Biomedical subjects

P A Fields

Publications and source records attributed to P A Fields.

At least 19 recordsLinked to original sources

PubMed2005

FDG-PET after two to three cycles of chemotherapy predicts progression-free and overall survival in high-grade non-Hodgkin lymphoma.

BACKGROUND: Less than 50% of all high-grade non-Hodgkin lymphoma (NHL) patients experience lasting disease-free survival. Risk-adapted treatment strategies require better tools for prediction of outcome. This investigation aimed to assess the value of positron emission tomography with 2-[18F]fluoro-2-deoxy-D-glucose (FDG-PET) after two to three cycles of chemotherapy for prediction of progression-free survival (PFS) and overall survival (OS). PATIENTS AND METHODS: One hundred and twenty-one patients with high-grade NHL underwent FDG-PET. The therapy response on FDG-PET was correlated to PFS and OS using Kaplan-Meier survival analysis. Cox regression analyses were employed to test for independence of known pretreatment prognostic factors. RESULTS: Fifty FDG-PET scans were negative, 19 scans showed minimal residual uptake (MRU), and 52 scans were positive. The estimated 5 year PFS was 88.8% for the PET-negative group, 59.3% for the MRU group, and 16.2% for the PET-positive group. Kaplan-Meier analyses showed strong associations between FDG-PET results and PFS (P <0.0001) and OS (P <0.01). Early interim FDG-PET was independent of the other prognostic factors. CONCLUSIONS: Early interim FDG-PET is an accurate and independent predictor of PFS and OS. An early assessment of chemotherapy response with FDG-PET could provide the basis for selection of patients for alternative therapeutic strategies.

Adult↗

PubMed2005

Prognostic value of interim FDG-PET after two or three cycles of chemotherapy in Hodgkin lymphoma.

BACKGROUND: Long-term survival from Hodgkin lymphoma (HL) is 80-90%, but the treatment has serious late adverse effects. Modern risk-adapted treatment requires accurate assessment of the patient's prognosis. This investigation assessed the value of positron emission tomography (PET) with 2-[18F]fluoro-2-deoxy-D-glucose (FDG-PET) after two or three cycles of chemotherapy for prediction of progression-free survival (PFS) and overall survival (OS). PATIENTS AND METHODS: A total of 85 patients with HL underwent FDG-PET after two or three cycles of chemotherapy. Median follow-up was 3.3 years. FDG-PET results were related to PFS and OS using Kaplan-Meier analysis. Regression analyses were employed to test for independence of established pretreatment prognostic factors. RESULTS: After two or three cycles of chemotherapy, 63 patients had negative FDG-PET scans, nine patients had minimal residual uptake (MRU) and 13 patients had positive scans. Three PET-negative patients and one patient from the MRU group relapsed. In the PET-positive group, nine patients progressed and two died. Survival analyses showed highly significant associations between early interim FDG-PET and PFS (P <0.0001) and OS (P <0.03). All advanced-stage patients with positive interim FDG-PET relapsed within 2 years. CONCLUSION: Early interim FDG-PET is an accurate and independent predictor of PFS and OS in HL. A positive interim FDG-PET is highly predictive of relapse in advanced-stage disease.

Adolescent↗

PubMed2005

Clinical use of relaxin to facilitate birth: reasons for investigating the premise.

In the United States, both medical and nonmedical factors have driven the cesarean section rate to over 26% of all deliveries. In addition to questions of increased cost associated with operative delivery, some have questioned the ethics of performing cesarean section for nonmedical reasons. Reduction of both the duration and the pain associated with vaginal delivery would likely bring about a decline in the rate of both medical and nonmedical cesarean sections. This chapter summarizes recent findings that support the premise that through its growth-promoting and softening effects on the cervix, short-term subcutaneous administration of pharmacologic amounts of relaxin to women at term holds promise as a means of reducing the duration and discomfort associated with delivery. Two recent studies conducted in pregnant rats demonstrated that the cervix is highly responsive to relaxin during the antepartum period and that short-term subcutaneous administration of the hormone to relaxin-deficient animals not only promotes growth and softening of the cervix, but also reduces the duration of labor and delivery. Moreover, recent human clinical trials examining the influence of 24 weeks of continuous subcutaneous administration of recombinant human relaxin for the treatment of scleroderma provided evidence not only that the human reproductive tract is responsive to relaxin, but also that the administration of the hormone does not cause serious adverse side effects. It is concluded that recent findings provide an impetus for an investigation into relaxin's potential for cervical remodeling and facilitating birth in women.

