New insights into sex chromosome evolution in anole lizards (Reptilia, Dactyloidae).

Anoles are a clade of iguanian lizards that underwent an extensive radiation between 125 and 65 million years ago. Their karyotypes show wide variation in diploid number spanning from 26 (Anolis evermanni) to 44 (A. insolitus). This chromosomal variation involves their sex chromosomes, ranging from simple systems (XX/XY), with heterochromosomes represented by either micro- or macrochromosomes, to multiple systems (X1X1X2X2/X1X2Y). Here, for the first time, the homology relationships of sex chromosomes have been investigated in nine anole lizards at the whole chromosome level. Cross-species chromosome painting using sex chromosome paints from A. carolinensis, Ctenonotus pogus and Norops sagrei and gene mapping of X-linked genes demonstrated that the anole ancestral sex chromosome system constituted by microchromosomes is retained in all the species with the ancestral karyotype (2n = 36, 12 macro- and 24 microchromosomes). On the contrary, species with a derived karyotype, namely those belonging to genera Ctenonotus and Norops, show a series of rearrangements (fusions/fissions) involving autosomes/microchromosomes that led to the formation of their current sex chromosome systems. These results demonstrate that different autosomes were involved in translocations with sex chromosomes in closely related lineages of anole lizards and that several sequential microautosome/sex chromosome fusions lead to a remarkable increase in size of Norops sagrei sex chromosomes.

Extensive homology of chicken macrochromosomes in the karyotypes of Trachemys scripta elegans and Crocodylus niloticus revealed by chromosome painting despite long divergence times.

We report extensive chromosome homology revealed by chromosome painting between chicken (Gallus gallus domesticus, GGA, 2n = 78) macrochromosomes (representing 70% of the chicken genome) and the chromosomes of a turtle, the red-eared slider (Trachemys scripta elegans, TSC, 2n = 50), and the Nile crocodile (Crocodylus niloticus, CNI, 2n = 32). Our data show that GGA1-8 arms seem to be conserved in the arms of TSC chromosomes, GGA1-2 arms are separated and homologous to CNI1p, 3q, 4q and 5q. In addition to GGAZ homologues in our previous study, large-scale GGA autosome syntenies have been conserved in turtle and crocodile despite hundreds of millions of years divergence time. Based on phylogenetic hypotheses that crocodiles diverged after the divergence of birds and turtles, our results in CNI suggest that GGA1-2 and TSC1-2 represent the ancestral state and that chromosome fissions followed by fusions have been the mechanisms responsible for the reduction of chromosome number in crocodiles.

Emergence of complex rearrangements at translocation breakpoints in a transgenic mouse; implications for mechanisms involved in the formation of chromosome rearrangements.

Cryptic complex rearrangements as a result of a reciprocal chromosome translocation have been characterised in a transgenic mouse strain. Analysis of the breakpoint junctions in our previous studies showed that the ada transgene was integrated at the breakpoint forming a fusion gene with Golga3 (Mea2). In this study, further detailed analysis around the translocation junctions revealed that the surrounding regions were composed of 13 fragments of defined transgenic chromosome origins over approximately 1.9-Mb areas. Exactly the same cluster structure of these 13 breakpoint fragments already existed in the second generation of the transgenic mice. Our results show that this highly complex rearrangement has been conserved as the incipient form without any additional changes for 18 years up to the present generation, suggesting simultaneous occurrence of multiple events in the founder mouse.

Gene arrangement at the Rhesus blood group locus of chimpanzees detected by fiber-FISH.

