Trimeresurus flavoviridis (Habu snake) venom aspartate-49-phospholipase A2 (Asp-49-PLA2) was reacted at pH 9.0 with a 2-fold molar excess of 2,4,6-trinitrobenzenesulfonate in the absence of Ca2+ and two trinitrophenylated derivatives were isolated by HPLC. One was a derivative modified at Lys-11 and its activity was mostly retained. The other was a derivative modified at both Lys-11 and Lys-72 and its activity was 40% that of unmodified enzyme. Trinitrophenylation of Lys-72 appeared to bring about a conformational disorder at the lipid-water interface recognition site and thus a reduction of activity. When the enzyme was modified in the presence of Ca2+, activity decreased at a rate much faster than that in the absence of Ca2+ and Lys-69 came to be modified. These results suggested that conformational displacement of Asp-49-PLA2 of a local to global type occurs upon the binding of Ca2+. The derivative modified at Lys-69 had 28% activity and existed as a monomer. This supports a previous assumption that Lys-69 participates in dimerization of group II Asp-49-PLA2s [Brunie et al. (1985) J. Biol. Chem. 260, 9742-9749] and shows that dimerization is not necessarily essential for activity manifestation.

Amino Acid Sequence↗

PubMedDate not supplied

Balloon dilatation for anastomotic stricture after upper gastro-intestinal surgery.

We report our study on the correlation between the types of anastomosis and the incidence of anastomotic stricture formation in the upper gastro-intestinal tract. Our experience with balloon dilatation is also reported. We examined the incidence of stricture formation among patients who had an anastomosis between the esophagus and stomach following subtotal esophagectomy for esophageal cancer, and esophagojejunostomy following proximal or total gastrectomy for gastric cancer in the past 17 years. Among 283 patients undergoing esophagojejunostomy, 7 cases of stricture (excluding 3 cases of cancer recurrence) were observed (conventional anastomosis 1.8%; stapling anastomosis 4.6%). There were 17 cases of stricture among 56 patients who had anastomosis between the esophagus and stomach following subtotal esophagectomy (conventional anastomosis 28.6%; stapling anastomosis 50.0%). One month or more after the operation, the diameter of the esophagojejunostomy was estimated using a barium study. The mean diameter of the anastomosis using the stapling method was 11.9 +/- 2.9 mm, whereas the mean diameter of serosubmucosal single layer hand-sewn anastomosis (Jourdan's) was 19.8 +/- 2.2 mm, and that of vertical mattress hand-sewn anastomosis was 19.0 +/- 2.0 mm. Balloon dilatation was used in 29 patients with anastomotic stricture of the upper gastro-intestinal tract (esophageal cancer, 19 patients, gastric cancer, 10 patients). With repeated dilatation, we were able to obtain satisfactory efficacy for benign strictures and there were no severe complications. We believe that balloon dilatation is an easy, safe and effective therapy for anastomotic stricture of the upper gastro-intestinal tract.

Anastomosis, Surgical↗

PubMedDate not supplied

Epidermoid cyst of an accessory spleen at the pancreatic tail: diagnostic value of MRI.

We report a rare case of an epidermoid cyst in an accessory spleen at the pancreatic tail. Only 12 cases have been reported. Among the different diagnostic modalities, magnetic resonance imaging was most useful for the differential diagnosis. Precise imaging of the cyst wall and its comparison with the surrounding organs are essential.

Adult↗

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A trimmed viral cap-independent translation enhancing sequence for rapid in vitro gene expression.

