Endometrial carcinoma recurrence according to race and ethnicity: An NRG Oncology/Gynecologic Oncology Group 210 Study.

Non-Hispanic black (NHB) women are more likely to experience an endometrial carcinoma (EC) recurrence compared to non-Hispanic white (NHW) women. The extent to which tumor characteristics, socioeconomic status (SES) and treatment contribute to this observation is not well defined. In the NRG Oncology/Gynecology Oncology Group (GOG) 210 Study we evaluated associations between race/ethnicity and EC recurrence according to tumor characteristics with adjustment for potential confounders. Our analysis included 3,199 NHW, 532 NHB and 232 Hispanic women with EC. Recurrence was documented during follow-up. We used Cox regression to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for associations between race/ethnicity and EC recurrence in models stratified by histologic subtype (low-grade endometrioid, high-grade endometrioid, serous, mixed cell, carcinosarcoma, clear cell) or stage (I, II, III) and adjusted for age, SES, body mass index, smoking status and treatment. In histologic subtype-stratified models, higher EC recurrence was noted in NHB women with low-grade endometrioid (HR = 1.94, 95% CI = 1.21-3.10) or carcinosarcomas (HR = 1.66, 95% CI = 0.99-2.79) compared to NHWs. In stage-stratified models, higher EC recurrence was noted among NHB women with stage I (HR = 1.48, 95% CI = 1.06-2.05) and Hispanic women with stage III disease (HR = 1.81, 95% CI = 1.11-2.95). Our observations of higher EC recurrence risk among NHB and Hispanic women, as compared to NHW women, were not explained by tumor characteristics, SES, treatment or other confounders. Other factors, such as racial differences in tumor biology or other patient factors, should be explored as contributors to racial disparities in EC recurrence.

Increased adult hippocampal neurogenesis is not necessary for wheel running to abolish conditioned place preference for cocaine in mice.

Recent evidence suggests that wheel running can abolish conditioned place preference (CPP) for cocaine in mice. Running significantly increases the number of new neurons in the hippocampus, and new neurons have been hypothesised to enhance plasticity and behavioral flexibility. Therefore, we tested the hypothesis that increased neurogenesis was necessary for exercise to abolish cocaine CPP. Male nestin-thymidine kinase transgenic mice were conditioned with cocaine, and then housed with or without running wheels for 32 days. Half of the mice were fed chow containing valganciclovir to induce apoptosis in newly divided neurons, and the other half were fed standard chow. For the first 10 days, mice received daily injections of bromodeoxyuridine (BrdU) to label dividing cells. On the last 4 days, mice were tested for CPP, and then euthanized for measurement of adult hippocampal neurogenesis by counting the number of BrdU-positive neurons in the dentate gyrus. Levels of running were similar in mice fed valganciclovir-containing chow and normal chow. Valganciclovir significantly reduced the numbers of neurons (BrdU-positive/NeuN-positive) in the dentate gyrus of both sedentary mice and runner mice. Valganciclovir-fed runner mice showed similar levels of neurogenesis as sedentary, normal-fed controls. However, valganciclovir-fed runner mice showed the same abolishment of CPP as runner mice with intact neurogenesis. The results demonstrate that elevated adult hippocampal neurogenesis resulting from running is not necessary for running to abolish cocaine CPP in mice.

Septin: a factor in plasma that opsonizes lipopolysaccharide-bearing particles for recognition by CD14 on phagocytes.

We have previously reported that lipopolysaccharide (LPS) binding protein (LBP) opsonizes endotoxin (LPS) for recognition by CD14 on phagocytes. Here we show that normal human plasma contains high titers of an activity that also binds LPS (Re, 595) and mediates recognition by CD14. Opsonization of LPS-coated particles with plasma enables the particles to be bound by phagocytes. Further, opsonization with plasma also enables subnanogram-per-milliliter concentrations of LPS to induce dramatic alterations in the function of leukocyte integrins on polymorphonuclear leukocytes and to induce secretion of tumor necrosis factor by monocytes, suggesting that opsonization by factors in plasma may be important in responses of cells to endotoxin. The opsonic activity in plasma appears distinct from LBP since it is not blocked by neutralizing antibodies against LBP. Surprisingly, the opsonic activity of plasma is not present in a single protein species, but at least two species must be combined to observe activity. Further, the opsonic activity of plasma for LPS is blocked by addition of protease inhibitors, suggesting that proteolytic activity or activities are required for opsonization. These properties are suggestive of the action of a protease cascade, but opsonic activity of plasma is not affected by blockade or depletion of either the complement or clotting cascades. We propose the name "septin" to describe this novel LPS-opsonizing activity in plasma.

