FadD19 of Rhodococcus rhodochrous DSM43269, a steroid-coenzyme A ligase essential for degradation of C-24 branched sterol side chains.

The actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode ß-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e., fadD17, fadD19, fadE26, fadE27, and ro04690DSM43269, by gene inactivation studies in mutant strain RG32 of Rhodococcus rhodochrous DSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9α-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of five kshA gene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32ΔfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion of fadD19 in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation of fadD17 in mutant RG32ΔfadD19 also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269 nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essential in vivo role in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with an in vivo role in sterol side chain degradation. The high similarity (67%) between the FadD19(DSM43269) and FadD19H37Rv enzymes further suggests that FadD19H37Rv has an in vivo role in sterol metabolism of Mycobacterium tuberculosis H37Rv.

Rigidity percolation in dispersions with a structured viscoelastic matrix.

This paper deals with rigidity percolation in composite materials consisting of a dispersion of mineral particles in a microstructured viscoelastic matrix. The viscoelastic matrix in this specific case is a hydrocarbon refinery residue. In a set of model random composites the mean interparticle surface-to-surface distance was controlled, changing particle volume fraction phi and particle number density independently. This was achieved by mixing two sets of monodisperse particles with widely differing radii (0.35 microm and 17.5 microm) with the matrix. A scaling exponent of 3.9 +/- 0.6 for the storage modulus G' vs phi- phi(c) was observed above a threshold phi(c) , in good agreement with theoretical values for rigidity percolation. It is found that at the rigidity-percolation threshold the pore structure, as characterized by the mean surface-to-surface distance for the filler, rather than the filler volume fraction, is similar for different types of composites. This behavior is explained from the internal structure of the viscoelastic matrix, which consists of fractal solid aggregates dissolved in a viscous medium; the effective radius of these aggregates and the mean surface-to-surface distance together determine whether or not the aggregates are capable of providing rigidity to the composite. The explanation is further supported by a qualitative comparison with effective-medium calculations. These indicate that the observed breakdown of time-temperature superposition near phi(c) is due to the appearance of a time scale characteristic for the mechanical interplay between the viscous binder phase and the purely elastic solid particles.

Genomic structure, chromosomal localization, and embryonic expression of the mouse homolog of PRCC, a gene associated with papillary renal cell carcinoma.

In a subset of papillary renal cell carcinomas a t(X;1)(p11;q21) chromosome translocation has repeatedly been reported. Positional cloning has demonstrated that, as a result of this translocation, the transcription factor TFE3 gene on the X-chromosome becomes fused to a novel gene, PRCC, on chromosome 1. Since as yet little is known about the function of PRCC, we sought to identify the mouse counterpart of the PRCC gene. Isolation and sequence analysis of a mouse Prcc cDNA revealed a high level of conservation between man and mouse, both at the nucleotide and protein level. As the human PRCC gene, the mouse Prcc gene is ubiquitously expressed. It shows low expression in all mouse fetal tissues examined. In addition, we identified a genomic cosmid clone containing the complete Prcc gene. The mouse Prcc gene consists of seven exons, all of which contain coding sequences. The small second exon, which was found to be located adjacent to the t(X;1) breakpoint in the human gene on chromosome 1, is also conserved between man and mouse. In mouse, Prcc is located on chromosome 3. These cDNA and genomic clones will be instrumental in the creation of mouse models for a further elucidation of the function of PRCC.

Renal oncocytoma with t(5;12;11), der(1)1;8) and add(19): "true" oncocytoma or chromophobe adenoma?

Renal oncocytomas reveal a considerable (cyto)genetic heterogeneity. At least 2 genetic subsets are currently recognized, characterized by (1) translocations involving breakpoint 11q13 and (2) the combined loss of chromosomes 1 and X/Y. We present a case of oncocytoma revealing a 3-way translocation involving breakpoint 11q13, a der(1)t(1;8) and an add(19). The der(1) resulted in loss of chromosome 1 sequences. Using fluorescence in situ hybridization, the 11q13 breakpoint of the present case proved to be slightly different from the one observed previously in 3 cases of renal oncocytoma. Whether the 11q13 breakpoint observed in our case resides in or near another gene remains to be elucidated.

Fusion of the transcription factor TFE3 gene to a novel gene, PRCC, in t(X;1)(p11;q21)-positive papillary renal cell carcinomas.

