Crystal structure of 1',1''-dimethyl-4'-(4-cholorophen-yl)di-spiro-[11H-indeno[1,2-b]quinoxaline-11,2'-pyrrolidine-3',3''-piperidin]-4''-one.

In the title compound, C30H27ClN4O, the central pyrrolidine ring adopts an envelope conformation with the methyl-ene C atom being the flap. The quinoxaline and indane rings are each essentially planar, with r.m.s. deviations of 0.027 (1) and 0.0417 (1) Å, respectively. The pyrrolidine ring forms dihedral angles of 88.25 (1) and 83.76 (1)° with the quinoxaline and indane rings, respectively. A weak intra-molecular C-H⋯N inter-action is observed. In the crystal, C-H⋯π inter-actions lead to supra-molecular chains along [101] that assemble in the ac plane. Connections along the b axis are of the type Cl⋯Cl [3.6538 (16) Å].

10-(4-Chloro-phen-yl)-14a-hy-droxy-12-methyl-8,9,9a,10,12,13,14,14a-octa-hydro-5H-10a,14-methano-indeno-[2',1':4,5]azepino[3,4-b]pyrrolizine-5,15(7H,11H)-dione.

The asymmetric unit of the title compound, C26H25ClN2O3, contains two independent mol-ecules (A and B). The conformation of the two mol-ecules differs essentially in the dihedral angle involving the two benzene rings. They are inclined to one another by 52.47 (10) in A and by 31.75 (11)° in B. In both mol-ecules, the six-membered piperidin-3-one rings have chair conformations. In mol-ecule A, all four five-membered rings have twist conformations. In mol-ecule B, only three of the four five-membered rings have twist conformations. The fourth, of the inden-1-one moiety, has an envelope conformation with the spiro C atom, bonded to the N atom of the pyrrolidine ring, as the flap. A weak intra-molecular O-H⋯N hydrogen bond occurs in each independent mol-ecule while a C-H⋯O inter-action is also observed in mol-ecule A. In the crystal, pairs of O-H⋯O hydrogen bonds link the mol-ecules, forming inversion dimers with graph-set motif R 2 (2)(12). These dimers are further inter-connected by C-H⋯O and C-H⋯π inter-actions, forming a three-dimensional network.

A clinical evaluation of a bioresorbable membrane and porous hydroxyapatite in the treatment of human molar class II furcations.

BACKGROUND: The ultimate goal of periodontal therapy is predictable regeneration of a functional attachment apparatus destroyed as a result of periodontitis. Reconstructive procedures have been used with varying success during the past decades to accomplish this goal. AIM: To evaluate whether the use of porous hydroxyapatite alone or a bioresorbable membrane alone would enhance the clinical results in the treatment of class II furcation defects in human lower molars. MATERIALS AND METHODS: Fifteen patients with chronic periodontitis, aged between 39 and 49 years, with a pair of similar bilateral class II furcation defects (classification of Hamp et al.) in mandibular first molars were selected. A split-mouth design was incorporated and the selected 30 furcation defects were assigned to one of the two treatment groups, i.e., Group I treated with a bioresorbable membrane from bovine-derived collagen guided tissue regeneration membrane and Group II treated using porous hydroxyapatite bone graft material on the contralateral sides. Evaluation of clinical parameters, probing depths and attachment levels, and radiographs was done preoperatively and 6 months postoperatively. RESULTS: Both the groups showed statistically significant mean reduction in probing depths and gain in clinical attachment levels and linear bone fill. Comparison between Group I and Group II showed insignificant difference. CONCLUSION: Within the limits of this study, both the treatment modalities are beneficial for the treatment of human mandibular class II furcation defects.

14a-Hy-droxy-12-methyl-10-(4-methyl-phen-yl)-8,9,9a,10,12,13,14,14a-octa-hydro-10a,14-methano-5H-indeno-[2',1':4,5]azepino[3,4-b]pyrrolizine-5,15(7H,11H)-dione.

