Madan L. Nagpal

Utility of 16S–23S rRNA spacer region methodology: how similar are interspace regions within a genome and between strains for closely related organisms?

Abstract Use of 16S–23S interspace region sequence variability, as a relatively new method, is becoming an important supplement to 16S rRNA sequencing as the standard for differentiating bacterial species. If interspace regions are as variable within a genome as between strains for closely related organisms, this limits the utility of the technique. Strains W23 and 168 represent two distinct genetic clusters within the species Bacillus subtilis . B. atrophaeus var. niger was selected as a member of a group of species closely related to B. subtilis . Comparison of the 10 rDNA operons, available from Genbank, for B. subtilis 168 shows three distinct types of interspace (ISR) regions. Two of the ten 16S–23S ISRs contain the sequences for isoleucine and alanine tRNA and are identical in sequence. The remaining eight ISRs lack tRNA sequences and have two distinct sizes. Variability among non-tRNA operons ranged from 97–100%. Counting the tRNA insert as one change, variability between tRNA and non-tRNA containing sequences ranged from 95.3–97%. The sequences of equivalent 16S–23S ribosomal operon interspace regions (ISRs) are highly conserved between W23 and 168 (99.9–100%). Thus the sequence differences between strains 168 and W23 are less than between multiple operons within 168. However, the sequence of an ISR from B. atrophaeus var. niger is quite distinct from any of the ISRs found in B. subtilis (range 88.2–91.6%). These observations are consistent with the previous suggestion that B . atrophaeus is distinct genetically from the B . subtilis sub-groups represented by W23 and 168 respectively. This is the first study to make sequence comparisons at the genome, strain and species level for the rRNA interspace region. Considerations of this issue will be important in using ISR methodology to differentiate other closely related bacterial species.

Restriction fragment length polymorphism of rRNA operons for discrimination and intergenic spacer sequences for cataloging of Bacillus subtilis sub-groups

Restriction fragment length polymorphism of rRNA operons (RFLP) and 16S-23S rRNA intergenic region (ISR) sequences of Bacillus subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. atrophaeus were compared. ISR sequences of the B. subtilis subspecies were extremely similar (W23 versus 168 rrn H, J, G,W; 96.8%; rrn D, E; 98.4%; rrnB; 97.9%) and, therefore, not useful for their differentiation. However, RFLP of rRNA operons of the B. subtilis subspecies were distinct in terms of numbers and organization within the genome (e.g. the 168 sub-group generally contained 8.3- and 8.0-kb fragments absent in the W23 sub-group). The more distantly related B. atrophaeus was distinct from both B. subtilis subspecies in terms of ISR sequence and rRNA operon number and organization. RFLP of rRNA operons discriminates the two sub-groups of Bacillus subtilis that are indistinguishable by ISR sequence. However, ISR sequence defines the relatedness of B. subtilis to other species (e.g. B. atrophaeus) within the genus Bacillus.

Effects of genistein, resveratrol, and quercetin on steroidogenesis and proliferation of MA-10 mouse Leydig tumor cells.

This study was performed to compare the effects of three well-known phytoestrogens such as genistein, resveratrol, and quercetin on steroidogenesis in MA-10 mouse tumor Leydig cells. Addition of genistein or resveratrol to MA-10 cells resulted in decreases in the cAMP-stimulated progesterone secretion, but quercetin had an opposite response. Steroidogenic acute regulatory (StAR) mRNA expression and StAR promoter activity in transiently transfected MA-10 cells were significantly reduced by genistein or resveratrol, but increased by quercetin. Genistein was found to inhibit MA-10 cell proliferation, while resveratrol and quercetin had no effect. Quercetin-induced increase in cAMP-stimulated progesterone secretion was reversed by ICI 182,780, an estrogen receptor (ER) antagonist. However, ICI 182,780 had no effect on cAMP plus quercetin-stimulated StAR promoter activity. To examine whether non-ER factors are associated with quercetin-stimulated progesterone production, we treated MA-10 cells with EGTA to deprive them of extracellular Ca(2+). We found that EGTA inhibited quercetin-plus cAMP-stimulated progesterone secretion and StAR promoter activity. Blocking of Ca(2+) influx through L- or T-type voltage-gated Ca(2+) channels with verapamil or mibefradil respectively, attenuated quercetin-stimulated progesterone secretion, while they had no effect on quercetin-plus cAMP-stimulated StAR promoter activity. Blocking of intracellular Ca(2+) efflux by sodium orthovanadate, a Ca(2+)-pump inhibitor, blocked quercetin- plus cAMP-stimulated progesterone secretion and StAR promoter activity in MA-10 cells. Finally, EGTA or vanadate reduced quercetin and cAMP-increased in StAR mRNA expression in MA-10 cells, while ICI 182,780 had no effect. Taken together, these results indicate that phytoestrogens have differential effects on steroidogenesis in MA-10 cells.

