Localization of the β-globin gene to 11p15 by in situ hybridization: Utilization of chromosome 11 rearrangements

Summary Chromosome preparations from four subjects, one normal 46,XY male and three patients with different rearrangements of chromosome 11:46,XX,del(11)(p11.2p15.1), 46,XY,inv(11)(p13q24.2), and 46,XY,rec(11)inv(11)(p13q24.2) pat, were utilized for in situ hybridization studies with a tritium-labeled cDNA probe containing a -globin insert. Using the hybridization technique described by Harper and Saunders (1981), there were 1–2 grains over each labeled metaphase. Of 360 cells scored, 88 were labeled over chromosome 11, band p15 (24%). Approximately half of the chromosome 11s labeled from the abnormal patients were the del(11) or inv(11). These results exclude the -globin locus from 11p11p14, since these bands were not present in the recent 11, and assign it to 11p15. This is in agreement with the recent exclusion data of de Martiville and Francke (1984) and Junien (1984), and suggestive assignment data of Morton et al. (1984).     

Production of 11α-hydroxysteroids from sterols in a single fermentation step by Mycolicibacterium smegmatis

11α-hydroxylated steroid synthons are one of the most important commercially pharmaceutical intermediates used for the production of contraceptive drugs and glucocorticoids. These compounds are currently produced by biotransformation using fungal strains in two sequential fermentation steps. In this work, we have developed by a rational design new recombinant bacteria able to produce 11α-hydroxylated synthons in a single fermentation step using cholesterol (CHO) or phytosterols (PHYTO) as feedstock. We have designed a synthetic operon expressing the 11α-hydroxylating enzymes from the fungus Rhizopus oryzae that was cloned into engineered mutant strains of Mycolicibacterium smegmatis that were previously created to produce 4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD) from sterols. The introduction of the fungal synthetic operon in these modified bacterial chassis has allowed producing for the first time 11αOH-AD and 11αOH-ADD with high yields directly from sterols in a single fermentation step. Remarkably, the enzymes of sterol catabolic pathway from M. smegmatis recognized the 11α-hydroxylated intermediates as alternative substrates and were able to efficiently funnel sterols to the desired hydroxylated end-products.     

Deficiencies of the müllerian ducts induced by norethindrone in the female chick embryo

Norethindrone produces two effects on müllerian ducts (MD) of female chick embryos. It induces the loss of the lower end of both ducts, as a result of a stop in their development, before 8 days. After 12 days NET causes regressions of the upper part of the MD particularly of the oviduct. NET like estrogens are the only known substances which present these both properties.     

β-Glucanase specific expression in the parotid gland of transgenic mice

The feasibility of using the pig parotid secretory protein promoter to drive the β-glucanase transgene expression in mouse parotid glands was examined in this study. The parotid gland-specific vector expressing β-glucanase gene (GLU, from Paenibacillus polymyxa CP7) was constructed. Transgenic mice were produced by the pronuclear microinjection. Both PCR and Southern blot analysis showed that the mice carried the β-glucanase gene and the β-glucanase gene could be stably inherited. Furthermore, RT-PCR and northern blot analysis indicated that it was specifically expressed in the parotid. The β-glucanase activity in the saliva was found to be 0.18 U/mL. After feeding a diet containing 2 % β-glucan, the average daily gain of transgenic was significantly higher than non-transgenic mice. The crude protein and crude fat concentration in faeces of transgenic mice were significantly reduced compared with that of the non-transgenic mice. These results suggest that the successful expression of foreign β-glucanase in the animal parotid would offer a promising biological approach to reduce the anti-nutritional effect of β-glucans in feed.     

Prostaglandin F2α as a potent excitant of the parasympathetic postganglionic neurons of the dog salivary gland

Prostaglandin F_2α (PGF_2α) (1–100 ng) and acetylcholine (ACh) (0.3–30 μg) injected selectively into the artery supplying the submaxillary gland of the dog produced salivation and an increase in blood flow. Both salivary and vascular responses to PGF_2α developed slowly and lasted long as compared with those to ACh. On a weight basis PGF_2α was about 1000 times more potent than ACh in producing salivation. Upon repeated injections of PGF_2α most glands developed moderate desensitization to PGF_2α but not to ACh. Both salivary and vascular responses to PGF_2α were abolished by infusion of tetrodotoxin ( or 0.1 or 0.2 μg/min), whereas those to ACh remained virtually unchanged. These results indicate that in the dog submaxillary gland PGF_2α causes salivation and vasodilation exclusively through excitation of the parasympathetic postganglionic neurons.     