We prepared a short (29 nucleotides) 5' UTR that enhanced cap-independent translation in a wheat germ translation system by trimming the tobacco etch virus 5' UTR. The trimmed sequence, designated as TE(37-65), was obtained from a conserved region among several potyviruses. The productivities of uncapped reporter mRNAs carrying the TE(37-65) sequence were comparable to those of capped counterparts, in that 5-20 microg of proteins were synthesized per 1 mL of translation reaction mixture. The ribosome that entered onto the TE(37-65) sequence precisely initiated polypeptide synthesis at the defined initiation codon, which ensures rapid and efficient protein truncation analyses. Moreover, the TE(37-65) sequence is short enough to be involved in a PCR primer, which allows a simple method for rapid gene expression, i.e., PCR amplification of a target gene and succeeding in vitro transcription and translation. As a demonstration, the rapid in vitro expression of rice cDNAs using the TE(37-65) sequence was also performed.

5' Untranslated Regions↗

PubMedDate not supplied

Application of an on-line turbidimeter for the automation of fed-batch cultures.

Recent advances in the technology of continuous in situ turbidity measurements in microbial cultivations are reviewed. Some characteristics of commercially available on-line laser turbidimeters are estimated in relation to requirements of sensors for microbial cultures. A system for the automation of fed-batch cultures using an on-line turbidimeter is proposed. Experimental data are presented concerning fed-batch cultures grown on synthetic and natural complex media. In situ turbidity data were found to be particularly useful for the automation of the feeding of natural complex nutrient solutions by converting the data into dry cell mass concentration and at the same time utilizing the instantaneous data on the culture broth volume through a computer.

Autoanalysis↗

PubMedDate not supplied

Insertion of stabilizing loci in vectors of T7 RNA polymerase-mediated Escherichia coli expression systems: a case study on the plasmids involving foreign phospholipase D gene.

Plasmids carrying stabilizing loci were used in the expression of phospholipase D (PLD) gene fused with pelB signal sequence by a recombinant strain of Escherichia coli BL21 (DE3) using T7 RNA polymerase mediated expression system. By checking the living cell number and the percentage of the plasmid-bearing cells, it was found that the plasmids involving PLD gene were not stable under noninduced conditions and that, after the induction, the number of plasmid-bearing cells were rapidly decreased to almost zero. Then, a biologically stabilizing locus such as par B, ccd, or par was inserted into the plasmids. The newly constructed plasmids were maintained very stably in the recombinant cells until the cells were induced. However, after the induction, almost all the recombinant cells were rapidly killed due to highly toxic PLD. Using the best one of the stabilized PLD-expressing plasmids, PLD production was improved 2-fold.

Amino Acid Sequence↗

PubMedDate not supplied

Characterization of proteins by nuclear magnetic resonance.

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Albumins↗

PubMedDate not supplied

Optical trapping and manipulation of single cells using infrared laser beams.

Use of optical traps for the manipulation of biological particles was recently proposed, and initial observations of laser trapping of bacteria and viruses with visible argon-laser light were reported. We report here the use of infrared (IR) light to make much improved laser traps with significantly less optical damage to a variety of living cells. Using IR light we have observed the reproduction of Escherichia coli within optical traps at power levels sufficient to give manipulation at velocities up to approximately 500 micron s-1. Reproduction of yeast cells by budding was also achieved in IR traps capable of manipulating individual cells and clumps of cells at velocities of approximately micron s-1. Damage-free trapping and manipulation of suspensions of red blood cells of humans and of organelles located within individual living cells of spirogyra was also achieved, largely as a result of the reduced absorption of haemoglobin and chlorophyll in the IR. Trapping of many types of small protozoa and manipulation of organelles within protozoa is also possible. The manipulative capabilities of optical techniques were exploited in experiments showing separation of individual bacteria from one sample and their introduction into another sample. Optical orientation of individual bacterial cells in space was also achieved using a pair of laser-beam traps. These new manipulative techniques using IR light are capable of producing large forces under damage-free conditions and improve the prospects for wider use of optical manipulation techniques in microbiology.

Animals↗

PubMedDate not supplied

Expression of cyclin kinase inhibitor p27(Kip1) in skin tumours of dogs.

