Estrogen therapy influence on periurethral vessels in postmenopausal incontinent women using Dopplervelocimetry analysis.

UNLABELLED: Lack of estrogen affects the urinary tract mainly by diminishing vascular, muscular and epithelial trophism, resulting in negative effects on continence in postmenopausal women. Therefore, the use of estrogens in these patients may revert these alterations and lead to an expressive improvement of the urinary symptoms. OBJECTIVE: Study the effect of topical estrogen therapy (conjugated equine estrogens, estriol or promestriene) in periurethral vessels detected by Dopplervelocimetric analysis using, as parameters: the number of vessels, resistance and pulsatility indexes, as well as the minimum diastolic value. METHODS: Forty-one postmenopausal women with stress urinary incontinence were randomized into three groups according to different types of topical estrogen received during 3 months. Group 1 received conjugated equine estrogens, group 2 received estriol and group 3 received promestriene. Periurethral Dopplervelocimetry analysis was done before estrogen administration and during treatment in all groups. RESULTS: We observed an increase in the number of the periurethral vessels in group 1 and group 2, being higher in group 1 than in group 2. The pulsatility index remained unchanged in all three groups. The resistance index at the periurethral vessels reduced only at the conjugated estrogen group (group 1). In this same group we noticed an increase in the mean minimal diastolic value, meaning a better periurethral vascularization. CONCLUSION: Topical conjugated equine estrogens and estriol were effective in increasing the number of periurethral vessels in postmenopausal women with urinary stress incontinence, with the conjugated equine estrogens being the most effective intervention studied.

Beneficial effect of pretreatment of islets with fibronectin on glucose tolerance after islet transplantation.

The scarcity of available islets is an obstacle for clinically successful islet transplantation. One solution might be to increase the efficacy of the limited islets. Isolated islets are exposed to a variety of cellular stressors, and disruption of the cell-matrix connections damages islets. We examined the effect of fibronectin, a major component of the extracellular matrix, on islet viability, mass and function, and also examined whether fibronectin-treated islets improved the results of islet transplantation. Islets cultured with fibronectin for 48 hours maintained higher cell viability (0.146 +/- 0.010 vs. 0.173 +/- 0.007 by MTT assay), and also had a greater insulin and DNA content (86.8 +/- 3.6 vs. 72.8 +/- 3.2 ng/islet and 35.2 +/- 1.4 vs. 30.0 +/- 1.5 ng/islet, respectively) than islets cultured without fibronectin (control). Absolute values of insulin secretion were higher in fibronectin-treated islets than in controls; however, the ratio of stimulated insulin secretion to basal secretion was not significantly different (206.9 +/- 23.3 vs. 191.7 +/- 20.2% when the insulin response to 16.7 mmol/l glucose was compared to that of 3.3 mmol/l glucose); the higher insulin secretion was thus mainly due to larger islet cell mass. The rats transplanted with fibronectin-treated islets had lower plasma glucose and higher plasma insulin levels within 2 weeks after transplantation, and had more favorable glucose tolerance 9 weeks after transplantation. These results indicate that cultivation with fibronectin might preserve islet cell viability, mass and insulin secretory function, which could improve glucose tolerance following islet transplantation.

Heat shock restores insulin secretion after injury by nitric oxide by maintaining glucokinase activity in rat islets.

Heat shock protein (hsp), including hsp70, has been reported to restore the glucose-induced insulin release suppressed by nitric oxide (NO). However, the mechanism underlying this recovery remains unclear. In the present study, we examine the effects, in rat islets, of heat shock on insulin secretion inhibited by a small amount of NO and also on glucose metabolism, the crucial factor in insulin release. Exposure to a higher dose (15 U/ml) of interleukin-1beta (IL-1beta) abolished the insulin release by stimulation of glucose or KCl in both control and heat shocked islets. In rat islets exposed to a lower dose (1.5 U/ml) of IL-1beta, insulin secretion in response to glucose, but not to glyceraldehydes (GA), ketoisocaproate (KIC), or KCl, was selectively impaired, concomitantly with lower ATP concentrations in the presence of 16.7 mM glucose, while such suppression of insulin secretion and ATP content was not observed in heat shock-treated islets. NO production in islets exposed to 1.5 U/ml IL-1beta was significantly, but only partly, decreased by heat shock treatment. The glucose utilization rate measurement using [5-3H]-glucose and [2-3H]-glucose and the glucokinase activity in vitro were reduced in islets treated with 1.5 U/ml IL-1beta. In heat shock-treated islets, glucose utilization and glucokinase activity were not affected by 1.5 U/ml IL-1beta. These data suggest that heat shock restores glucose-induced insulin release inhibited by NO by maintaining glucokinase activity and the glucose utilization rate in islets in addition to reducing endogenous NO production.