In the title compound, C27H28N2O3, each of the pyrrolidine rings adopts a twisted conformation, as does the cyclo-pentane ring. The indane ring has an r.m.s deviation of 0.0693 Å. The dihedral angle between the mean plane of the pyrrolizine ring and indane system is 82.58 (1)°. The piperidine ring has the methyl substituent in an equatorial position and adopts a twisted chair conformation. The mol-ecular structure is stabilized by a weak intra-molecular O-H⋯N inter-action.

1',1''-Dimethyl-4'-phenyl-dispiro-[11H-indeno-[1,2-b]quinoxaline-11,2'-pyrrolidine-3',3''-piperidin]-4''-one.

In the title compound, C30H28N4O, the central pyrrolidine ring adopts an envelope conformation with the CH2 C atom as the flap. The quinoxaline and indene ring systems are planar, with r.m.s. deviations of 0.0165 and 0.0181 Å, respectively. The pyrrolidine ring mean plane forms dihedral angles of 88.84 (1) and 86.14 (1)° with the quinoxaline and indene ring systems, respectively. A weak intra-molecular C-H⋯N inter-action is observed. In the crystal, C-H⋯O inter-actions lead to helical supra-molecular chains along the b axis having a C(9) motif.

1',1''-Dimethyl-4'-(4-methyl-phen-yl)di-spiro-[11H-indeno-[1,2-b]quinoxaline-11,2'-pyrrolidine-3',3''-piperidin]-4''-one.

In the title compound, C31H30N4O, the central pyrrolidine ring adopts an envelope conformation with the methyl-ene C atom being the flap. The quinoxaline and indane rings are each planar, having r.m.s. deviations of 0.030 and 0.050 Å, respectively. The pyrrolidine ring mean plane forms dihedral angles of 88.25 (1) and 83.76 (1)° with the quinoxaline and indane rings, respectively. Intra-molecular C-H⋯O and C-H⋯N inter-actions are observed. In the crystal, C-H⋯π inter-actions lead to helical supra-molecular chains along the b-axis direction.

Comparative study of nanoparticle-mediated transfection in different GI epithelium co-culture models.

Oral nonviral gene delivery is the most attractive and arguably the most challenging route of administration. To identify a suitable carrier, we studied the transport of different classes (natural polymer, synthetic polymer and synthetic lipid-polymer) of DNA nanoparticles through three well-characterized cellular models of intestinal epithelium (Caco2, Caco2-HT29MTX and Caco2-Raji). Poly(phosphoramidate-dipropylamine) (PPA) and Lipid-Protamine-DNA (LPD) nanoparticles consistently showed the highest level of human insulin mRNA expression and luciferase protein expression in these models, typically at least three orders of magnitude above background. All of the nanoparticles increased tight junction permeability, with PPA and PEI having the most dramatic transepithelial electrical resistance (TEER) decreases of (35.3±8.5%) and (37.5±1.5%) respectively in the first hour. The magnitude of TEER decrease correlated with nanoparticle surface charge, implicating electrostatic interactions with the tight junction proteins. However, confocal microscopy revealed that the nanoparticles were mostly uptaken by the enterocytes. Quantitative uptake and transport experiments showed that the endocytosed, quantum dot (QD)-labeled PPA-DNA nanoparticles remained in the intestinal cells even after 24h. Negligible amount of quantum dot labeled DNA was detected in the basolateral chamber, with the exception of the Caco2-Raji co-cultures, which internalized nanoparticles 2 to 3 times more readily compared to Caco2 and Caco2-HT29MTX cultures. PEGylation decreased the transfection efficacy by at least an order of magnitude, lowered the magnitude of TEER decrease and halved the uptake of PPA-DNA nanoparticles. A key finding was insulin mRNA being detected in the underlying HepG2 cells, signifying that some of the plasmid was transported across the intestinal epithelial layer while retaining at least partial bioactivity. However, the inefficient transport suggests that transcytosis alone would not engender a significant therapeutic effect, and this transport modality must be augmented by other means in vivo to render nonviral oral gene delivery practical.

