Tritrichomonas foetus induces apoptotic cell death in bovine vaginal epithelial cells.

Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis, a sexually transmitted disease leading to infertility and abortion. T. foetus infects the mucosal surfaces of the reproductive tract. Infection with T. foetus leads to apoptotic cell death of bovine vaginal epithelial cells (BVECs) in culture. An affinity-purified cysteine protease (CP) fraction yielding on sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band with an apparent molecular mass of 30 kDa (CP30) also induces BVEC apoptosis. Treatment of CP30 with the protease inhibitors TLCK (Nalpha-p-tosyl-l-lysine chloromethyl ketone) and E-64 [l-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane] greatly reduces induction of BVEC apoptosis. Matrix-assisted laser desorption ionization-time-of-flight MALDI-TOF mass spectrometry analysis of CP30 reveals a single peak with a molecular mass of 23.7 kDa. Mass spectral peptide sequence analysis of proteolytically digested CP30 reveals homologies to a previously reported cDNA clone, CP8 (D. J. Mallinson, J. Livingstone, K. M. Appleton, S. J. Lees, G. H. Coombs, and M. J. North, Microbiology 141:3077-3085, 1995). Induction of apoptosis is highly species specific, since the related human parasite Trichomonas vaginalis and associated purified CPs did not induce BVEC death. Fluorescence microscopy along with the Cell Death Detection ELISA(PLUS) assay and flow cytometry analyses were used to detect apoptotic nuclear condensation, DNA fragmentation, and changes in plasma membrane asymmetry in host cells undergoing apoptosis in response to T. foetus infection or incubation with CP30. Additionally, the activation of caspase-3 and inhibition of cell death by caspase inhibitors indicates that caspases are involved in BVEC apoptosis. These results imply that apoptosis is involved in the pathogenesis of T. foetus infection in vivo, which may have important implications for therapeutic interference with host cell death that could alter the course of the pathology in vivo.

Binding specificity of avian heat shock protein 108.

Chicken heat shock protein 108 (HSP108), the avian homolog of GRP94, was originally isolated from hen oviduct and binds Fe-ovotransferrin (Fe-OTf). The liver is also a rich source, and liver membranes bind Fe-OTf with a KD of 1.7 x 10(-7) M, a value similar to oviduct membranes. A competition assay, based on the binding of 125I-Fe-OTf to liver membranes, was utilized to examine the binding specificity of HSP108. Ovalbumin and avidin competed effectively, with KD's of 1.8 x 10(-7) M and 1.4 x 10(-7) M, respectively. Iron-free OTf bound with a 10-fold higher KD. Egg white lysozyme, chicken IgG, human transferrin, rabbit muscle actin, and porcine insulin do not bind. Neither do denatured ovalbumin or ovalbumin tryptic peptides. Thus, the binding activity of HSP108 is not restricted to Fe-OTf, nor is it universal.

Structure of human transferrin receptor oligosaccharides: conservation of site-specific processing.

The human transferrin receptor (TfR) has three N-linked oligosaccharides. A combination of site-directed mutagenesis and carbohydrate and protein chemistry was used to characterize the structures of the N-linked oligosaccharides and to map their locations. We find that the type of oligosaccharide at each position was unique for that particular site. Human TfR isolated from placentae was used to characterize the structure of the oligosaccharides found in the native TfR. Following digestion of purified TfR with trypsin, individual peptides were obtained via RP-HPLC and were assayed for monosaccharides by strong acid hydrolysis and HPAE-PAD. Peptides containing carbohydrate were subjected to amino acid sequencing to identify the specific Asn residue. The oligosaccharides at Asn 251 are of the complex type. HPAE-PAD and FACE analysis suggests that they are triantennary and trisialylated with core fucosylation. The glycopeptide containing the site at Asn 317 was obtained after limited tryptic digestion and RP-HPLC. FACE analysis reveals predominantly a family of sialylated hybrid oligosaccharides. The consensus sequences for each N-linked site were mutated in various combinations and the resultant TfRs expressed in mouse 3T3 cells. Endoglycosidase H digestion of the mutated TfRs indicates that the pattern of oligosaccharides is consistent with the type of oligosaccharides found at each position in human tissue and the glycosylation of one site does not directly affect the glycosylation of other sites. Previous studies indicated that the oliosaccharide at Asn 727 was high-mannose type [Hayes, G. R., et al. (1995) Glycobiology 5, 227-232]. These results indicate that the type of oligosaccharide found at each site is most dependent on the environment surrounding it.

