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Increased epithelial cell proliferation is associated with an increased risk of gastric carcinoma. Helicobacter pylori infection is an established risk factor for gastric cancer and the organism has recently been classified as a group I carcinogen by an IARC working group. In this study, we describe differences in gastric epithelial cell proliferation between a H. pylori eradicated group (n = 21) and a not eradicated group (n = 8) after anti-H. pylori eradication therapy to show that increased cell proliferation is associated with H. pylori infection. H. pylori infection was determined by rapid urease test and immunohistochemical method with anti-H. pylori polyclonal antibody. Gastric epithelial cell proliferation was assessed using immunohistochemical method using Ki-67 monoclonal antibody. Ki-67 positive cells in H. pylori associated chronic active gastritis were observed in the glandular neck and the upper portion of foveolar epithelium. Patients who cleared their H. pylori infections showed a significant decrease of Ki-67 labeling index after therapy (0.73 +/- 0.10 vs. 0.48 +/- 0.08, p < 0.01). By contrast, Ki-67 labeling index before and after treatment in patients who remained positive for H. pylori showed no significant difference (0.78 +/- 0.08 vs 0.74 +/- 0.10, p > 0.05). These results indicate that H. pylori infection increases the proliferation of gastric foveolar epithelium, which is reduced by the eradication therapy. We suggest that anti-H. pylori eradication therapy can prevent mucosal cell proliferation to be closely associated with gastric carcinogenesis.

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). Here we report the purification and characterization of one of the UCHs, called UCH-8, with 125I-labelled ubiquitin-alpha-NH-MHISPPEPESEEEEEHYC as a substrate. The purified UCH-8 behaved as a 240 kDa protein on a Superdex-200 column under non-denaturing conditions but as a 130 kDa polypeptide on analysis by PAGE under denaturing conditions, suggesting that the enzyme consists of two identical subunits. Thus this enzyme seems to be distinct in its dimeric nature from other purified UCHs that consist of a single polypeptide, except that UCH-6 is also a homodimer of 27 kDa subunits. UCH-8 was maximally active between pH 7.5 and 8, but showed little or no activity below pH 7 and above pH 9. Like other UCHs it was sensitive to inhibition by thiol-blocking agents such as N-ethylmaleimide, and by ubiquitin aldehyde. The purified UCH-8 hydrolysed not only ubiquitin-alpha-NH-protein extensions, including ubiquitin-alpha-NH-carboxy extension protein of 80 amino acid residues and ubiquitin-alpha-NH-dihydrofolate reductase, but also branched poly-ubiquitin that are ligated to proteins through epsilon-NH-isopeptide bonds. However, it showed little or no activity against poly-His-tagged di-ubiquitin, suggesting that UCH-8 is not involved in the generation of free ubiquitin from the linear poly-ubiquitin precursors. These results suggest that UCH-8 might have an important role in the production of free ubiquitin and ribosomal proteins from their conjugates as well as in the recycling of ubiquitin molecules after the degradation of poly-ubiquitinated protein conjugates by the 26 S proteasome.

We have previously shown that chick muscle extracts contain at least 10 different ubiquitin C-terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH-1 was partially purified by conventional chromatographic procedures using (125)I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate. The purified enzyme behaved as a 35-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consisted of a single polypeptide chain. It was maximally active at pHs between 8 and 9, but showed little or no activity at pH below 6 and above 11. Like other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide, and by ubiquitin-aldehyde. In addition to Ub-PESTc, UCH-1 hydrolyzed ubiquitin-alphaNH-protein extensions, including ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids, ubiquitin-alphaNH-dihydrofolate reductase, and poly-His-tagged di-ubiquitin. This enzyme was also capable of generating free ubiquitin from mono-ubiquitin-epsilonNH-protein conjugates and from branched poly-ubiquitin chains that are ligated to proteins through epsilon NH-isopeptide bonds. These results suggest that UCH-1 may play an important role in the generation of free ubiquitin from ubiquitin-ribosomal protein fusions and linear poly-ubiquitin, as well as in recycling of Ub molecules after degradation of poly-ubiquitinated protein conjugates by the 26S proteasome.

