Advances in sex determination in bats and its utility in wind-wildlife studies.

We developed a simple and reliable genetic method to determine sex in bats from the Vespertilionidae and Molossidae families. Polymerase chain reaction was used to amplify a portion of the introns within the zinc-finger-X (Zfx) and zinc-finger-Y (Zfy) genes. We designed primers to produce size variation between the Zfx and Zfy products that could be visualized using gel electrophoresis. Using an example from our wind-wildlife research, we show how sex data generated using this method are superior to sex data based on external morphology. Our method allows for the generation of sex data across a wide range of bats that can be used to address key questions in wildlife forensics, behavioural ecology, conservation and evolutionary biology.

Neurokinin-1 receptor resensitization precedes receptor recycling.

Following agonist binding, neurokinin-1 receptors undergo rapid desensitization followed by internalization and recycling. Desensitization requires receptor phosphorylation but does not require internalization, whereas resensitization is thought to require internalization and recycling. Our previous data, however, have suggested that, following activation and desensitization, the return of responsiveness to the neurokinin-1 agonist substance P (termed "resensitization") occurs hours before internalized receptors are recycled back to the plasma membrane. To further investigate this novel mechanism of neurokinin-1 receptor resensitization, we have studied the time courses of neurokinin-1 receptor responsiveness, recycling, and dephosphorylation by measuring cellular Ca(2+) responses, ligand-receptor binding, and receptor phosphorylation, respectively. Concentration-response curves and competition binding curves were obtained at various times following desensitization. The effects of the nonhydrolyzable GTP analog Gpp(NH)p on substance P binding were also studied to assess receptor-G protein coupling. After receptor activation and desensitization, Ca(2+) signaling in response to substance P occurred within 90 min, whereas the return of receptor binding required 240 min. Receptor dephosphorylation was greater than 90% complete 20 min after agonist washout. In addition, the return of substance P responsiveness coincided with a return in sensitivity of substance P binding to Gpp(NH)p, indicating a return in receptor-G protein coupling. These data show that the resensitization of responsiveness to substance P precedes receptor recycling. This may result from a conversion of nonfunctional neurokinin-1 receptors to functional receptors at the plasma membrane.

Tachykinin peptide-induced activation and desensitization of neurokinin 1 receptors.

The actions of four tachykinins on inhibition and desensitization of the M-current of bullfrog sympathetic neurons have been characterized. Radioligand binding parameters of the tachykinins were determined at a neurokinin receptor in a heterologous expression system. The correlation between binding, signaling and receptor regulation was investigated. A correlation between receptor binding and signaling was found between the peptides; however, their ability to produce desensitization was not correlated with binding and signaling. These results show that the ability of a tachykinin peptide to induce signal activation is not indicative of its ability to induce receptor regulation.

A novel mechanism of neurokinin-1 receptor resensitization.

Prolonged or repeated activation of many G protein-coupled receptors induces rapid desensitization followed by a period during which receptors are resensitized. In this study, concanavalin A (Con A) and monensin were used to investigate the mechanisms of desensitization and resensitization of the neurokinin-1 receptor. Con A inhibits internalization, whereas monensin prevents receptor recycling. The effects of Con A and monensin on desensitization, resensitization, receptor phosphorylation, endocytosis, and recycling of the neurokinin-1 receptor were assessed. Desensitization was defined as the decrease in the ability of substance P (SP) to elicit an intracellular Ca2+ response after a prolonged SP exposure. Resensitization was characterized as the return of SP responsiveness. Under control conditions, desensitization occurred after a 5-min exposure to agonist. Resensitization was evident 30 min after agonist washout. Neither monensin nor Con A prevented desensitization. Monensin completely inhibited resensitization, whereas Con A decreased but did not completely block resensitization. Receptor phosphorylation was increased after agonist activation and returned to basal levels after a recovery period. Neither Con A nor monensin altered the amount of agonist-specific receptor phosphorylation. Receptor binding analysis showed that plasma membrane receptors were internalized after a 5-min agonist exposure. Receptor recycling was not observed after a 1-h recovery period; however, resensitization was apparent. Taken together, these results suggest that rapid neurokinin-1 receptor desensitization can occur without receptor internalization and that resensitization occurs before receptor recycling.

Growth inhibition by cyclosporine A in vivo and in vitro.