The critical glycosylation site of human transferrin receptor contains a high-mannose oligosaccharide.

The human transferrin receptor (TfR) contains three N-linked oligosaccharides and glycosylation is required for the proper folding and function of the molecule. Earlier studies demonstrated that the oligosaccharide at Asn-727 is vital for the production of fully active TfR. The oligosaccharide(s) present at this site have been analysed using a combination of site-directed mutagenesis and chemical analysis. Wild-type TfR and mutants containing only the Asn-727 site or missing all three sites were transfected into mouse 3T3 cells and receptors were analysed by endo-N-acetylglucosaminidase H (Endo-H) digestion, SDS-PAGE and immunoblotting. These studies suggested that the Asn-727 site contains high-mannose or Endo-H-sensitive hybrid oligosaccharides. Glycosylation of Asn-727 found in the TfR purified from human placentae was analysed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and mass spectrometry following tryptic digestion, peptide purification via reverse-phase high-performance liquid chromatography (RP-HPLC) and peptide sequencing. HPAE-PAD showed the presence of a series of high-mannose oligosaccharides. Mass spectrometry confirmed these observations, but also showed the presence of an 80 Da anionic moiety on a fraction of the oligosaccharides.

A chicken transferrin binding protein is heat shock protein 108.

A previously described transferrin binding protein from chicken oviduct has been identified as a heat shock protein, HSP108, by microsequencing of RP-HPLC purified tryptic peptides. The protein purified from oviduct by SDS-PAGE or from liver by ovotransferrin-affinity chromatography reacts with a monoclonal antibody raised against HSP108. The cDNA sequence predicts of KDEL peptide at the carboxyl terminus, but the protein does not react with anti-KDEL monoclonal antibodies, suggesting that HSP108 is processed in a way that eliminates the KDEL epitope.

Identification of the O-linked glycosylation site of the human transferrin receptor.

The human transferrin receptor is a glycoprotein containing three N-linked and one O-linked glycosylation sites. Tryptic digestion of the receptor, followed by chromatography on BioGel P-2 and reverse-phase HPLC, yields a glycopeptide (amino acids 101-120) containing the O-linked site. Amino acid sequence analysis reveals that the site of O-glycosylation is Thr-104. Mass spectral analysis is consistent with the presence of a Gal-GalNAc core with predominantly two sialic acid residues.

Intermolecular phosphorylation of insulin receptor as possible mechanism for amplification of binding signal.

Clustering of cell-surface insulin receptors has led to the speculation that intermolecular phosphorylation of unoccupied receptors catalyzed by ligand-occupied receptors within the cluster could be a mechanism by which the insulin-binding signal is amplified. We examined whether insulin receptors can be phosphorylated by an intermolecular mechanism. In this study, we used highly purified insulin receptors isolated from rat liver plasma membranes and human placental membranes. Rat liver insulin receptors were "activated" by incubation with 10 nM insulin in the presence of ATP. Subsequent to removal of insulin by immunodepletion, these receptors were used as an enzyme source to study phosphorylation of unphosphorylated "substrate" human receptors. Initially, we found no evidence that the addition of activated rat receptors increased phosphorylation of human receptors, when assessed by immunoprecipitation with a human-specific monoclonal antibody. To examine the possibility that these negative results were due to insufficient receptor concentration, activated human receptors were mixed with unphosphorylated substrate receptors at concentrations up to 60 micrograms/ml. In this study, we found that addition of activated receptors resulted in increased phosphorylation of the substrate receptors at the highest concentrations employed. These are the first data indicating that insulin receptors per se are capable of intermolecular phosphorylation. In vivo, this could be the initial step in amplifying the insulin-binding signal.

Mapping of carbohydrate sites on the human insulin receptor.