Recently, with the widespread use of new imaging techniques, the diagnosis of autosomal dominant polycystic kidney disease (ADPKD) is increasing. To analyze the extrarenal manifestations of ADPKD in Korean patients, we retrospectively studied the clinical characteristics of 30 patients with ADPKD. Thirty Patients with ADPKD who had been diagnosed at Yongdong Severance Hospital from 1988 through 1994 were recruited for this study. All patients' past and family histories were re-evaluated, and charts and radiologic images were reviewed retrospectively. The male to female ratio was 9:21, and the age of initial diagnosis was 39.2 +/- 13.8 (mean +/- SD) years. In 15 cases (50%), ADPKD had been diagnosed by renal symptoms; in 8 cases (26.7%), by chance during evaluation of extrarenal diseases; in 5 cases (16.7%), by family screening; and in 2 cases (6.7%), by uremic symptoms. Extrarenal involvement included hepatic cysts (70%), pancreatic cysts (16.7%), splenic cysts (6.7%), thyroid cysts (6.7%), inguinal hernia (3.3%), and colonic diverticula (3.3%). In 5 cases (16.7%), cardiac valvular abnormalities were noted by echocardiography. Seven patients underwent hemodialysis, and the duration from the initial diagnosis to initiation of dialysis was 9.9 +/- 8.5 (mean +/- SD) years. We investigated the extrarenal manifestations of 30 cases of ADPKD in Koreans, which were also common and clinically important as renal manifestations. Renal cysts are only one of a myriad of renal and extrarenal manifestations of ADPKD. ADPKD should be managed systematically since this disorder is a systemic disease with clinically important involvement of the cardiovascular system, the gastrointestinal tract, the genitourinary system, and the musculoskeletal system.

The accuracy in target localization of CT, MRI, and digital angiography and the isocentric deviation of linear accelerator were investigated for stereotactic radiosurgery. Twenty-five slice images using CT and MRI were obtained out of geometrical phantom which was designed to produce exact 3-dimensional coordinates of several points within a 0.1 mm error range. These diagnostic images were transferred to a 3-D treatment planning system through ethernet. Measured 3-D coordinates of these images from the planning system were compared to known values by geometrical phantom. Anterior-posterior and lateral films were taken by digital angiography for measurement of spatial accuracy. The accuracy of gantry isocenter aligned by laser localizer was also measured. Lead ball was located at the isocenter of linear accelerator and x-ray films were taken by 4-MV photon beam with gantry angles of 0, 90, 180 and 270 degrees, Overall procedure from CT scanning to treatment was carried out using the geometrical phantom. The target accuracy was verified by high energy x-ray portal film. The accuracy of diagnostic machines were within 2.1 mm except MR-axial images. In case of linear accelerator, the deviation of isocenter was within 0.7 mm. Finally, the total isocentric deviation of overall procedure was 1.3 +/- 0.5 mm (using CT localization).

Multilocus enzyme electrophoresis was used to determine genetic relationships amongst 32 intestinal spirochaetes (Serpulina spp.) isolated from rats (17), rheas (7), chickens, (4), ducks (2), a swan (1) and a flamingo (1). The strains were divided into 20 electrophoretic types (ETs), with a mean genetic diversity per locus of 0.62. The results were compared with those previously published for porcine intestinal spirochaetes. One strain from a healthy rat, and three rhea strains which were recovered from cases of necrotizing typhlitis, were grouped in the same ETs as certain porcine strains of Serpulina hyodysenteriae. The rhea strains could be differentiated from these by pulsed-field gel electrophoresis. Fifteen of the rat strains were genetically and phenotypically closely related. In contrast the avian strains were genetically more heterogeneous, with pathogenic isolates located in three different genetic groups.

The fundamental event of cancer invasion and metastasis is the complicated interaction of cancer cells with host cells, in which event, a number of proteases and their inhibitors are involved. Matrix metalloproteinases are the potent proteases in degrading the basement membrane and extra cellular matrix and are inhibited by specific endogeneous inhibitors, tissue inhibitors of metalloproteinases-1(TIMP-1) and TIMP-2. The expression of mRNA for TIMP-1 and -2 was investigated by Northern blot analysis in specimens taken from 27 patients with primary gastric adenocarcinoma; 25 samples from the primary site, six from the metastatic lymph nodes and two from the peritoneal fluids. The expression for TIMP-1 and -2 was compared in primary gastric cancer tissues, metastatic lymph nodes and normal gastric mucosae. TIMP-1 mRNA was overexpressed in 24 (96%) out of 25 primary cancer tissues compared with the paired normal mucosae, while TIMP-2 was in 10 (40%). In six specimens of metastatic lymph nodes, TIMP-1 and -2 were overexpressed in 6 (100%) and 4 (67%) specimens, respectively. Of two specimens prepared from the peritoneal fluids, all specimens overexpressed TIMP-1 compared with the those of primary cancer tissues, while one (50%) specimen overexpressed TIMP-2. Immunohistochemical staining was done to investigate the localization of TIMP-1 and -2, demonstrating that the immunoreactivity for TIMP-1 and -2 was clearly detected in the cytoplasm of the stromal cells. These results suggest that both TIMP-1 and -2 are overexpressed by stromal cells in most of primary and some metastatic gastric cancer tissues and that TIMP-1 and TIMP-2, produced by stromal cells, may play an important role in inhibiting the proteolytic activity of matrix metalloproteinases originated from cancer cells, in gastric cancer.