Requirement for HLA-DR+ accessory cells in natural killing of cytomegalovirus-infected fibroblasts.

The role of HLA-DR+ cells in NK activity against CMV-infected FS4 foreskin fibroblasts and K562 erythroleukemia cells was examined. When nonadherent PBMC were depleted of either HLA-DR+ or Leu-11b+ cells by treatment with mAbs plus C, NK activity against CMV-FS4 target cells was markedly reduced. In contrast, depletion of HLA-DR+ cells had no effect on NK activity against K562 target cells. When HLA-DR-depleted cells were added to Leu-11b-depleted cells, NK activity against CMV-FS4 was restored. Negative selection experiments indicated that the HLA-DR+ cells contributing to NK activity against CMV-FS4 are not B or T cells, while negative and positive selection experiments excluded a role for monocytes. Experiments in which HLA-DR- and Leu-11b- cells were mixed in varying proportions indicated that NK(CMV-FS4) is mediated by Leu-11b+ cells, while HLA-DR+ cells provide an accessory function. Irradiation (50 GY) abolished the NK effector function of Leu-11b+ cells, but not the accessory function of HLA-DR+ cells. The NK activity against CMV-FS4 of HLA-DR- cells was restored by the addition of rIFN-alpha or of cell-free supernatants generated by coculturing PBMC or Leu-11b- cells with CMV-FS4. The ability of these supernatants to restore NK activity of HLA-DR- cells was completely abrogated by the addition of neutralizing amounts of antibody to IFN-alpha. In related experiments, neutralization of IFN-alpha in NK assays had little or no effect on NK activity against CMV-FS4, suggesting that the accessory function of HLA-DR+ cells might be mediated by alternative mechanisms in addition to the secretion of extracellular IFN-alpha.

Soluble CD14 participates in the response of cells to lipopolysaccharide.

CD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononuclear phagocytes and as a soluble protein in the blood. CD14 on the cell membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothelial-leukocyte adhesion molecule 1 expression by human umbilical vein endothelial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial cells. None of these cell types express mCD14, yet all respond to LPS in a serum-dependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14.

CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.

Estrogen receptor beta signaling through phosphatase and tensin homolog/phosphoinositide 3-kinase/Akt/glycogen synthase kinase 3 down-regulates blood-brain barrier breast cancer resistance protein.

Breast cancer resistance protein (BCRP) is an ATP-driven efflux pump at the blood-brain barrier that limits central nervous system pharmacotherapy. Our previous studies showed rapid loss of BCRP transport activity in rat brain capillaries exposed to low concentrations of 17-beta-estradiol (E2); this occurred without acute change in BCRP protein expression. Here, we describe a pathway through which sustained, extended exposure to E2 signals down-regulation of BCRP at the blood-brain barrier. Six-hour exposure of isolated rat and mouse brain capillaries to E2 reduced BCRP transport activity and BCRP monomer and dimer expression. Experiments with brain capillaries from estrogen receptor (ER)alpha and ERbeta knockout mice and with ER agonists and antagonists showed that E2 signaled through ERbeta to down-regulate BCRP expression. In rat brain capillaries, E2 increased unphosphorylated, active phosphatase and tensin homolog (PTEN); decreased phosphorylated, active Akt; and increased phosphorylated, active glycogen synthase kinase (GSK)3. Consistent with this, inhibition of phosphoinositide 3-kinase (PI3K) or Akt decreased BCRP activity and protein expression, and inhibition of PTEN or GSK3 reversed the E2 effect on BCRP. Lactacystin, a proteasome inhibitor, abolished E2-mediated BCRP down-regulation, suggesting internalization followed by transporter degradation. Dosing mice with E2 reduced BCRP activity in brain capillaries within 1 h; this reduction persisted for 24 h. BCRP protein expression in brain capillaries was unchanged 1 h after E2 dosing but was substantially reduced 6 and 24 h after dosing. Thus, E2 signals through ERbeta, PTEN/PI3K/Akt/GSK3 to stimulate proteasomal degradation of BCRP. These in vitro and in vivo findings imply that E2-mediated down-regulation of blood-brain barrier BCRP has the potential to increase brain uptake of chemotherapeutics that are BCRP substrates.