Prior to investigating the role of individual glycosylation sites in insulin receptor function, we are mapping the sites of glycosylation in the receptor. We report here a generally applicable methodology for the isolation and identification of glycosylation sites in cell surface glycoproteins. Human insulin receptors were labeled with [3H]-sugars using a CHO cell line transfected with the human receptor cDNA. Labelled receptors were mixed with receptors purified from human placental membranes and tryptic peptides prepared. Peptides were fractionated by gel filtration chromatography to limit the number of non-glycopeptides present. Peptides were then separated by reverse phase HPLC and glycopeptides identified by scintillation counting. Using this technique we have shown the insulin receptor to be glycosylated at Asn 397 and Asn 881. This increase the known number of occupied glycosylation sites to five.

Alterations of glucose transporter systems in insulin-resistant uremic rats.

To further define the cellular alteration(s) involved in the impaired glucose transport associated with chronic uremia, we examined the concentration and translocation of glucose transport systems in adipocytes isolated from partially nephrectomized uremic rats. Uremic animals, compared with matched controls, had increased blood urea nitrogen and serum insulin, whereas serum glucose was unchanged. In agreement with previous work, 125I-insulin binding to its receptor was unaltered and transport of 2-deoxy-D-glucose was decreased in both the absence (basal) and presence of a maximal (7 nM) insulin concentration by 44 and 35%, respectively. To assess the movement and concentration of glucose transport systems in various membrane fractions prepared from basal and insulin-treated (20 nM) uremic fat cells, the technique of D-glucose-inhibitable cytochalasin B binding was utilized. In plasma membranes isolated from these cells the concentration of glucose transporters was decreased by 16 (P less than 0.01) and 30% (P less than 0.005) in basal and insulin-treated cells, respectively. Concomitantly, microsomal membranes prepared from uremic cells treated in the absence and presence of insulin had a 28 (P less than 0.01) and 15% (P less than 0.05) decrease in concentration of glucose transport systems, respectively. Additionally, glucose transporter concentration was significantly decreased by 17% (P less than 0.025) in total membranes prepared from uremic cells. Thus, impairment of glucose transport in uremic fat cells can be attributed to a postbinding defect that, at least in part, results from a decrease in the total concentration of glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro effects of sulfonylurea on glucose transport and translocation of glucose transporters in adipocytes from streptozocin-induced diabetic rats.

The in vitro effects of the sulfonylurea glyburide on insulin binding and action were compared in adipocytes from control and nonketotic streptozocin-induced diabetic rats. Adipose tissue from control and diabetic animals was maintained in the absence or presence of 2 micrograms/ml glyburide for 20 h. Insulin binding and insulin-stimulated glucose transport were examined in adipocytes prepared from this tissue. As expected, insulin binding was increased in adipocytes from diabetic animals. Exposure of tissue to glyburide did not influence insulin binding in either control or diabetic cells. Glucose transport activity of diabetic cells, assessed with 2-deoxyglucose, was decreased 30-40% in both the absence (basal) and presence of insulin compared with controls. Glyburide potentiated insulin's effects in both control (15-20%) and diabetic (30-40%) adipocytes. As a result, glucose transport activity in glyburide-treated diabetic cells was restored to a level similar to that of control cells not exposed to the drug. The mechanism by which glyburide potentiated glucose transport activity was examined with the D-glucose-displaceable cytochalasin B-binding technique to measure glucose-transporter concentration in membranes prepared from control and diabetic adipocytes exposed to the drug. Adipocytes from this model of diabetes are known to have a decreased cellular content of glucose transporters. The concentration of glucose transporters was decreased by 31% in plasma membranes from insulin-treated diabetic cells. There were corresponding decreases in diabetic microsomal and total membrane fractions. There was also a 40% decrease in the translocation of transporters from the microsomes to the plasma membrane in response to insulin in diabetic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Intact adipocyte insulin-receptor phosphorylation and in vitro tyrosine kinase activity in animal models of insulin resistance.