The (X;1)(p11;q21) translocation is a recurrent chromosomal abnormality in a subset of human papillary renal cell carcinomas, and is sometimes the sole cytogenetic abnormality present. Via positional cloning, we were able to identify the genes involved. The translocation results in a fusion of the transcription factor TFE3 gene on the X chromosome to a novel gene, designated PRCC, on chromosome 1. Through this fusion, reciprocal translocation products are formed, which are both expressed in papillary renal cell carcinomas. PRCC is ubiquitously expressed in normal adult and fetal tissues and encodes a putative protein of 491 aa with a relatively high content of prolines. No relevant homologies with known sequences at either the DNA or the protein level were found.

Fine mapping of the 1q21 breakpoint of the papillary renal cell carcinoma-associated (X;1) translocation.

A combination of Southern blot analysis on a panel of tumor-derived somatic cell hybrids and fluorescence in situ hybridization (FISH) techniques was used to map a series of DNA markers relative to the 1q21 breakpoint of the renal cell carcinoma (RCC)-associated (X;1)-(p11;q21) translocation. This breakpoint maps between several members of the S100 family which are clustered in the 1q21 region and a conserved region between man and mouse containing the markers SPTA1-CRP-APCS-FcER1A-ATP1A2-APOA2. The location of the breakpoint coincides with the transition of a region of synteny of human chromosome 1 with mouse chromosomes 3 and 1.

Molecular cloning of the papillary renal cell carcinoma-associated translocation (X;1)(p11;q21) breakpoint.

A combination of Southern blot analysis on a panel of tumor-derived somatic cell hybrids and fluorescence in situ hybridization techniques was used to map YACs, cosmids and DNA markers from the Xp11.2 region relative to the X chromosome breakpoint of the renal cell carcinoma-associated t(X;1)(p11;q21). The position of the breakpoint could be determined as follows: Xcen-OATL2-DXS146-DXS255-SYP-t(X;1)-TFE 3-OATL1-Xpter. Fluorescence in situ hybridization experiments using TFE3-containing YACs and cosmids revealed split signals indicating that the corresponding DNA inserts span the breakpoint region. Subsequent Southern blot analysis showed that a 2.3-kb EcoRI fragment which is present in all TFE3 cosmids identified, hybridizes to aberrant restriction fragments in three independent t(X;1)-positive renal cell carcinoma DNAs. The breakpoints in these tumors are not the same, but map within a region of approximately 6.5 kb. Through preparative gel electrophoresis an (X;1) chimaeric 4.4-kb EcoRI fragment could be isolated which encompasses the breakpoint region present on der(X). Preliminary characterization of this fragment revealed the presence of a 150-bp region with a strong homology to the 5' end of the mouse TFE3 cDNA in the X-chromosome part, and a 48-bp segment in the chromosome 1-derived part identical to the 5' end of a known EST (accession number R93849). These observations suggest that a fusion gene is formed between the two corresponding genes in t(X;1)(p11;q21)-positive papillary renal cell carcinomas.

Distinct Xp11.2 breakpoints in two renal cell carcinomas exhibiting X;autosome translocations.

Several human renal cell carcinomas with X;autosome translocations have been reported in recent years. The t(X;1)(p11.2;q21) appears to be a specific primary anomaly, suggesting that tumors with this translocation form a distinct subgroup of RCC. Here we report two new cases, one with a t(X;10)(p11.2;q23), the other with a t(X;1)(p11.2;p34). The common breakpoint in Xp11.2 suggests that they belong to the above-mentioned subset of RCC. Using FISH in conjunction with X-specific YAC clones, we demonstrate that the two new cases exhibited distinct breakpoints within Xp11.2.

Interspecific sequence comparison of the muscle-myosin heavy-chain genes from Drosophila hydei and Drosophila melanogaster.

The muscle-myosin heavy-chain (mMHC) gene of Drosophila hydei has been sequenced completely (size 23.3 kb). The sequence comparison with the D. melanogaster mMHC gene revealed that the exon-intron pattern is identical. The protein coding regions show a high degree of conservation (97%). The alternatively spliced exons (3a-b, 7a-d, 9a-c, 11a-e, and 15a-b) display more variations in the number of nonsynonymous and synonymous substitutions than the common exons (2, 4, 5, 6, 8, 10, 12, 13, 14, 16, 17, and 19). The base composition at synonymous sites of fourfold degenerate codons (third position) is not biased in the alternative exons. In the common exons there exists a bias for C and against A. These findings imply that the alternative exons of the Drosophila mMHC gene evolve at a different, in several cases higher, rate than the common ones. The 5' splice junctions and 5' and 3' untranslated regions show a high level of similarity, indicating a functional constraint on these sequences. The intron regions vary considerably in length within one species, but the corresponding introns are very similar in length between the two species and all contain stretches of sequence similarity. A particular example is the first intron, which contains multiple regions of similarity. In the conserved regions of intron 12 (head-tail border) sequences were found which have the potential to direct another smaller mMHC transcript.

