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Biomedical subjects

C W Jones

Publications and source records attributed to C W Jones.

At least 37 records · Page 2Linked to original sources

Microperfusion studies on the permeability of retinal vessels. A new model demonstrating organic anion transport and a reabsorptive fluid flux.

We developed an experimental model to study the permeability of individual retinal vessels in vitro using microperfusion techniques adapted from kidney tubule studies. The retinal vessels were isolated by freehand dissection and mounted on a microperfusion apparatus. When inulin was perfused luminally, it was diluted to 80.2 +/- 2.3% of its initial concentration. However, no radioactive leak into the bath side was observed, suggesting that the dilution was due to fluid flux from bath to lumen. The dilution of fluorescein (81.9 +/- 3.8%) was in the same range as that of inulin, the reference marker. The extremely low lumen-to-bath fluorescein flux, 0.5 +/- 0.9 X 10(-12) mol/min/mm, increased by 68% when probenecid was added to the perfusate and by 210% when probenecid was placed in the bath. The effect was concentration-dependent. When placed in the bath, fluorescein moved rapidly across the retinal vessel walls, accumulating in the lumen to concentrations 40 times higher than in the bath. This movement from bath to lumen, which was much higher (13.6 +/- 0.3 X 10(-12) mol/min/mm) than the lumen-to-bath fluorescein flux for the same fluorescein concentration, decreased by adding probenecid to the bath. The kinetics of this unidirectional movement of fluorescein were consistent with a saturable active transport process. The fluid flux from bath to lumen across the retinal vessels, which was 6.3 +/- 1.0 nl/min/mm for perfusion rates of 6.6 +/- 0.2 nl/min, was temperature-dependent and was coupled to the fluorescein transport. Fluorescein stimulated the fluid flux by 17% when added to the perfusate and by 60% when added to the bath, and this effect could be reversed by probenecid. Our results showed an active transport of fluorescein in the rabbit retinal vessels coupled with net fluid flux from outside the vessels into the lumen.

Animals↗

Binding-protein-dependent lactose transport in Agrobacterium radiobacter.

Agrobacterium radiobacter NCIB 11883 was grown in lactose-limited continuous culture at a dilution rate of 0.045/h. Washed cells transported [14C]lactose and [methyl-14C]beta-D-thiogalactoside, a nonmetabolisable analog of lactose, at similar rates and with similar affinities (Km for transport, less than 1 microM). Transport was inhibited to various extents by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, by unlabeled beta-galactosides and D-galactose, and by osmotic shock. The accumulation ratio for methyl-beta-D-thiogalactoside was greater than or equal to 4,100. An abundant protein (molecular weight, 41,000) was purified from osmotic-shock fluid and shown by equilibrium dialysis to bind lactose and methyl-beta-D-thiogalactoside, the former with very high affinity (binding constant, 0.14 microM). The N-terminal amino acid sequence of this lactose-binding protein exhibited some homology with several other sugar-binding proteins from bacteria. Antiserum raised against the lactose-binding protein did not cross-react with two glucose-binding proteins from A. radiobacter or with extracts of other bacteria grown under lactose limitation. Lactose transport and beta-galactosidase were induced in batch cultures by lactose, melibiose [O-alpha-D-galactoside-(1----6)alpha-D-glucose], and isopropyl-beta-D-thiogalactoside and were subject to catabolite repression by glucose, galactose, and succinate which was not alleviated by cyclic AMP. We conclude that lactose is transported into A. radiobacter via a binding protein-dependent active transport system (in contrast to the H+ symport and phosphotransferase systems found in other bacteria) and that the expression of this transport system is closely linked to that of beta-galactosidase.

Amino Acid Sequence↗

Binding-protein-dependent sugar transport by Agrobacterium radiobacter and A. tumefaciens grown in continuous culture.