Solvent properties of ground substance studied by cryomicrodissection and intracellular reference-phase techniques.

Water, sodium, potassium, ATP, amino acids, and sugars are not uniformly distributed in Rana pipiens oocytes. Concentration differences exist between nucleus (germinal vesicle) and ooplasm and between animal and vegetal ooplasmic regions. The mechanisms responsible for these differences were investigated using intracellular reference-phase (iRP) analysis. The iRP is an artificial "organelle" that has the solvent properties of a dilute salt solution and is in diffusional equilibrium with water and solutes present in other cellular compartments. Ooplasm/iRP solute distributions show that ooplasm differs from ordinary aqueous solutions--exhibiting both solute exclusion and solute binding. Yolk platelets are an important cause of this behavior, largely because their proteins are present as hydrate crystals, which are rich in anionic sites and which interact intensely with associated water. Because of yolk's abundance, it obscures the solvent and binding properties of ooplasmic ground substance. The oocyte nucleus is yolk and organelle free and the nuclear envelope is readily permeable. Consequently, nucleus/iRP solute concentration differences reflect the binding and solvent properties of nuclear ground substance. Nucleoplasm binds approximately 19 meq of potassium. Furthermore, the monosaccharides, 3-O-methylglucose, L-glucose, and D-xylose, are selectively excluded, their nucleus/iRP concentration ratios averaging about 0.7; ratios for other solutes studied are unity. We interpret monosaccharide exclusion to mean that nuclear ground substance water is different in its "instantaneous" structure from ordinary saline water. Because of this difference, hydrogen bond interaction between nuclear water and certain sterically restricted solutes, of which ringed monosaccharides are examples, is reduced. Some implications of modified ground substance water and selective solute exclusion are discussed.

Stimulation of RNA and protein synthesis by intracellular insulin.

Like insulin-sensitive somatic cells, stage IV oocytes from Xenopus laevis increase their synthesis of RNA, protein, and glycogen in response to extracellular insulin. Synthesis of RNA and protein are also increased when oocytes are maintained under paraffin oil and insulin is microinjected into the cytoplasm. The effects of external and intracellular insulin are additive, suggesting separate mechanisms of action. Experiments with nuclei isolated under oil show that RNA synthesis can be stimulated by applying insulin to the nucleus directly. Thus, the nucleus appears to be one intracellular site of hormone action.

Antigens specific for human lymphocytic and myeloid leukemia cells: detection by nonhuman primate antiserums.

Primate antiserums to human leukemia cells can detect antigens specific for lymphocytic leukemia cells or antigens present on certain myeloid leukemia cells. The antigen specific for lymphocytic leukemia cells is destroyed by treatment with neuraminidase or trypsin. Tryptic digests of lymphocytic leukemia cells contain the antigen, which has a high molecular weight.

Actinomycin D: inhibition of respiration and glycolysis.

Actinomycin D inhibited respiration and anaerobic glycolysis of human leukemic leukocytes and lowered the adenosine triphosphate content of the cells. Inhibitory effects on respiration and on RNA synthesis could not be dissociated from one another over a wide range of drug concentrations. Actinomycin D also impaired protein synthesis, probably by decreasing the availability of adenosine triphosphate and by inhibiting messenger RNA.

Ingestion of crude oil: sublethal effects in herring gull chicks.