RFX2 is a candidate downstream amplifier of A-MYB regulation in mouse spermatogenesis.

BACKGROUND: Mammalian spermatogenesis involves formation of haploid cells from the male germline and then a complex morphological transformation to generate motile sperm. Focusing on meiotic prophase, some tissue-specific transcription factors are known (A-MYB) or suspected (RFX2) to play important roles in modulating gene expression in pachytene spermatocytes. The current work was initiated to identify both downstream and upstream regulatory connections for Rfx2. RESULTS: Searches of pachytene up-regulated genes identified high affinity RFX binding sites (X boxes) in promoter regions of several new genes: Adam5, Pdcl2, and Spag6. We confirmed a strong promoter-region X-box for Alf, a germ cell-specific variant of general transcription factor TFIIA. Using Alf as an example of a target gene, we showed that its promoter is stimulated by RFX2 in transfected cells and used ChIP analysis to show that the promoter is occupied by RFX2 in vivo. Turning to upstream regulation of the Rfx2 promoter, we identified a cluster of three binding sites (MBS) for the MYB family of transcription factors. Because testis is one of the few sites of A-myb expression, and because spermatogenesis arrests in pachytene in A-myb knockout mice, the MBS cluster implicates Rfx2 as an A-myb target. Electrophoretic gel-shift, ChIP, and co-transfection assays all support a role for these MYB sites in Rfx2 expression. Further, Rfx2 expression was virtually eliminated in A-myb knockout testes. Immunohistology on testis sections showed that A-MYB expression is up-regulated only after pachytene spermatocytes have clearly moved away from the tubule wall, which correlates with onset of RFX2 expression, whereas B-MYB expression, by contrast, is prevalent only in earlier spermatocytes and spermatogonia. CONCLUSION: With an expanding list of likely target genes, RFX2 is potentially an important transcriptional regulator in pachytene spermatocytes. Rfx2 itself is a good candidate to be regulated by A-MYB, which is essential for meiotic progression. If Alf is a genuine RFX2 target, then A-myb, Rfx2, and Alf may form part of a transcriptional network that is vital for completion of meiosis and preparation for post-meiotic differentiation.

Differential expression of Rfx1-4 during mouse spermatogenesis.

The regulatory factor X (RFX) family of transcription factors has been recently implicated in gene regulation during spermatogenesis. However, the relative expression of individual members during this developmental process is not completely characterized, particularly in the case of Rfx4, which has multiple transcript variants in the testis. We used reverse transcriptase-dependent real-time PCR, 5'-RACE cloning, and Western blotting to compare transcripts and protein levels for this family in cell populations from the three major phases of spermatogenesis (mitotic, meiotic, and haploid). Transcripts for Rfx1-4 were present at trace to low levels in spermatogonia prepared from 8-day-old mice. Transcripts for both Rfx2 and Rfx4 were elevated in mid-late pachytene spermatocytes; however, the dominant Rfx4 transcript present begins at a downstream exon and lacks the DNA binding domain. Transcripts for all four genes were elevated in early haploid cells (round spermatids). In these cells Rfx4 transcripts originate primarily from a newly described promoter with intron 1 but are expected to be translationally compromised due to a poorly situated start codon. Western blotting confirmed that RFX2 is greatly elevated beginning in meiosis and also confirmed that full-length RFX4 protein is not prevalent in mouse testis at any stage. These results imply that RFX2 is the most likely X box binding factor to influence novel gene expression during meiosis, that RFX1-3 may all play roles in haploid cells but that RFX4 is much less prevalent than implied by its high transcript levels.

Expression patterns of SP1 and SP3 during mouse spermatogenesis: SP1 down-regulation correlates with two successive promoter changes and translationally compromised transcripts.

