'98 Escherichia coli SWISS-2DPAGE database update

The combination of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d-top.html . Here we report recent progress in the development of the E. coli SWISS-2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman "sequence tag" approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.     

Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells

There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.     

Analysis of patients referred to the gastroenterology service of a VA medical center

We recorded clinical information over a 12-month period on consecutive consultations to the gastroenterology service of the Durham VA Medical Center. Of 902 consultations, 789 were prospectively collected. Eighty-five percent of the patients were between 40 and 70 years old. Seventy-five percent of the referrals were from the internal medicine service. The most frequent reasons for consultation were abdominal pain (19%), GI bleeding (active, 16%; occult, 9%), abnormal results of liver tests (18%), and request for a procedure (11%). Diseases of the liver (32%) and "peptic diseases" (30%) were the most common diagnoses. One or more procedures were done in 71% of consultations. When these data are compared with those of a practicing gastroenterologist, using an identical instrument, it is apparent that trainees' experience with structurally identifiable gastroenterologic disease and with a variety of procedures was similar in scope. There were, however, differences in that the physicians at the VA saw substantially fewer patients with so-called "functional" illness. If these data are applicable to other VA Medical Centers, then the training of physicians in gastroenterology at a VA Medical Center should probably be broadened.     

Diagnosis and epidural hematoma by brain scan and perfusion study: case report

By using the arterial and venous phases of an anterior cerebral perfusion study, which showed downward displacement of the sagittal sinus, and the finding of a "rim" on the delayed scans, the specific diagnosis of epidural hematoma was established.     

An educational strategy for occupational therapy community service

In response to the increasing trend toward community health care, a model of training that prepares students for community practice was incorporated into the occupational therapy curriculum at the University of Southern California. During academic training students are placed in a part-time community assignment where no occupational therapy services are offered. Training students for the role of community health specialist produces a dilemma for curriculum design. A balance must be achieved between providing traditional clinical content and providing the knowledge and expertise necessary for community practice. However, this training is considered necessary for maintaining the viability of the profession in a changing health system. Significantly, during the four-year period using this model, many graduates have sought employment in "nontraditional" community roles.     

A Stochastic and Dynamic Vehicle Routing Problem with Time Windows and Customer Impatience

In this paper, we study the problem of designing motion strategies for a team of mobile agents, required to fulfill request for on-site service in a given planar region. In our model, each service request is generated by a spatio-temporal stochastic process; once a service request has been generated, it remains active for a certain deterministic amount of time, and then expires. An active service request is fulfilled when one of the mobile agents visits the location of the request. Specific problems we investigate are the following: what is the minimum number of mobile agents needed to ensure that a certain fraction of service requests is fulfilled before expiration? What strategy should they use to ensure that this objective is attained? This problem can be viewed as the stochastic and dynamic version of the well-known vehicle routing problem with time windows. We also extend our analysis to the case in which the time service requests remain active is itself a random variable, describing customer impatience. The customers’ impatience is only known to the mobile agents via prior statistics. In this case, it is desired to minimize the fraction of service requests missed because of impatience. Finally, we show how the routing strategies presented in the paper can be executed in a distributed fashion.     

Effects of fibroblast growth factor-2 on longitudinal bone growth

In vivo, fibroblast growth factor-2 (FGF-2) inhibits longitudinal bone growth. Similarly, activating FGF receptor 3 mutations impair growth in achondroplasia and thanatophoric dysplasia. To investigate the underlying mechanisms, we chose a fetal rat metatarsal organ culture system that would maintain growth plate histological architecture. Addition of FGF-2 to the serum-free medium inhibited longitudinal growth. We next assessed each major component of longitudinal growth: proliferation, cellular hypertrophy, and cartilage matrix synthesis. Surprisingly, FGF-2 stimulated proliferation, as assessed by [3H]thymidine incorporation. However, autoradiographic studies demonstrated that this increased proliferation occurred only in the perichondrium, whereas decreased labeling was seen in the proliferative and epiphyseal chondrocytes. FGF-2 also caused a marked decrease in the number of hypertrophic chondrocytes. To assess cartilage matrix synthesis, we measured 35SO4 incorporation into newly synthesized glycosaminoglycans. Low concentrations (10 ng/ml) of FGF-2 stimulated cartilage matrix production, but high concentrations (1000 ng/ml) inhibited matrix production. We conclude that FGF-2 inhibits longitudinal bone growth by three mechanisms: decreased growth plate chondrocyte proliferation, decreased cellular hypertrophy, and, at high concentrations, decreased cartilage matrix production. These effects may explain the impaired growth seen in patients with achondroplasia and related skeletal dysplasias.     

A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production, and behavior

Two chemicals previously shown to have estrogenic activity, bisphenol A and octylphenol, were examined for their effects on accessory reproductive organs and daily sperm production in male offspring of mice fed these chemicals during pregnancy. These chemicals are used in the manufacture of plastics and other products, and have been detected in food and water consumed by animals and people. From gestation day 11-17 female mice were fed an average concentration (dissolved in oil) of bisphenol A or octylphenol of 2 ng/g body weight (2 ppb) and 20 ng/g (20 ppb). The 2 ppb dose of bisphenol A is lower than the amount reported to be swallowed during the first hour after application of a plastic dental sealant (up to 931 micrograms; 13.3 ppb in a 70 kg adult). We found that the 2 ng/g dose of bisphenol A permanently increased the size of the preputial glands, but reduced the size of the epididymides; these organs develop from different embryonic tissues. At 20 ng/g, bisphenol A significantly decreased efficiency of sperm production (daily sperm production per g testis) by 20% relative to control males. The only significant effect of octylphenol was a reduction in daily sperm production and efficiency of sperm production at the 2 ng/g dose. A new approach to studying physiologically relevant doses of environmental endocrine disruptors is discussed, particularly with regard to the development of the reproductive organs, the brain, and behavior.