Binding-protein-dependent sugar transport has been investigated in Agrobacterium radiobacter and A. tumefaciens. A. radiobacter contained two high-affinity glucose-binding proteins (GBP1 and GBP2) that additionally bound D-galactose (KD 0.26 microM) and D-xylose (KD 0.04 microM) respectively and were involved in the transport of these sugars. Partial sequencing of GBP1 and GBP2 showed that GBP2 exhibited significant homology with both the arabinose-binding protein (ABP) and the galactose-binding protein (GalBP) from Escherichia coli, whereas GBP1 exhibited significant homology only with ABP. Antiserum raised against GBP1 cross-reacted with GBP1 but not with GBP2, and vice versa. Anti-GBP1 and anti-GBP2 also cross-reacted with proteins corresponding to GBP1 and GBP2 respectively in A. tumefaciens, but little or no cross-reaction was observed with selected members of the Enterobacteriaceae, Rhizobiaceae and Pseudomonadaceae families grown under glucose limitation. GBP1 was less strongly repressed than GBP2 following batch growth of A. radiobacter on various carbon sources. The growth of A. radiobacter for more than approximately 10 generations in continuous culture under galactose or xylose limitation (D 0.045 h-1) led to the emergence of new strains which exhibited increased rates of glucose/galactose or glucose/xylose uptake, and which respectively hyperproduced GBP1 (strain AR18a) or GBP2 (strain AR9a). Similarly, growth of A. tumefaciens for more than approximately 15 generations under glucose or galactose limitation produced new strains which exhibited increased rates of glucose/xylose or glucose/galactose uptake and which respectively hyperproduced proteins analogous to GBP2 (strain AT9) or GBP1 (strain AT18a). It is concluded that growth of Agrobacterium species under carbon-limited conditions leads to the predictable emergence of new strains which specifically hyperproduce the transport system for the limiting nutrient. The GBP1-dependent system of A. radiobacter is unique amongst these transport systems in that the mutations that lead to its hyperproduction under carbon limitation render it least susceptible to repression by excess glucose during ammonia limitation, with the result that succinoglucan exopolysaccharide is produced from glucose at an enhanced rate.

Bacterial Proteins↗

Binding-protein-dependent glucose transport by Agrobacterium radiobacter grown in glucose-limited continuous culture.

Agrobacterium radiobacter NCIB 11883 was grown in glucose-limited continuous culture at low dilution rate. Whole cells transported glucose using an energy-dependent mechanism which exhibited an accumulation ratio greater than 2000. Three major periplasmic proteins were purified and their potential role as glucose-binding proteins (GBP) were investigated using equilibrium dialysis. Two of these, GBP1 (Mr 36,500) and GBP2 (Mr 33,500), bound D-glucose with high affinity (KD 0.23 and 0.07 microM respectively), whereas the third protein (Mr 30,500) showed no binding ability. Competition experiments using various analogues showed that those which differed from glucose at C-6 (e.g. 6-chloro-6-deoxy-D-glucose and 6-deoxy-D-glucose) variably decreased the binding of glucose to both GBP1 and GBP2, whereas those which differed at C-4 (e.g. D-galactose) were only effective with GBP1. The rate of glucose uptake and the concentration of the glucose-binding proteins increased in parallel during prolonged growth under glucose-limitation due to the emergence of new strains in which GBP1 (e.g. strain AR18) or GBP2 (e.g. strain AR9), but not both, was hyperproduced and accounted for at least 27% of the total cell protein. It is concluded that A. radiobacter synthesizes two distinct periplasmic binding proteins which are involved in glucose transport, and that these proteins are maximally derepressed during growth under glucose limitation.

Biological Transport↗

The relationship between glucose transport and the production of succinoglucan exopolysaccharide by Agrobacterium radiobacter.

