Regulating the onset of mitosis.

In eukaryotes, G2/M progression is mediated by activation of mitosis promoting factor (MPF). To ensure faithful chromosome segregation, the activity of key mitotic inducers and inhibitors are coupled with chromosome replication, spindle pole duplication, morphogenesis, and DNA damage. Evidence gathered in the past two years has underscored the importance of positioning MPF and its regulators in the proper place at the proper time to ensure orderly progression through the G2/M transition. Altering the spatial organization of G2/M regulators also contributes to prevention of mitosis following DNA damage.

Prenatal diagnosis and management of congenital subglottic stenosis associated with congenital esophageal atresia type C.

Most patients with congenital esophageal atresia (EA) have congenital tracheobronchial abnormalities, which may cause respiratory distress, be difficult to treat and have a poor prognosis. The authors report a neonate with EA and congenital subglottic stenosis (SGS) who exhibited severe respiratory distress immediately after birth. After emergency endotracheal intubation with a narrow endotracheal tube, the authors performed total correction of EA and anterior cricoid split (ACS) on day 1 of age. The postoperative course was uneventful. Some reports have stated that it is difficult to make a prenatal diagnosis when SGS is associated with EA and tracheoesophageal fistula (TEF). The anterior cricoid split technique may be suitable for managing moderate SGS even in neonates with EA. Partial resection of the hypertrophic cricoid cartilage is considered effective in preventing restenosis.

Myb-related fission yeast cdc5p is a component of a 40S snRNP-containing complex and is essential for pre-mRNA splicing.

Myb-related cdc5p is required for G(2)/M progression in the yeast Schizosaccharomyces pombe. We report here that all detectable cdc5p is stably associated with a multiprotein 40S complex. Immunoaffinity purification has allowed the identification of 10 cwf (complexed with cdc5p) proteins. Two (cwf6p and cwf10p) are members of the U5 snRNP; one (cwf9p) is a core snRNP protein. cwf8p is the apparent ortholog of the Saccharomyces cerevisiae splicing factor Prp19p. cwf1(+) is allelic to the prp5(+) gene defined by the S. pombe splicing mutant, prp5-1, and there is a strong negative genetic interaction between cdc5-120 and prp5-1. Five cwfs have not been recognized previously as important for either pre-mRNA splicing or cell cycle control. Further characterization of cwf1p, cwf2p, cwf3p, and cwf4p demonstrates that they are encoded by essential genes, cosediment with cdc5p at 40S, and coimmunoprecipitate with cdc5p. We further show that cdc5p associates with the U2, U5, and U6 snRNAs and that cells lacking cdc5(+) function are defective in pre-mRNA splicing. These data raise the possibility that the cdc5p complex is an intermediate in the assembly or disassembly of an active S. pombe spliceosome.

Myb-related Schizosaccharomyces pombe cdc5p is structurally and functionally conserved in eukaryotes.

Schizosaccharomyces pombe cdc5p is a Myb-related protein that is essential for G2/M progression. To explore the structural and functional conservation of Cdc5 throughout evolution, we isolated Cdc5-related genes and cDNAs from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens. Supporting the notion that these Cdc5 gene family members are functionally homologous to S. pombe cdc5(+), human and fly Cdc5 cDNAs are capable of complementing the temperature-sensitive lethality of the S. pombe cdc5-120 mutant. Furthermore, S. cerevisiae CEF1 (S. cerevisiae homolog of cdc5(+)), like S. pombe cdc5(+), is essential during G2/M. The location of the cdc5-120 mutation, as well as mutational analyses of Cef1p, indicate that the Myb repeats of cdc5p and Cef1p are important for their function in vivo. However, we found that unlike in c-Myb, single residue substitutions of glycines for hydrophobic residues within the Myb repeats of Cef1p, which are essential for maintaining structure of the Myb domain, did not impair Cef1p function in vivo. Rather, multiple W-to-G substitutions were required to inactivate Cef1p, and many of the substitution mutants were found to confer temperature sensitivity. Although it is possible that Cef1p acts as a transcriptional activator, we have demonstrated that Cef1p is not involved in transcriptional activation of a class of G2/M-regulated genes typified by SWI5. Collectively, these results suggest that Cdc5 family members participate in a novel pathway to regulate G2/M progression.

