Effects of intermittent 60-Hz high voltage electric fields on metabolism,activity, and temperature in mice

Transient effects of 100-kV/m extremely low frequency electric fields were studied in the white footed deermouse, Peromyscus leucopus. Gross motor activity, carbon dioxide production, oxygen consumption, and core body temperature were monitored before, during, and after intermittent field exposures (four hour-long exposures, at one-hour intervals). Thirty-four mice were exposed in cages with plastic floors floating above ground potential, and 21 mice were exposed in cages with grounded metal floor plates. The first field exposure produced an immediate, transient increase of activity and gas measures during the inactive phase of the circadian cycle. All measures returned to baseline levels before the second exposure and were not significantly changed throughout the remainder of the exposures. The rapid habituation of field-induced arousal suggests that significant metabolic changes will not be measured in experiments in which the interval between exposure and measurement is greater than two hours.     

Influence of day length and temperature on number of main stem leaves and time to flowering in lupin

A growth chamber experiment was carried out to investigate the influence of day length and temperature on the development of flowering in eight varieties of the three grain lupin species Lupinus albus (Wat and C3396), L. angustifolius (Gungurru, Polonez and W26) and L. luteus, (Juno, Radames and Teo). The plants were grown at two temperatures, 10°C and 18°C, in combination with five daylength regimes: 10, 14, 18, 24 h day at full light intensity and 10 h full light extended with 8 h low intensity light. Increased daylength decreased days from sowing to flowering in all varieties, but had little effect on thermal time to flowering in most varieties. However, C3396, W26 and Radames had a significantly longer thermal time to flowering at high, non‐vernalising temperature (18°C) at short daylengths. Low light intensity daylength extension did not significantly influence thermal time to flowering. For flower initiation, measured as number of leaves on the main stem three types of response were found. All varieties formed fewer leaves on the main stem at 10°C than at 18°C, although the two thermo‐neutral varieties of L. luteus, Juno and Teo, gave only a small response to temperature and daylength. In Polonez, Gungurru and Wat, low temperature decreased leaf number, but there was only a small response to changes in daylength. Three varieties, C3396, W26 and Radames, showed longer thermal time to flowering at 18°C with short daylengths. This could be explained by a greater number of main stem leaves formed at short daylength at non‐vernalising temperatures. Increased daylength decreased leaf number in these varieties, but never to a smaller number than for plants grown at 10°C. In these varieties, low intensity extension of the daylength had a similar (W26, Radames) or decreased (C3396) effect compared to full light extension. The hastening of time to flowering by long days could be separated into two effects: a high light energy effect hastened development by increasing the rate of leaf appearance in all varieties, while low light energy in thermo‐sensitive varieties was able to substitute for vernalisation by decreasing leaf number.     

Mutational analysis of the Escherichia coli phosphate-specific transport system, a member of the traffic ATPase (or ABC) family of membrane transporters. A role for proline residues in transmembrane helices.

The Escherichia coli Pst system is a periplasmic phosphate permease. A mutational analysis of the requirement for function of specific charged residues or proline residues in the two hydrophobic subunits (PstC and PstA) has been carried out. No residues, among 19 charged residues altered, were found to be essential for phosphate uptake, although some alterations resulted in partial effects. Evidence was obtained that the 3 residues, R220 in the PstA protein and R237 and E241 in the PstC protein, previously shown to be required for phosphate transport (Cox, G. B., Webb, D., Godovac-Zimmermann, J., and Rosenberg, H. (1988) J. Bacteriol. 170, 2283-2286; Cox, G. B., Webb, D., and Rosenberg, H. (1989) J. Bacteriol. 171, 1531-1534), interact with each other. A feature of the proposed structures of the PstA and PstC proteins was 2 pairs of proline residues in putative transmembrane helices 3 and 4. While individual substitutions of these proline residues by leucine resulted in loss of phosphate transport activity substitution by alanine only had partial effects. However, if the proline to alanine changes were paired then, depending on the particular subunit, markedly different effects were obtained. The double mutation in the PstA protein resulted in a permanently "closed" system, whereas the double mutation in the PstC protein resulted in a permanently "open" transport system.     

Isolation and characterization of heparan sulfate proteoglycans produced by cloned rat microvascular endothelial cells.

We have isolated heparan sulfate proteoglycans (HSPGs) from cloned rat microvascular endothelial cells using a combination of ion-exchange chromatography, affinity fractionation with antithrombin III (AT III), and gel filtration in denaturing solvents. The anticoagulantly active heparan sulfate proteoglycans (HSPGact) which bind tightly to AT III bear mainly anticoagulantly active heparan sulfate (HSact) whereas the anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact) possess mainly anticoagulantly inactive heparan sulfate (HSinact). HSact and HSinact were also isolated by a combination of ion-exchange chromatography, treatment with protease and chondroitin ABC lyase, and affinity fractionation with AT III. HSact and HSinact have molecular sizes of about 25-30 kDa with the same overall composition of monosaccharides except that HSact exhibits about nine glucuronsyl 3-O-sulfated glucosamines/chain whereas HSinact possesses about three glucuronsyl 3-O-sulfated glucosamines/chain. Direct isolation of the AT III-binding site of HSact by exposing carbohydrate chains to Flavobacterium heparitinase in the presence of protease inhibitor revealed only a single interaction site which contained two to three glucuronsyl 3-O-sulfated glucosamine residues. The core proteins of HSPGact and HSPGinact were isolated by treatment with Flavobacterium heparitinase and purification by ion-exchange chromatography. The molecular sizes of the core proteins were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their primary structures were examined by cleavage with trypsin or endopeptidase Glu-C as well as separation of peptides by reverse-phase high performance liquid chromatography. The results showed that both sets of core proteins exhibited three major components with molecular sizes of 50, 30, and 25 kDa, respectively. The 25-kDa species appears to be a proteolytic degradation product of the 30-kDa species. The peptide mapping revealed that HSPGact and HSPGinact possess extremely similar core proteins.     

