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It is generally accepted that bacteria in or outside the root canal are the reason for apical periodontitis and endodontic failures. This case report presents a 60-year-old woman with a periapical lesion and a fistulous tract which did not respond to conventional root canal treatment. During periapical surgery, granulomatous tissue was removed and a calculus-like deposit was observed on the root surface. A radicular cyst was diagnosed. Nine months after this calculus-like deposit had been removed and the cyst enucleated, complete recovery of the bone had occurred. It is suggested that the presence of the calculus-like deposit at the apex of the tooth or its effects may in part have delayed the healing of the periapical inflammation in spite of apparently adequate endodontic treatment.

To define the role of the Cys residues in the ATP-dependent HslVU protease, mutagenesis was performed to replace either Cys261 or Cys287 in HslU with Val and Cys159 in HslV with Ser or Ala. Whereas HslU/C261V could hydrolyze ATP and support the ATP-dependent proteolytic activity of HslV as well as the wild-type HslU, HslU/C287V could not hydrolyze ATP. Nevertheless, HslU/C287V could support the HslV-mediated proteolysis by forming the HslVU complex in the presence of ATP but not its absence, indicating that ATP binding but not its hydrolysis is essential for proteolysis. Whereas treatment of N-ethylmaleimide (NEM) resulted in dissociation of the oligomeric HslU into monomers, the C261V mutation, but not C287V, prevented the NEM effect. These results suggest that Cys261 is involved in oligomerization and that Cys287 is related to the ATPase function of HslU. NEM also dissociated the dodecameric HslV into monomers, and this effect could be prevented by either the C159S or C159A mutation, suggesting the involvement of Cys159 in oligomerization of HslV. Moreover, either mutation abolished both the basal and HslU-activated proteolytic activity of HslV and its ability to activate the HslU ATPase and to form the HslVU complex, indicating that Cys159 is essential for the proteolytic activity of HslV and its interaction with HslU. These results suggest that the Cys residues play an important role in maintaining the structure and function of the HslVU protease.

The p53 tumor suppressor gene encodes a transcriptional activator whose targets include genes that regulate cell cycle progression and apoptosis. Since we have shown that a critical event in the life history of the chondrocyte is programmed cell death, we asked the question: does loss of the p53 gene influence skeletogenesis? Female p53(+/-) mice were mated with p53(+/-) male mice and 17-day-old fetal mice were studied. Exencephaly was the most profound skeletal defect of the p53 null mutation. This defect was due to failure of formation of the bones that comprise the mouse calvarium. There was also loss of the hyoid bone, and defective mineralization of the manubrium sternum and the terminal phalanges. In the homozygous state (-/-), in the absence of exencephaly, the number of skeletal deformities was markedly reduced. Aside from the gross changes associated with null status, the mutants exhibited alterations in bone length and width. Small differences in the size and orientation of the mineral crystals in embryonic bone, as evaluated by small-angle X-ray scattering, were found to disappear after birth. To explain these observations, we evaluated the extent of apoptosis in the tibial growth plates using the TUNEL stain. In the growth plate of the p53(-/-) homozygote, there was minimal labeling of the hypertrophic layer. Since the p53(-/-) TUNEL stain pattern at 17 days was very similar to the pattern of labeling of the p53(+/+) at 15 days, we concluded that the growth defect reflected a delay in cartilage maturation rather than a change in chondrocyte phenotype. On this basis, we predict that after birth, in mice that survive, differences in bone length would become minimal, and at maturity, the length of the long bones of (+/+) and (-/-) mice would be similar.

The ATP-dependent casein hydrolysis by protease Ti (ClpAP) has been shown to be inhibited by sulfhydryl blocking agents, such as N-ethylmaleimide (NEM), when preincubated with ClpA but not with ClpP. To define the role of three Cys residues in ClpA, site-directed mutagenesis was performed to substitute each of them with Ser or Ala. None of the mutations showed any effect on the ATPase activity of ClpA or its ability to support the casein degradation by ClpP. However, NEM could no longer block the ability of ClpA/C47S or ClpA/C47A in supporting the ClpP-mediated proteolysis, unlike that of ClpA, ClpA/C203S, or ClpA/C243S. Furthermore, in the presence of NEM, casein could stimulate the ATPase activities of ClpA/C47S and ClpA/C47A and protect from their degradation by ClpP, but not of the other ClpA proteins. These results suggest that the inhibitory effect of NEM is due to prevention of the interaction of ClpA with casein by introduction of a bulky alkyl group to Cys47, but not linked to the catalytic function of the ATPase.