Evaluation of ionic pollutants of acid fog and rain using a factor analysis and back trajectories.

Fog and rain water samples were collected at the same time in the Akita Hachimantai mountain range in northern Japan from June to September in 1998 and 1999. The various ion concentrations in these samples were analyzed, and the fog droplet sizes were measured for each fog event. As the fog droplet size increased, the ion concentration decreased. The slope of log-log plots of the concentration versus the droplet size differed with the kind of ion. In order to characterize the air pollutant, moreover, these data were quantitatively analyzed by an oblique rotational factor analysis. We found that three factors were extracted as the air pollutant source: (NH4)2SO4, acids (HNO3 + H2SO4) and sea-salt. Combining the factor analysis with the 72 h back-trajectory at 850 hPa level, we found that the contribution of each factor varied with the transport pattern of air masses.

Augmentation of basal insulin release from rat islets by preexposure to a high concentration of glucose.

We have found that preexposure to an elevated concentration of glucose reversibly induces an enhancement of basal insulin release from rat pancreatic islets dependent on glucose metabolism. This basal insulin release augmented by priming was not suppressed by reduction of the intracellular ATP or Ca(2+) concentration, because even in the absence of ATP at low Ca(2+), the augmentation was not abolished from primed electrically permeabilized islets. Moreover, it was not inhibited by an alpha-adrenergic antagonist, clonidine. A threshold level of GTP is required to induce these effects, because together with adenine, mycophenolic acid, a cytosolic GTP synthesis inhibitor, completely abolished the enhancement of basal insulin release due to the glucose-induced priming without affecting the glucose-induced increment in ATP content and ATP-to-ADP ratio. In addition, a GDP analog significantly suppressed the enhanced insulin release due to priming from permeabilized islets in the absence of ATP at low Ca(2+), suggesting that the GTP-sensitive site may play a role in the augmentation of basal insulin release due to the glucose-induced priming effect.

PQBP-1/Npw38, a nuclear protein binding to the polyglutamine tract, interacts with U5-15kD/dim1p via the carboxyl-terminal domain.

PQBP-1 was identified as a binding protein to the polyglutamine tract present in various transcription-related factors and causative genes for neurodegenerative disorders. This novel gene contains at least two functional domains, WW domain and carboxyl-terminal domain (CTD), strictly conserved beyond species. Although human PQBP-1 additionally contains the polar amino acid-rich domain by which it binds to the polyglutamine tract, genuine physiological function(s) have not been clarified. In this study, we showed that U5-15kD, human homologue of fission yeast dim1p, is a partner molecule of PQBP-1 binding to CTD. This finding suggests physiological functions of PQBP-1 in splicing, cell cycle, and ubiquitination, through which we can speculate the pathological roles of PQBP-1 in triplet repeat diseases.

The insulinotropic mechanism of the novel hypoglycaemic agent JTT-608: direct enhancement of Ca(2+) efficacy and increase of Ca(2+) influx by phosphodiesterase inhibition.

We examined the effects of the novel hypoglycaemic agent JTT-608 [trans-4-(4-methylcyclohexyl)-4-oxobutyric acid] on insulin secretion using rat pancreatic islets, and analysed the mechanism of its effect. JTT-608 augmented 8.3 mM glucose-induced insulin secretion dose-dependently, and there was a stimulatory effect of 100 microM JTT-608 at both moderate and high concentrations (8.3, 11. 1 and 16.7 mM) of glucose, but not at low concentrations (3.3 and 5. 5 mM). In perifusion experiments, both phases of insulin release were enhanced, and the effect was eliminated 10 min after withdrawal of the agent. In the presence of 200 microM diazoxide and a depolarizing concentration (30 mM) of K(+), there was an augmentation of insulin secretion by 100 microM JTT-608, not only under high levels of glucose but also under low levels, and the effects were abolished by 10 microM nitrendipine. JTT-608 augmented insulin secretion from electrically permeabilized islets in the presence of stimulatory concentrations (0.3 and 1.0 microM) of Ca(2+), and the intracellular Ca(2+) concentration ([Ca(2+)](i)) response under 16.7 mM glucose, 200 microM diazoxide, and 30 mM K(+) was also increased. The cyclic AMP content in the islets was increased by 100 microM JTT-608, and an additive effect to 1 microM forskolin was observed, but not to 50 microM 3-isobutyl-1-methylxanthine (IBMX). JTT-608 inhibited phosphodiesterase (PDE) activity dose-dependently. We conclude that JTT-608 augments insulin secretion by enhancing Ca(2+) efficacy and by increasing Ca(2+) influx. This appears to be a result of the increased intracellular cyclic AMP concentration due to PDE inhibition.