The Rhesus (Rh) blood group system in humans is encoded by two genes with high sequence homology. These two genes, namely, RHCE and RHD, have been implied to be duplicated during evolution. However, the genomic organization of Rh genes in chimpanzees and other nonhuman primates has not been precisely studied. We analyzed the arrangement of the Rh genes of chimpanzees (Pan troglodytes) by two-color fluorescence in situ hybridization on chromatin DNA fibers (fiber-FISH) using two genomic DNA probes that respectively contain introns 3 and 7 of human RH genes. Among the five chimpanzees studied, three were found to be homozygous for the two-Rh-gene type, in an arrangement of Rh (5'-->3') - Rh (3'<--5'). Although a similar gene arrangement can be detected in the RH gene locus of typical Rh-positive humans, the distance between the two genes in chimpanzees was about 50 kb longer than that in humans. The remaining two chimpanzees were homozygous for a four-Rh-gene type, in an arrangement of Rh (5'-->3') - Rh (3'<--5') - Rh (3'<--5') - Rh (3'<--5') within a region spanning about 300 kb. This four-Rh-gene type has not been detected in humans. Further analysis of other great apes showed different gene arrangements: a bonobo was homozygous for the three-Rh-gene type; a gorilla was heterozygous for the one-Rh- and two-Rh-gene types; an orangutan was homozygous for the one-Rh-gene type. Our findings on the intra- and interspecific genomic variations in the Rh gene locus in Hominoids would shed further light on reconstructing the genomic pathways of Rh gene duplication during evolution.

Chromosome homology between chicken (Gallus gallus domesticus) and the red-legged partridge (Alectoris rufa); evidence of the occurrence of a neocentromere during evolution.

Chromosome-specific paints from macrochromosomes 1-9 and Z of the chicken were hybridised to metaphases of the red-legged partridge and revealed no inter-chromosomal rearrangements. The results from chromosome painting are similar to previous studies on the Japanese quail but different from findings in guinea fowl and several species of pheasant. The difference in centromere position in chicken and partridge chromosome 4, previously assumed to be the result of an inversion, was confirmed. However, FISH mapping of BAC clones from chicken chromosome 4 revealed that the order of loci was the same in both species, indicating the occurrence of a neocentromere during divergence.

The genomic structure and expression of MJD, the Machado-Joseph disease gene.

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder that is clinically characterized by cerebellar ataxia and various associated symptoms. The disease is caused by an unstable expansion of the CAG repeat in the MJD gene. This gene is mapped to chromosome 14q32.1. To determine its genomic structure, we constructed a contig composed of six cosmid clones and eight bacterial artificial chromosome (BAC) clones. It spans approximately 300kb and includes MJD. We also determined the complete sequence (175,330bp) of B445M7, a human BAC clone that contains MJD. The MJD gene was found to span 48,240bp and to contain 11 exons. Northern blot analysis showed that MJD mRNA is ubiquitously expressed in human tissues, and in at least four different sizes; namely, 1.4, 1.8, 4.5, and 7.5kb. These different mRNA species probably result from differential splicing and polyadenylation, as shown by sequences of the 21 independent cDNA clones isolated after the screening of four human cDNA libraries prepared from whole brain, caudate, retina, and testis. The sequences of these latter clones relative to the MJD gene in B445M7 indicate that there are three alternative splicing sites and eight polyadenylation signals in MJD that are used to generate the differently sized transcripts.

Genomic anatomy of a premier major histocompatibility complex paralogous region on chromosome 1q21-q22.

Human chromosomes 1q21-q25, 6p21.3-22.2, 9q33-q34, and 19p13.1-p13.4 carry clusters of paralogous loci, to date best defined by the flagship 6p MHC region. They have presumably been created by two rounds of large-scale genomic duplications around the time of vertebrate emergence. Phylogenetically, the 1q21-25 region seems most closely related to the 6p21.3 MHC region, as it is only the MHC paralogous region that includes bona fide MHC class I genes, the CD1 and MR1 loci. Here, to clarify the genomic structure of this model MHC paralogous region as well as to gain insight into the evolutionary dynamics of the entire quadriplication process, a detailed analysis of a critical 1.7 megabase (Mb) region was performed. To this end, a composite, deep, YAC, BAC, and PAC contig encompassing all five CD1 genes and linking the centromeric +P5 locus to the telomeric KRTC7 locus was constructed. Within this contig a 1.1-Mb BAC and PAC core segment joining CD1D to FCER1A was fully sequenced and thoroughly analyzed. This led to the mapping of a total of 41 genes (12 expressed genes, 12 possibly expressed genes, and 17 pseudogenes), among which 31 were novel. The latter include 20 olfactory receptor (OR) genes, 9 of which are potentially expressed. Importantly, CD1, SPTA1, OR, and FCERIA belong to multigene families, which have paralogues in the other three regions. Furthermore, it is noteworthy that 12 of the 13 expressed genes in the 1q21-q22 region around the CD1 loci are immunologically relevant. In addition to CD1A-E, these include SPTA1, MNDA, IFI-16, AIM2, BL1A, FY and FCERIA. This functional convergence of structurally unrelated genes is reminiscent of the 6p MHC region, and perhaps represents the emergence of yet another antigen presentation gene cluster, in this case dedicated to lipid/glycolipid antigens rather than antigen-derived peptides.