The aims of our study were (1) to describe the ultrasonographic findings of the injured medial collateral ligament of the knee and (2) to assess the usefulness of ultrasonography in both evaluating the presence and location of the medial collateral ligament injuries and predicting the prognoses for the patients. The study group consisted of 16 patients with the clinical diagnosis of medial collateral ligament injury. We also examined 20 knee joints in 10 volunteers who had no history of injury to the knee. To predict prognoses, we divided the patients into two groups on the basis of the location of medial collateral ligament injuries. A thickened and heterogeneously hypoechoic appearance of the medial collateral ligament was considered abnormal. The normal medial collateral ligament was a thin and moderately homogeneous hypoechoic band with an average thickness of 4.3 mm (range, 3.3 to 5.6 mm) at the femoral attachment and of 2.3 mm (range, 1.3 to 3.2 mm) at the tibial attachment. All injured medial collateral ligament structures were thickened and heterogeneously hypoechoic. According to the criteria for grading of medial collateral ligament injury with stress view, 10 patients had grade III medial collateral ligament tears, three had grade II tears, and none had grade I injuries. In 15 (94%) of 19 knees, a correct diagnosis could be made with ultrasonography. Ultrasonography is useful in evaluating isolated medial collateral ligament injuries and in predicting patient outcome on the basis of the location of the medial collateral ligament injuries.

Chemostats supplied with limited lactose were used to ask whether it was possible to generate and isolate any mutant of Escherichia coli lactose permease which allowed cells to grow faster. The permease and beta-galactosidase activities of the chemostat culture initially rose together to reach a plateau. After 30 days, the former underwent a second increase alone. From this culture, a faster-growing mutant was isolated. Its permease gene was cloned, sequenced, and found to have a single base pair changed. Thymine at position 199 was changed to guanine, resulting in serine 67 being substituted by alanine. Cells bearing this mutant in the plasmid could grow faster than parents in 10 microM lactose. The Km of the mutant permease toward lactose was 1.4 mM, about half of the wild-type value. Thus, a mutant with higher affinity for substrate could be selected from the chemostat.

An immunohistochemical stain to the c-ErbB-2 protein was performed in 225 paraffin-embedded tissue blocks from patients with locally advanced gastric cancer who underwent curative resection. The overexpression of the c-ErbB-2 protein was observed in 27.4% of the patients. The c-ErbB-2 positivity showed a statistically significant correlation with nodal status and stage. The patients with an overexpression of the c-ErbB-2 protein had a tendency to a shorter survival than those without, but it was not statistically significant (p = 0.08). The 5-year survival rate after surgery was 54% in the negative staining group to the c-ErbB-2 protein and 49% in the positive staining group. This suggests that the c-ErbB-2 protein has a possible role in lymph node metastasis. Therefore overexpression of the c-ErbB-2 protein is a useful indicator of disease progression in gastric carcinoma patients who received curative surgery.

A 15-year-old boy developed back pain and sciatica after a minor trauma. Magnetic resonance imaging revealed lumbar spinal subdural hematoma. After repeated lumbar spinal puncture and drainage of hemorrhagic fluid, spinal subdural hematoma was resolved completely. The benefits of conservative treatment by lumbar spinal puncture are discussed.

Mutations in the mouse diabetes (db) gene result in obesity and diabetes in a syndrome resembling morbid human obesity. Previous data suggest that the db gene encodes the receptor for the obese (ob) gene product, leptin. A leptin receptor was recently cloned from choroid plexus and shown to map to the same 6-cM interval on mouse chromosome 4 as db. This receptor maps to the same 300-kilobase interval as db, and has at least six alternatively spliced forms. One of these splice variants is expressed at a high level in the hypothalamus, and is abnormally spliced in C57BL/Ks db/db mice. The mutant protein is missing the cytoplasmic region, and is likely to be defective in signal transduction. This suggests that the weight-reducing effects of leptin may be mediated by signal transduction through a leptin receptor in the hypothalamus.

Multilocus enzyme electrophoresis (MEE) analysis and comparisons of nearly complete 16S rRNA gene sequences (1416 nucleotide positions) were used to evaluate phylogenetic relationships among Serpulina hyodysenteriae strain B78T, S. innocens strain B256T, Brachyspira aalborgi strain 513AT, and eight uncharacterised strains of swine, avian, and human intestinal spirochaetes. From MEE analysis, nine strains could be assigned to five groups containing other intestinal spirochaetes (genetic distances between groups = 0.6-0.9). Chicken spirochaete strain C1 and B. aalborgi 513AT represented unique electrophoretic types and formed their own MEE groups. Despite MEE differences, the 11 strains had highly similar (96.3-99.9%) 16S rRNA sequences. These findings point out limitations of both MEE analysis and 16S rRNA sequence comparisons when used as solitary techniques for classifying intestinal spirochaetes related to Brachyspira/Serpulina species.

