Antibiotic Cycling Reverts Extensive Drug Resistance in Burkholderia multivorans.

Antibiotic collateral sensitivity, in which acquired resistance to one drug leads to decreased resistance to a different drug, occurs in Burkholderia multivorans. Here, we observed that treatment of extensively drug-resistant variants evolved from a cystic fibrosis (CF) sputum sample isolate with either meropenem or sulfamethoxazole-trimethoprim, depending on past resistance phenotypes, resulted in increased sensitivity to five different classes of antibiotics. We further identified mutations, including putative resistance-nodulation-division efflux pump regulators and uncharacterized pumps, that may be involved in this phenotype in B. multivorans.

The Response to Gonadotropin-Releasing Hormone and hCG in Men with Prior Chronic Androgen Steroid Abuse and Clinical Hypogonadism.

Androgens were initially developed to improve anabolism for therapeutic purposes. An observed side effect is a sustained inability to regain normal gonadal function after long-term use. This study was designed to evaluate the response to a standard GnRH (gonadotropin-releasing hormone) test (100 µg) followed by an hCG (human chorionic gonadotropin) test to evaluate the HPG (hypothalamic-pituitary-gonadal) axis in a subgroup of men with former androgen use (FAU, n=13, mean age 38±8 years) with secondary hypogonadotropic hypogonadism and total serum testosterone levels below 10 nmol/l. For comparison, healthy men (n=8, mean age 41±5 years) and untreated men with idiopathic hypogonadotropic hypogonadism (IHH, n=5, mean age 26±8 years) were included. Five of 13 FAU males had an LH (luteinizing hormone) peak after GnRH over 9.6 U/l, the 5(th) percentile of normal reference controls. None of the 13 FAU males reached a testosterone response above 16.0 nmol/l after the 72-h hCG stimulation test, the lowest recorded value for healthy male controls. The IHH patients responded to GnRH with an LH peak after 45 min, while the FAU males and healthy controls had an LH peak after 30 min. After hCG stimulation, the IHH patients increased mean testosterone level to 16.8 nmol/l (median 15.0 nmol/l), significantly higher than the FAU males, p<0.05. Current data support that GnRH and 72-h hCG stimulation tests may be valuable clinical tools to evaluate the HPG axis in adults with previous history of complex androgen abuse, and may provide valuable information in clinical management of these men.

Enhancing 1 alpha-hydroxylase activity with the 25-hydroxyvitamin D-1 alpha-hydroxylase gene in cultured human keratinocytes and mouse skin.

1 alpha,25-Dihydroxyvitamin D(3) (1 alpha,25(OH(2))D(3)) and its analogs are used to treat psoriasis because of their potent antiproliferative activity. They have the potential for causing hypercalcemia, however, and patients often become resistant to the drug. We examined the feasibility of enhancing the cutaneous production of 1 alpha,25(OH(2))D(3) using a human 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-OHase) plasmid. The 1 alpha-OHase gene was fused to the green fluorescent protein gene (1 alpha-OHase-GFP) driven by the cytomegalovirus promoter. Transfection of cultured normal human keratinocytes with the 1 alpha-OHase-GFP plasmid resulted in a marked increase in the expression of 1 alpha-OHase-GFP in the mitochondria. Transfection of keratinocytes with 1 alpha-OHase-GFP or 1 alpha-OHase plasmids in vitro enhanced the 1 alpha-OHase activity substantially and increased the sensitivity of the keratinocytes to the antiproliferative effect of 25(OH)D(3). The 1 alpha-OHase-GFP plasmid was topically applied to shaved C57/BL6 mice. Twenty-four hours after topical application, immunohistochemical analysis of the skin for 1 alpha-OHase-GFP revealed the presence of 1 alpha-OHase-GFP in the epidermis and epidermal appendages including the hair follicles. The results from this study offer a unique new approach for the topical treatment of hyperproliferative disorders such as psoriasis and skin cancer using the 1 alpha-OHase gene that could locally increase the production of 1 alpha,25(OH(2))D(3) without causing hypercalcemia or resistance.