Bipolarization and poleward flux correlate during Xenopus extract spindle assembly.

We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux.

Identification and characterization of Schizosaccharomyces pombe asp1(+), a gene that interacts with mutations in the Arp2/3 complex and actin.

The Arp2/3 complex is an essential component of the actin cytoskeleton in yeast and is required for the movement of actin patches. In an attempt to identify proteins that interact with this complex in the fission yeast Schizosaccharomyces pombe, we sought high-copy suppressors of the S. pombe arp3-c1 mutant, and have identified one, which we have termed asp1(+). The asp1(+) open reading frame (ORF) predicts a highly conserved protein of 921 amino acids with a molecular mass of 106 kD that does not contain motifs of known function. Neither asp1(+) nor its apparent Saccharomyces cerevisiae ortholog, VIP1, are essential genes. However, disruption of asp1(+) leads to altered morphology and growth properties at elevated temperatures and defects in polarized growth. The asp1 disruption strain also is hypersensitive to Ca+ ions and to low pH conditions. Although Asp1p is not stably associated with the Arp2/3 complex nor localized in any discrete structure within the cytoplasm, the asp1 disruption mutant was synthetically lethal with mutations in components of the Arp2/3 complex, arp3-c1 and sop2-1, as well as with a mutation in actin, act1-48. Moreover, the vip1 disruption strain showed a negative genetic interaction with a las17Delta strain. We conclude that Asp1p/Vip1p is important for the function of the cortical actin cytoskeleton.

Construction of vectors and a genomic library for use with his3-deficient strains of Schizosaccharomyces pombe.

The construction of vectors for use in Schizosaccharomyces pombe using the his3+ gene as a selectable marker is described. In addition, we report the construction of a genomic library in a his3(+)-containing shuttle vector to facilitate the cloning of genes by complementation of mutant function in strains defective for His3 activity.

Serum vitamin E levels in children with corrected biliary atresia.

In 37 children with long-standing cholestasis who had undergone a Kasai's procedure (double Roux-en-Y hepatic portoenterostomy), serum vitamin E levels were determined. In addition, serum bile acid levels were simultaneously tested as a marker of cholestasis. Eighteen of 37 children had vitamin E levels of less than 0.50 mg/100 ml, and two showed neurological abnormalities including hypoactive deep tendon reflexes and ataxia. Serum vitamin E levels were inversely correlated with serum bile acid levels (p less than 0.01). Older patients have mild cholestasis and high serum vitamin E levels in comparison with younger ones. Improvement in bile excretion into the intestinal tract with age seemed to be responsible for an increase of serum vitamin E levels. Oral supplements of alpha-tocopherol in doses of 5 to 10 mg/kg/day were needed to maintain the normal serum vitamin E levels in postoperative infants.

Biliary atresia. A surgical perspective.

The combination of portoenterostomy with subsequent liver transplantation is the treatment of choice for patients with biliary atresia. It is important, however, to attempt to keep the patient's own organ by continuing efforts to achieve the best possible results with portoenterostomy. Additional basic research, perhaps concerning on the role of cytokines and apoptosis in the control of biliary atresia, may provide insight into possible new medical strategies for treating patients with biliary atresia. For example, in addition to portoenterostomy, control of apoptosis at various cellular levels and of bile duct cell proliferation and maturation by manipulation of the growth factors and cytokines may become part of future treatment modalities. Another direction of research should be the control of fibrogenesis, which might be accomplished by blocking TGF-beta 1 and platelet-derived growth factor and by HGF gene therapy. The author's current strategy for surgical treatment for patients with biliary atresia include (1) early diagnosis, including prenatal diagnosis and broader use of mass screening programs, (2) hepatic portoenterostomy, without stoma formation; (3) close postoperative care, especially for prevention of postoperative cholangitis; (4) revision of portoenterostomy only in selected cases; (5) early liver transplantation in patients with absolutely failed portoenterostomy; (6) avoidance of laparotomy for the treatment of esophageal varices and hypersplenism; (7) consideration of exploratory laparotomy or primary liver transplantation for patients with advanced liver disease at the time of referral. The development of new treatment modalities based on the understanding of the pathogenesis of the disease, and especially on the biology of intrahepatic bile ducts and hepatic fibrosis, is essential.

Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity in ganglioneuroblastoma and neuroblastoma.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a 38 amino acid peptide originally isolated from ovine hypothalamus. It has a potent stimulatory action on adenylate cyclase in the rat pituitary. The presence of PACAP was studied in the tumor tissues of ganglioneuroblastoma and neuroblastoma by radioimmunoassay and immunocytochemistry. Immunocytochemical studies showed positive immunostaining in 4 out of 7 ganglioneuroblastomas and 4 out of 6 neuroblastomas. Immunoreactive PACAP concentrations in tissues of 3 ganglioneuroblastomas ranged from 14.5 to 27.8 pmol/g wet weight (20.0 +/- 5.7 pmol/g wet weight, mean +/- S.D.) and the concentration in one neuroblastoma tissue was 111.0 pmol/g wet weight. Reverse phase high performance liquid chromatography of the tumor tissue extract of ganglioneuroblastoma showed a peak eluting in the position of PACAP1-38 and smaller broad peaks eluting later. These results indicated that high concentrations of immunoreactive PACAP were present in the tumor tissues of ganglioneuroblastoma and neuroblastoma, and suggest the possibility that this peptide plays a pathophysiological role in some ganglioneuroblastomas and neuroblastomas.

Disorder of bile acid metabolism in children with short bowel syndrome.

The profile of fecal bile acids was examined in 13 children with short bowel syndrome; 7 of the 13 did not have diarrhea and the other 6 had intractable diarrhea. In children without diarrhea, no severe fat malabsorption was recognized, and the content of total bile acids in the feces was within the normal range or slightly higher. The ratio of primary to total bile acids showed various patterns. In children with intractable diarrhea, in contrast, fat malabsorption was observed and the fecal content of total bile acids in these patients was more than ten times higher than that of the control group, primary bile acids accounting for more than 95% of the total bile acids and taurine- or glycine-conjugated bile acids for 10%. In the children with intractable diarrhea, the values for the D-xylose absorption test were lower than the normal range. These results suggested that, in children with short bowel syndrome with diarrhea, the loss of bile acids was strongly associated with a decrease in the actual absorptive surface area of the residual small intestine, and the growth of the normal bacterial flora was disturbed in the residual intestine. Some children with or without diarrhea also had hyper bile acidemia. Ursodeoxycholic acid was not effective for the treatment of hyper bile acidemia or fat malabsorption.

Immunohistochemistry of DNA fragmentation factor in human stomach and colon: its correlation to apoptosis.