Many patients with carcinoma of the pancreas die because their disease is not detected until late in its course. Methods that detect these cancers earlier will improve patient outcome. Over 80% of pancreatic carcinomas contain mutations in codon 12 of the K-ras gene. Screening duodenal fluid for these mutations may lead to early detection of these cancers and assist in establishing a diagnosis of pancreatic carcinoma.Polymerase chain reaction (PCR), with and without restriction enzyme-mediated mutant enrichment, was performed on DNA isolated from duodenal fluid specimens from 61 patients who underwent pancreaticoduodenectomy (Whipple's operation) for either periampullary cancer or a benign condition of the pancreas. Representative sections of pancreas pathology (primary carcinoma, benign tumor, or chronic pancreatitis) from the patients with duodenal fluid specimens containing amplifiable DNA were also analyzed for K-ras mutations. Wild-type and mutant K-ras were detected by hybridization of the PCR products with K-ras codon 12 mutant and wild-type specific probes.Seven of the 61 duodenal fluid specimens contained DNA that did not amplify. Thirteen (24% of the 54 duodenal fluid specimens with amplifiable DNA and 21% of the total of 61 specimens) contained activating point mutations at codon 12 of the K-ras gene. Mutations were detected in 13 of the 51 duodenal fluid specimens from patients with cancer (sensitivity, 25%), whereas mutations were not detected in any of the 9 amplifiable duodenal fluid specimens from patients with benign conditions of the pancreas (specificity, 100%). One duodenal fluid specimen from a patient with adenocarcinoma of the pancreas had two different K-ras mutations. DNA from three of the primary carcinomas did not amplify or was not available. Twenty-nine (69%) of the 42 primary tumors with amplifiable DNA contained K-ras mutations, whereas 3 (30%) of the 10 pancreata with benign conditions harbored mutations. Twenty-two (65%) of 34 ductal adenocarcinomas of the pancreas with amplifiable DNA had K-ras mutations. It is noteworthy that the same mutation was present in both the duodenal fluid and the primary carcinomas of 11 (92%) of the 12 patients who had primary tumors with amplifiable DNA as well as K-ras mutations in their duodenal fluid specimens.The identification of genetic alterations in cancer-causing genes in duodenal fluid may form the basis for the development of new approaches to the detection of carcinoma of the pancreas. Some pancreata without cancer, however, may also harbor K-ras mutations, potentially limiting the specificity of K-ras-based tests.

Massive endobronchial bleeding during extracorporeal circulation was encountered in a patient during aortic dissection operation. The use of high frequency jet ventilation resulted in successful staunching of the bleeding, thus avoiding the need of pneumonectomy or lobectomy.

The ClpP component Clp protease from Escherichia coli has been crystallized and examined by X-ray crystallography and self-rotation function calculations. The crystal belongs to the monoclinic space group P2(1) with unit cell dimensions of a = 196.9 A, b = 104.3 A, c = 162.4 A and beta = 98.3 degrees. The X-ray diffraction pattern extends at least to 2.5 A Bragg spacing when exposed to CuK alpha X-rays. Self-rotation function analyses indicate that the ClpP oligomer has 72-point group symmetry. This symmetry suggests that the ClpP oligomer is a tetradecamer, (ClpP)14, consisting of two heptamers, (ClpP)7 stacked on top of each other in a head-to-head fashion. The measurement of crystal density indicates that two independent copies of the ClpP oligomers are present in the asymmetric unit, giving a crystal volume per protein mass (VM) of 2.73 A3/Da and a solvent content of 54.9% (v/v). Self-rotation function calculations are consistent with the presence of two ClpP tetradecamers in the asymmetric unit. The Patterson function suggests that a translation of x = 0.5 and y = 0.5 relates a pair of ClpP oligomers in one asymmetric unit to another pair in the other asymmetric unit. And the two independent tetradecamers in one asymmetric unit are related by a relative rotation of about 18 degrees around the 7-fold axis.

The ATP-dependent protease Ti(Clp) consists of two different multimeric components: ClpA containing ATP-cleaving sites and ClpP, with serine active sites for proteolysis. Here we summarize the most recent results on the structure and function of protease Ti. (1) The clpA gene has dual translational initiation sites and therefore encodes two polypeptides with sizes of 84 and 65 kDa. The abbreviated form of ClpA may play an important role in regulation of the ATP-dependent proteolysis, since it inhibits the ability of the 84-kDa ClpA in supporting the ClpP-mediated protein breakdown and the autodegradation of the 84-kDa ClpA. (2) ClpA contains two highly conserved sequences for ATP-binding: the first site is essential for oligomerization and the second site is responsible for ATP hydrolysis. (3) ATP hydrolysis by ClpA is required not only for assembly of the ClpA/ClpP complex but also for its rapid dissociation.