Glucose intolerance caused by a defect in the entero-insular axis: a study in gastric inhibitory polypeptide receptor knockout mice.

Mice with a targeted mutation of the gastric inhibitory polypeptide (GIP) receptor gene (GIPR) were generated to determine the role of GIP as a mediator of signals from the gut to pancreatic beta cells. GIPR-/- mice have higher blood glucose levels with impaired initial insulin response after oral glucose load. Although blood glucose levels after meal ingestion are not increased by high-fat diet in GIPR+/+ mice because of compensatory higher insulin secretion, they are significantly increased in GIPR-/- mice because of the lack of such enhancement. Accordingly, early insulin secretion mediated by GIP determines glucose tolerance after oral glucose load in vivo, and because GIP plays an important role in the compensatory enhancement of insulin secretion produced by a high insulin demand, a defect in this entero-insular axis may contribute to the pathogenesis of diabetes.

An insulinotropic effect of vitamin D analog with increasing intracellular Ca2+ concentration in pancreatic beta-cells through nongenomic signal transduction.

The effect of 1alpha,25-dihydroxylumisterol3 (1alpha,25(OH)2lumisterol3) on insulin release from rat pancreatic beta-cells was measured to investigate the nongenomic action of vitamin D via the putative membrane vitamin D receptor (mVDR). 1Alpha,25(OH)2lumisterol3, a specific agonist of mVDR, dose-dependently augmented 16.7 mM glucose-induced insulin release from rat pancreatic islets and increased the intracellular Ca2+ concentration ([Ca2+]i), though not increasing Ca2+ efficacy in the exocytotic system. These effects were completely abolished by an antagonist of mVDR, 1beta,25-dihydroxyvitamin D3 (1beta,25(OH)2D3), or by a blocker of voltage-dependent Ca2+ channels, nitrendipine. Moreover, both [Ca2+]i elevation, caused by membrane depolarization, and sufficient intracellular glucose metabolism are required for the expression of these effects. 1Alpha,25(OH)2lumisterol3, therefore, has a rapid insulinotropic effect, through nongenomic signal transduction via mVDR, that would be dependent on the augmentation of Ca2+ influx through voltage-dependent Ca2+ channels on the plasma membrane, being also linked to metabolic signals derived from glucose in pancreatic beta-cells. However, further investigations will be needed to discuss physiologically the meaning of insulinotropic effects of vitamin D through mVDR.

Regulation of intracellular ATP concentration under conditions of reduced ATP consumption in pancreatic islets.

ATP is the most important factor in glucose-induced insulin secretion in pancreatic beta-cells, but examination of intracellular differences in ATP concentration is difficult because ATP production and consumption occur simultaneously. In the present study, we measured the ATP concentration under the condition of a reduced ATP requirement by omitting extracellular Ca(2+) and inhibiting Na-K ATPase. The ATP concentration in islets incubated with 16.7 mM glucose in the absence of Ca(2+) for 30 min was increased by about 1. 9-fold more than in the presence of Ca(2+). The increment was extracellular Ca(2+)-dependent, and was completely abolished by the metabolic inhibitors dinitrophenol and iodoacetic acid. The Ca channel blockers including nitrendipine and Ni(2+) did not affect the ATP concentration in islets incubated with 16.7 mM glucose in the presence of Ca(2+). However, when thapsigargin and suramin, inhibitors of Ca-ATPase at the endoplasmic reticulum, were added to Ca channel blockers in the presence of ambient Ca(2+), the intraislet ATP content was increased, similarly to that under Ca-free conditions. But thapsigargin did not further augment the ATP concentration in the islet with 16.7 mM glucose in the absence of Ca(2+). On the other hand, the suppression of Na-K ATPase by ouabain rather reduced the ATP concentration augmented by omission of extracellular Ca(2+). In addition, vanadate, a blocker of Ca-ATPase at the plasma membrane, failed to increase the ATP concentration in the islets. These data suggest that the increment of ATP concentration in the absence of Ca(2+) is attributable to the reduced ATP requirement due to stopping of the Ca-ATPase activity at the endoplasmic reticulum, and that the intracellular ATP concentration is differently regulated by Na-K ATPase at plasma membrane and by Ca-ATPase at endoplasmic reticulum.