Agrobacterium radiobacter NCIB 11883 was grown in ammonia-limited continuous culture at low dilution rate with glucose as the carbon source. Under these conditions the organism produced an extracellular succinoglucan polysaccharide and transported glucose using the same periplasmic glucose-binding proteins (GBP1 and GBP2) as during glucose-limited growth. Transition from glucose- to ammonia-limited growth was accompanied by a very rapid decrease in glucose uptake capacity, whereas the glucose-binding proteins were diluted out much more slowly (t1/2 approximately 1 h and 14 h respectively). Although the rate of glucose uptake and the concentrations of GBP1 and GBP2 were much lower during ammonia limitation, the activities of enzymes involved in the early stages of glucose metabolism and in the production of succinoglucan precursors were essentially unchanged. Glucose transport was also investigated in two new strains of A. radiobacter which had been isolated following prolonged growth under glucose limitation. Glucose uptake by strain AR18 was significantly less repressed during ammonia limitation compared with either the original parent strain or strain AR9, and this was reflected both in its relatively high concentration of GBP1 and in its significantly higher rate of succinoglucan synthesis. Flux control analysis using 6-chloro-6-deoxy-D-glucose as an inhibitor of glucose transport showed that the latter was a major kinetic control point for succinoglucan production. It is concluded that glucose uptake by A. radiobacter, particularly via the GBP1-dependent system, is only moderately repressed during ammonia-limited growth and that the organism avoids the potentially deleterious effects of accumulating excess glucose by converting the surplus into succinoglucan.

Ammonia↗

The apparent oxidation of NADH by whole cells of the methylotrophic bacterium Methylophilus methylotrophus. A cautionary tale.

Previous reports that whole cells of Methylophilus methylotrophus oxidase exogenous NADH have been investigated. Essentially identical rates of oxygen consumption were observed following the addition of methanol or NADH to whole cells. Both activities were inhibited by EDTA and hydroxylamine, but not by HQNO, and exhibited similar pH optima. Analyses of the reaction stoichiometry with NADH as substrate showed that the expected amount of oxygen was consumed, but also revealed acidification (instead of alkalinisation) and no oxidation of NADH. Further studies showed that commercial NADH is contaminated with ethanol which is oxidised to acetic acid by the low specificity methanol oxidase system present in this organism. The oxidation of exogenous NADH by whole cells of M. methylotrophus reported previously is therefore spurious.

Bacteria↗

Duration of antibiotic prophylaxis. An experimental study.

An animal wound model was used to evaluate single dose cefazolin, multiple dose cefazolin, and single dose cefonicid in the prevention of wound infection. Incisions made in Swiss-Webster mice were contaminated with either Staph. aureus (1.94 X 10(8) colony forming units) or E. coli (4.39 X 10(8) colony forming units). Five experimental groups were studied. Group I encompassed control animals given saline solution, Group II animals given 10 mg cefazolin preoperatively, Group III animals given 10 mg of cefazolin preoperatively and postoperatively, Group IV animals given 10 mg of cefonicid preoperatively, and Group V animals given 20 mg of cefonicid preoperatively. All medications were given by intraperitoneal injection. Antibiotics were given 1 hour before operation. Postoperative doses were given 4 hours after operation. Incisions were opened 48 hours after surgery and wound bacterial concentrations were determined. After both Staph. aureus and E. coli contamination, each of the four cephalosporin regimens significantly reduced the mean wound bacterial concentrations compared with that of the control animals (p less than 0.001). Each of the four cephalosporin regimens also significantly reduced the number of infected wounds compared with that of the control subjects (p less than 0.001). No significant differences were noted among the four antibiotic regimens with respect to mean wound bacterial concentration or infection rate. In the context of this model, a single dose of cefazolin seems to be equally effective as multiple doses of the drug for surgical prophylaxis. Extended half-life cephalosporins, like cefonicid, do not appear to be more effective than a single dose of cefazolin, which is a much less expensive antibiotic.

Animals↗

Anaerobic coverage for wound prophylaxis. Comparison of cefazolin and cefoxitin.

An experimental wound model has been used to evaluate the effectiveness of cefazolin and cefoxitin in the prevention of wound infection. Incisions were contaminated with Staph. aureus, E. coli, or a standardized fecal suspension. Regardless of the contaminant employed, the prophylactic use of either cefazolin or cefoxitin yielded lower wound bacterial concentrations and fewer infections compared with treatment with placebo. Cefazolin proved just as effective as cefoxitin in preventing infection when wounds were contaminated with Staph. aureus or E. coli. Although cefoxitin is the only cephalosporin that offers anaerobic coverage, its prophylactic administration when wounds were contaminated with a standardized fecal suspension did not significantly alter wound bacterial concentrations or infection rates compared with cefazolin. The data from our animal wound model suggest that prophylactic anaerobic coverage is not necessary.