DNA fragmentation factor (DFF) is an important factor in the pathway leading to apoptosis, which is activated by caspase-3 and is involved in the formation of nuclear DNA fragments. DFF is a heterodimic protein of 40kDa and 45kDa that becomes activated when DFF is cleaved by caspase-3. Of the two enzymatically cleaved fragments of DFF, it is the 40kDa fragment (DFF40) that is the active component of DFF and is responsible for triggering chromatin condensation when incubated with nuclei. However, the topological correlation between apoptosis and DFF expression in human tissues has not been examined. Therefore, in this study, we first immunolocalized DFF in non-neoplastic mucosa, hyperplastic polyp, adenoma and carcinoma of human stomach and colon. We then examined apoptosis in serial tissue sections. Labeling index (LI) of DFF and TUNEL positive cells in the same areas of serial tissue sections were obtained using computer-assisted image analysis. In the stomach, the DFF LI in non-neoplastic mucosa (9.8 +/- 5.0%, n = 3) and carcinoma (18.2 +/- 3.6, n = 3) were significantly lower than that of hyperplastic polyp (73.3 +/- 9.2%, n = 3) and adenoma (66.5 +/- 18.3%, n = 3) [p < 0.0001]. In colon, the DFF LI in non-neoplastic mucosa (10.2 +/- 6.4%, n = 3) was significantly lower than that of hyperplastic polyp (56.0 + 34.7%, n = 3) [p = 0.0013] and adenoma (30.1 +/- 16.3%, n = 3) [p = 0.0037]. Cells positive for DFF were much more widely distributed than TUNEL positive cells in both non-pathologic and pathologic mucosa of human stomach and colon. Notably, DFF positive cells were present beneath the TUNEL positive cells in non-pathological gastric and colonic epithelium. In addition, there was a significant positive correlation between DFF and TUNEL LIs in human stomach and colon [p < 0.0001]. These results suggest that DFF may be involved in the process of apoptosis in human gastric and colonic mucosa.

Biliary atresia.

Although the prognosis of biliary atresia has been dramatically improved in the era of liver transplantation, the Kasai operation is still the first line of surgical treatment. Successful hepatic portoenterostomy depends on early diagnosis and surgery, adequate surgical technique, prevention of cholangitis, and precise postoperative management.

Biliary atresia.

Although biliary atresia is characterized by luminal obstruction of the extrahepatic bile ducts, the etiology and the pathophysiology of the liver are still controversial. The prognosis of biliary atresia has been improved after the introduction of Kasai's hepatic portoenterostomy, but there are still many problems to be solved in the treatment of this disease. Successful results of hepatic portoenterostomy depend on early diagnosis and operation, adequate operative technique, prevention of postoperative cholangitis, and precise postoperative management. However, we are on the verge of a new era in the therapy of biliary atresia combining portoenterostomy with liver transplantation.

Do we still need to collect stool? Evaluation of visualized fatty acid absorption: experimental studies using rats.

BACKGROUND: Short-gut syndrome is likely to impair enteric fat utilization. This study was undertaken to develop a clinical test of lipid absorption without fecal collection. METHODS: The absorption of enterally fed radioactive long-chain fatty acid, beta-methyl-p-(123I)-iodophenylpentadecanoic acid was investigated with continuous chyle collection in rats. The changes in excretion and time-dependent biodistribution of radioactivity of the enterally fed agent were assessed in normal control animals. Similarly, sequential urinary excretion and biodistribution were studied along with scintigraphy using sham-operated and short-gut animals. RESULTS: Approximately 64% of the enterally fed radioactivity was recovered in the collected chyle (24 hours). A comparison of normal control, sham-operated, and short-gut animals showed significantly less urinary and greater fecal excretions of radioactivity in short-gut animals. With the use of sequential scintigraphy, the small intestine, whole-body soft tissues, and urinary bladder were well visualized in sham-operated animals, whereas the large intestine and feces were demonstrated earlier in short-gut animals. CONCLUSIONS: Our results suggest that enteral feeding of the agent might be feasible for determining lipid absorption from the the dynamic changes of radioactivity in visualized abdominal organs and in urine.

Choline acetyltransferase activity and evoked spinal cord potentials for diagnosis of brachial plexus injury.

The purpose of this study is to investigate the diagnostic value of evoked spinal cord potentials (ESCPs) and choline acetyltransferase (CAT) activity during exploration of injuries to the brachial plexus. We assessed 25 spinal roots in 19 patients. The results of the two investigations were consistent in all except two roots. Although assessment of ESCPs is easy and quick, it mainly records the nerve potentials along the sensory pathway. Although measurement of CAT activity needs a specimen of the nerve and the availability of a radioisotope laboratory, it gives direct information regarding the motor function of ventral spinal roots. These two techniques should be complementary to each other in order to achieve a more accurate diagnosis.