Comparative FISH mapping of the ancestral fusion point of human chromosome 2.

It is known that human chromosome 2 originated from the fusion of two ancestral primate chromosomes. This has been confirmed by chromosome banding and fluorescence in-situ hybridization (FISH) with human chromosome-2-specific DNA libraries. In this study, the order of 38 cosmid clones derived from the human chromosome region 2q12-q14 was exactly determined by high-resolution FISH in human chromosome 2 and its homologous chromosomes in chimpanzees (Pan trogrodydes, 2n=48) and cynomolgus monkeys (Macacafascicularis, 2n = 42). This region includes the telomere-to-telomere fusion point of two ancestral ape-type chromosomes. As a result of comparative mapping, human chromosome region 2q12-q14 was found to correspond to the short arms of chimpanzee chromosomes 12 and 13 and cynomolgus monkey chromosomes 9 and 15. It is noted that no difference was detected in the relative order of the cosmid clones between human and chimpanzee chromosomes. This suggests that two ancestral ape-type chromosomes fused tandemly at telomeres to form human chromosome 2, and the genomic organization of this region is thought to be considerably conserved. In the cynomolgus monkey, however, the order of clones in each homologue was inverted. In addition to cosmid mapping, two chromosome-2-specific yeast artificial chromosome (YAC) clones containing the fusion point were identified by FISH.

Shifts in herbicide tolerance in paddy field periphyton following herbicide application

Herbicide susceptibility was examined in various algal strains isolated from herbicide-treated and untreated paddy fields. Diatom strains showed obvious tolerance to the herbicides simetryn and pretilachlor irrespective of herbicide exposure, while the susceptibility of green algae mostly reflected their history of herbicide exposure. Cross-resistance was also induced in some algae. Rapid development of herbicide tolerance indicated that the tolerance seemed to be conferred by tolerant strains already present in the communities. Thus, changes in genetic composition within a population seem to be the main mechanism by which paddy periphyton gain herbicide tolerance.

Gene arrangement of the killer cell inhibitory receptor family on human chromosome 19q13.4 detected by fiber-FISH.

Human chromosome 19q13.4 has recently been revealed to be a remarkable region harboring multiple receptor genes of the immunoglobulin (Ig) superfamily differentially expressed on hematopoietic cell lineages. Over the past few years, more than 50 cDNAs have been cloned for the natural killer cell inhibitory receptor (KIR) gene family, which possess two or three Ig-like domains in the extracellular region. In this study, using two genomic DNA probes containing intron sequences of genes corresponding to the two- and three-domain types, we applied two-color-fluorescence in situ hybridization on stretched DNA fiber preparations (fiber-FISH). As a result, 11 positions homologous to KIR genes were found as a cluster within a range of approximately 120 kilobases on a chromatin fiber from human chromosome 19.

Erythromycin derivative improves gastric emptying and insulin requirement in diabetic patients with gastroparesis.

OBJECTIVE: To evaluate the effect of the erythromycin derivative EM523L on gastric emptying and postprandial insulin requirement in insulin-dependent diabetic patients with severe gastroparesis. RESEARCH DESIGN AND METHODS: In six IDDM patients with severe gastroparesis (two men and four women, mean age 44.5 years [range 36-53]), the insulin infusion pattern during feedback control with an artificial endocrine pancreas device (Biostator) after intake of a test meal, the retention rate of residual isotope ([99m]Tc-labelled Sn-colloid) in the stomach, and the time-concentration curve of plasma acetaminophen as the marker for liquid emptying were studied with EM523L or a control placebo RESULTS: Time courses of insulin infusion rates peaked within 120 min after intake of the test meal in the EM523L phase, whereas no apparent peak rates were observed in the control phase. The total amount of insulin required in the first 90 min postprandial was significantly greater in the EM523L phase than in the control phase. EM523L significantly decreased the residual isotope ratio in the stomach at > or =50 min postprandial and increased the plasma acetaminophen concentrations at 30-120 min postprandial, compared with respective values in the control phase. CONCLUSIONS: Preliminary results obtained from a small number of patients suggest that EM523L or erythromycin analogs, which have agonistic activity to motilin receptors as well as no antibacterial effect, may be useful to accelerate gastric emptying and improve insulin requirement patterns, thereby establishing more stable glycemic control.