The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to beta-type subunits of the 20 S proteasomes, and HslU, a 50-kDa protein related to the ATPase ClpX. We have recently shown that HslV and HslU can function together as a novel ATP-dependent protease, the HslVU protease. We have now purified both proteins to apparent homogeneity from extracts of E. coli carrying the hslVU operon on a multicopy plasmid. HslU by itself cleaved ATP, and pure HslV is a weak peptidase degrading certain hydrophobic peptides. HslU dramatically stimulated peptide hydrolysis by HslV when ATP is present. With a 1:4 molar ratio of HslV to HslU, approximately a 200-fold increase in peptide hydrolysis was observed. HslV stimulated the ATPase activity of HslU 2-4-fold, but had little influence on the affinity of HslU to ATP. The nonhydrolyzable ATP analog, beta,gamma-methylene-ATP, did not support peptide hydrolysis. Other nucleotides (CTP, dATP) that were slowly hydrolyzed by HslU allowed some peptide hydrolysis. Therefore, ATP cleavage appears essential for the HslV activity. Upon gel filtration on a Sephacryl S-300 column, HslV behaved as a 250-kDa oligomer (i.e. 12-14 subunits), and HslU behaved as a 100-kDa protein (i.e. a dimer) in the absence of ATP, but as a 450-kDa multimer (8-10 subunits) in its presence. Therefore ATP appears necessary for oligomerization of HslU. Thus the HslVU protease appears to be a two-component protease in which HslV harbors the peptidase activity, while HslU provides an essential ATPase activity.

AIMS: To investigate the prevalence of K-ras codon 12 point mutations in ampullary neoplasms, to explore their clinical usefulness, and to test whether the detection of these mutations could be used to identify ampullary malignancies at an early stage. METHODS: Forty one tumour specimens from 28 patients with ampullary neoplasms were analysed for activating point mutations in K-ras codon 12 using a sensitive polymerase chain reaction (PCR) based assay. RESULTS: Eleven (39%) of the 28 primary tumours harboured point mutations in K-ras. Mutations were identified in seven (41%) of the 17 carcinomas and four (36%) of the 11 adenomas. Four of the possible six permutations in codon 12 were found in these 11 samples. This spectrum of mutations is different from pancreatic carcinoma but resembles that of colorectal neoplasms. Cytological brush specimens were available in 11 cases, and in all of these specimens, the K-ras status in the primary tumour and brush specimens was identical. CONCLUSIONS: K-ras codon 12 point mutations occur in about 40% of ampullary neoplasms at a relatively early stage in tumorigenesis. The pattern of mutations in these tumours resembles that of the adenoma-carcinoma sequence in the colorectum. These results indicate that ampullary neoplasms can be detected at an early stage by searching for genetic alterations in the K-ras oncogene in cytological brush specimens.

Protease Ci, a cytoplasmic metalloprotease in Escherichia coli, has been purified to apparent homogeneity by conventional chromatographic procedures using 125I-labeled oxidized insulin B-chain as a substrate. The purified enzyme behaves as a 54-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It is inhibited by metal-chelating agents, including o-phenanthroline and NaCN, but not by inhibitors of serine proteases or thiol-blocking agents. Furthermore, protease Ci was found to contain 1.1 mol of zinc per mol of the enzyme upon analysis by HR ICP mass spectroscopy. Thus, protease Ci must be a zinc metalloprotease. Among the polypeptides tested as substrates, oxidized insulin B-chain and glucagon are most rapidly hydrolyzed. Intact insulin is a much poorer substrate than oxidized insulin B-chain, even though the affinity of the enzyme to intact insulin is approximately 100-fold greater than that to the B-chain. Since unlabeled oxidized insulin A-chain is capable of inhibiting the hydrolysis of 125I-labeled insulin B-chain, it also appears to be a substrate. Protease Ci also degrades lysozyme and lactalbumin, although to a much lesser extent than oxidized insulin B-chain. However, it shows little or no activity against proteins larger than 15 kDa (e.g. ovalbumin and denatured bovine serum albumin). Hydrolysis of oxidized insulin B-chain followed by amino acid composition analyses of the cleavage products reveals that as many as 10 of its 29 peptide bonds are hydrolyzed by protease Ci. This ability to hydrolyze relatively small polypeptides suggests that protease Ci may catalyze the later steps in the pathway for intracellular protein breakdown.

The clpA gene that encodes the ATPase subunit of the ATP-dependent protease Ti (Clp) in Escherichia coli contains a putative internal translational initiation site. Here we show that mutagenesis of its 5'-end AUG codon resulted in an exclusive synthesis of the 65-kDa protein (ClpA65), while mutation at the internal 169th AUG codon (Met) to ACG (Thr) produced only the 84-kDa protein (ClpA84T). On the other hand, the cells carrying the wild-type clpA gene produced both the 84- and 65-kDa proteins (ClpA84/65). While the purified ClpA84T and ClpA84/65 hydrolyzed ATP nearly as well as the 84-kDa ClpA alone (ClpA84), ClpA65 cleaved ATP at a rate less than 5% of that by ClpA84. Unlike ClpA84 and ClpA84T, ClpA65 could not support the casein-degrading activity of ClpP. Furthermore, ClpA65 inhibited the proteolysis by the mixture of ClpP with ClpA84 or ClpA84T but not that with ClpA84/65, which could support the proteolytic activity of ClpP only about 40% as well as ClpA84. Nevertheless, ClpA65 showed little or no effect on the basal or protein-activated ATPase activity of ClpA84, ClpA84T, or ClpA84/65 alone or in the presence of ClpP. These results suggest that ClpA65 may interfere the interaction of ClpA84 or ClpA84T with ClpP and, hence, impair their assembly into an active form of the ATP-dependent protease Ti.