We evaluated the possibility that impaired insulin-receptor kinase activity contributes to insulin resistance by examining in vitro receptor tyrosine kinase activity and in situ receptor phosphorylation in four models of insulin resistance. Adipocytes from streptozocin-induced nonketotic diabetic (STZ-D), glucocorticoid-treated, fasted, and chronically uremic rats showed reduced basal and maximally insulin-stimulated 2-deoxy-D-glucose transport compared with matched controls. Adipocytes from these models were also resistant to stimulation of hexose transport by hydrogen peroxide, a postbinding insulin mimicker. Changes in the number of insulin receptors per cell could not account for these alterations in transport. Cell surface 125I-labeled insulin binding was 142% of control in STZ-D and 129% with fasting and unchanged in glucocorticoid excess and chronic uremia. Insulin-stimulated tyrosine kinase was measured by means of a synthetic substrate, Glu80Tyr20. Partially purified receptors from these resistant models had unaltered kinase activity when normalized to soluble 125I-insulin binding. In situ stimulation of receptor phosphorylation by 7 and 100 nM insulin was determined after equilibration of adipocytes with 32PO4. Compared with matched controls, these intact cells, from all four resistant models, had insulin-stimulated receptor phosphorylation that was unchanged per unit of cell surface binding. Similar to results with insulin, hydrogen peroxide stimulation of in situ receptor phosphorylation was unchanged in each model. Thus, both in vitro and in situ measures of receptor phosphorylation suggest that the cellular alterations leading to insulin resistance in these adipocytes resides beyond phosphorylation of the insulin receptor.

Role of insulin receptor phosphorylation in the insulinomimetic effects of hydrogen peroxide.

The oxidant H2O2 has many insulin-like effects in rat adipocytes. To determine whether these effects could be mediated by the tyrosine kinase activity of the insulin receptor, the ability of H2O2 to stimulate receptor phosphorylation in intact adipocytes and partially purified insulin receptors has been examined. Phosphorylation of the beta subunit of the insulin receptor was increased approximately 2-fold by treatment of intact cells with 3 mM H2O2, a concentration that maximally stimulates 2-deoxyglucose uptake. Stimulation of receptor phosphorylation was rapid, reaching maximal levels within 5 min, and preceded activation of glucose transport. Phosphoamino acid analysis of insulin receptors from H2O2-treated adipocytes showed that 32P incorporation into phosphotyrosine and phosphoserine residues of the beta subunit was enhanced. Furthermore, partially purified receptors from H2O2-treated cells exhibit increased tyrosine kinase activity, as measured by phosphorylation of the peptide Glu80Tyr20. In contrast, the direct addition of H2O2 to partially purified insulin receptors did not stimulate tyrosine kinase activity or insulin receptor autophosphorylation. This was not due to breakdown of H2O2 or oxidation of ATP or the required divalent cations. To define the factors involved in H2O2's effect, we have examined receptor phosphorylation in fat cell homogenates and purified plasma membranes. Although insulin stimulated receptor phosphorylation in both of these systems, H2O2 was only effective in the cell homogenates. These data demonstrate that, under certain conditions, H2O2 stimulates insulin receptor phosphorylation and tyrosine kinase activity, suggesting that the insulin-like effects of H2O2 may be mediated by stimulation of insulin receptor phosphorylation. This does not appear to be a direct effect of H2O2 on the insulin receptor and requires nonplasma membrane cellular constituents.

Effect of chlorpropamide on glucose transport in rat adipocytes in the absence of changes in insulin binding and receptor-associated tyrosine kinase activity.

In an attempt to elucidate the cellular mechanism(s) by which sulfonylureas exert their extrapancreatic hypoglycemic effects, various parameters of insulin action were examined in vitro, using rat adipocytes maintained in a biochemically defined medium. Cells were maintained for 20 hours in the absence or presence of 175 micrograms/mL chlorpropamide and insulin binding, hexose transport, glucose metabolism, and insulin receptor tyrosine kinase activity were compared. Chlorpropamide treatment had no effect on insulin binding, altering neither receptor number nor affinity. However, the sulfonylurea did enhance 2-deoxyglucose transport in both the absence (17%, P less than .01) and presence (20%, P less than .01) of insulin. Furthermore, glucose metabolism as measured by the conversion of glucose (0.2 mmol/L) to CO2 and total lipids was also significantly increased by chlorpropamide treatment in both the absence (30%, P less than .01) and presence (31%, P less than .05) of insulin. Potentiation of insulin-stimulated transport or metabolism was not explained by an increase in the basal state alone because the incremental responses to 40 ng/mL insulin were potentiated by 19% (P less than .01) and 25% (P less than .05), respectively. Activity of the insulin receptor kinase was unchanged as evaluated by autophosphorylation of partially purified receptors, phosphorylation of an artificial substrate and by phosphorylation of the receptor in situ. These studies demonstrate that the sulfonylurea, chlorpropamide, stimulates glucose transport and potentiates insulin's effect on this process by acting at a site(s) beyond insulin receptor binding and phosphorylation.