The plasminogen and plasmin system, which is mainly regulated by urokinase-type plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1), is generally believed to play a role in cancer invasion and metastasis. This study was conducted to investigate the role of uPA, uPAR and PAI-1 in the invasion and metastasis of gastric adenocarcinoma. The expression of mRNAs for uPA and PAI-1 was determined by Northern blot analysis in nine primary gastric cancer tissues, nine paired metastatic lymph nodes and normal gastric mucosa. The mRNA of uPA was not or faintly detected in normal mucosa, while the expression was increased in both primary gastric cancer tissues and metastatic lymph nodes to a similar degree. The mRNA expression for PAI-1 in the gastric cancer tissues was not different from that in the paired metastatic lymph nodes and normal mucosae. uPAR was determined by immunohistochemical staining, demonstrating that five (56%) and six (67%) out of nine primary gastric cancer tissues and nine paired metastatic lymph nodes were positive, respectively and the intensity was stronger in metastatic lymph nodes. The results support the concept that most gastric cancer cells may have an innately moderate level of uPA and uPAR, and that increase of uPAR expression can be considered to be closely associated with cancer invasion and metastasis.

Site-directed mutagenesis and second-site suppressor analysis have proven to be useful approaches to examine the role of charged amino acids in the structure and function of the lactose carrier of Escherichia coli. A lactose carrier mutant Glu-325 --> Ser failed to ferment melibiose and showed white clones on melibiose MacConkey indicator plates. Several red revertants were isolated from these plates. Two of these revertants showed a double mutation, the original mutation (Glu-325 --> Ser) plus His-322 --> Asp. Seven revertants showed a second site mutation His-322 --> Asn. Although the second site revertants failed to accumulate sugars they do show more rapid uptake of melibiose into cells containing alpha-galactosidase than the original mutant Glu-325 --> Ser. The complete loss of transport activity due to the removal of the negative charge at 325 can be partially compensated for by the introduction of a new negative charge at 322. A site-directed double mutant His-322 --> Asn/Glu-325 --> Asn showed a greater rate of lactose uptake (Vmax) than either of the single mutants His-322 --> Asn or Glu 325 --> Asn. It was concluded that there is some type of physiological interaction (possibly a salt bridge) between His-322 and Glu-325.

Background: Scintigraphy with 111In (indium-111)-oxine or 99mTechnetium-hexamethylpropyleneamine oxime (99mTc-HMPAO) labeled leukocytes has been used to differentiate brain abscess from brain tumor. However, there are false positive or false negative results from planar scintigraphic images. So a more specific and sensitive scintigraphic technique needs to be developed., Methods: Planar and single photon emission computed tomography (SPECT) images were obtained and reviewed in 14 patients with intracerebral ring-enhancing lesions on computed tomography (CT) or magnetic resonance imaging (MRI). In all patients, diagnosis was confirmed by histopathologic examination. The effect of steroids or antibiotics on scintigraphic finding was examined., Results: Abscess was confirmed in six patients, tumor in six, tuberculoma and cysticercosis in one each, respectively. In all the patients with abscess, SPECT showed increased focal activity irrespective of steroid or antibiotic therapy. Increased radioactivity, which could not be detected on planar images, could be identified in two patients with abscess. In three of the six patients with tumor, radiolabeled leukocytes did not accumulate in the tumor. In the other three patients with tumor, SPECT showed focally increased activity that was less intense than the activity shown in the patients with abscess., Conclusions: 99mTc-HMPAO labeled leukocyte SPECT is useful for the differential diagnosis of intracerebral ring-enhancing lesions, and the use of steroids or antibiotics does not influence the sensitivity of SPECT. Diagnostic sensitivity of scintigraphy with labeled leukocytes could be improved by SPECT in addition to planar image.

We have discovered that the sulfonation of polystyrene can form a gradient-index medium useful for lightfocusing purposes. We found that the refractive index of sulfonated polystyrene varies with the degree of sulfonation and that the refractive index of the fully sulfonated polystyrene decreased by approximately 0.06 at 0.633 microm from that of pure polystyrene.

Background: The syndrome of cancer cachexia can have a significant impact on response to therapy as well as on survival in cancer patients. Therapies directed at metabolic perturbations in cachectic patients are dependent on nutritional repletion and maintenance of adequate amino acid substrate levels. This study compares the ability of oral feeding, enteral nutrition, and total parenteral nutrition to alter plasma amino acid levels in cancer patients., Methods: Patients with esophageal cancer were stratified by weight loss. Patients with < 20% weight loss were randomized to continue an ad libitum oral diet (group I) or to receive total parenteral nutrition (group II) for 2 weeks; patients with > 20% weight loss were randomized to receive either enteral nutrition (group III) or total parenteral nutrition (group IV) for 2 weeks. Plasma amino acid levels were measured before the study and again after 2 weeks of nutrition support., Results: Before therapy, there was no difference between the groups in total or essential amino acid levels; however, patients in all groups had significantly lower total amino acid levels compared with those of normal controls. After 2 weeks of treatment, patients in group I and III showed no difference in individual, essential, or total amino acid levels. However, patients in groups II and IV showed significant increases in a number of individual amino acids as well as in essential and total amino acid levels after 2 weeks of TPN., Conclusions: Patients with esophageal cancer demonstrated significant alterations in amino acid profiles compared with those of normal controls. Total parenteral nutrition was superior to ad libitum oral feeding and jejunostomy feeding in repleting plasma amino acid levels.