Human chorionic gonadotropin induces interleukin-1 gene expression in rat Ley dig cells in vivo

Abstract Luteinizing hormone (LH)/human chorionic gonadotropin (hCG) causes inflammatory-type responses in the testis. In the present study, we evaluated the effects of hCG on Leydig cell interleukin-1 (IL-1) gene expression. Using monoclonal antibody (ED2) staining for macrophages, our Leydig cell preparations had no significant contamination with macrophages. When purified Leydig cells from normal rats were cultured for 24 h and then treated with IL-1β (1–100 ng/ml) for 6 h, IL-1β induced dose-dependent increases in IL-1β mRNA levels. IL-1β also induced IL-lα mRNA accumulation; however, the level of IL-1α mRNA was much lower than that of IL-1β mRNA. When rats were treated with either saline or hCG (5 units i.p.), hCG markedly induced IL-1β mRNA accumulation in purified Leydig cells at 6 h which persisted for over 24 h. However, hCG had no direct effect on purified Leydig cell or crude interstitial cell IL-1 mRNA levels. Our results suggest that inflammatory effects of hCG in vivo may be mediated by increased IL-1 gene expression in Leydig cells.

Recombinant murine tumor necrosis factor-alpha inhibits cholesterol side-chain cleavage cytochrome P450 and insulin-like growth factor-I gene expression in rat Leydig cells

The purpose of the present study was to evaluate the effects of murine recombinant tumor necrosis factor-alpha (TNF-alpha) on rat Leydig cell function. In primary cultures of Leydig cells, we found that in the presence of hCG (10 ng/ml), testosterone levels were markedly elevated, 69.3 +/- 3.1 ng/10(6) cells/h (mean + SE). TNF-alpha in a concentration of 1 ng/ml markedly inhibited testosterone biosynthesis (a 69% reduction; p < 0.01) and 100 ng/ml of TNF-alpha almost completely inhibited testosterone formation (p < 0.001). TNF-alpha (10 ng/ml) inhibited hCG (0.1, 1 and 10 ng/ml)-induced testosterone formation by 63%, 67% and 61%, respectively. TNF-alpha (10 ng/ml) also markedly inhibited 8-bromo cAMP-induced testosterone formation from 76 +/- 9 ng/10(6) cells/h to 4.9 ng/10(6) cells/h. This indicates that the major effect of TNF-alpha is at steps beyond LH receptor site. To further evaluate the site(s) of action of TNF-alpha, we evaluated its effect on the conversion of precursor steroids to testosterone. We found that the addition of 20-hydroxy-cholesterol could not reverse inhibitory effects of TNF-alpha on hCG-induced testosterone formation. TNF-alpha had no effect on the conversions of pregnenolone, 17-OH-pregnenolone, DHEA and androstenedione to testosterone. This indicates that the major effect of TNF-alpha is at the key steroidogenic enzyme, P450scc. We reported previously that human recombinant TNF-alpha had no effect on hCG-induced testosterone formation but did enhance the inhibitory effects of human recombinant IL-1beta. In the present study, we demonstrated that both murine TNF-alpha and human IL-1beta were potent inhibitors of hCG-induced testosterone formation. IL-1beta alone in concentrations of 0.1, 1 and 10 ng/ml inhibited testosterone formation by 45%, 62% and 91%, respectively, in the presence of TNF-alpha (10 ng/ml), IL-1beta in a concentration as low as 0.1 ng/ml completely blocked hCG-induced testosterone formation. We next evaluated the effect of TNF-alpha on P450scc gene expression. There was no constitutively expressed P450scc mRNA in Leydig cells after 24 h in culture. In response to hCG, there was a 33-fold increase in the P450scc mRNA level. Both TNF-alpha and IL-1beta inhibited hCG-induced expression of P450scc mRNA. Finally, the effect of TNF-alpha on IGF-I gene expression was investigated since IGF-I enhances Leydig cell androgen formation and IGF-I gene is expressed in high levels in Leydig cells. TNF-alpha inhibited both large (7.4 kb) and small species (0.8-1.2 kb) IGF-I mRNA levels in a dose-dependent manner. In conclusion, murine TNF-alpha is a potent inhibitor of Leydig cell function. TNF-alpha inhibited both P450scc and IGF-I mRNA gene expression.