Studies of 11β-hydroxylation by beef adrenal mitochondria

The 11β-hydroxylations of androstenedione (Δ⁴A), 11-deoxycortisol (S) and deoxycorticosterone (DOC) were studied using mitochondria from calf or heifer adrenal tissue. Standard assay conditions were: non-radioactive androstenedione (30.0μM) 11-deoxycortisol (24.5 μM) or deoxycorticosterone (26.0 μM) plus 6.0 × 10⁴ d.p.m. ¹⁴C-steroid, 0.2 mM NADPH, 1.0 mM Mg²⁺ 1.0 mM Ca²⁺ and the mitochondrial fraction equivalent to 20 mg of adrenal tissue in a final vol. of 3ml of 0.1 M HEPES buffer. pH 7.4. Incubations were performed at 37°C for 4min. Product formation under these conditions was identical to product formation measured when the NADPH and Ca²⁺ were replaced with 10mM malate. The 11β-hydroxylation of Δ⁴A showed a requirement for NADPH and oxygen, indicating that the enzyme involved is a mixed-function oxidase. The K_m values for calf adrenal mitochondria were 3.8, 8.5 and 8.0μM for Δ⁴A, S and DOC, respectively. For heifer adrenal mitochondria, the K_m values were 12 and 15μM respectively, for Δ⁴A and S. Competition studies in which equal amounts of two substrates were incubated simultaneously, revealed that Δ⁴A, S and DOC did not compete for the same enzymatic site, but were hydroxylated to the same degree in the presence or absence of each of the other two precursors. The 11β-hydroxylations of S and DOC were stimulated by Mg²⁺ at a concentration of 1.0 mM, while the 11β-hydroxylation of Δ⁴A was inhibited by this concentration of Mg²⁺. In experiments in which the mitochondria were preheated at 50°C for 6 min, the 11β-hydroxylation of Δ⁴A, under standard assay conditions, was 96% of the unheated value, while the 11β-hydroxylation of S and DOC was 77 and 59%, respectively, of the unheated values. These studies indicate that there are three substrate specific 11β-hydroxylases in beef adrenal mitochondria.     

The effect of zinc on the 5α-reduction of testosterone by the hyperplastic human prostate gland

The present studies were performed to evaluate the role of zinc in the regulation of testosterone 5α-reduction by the 800 g supematants prepared from human benign prostate hyperplasia specimens. The results show that when zinc is added at low concentrations the 5α-reduction of testosterone is increased but at higher cation concentrations the metabolism is significantly inhibited. This decrease was mediated by both a non-competitive inhibition of the binding of testosterone to the 5α-reductase enzyme and by a reduction in the formation of the NADPH cofactor. We have also demonstrated that the decreased synthesis of NADPH was produced by a competitive inhibition of both G6P and NADP binding to the G6PD enzyme. The data also suggests that the increase in testosterone metabolism observed at low zinc concentrations does not produce any changes in the binding of testosterone to the 5α-reductase enzyme. In spite of the above observations we were unable to establish any correlation between the endogenous zinc content of the tissue and the the in vitro capacity of the BPH samples to 5α-reduce testosterone. The present study suggests a possible physiological role for the regulation of testosterone metabolism by zinc in the human prostate gland.     

The apical Na+–HCO3− cotransporter Slc4a7 (NBCn1) does not contribute to bicarbonate transport by mouse salivary gland ducts

The HCO₃ ⁻ secretion mechanism in salivary glands is unclear but is thought to rely on the co-ordinated activity of multiple ion transport proteins including members of the Slc4 family of bicarbonate transporters. Slc4a7 was immunolocalized to the apical membrane of mouse submandibular duct cells. In contrast, Slc4a7 was not detected in acinar cells, and correspondingly, Slc4a7 disruption did not affect fluid secretion in response to cholinergic or β-adrenergic stimulation in the submandibular gland (SMG). Much of the Na ⁺-dependent intracellular pH (pH _i) regulation in SMG duct cells was insensitive to 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, S0859, and to the removal of extracellular HCO ₃ ⁻. Consistent with these latter observations, the Slc4a7 null mutation had no impact on HCO ₃ ⁻ secretion nor on pH _i regulation in duct cells. Taken together, our results revealed that Slc4a7 targets to the apical membrane of mouse SMG duct cells where it contributes little if any to pH _i regulation or stimulated HCO ₃ ⁻ secretion.     

Intercellular junctions in the hypodermis,salivary gland and gené's organ of the cattle tick,Boophilus microplus

Summary The intercellular junctions that occur in the hypodermis, Gené's organ, and the salivary glands of the tick, B. microplus, are described. The epithelial cells of the hypodermis are connected by spot desmosomes and septate junctions and the secretory cells of Gené's organ by septate and gap junctions. The cap cells in the alveoli of the salivary gland connect to adjacent cells by gap junctions, hemidesmosomes and septate junctions into which microtubules are inserted.The authors would like to thank Mr. R. Lamb for preparing the plates. M.W.J. Megaw was supported by an S.R.C. Studentship     

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Patients with Sjögren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (Vλ2E) and variable kappa chain genes (VκA27). Moreover, clonally related V_L chain rearrangements were identified; namely, VκA27–Jκ5 and VκA19–Jκ2 in the parotid gland, and Vλ1C–Jλ3 in the parotid gland and the peripheral blood. Vκ and Vλ rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V_L chain genes caused by selection and clonal expansion of B cells expressing particular V_L genes. In addition, the data document an accumulation of B cells bearing mutated V_L gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.     