Because of their prominent roles in regulation of gene expression, it is important to understand how levels of Krüpple-like transcription factors SP1 and SP3 change in germ cells during spermatogenesis. Using immunological techniques, we found that both factors decreased sharply during meiosis. SP3 declined during the leptotene-to-pachytene transition, whereas SP1 fell somewhat later, as spermatocytes progressed beyond the early pachytene stage. SP3 reappeared for a period in round spermatids. For Sp1, the transition to the pachytene stage is accompanied by loss of the normal, 8.2-kb mRNA and appearance of a prevalent, 8.8-kb variant, which has not been well characterized. We have now shown that this pachytene-specific transcript contains a long, unspliced sequence from the first intron and that this sequence inhibits expression of a reporter, probably because of its many short open-reading frames. A second testis-specific Sp1 transcript in spermatids of 2.4 kb also has been reported previously. Like the 8.8-kb variant, it is compromised translationally. We have confirmed by Northern blotting that the 8.8-, 8.2-, and 2.4-kb variants account for the major testis Sp1 transcripts. Thus, the unexpected decline of SP1 protein in the face of continuing Sp1 transcription is explained, in large part, by poor translation of both novel testis transcripts. As part of this work, we also identified five additional, minor Sp1 cap sites by 5' rapid amplification of cDNA ends, including a trans-spliced RNA originating from the Glcci1 gene.

Insight into Trichoderma reesei's genome content, organization and evolution revealed through BAC library characterization.

Trichoderma reesei is an important industrial fungus known for its ability to efficiently secrete large quantities of protein as well as its wide variety of biomass degrading enzymes. Past research on this fungus has primarily focused on extending its protein production capabilities, leaving the structure of its 33 Mb genome essentially a mystery. To begin to address these deficiencies and further our knowledge of T. reesei's secretion and cellulolytic potential, we have created a genomic framework for this fungus. We constructed a BAC library containing 9216 clones with an average insert size of 125 kb which provides a coverage of 28 genome equivalents. BAC ends were sequenced and annotated using publicly available software which identified a number of genes not seen in previously sequenced EST datasets. Little evidence was found for repetitive sequence in T. reesei with the exception of several copies of an element with similarity to the Podospora anserina transposon, PAT. Hybridization of 34 genes involved in biomass degradation revealed five groups of co-located genes in the genome. BAC clones were fingerprinted and analyzed using fingerprinted contigs (FPC) software resulting in 334 contigs covering 28 megabases of the genome. The assembly of these FPC contigs was verified by congruence with hybridization results.

RFX2 is a potential transcriptional regulatory factor for histone H1t and other genes expressed during the meiotic phase of spermatogenesis.

H1t is a novel linker histone variant synthesized in mid- to late pachytene spermatocytes. Its regulatory region is of interest because developmentally specific expression has been impressed on an otherwise ubiquitously expressed promoter. Using competitive band-shift assays and specific antisera, we have now shown that the H1t-60 CCTAGG palindrome motif region binds members of the RFX family of transcriptional regulators. The testis-specific binding complex contains RFX2, probably as a homodimer. Other DNA-protein complexes obtained from testis as well as somatic organs contain RFX1, primarily as a heterodimer. Western blots confirmed that RFX2 expression is greatly enhanced in adult testis and that RFX2 is equally prominent in highly enriched populations of late pachytene spermatocytes and round spermatids. Immunohistochemistry carried out on mouse testis showed that RFX2 is strongly expressed in pachytene spermatocytes, remains high in early round spermatids, and declines only in advance of nuclear condensation. Maximum expression correlates well with the appearance of H1t. In contrast, RFX1 immunoreactivity in germ cells was only detected in late round spermatids. RFX-specific band complexes were also identified for both the mouse lamin C2 and Sgy promoters, using either testis nuclear extracts or in vitro-synthesized RFX2. These results call attention to RFX2 as a transcription factor with obvious potential for the regulation of gene expression during meiosis and the early development of spermatids.

The rat histone H1d gene has intragenic activating sequences that are absent from the testis-specific variant H1t.