Effects of metformin on insulin receptor tyrosine kinase activity in rat adipocytes.

The cellular mechanism(s) by which the biguanide, metformin, exerts its antihyperglycaemic effect was investigated. Rat adipocytes were either treated acutely (2 h) or maintained in a biochemically defined medium (20 h) in the presence or absence of metformin (1 X 10(-4) mol/l). Exposure to the drug resulted in a significant enhancement (p less than 0.01) of hexose transport in both the absence (basal) and presence of insulin. Stimulation of transport was not explained by the increase in the basal state alone, since the incremental response to maximally effective concentrations of insulin was significantly enhanced p less than 0.025. Insulin-receptor tyrosine kinase activity was examined under the same experimental conditions. Activity of the kinase was unaltered as evaluated by phosphorylation of an artificial substrate and by phosphorylation of the receptor in situ. Furthermore, in this investigation neither insulin receptor number nor affinity was changed in adipose tissue treated with metformin. These studies indicate that metformin potentiates the effect of insulin on glucose transport at a site(s) beyond insulin receptor binding and phosphorylation.

The role of cell surface sialic acid in insulin receptor function and insulin action.

Removal of cell surface sialic acid from adipocytes with neuraminidase inhibits insulin action. Here, we have examined the effects of mild neuraminidase treatment (5 milliunits/ml, 12 degrees C, 15 min) on insulin receptor structure and function. Neuraminidase treatment sufficient to cause greater than 90% loss of insulin stimulatable lipogenesis had no effect on 125I-insulin binding, tyrosine kinase activity of partially purified insulin receptors, insulin receptor phosphorylation in intact cells, or insulin-induced receptor internalization. However, recycling of the insulin receptor to the plasma membrane was inhibited by 50%. Recycled receptors in neuraminidase-treated cells were unable to mediate insulin action in contrast to recycled receptors from non-neuraminidase-treated cells. Furthermore, when insulin receptors were protected from exposure to neuraminidase, by inducing receptor internalization prior to neuraminidase treatment, the cells were still unable to respond to insulin. Analysis of the alpha and beta subunits of the receptor from neuraminidase-treated cells, affinity-labeled with 125I-insulin, or labeled by autophosphorylation, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis failed to indicate any changes in the holoreceptor or the individual subunits. This suggests there was no detectable release of sialic acid from the receptor. From this data we conclude that loss of sialic acid from nonreceptor glycoconjugates leads to loss of insulin action and inhibition of receptor recycling. The insulin receptor does not appear to be involved in this inhibitory effect. These findings suggest that an uncharacterized plasma membrane glycoprotein is essential in transmitting the "signal" of insulin binding to the cellular effector system.

Insulin receptor cycling and insulin action in the rat adipocyte.

The possibility that the insulin receptor of adipocytes undergoes cycling was examined by a method involving pronase digestion at 12 degrees C, followed by insulin binding studies to determine receptor location and quantity. In the absence of insulin treatment, the amount of internal receptors (i.e. protected from pronase) was 10% of total receptor content. Following a 30-min insulin treatment (0.1 microM) at 37 degrees C, the internal receptor content increased 2-fold (206 +/- 12% of control, 100%). This effect was rapid, and maximum internalization was approached by 5 min of insulin treatment. Warming pronase-digested cells to 37 degrees C allowed the internal receptors to move to the cell surface. This movement was rapid also, and expansion of the internal pool by insulin pretreatment provided a 2.4-fold increase in the reinsertion of cell-surface receptors (238 +/- 28% of nontreated cells, 100%). Insulin-pretreated and nontreated cells had approximately 13 and 6%, respectively, of their original cell-surface receptor content, i.e. their content before pronase digestion. These receptors appeared intact after the cycling process, as judged by affinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the receptor and its binding subunit. The ability of the recycled receptor to respond to insulin was examined by studies of glucose incorporation into lipids and the inhibition of isoproterenol-stimulated lipolysis. Cells pretreated with insulin and allowed to recycle (e.g. 13% of normal receptor content) were 2-3-fold more responsive and 7-fold more sensitive to subsequent insulin stimulation than nontreated cells (e.g. 6% of normal receptor content), indicating that the recycled receptors are biologically active and coupled to cellular effector systems.