Skin tumours (n=148) of epidermal or hair follicle origin were examined immunohistochemically to determine the expression of p27(Kip1)(p27), a cyclin-dependent kinase inhibitor (CDKI), and of Ki-67. In normal skin, a large number of basal cells of the epidermis and hair follicles were positive for Ki-67 and many suprabasal epithelial cells were positive for p27. Most of the hair matrix cells were positive for Ki-67 but negative for p27. Hair papillae were strongly positive for p27. Squamous cell carcinomas had a p27 positive index (PI) significantly lower than that of trichoepitheliomas (P<0.005), basal cell tumours (P<0.05) and intracutaneous cornifying epitheliomas (P<0.001). In contrast, Ki-67 PIs of squamous cell carcinomas and pilomatrixomas were significantly higher than those of trichoepitheliomas, basal cell tumours and intracutaneous cornifying epitheliomas (P<0.01 to P<0.001). No significant difference was observed between the Ki-67 PI values of squamous cell carcinomas and pilomatrixomas. The results suggested that p27 is capable of suppressing cell proliferation in the differentiation of normal canine skin. In spite of being a benign neoplasm, pilomatrixomas had a low p27 expression; this may be a reflection of the proliferative potential of the hair matrix. The expression of p27 may be a useful marker for the analysis of cell kinetics.

Animals↗

PubMedDate not supplied

Heredity versus abnormalities in pregnancy and delivery as risk factors for different types of comitant strabismus.

PURPOSE: To elucidate risk factors for different types of comitant strabismus, the incidence of heredity and abnormalities in pregnancy and delivery was compared among different types of strabismus. METHODS: Between May 1998 and January 1999, a prospective clinical study of 500 consecutive patients with comitant strabismus at a referral-based university hospital was performed using data collected from questionnaires and interviews. Inclusion criteria were infantile esotropia (168 patients), accommodative and partially accommodative esotropia (97 patients), microesotropia (15 patients), acquired esotropia (12 patients), intermittent or constant exotropia (205 patients), and congenital exotropia (3 patients). Exclusion criteria were strabismus associated with systemic and central nervous system abnormalities and organic eye diseases. RESULTS: Family history was significantly more prevalent in intermittent or constant exotropia and accommodative or partially accommodative esotropia than in infantile esotropia (P<.0001 and P=.0267, respectively, Fisher's exact test). In contrast, abnormalities in pregnancy and delivery were noted at a significantly higher rate in infantile esotropia than in accommodative or partially accommodative and in intermittent or constant exotropia (P=.003 and P=.0215, respectively). Patients with infantile esotropia were significantly younger at the survey than those with the other two types of strabismus (P=.0004 and P<.0001). No significant difference was found in the number of siblings, birthweight, maternal age at delivery, or maternal cigarette smoking or alcohol drinking among these three types of strabismus. CONCLUSION: Heredity and abnormalities in pregnancy and delivery are two major risk factors for comitant strabismus and contribute at different levels to the development of infantile esotropia, accommodative or partially accommodative esotropia, and intermittent or constant exotropia.

Child↗

PubMedDate not supplied

Fluid dynamic characteristics of monopivot magnetic suspension blood pumps.

A monopivot magnetic suspension blood pump is a centrifugal pump under development with a magnetic suspension and a ceramic pivot to support the impeller with minimum contact. The pump size has been reduced by implementing a direct impeller drive mechanism in place of a magnetic coupling and motor. Flow visualization studies revealed that high shear, which seems to be closely related to hemolysis, concentrates in boundary layers near the walls. This implies that fluid dynamic shear can be reduced not by widening the gap, but by reducing the impeller velocity. Therefore, compared with the results of the previous semi-open curved vane impeller model, impeller velocity was reduced by 30% with a closed impeller having radial straight vanes, and smaller impeller/housing gaps. The volute shape around the impeller tip was also changed such that the outflow from the impeller enters along the center plane of the volute. To examine the effect of the improvements, hemolysis testing was conducted and found that the newly developed closed impeller model generated a lower level of hemolysis than the previous semi-open impeller model.

Animals↗