Study of gastric emptying in patients with pancreatic diabetes (chronic pancreatitis) using acetaminophen and isotope.

The gastric emptying function tests were carried out in eight patients with pancreatic diabetes, who were classified into two groups according to the coefficient of variation in the R-R interval in ECG (C.V. R-R) on the normal subjects: < or = the mean - 2SD (the autonomic nerve dysfunction group: AND+ group) and > the mean - 2SD (the autonomic nerve normal group: AND- group). Both the gastric emptying of liquid food by the acetaminophen method and that of solid food by the isotope method were significantly reduced in the AND+ group than in the AND- and normal groups. In addition, a significant correlation was found between the C.V. R-R and the serum acetaminophen concentration (a 45 min value) and the % gastric retention of isotope (a 120 min value). The above results demonstrated that even pancreatic diabetes might be complicated by gastroparesis diabeticorum among autonomic nerve dysfunction. There was a close relation of delayed gastric emptying to the C.V. R-R in ECG or an index of the vagus nerve function.

Steatorrhea in Japanese patients with chronic pancreatitis.

Fecal fat excretion, fecal mass, fecal fat concentration, and the coefficient of fat absorption were evaluated in 31 normal Japanese subjects with a mean fat consumption of 61.8 g and compared with the values in 43 Japanese patients with chronic pancreatitis (CP) with a fat consumption of 40.2 g. Fecal fatty acids were analyzed by the gas chromatographic method. Fecal fat excretion by normal individuals was 1.7 +/- 1.0 (Mean +/- SD) g/day (range, 0.4-4.9 g/day). Steatorrhea was therefore defined as fecal fat excretion that exceeded 5.0 g/day. The patients with CP were divided into three groups: non-steatorrhea (fecal fat < 5.0 g/day), chemical steatorrhea (fecal fat > or = 5 g/day and no appearance of fatty stool), and manifest steatorrhea (evaluated from the appearance of fatty stool). In addition, we investigated the correlation between fecal fat excretion and pancreatic exocrine function, obtained by the pancreozymin-secretin (or secretin) test in 24 controls and 30 CP patients. Fecal fat excretion by CP patients was 9.1 +/- 8.8 g/day, which was significantly higher (P < 0.01) than that of controls. There were 28 (65%) CP patients with steatorrhea. Of these, 15 (35%) showed chemical steatorrhea and 13 (30%) manifest steatorrhea. In CP patients, the fecal mass (250.5 +/- 133.6 g vs control, 125.6 +/- 52.5 g), fecal fat concentration (3.40 +/- 2.16 g% vs control, 1.48 +/- 0.89 g%), and coefficient of fat absorption (77.3 +/- 20.2% vs control, 97.2 +/- 1.7%) all showed significant differences from the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

No negative feedback regulation between plasma CCK levels and luminal tryptic activities in patients with pancreatic insufficiency.

The study was conducted on five healthy subjects and six patients with calcifying pancreatitis (CP) and steatorrhea. Following overnight fasting, one tube each was placed in the stomach and the upper of the small intestine, respectively. Through the gastric tube, a test meal that included 30 g of fat (total calories, 625 kcal, 500 mL) was infused over a span of 30 min. Every 30 min (up to 150 min), fluid samples in the upper small intestine were collected and chilled, and the amylase, trypsin, and lipase levels were determined. In addition, in the case of the CP patients, a high-potency pancreatin preparation was infused into the stomach together with the test meal. In order to determine the plasma CCK level, blood sample were collected before test meal infusion and at 10, 20, 30, 45, 60, 90, 120, and 150 min subsequent to infusion. The plasma CCK was extracted using a Sep-Pak C-18 cartridge and analyzed with radioimmunoassay using an OAL-656 antibody. The result was converted to the CCK-8 level and expressed in pg/mL. The enzyme activities in the upper small intestine of the CP patients after test meal administration amounted to 22.8 (amylase), 10.8 (trypsin), and 16.9% (lipase) compared with the corresponding figures for the normal subjects. Following administration of a high-potency pancreatin in patients with CP, enzyme activities in the upper small intestine increased to 132.2 (amylase), 38.7 (trypsin), and 45.3% (lipase) compared with levels in the normal subjects. However, the healthy subjects and the CP patients, both with and without treatment with supplementary exogenous enzymes, all exhibited similar profiles in the plasma CCK response to stimuli. Based on these findings, we concluded that a negative feedback mechanism does not exist between the tryptic activity of the upper small intestine and the CCK secretory response in patients with chronic pancreatitis.