Intracellular Mg2+ surge follows Ca2+ increase during depolarization in cultured neurons.

The intracellular magnesium and calcium concentrations in cultured dorsal root ganglion neurons were measured using a fluorescent Mg2+ indicator, Mag-Fura-2 and a Ca2+ indicator, Fura-2, respectively. The magnesium concentration in the cytoplasm was higher than that in the nuclei at rest; 0.68+/-0.10 mM (mean+/-S.E.M., n=7) in the cytoplasm and 0.11+/-0.05 mM in the nucleus. When depolarized by a 60 mM KCl solution, the magnesium concentration increased remarkably in the cytoplasm; 1.52+/-0.26 mM (n=7) in the cytoplasm and 0.25+/-0. 12 mM in the nucleus. This is in contrast to a Ca2+ increase due to depolarization in which the increase was remarkable also in the nucleus. The Mg2+ response displayed a rapid spontaneous recovery even in the presence of the high K+ solution. The Ca2+ response, on the other hand, accompanied a slow recovery 'plateau'. Simultaneous measurements of Mg2+ and Ca2+ by a double-labeling experiment revealed that the Ca2+ concentration started to rise 0.46+/-0.05 s (n=32) earlier, and it reached its peak 1.38+/-0.12 s (n=32) earlier than Mg2+. These results support the scheme of 'calcium induced magnesium release', that the depolarization-induced elevation of the Ca2+ concentration causes an increase in the Mg2+ concentration in the cytoplasm.

Nisoldipine improves the impaired erythrocyte deformability correlating with elevated intracellular free calcium-ion concentration and poor glycaemic control in NIDDM.

AIMS: To explore the mechanisms underlying the impaired erythrocyte deformability (RBC-df) in diabetic patients, the relationship between erythrocyte intracellular free calcium-ion concentration ([Ca2+]i) and RBC-df, and the effects of Ca2+-channel blocker on [Ca2+]i and RBC-df were evaluated. METHODS: Forty-eight patients with NIDDM and 24 control subjects were enrolled in this study. [Ca2+]i was determined using fura-2, and RBC-df by filtration method expressed as Deformability Index (DI). Erythrocytes were treated with nisoldipine to evaluate the effects of a Ca2+-channel blocker. RESULTS: [Ca2+]i was significantly higher (82.6 (78.0-87.2) vs 76.6 (74.3-81.2) nmol lRBC-1, P<0.001), and DI was significantly lower (0. 14 (0.09-0.28) vs 0.22 (0.16-0.28), P<0.01) in NIDDM than in controls. There was a significant correlation between HbA1c and [Ca2+]i (r=0.38, P<0.01), between HbA1c and DI (r=-0.51, P<0.01), and between [Ca2+]i and DI (r=-0.42, P<0.01). Stepwise multiple regression analysis revealed HbA1c and [Ca2+]i as independent determinants for the impaired RBC-df. Nisoldipine treatment in vitro significantly decreased [Ca2+]i, and significantly improved RBC-df. CONCLUSIONS: These data indicate that the impaired RBC-df in NIDDM may at least partly be attributed to the elevated [Ca2+]i and poor glycaemic control. In addition, favorable effects of a Ca2+-channel blocker on both [Ca2+]i and RBC-df have been demonstrated.

[Rare intra-abdominal complications of a ventriculoperitoneal shunt: report of three cases].