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Patients with Sj?gren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunoglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunoglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (V lambda 2E) and variable kappa chain genes (V kappa A27). Moreover, clonally related V(L) chain rearrangements were identified; namely, V kappa A27-J kappa 5 and V kappa A19-J kappa 2 in the parotid gland, and V lambda 1C-J lambda 3 in the parotid gland and the peripheral blood. V kappa and V lambda rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V(L) chain genes caused by selection and clonal expansion of B cells expressing particular V(L) genes. In addition, the data document an accumulation of B cells bearing mutated V(L) gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.     

The incorporation of acetate,stearate and d(?)-β-hydroxybutyrate into milk fat by the isolated perfused mammary gland of the goat

1. Mammary glands of lactating goats were perfused for 12.5-15hr. with heparinized whole blood and infused with a substrate mixture of glucose, acetate and amino acids (and sometimes chylomicra) containing either [1-(14)C]acetate, d(-)-beta-hydroxy[1-(14)C]butyrate or [U-(14)C]stearate. 2. There was a substantial net uptake of acetate by the glands and transfer of radioactivity into milk fat. Acetate was extensively utilized for the synthesis of milk fatty acids of chain length up to C(14) and to a smaller extent for the synthesis of palmitate. 3. There was a small and variable net uptake of stearate and beta-hydroxybutyrate and negligible oxidation of these substrates. However, tissue uptake was demonstrated by a substantial fall in specific radioactivity across the glands and an extensive transfer of radioactivity into milk fatty acids. 4. With beta-hydroxybutyrate the labelling of milk fat was very similar to that with acetate, but the distribution of radioactivity suggested a cleavage into C(2) fragments of about 40%. 5. Labelled stearate gave rise to highly labelled stearate and oleate in the milk fat. Small amounts of radioactivity were detected in stearate of plasma triglycerides and oleate of plasma free fatty acids. 6. In experiments where there was a decline in milk-fat secretion late in perfusion, the milk fatty acids showed a marked decline in the proportion of stearate and oleate and a rise in the proportion of myristate and palmitate. This did not occur in experiments where milk-fat secretion was maintained at a higher level. 7. The present results confirm that there is a large pool of long-chain fatty acids in mammary tissue that can act as an endogenous source of these substrates.     

An annotated catalogue of salivary gland transcripts in the adult female mosquito, Ædes ægypti*

Background

The number of completely sequenced plastid genomes available is growing rapidly. This array of sequences presents new opportunities to perform comParative analyses. In comParative studies, it is often useful to compare across wide phylogenetic spans and, within angiosperms, to include representatives from basally diverging lineages such as the genomes reported here: Nuphar advena (from a basal-most lineage) and Ranunculus macranthus (a basal eudicot). We report these two new plastid genome sequences and make comparisons (within angiosperms, seed plants, or all photosynthetic lineages) to evaluate features such as the status of ycf15 and ycf68 as protein coding genes, the distribution of simple sequence repeats (SSRs) and longer dispersed repeats (SDR), and patterns of nucleotide composition.

Results

The Nuphar [GenBank:NC_008788] and Ranunculus [GenBank:NC_008796] plastid genomes share characteristics of gene content and organization with many other chloroplast genomes. Like other plastid genomes, these genomes are A+T-rich, except for rRNA and tRNA genes. Detailed comparisons of Nuphar with Nymphaea, another Nymphaeaceae, show that more than two-thirds of these genomes exhibit at least 95% sequence identity and that most SSRs are shared. In broader comparisons, SSRs vary among genomes in s of abundance and length and most contain repeat motifs based on A and T nucleotides.

Conclusion

SSR and SDR abundance varies by genome and, for SSRs, is proportional to genome size. Long SDRs are rare in the genomes assessed. SSRs occur less frequently than predicted and, although the majority of the repeat motifs do include A and T nucleotides, the A+T bias in SSRs is less than that predicted from the underlying genomic nucleotide composition. In codon usage third positions show an A+T bias, however variation in codon usage does not correlate with differences in A+T-richness. Thus, although plastome nucleotide composition shows "A+T richness", an A+T bias is not apparent upon more in-depth analysis, at least in these aspects. The pattern of evolution in the sequences identified as ycf15 and ycf68 is not consistent with them being protein-coding genes. In fact, these regions show no evidence of sequence conservation beyond what is normal for non-coding regions of the IR.     

