Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

An abundant, highly conserved tonoplast protein in seeds

We have isolated the membranes of the protein storage vacuoles (protein bodies) from Phaseolus vulgaris cotyledons and purified an integral membrane protein with M_r 25,000 (TP 25). Antiserum to TP 25 recognizes an abundant polypeptide in the total cell extracts of many different seeds (monocots, dicots, and a gymnosperm), and specifically labels the vacuolar membranes of thin-sectioned soybean embryonic axes and cotyledons. TP 25 was not found in the starchy endosperm of barley and wheat or the seed coats of bean but was present in all seed parts examined that consist of living cells at seed maturity. The abundance of TP 25 was not correlated with the amount of storage protein in seed tissue, and the protein was not found in leaves that accumulate leaf storage protein. On the basis of its abundance, evolutionary conservation, and distribution in the plant, we propose that TP 25 may play a role in maintaining the integrity of the tonoplast during the dehydration/rehydration sequence of seeds.     

Immediate breast reconstruction: reducing the risks

One-hundred and sixty-five consecutive immediate breast reconstructions in 157 patients were reviewed. Reconstructions were performed with tissue expanders (53 percent) or immediate gel prostheses (47 percent). Immediate reconstruction was associated with an 18 percent rate of implant loss. Certain risk factors were identified at the p less than 0.05 level using immediate gel implants: failure to achieve complete muscle coverage of the implant, smoking at the time of surgery, initial gel implants of 400 ml or more volume, and age. Expander loss was increased by detaching the pectoralis major (p less than 0.05) and probably by lack of complete muscle coverage in general. Chemotherapy, history of previous smoking, and clinical stage of the carcinoma did not seem to affect reconstructive success. Smoking and patient age should be considered during patient selection for immediate reconstruction. Muscle coverage of the prosthesis should always be attempted. Muscle coverage is mandatory in the smoker. Gel implants of 400 ml or more volume are to be avoided at the initial operation. This approach should enable all surgeons to achieve lower rates of implant loss.     

Development ofBradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (Glycine max [L.] Merrill)

Summary The early events in the development of nodules induced byBradyrhizobium japonicum were studied in serial sections of a wild type (cv. Bragg), a supernodulating mutant (nts 382) and four non-nodulating mutants (nod49, nod139, nod772, andrj ₁) of soybean (Glycine max [L.] Merrill). Cultivar Bragg responded to inoculation in a similar manner to that described previously for cv. Williams; centres of sub-epidermal cell divisions were observed both with and without associated infection threads and most infection events were blocked before the formation of a nodule meristem. The non-nodulating mutants (nod49, nod772, andrj ₁) had, at most, a few centres of sub-epidermal cell divisions. In general, these were devoid of infection threads and did not develop beyond the very early stages of nodule ontogeny. Sub-epidermal cell divisions or infection threads were never observed on mutant nodl39. This mutant is not allelic to the other non-nodulating mutants and represents a defect in a separate complementation group or gene that is required for nodulation. The supernodulating mutant nts382, which is defective in autoregulation of nodulation, had a similar number of sub-epidermal cell divisions as the wild-type Bragg, but a much greater proportion of these developed to an advanced stage of nodule ontogeny. Mutant nts382, like Bragg, possessed other infection events that were arrested at an early stage of development. The results are discussed in the context of the progression of events in nodule formation and autoregulation of nodulation in soybean.Abbreviations nts nitrate tolerant symbiosis - RT root tip (i.e., position of the tap root tip at the time of inoculation) - SERH shortest emerging root hair (i.e., position of the shortest emerging root hair on the tap root at the time of inoculation) - SCD subepidermal cell divisions     

54Mn absorption and excretion in rats fed soy protein and casein diets

Rats were fed diets containing either soy protein or casein and different levels of manganese, methionine, phytic acid, or arginine for 7 days and then fed test meals labeled with 2 microCi of 54Mn after an overnight fast. Retention of 54Mn in each rat was measured every other day for 21 days using a whole-body counter. Liver manganese was higher (P less than 0.0001) in soy protein-fed rats (8.8 micrograms/g) than in casein-fed rats (5.2 micrograms/g); manganese superoxide dismutase activity also was higher in soy protein-fed rats than in casein-fed rats (P less than 0.01). There was a significant interaction between manganese and protein which affected manganese absorption and biologic half-life of 54Mn. In a second experiment, rats fed soy protein-test meals retained more 54Mn (P less than 0.001) than casein-fed rats. Liver manganese (8.3 micrograms/g) in the soy protein group was also higher than that (5.7 micrograms/g) in the casein group (P less than 0.0001), but manganese superoxide dismutase activity was unaffected by protein. Supplementation with methionine increased 54Mn retention from both soy and casein diets (P less than 0.06); activity of manganese superoxide dismutase increased (P less than 0.05) but liver manganese did not change. The addition of arginine to casein diets had little effect on manganese bioavailability. Phytic acid affected neither manganese absorption nor biologic half-life in two experiments, but it depressed liver manganese in one experiment. These results suggest that neither arginine nor phytic acid was the component in soy protein which made manganese more available from soy protein diets than casein diets.     