The gene coding for the lactose carrier of Citrobacter freundii was cloned into the plasmid pBR322. The gene was sequenced and the amino acid sequence was found to be 70% identical to the lactose carrier of E. coli. All of the charged residues in the membrane spanning region were conserved. The sugar specificity is somewhat different from that of E. coli. The C. freundii carrier has less activity for lactose and more activity for o-nitrophenyl-galactose (ONPG) than the carrier of E. coli.

Multilocus enzyme electrophoresis was used to assess genetic relationships amongst 175 isolates of anaerobic intestinal spirochaetes, including 72 isolates from individuals living in different parts of the world, 102 from pigs and one from a dog. Amongst porcine isolates belonging to the genus Serpulina, a possible new species was identified. All but one of the isolates from man were clustered with the canine isolate and 59 porcine isolates in a distinct group that we have previously called "Anguillina coli". The human and animal spirochaetes in this group had four-to-six axial flagella and most were recovered from individuals with diarrhoea. They included a strain of the so-called "Serpulina jonesii", that was not a true serpulina. These 71 human isolates were distributed into 44 electrophoretic types and had a mean genetic diversity of 0.32. These were further divided into 26 clonal groups. Three of these clones also contained porcine isolates, one of which was strain P43/6/78, the agent of porcine intestinal spirochaetosis. Four of the clones contained human isolates from different sources. One included isolates from Western Australian Aboriginal children and from Italian adults, and the other three contained isolates from Western Australian Aboriginal children and from homosexual males in Sydney, New South Wales. There were no known connections between these human populations. The other spirochaete of human origin was Brachyspira aalborgi, which was distinct from isolates in the genera Serpulina and "Anguillina". Both B. aalborgi and "A. coli" have been associated with human cases of intestinal spirochaetosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Medulloblastoma, once a tumor with a dismal prognosis, is one of the most common primary brain tumors of childhood. As the methods of treatment have been continuously refined, the outcome has improved remarkably during the last few decades. The outcome of 78 medulloblastoma patients, which were managed from 1972 to 1992 at the Department of Neurosurgery of Seoul National University Hospital, were analyzed to calculate the 3-year and 5-year survival rates (3yS and 5yS). Of those, 52 cases which were treated after July 1982 were studied 1) to calculate the 3yS and 5yS, 2) to figure out the prognostic factors of survival, and 3) to investigate the role of adjuvant chemotherapy ('8-drugs-in-a-day' protocol: CCNU, cisplatin, vincristine, hydroxyurea, procarbazine, cytosine arabinoside, methylprednisolone and cyclophosphamide). The 3yS and 5yS of the 78 patients were 57.4% and 47.3%, respectively. Of the 52 patients treated after July 1982, the 3yS and 5yS were 67.8% and 64.1%, respectively. The latest recurrence was at 56 months after surgery. All the recurrences were within the risk period of Collins' rule. Of the prognostic factors studied by univariate analysis (age, sex, Chang's classification T- and M-stages, extent of surgical removal, and chemotherapy), Chang's classification M-stage and sex were the statistically significant factors (p = 0.028 and 0.024 respectively). On multivariate analysis, only the M-stage was statistically significant (p = 0.004). Adjuvant chemotherapy had different influences in different patient groups. Only in the 'poor risk' group, did adjuvant chemotherapy have a strong tendency to better outcome (p = 0.069). Further data collection and analysis will lead to better treatment modalities and better outcome for this most common primary malignant brain tumor in childhood.

It is believed that there are several charged amino acid residues in membrane-spanning alpha-helices of the lactose carrier of Escherichia coli. Evidence has previously been presented for two different salt bridges in membrane-spanning regions of the lactose carrier. One of these involves an interaction between Asp-237 and Lys-358; another involves interaction between Asp-240 and Lys-319. Additional studies of Lys-319 suggest that it may interact with Glu-269 as well as Asp-240. A cell containing the LacY gene with the mutation Lys-319-->Asn failed to ferment melibiose and after several days melibiose-positive mutants arose on indicator plates. These revertants showed second site mutations which replaced Asp-240 by neutral amino acids (Val or Gly). In addition, a second site mutation showed Glu-269 changed to Asn. Cells containing the mutation Lys-319-->Leu also failed to ferment melibiose and melibiose-positive revertants showed Asp-240-->Ala and Asp-240-->Tyr as well as Tyr-236-->Phe and His-322-->Arg. Second site revertants were also sought from the mutant Glu-269-->Asn which grew poorly on melibiose minimal plates. Melibiose-positive revertants included the double mutant Gln-269/Asn-319. All of the Glu-269-->Asn mutants were extremely defective in transport. It was concluded that Lys-319 interacts with Glu-269 and Asp-240 probably as salt bridges.