A single small oral dose of Kuwait or South Louisiana crude oil caused cessation of growth, osmoregulatory impairment, and hypertrophy of hepatic, adrenal, and nasal gland tissue in herring gull chicks living in a simulated marine environment. These findings suggest that ingesting crude oil causes multiple sublethal effects that might impair a bird's ability to survive at sea.

Comparing the Standard and Electronic Versions of the Alzheimer's Disease Assessment Scale - Cognitive Subscale: A Validation Study.

The Alzheimer's Disease Assessment Scale (ADAS-Cog) has become the de facto gold-standard for assessing the efficacy of putative anti-dementia treatments. There has been an increasing interest in providing greater standardization, automation, and administration consistency to the scale. Recently, electronic versions of the ADAS-Cog (eADAS-Cog) have been utilized in clinical trials and demonstrated significant reductions in frequency of rater error as compared to paper. In order to establish validity of the electronic version (eADAS-Cog), 20 subjects who had received a diagnosis of probable Alzheimer's disease (AD) at a private US Memory Clinic completed a single-center, randomized, counterbalanced, prospective trial comparing a version of the eADAS-Cog to the standard paper scale. Interclass Correlation Coefficient on total scores and Kappa analysis on domain scores yielded high agreement (0.88 - 0.99). Effects of order and mode of administration on ADAS-Cog total scores did not demonstrate a significant main effect. Overall, this study establishes adequate concurrent validity between the ADAS-Cog and eADAS-Cog among an adult population with diagnosed AD.

Membrane-bound immunoglobulins on human leukemic cells. Evidence for humoral immune responses of patients to leukemia-associated antigens.

Immunoglobulins were detected on the membranes of human leukemic cells by a microcytotoxicity technique. A significant percentage of lymphocytes from normal donors failed to react with goat antisera to human heavy chain determinants or to lambda-light chains. Lymphocytes from some normal donors, however, did react with antisera to k-light chains. A high percentage (50-90) of cells from some leukemia patients were killed by antisera to light chains and by one or more antisera to heavy chain determinants. Trypsin treatment of leukemic cells resulted in a loss of cytotoxic activity with all immunoglobulin antisera. Reactivity with the k-light chain antiserum was detectable 2 h after trypsinization of chronic myeloid leukemic (CML) cells and 8 h after treatment of acute lymphocytic leukemic (ALL) cells. Reactivity with the antisera to heavy chain determinants and lambda-light chains could not be detected 8 and 48 h after trypsinization of CML and ALL cells, respectively. The cytotoxic activity of the immunoglobulin antisera to heavy chains was abolished by absorption with the specific immunoglobulin used to define the antisera by precipitation. Eluates (pH 3.2) prepared from leukemic cells which reacted by cytotoxicity with the immunoglobulin antisera were shown to contain immunoglobulins of different heavy chain classes. In addition, some of the eluates had cytotoxic antibody activity to human leukemia cells. The specificity of the eluted antibodies is similar to the specificity previously described for cytophilic antibodies from leukemic patients and nonhuman primate antisera to human leukemia cells. The possible in vitro detection and in vivo significance of the eluted non-complement-fixing antibodies is considered.

Annual report to the nation on the status of cancer, 1973-1996, with a special section on lung cancer and tobacco smoking.

BACKGROUND: The American Cancer Society, the National Cancer Institute (NCI), and the Centers for Disease Control and Prevention (CDC), including the National Center for Health Statistics (NCHS), provide the second annual report to the nation on progress in cancer prevention and control, with a special section on lung cancer and tobacco smoking. METHODS: Age-adjusted rates (using the 1970 U.S. standard population) were based on cancer incidence data from NCI and underlying cause of death data compiled by NCHS. The prevalence of tobacco use was derived from CDC surveys. Reported P values are two-sided. RESULTS: From 1990 through 1996, cancer incidence (-0.9% per year; P = .16) and cancer death (-0.6% per year; P = .001) rates for all sites combined decreased. Among the 10 leading cancer incidence sites, statistically significant decreases in incidence rates were seen in males for leukemia and cancers of the lung, colon/rectum, urinary bladder, and oral cavity and pharynx. Except for lung cancer, incidence rates for these cancers also declined in females. Among the 10 leading cancer mortality sites, statistically significant decreases in cancer death rates were seen for cancers of the male lung, female breast, the prostate, male pancreas, and male brain and, for both sexes, cancers of the colon/rectum and stomach. Age-specific analyses of lung cancer revealed that rates in males first declined at younger ages and then for each older age group successively over time; rates in females appeared to be in the early stages of following the same pattern, with rates decreasing for women aged 40-59 years. CONCLUSIONS: The declines in cancer incidence and death rates, particularly for lung cancer, are encouraging. However, unless recent upward trends in smoking among adolescents can be reversed, the lung cancer rates that are currently declining in the United States may rise again.