Animals↗

PubMed2004

The extent to which relaxin promotes proliferation and inhibits apoptosis of cervical epithelial and stromal cells is greatest during late pregnancy in rats.

Relaxin promotes marked growth of the cervix during the second half of rat pregnancy, and this growth is accompanied by an increase in both epithelial and stromal cells. The objective of this study was to test the hypothesis that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical cells is greatest during late pregnancy in rats. The influence of neutralization of circulating relaxin by iv injection of 5 mg monoclonal antibody against rat relaxin (MCA1) was examined at 3-d intervals throughout the second half of pregnancy. Controls were injected with either 5 mg monoclonal antibody against fluorescein or 0.5 ml PBS vehicle. To evaluate cell proliferation, 5'-bromo-2-deoxyuridine was injected sc 8 h before cervixes were collected. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling and electron microscopy were used to detect apoptotic cells. Neutralization of relaxin with MCA1 decreased the rate of proliferation and increased the rate of apoptosis of cervical cells by d 13. However, the extent to which relaxin influenced these processes was greatest and dramatic by late pregnancy. In MCA1-treated rats on d 22 of pregnancy, the rates of proliferation of both epithelial and stromal cells were less than 20% those in controls, and the rates of apoptosis in epithelial cells and stromal cells were more than 10- and 3-fold, respectively, greater than those in controls. In conclusion, this study provides evidence that the extent to which relaxin promotes proliferation and inhibits apoptosis of cervical epithelial and stromal cells is greatest during late pregnancy.

Animals↗

PubMed2003

Localization of the iron transport proteins Mobilferrin and DMT-1 in the duodenum: the surprising role of mucin.

There are two pathways for inorganic iron uptake in the intestine, the ferric pathway, mediated by the key protein mobilferrin, and the ferrous pathway, mediated by DMT-1. Previous studies reported that the amount of DMT-1 increased in the intestinal mucosa in iron deficiency and the increase was seen in the apical portion of the villus of the duodenal mucosa. Mobilferrin did not quantitatively increase but became localized at the cell membrane. However, studies on fresh tissue have not previously been performed and the localization to the microvillae has not been demonstrated. In order to more definitively localize these proteins immunofluorescent and electron microscopic studies were undertaken. Samples were also subjected to biochemical analysis and Western analysis. In iron-deficient animals both DMT-1 and Mobilferrin were concentrated in the apical surface of the villae. Electron microscopy revealed that the majority of this increase in the amount of these proteins near the luminal surface was due to increased binding of the proteins to mucin in vesicles near the surface. A significant portion of the iron transport proteins was localized in the goblet cells and outside the cell in the luminal mucin, as demonstrated by immunofluorescence, electron microscopy, and isolation of the mucin by cesium chloride gradient centrifugation and Western analysis. A new model for the transport of metal ions was suggested. The metal transport proteins travel from vesicles inside the cell out to the lumen mucin. This increases the surface area and allows a greater portion of the lumen contents to be exposed to the binding proteins. Once the metal is bound to the externalized protein it is internalized into the cell. This explains many of the unique properties of the iron-binding proteins and suggests that it may be a more general model for the absorption of other nutrients.

Animals↗

PubMed2002

Non-Hodgkin's lymphomas: clinical governance issues.

Every patient in every part of the world has the right to expect the best possible quality of care from health care providers. Non-Hodgkin's lymphomas (NHL) are an extremely heterogeneous group of conditions which require important decisions to be taken at many points along the treatment pathway. To get this right every time requires that high-quality standards are instituted and adhered to, so that the best possible outcome is achieved. In the past this has not always been the case because of the failure of clinicians sometimes to adhere to an optimal management plan. In 1995, the UK government commissioned an inquiry into the running of cancer services in the United Kingdom, which culminated in a series of recommendations to improve them. Subsequently, these recommendations were implemented as objectives of the NHS Cancer Plan which is the framework by which the UK government wishes to improve cancer services. Concurrently another general concept has emerged which is designed to ensure that the highest quality standards may be achieved for all patients across the whole National Health Service (NHS). This concept, termed 'clinical governance', brings together a corporate responsibility of all health care workers to deliver high quality standards, in the hope that this will translate into better long-term survival of patients with malignant disease. This chapter focuses on the issues surrounding clinical governance and how the principles of this concept relate to non-Hodgkin's lymphomas.

Humans↗

PubMed2001

Posttranslational modifications of recombinant myotube-synthesized human factor IX.

Recent data demonstrate that the introduction into skeletal muscle of an adeno-associated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical gamma-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and gamma-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX.