The histological change of the biliary mucosa in clonorchiasis is characterized as adenomatous hyperplasia, and cross-sectioned mucosa looks like intestinal mucosa. In addition to the glandular hyperplasia, the metaplasia of mucin secreting cells is also known. The present study investigated the presence of intestinal secretion from the biliary mucosal cells of rabbits and rats with Clonorchis sinensis infection. The rabbit was infected with 300 and the rat was infected with 100 metacercariae of C. sinensis. A part of the animals were followed up after praziquantel treatment. The rabbit livers were prepared for histochemistry to observe any endocrine secretion and the bile duct mucosa of the mice was processed for the activity of brush border membrane (BBM)-bound enzymes of the small intestine. Immunohistochemistry with the polyclonal antibodies and biotin-streptavidin-peroxidase staining kit showed no positive cells for gastrin and secretin, but a few cells were positive for serotonin. The proliferated biliary mucosa of the mice revealed no activity of disaccharidases and aminopeptidase. Only alkaline phosphatase activity was found both in the control and the infected. The hyperplastic biliary mucosal cells showed no gastrointestinal secretory functions. The serotonin secreting cells may be one of the inflammatory cells.

Twenty-nine intestinal spirochetes isolated from Australian aboriginal children and six strains from Italian adults (HRM1, -2, -4, -5, -7, and -14) were genetically examined at 15 enzyme loci by using multilocus enzyme electrophoresis. Results were compared with those previously obtained for 188 porcine intestinal spirochetes. DNA from human strain HRM7 and porcine strain Serpulina hyodysenteriae P18A were also radioactively labeled and hybridized with DNA from 12 other human and porcine intestinal spirochetes. Both the multilocus enzyme electrophoresis and hybridization techniques demonstrated that the human spirochetes were not S. hyodysenteriae. They belonged to another distinct genetic group of spirochetes that included P43/6/78, the bacterium recovered from the first recorded case of porcine intestinal spirochetosis. Bacteria in this distinct group also differed from Serpulina spp. in possessing only four, five, or occasionally six axial filaments, being slightly thinner, and having more pointed ends. These findings add further weight to the possibility that human intestinal spirochetes may act as enteric pathogens.

Although it is energetically extremely unfavorable to have charged amino acid residues of a polypeptide in the hydrophobic environment of the membrane phospholipid bilayer, a few such charged residues are found in membrane-spanning regions of membrane proteins. Ion pairs (salt bridges) would be much more stable in low dielectric media than single ionized residues. This paper provides indirect evidence for a salt bridge between Asp-240 and Lys-319 in the lactose carrier of Escherichia coli. When Asp-240 was changed to alanine by site-directed mutagenesis, there was a loss of the ability to accumulate methyl-beta-D-thiogalactopyranoside (TMG), melibiose, or lactose. Fast-growing revertants were isolated on melibiose minimal agar plates. Two second-site revertants were isolated: Asp-240-->Ala plus Gly-268-->Val and Asp-240-->Ala plus Lys-319-->Gln. These revertants showed extremely poor accumulation of TMG, melibiose, and lactose, but showed significant "downhill" lactose entry into beta-galactosidase-containing cells with sugar concentrations of 2 and 5 mM. It is concluded that there is some important interaction between Asp-240 and Lys-319, possibly a salt bridge.

Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains. The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside. Most of the amino acid substitutions in the regions of the putative loops (between transmembrane alpha helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity. In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found. The substitution of proline for Trp-33 (in the region of the first alpha helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity. Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine. It is concluded that Trp-33 may be involved directly or indirectly in sugar recognition.

Intestinal spirochaetal bacteria were isolated from 59 of 181 (32.6%) faecal samples obtained from Aboriginal children and a few adults living in communities in the Kimberley region in the north of Western Australia. Colonization was more common in young Aborigines between 2 and 18 years of age than it was in adults, or in babies and children less than 2 years of age. Three of 22 Aboriginal children who were sampled on two consecutive years were colonized on both occasions. None of four other children were found to be consistently colonized with the bacteria when sampled on three sequential years, but three were positive on two consecutive visits and the other child was positive on the first and third sampling. Most Aboriginal children had abnormal or watery stools, and both abnormal and watery stool samples were significantly more likely to contain spirochaetes than were normal samples. However, it was not possible to prove that the spirochaetes were the cause of the diarrhoea. In contrast, spirochaetes were only recovered from 8 of 695 (1.2%) faecal samples that were obtained from other mainly non-Aboriginal children and adults in Western Australia or the Northern Territory of Australia, even though most of these individuals were suffering from gastrointestinal disturbances.