The hospice at Mackay Memorial Hospital was established in February 1990. A group of team workers including physicians, nurses, social workers and the clergy were involved in this holistic care program for terminal cancer patients. Four hundred and seventy-nine patients were eligible for the program up to February 1993. Regarding duration of stay, 62.5% of patients resided for 14 days. Those surviving under 90 days constituted 75.5% of patients. Fifty-one point eight percent of patients died in the hospice and 18.2% died at home soon after being discharged from the hospice. Pain is the most common symptom among the patients. Treatment strategies vary according to the three-step-ladder protocol designed by WHO. Total pain relief was achieved in 80% of patients. Opportune private talking and family conferences formed the basis of the "peer model". Through this model, treatment decisions including physical, psychosocial and spiritual issues were made. Before the peer model, only 36 (10.3%) patients agreed with the idea of hospice care, while 257 (73.6%) patients agreed after the model was established. Awareness of dying was evident in 412 (86%) patients. Two hundred and eighty (68%) patients became aware of the prospect of death through guessing, while the other 132 (32%) patients were informed by medical staff. Problems encountered by the team workers included 1) needs in education and training, 2) psychological pressure, 3) management of loss and grief, 4) needs in supportive system and 5) troubles caused by families' lying to patients. The team workers were satisfied with the quality of care in 38.4% of patients and fairly satisfied with 30.7% of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The importance of the P1 reactive site for the specificity of ecotin on target proteases was examined by site-directed mutagenesis. The replacement of Met at the P1 site with Ile, Arg, Glu, or Tyr showed little or no effect on the ability of ecotin to inhibit trypsin. Similar results were obtained for chymotrypsin, except that its replacement with Glu caused about 40% reduction of the inhibitory activity of ecotin. On the other hand, the replacement of the Met residue with Arg, Tyr, or Glu dramatically reduced its ability to inhibit elastase, while that with Ile showed little or no effect. Nevertheless, elastase could be completely inhibited upon incubation with excess amounts of the mutant ecotin containing Arg, Glu, or Tyr. Moreover, all the mutant forms of ecotin could be cleaved at the mutated P1 site upon incubation with trypsin at pH 3.75. In addition, the replacement of a Cys residue in the disulfide bridge with Ser showed little or no effect on the ability of ecotin to inhibit trypsin, chymotrypsin, or elastase. However, the mutant ecotin containing Ser was more sensitive to inactivation by heating at 100 degrees C than the wild-type inhibitor. Furthermore, the wild-type ecotin whose disulfide bond had been reduced and alkylated was also more easily inactivated by heat treatment than the untreated control. These results strongly suggest that the P1 site of ecotin is not crucial for its specificity on target proteases and that the disulfide bridge in ecotin appears to play an important role in maintenance of its structural stability.

Nitric oxide (NO) is a messenger molecule of vascular endothelial cells, macrophages, and neurons. Here, we demonstrate that the activity of NO synthase increases transiently but dramatically in chick embryonic myoblasts that are competent for fusion. This activity requires Ca2+, calmodulin, and NADPH. In addition, the increase in NO synthase activity coincides with an increase in cellular cGMP level. Furthermore, NO generated by treatment with sodium nitroprusside induces precocious myoblast fusion, while treatment with NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase, or methylene blue, an inhibitor of guanylate cyclase, delays fusion. These results provide the first evidence for a strong association of NO with myoblast fusion.

Myoblast surface proteins with binding activity toward the N-terminal 29-kDa fragment of fibronectin were identified by two different experimental techniques: one involves radioiodination of the cell surface proteins, followed by solubilization with Triton X-100 and affinity purification on a Sepharose column conjugated with the 29-kDa fragment, and the other involves cross-linking of the 29-kDa fragment to the cells metabolically labeled with [35S]methionine, followed by immunoprecipitation with anti-29-kDa IgG. Both approaches revealed that primary cultures of chick myoblasts contain the 66- and 48-kDa proteins that bind to the 29-kDa fragment. These binding proteins were then purified to apparent homogeneity by two successive chromatographies of the solubilized extracts of 12-day-old embryonic muscle on wheat germ agglutinin-agarose and 29-kDa fragment-Sepharose columns. However, the 48-kDa protein was found to be derived from contaminating fibroblasts upon immunoblot analysis of the myogenic cell lines, rat L8E63 and mouse C2A3, and cultured fibroblasts using the antibody raised against the 66-kDa protein. Anti-66-kDa IgG inhibited the binding of the 125I-29-kDa protein to the primary culture of myoblasts in a dose-dependent manner. On the other hand, the same antibody showed little or no effect on the initial binding of 125I-fibronectin to the cell surface, but dramatically inhibited its incorporation into deoxycholate-insoluble matrices. Furthermore, Fab fragments of anti-66-kDa IgG completely blocked the incorporation of fluoresceinated fibronectin into matrices but not its binding to the cell surface. These results suggest that fibronectin matrix assembly is mediated at least in part by the interaction of the 66-kDa protein with the N-terminal type I domain of fibronectin.