1-Carboxyglutamic Acid↗

PubMed2001

Risk and prevention of anti-factor IX formation in AAV-mediated gene transfer in the context of a large deletion of F9.

The safety of several gene therapy approaches for treatment of the severe, X-linked bleeding disorder hemophilia is currently being evaluated in early phase clinical trials. One strategy seeks to correct deficiency of functional coagulation factor IX (hemophilia B) by intramuscular (IM) administration of an adeno-associated viral (AAV) vector. A potentially serious complication of any treatment for hemophilia is formation of inhibitory antibodies against the coagulation factor protein, a risk that increases in the setting of null mutations in the factor IX gene (F9). Here, we describe hemophilia B mice with a large F9 deletion that form inhibitors within 1 to 2 months after IM administration of an AAV vector expressing mouse F9 or after repeated intravenous infusion of mouse F9 concentrate. In both cases, inhibitors are primarily IgG1 immunoglobulins representing a Th2-driven humoral immune response. We further demonstrate that anti-mouse F9 antibody formation in the gene-based approach can be reduced by transient immune modulation at the time of vector administration. Moreover, this maneuver resulted in complete absence of anti-mouse F9 and sustained expression of functional mouse F9 in some hemophilia B mice, particularly in those animals treated with the immunosuppressive drug cyclophosphamide. These data have direct relevance for design of clinical trials and strategies aimed at avoiding immune responses against a secreted transgene product.

Animals↗

PubMed2001

Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males.

A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.

Animals↗

PubMed2001

Review: Protein function at thermal extremes: balancing stability and flexibility.

No organism can survive across the entire temperature range found in the biosphere, and a given species can rarely support active metabolism across more than a few tens of degrees C. Nevertheless, life can be maintained at surprisingly extreme temperatures, from below -50 to over 110 degrees C. That proteins, which are assembled with the same 20 amino acids in all species, can function well at both extremes of this range illustrates the plasticity available in the construction of these macromolecules. In studying proteins from extremophiles, researchers have found no new amino acids, covalent modifications or structural motifs that explain the ability of these molecules to function in such harsh environments. Rather, subtle redistributions of the same intramolecular interactions required for protein stabilization at moderate temperatures are sufficient to maintain structural integrity at hot or cold extremes. The key to protein function, whether in polar seas or hot springs, is the maintenance of an appropriate balance between molecular stability on the one hand and structural flexibility on the other. Stability is needed to ensure the appropriate geometry for ligand binding, as well as to avoid denaturation, while flexibility is necessary to allow catalysis at a metabolically appropriate rate. Comparisons of homologous proteins from organisms spanning a wide range of thermal habitats show that adaptive mutations, as well as stabilizing solutes, maintain a balance between these two attributes, regardless of the temperature at which the protein functions.

Amino Acid Sequence↗

PubMed2001

Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice.

Inbred immunocompetent C57BL/6 mice have been a favored strain to study transgene expression of human blood coagulation factor IX (hF.IX) from viral vectors because systemic expression of the secreted protein is not limited by antibody responses following intravenous (i.v.) injection of vector. For example, i.v. injection of an adenoviral (Ad) vector results in sustained expression of hF.IX in normal or hemophilic C57BL/6 mice, while anti-hF.IX antibodies rapidly emerge in other strains (Gene Therapy 4: 473; Blood 91: 784). To investigate these observations further, we injected naive C57BL/6 mice and C57BL/6 mice with pre-existing anti-hF.IX with Ad-hF.IX vector via peripheral vein. All mice expressed hF.IX antigen without detectable anti-hF.IX, even when challenged with hF.IX in different immunogenic settings at later time points. Moreover, in mice with pre-existing immunity, anti-hF.IX titers diminished to undetectable levels after i.v. administration of Ad-hF.IX. Lymphocytes from mice that had received Ad-hF.IX i.v. failed to proliferate when stimulated with hF.IX in vitro after the animals had been repeatedly challenged with hF.IX protein formulated in complete Freund's adjuvant. Thus, absence of anti-hF.IX in C57BL/6 mice after i.v. injection of Ad vector is not due to ignorance to the foreign transgene product. Similar experiments in other strains showed that immune tolerance to hF.IX does not correlate with the strain haplotype or expression of IL-10 cytokine. Given the well-documented immunogenicity of the first-generation adenoviral vector, data from C57BL/6 mice may therefore grossly underestimate immunological consequences in certain gene therapy protocols.

Adenoviridae↗

PubMed2001

Intrinsic versus extrinsic stabilization of enzymes: the interaction of solutes and temperature on A4-lactate dehydrogenase orthologs from warm-adapted and cold-adapted marine fishes.