Leader peptidase of Escherichia coli spans the plasma membrane twice with its amino terminus on the periplasmic surface of the membrane and its large carboxyl-terminal domain protruding into the periplasm. To monitor the transfer of the amino terminus of leader peptidase to the periplasm, we have constructed a fusion protein between the 18-residue amino-terminal periplasmic domain of Pf3 bacteriophage coat protein and the beginning of leader peptidase. We find that neither the SecA or SecY proteins nor a transmembrane electrochemical potential is required for insertion of the amino terminus, while the transfer of the carboxyl-terminal domain of leader peptidase has these requirements. The first 35 residues of leader peptidase, which include the first hydrophobic domain and the carboxyl-terminal positively charged cluster, are sufficient to insert the amino terminus. When positively charged residues are introduced before the first transmembrane segment, translocation of the amino terminus is abolished. These studies in protein membrane topogenesis, showing that there are different requirements for amino and carboxyl termini insertion, indicate that multiple mechanisms exist even within the same protein.

Leader peptidase cleaves the leader sequence from the amino terminus of newly made membrane and secreted proteins after they have translocated across the membrane. Analysis of a large number of leader sequences has shown that there is a characteristic pattern of small apolar residues at -1 and -3 (with respect to the cleavage site) and a helix-breaking residue adjacent to the central apolar core in the region -4 to -6. The conserved sequence pattern of small amino acids at -1 and -3 around the cleavage site most likely represents the substrate specificity of leader peptidase. We have tested this by generating 60 different mutations in the +1 to -6 domain of the M13 procoat protein. These mutants were analyzed for in vivo and in vitro processing, as well as for protein insertion into the cytoplasmic membrane. We find that in vivo leader peptidase was able to process procoat with an alanine, a serine, a glycine, or a proline residue at -1 and with a serine, a glycine, a threonine, a valine, or a leucine residue at -3. All other alterations at these sites were not processed, in accordance with predictions based on the conserved features of leader peptides. Except for proline and threonine at +1, all other residues at this position were processed by leader peptidase. None of the mutations at -2, -4, or -5 of procoat (apart from proline at -4) completely abolished leader peptidase cleavage in vivo although there were large effects on the kinetics of processing.(ABSTRACT TRUNCATED AT 250 WORDS)

Leader peptidase (Lep) is a central component of the secretory machinery of Escherichia coli, where it serves to remove signal peptides from secretory proteins. It spans the inner membrane twice with a large C-terminal domain protruding into the periplasmic space. To investigate the importance of the different structural domains for the catalytic activity, we have studied the effects of a large panel of Lep mutants on the processing of signal peptides, both in vivo and in vitro. Our data suggest that the first transmembrane and cytoplasmic regions are not directly involved in catalysis, but that the second transmembrane region and the region immediately following it may be in contact with the signal peptide and/or located spatially close to the active site of Lep.

Two Australian isolates of Treponema hyodysenteriae which did not fit within the current serological grouping system for these bacteria were examined by agarose gel double immunodiffusion tests (AGDP). Isolate Vic1 was serologically unique, and we propose that it becomes the type organism for a new sixth serological group of T. hyodysenteriae (Group F). Isolate Q1 was unusual in that lipopolysaccharide (LPS) extracted from it reacted strongly in AGDP with serum raised against the type organism for serogroup D (A1), and also weakly with serum raised against the type organism for serogroup B (WA1). The nature of this cross-reactivity was examined by using cross-absorbed antisera in AGDP, and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The pattern of serological cross-reactivity between Q1, A1 and WA1 was complex and was not fully defined, but the isolate Q1 apparently shared low molecular weight 'serogroup' LPS antigens with A1, and shared higher molecular weight LPS antigens with WA1. On this basis Q1 was designated as belonging to serogroup D, although it was recommended that this be qualified as D (B) to indicate the presence of weak cross-reactivity with serogroup B. Such serological cross-reactivity may have significance in relation to the development of immunity to T.hyodysenteriae. Isolate Q1 may be a potentially useful organism for vaccine development because of its ability to induce a good serological response to LPS of treponemes from both serogroups D and B.

Previous studies have shown that expression of adenovirus type 2 (Ad2) is restricted in epidermal keratinocytes in what appears to be a differentiation specific manner. We have analyzed the relationship between keratinocyte differentiation and Ad2 early and late gene expression. Cultured epidermal keratinocytes infected with Ad2 were fractionated in density gradients of Ficoll 400 to enrich for populations of nondifferentiated cells and differentiated cells. Analysis of these populations revealed that both populations supported early Ad2 gene expression but restricted Ad2 late gene expression. The restriction to late gene expression differed in the two cell populations: Nondifferentiated keratinocytes did not support production of high levels of Ad2 capsid proteins, whereas differentiated keratinocytes supported synthesis of Ad2 capsid proteins but restricted Ad2 expression at a later step that normally leads to production of high titers of progeny virus. The changing restriction to Ad2 expression during keratinocyte differentiation may have resulted from changes in cellular components that play a role in cell differentiation.