Abnormalities of fragile histidine triad genomic and complementary DNAs in cervical cancer: association with human papillomavirus type.

BACKGROUND: Chromosome 3p14.2 contains FRA3B, the most active chromosome breakage site in the human genome. The fragile histidine triad (FHIT) gene, a putative tumor suppressor gene, overlaps FRA3B. Human papillomavirus (HPV), a known cofactor in cervical carcinogenesis, can integrate into FRA3B. We examined abnormalities in FHIT and its RNA transcripts in cervical cancer cell lines and tumors. We also investigated the relationship between loss of heterozygosity (LOH) in FHIT/FRA3B and the presence of oncogenic HPV types. METHODS: Eleven cell lines, 40 tumors (20 fresh and 20 archival), and 10 normal cervical epithelia were examined. Two intragenic polymorphic markers (D3S1300 and D3S4103) and the polymerase chain reaction (PCR) were used to examine FHIT LOH. Reverse transcription-PCR (RT-PCR) analysis and single-strand conformation polymorphism analysis of RT-PCR products were used to characterize FHIT transcripts. Oncogenic HPV types were identified by PCR, using general and type-specific primers. RESULTS: All normal epithelia, 19 of 20 fresh tumors and nine of 11 cell lines expressed wild-type and, occasionally, exon 8-deleted FHIT transcripts. Additional aberrant FHIT transcripts were seen in nine of 20 fresh tumors and in seven of 11 cell lines. DNA sequencing of the aberrant transcripts revealed a variety of insertions and deletions but no point mutations. Three cell lines also had homozygous FHIT deletions. Oncogenic HPV types (i.e., 16, 18, 31, and 33) were detected in 18 of 20 archival tumors, and, in these tumors, LOH within FHIT was identified in nine of 16 informative cases. HPV 16 was found to be associated with LOH in the FHIT/FRA3B region (P = .041). CONCLUSION: FHIT/FRA3B is frequently altered in cervical cancer, demonstrating LOH, occasional homozygous deletions, and frequent aberrant transcripts not found in normal epithelia. However, the presence of wild-type transcripts and the lack of protein-altering point mutations raise questions about FHIT's function as a classic tumor suppressor gene in cervical tissue.

Behavioral deficits induced by third-trimester equivalent alcohol exposure in male C57BL/6J mice are not associated with reduced adult hippocampal neurogenesis but are still rescued with voluntary exercise.

Prenatal alcohol exposure can produce permanent alterations in brain structure and profound behavioral deficits. Mouse models can help discover mechanisms and identify potentially useful interventions. This study examined long-term influences of either a single or repeated alcohol exposure during the third-trimester equivalent on survival of new neurons in the hippocampus, behavioral performance on the Passive avoidance and Rotarod tasks, and the potential role of exercise as a therapeutic intervention. C57BL/6J male mice received either saline or 5g/kg ethanol split into two s.c. injections, two hours apart, on postnatal day (PD)7 (Experiment 1) or on PD5, 7 and 9 (Experiment 2). All mice were weaned on PD21 and received either a running wheel or remained sedentary from PD35-PD80/81. From PD36-45, mice received i.p. injections of 50mg/kg bromodeoxyuridine (BrdU) to label dividing cells. Behavioral testing occurred between PD72-79. Number of surviving BrdU+ cells and immature neurons (doublecortin; DCX+) was measured at PD80-81. Alcohol did not affect number of BrdU+ or DCX+ cells in either experiment. Running significantly increased number of BrdU+ and DCX+ cells in both treatment groups. Alcohol-induced deficits on Rotarod performance and acquisition of the Passive avoidance task (Day 1) were evident only in Experiment 2 and running rescued these deficits. These data suggest neonatal alcohol exposure does not result in long-term impairments in adult hippocampal neurogenesis in the mouse model. Three doses of ethanol were necessary to induce behavioral deficits. Finally, the mechanisms by which exercise ameliorated the neonatal alcohol induced behavioral deficits remain unknown.