Effects of 3 days unloading on molecular regulators of muscle size in humans.

Changes in skeletal muscle mass are controlled by mechanisms that dictate protein synthesis or degradation. The current human study explored whether changes in activation of the phosphoinositide 3-kinase (PI3K)-Akt1, p38, myostatin, and mRNA expression of markers of protein degradation and synthesis occur soon after withdrawal of weight bearing. Biopsies of the vastus lateralis muscle (VL) and soleus muscle (Sol) were obtained from eight healthy men before and following 3 days of unilateral lower limb suspension (ULLS). Akt1, Forkhead box class O (FOXO)-1A, FOXO-3A, p38, and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) phosphorylation and protein levels and myostatin protein level were analyzed by Western blot. Levels of mRNA of IGF1, FOXO-1A, FOXO-3A, atrogin-1, MuRF-1, caspase-3, calpain-2, calpain-3, 4E-BP1, and myostatin were measured using real-time PCR. The amounts of phosphorylated Akt1, FOXO-1A, FOXO-3A, and p38 were unaltered (P>0.05) after ULLS. Similarly, mRNA levels of IGF1, FOXO-1A, FOXO-3A, caspase-3, calpain-2, and calpain-3 showed no changes (P>0.05). The mRNA levels of atrogin-1 and MuRF-1, as well as the mRNA and protein phosphorylation of 4E-BP1, increased (P<0.05) in VL but not in Sol. Both muscles showed increased (P<0.05) myostatin mRNA and protein following ULLS. These results suggest that pathways other than PI3K-Akt stimulate atrogin-1 and MuRF-1 expression within 3 days of ULLS. Alternatively, transient changes in these pathways occurred in the early phase of ULLS. The increased myostatin mRNA and protein expression also indicate that multiple processes are involved in the early phase of muscle wasting. Further, the reported difference in gene expression pattern across muscles suggests that mechanisms regulating protein content in human skeletal muscle are influenced by phenotype and/or function.

Wilms' tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression.

Products of steroidogenic factor 1 (SF-1) and Wilms' tumor 1 (WT1) genes are essential for mammalian gonadogenesis prior to sexual differentiation. In males, SF-1 participates in sexual development by regulating expression of the polypeptide hormone Müllerian inhibiting substance (MIS). Here, we show that WT1 -KTS isoforms associate and synergize with SF-1 to promote MIS expression. In contrast, WT1 missense mutations, associated with male pseudohermaphroditism in Denys-Drash syndrome, fail to synergize with SF-1. Additionally, the X-linked, candidate dosage-sensitive sex-reversal gene, Dax-1, antagonizes synergy between SF-1 and WT1, most likely through a direct interaction with SF-1. We propose that WT1 and Dax-1 functionally oppose each other in testis development by modulating SF-1-mediated transactivation.

25-hydroxyvitamin D-1alpha-hydroxylase in normal and malignant colon tissue.

Vitamin D affects calcium metabolism and prevents proliferation of colon cells in vitro. In human beings the main circulating form of vitamin D is 25-hydroxyvitamin D; to regulate calcium homoeostasis, this form must be converted to 1alpha, 25-dihydroxyvitamin D by 1alpha-hydroxylation in the kidney with 25-hydroxyvitamin D-1alpha-hydroxylase. Cultured transformed colon cancer cells can convert 25-hydroxyvitamin D(3) to 1alpha,25-dihydroxyvitamin D(3). We identified messenger RNA (mRNA) for 25-hydroxyvitamin D-1alpha-hydroxylase in normal colon tissue and in malignant and adjacent normal colon tissue. These findings support the notion that vitamin D might have a role in cell growth regulation and cancer protection, and might be the explanation for why the risk of dying from colorectal cancer is highest in areas with the least amount of sunlight.