea pig and human IL-5 (EC50 = 1.9, 2200 and 720 pM, respectively). Recombinant gpIL-5 binds to the gpIL-5r with high affinity, similar to that seen with the human ligand-receptor pair. gpIL-5r and hIL-5r do not distinguish between the three IL-5 orthologs whereas mIL-5r has restricted specificity for its cognate ligand.     

Prospective analysis of psychosocial outcomes in breast reconstruction: one-year postoperative results from the Michigan Breast Reconstruction Outcome Study

In the past decade, changing attitudes toward breast reconstruction among both patients and providers have led a growing number of women to seek breast reconstruction after mastectomy. Although investigators have documented the psychological, social, emotional, and functional benefits of breast reconstruction, little research has evaluated the effects of procedure choice on these outcomes. The current study prospectively evaluated and compared psychosocial outcomes for three common options for mastectomy reconstruction: tissue expander/implant, pedicle TRAM, and free TRAM techniques. In a prospective cohort design, patients undergoing postmastectomy reconstruction for the first time with expander/implant, pedicle TRAM, or free TRAM procedures were recruited from 12 centers and 23 plastic surgeons in the United States and Canada. Before reconstruction and at 1 year after reconstruction, patients were evaluated by a battery of questionnaires consisting of both generic and condition-specific surveys. Outcomes assessed included emotional well-being, vitality, general mental health, social functioning, functional well-being, social well-being, and body image. Baseline (preoperative) scores and the change in scores (the difference between postoperative and preoperative scores) were compared across procedure types using t tests and analysis of covariance. Preoperative and 1-year postoperative surveys were obtained from 273 patients. Procedure type was reported in 250 patients, of whom 56 received implant reconstructions, 128 pedicle TRAM flaps, and 66 free TRAM flaps. A total of 161 immediate and 89 delayed reconstructions were performed. Among women receiving immediate reconstruction, significant improvements were observed in all psychosocial variables except body image. However, no significant effects of procedure type on these changes over time existed. Similarly, delayed reconstruction patients had significant increases in emotional well-being, vitality, general mental health, functional well-being, and body image. Although the choice of reconstructive technique did not significantly impact most of these outcomes, significant differences existed among procedure types for three psychosocial subscales. Patients undergoing delayed expander/implant reconstructions reported greater improvements in vitality and social well-being relative to women receiving delayed TRAM procedures. By contrast, delayed TRAM patients noted significantly greater gains in body image compared with women choosing delayed expander-implant reconstruction. The authors conclude that both immediate and delayed breast reconstructions provide substantial psychosocial benefits for mastectomy patients. Although the choice of reconstructive procedure does not seem to significantly affect improvements in psychosocial status with immediate reconstruction, our data suggest that procedure type does have a significant effect on gains in vitality and body image for women undergoing delayed reconstruction.     

Implementation and evaluation of a clinical pathway for TRAM breast reconstruction

Among strategies recently proposed to reduce practice variation, promote quality, and control costs in health care delivery, the concept of the clinical pathway has received considerable attention. Because transverse rectus abdominis musculocutaneous (TRAM) breast reconstruction is a common and often costly intervention, this institution sought to evaluate cost and quality outcomes of a clinical pathways program for this procedure. The TRAM reconstruction clinical pathway was implemented in April of 1996 to standardize postoperative care in this patient population. Outcomes of consecutive pathway cases for the first 14 months of the program were assessed in a retrospective cohort design, by using all nonpathway TRAM cases from the 18 months immediately before pathway implementation as controls. Outcomes assessed included length of hospital stay, postoperative complications, total postoperative charges, and total postoperative costs in relative value units. Data on these dependent variables were collected from hospital charts and billing records. The effects of pathway implementation on the outcomes of interest were analyzed by using analysis of covariance to control for potential confounding by other independent variables, including surgical site (unilateral versus bilateral reconstructions), technique (pedicle versus free TRAMs), timing (immediate versus delayed reconstructions), and patient age. Finally, a comparison of variances in the outcomes of interest between the two groups was analyzed by using an Ftest. For all statistical tests, p values of < or = 0.05 were considered significant. Twenty-nine patients were treated in the TRAM pathway group, whereas the control population included 40 nonpathway patients. After implementation of the TRAM pathway, length of stay decreased from 6.0 to 5.2 days; total postoperative charges were reduced from $8587 to $7744; and total postoperative relative value unit utilization declined from 1686 to 1104. Analysis of covariance showed that the decreases in length of hospital stay and relative value units in the TRAM pathway were statistically significant (p = 0.05 and p = 0.007, respectively). By contrast, no significant increase in complications was observed after pathway implementation. Variability in the TRAM pathway group, as measured by SD, decreased significantly for both length of hospital stay (p = 0.039) and relative value units (p = 0.023). Implementation of the TRAM reconstruction clinical pathway resulted in significant declines in length of hospital stay and total costs. These decreases in resource utilization had no significant effect on postoperative complication rates. Although additional research is needed to further assess the impact of clinical pathways, this approach offers considerable promise for improving the cost-effectiveness of health care.