Variation in 16S-23S rRNA intergenic spacer regions among Bacillus subtilis 168 isolates.

The genome of the Bacillus subtilis 168‐type strain contains 10 ribosomal RNA (rRNA) operons. In the intergenic spacer region (ISR) between the 16S and 23S rRNA genes, five rRNA operons, rrnI‐H‐G and rrnJ‐W, lack a trinucleotide signature region. Precise determination of molecular weight (MW), using electrospray mass spectrometry (MS), of the polymerase chain reaction (PCR) products from a segment of the ISR from the 168‐type strain and B. subtilis 168‐like strain 23071 demonstrated 114 and 111 basepair (bp) PCR products (due to the presence or absence of the insert in the operons) as predicted from sequence. However, PCR of the ISR segment for five other B. subtilis 168 isolates generated only a 114 bp PCR product, suggesting the presence of the trinucleotide signature region in all rRNA operons for these strains. Additional genetic variability between the seven B. subtilis 168 isolates was demonstrated by restriction fragment length polymorphism (RFLP) of the rRNA operons, with three distinct patterns found upon Southern blot analysis. The 168‐type strain and three others (23066, 23067, and 23071) exhibited the same Southern pattern. Thus, operon deletion is not responsible for the absence of a 111 bp product on MS analysis for strains 23066 and 23067. Restriction analysis confirmed the presence of the trinucleotide signature region in the ISR of all rRNA operons for five B. subtilis 168 isolates; sequencing of rrnW/H from a representative strain also upheld this finding. These results help provide a better understanding of variations in sequence, operon number and chromosomal organization, both within a genome and among isolates of B. subtilis subgroup 168. It is also hypothesized that the presence of the trinucleotide insert in certain rRNA operons may play a role in rRNA maturation and protein synthesis.

Characterization of a continuous smooth muscle cell line derived from rabbit aorta

SummaryA spontaneously arising continuous cell line (Rb-1) derived from collagenase-elastase digested rabbit aorta has been propagated in vitro for over 100 passages. During this period, the Rb-1 cells remained spindle-shaped and formed regularly oriented parallel bundles. After Passage 50, Rb-1 cells were found to the serum-independent in their growth and reached higher saturation density than rabbit aorta smooth muscle cells. Alpha-actin and desmin filaments were detected by immunostaining in Rb-1 cells and early passage of rabbit aorta smooth muscle cells. The proportion of alpha-actin transcripts in Rb-1 cells was lower than that of transcripts for beta- and gamma-actins. The modal chromosome number was maintained at 44 between Passages 11 and 60, and two marker chromosomes were constantly present Infection of Rb-1 cells with two strains of herpes simples virus type 1 resulted in high titers of virus, whereas a herpes simplex virus type 2 temperature-sensitive mutant replication only at the permissive temperature. The Rb-1 cell line could be used for the study of vascular smooth muscle cell proliferation and their interaction with viruses.