Three cases of rare intra-abdominal complications of ventriculoperitoneal shunt (VPS) surgery are reported. Case 1 was a 32-year-old male who had undergone VPS surgery for hydrocephalus following meningitis on July 10, 1980. Two weeks later he developed fever and a cystic mass about 10 cm in diameter in the right hypochondrium. Shuntography and a barium enema study demonstrated a pseudocyst at the distal end of the shunt. The cyst wall was excised, the peritoneal tube removed, and VPS converted to a ventriculoatrial route following which the pseudocyst resolved. Case 2 was a 49-year-old female who developed hydrocephalus following subarachnoid hemorrhage, and VPS surgery was performed on March 10, 1989. Two weeks later, she developed fever and right upper abdominal pain. Abdominal x-ray and CT scan revealed a right subdiaphragmatic abscess. The abscess was drained and the shunt system was removed on April 4. VPS was placed again on April 21 without further complications. She was symptom free for the next 7 years. Case 3 was a 57-year-old female who presented in a semicomatose state after falling from bed on May 5, 1995. CT scan showed left-sided acute subdural hematoma (ASDH) for which surgery was performed. Her neurological status improved postoperatively. She eventually developed hydrocephalus and left-sided subdural effusion for which right VPS and left subduroperitoneal shunt (SPS) surgery was performed on January 25, 1996. The peritoneal end of the tube of the SPS protruded out of the anus one and a half year after shunt placement. The entire SPS system was removed as there was no more collection in the subdural space. We reviewed the literature and discussed the pathophysiology involved in the development of intraabdominal complications following VPS.

Thyroxine (T4) metabolism in an athyreotic patient who had taken a large amount of T4 at one time.

As we had an opportunity to take blood samples from a totally thyroidectomized patient who had attempted suicide by taking 2,000 microg of Levothyroxine (L-T4), the serum levels of thyroid hormones were sequentially measured to investigate the metabolism of circulating thyroid hormones in an athyreotic human. The serum concentrations of most thyroid hormones reached a peak on the second day, but the serum T3 level showed a peak one day later. The maximum concentrations of T4 (315 microg/l), FT4 (48.8 ng/l) and rT3 (0.80 microg/l) were very high, while the peak T3 level (1.92 microg/l) did not exceed the upper limit of the normal range. The serum T4 and rT3 levels returned to their normal range 13-17 days after the suicide attempt. The TSH level was suppressed rapidly and reached its nadir (0.044 mU/l) on the 6th day. During this period, the T1/2 and MCR of serum T4 were 10.4 days and 0.64 l/day, respectively, which values were almost equivalent to those observed during 15 days after discontinuation of the maintenance L-T4 therapy. In summary, the oral intake of a large amount of L-T4 at one time does not induce a proportional increase in the T3 level in an athyreotic person. The MCR of serum T4 is decreased and the T1/2 of serum T4 is prolonged, probably due to the lack of intrathyroidal deiodination. These findings support the conclusion that the D1 activity in the thyroid is one of the major determinants in the metabolic clearance of serum T4.

Ouabain suppresses ATP elevation in response to fuel secretagogues in pancreatic islets.

The alterations of the ATP concentration in response to fuel secretagogues such as glucose, glyceraldehyde, and ketoisocaproate (KIC) were investigated in a single islet. The intraislet ATP concentration was transiently elevated and then decreased to a level slightly higher than basal. To asses the ATP content under conditions of reduced ATP consumption, the Na-K pump blocker ouabain was used. The elevation of ATP concentration was found unexpectedly to be suppressed under ouabain in the islet, even when incubated with any of the secretagogues. High glucose did not elevate the intracellular creatine phosphate during incubation with ouabain. Since the suppression rate for the intraislet ATP elevation was considerably smaller with KIC than with glyceraldehyde and glucose, we conclude that ouabain inhibits ATP production, at least in part, in the glycolytic pathway through a feedback mechanism.

Necessity of endogenous GTP derived from glucose-6-phosphate for insulin secretion augmented by glucose under protein kinase A activation.

To investigate the possible involvement of some intracellular metabolic signaling other than the ATP derived from glucose metabolism under protein kinase A (PKA) activation, we measured the insulin secretory capacity stimulated by glucose and other fuel secretagogues using diazoxide-treated pancreatic islets. Under these conditions, we found a signal from a site proximal to glyceraldehyde-3-phosphate (GA-3-P) in the glycolysis to be necessary for glucose-induced insulin secretion. By using several different glycolytic enzyme inhibitors, we found that this proximal signal is derived from glucose-6-phosphate (G-6-P), and that metabolic signaling distal to GA-3-P also is necessary. Mycophenolic acid completely inhibited the augmented glucose-induced insulin secretion, which guanosine could reverse, indicating that the proximal signaling is coupling with endogenous GTP production. In this novel system of metabolic signaling, endogenous GTP derived from G-6-P in the glycolysis elicits the augmentation of glucose-induced insulin secretion under PKA activation in diazoxide-treated pancreatic islets.