The subunit patterns of the proteasomes, that were purified from muscle, liver and brain, were found to be significantly different from one another. Furthermore, the proteasomes from adult and embryonic tissues of the same types also differed from each other in their subunit patterns. In addition, the specific activities of the purified proteasomes for peptide-cleavage, but not for casein-hydrolysis, appeared to be varied among the enzymes isolated from the different tissues. Thus, expression of a large number of proteasome subunits appears to be tissue-specific and under developmental control, although its relation with the multicatalytic activities of the proteasomes remains unclear.

The heat shock protein ClpB in Escherichia coli is a protein-activated ATPase and consists of two proteins with sizes of 93 and 79 kDa. By polymerase chain reaction-aided site-directed mutagenesis, both the proteins have been shown to be encoded by the same reading frame of the clpB gene, the 93-kDa protein (ClpB93) from the 5'-end AUG translational initiation site and the 79-kDa protein (ClpB79) from the 149th codon (an internal GUG start site). Both the purified ClpB93 and ClpB79 proteins behave as tetrameric complexes with a very similar size of about 350 kDa upon gel filtration on a Superose-6 column. Both appear to be exclusively localized to the cytosol of E. coli. Both show inherent ATPase activities and have an identical Km of 1.1 mM for ATP. The ATPase activity of ClpB93 is as markedly stimulated by proteins, including casein and insulin, as that of wild-type ClpB, but the same proteins show little or no effect on ClpB79. Because ClpB79 lacks the 148 N-terminal sequence of ClpB93 but retains the two consensus sequences for adenine nucleotide binding, the N-terminal portion appears to contain a site(s) or domain(s) responsible for protein binding. Furthermore, ClpB79 is capable of inhibiting the casein-activated ATPase activity of ClpB93 in a dose-dependent manner but without any effect on its inherent ATPase activity. In addition, ClpB93 mixed with differing amounts of ClpB79 behave as tetrameric molecules, although its protein-activated ATPase activity is gradually reduced. These results suggest that tetramer formation between ClpB93 and ClpB79 may be responsible for the inhibition of the activity.

7607 Background: E2501 was a randomized discontinuation phase II study of sorafenib (S) versus placebo (P) in patients (pts) with metastatic NSCLC who have failed at least two prior chemotherapy re...

The clpB gene in Escherichia coli encodes a heat-shock protein that is a close homolog of the clpA gene product. The latter is the ATPase subunit of the multimeric ATP-dependent protease Ti (Clp) in E. coli, which also contains the 21-kDa proteolytic subunit (ClpP). The clpB gene product has been purified to near homogeneity by DEAE-Sepharose and heparin-agarose column chromatographies. The purified ClpB consists of a major 93-kDa protein and a minor 79-kDa polypeptide as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Upon gel filtration on a Superose-6 column, it behaves as a 350-kDa protein. Thus, ClpB appears to be a tetrameric complex of the 93-kDa subunit. The purified ClpB has ATPase activity which is stimulated 5-10-fold by casein. It is also activated by insulin, but not by other proteins, including globin and denatured bovine serum albumin. ClpB cleaves adenosine 5'-(alpha,beta-methylene)-triphosphate as rapidly as ATP, but not adenosine 5'-(beta,gamma-methylene)-triphosphate. GTP, CTP, and UTP are hydrolyzed 15-25% as well as ATP. ADP strongly inhibits ATP hydrolysis with a Ki of 34 microM. ClpB has a Km for ATP of 1.1 mM, and casein increases its Vmax for ATP without affecting its Km. A Mg2+ concentration of 3 mM is necessary for half-maximal ATP hydrolysis. Mn2+ supports ATPase activity as well as Mg2+, and Ca2+ has about 20% their activity. Anti-ClpB antiserum does not cross-react with ClpA nor does anti-ClpA antiserum react with ClpB. In addition, ClpB cannot replace ClpA in supporting the casein-degrading activity of ClpP. Thus, ClpB is distinct from ClpA in its structural and biochemical properties despite the similarities in their sequences.

