Production of F cells in sickle cell anemia: regulation by a genetic locus or loci separate from the beta-globin gene cluster

Levels of fetal hemoglobin (HbF) bearing reticulocytes (F reticulocytes) range from 2% to 50% in patients with sickle cell (SS) anemia. To learn whether any portion of such variation in F cell production is regulated by loci genetically separable from the beta- globin gene cluster, percentages of F reticulocytes were compared in 59 sib pairs composed solely of SS members, including 40 pairs from Jamaica and 19 from the United States. We reasoned that differences in F reticulocyte levels might arise (1) from any of several kinds of artifact, (2) via half-sib status, or (3) because one or more genes regulating F cell production segregate separately from beta S. We minimized the role of artifact by assay of fresh samples from 84 SS individuals, including both members of 38 sib pairs. In 78 of the 84 subjects, serial values for percent F reticulocytes fell within 99.9% confidence limits or were alike by t test (P greater than or equal to .05). This left 32 sib pairs for which F reticulocyte levels in each member were reproducible. When sib-sib comparisons were limited to these 32 pairs, percentages of F reticulocytes were grossly dissimilar within 12 Jamaican and 3 American sibships. Within them, the probability that sibs were alike was always less than or equal to .005 and usually less than or equal to 10(-4). We next minimized the contribution of half-sibs among Jamaicans by a combination of paternity testing and sib-sib comparison of beta-globin region DNA restriction fragment length polymorphisms, especially among discordant pairs. We thereafter concluded that at least seven to eight Jamaican pairs were composed of reproducibly discordant full sibs. There is thus little doubt that there are genes regulating between-patient differences in F cell production that are separate from the beta-globin gene cluster. Still unanswered is (1) whether or not these genes are actually linked to beta S, (2) why F reticulocyte levels in Americans tend to be lower than in Jamaicans, and (3) whether or not differences in F cell production among SS patients are regulated by several major loci or by only one.     

Transport of free and conjugated steroids from the boar testis in lymph, venous blood and rete testis fluid

In 12 anaesthetized boars the concentrations of oestrone sulphate and dehydroepiandrosterone sulphate (DHAS) were 15- to 35-fold higher in lymph collected from a vessel in the spermatic cord than in testicular venous blood plasma from a vein in the spermatic cord. The concentrations of testosterone, total unconjugated oestrogens and dehydroepiandrosterone (DHA) were about twofold higher in lymph. The concentrations of all steroids studied were higher in testicular venous blood plasma than in arterial blood plasma (testosterone about sixfold; total unconjugated oestrogens about fourfold; oestrone sulphate about threefold; DHA and DHAS about twofold), but the concentrations of testosterone, total unconjugated oestrogens and oestrone sulphate in rete testis fluid were comparable to those in arterial blood plasma. Lymph flow from the pig testis was about 7% of plasma flow so that about 80% of the oestrone sulphate and DHAS produced by the testis leaves the organ in the lymph; the comparable values for testosterone, total unconjugated oestrogen and DHA were about 20%. In the 90-min period following an injection of human chorionic gonadotrophin there were substantial increases in the concentration of testosterone and smaller increases in the other steroids in arterial and spermatic venous blood plasma and in testicular lymph, but not in rete testis fluid; there were also small increases in lymph flow, but no change in blood flow.     

The membrane protein ATPase class I type 8B member 1 signals through protein kinase C zeta to activate the farnesoid X receptor

Prior loss-of-function analyses revealed that ATPase class I type 8B member 1 [familial intrahepatic cholestasis 1 (FIC1)] posttranslationally activated the farnesoid X receptor (FXR). Mechanisms underlying this regulation were examined by gain-of-function studies in UPS cells, which lack endogenous FIC1 expression. FXR function was assayed in response to wild-type and mutated FIC1 expression constructs with a human bile salt export pump (BSEP) promoter and a variety of cellular localization techniques. FIC1 overexpression led to enhanced phosphorylation and nuclear localization of FXR that was associated with FXR-dependent activation of the BSEP promoter. The FIC1 effect was lost after mutation of the FXR response element in the BSEP promoter. Despite similar levels of FIC1 protein expression, Byler disease FIC1 mutants did not activate BSEP, whereas benign recurrent intrahepatic cholestasis mutants partially activated BSEP. The FIC1 effect was dependent on the presence of the FXR ligand, chenodeoxycholic acid. The effect of FIC1 on FXR phosphorylation and nuclear localization and its effects on BSEP promoter activity could be blocked with protein kinase C zeta (PKC zeta) inhibitors (pseudosubstrate or small interfering RNA silencing). Recombinant PKC zeta directly phosphorylated immunoprecipitated FXR. The mutation of threonine 442 of FXR to alanine yielded a dominant negative protein, whereas the phosphomimetic conversion to glutamate resulted in FXR with enhanced activity and nuclear localization. Inhibition of PKC zeta in Caco-2 cells resulted in activation of the human apical sodium-dependent bile acid transporter promoter. Conclusion: These results demonstrate that FIC1 signals to FXR via PKC zeta. FIC1-related liver disease is likely related to downstream effects of FXR on bile acid homeostasis. Benign recurrent intrahepatic cholestasis emanates from a partially functional FIC1 protein. Phosphorylation of FXR is an important mechanism for regulating its activity.     