We examined the effects of temperature and stabilizing solutes on A4-lactate dehydrogenase (A4-LDH) from warm- and cold-adapted fishes, to determine how extrinsic stabilizers affect orthologs with different intrinsic stabilities. Conformational changes during substrate binding are rate-limiting for A4-LDH, thus stabilization due to intrinsic or extrinsic factors leads to decreased activity. A4-LDH from a warm-temperate goby (Gillichthys mirabilis), which has lower values for kcat and the Michaelis constant for pyruvate ( K m PYR), was intrinsically more stable than the orthologs of the cold-adapted Antarctic notothenioids Parachaenichthys charcoti and Chionodraco rastrospinosus, as shown by a higher apparent transition ('melting') temperature (Tm(APP)). We used four solutes, glycerol, sucrose, trimethylamine-N-oxide and poly(ethylene glycol) 8000, which stabilize proteins through different modes of preferential exclusion, to study temperature-solute interactions of the three orthologs. Changes in Tm(APP) were similar for all orthologs in each solute tested, but the catalytic rate of G. mirabilis A4-LDH was decreased most by solutes and increased most by temperature. In contrast, the K m PYR values of the Antarctic orthologs were more affected than that of the goby by both solutes and temperature. We conclude that (a) preferential exclusion of solutes functions within the native state of A4-LDH to favor conformational microstates with minimal surface area; (b) the varied effects of the different solutes on the kinetic properties are due to the interaction between this nonspecific stabilization and the differing intrinsic stabilities of the orthologs; (c) the catalytic rates of A4-LDH orthologs are equally affected by stabilizing solutes, if measurements are made at physiologically appropriate temperatures; and (d) global stability and localized flexibility of these A4-LDH orthologs may evolve independently.

Animals↗

PubMed2001

Evidence that relaxin inhibits apoptosis in the cervix and the vagina during the second half of pregnancy in the rat.

The growth of the cervix and vagina that occurs during the second half of rat pregnancy is accompanied by an increase in both epithelial and stromal cells. Neither the mechanism(s) that regulates this accumulation of cells nor its hormonal control is known. To test the hypothesis that the rate of apoptosis declines during the second half of pregnancy, cervices and vaginas were collected on days 5, 10, 15, 18, and 21 of pregnancy. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling was used to detect apoptotic cells. The rate of apoptosis declined (P < 0.05) in epithelial and stromal cells in both the cervix and vagina during the second half of pregnancy, when blood levels of relaxin are increasing. To test the hypothesis that relaxin inhibits apoptosis, cervices and vaginas were collected 6, 12, 24, 48, and 72 h after the neutralization of endogenous relaxin, on days 19-21 of pregnancy, with a monoclonal antibody for rat relaxin. Both the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling method and electron microscopy were used to detect apoptotic cells. Withdrawal of relaxin caused an increase in the rate of apoptosis in both the cervix and the vagina (P < 0.05). It is concluded that the rate of apoptosis declines in the cervix and the vagina during the second half of rat pregnancy, and that relaxin likely contributes to this process.

Animals↗

PubMed2000

Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter.

Hemophilia B is caused by the absence of functional coagulation factor IX (F.IX) and represents an important model for treatment of genetic diseases by gene therapy. Recent studies have shown that intramuscular injection of an adeno-associated viral (AAV) vector into mice and hemophilia B dogs results in vector dose-dependent, long-term expression of biologically active F.IX at therapeutic levels. In this study, we demonstrate that levels of expression of approximately 300 ng/mL (6% of normal human F.IX levels) can be reached by intramuscular injection of mice using a 2- to 4-fold lower vector dose (1 x 10(11) vector genomes/mouse, injected into 4 intramuscular sites) than previously described. This was accomplished through the use of an improved expression cassette that uses the cytomegalovirus (CMV) immediate early enhancer/promoter in combination with a 1.2-kilobase portion of human skeletal actin promoter. These results correlated with enhanced levels of F.IX transcript and secreted F.IX protein in transduced murine C2C12 myotubes. Systemic F.IX expression from constructs containing the CMV enhancer/promoter alone was 120 to 200 ng/mL in mice injected with 1 x 10(11) vector genomes. Muscle-specific promoters performed poorly for F.IX transgene expression in vitro and in vivo. However, the incorporation of a sequence from the alpha-skeletal actin promoter containing at least 1 muscle-specific enhancer and 1 enhancer-like element further improved muscle-derived expression of F.IX from a CMV enhancer/promoter-driven expression cassette over previously published results. These findings will allow the design of a clinical protocol for therapeutic levels of F.IX expression with lower vector doses, thus enhancing efficacy and safety of the protocol. (Blood. 2000;95:2536-2542)

Actins↗

PubMed2000

Role of vector in activation of T cell subsets in immune responses against the secreted transgene product factor IX.