Expression and Regulation of Interferon-γ-Inducible Protein 10 Gene in Rat Leydig Cells1

In the present study, we report the cloning of a gene that is differentially expressed in normal adult rat Leydig cells and whose expression is inhibited by hCG but is induced by interferon-γ (IFNγ). DNA sequence analysis identified this gene as rat IFNγ-inducible protein 10 (IP-10), a member of the -C-X-C- chemokine superfamily of proinflammatory cytokines. High levels of IP-10 messenger RNA (mRNA) were constitutively expressed in freshly isolated and primary cultured Leydig cells. hCG inhibited this expression in a dose-dependent manner. The addition of 1 ng/ml hCG inhibited IP-10 mRNA levels more than 80%. Conversely, IP-10 mRNA levels were markedly increased in response to murine interleukin-1α, murine tumor necrosis factor-α, and murine IFNγ by 3.3-, 10-, and 26-fold, respectively. Concomitant addition of murine interleukin-1α, murine tumor necrosis factor-α, and murine IFNγ synergistically increased IP-10 mRNA levels by 58-fold. Furthermore, in addition to one previously described rat IP-10 mRNA tra...

Transformation and immortalization of Leydig cells from the Sprague-Dawley rat by an early genetic region of simian virus 40 DNA

Two transformed cell lines of rat Leydig cells were established by transfection of primary cells with the transforming region of simian virus (SV40) DNA. Normal adult Leydig cells are non-proliferating cells and cease to grow after the first trypsinization for cell culturing. The cell lines. NWL2 and NWL15, continued to proliferate and subsequently needed subculturing every 2 weeks (split ratio 1:2). No crisis was observed after 35 passages for 18 months. Nile red staining showed the presence of lipid droplets in both normal and transformed cells, although the transformed cells had 2–3-fold higher amounts than the normal cells. The integration of T-antigen DNA has taken place in at least 2 and 1 sites in NWL2 and NWL15, respectively. Both cell lines expressed T-antigen mRNA. The cell lines expressed luteinizing hormone receptor (LH-R) (a Leydig cellspecific gene), insulin-like growth factor-I, insulin-like growth factor-I receptor (IGF-I-R) and insulin-like growth factor binding protein-2 (IGFBP-2) genes. The amounts of transcripts of LH-R were lower in the transformed cells as compared to the normal cells. The IGF-I-R mRNA levels were comparable to those of the normal Leydig cells. NWL2 and NWL15 cells also expressed IGF-I mRNA although to a lesser extent than the normal Leydig cells. IGFBP-2 mRNA levels were much higher in both the transformed cell lines than in the normal Leydig cells. The transformed cells were evaluated for the expression of P450scc, which catalyzes the conversion of cholesterol to pregnenolone. The transformed Leydig cells expressed very low levels of P450scc as compared to normal Leydig cells. We found previously that P450scc mRNA in Leydig cells after 24h in culture decreased to undetectable levels. the NWL2 and NWL15 cells are promising models for the study of the regulation of Leydig cell function at the molecular level.

Immortalization of Rabbit Vascular Smooth Muscle Cells after Transfection with a Fragment of the BglII N Region of Herpes Simplex Virus Type 2 DNA

Rabbit arterial smooth muscle cells were transfected with the pBC24neo plasmid DNA which consists of a sequence of 790 base pairs from the BglII N fragment of herpes simplex virus type 2 DNA linked to the neo resistance gene. Selection in G418-containing medium resulted in eleven immortalized clones which showed increased growth rate and saturation densities. One of the eleven clones maintained the transforming DNA sequence integrated in the cellular genome and showed continuous resistance to G418, but Southern blot analysis of the other ten immortalized clones did not detect pBC24neo DNA sequences. Possible mechanisms of herpes simplex virus induced immortalization and their implications in the development of atheromatous plaques are discussed.