A protein capable of inhibiting trypsin and a number of other serine proteases was purified from chicken skeletal muscle. It has an apparent molecular weight of 64,000 as determined by gel filtration. The inhibitor molecule binds trypsin at a molar ratio of 1:1 to form a stable complex, in which trypsin can be completely inhibited. In this complex, the inhibitor is extensively digested by trypsin but retains its inhibitory activity and tertiary structure by intramolecular disulfide bonds. In addition, its activity was found to markedly increase during development of embryonic muscle. The physiological role of this inhibitor, however, remains unknown.

An implantable drug delivery system will play an important role for patients who need chemotherapy or prolonged total parenteral nutrition in the future. We implanted 72 injection capsules in 74 patients who were undergoing aggressive chemotherapy or total parenteral nutrition programs. These capsules were implanted in different positions, including central access in 67 cases, peritoneal cavity in 1, hepatic artery in 2, portal vein in 1 and intrathecal space in 1. The actual median functional survival of the injection capsules was 12.3 months (range, 2 months to 4 years). The total cumulative puncture numbers were 1651 (range, 5 to 153; median 23). There was no unsuccessful instance in infusion or injection. To date, 27 patients still survive. The complication rate was about 7% including 1 sepsis, 1 skin infection, 1 catheter thrombosis, 1 dislodgement of reservoir and 1 catheter-reservoir disengagement, which were all corrected by a secondary operation without mortality. Patency of the Port-A-Cath system was maintained using a regular flushing schedule once every 30 days. The results of this study suggest that the implantable drug delivery system can provide a safe and reliable method for venous access or even arterious access in patients requiring intermittent or prolonged intravenous therapy.

The transfer function formulation of constrained distributed systems is presented. The methodology is illustrated for subsystems under pointwise constraints and distributed systems consisting of multiple subsystems. A general displacement method (GDM) is used to determine the eigensolution of the constrained systems. It is shown that GDM requires only the natural boundary conditions (force constraints) be imposed at the subsystem interface. The methodology is applied to two examples. Curve veering and mode localization phenomena are found in an elastic structure on an elastic foundation.

From January 1979 to September 1985, a total of 125 patients preoperatively staged as International Federation of Gynecology and Obstetrics (FIGO) Ib uterine cervical carcinoma were proven to have either microscopic involvement of the surgical margin, parametrium and/or regional lymph node metastasis histopathologically after radical hysterectomy and pelvic lymphadenectomy. All of these patients were treated postoperatively with radiotherapy because of the above indications. Based on indications of postoperative radiotherapy, patients were divided into 3 major subgroups according to the microscopic involvement: group A patients (50) with microscopic evidence of regional lymph node metastasis only; group B patients (59) with microscopic evidence of parametrial involvement; and group C patients (16) with microscopic infiltration of the surgical margin involving the vaginal cuff. All patients were treated with external irradiation using a Cobalt-60 teletherapy machine. The overall actuarial 5-year survival rate was 62% with a 77% 5-year survival rate for group A, a 50% rate for group B and a 75% rate for group C. Further analysis of the prognostic factors revealed that those with regional lymph node involvement of 4 or less had a better chance of survival than those with lymph node involvement of more than 4. Also those with poorly differentiated squamous cell carcinoma had a lower 5-year survival rate than those with moderately or well-differentiated squamous cell carcinoma. Univariate analysis revealed that hemoglobin values, age, and the time interval from surgery to radiotherapy were not significant prognostic factors.

The proteolytic activities of the 20 S proteasome were found to change in their levels during the development of chick embryonic muscle. The peptide-cleaving activities against N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-beta-naphthylamide gradually decreased with the time of development. On the other hand, the casein-degrading activity in the presence of poly-L-lysine markedly increased from embryonic day 11 and reached a maximal level by day 17. These changes appeared to be tissue-specific because little or no change in any of the proteolytic activities was observed with developing embryonic brain, while dramatic alterations occurred in the extents of the peptide hydrolyses in liver. Furthermore, a number, but not all, of the proteasome subunits in embryonic muscle were changed in their amounts during the development. These results suggest that the alterations in the proteasome activities and subunit pattern are developmentally regulated and may be correlated.

Twenty-four primiparous Holstein cows were fed corn silage:grain (1:1, DM basis) and hay (.9 kg/d) beginning 30 d prepartum and through wk 18 of lactation. Ten grams of Biomate® Yeast Plus (5 x 10 9 cfu of Saccharomyces cerevisiae /g) were top-dressed on the a.m. allotment of corn silage:grain fed to 12 cows. Corn silage:grain was restricted during prepartum and thereafter fed for ad libitum intake. Cows fed supplemental yeast peaked earlier and had a higher milk yield compared with control cows (wk 7, 29.5 kg/d vs. wk 11, 28.7 kg/d). Digestibilities of protein and cellulose were improved in cows fed supplemental yeast, contributing to a greater DMI during the first 6 wk of lactation and a higher average milk yield through wk 18 of lactation compared with control cows (27.2 vs. 26.0 kg/d).