Defining immune responses against the secreted transgene product in a gene therapy setting is critical for treatment of genetic diseases such as hemophilia B (coagulation factor IX deficiency). We have previously shown that intramuscular administration of an adeno-associated viral (AAV) vector results in stable expression of therapeutic levels of factor IX (F.IX) and may be associated with humoral immune responses against F.IX. This study demonstrates that intramuscular injection of an AAV vector expressing F.IX fails to activate F.IX-specific cytotoxic T lymphocytes (CTLs) in hemostatically normal or in hemophilia B mice, so that there is an absence of cellular immune responses against F.IX. However, transgene-derived F.IX can cause B cell responses characterized by production of T helper cell-dependent antibodies (predominantly IgG1, but also IgG2 subclasses) resulting from activation of CD4+ T helper cells primarily of the Th2 subset. In contrast, administration of an adenoviral vector efficiently activated F.IX-specific CTLs and T helper cells of both Th1 and Th2 subsets, leading to inflammation and destruction of transduced muscle tissue and activation of B cells as well. Therefore, vector sequences fundamentally influence T cell responses against transgene-encoded F.IX. In conclusion, activation of the immune system in AAV-mediated gene transfer is restricted to pathways mediated by F.IX antigen presentation through MHC class II determinants resulting in T and B cell responses that are more comparable to responses in the setting of protein infusion rather than of viral infection/gene transfer.

Adenoviridae↗

PubMed1999

Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.

Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

Animals↗

PubMed1999

B-chain sequence and in situ hybridization of the rabbit placental relaxin-like gene product.

We reported that the nucleotide sequence of a cDNA generated from rabbit placental poly(A)(+) RNA using porcine preprorelaxin primers was identical to SQ10, a product of squamous differentiated tracheal epithelial cells. However, these results did not confirm that SQ10 was the biologically active rabbit relaxin that had been isolated previously yet not sequenced. In this study, a 7-kDa protein isolated from rabbit placentas exhibited relaxin bioactivity and cross-reacted with a porcine relaxin antiserum. A partial amino acid sequence of this protein revealed a sequence identical to that of SQ10. Although the amino acid sequence of the putative relaxin receptor-binding domain found in the B chain of relaxin was modified in SQ10 from CGRDYVR to CRNDFVR, the placental protein was bioactive. These results suggest that SQ10 is the rabbit relaxin. In situ hybridization, using an SQ10 riboprobe, indicated radiolabeling in the syncytiotrophoblast cells of the rabbit placenta. The pattern of labeling corresponded with the immunohistochemical staining for relaxin observed with use of a porcine relaxin antiserum. These results indicate that the syncytiotrophoblast cells are a site of synthesis for SQ10 and that the immunostaining is not solely the result of sequestering SQ10 through receptor-mediated endocytosis. A potential role for relaxin in implantation is discussed.

Amino Acid Sequence↗

PubMed1998

Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes.

To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A4 (A4-LDH) from nine Antarctic (-1.86 to 1 degree C) and three South American (4 to 10 degree C) notothenioid teleosts. Higher Michaelis-Menten constants (Km) and catalytic rate constants (kcat) distinguish orthologs of Antarctic from those of South American species, but no relationship exists between adaptation temperature and the rate at which activity is lost because of heat denaturation. In all species, active site residues are conserved fully, and differences in kcat and Km are caused by substitutions elsewhere in the molecule. Within geographic groups, identical kinetic properties are generated by different substitutions. By combining our data with A4-LDH sequences for other vertebrates and information on roles played by localized conformational changes in setting kcat, we conclude that notothenioid A4-LDHs have adapted to cold temperatures by increases in flexibility in small areas of the molecule that affect the mobility of adjacent active-site structures. Using these findings, we propose a model that explains linked temperature-adaptive variation in Km and kcat. Changes in sequence that increase flexibility of regions of the enzyme involved in catalytic conformational changes may reduce energy (enthalpy) barriers to these rate-governing shifts in conformation and, thereby, increase kcat. However, at a common temperature of measurement, the higher configurational entropy of a cold-adapted enzyme may foster conformations that bind ligands poorly, leading to high Km values relative to warm-adapted orthologs.

Adaptation, Physiological↗