Animals↗

Ocular fluorophotometry in the normal- and diabetic monkey.

Normal- and diabetic rhesus monkeys without retinopathy demonstrable by ophthalmoscopy or fluorescein angiography were examined with ocular fluorophotometry to detect alterations in their blood-ocular barriers. All vitreous fluorophotometry values were corrected for fluorescence attributable to background levels and then normalized to a blood fluorescein level of 10 micrograms ml-1. Reproducibility studies demonstrated an average coefficient of variation of 0.17 for all animals combined. Insulin-dependent monkeys, both pancreatectomized and streptozotocin-treated, demonstrated significantly higher posterior vitreous fluorescence levels than either control animals or monkeys treated with streptozotocin that were not insulin-dependent. These results cannot be attributed to differences in fluorescein binding or to vitreous abnormalities. However, 14 out of 24 (58%) of the insulin-dependent animals exhibited posterior vitreous fluorescence values within two standard deviations of the control mean. No correlation was apparent between the vitreous values and age or duration of treatment. No difference in anterior chamber concentrations was found between groups after correction. Our results indicate that alterations in blood-retinal barrier can occur in insulin-dependent diabetic monkeys before development of retinopathy.

Aging↗

Mass-spectrometric assay of tocainide in serum.

This mass-spectrometric method for assaying tocainide in serum is specific, reproducible, and sensitive, and sample preparation is rapid. The drug is isolated from serum by liquid-solid extraction on a Baker C18 disposable column. Underivatized drug is separated by elution on a 0.20 mm X 25 m fused-silica capillary column coated with 5% phenylmethylsilicone, then quantified by mass-selective detection (selected ion monitoring). Sample size is 1 mL of serum, but smaller volumes may be used. Mean analytical recovery of the drug from the disposable columns is 75%. Commonly used antiarrhythmics, sedatives, or hypnotics do not interfere. The run-to-run CV at 3.7 mg/L is 4.0%, 3.0% at 10.5 mg/L.

Gas Chromatography-Mass Spectrometry↗

Reducing the cancer risk of 239Pu by chelation therapy.

Groups of adult female C57BL/Do mice were injected intraperitoneally with graded activities of monomeric 239Pu(IV) citrate at 10 weeks of age. Beginning 3 days after plutonium injection, some mice received repeated subcutaneous injections of Zn Na3 diethylenetriaminepentaacetate (Zn-DTPA). Each injection was 37 mumol Zn-DTPA/kg body weight. To evaluate protection from bone sarcoma, brief, intermediate, or protracted chelation therapies were administered to groups of mice. The brief chelation therapy covered a 2-week period, the intermediate therapy 2 months, and the protracted therapy 1 year. The mice were followed throughout life and examined for bone sarcoma. Both skeletal dose and bone sarcoma risk were reduced by chelation. The bone sarcoma incidences in the mice given chelation treatments generally fell below the dose-response curve for the mice not given DTPA, indicating that the cancer risk was reduced more than that corresponding to the decreased skeletal dose. This results suggests that Zn-DTPA preferentially removed Pu from the most carcinogenic locations in the skeleton, such as on bone surfaces near living cells.

Animals↗

Methods of splenic preservation and their effect on clearance of pneumococcal bacteremia.