Induction of protease La was found to increase to higher extent in E. coli that had been treated with canavanine for longer period. However, hydrolysis of canavanine-containing proteins occurred rapidly but at nearly an identical rate regardless of the period of canavanine-treatment. Exposure of E. coli to heat also raised the level of protease La but showed little effect on overall rate of proteolysis. These results suggest that induction of protease La under stress occurs as a part of heat shock response but not necessarily for elimination of denatured or abnormal proteins.

The YC7-alpha gene encoding a subunit of yeast proteasomes named PRS2 has been isolated and sequenced. Southern blot analysis after electrophoretic separation of yeast chromosomal DNAs showed that the PRS2 gene is located in chromosome VII, unlike the PRS1 gene for subunit YC1, is located on chromosome XV. Surprisingly, the overall structure of the PRS2 gene was found to be identical to that of the suppressor scl1+ gene restoring the SCL1-1 mutation that suppresses crl3 cycloheximide-resistant, temperature-sensitive lethality. The identity of these two genes indicates that the suppressive role of the scl1+ gene product on crl3 mutation could be attributed to proteasomal function.

Large-area, vertically aligned silicon nanowire (SiNW) arrays have been successfully synthesized in an aqueous solution containing AgNO 3 and HF on (001)Si substrates. The as-synthesized SiNWs were determined to be perfectly single crystalline with the axis of the wire parallel to the [001] direction. The typical widths of the SiNWs were in the range of 30-200 nm. The lengths of SiNWs could be tuned from several to tens of micrometers by adjusting the synthesis temperature and time. We measured the formation rate at different reaction temperatures. The activation energy for linear growth of the SiNWs, as obtained from an Arrhenius plot, was found to be about 0.36 eV. In addition, the Si substrates with highly oriented SiNW arrays were found to exhibit significant hydrophobic properties. The water contact angles of the HF-treated SiNW arrays were measured to be about 120-148°, much greater than that with a flat silicon wafer surface (∼85 to 88°).

KMnO4 and N-ethylmaleimide at low concentrations (i.e., below 0.2 and 1.5 mM, respectively) are known to interact specifically with four to five sulfhydryl residues per Ca2+/Mg2(+)-ATPase molecule in sarcoplasmic reticulum. Purified calpain preferentially hydrolyzes the ATPase that was treated with either agent but not the native form of the enzyme. Exposure to each agent with increasing concentrations results in a greater loss of the ATPase activity and renders the enzyme more susceptible to calpain. In addition, beta,r-methylene-ATP, when added during the treatment of KMnO4 or N-ethylmaleimide, can partially protect the ATPase against the degradation. These results suggest that the covalent modification at the specific sulfhydryl residues in sarcoplasmic reticulum ATPase may mark the enzyme for degradation by intracellular proteinases, such as calpain.

The cDNAs encoding two major subunits, named YC1 and YC7-alpha, of yeast proteasomes (multicatalytic proteinase complexes) were isolated and sequenced. As deduced from their nucleotide sequences, YC1 and YC7-alpha consist of 288 and 252 amino acid residues with calculated molecular weights of 31,534 and 27,999, respectively. They showed marked sequence homology to other eukaryotic proteasome components, suggesting that proteasomes are composed of a family of subunits with the same evolutional origin. To obtain information on the physiological role of proteasomes, we disrupted the chromosomal genes of YC1 and YC7-alpha of yeast cells independently, using isolated cDNA clones. Disruption of the coding region of one copy of the YC1 gene in diploid yeast created a recessive lethal mutation, but disruption of the 3'-noncoding region of the gene had no effect on cell proliferation. Disruption of the YC7-alpha gene also had a lethal effect on haploid yeast cells. These findings demonstrated that YC1 and YC7-alpha are both encoded by a single copy gene and that these genes are essential for proliferation of yeast cells.