Characterization of proteoglycan-reactive T cell lines and hybridomas from mice with proteoglycan-induced arthritis.

Hyperimmunization with chondroitin sulfate-depleted fetal human cartilage proteoglycan (HFPG) leads to the development of peripheral arthritis and spondylitis in BALB/c mice. Chondroitin-sulfate-depleted adult human cartilage proteoglycan (HAPG) is much less effective at inducing arthritis. These observations suggest age differences in the presence of arthritogenic proteoglycan (PG) epitopes. Earlier studies from this laboratory have indicated an important role for PG-reactive T cells in the pathogenesis of this arthritis model. To investigate further the cellular immunity to PG in mice, two T cell lines, JY.A and JY.D, and two T cell hybridomas, TH5 and TH14, were isolated from mice with PG-induced arthritis and characterized. Two patterns of reactivity to PG emerged from the analysis of these T cells. One pattern, as demonstrated by the T cell line JY.D and the two T cell hybridomas, TH5 and TH14, was characterized by reactivity to HFPG, HAPG, chondroitin sulfate-depleted bovine cartilage PG, the G1 domain (hyaluronate binding region) of bovine cartilage PG and bovine link protein. The epitope(s) recognized by these T cells appear to be part of the homologous regions shared between the G1 domain and the link protein. The second pattern of reactivity, as demonstrated by the T cell line JY.A, was characterized by reactivity to HFPG but not to HAPG or the other PG Ag or bovine link protein. All the T cell lines and hybridomas had a CD4+, CD8- phenotype, possibly belonged to the TH1 subset (IL-2+, IL-4-), and were MHC class II restricted. These studies indicate that HFPG has T cell epitopes in common with HAPG (such as in the G1 domain) and different than those in HAPG. The significance of this data in terms of PG structure, changes with age, and induction of arthritis remains to be established.     

Management of reproduction in dairy heifers based on the synchronization of estrous cycles

Israeli-Holstein breed dairy heifers (n = 571), 13 to 15 mo old, were utilized in two experiments. In Experiment 1, the reproductive performance of synchronized heifers was compared with that of untreated controls. The heifers in both groups were inseminated following the detection of estrus. In Experiment 2, all heifers were synchronized and inseminated following the detection of estrus. Half of the animals in this experiment also received one or two fixed-time inseminations 72 and 96 h after the last synchronization treatment. Synchronization of estrous cycles was performed by two prostaglandin F(2alpha) (PG) injections given 12 d apart. In the control group of Experiment 1, observation of estrous behavior and insemination of heifers detected in estrus were carried out daily throughout the experiment. In the synchronized groups of Experiments 1 and in 2, the management of reproduction consisted of estrus detection followed by the insemination of heifers in estrus carried out only during 6 d of every 3 wk. Five days following the second PG injection, 86% of the heifers were detected in estrus, 71% of them at 49 to 96 h after treatment. In Experiment 1, age at first insemination, age at conception, and conception rate were, respectively, 425 d, 446 d and 54% in the control group vs 432 d (P<0.02), 449 d and 62% in the PG-treated group. In Experiment 2, the respective figures were 436 d, 462 d and 59% in the group inseminated following the detection of estrus vs 427 d (P<0.002), 464 d and 51% (P<0.05) in the group in which heifers were inseminated at estrus and also received one or two fixed-time inseminations.     

bcr/abl and src but not myc and ras replace v-abl in lymphoid transformation.

Lymphoid cells transformed by temperature-sensitive Abelson virus die at the nonpermissive temperature. This property was exploited to show that bcr/abl and v-src but not myc and ras can replace the transforming signal of v-abl, a result suggesting that the former but not the latter oncogenes transform lymphoid cells via a similar pathway.     

Epidermis generated in vitro: practical considerations and applications

The technology for culture of epidermis is one of the most advanced to date for generation of a tissue in vitro. Cultured epidermis is already used for a number of applications ranging from use as a permanent skin replacement to use as an organotypic culture model for toxicity testing and basic research. While simple epidermal sheets have been grafted successfully, more advanced models for skin replacement consisting of both dermal and epidermal components are in development and being tested in a number of laboratories. One of the most advanced in vitro models is the living skin equivalent, an organotypic model consisting of a collagen lattice contracted and nourished by dermal fibroblasts overlaid with a fully formed epidermis.