Enhancement of the anabolic effects of growth hormone and insulin-like growth factor I by use of both agents simultaneously.

The use of growth hormone (GH) as an anabolic agent is limited by its tendency to cause hyperglycemia and by its inability to reverse nitrogen wasting in some catabolic conditions. In a previous study comparing the anabolic actions of GH and IGF-I (insulin-like growth factor I), we observed that intravenous infusions of IGF-I (12 micrograms/kg ideal body wt [IBW]/h) attenuated nitrogen wasting to a degree comparable to GH given subcutaneously at a standard dose of 0.05 mg/kg IBW per d. IGF-I, however, had a tendency to cause hypoglycemia. In the present study, we treated seven calorically restricted (20 kcal/kg IBW per d) normal volunteers with a combination of GH and IGF-I (using the same doses as in the previous study) and compared its effects on anabolism and carbohydrate metabolism to treatment with IGF-I alone. The GH/IGF-I combination caused significantly greater nitrogen retention (262 +/- 43 mmol/d, mean +/- SD) compared to IGF-I alone (108 +/- 29 mmol/d; P < 0.001). GH/IGF-I treatment resulted in substantial urinary potassium conservation (34 +/- 3 mmol/d, mean +/- SE; P < 0.001), suggesting that most protein accretion occurred in muscle and connective tissue. GH attenuated the hypoglycemia induced by IGF-I as indicated by fewer hypoglycemic episodes and higher capillary blood glucose concentrations on GH/IGF-I (4.3 +/- 1.0 mmol/liter, mean +/- SD) compared to IGF-I alone (3.8 +/- 0.8 mmol/liter; P < 0.001). IGF-I caused a marked decline in C-peptide (1,165 +/- 341 pmol/liter; mean +/- SD) compared to the GH/IGF-I combination (2,280 +/- 612 pmol/liter; P < 0.001), suggesting maintenance of normal carbohydrate metabolism with the latter regimen. GH/IGF-I produced higher serum IGF-I concentrations (1,854 +/- 708 micrograms/liter; mean +/- SD) compared to IGF-I only treatment (1,092 +/- 503 micrograms/liter; P < 0.001). This observation was associated with increased concentrations of IGF binding protein 3 and acid-labile subunit on GH/IGF-I treatment and decreased concentrations on IGF-I alone. These results suggest that the combination of GH and IGF-I treatment is substantially more anabolic than either IGF-I or GH alone. GH/IGF-I treatment also attenuates the hypoglycemia caused by IGF-I alone. GH/IGF-I treatment could have important applications in diseases associated with catabolism.

CD44 antibodies inhibit osteoclast formation.

Osteoclast differentiation is a complex process requiring multiple factors and sequential regulation. We have determined that CD44, a cell surface glycoprotein that is known to function as an adhesion receptor, is involved in this process. By immunocytochemistry, we show that CD44 is expressed in mouse osteoclasts that develop in primary cultures of bone marrow cells treated with 1 alpha, 25-dihydroxyvitamin D3. Monoclonal antibodies to CD44 inhibit osteoclast formation in bone marrow cultures in a dose- and time-dependent manner. In contrast, CD44 Fab monomer antibodies have no effect on osteoclast development, suggesting that the inhibition of differentiation by the whole antibodies is facilitated by cross-linking of CD44 molecules. Cocultures of spleen cells and ST2 bone marrow stromal cells indicate that hematopoietic cells mediate the CD44 antibody inhibitory effect. CD44 antibodies do not inhibit osteoclast resorption of calcified matrix, indicating that CD44 is not absolutely required for resorption activity. These observations demonstrate that CD44 may play a role in osteoclast formation and suggest mechanisms by which CD44 antibody effects are mediated.

Genomic sequence of the DAX1 gene: an orphan nuclear receptor responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism.

The gene responsible for X-linked adrenal hypoplasia congenita, DAX1, encodes a member of the nuclear hormone receptor superfamily. We sequenced 8851 bp that contained the DAX1 genomic region. The DAX gene was composed of two exons and one 3.4-kilobase intron. Putative TATA and GC boxes and a putative steroidogenic factor 1 response element were present in the 5'-flanking region. Two potentially polymorphic short tandem repeats were identified. The first exon encoded two putative novel zinc finger motifs within a putative DNA binding domain and part of the ligand binding domain, and the second exon encoded the remainder of the ligand binding domain. Although the putative DNA binding domain of DAX1 does not contain substantial sequence similarity to other nuclear hormone receptor superfamily members, the putative ligand binding domain had remarkable similarity to other family members. Single-strand conformational polymorphism analysis permitted identification of three new mutations in DAX1. In conclusion, single-strand conformational polymorphism analysis facilitates identification of mutations in the DAX1 gene, and the short tandem repeats may permit linkage analysis in families in which mutations are not yet identified. We speculate that DAX1 may be the most primitive member of the nuclear hormone receptor superfamily identified in mammals.

Prevention of dry socket with clindamycin. A retrospective study.