How many Samoans? An evaluation of the 1980 census count of Samoans in the United States.

"This paper reports the results of applying demographic methods, in combination with assumptions about vital rates and migration, to estimate the total Samoan population of the United States in 1980." The authors conclude that the most plausible estimate of the Samoan population in the United States in 1980 is between 46,600 and 50,800. These estimates are 10 to 17.5 percent higher than the census count.

Stimulation of lipid-linked oligosaccharide assembly during oviduct differentiation.

Regulation of dolichyl phosphate-linked oligosaccharide assembly has been studied during the course of diethylstilbestrol-induced chick oviduct differentiation. Oviduct membranes from treated chicks form 4.6 times as much GlcNAc-P-P-Dol and GlcNAc2-P-P-Dol upon incubation with UDP-[14C]GlcNAc and MgCl2 than do membranes from untreated chicks. Assembly of oligosaccharide-lipid was studied by incubation of membranes with purified exogenous [14C]GlcNAc2-P-P-Dol and GDP-Man. Man transfer required a divalent cation (10 mM Mg2+) and detergent (0.5% Nonidet P-40 is optimal) and occurs in the presence of amphomycin (500 micrograms/ml). The apparent Km for GDP-Man is 1 microM and for [14C]GlcNAc2-P-P-Dol is 0.45 microM. The products are a series of sequentially formed dolichyl pyrophosphate-linked saccharides up to Man5GlcNAc2, the first of which is Man beta 1,4GlcNAc2. The same products are formed either in the presence or absence of amphomycin. Conversion of GlcNAc2-P-P-Dol to higher oligosaccharides is stimulated 3-fold by estrogen treatment of chicks. Similarly, the conversion of partially purified exogenously added Man beta-[14C]GlcNAc2-P-P-Dol is 4.6-fold higher after diethylstilbestrol treatment.

Microcomputers and minipopulations: the 1981 Cook Islands census.

PIP: This paper presents some of the background behind the installation and use of a microcomputer for census processing and briefly reviews the results of the 1981 census in the light of the Cook Islands' special demographic situation. Since independence from New Zealand in 1965, the Cook Islands has gradually developed a capacity for statistics gathering in general and census taking in particular. The 1981 quinquennial census represents a watershed in the development of local personnel without the aid of external advisors; it was the 1st census to be processed within the Cook Islands by means of a computer. Both the installation of the microcomputer and the processing of the 1981 census are counted as successes. This is due to a number of factors, namely: a long period of careful planning procedures which preceded the choice of system and installation; the enthusiasm of the local staff closely involved in the planning; the emphasis on training; tests of the equipment before installation by computer practitioners with knowledge of local needs and capacities; reasonable goal setting; and the supply, with the system, of adequate spare parts tools and maintenance and training manuals. Like many island populations, that of the Cook Islands is characterized by considerable instability as illustrated by the 1981 census results. As the rate and direction of population change varies from island to island, generalizations across the nation as a whole are difficult. For example, while some of the northern atolls experienced population decline during the latest intercensal period, most had their popultion increase in 1981. All the southern islands declined during the 1976-81 period at annual rates varying from 0.6% to 3.2%. The explanation for these patterns of change lies primarily in the different rates and direction of external migration on each island, as Cook Islanders are exempt from immigration regulations to New Zealand and Australia. In recent years, women have formed a majority of the total emigration stream, affecting the sex ratio which increased from 105 in 1976 to 107 in 1981. When combined with birth registration data, the 1981 census results confirm that the fertility transition is well under way in the Cook Islands. This pattern appears to support the argument that family planning programs can bring down fertility independently of significant changes in systems of production. Nevertheless, family planning services alone cannot explain the decline in fertility; women's status has been improving and the circulation of population between the Cook Islands and New Zealand has undoubtedly helped to diffuse modern norms with respect to family size, the deliberate control of conception, and the active participation of women in reproductive decision-making. The general pattern of rapid population decline could have important consequences for the Cook Islands' economy and society and raises questions as to the economic viability of some of these small and remote island communities.^ieng