Identification of a yeast artificial chromosome that spans the human papillary renal cell carcinoma-associated t(X;1) breakpoint in Xp11.2.

Recently, a specific chromosome abnormality, t(X;1)(p11;q21), was described for a subgroup of human papillary renal cell carcinomas. The translocation breakpoint in Xp11 is located in the same region as that in t(X;18)(p11;q11)-positive synovial sarcoma. We used fluorescence in situ hybridization (FISH) and somatic cell hybridization techniques to demonstrate 1) that the Xp11 translocation breakpoint in papillary renal cell carcinoma differs from that observed in synovial sarcoma and has a more proximal location, and 2) that an ornithine aminotransferase (OAT)L2 containing yeast artificial chromosome (YAC) spans the X;1 translocation. This YAC provides an ideal starting point from which the breakpoint itself and the gene(s) involved can be isolated and characterized.

Localization of the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei by fluorescence in situ hybridization.

We have used fluorescence in situ hybridization to map the positions of the different repetitive DNA sequences from the region forming the lampbrush loop pair Nooses on the Y chromosome of Drosophila hydei. This region harbours a megabase cluster of tandemly organized repeats of the Y-specific ay1 family and a megabase cluster of tandem repeats of the related Y-specific YsI family. In addition, ay1 repeats also occur in short blocks that are interspersed by other repetitive DNA sequences that we call Y-associated, since they have additional copies on other chromosomes. Using specific probes for ay1, YsI and Y-associated DNA sequences, we show that there is one large proximal cluster of YsI repeats and one, more distally located, large cluster of ay1 repeats. The Y-chromosomal copies of the Y-associated sequences are located in the most distal part of the ay1 cluster. This is consistent with the juxtaposition of ay1 and Y-associated sequences in more than 300 kb of cloned genomic DNA. Since both ay1 and Y-associated sequences have been shown to be transcribed in the Nooses, the lampbrush loop is formed in a distal region of the short arm of the Y chromosome, adjacent to the terminally located nucleolus organizer region. The clusters of homogeneous ay1 and YsI repeats are of no functional significance for the formation of the lampbrush loop.

Phase control of ultradian feeding rhythms in the common vole (Microtus arvalis): the roles of light and the circadian system.

In their ultradian (2- to 3-hr) feeding rhythm, common voles show intraindividual synchrony from day to day, as well as interindividual synchrony between members of the population, even at remote distances. This study addresses the question of how resetting of the ultradian rhythm, a prerequisite for such synchronization, is achieved. Common voles were subjected to short light-dark cycles (1 hr darkness with light varying between 0.7 and 2.5 hr); to T cycles (long light-dark cycles in the circadian range--16 hr darkness and 3-13 hr light); to light pulses (15 min) during different circadian and ultradian phases; and to addition of D2O to the drinking water (25%). Short light-dark cycles and D2O were also applied to voles without circadian rhythmicity, after lesions of the suprachiasmatic nuclei. In these experiments, four hypotheses on synchronization of ultradian rhythmicity were tested: (I) synchronization by a direct response to light; (II) synchronization via the circadian system with multiple triggers, here called "cogs," each controlling a single ultradian feeding bout; and (III and IV) synchronization via the circadian system with a single "cog," which resets an ultradian oscillator and either (III) originates directly from the circadian pacemaker, or (IV) is mediated via the overt circadian activity rhythm. Short light-dark cycles failed to entrain ultradian rhythms, either in circadian-rhythmic or in non-circadian-rhythmic voles; light pulses did not cause phase shifts; and in extreme T cycles no stable phase relationship with light could be demonstrated. Thus, Hypothesis I was rejected. Changes in the circadian period (tau) were generated as aftereffects of light pulses, by entrainment in various T cycles, and by the addition of D2O to the drinking water. These changes in tau did not lead to parallel, let alone proportional, changes in the ultradian period. This excluded Hypothesis II. Both in T-cycle experiments and in the D2O experiments with circadian-rhythmic voles, the phase of ultradian feeding bouts was locked to the end of circadian activity rather than to the most prominent marker of the pacemaker, the onset of circadian activity. This was not expected under Hypothesis III, but was consistent with entrainment via activity (Hypothesis IV). On the basis of these experiments, we conclude that the most likely mechanism of ultradian entrainment is that of a light-insensitive ultradian oscillator, reset every dawn by the termination of the activity phase controlled by the circadian pacemaker, which is itself entrained by the light-dark cycle. Neither in circadian-rhythmic nor in non-circadian-rhythmic voles was the period of the feeding rhythm lengthened by administration of D2O. This insensitivity to deuterium is exceptional among biological rhythms.