Clindamycin and other agents were compared for efficacy in preventing the entity "dry socket." A total 765 patients were treated with clindamycin, per os, and 408 patients were treated with other antibiotics or were non-treated controls. All patients underwent surgical removal of impacted mandibular third molars. The incidence of dry socket in untreated control and in non-clindamycin antibiotic-treated patients varied from 15 to 31 percent, while in those patients receiving clindamycin, the incidence was 0.65 percent. The results demonstrate a remarkable effectiveness of clindamycin in reducing the incidence of dry socket following surgical removal of impacted mandibular third molar.

Identification of a glucocorticoid-responsive element in Epstein-Barr virus.

Immortalization of B lymphocytes by Epstein-Barr virus (EBV) is complex and poorly understood. However, some evidence suggests that glucocorticoids influence this process. We identified a glucocorticoid-responsive element in the BamHI C fragment of EBV which we call ES-1. In glucocorticoid-treated cells, ES-1 enhanced chloramphenicol acetyltransferase gene expression from the herpes simplex virus thymidine kinase promoter, as well as the EBV Bam-C promoter, from which several latent viral gene products are transcribed. By Northern blot analysis, glucocorticoid treatment enhanced transcription from the Bam-C promoter in Jijoye cells, a Burkitt's lymphoma cell line. In addition, the DNA-binding domain of the glucocorticoid receptor bound specifically to the ES-1 region. These glucocorticoid effects on the Bam-C promoter region may provide some insight into the process of EBV immortalization.

Androgen and glucocorticoid receptors interact with insulin degrading enzyme.

We recently identified a protein, designated receptor accessory factor (RAF), that interacts directly with and enhances the DNA binding of androgen (AR) and glucocorticoid (GR) receptor peptide fragments. To determine its identity, RAF was purified from HeLa cell extracts by anion-exchange and DNA affinity chromatography. RAF activity co-purified with a 110-kDa protein, the partial amino acid sequence of which shares 97.5% identity with insulin degrading enzyme (IDE), a metalloendoprotease implicated in the intracellular degradation of insulin. The identity of RAF was confirmed in gel shift assays by demonstrating that AR.RAF and GR.RAF DNA complexes shifted to a slower mobility in the presence of an IDE antibody. Purified preparations of RAF had insulin degrading activity, and this activity was inhibited by AR. In addition, the interaction of AR or GR with RAF was competed by insulin and bacitracin, a competitive inhibitor of IDE. The interactions of AR and GR with IDE may have important implications for both insulin- and steroid-mediated signaling.

Receptor accessory factor enhances specific DNA binding of androgen and glucocorticoid receptors.

Protein-protein interactions are common among transcriptional activators and may have important consequences for gene regulation. Using the mobility shift assay, we have identified a factor that enhances specific DNA binding of truncated rat androgen (AR) and glucocorticoid (GR) receptors by 25- and 6-fold, respectively, through the formation of heteromeric complexes. This factor, designated receptor accessory factor, or RAF, also potentiates DNA binding of full-length human GR. RAF is temperature and trypsin sensitive and is present in a variety of cultured mammalian cells. By gel filtration RAF has a predicted molecular mass of 130 kDa. RAF enhancement of AR-DNA binding is optimal with androgen response element DNA. RAF appears to interact directly with AR because 1) deoxycholate, which interferes with protein-protein but not protein-DNA interactions, prevents RAF.AR.DNA complex formation, 2) RAF activity is recovered from an androgen response element DNA affinity column only in the presence of AR, and 3) RAF increases the size of an AR.DNA complex by gel filtration. Mutagenesis of truncated AR fragments indicates that a region in the NH2-terminal domain is required for RAF to enhance AR-DNA binding. The interaction of RAF with AR and GR suggests that RAF might influence the ability of these nuclear receptors to activate transcription.

Specificity of simple hormone response elements in androgen regulated genes.

Androgen (AR) and glucocorticoid (GR) receptors recognize a family of 15 base pair partial palindromic hormone response elements (HRE). We have studied receptor interactions with several HREs from androgen regulated genes to determine their potential to mediate a selective androgen response. Synthetic oligonucleotides corresponding to the elements were analysed for receptor binding and steroid dependent transcriptional enhancer activities. Each HRE contained the 3' half-site sequence (5'-TGTNCT-3') of the glucocorticoid response element (GRE) consensus sequence. HREs that countained the 5' half-site GRE consensus sequence (5'-A/GGNACA/G-3') had the strongest and-rogen response element (ARE) and GRE activities. In methylation interference assays, AR and GR interacted with identical base contact sites in the response elements. Two elements that deviated from the GRE consensus sequence by a single optimal base in the 5' half, had reduced ARE activity with no significant change in GRE activity and displayed lower binding of AR than GR in mobility shift assays using purified DNA binding domain peptides. Transfections with AR/GR and GR/AR chimeras containing the N-terminal domain of one receptor linked to the DNA-binding and C-terminal domains of the other suggested that N-terminal domain functions of GR also contributed to the greater GRE than ARE activities of the response elements.