Sox9+ cells are required for salivary gland regeneration after radiation damage via the Wnt/β-catenin pathway

Radiotherapy for head and neck cancer can cause serious side effects, including severe damage to the salivary glands, resulting in symptoms such as xerostomia, dental caries, and oral infection. Because of the lack of long-term treatment for the symptoms of xerostomia, current research has focused on finding endogenous stem cells that can differentiate into various cell lineages to replace lost tissues and restore functions. Here, we report that Sox9⁺ cells can differentiate into various salivary epithelial cell lineages under homeostatic conditions. After ablating Sox9⁺ cells, the salivary glands of irradiated mice showed more severe phenotypes and the reduced proliferative capacity. Analysis of online single-cell RNA-sequencing data reveals the enrichment of the Wnt/β-catenin pathway in the Sox9⁺ cell population. Furthermore, treatment with a Wnt/β-catenin inhibitor in irradiated mice inhibits the regenerative capability of Sox9⁺ cells. Finally, we show that Sox9⁺ cells are capable of forming organoids in vitro and that transplanting these organoids into salivary glands after radiation partially restored salivary gland functions. These results suggest that regenerative therapy targeting Sox9⁺ cells is a promising approach to treat radiation-induced salivary gland injury.     

Regeneration of the progesterone 11α-hydroxylase of Rhizopus nigricans by the use of periodate

Summary The cell-free progesterone 11-hydroxylase enzyme of Rhizopus nigricans can be directly regenerated by periodate oxidation. This permits action of the enzyme over a period of hours with an activity similar to that in the presence of an NADPH generating system.     

Modulation of α2β1 integrin changes during mammary gland development by β-oestradiol

In order to study the role of cell–matrix interactions in mammary gland function, temporal changes in α₂β₁ integrin, the major receptor for collagen and the influence of β-oestradiol on its level and distribution in rat mammary gland at different stages of development were studied. The level of α₂β₁ integrin determined by ELISA, was found to be high during different days of pregnancy, while in the lactating stage, it was significantly reduced. By immunocytochemical analysis, α₂β₁ integrin was found to be localized towards the luminal side of acinar cells, both in the virgin and midpregnant stage, while it was not detected in the lactating stage. The possible role of hormones in modulating the level of integrin was examined in both in vitro and in vivo experiments using β-oestradiol. Supplementing β-oestradiol to isolated mammary epithelial cells from both virgin and lactating glands caused a concentration dependent increase in the incorporation of [³⁵S]methionine into α₂β₁ integrin associated with the cells. Administration of β-oestradiol to virgin and lactating glands caused about 1.4–4-fold increase in the level of α₂ integrin, indicating that upregulation of integrin during pregnancy may be due to oestrogen and as the oestrogen level falls during lactating phase, downregulation of α₂β₁ integrin occurs. Treatment with β-oestradiol also resulted in the appearance of α₂β₁ integrin in the acinar region of the lactating tissue, while in the untreated controls no staining for integrin was seen. These results indicate that oestrogen, apart from directly affecting the cellular activity, can influence mammary tissue function by affecting cell–ECM interactions through the modulation of integrin receptors for matrix proteins.     

Impaired oxidoreduction by 11β-hydroxysteroid dehydrogenase 1 results in the accumulation of 7-oxolithocholic acid

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) mediates glucocorticoid activation and is currently considered as therapeutic target to treat metabolic diseases; however, biomarkers to assess its activity in vivo are still lacking. Recent in vitro experiments suggested that human 11β-HSD1 metabolizes the secondary bile acid 7-oxolithocholic acid (7-oxoLCA) to chenodeoxycholic acid (CDCA) and minor amounts of ursodeoxycholic acid (UDCA). Here, we provide evidence from in vitro and in vivo studies for a major role of 11β-HSD1 in the oxidoreduction of 7-oxoLCA and compare its level and metabolism in several species. Hepatic microsomes from liver-specific 11β-HSD1-deficient mice were devoid of 7-oxoLCA oxidoreductase activity. Importantly, circulating and intrahepatic levels of 7-oxoLCA and its taurine conjugate were significantly elevated in mouse models of 11β-HSD1 deficiency. Moreover, comparative enzymology of 11β-HSD1-dependent oxidoreduction of 7-oxoLCA revealed that the guinea-pig enzyme is devoid of 7-oxoLCA oxidoreductase activity. Unlike in other species, 7-oxoLCA and its glycine conjugate are major bile acids in guinea-pigs. In conclusion, the oxidoreduction of 7-oxoLCA and its conjugated metabolites are catalyzed by 11β-HSD1, and the lack of this activity leads to the accumulation of these bile acids in guinea-pigs and 11β-HSD1-deficient mice. Thus, 7-oxoLCA and its conjugates may serve as biomarkers of impaired 11β-HSD1 activity.