Thrombolysis using liposomal-encapsulated streptokinase: an in vitro study

The clot-lysing ability of streptokinase (SK) was examined using membrane-bound thrombi. Encapsulation of SK in large unilamellar phospholipid vesicles (liposomes) resulted in entrapping approximately 30% of its original activity. Measurements of streptokinase activity for liposomal-encapsulated streptokinase (LESK) indicated little loss of activity or leakage in Tris-buffered saline over a 24-hr period at temperatures of 4 and 23 degrees C. However, incubation of free SK and LESK in platelet-poor plasma (PPP) at 37 degrees C resulted in a decrease of SK activity. The retention of SK activity in LESK was considerably higher than that of unentrapped SK. Clot-dissolving time (CDT) was measured by monitoring the pressure drop during slow filtration in plasma through membrane-bound thrombi. The results indicated that both LESK and free SK were able to activate the fibrinolytic system. Without prior incubation in PPP at 37 degrees C, the CDT of a SK and PPP mixture (SK/PPP) was 10.7 +/- 1.9 min (n = 12), while that of a LESK and PPP mixture (LESK/PPP) was 12.4 +/- 1.7 min (n = 12). The CDT-detected clot-lysing abilities of both SK and LESK were diminished by incubation in PPP, but to different extents. After 15- and 30-min incubations, the CDT of SK/PPP increased significantly to 15.5 +/- 1.5 and 24.1 +/- 2.4 min (n = 5, P less than 0.05), respectively. In contrast, the CDT of LESK/PPP increased to 13.3 +/- 0.8 min (n = 5) after 15 min of incubation and to 16.0 +/- 1.1 min (n = 5, P less than 0.05) after a 30-min incubation. These results suggest that entrapment of SK in liposomes preserves the thrombolytic potential of the plasminogen activator by limiting its exposure to the components of the plasma.     

Inhibition by prostaglandin E2 of neurotransmission in rabbit but not human bronchus--a calcium-related mechanism?

Prostaglandins have been implicated in the development of airway hyperresponsiveness, and this may be mediated via modulation of neurotransmission. We compared the effects of prostaglandin E2 on the contractile response to electrical field stimulation in rabbit and human bronchus. Prostaglandin E2 produced marked inhibition in rabbit bronchus (mean % inhibition 35 +/- 17, P less than 0.05) but was without effect in human bronchus. The inhibition in rabbit bronchus was not the result of a direct effect on muscle tone and the site of action is likely to be pre-synaptic since prostaglandin E2 had only minor effects on exogenous acetylcholine. Since prostaglandins are known to affect calcium mobilization, we compared the dependence of cholinergic stimulation on the calcium voltage dependent channel (VDC) in the two species. Cholinergic stimulation was dependent on the VDC in rabbit but not human bronchus and this may be an explanation for the different effects of prostaglandin E2 in the two species.     

Effect of castration on plasma luteinizing hormone in postpubertal boars

Two experiments were conducted to test the working hypothesis that mean plasma concentrations of luteinizing hormone (LH) increase as a result of an increase in the frequency and amplitude of the pulsatile releases of LH in postpubertal boars after removal of gonadal steroid hormones by castration. It was further hypothesized that these changes in secretion of LH would be the result of changes in sensitivity of the pituitary to gonadotropin releasing hormone (GnRH). In Experiment 1, plasma LH was monitored in 10 postpubertal crossbred boars (13 to 14 mo old and weighing 159 +/- 6.0 kg) at 12-min intervals for 6 h before and 1 h after GnRH (375 ng/kg of body weight) on Days -1, 7, 14, 21 and 29 relative to castration. In Experiment 2, plasma LH was monitored in four castrated and five intact postpubertal boars (11 to 12 mo old and weighing 150 +/- 5.1 kg) after each of three doses of GnRH (94, 188 and 375 ng/kg) were administered to each animal. Sample collection occurred 5 wk after castration. Mean LH and frequency of pulsatile releases of LH increased as a result of castration (P<0.0001), with changes evident by Day 7 after castration. However, the amplitude of the LH pulses increased minimally after castration (P<0.10). The response to exogenous GnRH increased throughout Experiment 1 (P<0.0001), even though the amplitude of the pulsatile releases of LH (response to endogenous GnRH) did not change. Castrated animals in Experiment 2 had a greater response of LH to GnRH stimulation than intact boars (P<0.05). The dose-response curve of castrated animals was not parallel (P<0.001) to that of intact boars, and indicated that sensitivity of the pituitary to GnRH had increased in the absence of gonadal steroids. Thus, the hypotheses stated above can be accepted with the exception that castration may have a minimal effect on LH pulse amplitude. Based on the results of these experiments, we suggest that gonadal steroid hormones modulate both the size of releasable stores of LH and pituitary sensitivity to GnRH in boars.     

Cells that overproduce protein kinase C are more susceptible to transformation by an activated H-ras oncogene.

We recently developed rat fibroblast cell lines that stably overproduce high levels of the beta 1 form of protein kinase C (PKC). These cells display several disorders in growth control and form small microscopic colonies in agar. In the present study we demonstrate that one of these cell lines, R6-PKC3, is extremely susceptible to transformation by an activated human bladder cancer c-H-ras oncogene (T24). Compared with control cell line R6-C1, T24-transfected R6-PKC3 cells yielded a 10-fold increase in the formation of large colonies in agar. Cell lines established from these colonies displayed a highly transformed morphology, expressed the T24-encoded p21 ras protein, continued to express high levels of PKC, and were highly tumorigenic in nude mice. These results provide genetic evidence that PKC mediates some of the effects of the c-H-ras oncogene on cell transformation. Data are also presented suggesting that optimum synergistic effects between c-H-ras and PKC require critical levels of their respective activities. These findings may be relevant to the process of multistage carcinogenesis in tissues containing cells with an activated c-H-ras oncogene.