The intravascular clearance of type 3 Streptococcus pneumoniae was studied in Sprague-Dawley rats. Sham celiotomy was performed on 20 animals while another 20 rats underwent splenectomy. Four weeks later, bacteremia was induced by intraperitoneal (IP) injection of S. pneumoniae. Serial cultures of peripheral blood were obtained. Splenectomy produced significant impairment of intravascular clearance of bacteria compared to that noted among control animals (p less than 0.01). Eighty animals were divided into four equal groups: I--splenectomy, II--50% splenectomy with the upper half left in situ connected to the short gastric vessels, III--50% splenectomy with the lower half left in situ connected to the hilar vessels, and IV--splenectomy with implantation of splenic fragments. Pneumococcus was administered IP 16 weeks later. Rats were killed 6 hours after bacterial challenge. Residual splenic tissue was weighed. There was significantly less splenic tissue in Groups II-IV than noted in sham animals after 16 weeks (p less than 0.01). The type of partial splenectomy did not significantly affect the weight of residual splenic tissue 16 weeks later. Implantation did yield viable splenic tissue, though the amount proved significantly less than that resulting from either type of partial splenectomy (p less than 0.01). Mean bacterial counts with time for short gastric (Group II) and hilar (Group III) remnant animals were significantly different from those for the asplenic (Group I) rats (p less than 0.02 and p less than 0.001, respectively). Animals with splenic implants (Group IV) were not significantly different from asplenic rats (Group I). Animals with hilar splenic remnants proved significantly different from those with short gastric splenic remnants (p less than 0.01). Partial splenectomy offers protection against pneumococcal bacteremia, though preservation of the hilar blood supply affords the most benefit. The utility of splenic implantation remains unproven.

Animals↗

Effect of Zn-DTPA therapy on the maternal transfer of 241Am or 237Pu to young mice.

Newborn mice exposed to 241Am or 237Pu during gestation and whose mothers were given Zn-DTPA at various times before parturition contained less radioactivity than comparable newborn mice given identical 241Am or 237Pu exposure without Zn-DTPA treatment of the mothers. There was no net increase in radionuclide transfer from mothers to unborn young as a result of maternal administration of Zn-DTPA, regardless of the pregnancy stage during which the radioactivity and decorporation treatments were given. Administration of 241Am followed after three days by extended Zn-DTPA therapy resulted in lower fetal content of radioactivity in litters conceived long after or soon after maternal exposure to 241Am. These data indicate that the risk from Am and Pu to unborn young can be reduced by Zn-DTPA treatment of the pregnant mother and of the female who becomes pregnant subsequently. This information may prove to be of significance with the increasing probability of Am or Pu exposure to women in the nuclear industry who could be pregnant at the time or who may conceive a child in the future.

Americium↗

Inadequate tetanus protection among the rural elderly.

Three cases of tetanus occurring in a community in southern West Virginia in five years prompted a survey of the immunization status of the population of this region. An immunization history was obtained from 540 consecutive patients seen at three different health care facilities. Of these, 386 (71.5%) had received prior tetanus protection, 65 (12%) had never been immunized, and 89 (16.5%) were uncertain or had received incomplete immunization. Of the 65 nonimmunized patients, 54 (83%) were older than 50 and only 11 (17%) were younger. Compared to the total group sampled, significantly more nonimmunized patients (both male and female) were older than 50 (P less than .001). Also, significantly more of these never immunized individuals lived in rural areas (P less than .001). In a group of 222 patients identified as being at increased risk of having tetanus, 65 (29%) had never been immunized, 89 (40%) were of uncertain or incomplete status, and 68 (30.5%) had been immunized more than ten years previously. One hundred twenty (54%) high-risk patients were older than 50 and 103 (46%) were younger. Compared to the entire population sampled, high-risk patients included significantly more who were older than 50 (P less than .001). Also, significantly more high-risk patients lived in rural areas (P less than .007). When treating injured patients, it is important to recall that many older adults living in rural areas are not adequately immunized against tetanus. Such patients should receive human tetanus immunoglobulin, as well as tetanus toxoid.

Adolescent↗

Strategic interventions within a no-treatment frame.

It is the thesis of this paper that there are some problem contexts in which the risk for either no-response or negative effects of therapy are considerably greater than the probability of positive outcomes, despite the competence level of the therapist or special characteristics of the client system. It is these contexts that warrant the inclusion of a no-treatment option among therapists' current range of treatment choices. Rather than turning client systems away in an absolute sense, however, no-treatment is presented as a frame in which therapists are not obligated to help change a problem but may continue to have some form of contact with the client-system. Four no-treatment prescriptions are presented that can be employed strategically either to increase available opportunities for therapist leverage within a problem context, or to amplify competence in pivotal family members when the problem context demands immediate competent responses. Case studies are presented to highlight the contextual information used in making no-treatment decisions.

Adolescent↗