The characterization of polar corticosteroids in the urine of the macaque monkey (macaca fascicularis) and the baboon (papio hamadryas).

Computerised gas chromatography-mass spectrometry was employed in the identification of polar corticosteroid metabolites excreted in the urine from the macaque monkey (Macaca fascicularis) and the baboon (Papio hamadryas). The following steroids were identified in significant amounts in the urine from both species: 3alpha,17alpha,20alpha, 21-tetrahydroxy-5beta-pregnan-11-one; 3alpha,17alpha,20beta,21-tetrahydroxy-5beta-pregnan-11-one; 5beta-pregnane-3alpha,11beta,17alpha,20alpha,21-pentol; 5beta-pregnane-3alpha,11beta,17alpha,20beta-pentol; 5alpha-pregnane-3beta,11beta,17alpha,20beta,21-pentol. 11beta,17alpha,21-Trihydroxy-4-pregnene-3,20-dione (cortisol), 11beta,17alpha,20beta,21-tetrahydroxy-4-pregnen-3-one and 11beta,17alpha,20beta,21-tetrahydroxy-5xi-pregnan-3-one were identified in macaque monkey urine. Two steroids, 17alpha,20beta,21-trihydroxy-4-pregnane-3,11-dione and 17alpha,20alpha,21-trihydroxy-4-pregnene-3,11-dione were excreted as major C21 metabolites in the baboon but were not identified in the urine from the macaque monkey. 3beta-Hydroxy-5alpha-pregnane metabolites were identified in the urine from both species. All these steroids were excreted conjugated to glucuronic acid, evidenced by their recovery after hydrolysis with beta-glucuronidase enzyme. An efficient 20beta-reduction of corticosteroids in both species is apparent, and the excretion pattern of polar steroid metabolites in the two species was shown to be similar.     

Possible interaction of genotypes at cystathionine β-synthase and methylenetetrahydrofolate reductase (MTHFR) in neural tube defects

Neural tube defects are a common, complex disorder with genetic and environmental components to risk. We investigated the previously reported interaction between homozygosity for the thermolabile variant at the methylenetetrahydrofolate reductase and heterozygosity for the 844ins68 allele at the cystathionine beta-synthase loci in cases with lumbosacral myelomeningocele and their parents. Using control allele frequencies from our sample pooled with those published in the literature, we confirm a marginally significant interaction at these two loci. This finding suggests that additional, larger studies are warranted to investigate this possible interaction in more detail.     

Whole-body heat exposure induces membrane changes in spermatozoa from the cauda epididymidis of laboratory mice

This study was carried out to determine if exposure to hot environmental temperatures had a direct, detrimental effect on sperm quality. For this the effect of whole-body heat exposure on epididymal spermatozoa of laboratory mice was investigated. C57BL/6 mice （n = 7） were housed in a microclimate chamber at 37℃-38℃ for 8 h per day for three consecutive days, while control mice （n = 7） were kept at 23℃-24℃. Cauda epididymal spermatozoa were obtained 16 h after the last heat treatment. The results showed that sperm numbers were similar in the two groups （P = 0.23）, but after heat treatment, a significant reduction in the percentage of motile sperm was present （P 〈 0.0001）. Membrane changes of the spermatozoa were investigated by staining with phycoerythrin （PE）- conjugated Annexin V, which detects exteriorization of phosphotidylserine from the inner to the outer leaflet of the sperm plasma membrane, and 7-aminoactinomycin D （7-AAD）, which binds to the sperm nucleus when the plasma membrane is damaged. The percentage of spermatozoa showing positive staining with Annexin V-PE or 7-AAD or both, was significantly higher （P 〈 0.05） in heat-exposed mice compared with controls. These results show that whole-body heat exposure to 37℃-38℃ induces membrane changes in the epididymal spermatozoa of mice, which may lead to apoptosis.