Copper kinetics in infantile hepatobiliary disease.

Copper metabolism was investigated in 33 infants with hepatobiliary disease. In 25 paients with biliary atresia, 37-135 days old, hepatic copper ranged from 5 to 133 microgram Cu/g wet weight (mean:43.3 microgram). In over two-thirds of the liver samples copper content was elevated above normal. There was no correlation between hepatic copper concentration and patients' age or degree of liver fibrosis. The mean hepatic copper content was also elevated in six infants with other hepatobiliary diseases (mean:41.6 microgram). Serial assays of copper excretion in bile were made in 25 infants having Kasai procedures for biliary atresia. The average daily excretion of biliary copper in 12 patients with successful operations was 3.3 to 33.7 microgram (mean:15.1 microgram), whereas in 13 patients without postoperative bile drainage, the daily values were 0.3-6.4 microgram (mean:2.7 microgram) (p < 0.0001). In five patients with active bile excretion who had repeated liver biopsies there was a steady decrease in hepatic copper concentration. The results indicate that derangement of copper homeostasis occurs frequently in infants with hepatobiliary disease and that in patients with biliary atresia successful reestablishment of bile flow effects a return toward normal copper metabolism probably because of enhanced biliary excretion.

Cholangiographic study of the pancreaticobiliary ductal junction in biliary atresia.

The size and arrangement of each component of pancreaticobiliary ductal junction was investigated in 28 cases with biliary atresia in which cholangiography allowed visualization of the common bile duct, pancreatic duct, and duodenum and in which relevant data were available. As controls, seven cases of neonatal hepatitis and eight cases of intrahepatic bile duct hypoplasia were also studied. The following results were obtained. (1) The diameter of the common bile duct was shortest in biliary atresia, the value corresponding to about one-third of that for neonatal hepatitis. (2) The common bile duct was visualized in 100% of cases of intrahepatic bile duct hypoplasia and 78% of cases of biliary atresia, whereas the corresponding percentage was only 43% for neonatal hepatitis. The mean length of the common channel in cases of biliary atresia and that of intrahepatic bile duct hypoplasia was about 5 mm, which was longer than that of neonatal hepatitis. (3) The pancreatic duct type, in which the pancreatic duct merged into the bile duct, was frequent in cases of biliary atresia.

Follow-up studies of long term survivors after hepatic portoenterostomy for "noncorrectible" biliary atresia.

Fourteen patients with "noncorrectable" biliary atresia are living without jaundice for more than 2 yr after hepatic portoenterostomy or its modification. Retardation of physical growth was observed in one of them, and mental retardation in another, both of which seemed irrelevant to biliary atresia. Serial tests for liver function after operation revealed early recovery of serum bilirubin, transminase, and turbidity, and delayed improvement of alkaline phosphatase. Postoperative needle biopsy of the liver disclosed that changes in hepatic parenchyma and ductular proliferation were rapidly improved after successful operation. Improvement of fibrosis of the liver was delayed, and it was not satisfactory in patients whose preoperative changes in the liver were severe or in whom ascending cholangitis had been a frequent complication. Histologic features of hepatic cirrhosis were observed in the liver in three cases, in two of which there had been frequent episodes of cholangitis. Only one of these showed clinical signs of portal hypertension. Functional and morphologic cure can be achieved in "noncorrectable" biliary atresia by hepatic portoenterostomy or its modifications, although varying degree of hepatic fibrosis may remain according to severity of preoperative changes of the liver and postoperative complication of ascending cholangitis.

Development of the lung in fetal rabbits with experimental diaphragmatic hernia.

Congenital diaphragmatic hernias were successfully produced in fetal rabbits. The lungs of these animals remained small and hypoplastic on gross examination, although histologically they have matured significantly from the original pseudoglandular stage. It is our impression that the so-called hypoplastic lung in diaphragmatic hernias is small in size but potentially functioning when relieved of compression.