In some cases core histone genes in the mouse depend on intragenic sequence elements for high level expression [Gene 176 (1996) 1]. Here we report that the highly expressed gene for rat linker histone H1d also contains an intragenic activating region (IAR). Using transient transfection assays in mouse fibroblast NIH3T3 cells, we showed that rat H1d contains a downstream region (+21 to +116) that imparts a two- to threefold up-regulation of fused reporters. This region also activated expression when moved to the promoter region, though the effect was dependent on its distance from other promoter elements. The IAR contains sequence homologies to the core alpha and Omega elements identified as functional protein binding sites within the mouse H3.2 coding region activating sequence (CRAS). A pair of Omega elements (+32 and +66) accounts for the activating effect of the H1d intragenic region as shown by targeted mutations as well as stepwise deletions. The H1d and H3.2 Omega sequences bound similar and perhaps identical proteins by gel shift analysis. The H1d alpha-like sequence at +56 overlaps the translational start codon and was therefore not mutated. Like the mouse H3.2 alpha element, it bound transcription factor YY1 in gel shift assays. H1t, the gene for the testis-specific linker histone, did not demonstrate an IAR. While H1t has a similar alpha sequence and did bind YY1, it lacks the Omega homologies of H1d. Sequence comparison shows that the YY1/alpha site as well as the adjacent Omega site are likely present in genes for other standard H1 variants, but that the +32 Omega site in the 5' untranslated region (UTR) of H1d is unique. We conclude that the +32 and +66 Omega sequences of the rat H1d gene contribute significantly to its high-level expression.

Catalytic roles of yeast GSK3beta/shaggy homolog Rim11p in meiotic activation.

In Saccharomyces cerevisiae, many meiotic genes are activated by a heteromeric transcription factor composed of Ime1p and Ume6p. Ime1p-Ume6p complex formation depends upon the protein kinase Rim11p, which interacts with and phosphorylates both Ime1p and Ume6p in vitro. Rim11p may promote complex formation through its phosphorylation of Ime1p and Ume6p or simply through its interaction with both proteins. Here, we characterize mutant Ime1p derivatives that interact with Rim11p but are not phosphorylated in vitro. These mutant proteins are also defective in interaction with Ume6p. These results argue that Ime1p must be phosphorylated to interact with Ume6p. Our genetic observations suggest that Ime1p tyrosine residues are among the Rim11p phosphoacceptors, and we find that Ime1p reacts with an anti-phosphotyrosine antibody. Ime1p and Rim11p have been thought to act only through Ume6p, but we find that Ime1p and Rim11p promote meiosis at a very low level in the absence of Ume6p. A nonphosphorylatable mutant Ime1p derivative promotes sporulation through this Ume6p-independent pathway, as does a mutant Rim11p derivative that fails to interact with Ime1p. Therefore, Ime1p and Rim11p have two genetically separable functions in the sporulation program. However, catalytic activity of Rim11p is required for sporulation in the presence or absence of Ume6p.

Isolation of the murI gene from Brevibacterium lactofermentum ATCC 13869 encoding D-glutamate racemase.

The murI gene encoding D-glutamate racemase plays an important role in the biosynthesis of D-glutamic acid, an essential component of cell wall peptidoglycan of almost all eubacteria. A DNA fragment that could rescue the auxotrophy of D-glutamic acid in the Escherichia coli murI mutant strain WM335 was isolated from Brevibacterium lactofermentum ATCC 13869 belonging to the coryneform bacteria. DNA sequencing reveals that it encodes a protein of 284 amino acid residues, which shows a high level of homology with D-glutamate racemases from several other bacteria.

Interaction of yeast repressor-activator protein Ume6p with glycogen synthase kinase 3 homolog Rim11p.

