Developmentally regulated and lineage-specific rearrangement of T cell receptor Valpha/delta gene segments.

To quantitate the frequency of Valpha/delta gene utilization by TCRgammadelta T cells we have generated a large panel of gammadelta T cell hybridomas and characterized their productive VDJ rearrangements. Using three novel mAb specific for the Vdelta5 chain and for several members of the Vdelta6 subfamily together with previously described Valpha- and Vdelta-specific mAb we have also quantitated the frequency of gammadelta and alphabeta cells expressing those Valpha/delta gene segments and located in different anatomical sites. We have also characterized the members of the Vdelta7/ADV10 subfamily expressed in C57BL/6 mice and analyzed the representation of individual ADV10 gene segments in alphabeta and gammadelta cells, as well as in precursor cells, in a situation in which TCR-dependent selection is negligible. Our results show that (i) although many Valpha/delta gene segments have the potential to rearrange to either Ddelta and Jdelta segments or to Jalpha segments, only a limited number of Valpha/delta gene segments are expressed by a quantitatively important fraction of gammadelta cells; (ii) such restricted usage of a limited number of Vdelta gene segments by gammadelta cells is mainly established at the level of V(D)J rearrangement, and (iii) there is very little overlap between Valpha/delta gene segments expressed by gammadelta and alphabeta cells.

Male-specific transcription initiation of the C4-Slp gene in mouse liver follows activation of STAT5.

The mouse genes encoding the constitutively expressed complement component C4 and its closely related isoform C4-Slp (sex-limited protein), which is expressed only in male animals of several strains, provide a unique model to study sequence elements and trans-acting factors responsible for androgen responsiveness. Our previous studies have shown that hormonal induction of C4-Slp is mediated by a sex-specific pattern of growth hormone secretion. Promoter analyses in vitro have led to contradictory conclusions concerning the significance of C4-Slp-specific sequences in the 5' flanking region. Mutant mice carrying the H-2(aw18) haplotype, which is characterized by a large deletion in the S region covering the C4 and 21-OHase A genes, permit the direct in vivo analysis of C4-Slp transcription, unhindered by the presence of C4. Run-on analysis of transcription in liver nuclei of males and females of this strain demonstrated a 100-fold higher transcriptional activity in males, essentially determined at the transcription initiation level. The androgen dependence of transcription initiation was confirmed by run-on analysis of testosterone-treated females, where transcriptional activity started after 6 days of androgen treatment and reached male levels after 20 days. Conversely, the growth hormone-regulated activity of transcription factor STAT5 was already detected in liver nuclei after 48 hr of androgen treatment. Furthermore, we demonstrate that activated STAT5 recognizes in vitro two upstream gamma interferon-activated sequence (GAS) elements of the C4-Slp gene, centered at positions -1969 and -1831. We postulate that binding of STAT5 to these C4-Slp-specific GAS elements plays a crucial role in the chromatin remodelings that lead to transcriptional competence of the C4-Slp gene in the liver.

Behavioral changes in fasting emperor penguins: evidence for a "refeeding signal" linked to a metabolic shift.

This study examines the relationships between metabolic status and behavior in spontaneously fasting birds in the context of long-term regulation of body mass and feeding. Locomotor activity, escape behavior, display songs, body mass, and metabolic and endocrine status of captive male emperor penguins were recorded during a breeding fast. We also examined whether body mass at the end of the fast affected further survival. The major part of the fast (phase II) was characterized by the maintenance of a very low level of locomotor activity, with almost no attempt to escape, by an almost constant rate of body mass loss, and by steady plasma levels of uric acid, beta-hydroxybutyrate, and corticosterone. This indicates behavioral and metabolic adjustments directed toward sparing energy and body protein. Below a body mass of approximately 24 kg (phase III), spontaneous locomotor activity and attempts to escape increased by up to 8- and 15-fold, respectively, and display songs were resumed. This probably reflected an increase in the drive to refeed. Simultaneously, daily body mass loss and plasma levels of uric acid and corticosterone increased, whereas plasma levels of beta-hydroxybutyrate decreased. Some experimental birds were seen again in following years. These findings suggest that at a threshold of body mass, a metabolic and endocrine shift, possibly related to a limited availability of fat stores, acts as a "refeeding signal" that improves the survival of penguins to fasting.

Hormonal counterregulation failure in rats is related to previous hyperglycaemia-hyperinsulinaemia.

Hyperglycaemia and hyperinsulinaemia were induced in rats by a continuous 48-h infusion with glucose. Discontinuation of glucose infusion resulted in marked, persistent hypoglycaemia. To further delineate the mechanism underlying this condition, we measured counterregulatory hormone levels, in vivo glucose kinetics (glucose production = rate of appearance = Ra; glucose utilization = rate of disappearance = Rd), and in vitro gluconeogenesis during the 48-h postinfusion period. Prior to cessation of glucose infusion, Rd was increased 6-fold when compared to control rats, whereas Ra was totally abolished. During the first hour after the end of glucose infusion, Ra increased and Rd decreased (but was still higher than Ra), inducing hypoglycaemia which stabilized after 1 h at ¿¿126¿¿3.5 mmol/l when both Ra and Rd became equal. Despite hypoglycaemia, plasma glucagon and catecholamine levels did not increase during the 3-to 36-h time interval. The increase in Ra during the first hour post-infusion was not related to changes in counterregulatory hormone response. The increase in glucose production was accounted for by glycogenolysis, as shown by total depletion in liver glycogen within 6 h and thereafter by gluconeogenesis. In vitro experiments using isolated hepatocytes suggested that gluconeogenesis was supported during the first 24 h by substrates entering the pathway beyond the step catalysed by the PEPCK enzyme. Thereafter, lactate became the major substrate, and this condition was associated with a progressive rise in glucagon concentration. It is concluded that 48 h of hyperglycaemia/hyperinsulinaemia resulted in a failure of counterregulatory hormonal response to hypoglycaemia. Yet, despite this lack of counterregulatory response, hepatic gluconeogenesis was stimulated in response to hypoglycaemia.

Recognition of the E-C4 element from the C4 complement gene promoter by the upstream stimulatory factor-1 transcription factor.

Activation of complement gene expression plays a major role in the response to antigenic challenge. The induction of complement synthesis occurs primarily in liver and in macrophages and is mediated, at least in part, by increased transcription of the complement genes. For example, transcription of the C4 complement gene, which plays a crucial role in the complement pathway, is induced in response to acute inflammation or tissue injury. Previous work has defined the elements present in the C4 complement gene promoter that are required for its expression. Particularly important is an E-box motif, E-C4, that is conserved between the mouse, human, and rat promoters and that directed up to 90% of transcription from the mouse C4 promoter. Here we have purified the E-C4-binding factor to homogeneity using a novel and rapid affinity purification procedure. Following N-terminal microsequencing and subsequent isolation of the corresponding cDNA, the factor binding the E-C4 element was identified as upstream stimulatory factor-1 (USF-1), a basic helix-loop-helix-leucine zipper transcription factor. We also show for the first time that in vivo USF-1 is a phosphoprotein, but that phosphorylation of USF-1 is severely reduced in cells in culture. Moreover, the phosphorylated form of USF-1 binds DNA preferentially, indicating that phosphorylation may enhance the ability of USF-1 to bind DNA. The implications of USF-1 phosphorylation for C4 complement gene expression and transcription regulation are discussed.