Occurrence of a cytochrome P-450-containing mixed-function oxidase system in the pond snail, Lymnaea stagnalis.

1. The occurrence of an as yet unidentified cytochrome P-450 in the microsomal fraction of the digestive gland of the snail, Lymnaea stagnalis was studied. 2. Studies in vivo and in vitro (digestive gland homogenates or the 170,000g fraction) of the cytochrome P-450-mediated metabolism of substrates such as biphenyl, pentoxy- and ethoxy-resorufin and aminopyrine have been made. 3. Cytochrome P-450 concentration in the digestive gland calculated from CO-difference spectra was 0.30 +/- 0.05 nmol/g tissue. This amount was not increased either by phenobarbital or by 3-methylcholanthrene. 4. Aniline binding spectra resembled normal type II spectra, while type I model substrates such as hexobarbital and 2,2'-dichlorobiphenyl showed type II- or reversed type I-like spectra. 5. O-Deethylation of 7-ethoxyresorufin did not occur, but 7-pentoxyresorufin O-depentylation activity (80.4 +/- 28.6 pmol resorufin/g per hour) and aminopyrine N-demethylation activity (375 +/- 96 pmol formaldehyde/g per minute) were demonstrated. 6. 4-Hydroxybiphenyl was the major metabolite of biphenyl, while minor amounts of 2-hydroxybiphenyl were formed (in vivo: 63.7 nmol 4-hydroxybiphenyl and 3.33 nmol 2-hydroxybiphenyl per snail per 24 h, after an oral dose of 778.2 nmol biphenyl; in vitro 118 +/- 21 pmol and 21 +/- 9 nmol, respectively (digestive gland homogenate/mg protein, per hour). 7. The results indicate that the isoenzymes involved in the observed MFO activities resemble isoenzymes P-450b/e.

Comparative toxicokinetics of 2,2'- and 4,4'-dichlorobiphenyls in the pond snail Lymnaea stagnalis (L.).

Pond snails (Lymnaea stagnalis (L.)) were treated with 2,2'-dichlorobiphenyl (DCB) or 4,4'-DCB, to examine the toxicokinetic profile of these compounds. Snails were treated orally with 210 micrograms 4,4'-DCB (impregnated on food) for 14 hr, or snails were injected with 50 micrograms of 2,2'-DCB or 4,4'-DCB in the foot. At different times after starting feeding or injection, tissues (albumen gland, digestive gland and digestive tube, central nervous system, remainder parts), hemolymph and faeces were analyzed for unchanged 2,2'- or 4,4'-DCB. The results showed that in case of oral administration of 4,4'-DCB after 144 hr, 97.5% of the dose was excreted unchanged in the faeces. During the first 48 hr 4,4'-DCB was found in all tissues. Thereafter, an exponential elimination was found (the rate constant of elimination (kel) varied from 0.010-0.021 per hr, t1/2 from 33-60 hr and the apparent clearance from 0.02-0.3 g/hr for the different tissues). After injection, the compounds were found in all the above mentioned tissues, especially in the digestive gland. There was a clear difference between snails injected with 2,2'- and 4,4'-DCB. Firstly, kel for 2,2'-DCB was higher (0.028 per hr vs 4,4'-DCB: 0.001 per hr). Secondly, 2,2'-DCB was lethal; 63% of the animals died after 72 hr.

Depolarization-induced release of [(3)H]histamine by high potassium concentrations, electrical stimulation and veratrine from rat brain slices after incubation with the radiolabelled amine.

Brain slices obtained from neocortex, hypothalamus or hippocampus were incubated with [(3)H]histamine and subsequently superfused and exposed to different depolarizing stimuli, viz. high K(+)-concentrations, electrical field stimulation and veratrine. K(+)-induced release of tritium was completely calcium-dependent and its magnitude depended on the K(+)-concentration, with maximal release being reached at 56 mM K(+). Electrically-evoked release of tritium increased with increasing frequencies and reached its maximum at about 20 Hz. The electrically-evoked release appeared to be totally calcium-dependent and it was strongly inhibited by tetrodotoxin. Veratrine (5-100 ?M) also induced a release of tritium; maximal release was obtained at 100 ?M veratrine. Veratrine-induced release was partially calcium-dependent and was strongly reduced by tetrodotoxin. Taken together the data indicate that the depolarization-induced release of tritium from brain slices pre-labelled with [(3)H]histamine, represents [(3)H]histamine release from neurons and not from either mast cells or glial cells. It remains to be established whether these neurons are specifically histaminergic.