We examine the effect of keratinocyte differentiation upon transient expression of a nonepithelial gene following DNA-mediated transfer. Cultures of primary epidermal keratinocytes were transfected with the reporter gene, chloramphenicol acetyltransferase (CAT). The CAT gene was linked at the 5' end to the long terminal repeat (LTR) regulatory sequences from Rous sarcoma virus, and gene transfer was accomplished by the calcium phosphate coprecipitation method. Transfected cells were fractionated on Ficoll 400 density gradients. The major finding of this study was that the larger, more differentiated cells displayed five- to seven-fold higher levels of CAT activity per cell than the smaller, less differentiated cells. The higher levels of CAT activity did not result from greater uptake of DNA because cells of all gradient fractions contained one to two copies of plasmid DNA per cell. Furthermore, the CAT gene linked to the regulatory sequences from another virus, SV40, gave the same result. We conclude that the CAT gene, when controlled by these viral regulatory sequences, is expressed more efficiently in differentiated keratinocytes. These results have important implications for the interpretation of future studies of gene expression in transfected keratinocytes.

DNA sequence analysis of a cloned beta-globin gene from a Chinese patient with beta-thalassemia revealed a single nucleotide substitution (A leads to G) within the ATA box homology and 28 base pairs upstream from the cap site. The patient was homozygous for this particular allele based on restriction mapping at nine different polymorphic sites in the beta-globin gene cluster. Upon transient expression in HeLa cells this gene directed the production of 3-5-fold less beta-globin mRNA than the normal beta-gene. In RNA isolated from the patient's erythroid cells beta-RNA was 10-fold less abundant relative to alpha-RNA than normal, indicating close approximation of the heterologous cell expression results and the in vivo state. These findings support the validity of such transient expression assays for analysis of phenotypes associated with naturally occurring mutant genes and establish the functional significance of nucleotide substitutions at position -28 for human beta-globin gene transcription.

Sixty-five patients with suspected pulmonary embolism were studied prospectively with both Kr-81m and Xe-133 ventilation imaging and Tc-99m MAA perfusion imaging. The krypton images, perfusion scintigrams and chest radiographs were read independently of the xenon images, perfusion scintigrams and chest radiographs by three observers. The studies of 53 patients were interpreted as normal or as indicative of a low or intermediate probability for pulmonary embolism with both gases. One study indicated intermediate probability with Xe-133 due to diffuse, severe xenon retention but low probability with Kr-81m because of close ventilation-perfusion correspondence. The studies of 9 patients indicated a high probability of embolism with both gases, while those of two additional patients (one with emboli at angiography) indicated a high probability only with Kr-81m. While essential agreement between Xe-133 and Kr-81m ventilation imaging was found in most patients, the significant difference in interpretation in 2 of 11 patients with probable pulmonary embolism suggests that a controlled, prospective trial with pulmonary angiography is warranted before Kr-81m is employed for routine clinical use.

We have determined haplotypes for the known restriction site polymorphisms in the beta-globin gene cluster in two families of European ancestry containing individuals who are heterozygous for hemoglobin E. In both families, the beta E mutation is associated with a haplotype not previously found among the haplotypes of beta E chromosomes in Southeast Asia. Moreover, in one family, the mutation is present in a beta-gene framework not found in beta E chromosomes of Southeast Asia. These data provide further evidence of multiple independent origins of the beta E mutation in human populations.

We have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA fragment that maps greater than 40 kilobases (kb) 5' to the epsilon-gene as a probe, reduced amounts of normal fragments were found in the DNA of affected family members. Similar analysis using radiolabeled DNA fragments located 3' to the beta-globin cluster has shown that the deletion extends more than 17 kb 3' to the beta-gene, but terminates before the 3' endpoint of the Ghanian HPFH deletion. Hence, this gamma delta beta-thalassemia deletion eliminates over 105 kb of DNA and is the first report of a deletion of the entire beta-globin gene cluster.

Increasing the pH of local anesthetics with sodium bicarbonate has been reported to hasten their onset of action. The purpose of this study was to compare the onset and duration of epidural analgesia with the use of sodium bicarbonate and tromethamine to increase the pH of 2% chloroprocaine (2CP). Five groups of patients were studied: Group I received 2CP; Group II received 2CP buffered to a pH of 7.1 with tromethamine; Group III received 2CP buffered to a pH of 7.1 with sodium bicarbonate; Group IV received 2CP buffered to a pH of 7.7 with tromethamine; and Group V received 2CP buffered to a pH of 7.7 with sodium bicarbonate. The final pH and PCO2 of each solution were measured. Time to onset of analgesia was significantly delayed with either of the tromethamine buffered groups (II [5.6 +/- 1.0 minutes] and IV [5.4 +/- 0.4 minutes]) when compared with data from the unbuffered control (I [4.4 +/- 0.1 minutes]) and the sodium bicarbonate buffered (III [4.5 +/- 0.8 minutes] groups and Group V [2.7 +/- 0.9 minutes]). Only when sodium bicarbonate buffer adjusted to pH 7.7 (Group IV) was onset significantly more rapid than the unbuffered 2CP (I) and tromethamine buffered 2CP (II and IV). Multiple regression analysis revealed that onset times were significantly related to both pH and PCO2. The coefficient of determination for this model was 0.5156.(ABSTRACT TRUNCATED AT 250 WORDS)