The effect of freeze-drying on the quaternary structure of L-asparaginase from Erwinia carotovora.

L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Erwinia carotovora undergoes extensive dissociation from active tetramer to inactive monomers when freeze-dried. The monomeric state is stabilized by reconstitution of the freeze-dried enzyme with buffers of high pH and high ionic strength. Some compounds, particularly sugars and sugar derivatives, prevent dissociation on freeze-drying, whereas others, such as urea and chaotropic ions, increase dissociation. The effects of additives are not related to water retention. The dissociation is completely reversible on reconstitution at neutral pH, but the alkali-stabilized monomer only partially reassociates when the pH is brought back to neutrality.

Multiple effects of mercuric chloride on hexose transport in Xenopus oocytes.

HgCl(2) had both stimulatory and inhibitory effects on [(3)H]2-deoxyglucose (DG) uptake in Xenopus laevis oocytes. The Hg dose response was complex, with 0.1-10 microM Hg increasing total DG uptake, 30-50 microM Hg inhibiting, and concentrations >100 microM increasing uptake. Analyses of the effects of Hg on DG transport kinetics and cell membrane permeability indicated that low concentrations of Hg stimulated mediated uptake, intermediate concentrations inhibited mediated uptake, but high Hg concentrations increased non-mediated uptake. 10 microM Hg increased the apparent V(max) for DG uptake, but caused little or no change in apparent K(m). Phenylarsine oxide prevented the increase in DG uptake by 10 microM Hg, suggesting that the increase was due to transporter recruitment. Microinjecting low doses of HgCl(2) into the cell increased mediated DG uptake. Higher intracellular doses of Hg increased both mediated and non-mediated DG uptake. Both insulin and Hg cause cell swelling in isotonic media and, for insulin, this swelling has been linked to the mechanism of hormone action. Osmotically swelling Xenopus oocytes stimulated DG transport 2-5-fold and this increase was due to an increased apparent V(max). Exposing cells to 10 microM Hg or 140 nM insulin both increased cellular water content by 18% and increased hexose transport 2-4-fold. These data indicate that low concentrations of Hg and insulin affect hexose transport in a similar manner and that for both an increase cellular water content could be an early event in signaling the increase in hexose transport.

Comparative study on conformational stability and subunit interactions of two bacterial asparaginases.

The denaturation and reconstitution of Erwinia carotovora and Escherichia coli L-asparaginases has been followed by optical rotatory dispersion, circular dichroism and analytical ultracentrifugation. Denaturation in urea results in dissociation of the native enzyme (mol. wt. 140 000 approx.) to produce unfolded subunits (mol. wt. 35 000 approx.); the Erwinia L-asparaginase subunits can be refolded by dilution or dialysis in alkaline conditions, pH 10.5, without aggregation to the active tetramer, to give a rather unstable solution of a monomer possibly in equilibrium with dimer. These alkaline-reconstituted subunits undergo a conformational change to a more ordered state in the presence of sodium dodecylsulphate, similar to those produced by the action of sodium dodecylsulphate on the native enzyme. If the denatured subunits are reconstituted in the pH range 5.0-7.5, the enzymically active tetramer is reformed in up to 80% yield, depending upon the conditions of temperature and concentration. Kinetic data for these various transitions suggest that dissociation is a rate-limiting step while conformational changes of the polypeptide chains are relatively much more rapid. The possible significance of these different rates of change to therapeutic considerations is discussed.