Depolarization triggers intracellular magnesium surge in cultured dorsal root ganglion neurons.

Intracellular magnesium concentration ([Mg2+]i) of cultured dorsal root ganglion (DRG) neurons was measured using the magnesium indicator Mag-Fura-2/AM. [Mg2+]i was 0.48 +/- 0.08 mM (mean +/- SEM, n = 23) at rest, and it increased 3-fold by depolarization with a 60-mM K+ solution. The [Mg2+]i increase was observed in the absence of extracellular Mg2+, but the increase disappeared in the absence of extracellular Ca2+. 50 microM cadmium or 100 microM verapamil, a Ca2+ channel blocker, also diminished the rise of [Mg2+]i. The additional measurement of an intracellular Ca2+ concentration ([Ca2+]i) indicated that the [Mg2+]i rise requires a threshold concentration of [Ca2+]i to be reached; above 60 nM. The present results indicate that depolarization induces a Ca2+-influx through voltage dependent Ca channels and this causes the release of Mg2+ from intracellular stores into the cytoplasm.

Beneficial effect of long-term combined treatment with voglibose and pioglitazone on pancreatic islet function of genetically diabetic GK rats.

Effects of voglibose (an alpha-glucosidase inhibitor) and pioglitazone (an insulin sensitizer) on glycemic control and on the function of pancreatic islets were evaluated using Goto-Kakizaki (GK) rats with non-insulin-dependent diabetes mellitus (NIDDM). Five week administration (8-13 weeks of age in GK rats) of voglibose alone (added to the chow at a concentration of 10 ppm), pioglitazone alone (10 mg/kg daily p.o.), or both of the agents together significantly improved fasting plasma glucose levels and those at 120 min in oral glucose tolerance tests. Insulin secretory capacity in response to glucose of the isolated islets, assessed by batch incubation, was significantly improved in the voglibose and in the voglibose plus pioglitazone groups. Eight-week administration (5-13 weeks of age) of voglibose and voglibose plus pioglitazone successfully lowered the fasting levels of plasma glucose and triglyceride. The glucose-responsiveness in insulin release from the islets was also significantly recovered by the therapy. The treatment increased the insulin content of the islets to almost twice that in untreated controls. Thus, treatment by these drugs can not only effectively ameliorate the metabolic derangement of NIDDM in GK rats, but it can also restore the deteriorated islet function, possibly through protection from glucose toxicity.

Determination of the entire sequence of turtle CR1: the first open reading frame of the turtle CR1 element encodes a protein with a novel zinc finger motif.

CR1 elements are a family of retroposons. They are classified as long interspersed elements (LINEs) or non-long-terminal-repeat (non-LTR) retrotransposons, and they have been found in the genomes of many vertebrates. However, they have been only partially characterized, and only a 2-kb region of the 3' end of chicken CR1 has been sequenced. In the present study, we determined the entire consensus sequence of CR1 elements in the turtle genome, designated PsCR1. The first open reading frame (ORF1) of PsCR1 has two unusual arrangements of Cys residues. One of them includes a zinc finger motif, CX2CX14CX2C. The putative zinc finger has cysteine residues with identical spacing and a similar amino acid composition to those found in the species-specific transcription initiation factors SL1 and TIF-IB. The 5' untranslated region (5' UTR) of PsCR1 contains a sequence similar to part of the human L1 promoter, L1 site A, and several cis elements of the type found in eukaryotic genes. Within a region of about 500 bp, there are nine "E boxes," cis elements that are recognized by the basic helix-loop-helix (bHLH) family of proteins. This observation raises the possibility that cellular transcription factors that bind to these sequences might act in concert to regulate the expression of PsCR1. The extent of the sequence divergence of the 3' UTR of CR1 between species was found to be lower than the rate of nonsynonymous substitutions per site in ORF2, suggesting that a strict functional constraint must exist for this region. This result strongly suggests that the conserved 3'-end sequence of CR1 is the recognition site for the reverse transcriptase of CR1. A discussion is presented of a possible mechanism for the integration of CR1 elements and also of the intriguing possible recruitment of the reverse transcriptase for the retroposition of SINEs.