Effects of pancreatic digestive enzymes, sodium bicarbonate, and a proton pump inhibitor on steatorrhoea caused by pancreatic diseases.

Forty-five patients with pancreatic steatorrhoea (27 with calcified pancreatitis, 13 with non-calcified pancreatitis, two with pancreaticoduodenectomy, one with total pancreatectomy, and two with pancreatic cancer) were divided into four groups and given the following medication for 2 to 4 weeks: 4 to 6 g/day of sodium bicarbonate (group I); 9 g/day of high-lipase pancreatin (lipase, 56,600 U/g, Fédération Internationale Pharmaceutique (FIP); group II); 12 to 24 tablets or 9.0 g of commercial pancreatic enzyme preparations (group III); or 50 mg of omeprazole (group IV). Faecal fat excretion was evaluated before and after drug administration. Faecal fat excretion was reduced by 2.9 g (range, 1.7 to 5.0 g) in group I; 8.8 g (range, 2.9 to 39.9 g) in group II; 10.8 g (range, 2.3 to 21.8 g) in group III; and 4.3 g (range, 3.6 to 5.6 g) in group IV. The pancreatic digestive enzyme preparation was more effective than sodium bicarbonate and agents that raise the pH of the upper small intestine (such as proton-pump inhibitors) in reducing faecal fat excretion. The results indicate that all of the preparations used are effective against mild pancreatic steatorrhoea. If the condition is more advanced, however, a massive dosage of pancreatic digestive enzyme and possibly the combined use of an agent to raise the pH of the upper small intestine are likely to be effective.

Effects of the triazine herbicide, simetryn, on freshwater plankton communities in experimental ponds.

The effects of a triazine herbicide, simetryn, on freshwater phyto- and zooplankton communities and water chemistry were investigated using outdoor experimental ponds. Simetryn was applied at a concentration of 0.1 or 1.0 mg l(-1) 18 days after the start of the experiment. Simetryn treatments reduced pH, dissolved oxygen concentration, chlorophyll a concentration and photosynthetic rate in the pond water, whereas they increased the dissolved nutrient concentration. In the phytoplankton community Volvocales and Cryptophyceae were clearly reduced by the herbicide application. In the 0.1 mg l(-1) simetryn-treated ponds, photosynthetic rate of the phytoplankton community recovered slightly about two weeks after the treatment, which coincided with recovery of centric diatoms and Cryptophyceae, whereas it was completely suppressed until the end of the experiment in the 1.0 mg l(-1) simetryn-treated ponds. The decrease in zooplankton density in the treated ponds was not due to the direct toxic effects of simetryn but to indirect effects resulting from reduced algal productivity which in turn reduced the food supply.

Effects of the organophosphorus insecticide fenthion on phyto- and zooplankton communities in experimental ponds.

The organophosphorus insecticide fenthion was applied to experimental ponds and its effects on phyto- and zooplankton communities were analysed. The sensitivity to fenthion differed among cladoceran and rotiferan species according to the following order: Daphnia galeata > Monia micrura > Bosmina fatalis >/= Polyarthra trigla > Keratella valga. The results suggest that large zooplankton species tend to be more sensitive to fenthion than small ones, large cladocerans more sensitive than small cladocerans, and cladocerans more sensitive than rotifers. The application of fenthion induced an increase in the density of rotifers and phytoplankton. This was a secondary effect of the chemical, which directly depressed cladoceran populations and consequently released rotifers and phytoplankton from competition with, and grazing by cladocerans.