Cyclosporine A is a commonly used immunosuppressant in organ transplant. However, in addition to its nephro- and hepato-toxicity, we now report that it also exerts growth inhibiting effects in vivo and in vitro. Rats treated with daily Cyclosporine A I.P. injections of 10 mg or 20 mg/kg body weight gained weight at significantly lower rates than controls over a six week period. This growth retardation was completely reversible. By 6 weeks post treatment, the body weights of the treated animals were the same as controls. In contrast, rats treated with lower doses of cyclosporine A (5 mg/kg) did not suffer any growth retardation throughout. In vitro, rat primary cultured fibroblasts treated with greater than 1 microgram/ml Cyclosporine A suffered 25-60% cell loss in the post-log phase but the cytotoxic effect was not apparent during the log phase of growth. Fibroblasts treated with less than or equal to 1 microgram/ml of Cyclosporine A did not suffer any cell loss. In conclusion, Cyclosporine A exerts growth inhibitory effects in vivo when given at greater than 5 mg/kg body weight and cytotoxic effects in vitro at greater than 1 microgram/ml.

Suppression of immunological response against a novel gene product delivered by implants of genetically modified fibroblasts.

We have recently demonstrated the feasibility of genetically modifying autologous primary rat fibroblasts to deliver in vivo a foreign gene product, human growth hormone. However, in this model for gene replacement therapy, all recipient animals developed extremely high titres of antibodies against human growth hormone within two weeks of grafting. We now report on two approaches to suppress this immune-response. First, rats implanted with human growth hormone-secreting rat fibroblasts were treated with an immunosuppressant, cyclosporine A, at 20 mg/kg body weight per day. The production of anti-human growth hormone antibodies in the treated animals was completely blocked during the 12-week course of treatment. Secondly, by using immunologically immature neonatal rats as recipients, the rapid antibody response to the human growth hormone was also avoided. However, after a delay of one month, these rats also developed an extremely high titre of antibodies against the human growth hormone. In comparison, rats in the adolescent, mature and aged groups developed and maintained high titres of antibodies soon after implantation. Therefore, antigenic response against novel gene products can be suppressed either totally by cyclosporine A or temporarily in neonatal animals. The combination of early implantation and subsequent immuno-suppression should be considered in somatic gene therapy for those patients who are negative for cross-reacting-material and may be expected to mount an antigenic response to the replacement gene product.

Serological evaluation of a measles, mumps, and rubella vaccine.

Combined measles, mumps, and rubella vaccination is soon to become available in Britain in the routine immunisation programme. A controlled study was performed in 319 children, aged 13 months, to assess the antibody response and clinical reactions to a new combined measles, mumps, and rubella vaccine in comparison with a single component measles vaccine. In the children who received the combined vaccine, seroconversion was established in 93% for measles, 99% for rubella, and 100% for mumps. In the children who received the single measles vaccine, seroconversion was established in 92% for measles and in none for rubella and mumps. There was no increase in clinical reactions after the measles, mumps, and rubella vaccine compared with the measles vaccine. These results suggest that this combined vaccine would be effective and safe in a British population and give further support for the introduction of the combined measles, mumps, and rubella vaccine to Britain.

Proximal and distal tubular activity in chronically catheterized fetal sheep compared with the adult.

Renal function was studied in unanaesthetized fetal sheep aged 112-120 and 126-132 days and in adult nonpregnant ewes. The clearance of lithium was used to measure proximal and distal fractional sodium reabsorption. In five nonpregnant adult sheep, 80.6 +/- 1.7% (SE) of the filtered sodium load was reabsorbed proximally and 18.2 +/- 1.53% distally. This was different from all groups of fetal sheep (p less than 0.001). In younger fetuses, proximal fractional sodium reabsorption was less (51.3 +/- 2.3% (SE), p less than 0.05) and distal fractional sodium reabsorption greater (42.4 +/- 2.3% (SE), p less than 0.05) than older fetuses (126-132 days old) in which 61.4 +/- 2.4% (SE) was reabsorbed proximally and 33.6 +/- 2.5% (SE) distally. In another group of fetuses aged 125-137 days, in which proximal tubular sodium reabsorption was measured after distal tubular blockade, proximal fractional sodium reabsorption was 57.8 +/- 2.95% (SE) and distal fractional sodium reabsorption, 38.7 +/- 2.64% (SE). In adult sheep there was no relationship between distal tubular sodium reabsorption and glomerular filtration rate, i.e., proximal tubular function was responsible for glomerulotubular balance. However, in the fetuses, both proximal and distal tubular sodium reabsorption contributed to glomerulotubular balance. Thus in fetal life, the proximal tubule participates to a lesser extent in reabsorbing the filtered sodium load possibly because its function is suppressed by its relatively "volume-expanded" state or because it is functionally immature. Therefore, a greater proportion is reabsorbed distally and the distal nephron participates under physiological conditions in glomerulotubular balance.