Meiosis and expression of early meiotic genes in the budding yeast Saccharomyces cerevisiae depend upon Rim11p, Ume6p, and Ime1p. Rim11p (also called Mds1p and ScGSK3) is a protein kinase related to glycogen synthase kinase 3 (GSK3); Ume6p is an architectural transcription factor; and Imelp is a Ume6p-binding protein that provides a transcriptional activation domain. Rim11p is required for Ime1p-Ume6p interaction, and prior studies have shown that Rim11p binds to and phosphorylates Ime1p. We show here that Rim11p binds to and phosphorylates Ume6p, as well. Amino acid substitutions in Ume6p that alter a consensus GSK3 site reduce or abolish Rim11p-Ume6p interaction and Rim11p-dependent phosphorylation, and they cause defects in interaction between Ume6p and Ime1p and in meiotic gene expression. Therefore, interaction between Rim11p and Ume6p, resulting in phosphorylation of Ume6p, is required for Ime1p-Ume6p complex formation. Rim11p, like metazoan GSK3beta, phosphorylates both interacting subunits of a target protein complex.

Radionuclide studies in the evaluation of urinary tract infections.

OBJECTIVE: To estimate the prevalence of vesicoureteral reflux (VUR) and renal scarring in children presenting with culture proven urinary tract infections (UTI). DESIGN: Descriptive study. SETTING: Tertiary care hospital-based study. SUBJECTS: Thirty-two children with proven UTI were evaluated by means of an abdominal ultrasonogram (USG), Technetium-99m Dimercapto Succinic Acid (DMSA) scan and Direct Radionuclide Cystography (DRCG). A micturating cystourethrogram (MCU) was performed to rule out any structural abnormality and to grade VUR. RESULTS: A total of 64 renal units in 32 children were evaluated. DMSA scan showed scarring in 27 renal units (42.2%) in 16 patients. Bilateral renal scarring was more common in older (> 2 yr) children as compared to younger ones (89% Vs 43%; p < 0.05). USG detected abnormalities in 13 renal units (20.3%) in 7 cases. VUR was detected in 37.5% of children of all age group by DRCG. In contrast, MCU showed evidence of VUR in only 13/20 renal units with a sensitivity of 65% as compared to DRCG and did not pick up any additional VUR that could have been missed on the DRCG. Only 3/9 in < 2 yr, in contrast to 10/11 in > 2 yr were positive for VUR on MCU (p < 0.05). However, MCU detected evidence of cystitis in 3 children and a bladder diverticulum in one patient. CONCLUSION: Wherever available, DMSA scan should be considered as a part of the first line investigations in any patient presenting with UTI. DRCG can also be performed in the same sitting to screen for the presence of reflux particularly for girls.

Simultaneous determination of Ru(III) and Rh(III) using octadecyldithiocarbamate by first-derivative spectrophotometry.

A first-derivative spectrophotometric method is described for the simultaneous determination of Ru(III) and Rh(III) using octadecyl dithiocarbamate. The complexes are insoluble in water, but easily extractable into chloroform. Quantitative determination of Ru(III) and Rh(III) is possible in the ranges 0.5-6.0 mug ml(-1) and 1.0-10.0 mug ml(-1) respectively, with a standard deviation of +/-0.10. A statistical evaluation of the experimental results is reported.

ACPR, a STE12 homologue from Candida albicans, is a strong inducer of pseudohyphae in Saccharomyces cerevisiae haploids and diploids.

ACPR from Candida albicans encodes a protein antigenically related to the secretory acid proteinase of this yeast. Its amino terminal domain is highly similar to the amino terminal, DNA-binding domain of STE12 of Saccharomyces cerevisiae. STE12 is involved in mating of haploids and in pseudohyphae formation in diploids. ACPR, or its DNA-binding domain swapped into STE12, can support pseudohyphae formation in S. cerevisiae diploids. However, unlike STE12, these constructs affect the budding pattern and induce pseudohyphae formation in S. cerevisiae haploids as well, and this induction is independent of the nitrogen status of the medium. ACPR appears to be a stronger inducer of pseudohyphae than STE12 and is likely to be involved in the formation of pseudohyphae and hyphae in C. albicans.