Decreased counterregulatory hormone responses to insulin-induced hypoglycemia in patients with pancreatic diabetes having autonomic neuropathy.

Thirteen patients with pancreatic diabetes caused by calcifying pancreatitis were divided into 2 groups; 5 with diabetic autonomic neuropathy [AN(+) group] and 8 without [AN(-) group]. They were subjected to an insulin-induced hypoglycemic stress test to evaluate their blood pancreatic glucagon, adrenalin, and cortisol responses. When a blood glucose level below 45 mg/100 ml was defined to be hypoglycemia, all the patients in the AN(-) group exhibited peripheral adrenalin responses, with a significant increase (mean, 19.0 times the basal level) in the blood adrenalin level. Among the AN(+) group, on the other hand, central nervous symptoms became evident rather than the peripheral adrenalin response (the blood adrenalin level hardly exceeded the basal level). With the exception of a single patient, none exhibited responses in the blood pancreatic glucagon levels. Only one patient showed a minimal cortisol response but the remaining 12 reacted normally in the cortisol release. The findings are summarized as follows: in pancreatic diabetes, insulin-induced hypoglycemia causes little change in pancreatic glucagon secretion; when the condition is complicated with autonomic neuropathy, central nervous symptoms develop while the blood adrenalin level hardly increases. These findings indicated that patients with pancreatic diabetes complicated with diabetic autonomic neuropathy have a risk of lapsing into an acute hypoglycemic coma and difficulty in recovering from the hypoglycemic state.

Increased plasma gluconeogenic and system A amino acids in patients with pancreatic diabetes due to chronic pancreatitis in comparison with primary diabetes.

We examined the plasma amino acid profiles of 17 patients with pancreatic diabetes in comparison with those of 14 healthy subjects and 16 patients with primary diabetes of similarly poor glycemic control. We also measured fasting plasma glucagon and free insulin levels in patients with pancreatic diabetes and in those with primary diabetes. The fasting plasma amino acid level was highest in patients with pancreatic diabetes. Furthermore, the plasma glucagon level in patients with pancreatic diabetes was significantly low, and negative correlations were found between glucagon and glucagon-related System A and gluconeogenic amino acids. On the other hand, the levels of branched-chain amino acids (BCAA) and System L amino acids, which are subjected to change in the state of diabetes mellitus, increased in patients with pancreatic diabetes. From the above results, we determined that the amino acid profile in patients with pancreatic diabetes resembled that in patients with total pancreatectomy. It is our opinion that the increases of amino acid levels (gluconeogenic and System A amino acids) in patients with pancreatic diabetes is not dependent on maldigestion/malabsorption under treatment, but is mainly dependent on glucagon deficiency.

Altered postprandial insulin requirement in IDDM patients with gastroparesis.

OBJECTIVE: To evaluate the effect of gastric emptying on postprandial insulin requirement in insulin-dependent diabetes mellitus (IDDM) patients with and without gastroparesis. RESEARCH DESIGN AND METHODS: Postprandial insulin requirement and gastric emptying were simultaneously evaluated in five IDDM patients with gastroparesis and in six control IDDM patients without gastroparesis. Postprandial insulin requirement after test-meal intake was assessed by measuring the insulin infusion rate during a 4-h feedback control with an artificial endocrine pancreas device (Biostator, Life Science Instruments, Miles, Elkhart, IN). Gastric solid and liquid emptyings were evaluated during the Biostator study by measuring the disappearance rate of 99mTc in the stomach and in the time course of plasma acetaminophen concentration, respectively. RESULTS: Total insulin requirement during the first 120 min after the test-meal intake was significantly lower in the gastroparetic patients than in the control patients. The gastroparetic patients showed no apparent postprandial peak for insulin infusion rate during the 4-h study, although the peak rate was observed within 120 min after the test-meal intake in the control patients. The disappearance of 99mTc in the stomach was significantly slower, and plasma acetaminophen concentrations were significantly lower in the gastroparetic patients compared with those in the control patients, respectively. CONCLUSIONS: The results suggest that IDDM patients with gastroparesis, accompanied by impaired solid and liquid emptying, have an altered postprandial insulin requirement.