A total of 43 breast cancer patients were treated at the Department of Radiation Oncology, Mackay Memorial Hospital from January 1978 to December 1987 for postoperative local-regional recurrence. Twenty-six patients developed local recurrence within 12 months after surgery and 17 patients developed local recurrence after 12 months. The mean age for this group of patients was 52 years (range: 27-82). There were 26 postmenopausal and 17 premenopausal patients. Twenty-four patients had received chemotherapy with cyclophosphamide, methotrexate and 5-fluorouracil (CMF) regimen. Four patients were excluded from this study due to incompletion of radiotherapy. Sites of local recurrence in the remaining 39 patients included: chest wall, single (8) and multiple (4); axilla (7); supraclavicular area (5); chest wall with regional node (11); and multiple regional nodes (4). All were free of distant metastasis at the initiation of radiotherapy. A mean tumor dose of 5000 cGy in 28 fractions was given over a 6-week period to the chest wall and draining lymphatics, using an AECL Theratron 80 or a CGR Alcyon MEV Cobalt-60 unit. The overall local control rate was 72%, and the 5-year local control rate was 59%. Nine patients experienced a second local recurrence, with a median interval of 8 months after radiotherapy. Four of them (4/9) also showed evidence of systemic disease at the same time. Distant metastasis, the major cause of mortality, appeared in 49% of the patients with a median interval of 22.8 months after radiotherapy. The three leading sites of distant metastasis were the bones (42%), lungs (37%) and liver (16%).(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebral metastasis is a common manifestation of lung cancer. Presenting signs and symptoms are often grave, and consequently often result in patients being debilitated for the rest of their lives. Radiotherapy has been used to treat a majority of these cases, and is considered as the treatment of choice. At Mackay Memorial Hospital, we collected from 1982 to 1985, the records of 42 lung cancer patients with brain metastasis. All the cases had a histological diagnosis of primary lung cancer; most of them were squamous cell carcinoma and adenocarcinoma; only two cases were small cell carcinoma. The diagnosis of brain metastasis was established by computed tomographic scans of the brain and radioisotopic brain scans. Of the 42 cases, 22 received palliative radiotherapy from a cobalt-60 teletherapy machine to the whole brain for a total dose of 30 Gray (Gy) in 10-15 fractions over a time span of 2 to 3 weeks, while the remaining patients only received medical treatment (e.g. cranial decompression with mannitol, steroids, etc.). Most of the patients have already died. In our study, those who received radiotherapy attained considerable palliation of their symptoms and signs, including improvement of their general performance status and neurological function. Although the treatment did not prolong the patient's survival, it did decrease considerably the disability caused by the metastatic disease.

Electrophoresis gel analysis is a viable technique for determining the purity of hybrid corn seeds. Visually analyzing the electrophoretic gel images is a very tedious and time-consuming task. In this paper, a computer vision system integrating image processing and pattern recognition techniques with domain-specific structural information to automate the electrophoresis gel scoring procedure is presented. A set of image processing algorithms are developed to perform extraction of the region of interest, segmentation of samples, identification of bands within samples, and final classification of different types of seeds.The image processing algorithms utilize the structural information and operator expertise to achieve high classification rate with fuzzy and incomplete information contained on the electrophoresis gels. The developed technique clearly demonstrates the potential of using computer vision in automating the gel scoring procedure. The developed technology may also be extended to other areas such as general one-dimensional electrophoresis and high performance thin layer chromatography.

The active-matrix driven diode-type field emission display (FED) was fabricated using carbon nanotubes (CNTs) as electron emission sources. The a-Si TFT and screen-printed CNT film were successfully integrated for the active-matrix cathode. We fabricated the first prototype AM-CNT FED panel as 3-inch in diagonal with 96×64 pixels. The driving voltage of AM-CNT FED was below 25 V and the operation was well stabilized by the control of driving TFT.

For the reliable operation in Active-Matrix Field Emission Display (AMFED), the modification of the structure in field emitter cathode was proposed. A resistor bridge over field emitter gate electrode was added, which gave short-failure tolerance to the cathode. A light-shield electrode was also incorporated to minimize the off-leakage current of active-control transistor. The prototype AMFED composed of a-Si TFT and Mo-tip emitter array was fabricated and its operation was successfully demonstrated.

The product of the lon (capR or deg) gene in Escherichia coli is protease La, an ATP-dependent protease with a linked ATPase activity. Unlike most lon mutations, capR9 is dominant over the wild type under certain conditions. When protease La was isolated from R9 cells and from a recessive capR- strain using DEAE-cellulose chromatography, the mutant enzymes showed about 50% of the wild type activity. Unlike the wild type, the R9 and R- proteases were inhibited by addition of NaCl (less than 0.1 M). In addition, the R9, but not the R-, material inhibited protelysis by normal protease La, and this effect may account for its dominant phenotype. When isolated by phosphocellulose chromatography, the R9 protein lost proteolytic activity but still inhibited the wild type enzyme. This inhibitory activity was purified to near homogeneity using DEAE-cellulose and heparin-agarose chromatography, and corresponded to the 94,000-dalton R9 gene product. At different concentrations, it inhibited ATP-dependent casein degradation and casein-stimulated ATP hydrolysis to a similar extent. Thus, rates of ATP and protein cleavage remained proportional. Similar inhibition of the wild type protease was observed in the presence of DNA which stimulates both protein and ATP hydrolysis. Half-maximal inhibition was observed with approximately a 1:1 ratio of the R9 to the wild type protein. The subunit sizes of the R9 and the wild type protease were indistinguishable but they differed in isoelectric points. Upon gel filtration, both eluted as tetramers (450,000 daltons) in the absence of salt. However, with 0.1 M NaCl, the wild type protease La remained as a tetramer, but the R9 protein dissociated into dimers and monomers and became a more effective inhibitor. After mixing with R9 protein, 3H-labeled protease La remained tetrameric, though it had lost activity. These findings suggest that tetramer formation between the wild type and defective R9 subunits is responsible for the inhibition of the proteolytic and ATPase activities.