The effects of growth factors on tRNALys modification.

The tRNALys population from tissue culture cells contains several isoaccepting species which are not present in the tRNALys population from tissue sources. These isoacceptors were isolated from mouse LM cells and tested for their coding properties in ribosomal binding assays and their ability to incorporate lysine into protein in a reticulocyte lysate. tRNALys5A and tRNALys6 eluted in the area of tRNALys5. All three species coded preferentially for AAA and bound with equal efficiency. tRNALys1, tRNALys3, tRNALys4, and tRNALys6 all transferred lysine into protein at a slower rate than tRNALys2 and tRNALys5, which are the native species. Several purified growth factors were tested for their ability to affect the levels of tRNALys4, a tRNA possibly associated with cell division. When Balb/c 3T3 cells were grown in medium containing plasma instead of serum, there was a decrease in tRNALys2, tRNALys3, tRNALys4 and an increase in tRNALys5 and tRNALys6. The addition of either FGF or PDGF returned the tRNALys profile to normal. The extent of the tRNALys changes depended upon the concentration of growth factor added. FGF was able to cause a 35% decrease in the tRNALys5 peak with a corresponding increase in tRNALys2 within 1 h of the addition of the factor. These data suggest that competence factors have the ability to stimulate the modification of specific tRNALys isoacceptors.

Perturbation of the mitochondrial lysine tRNA population by virus-induced transformation or stress of mammalian cells: functional properties and nucleotide sequence of a mitochondrially associated lysine tRNA.

Eleven isoaccepting lysine tRNAs from mammalian sources are demonstrable by RPC-5 chromatography and polyacrylamide gel electrophoresis. The appearance and amounts of these isoacceptors varies with the source and growth state of cells. One isoacceptor, tRNALys6, observed in preparations of tRNA from some virus-transformed cells in culture, has been characterized by determining functional properties, cellular location, and its nucleotide sequence. tRNALys6 responds primarily to the lysine codon AAA, but it is not used efficiently in a wheat germ translational system in vitro. Compared with lysine isoacceptors 1, 2, 4, 5a, and 5, [3H]lysine appears in vivo in tRNALys6 with a delay of about 3 h. This delay may in part be a result of a less functional tRNA, but a compartmented state of tRNALys6 also appears to be important. tRNALys6 is associated with mitoplasts prepared from KA31 fibroblasts. The nucleotide sequence of tRNALys6 was determined by rapid postlabeling procedures involving limited hydrolysis in formamide, 32P-labeling of 5' ends of fragments with polynucleotide kinase, separation of the nested set of fragments in polyacrylamide denaturing gels, release of 5'-labeled nucleotides with RNase T2, and identification of the released nucleotides by chromatography on PEI cellulose. Confirmation of the positions of major nucleotides was done by using limited digestions by RNases of tRNALys6 labeled with 32P on the 3' terminus in a gel readout procedure. The nucleotide sequence of tRNALys6 differs from that of cytoplasmic lysine tRNAs and mammalian mitochondrial lysine tRNAs. It contains U*, an unidentified modified uridine occurring in the anticodon of some mitochondrial tRNAs. tRNALys6 appears to occur in very limited amounts, or not at all, in most cells unless stressed, but when present it is associated with mitochondria, although it is probably coded in the nucleus.

Smooth-muscle physiology.

In the last several years, significant advances have been made in the understanding of bladder smooth muscle physiology. This article provides a summary for the clinician of current knowledge about the detrusor smooth muscle cell structure, function, and the relationship of structure to function in terms of bladder storage and physical properties such as compliance. The integration of this basic science knowledge into clinical practice is illustrated in discussion of two common disorders: detrusor instability, and outflow obstruction.

Molecular aspects of bladder outlet obstruction.

In an animal model of obstruction, increasing load induces significant smooth muscle hypertrophy which is associated with a down-regulation of myosin heavy chain expression. This undoubtedly contributes to the decreased smooth muscle contractility seen in this model. Moreover, obstruction-induced hypertrophy leads to the development of a dedifferentiated smooth muscle phenotype, as evidenced by a revision of the cell to fetal (of non-muscle) gene expression patterns. Similar alterations are seen in atherosclerotic vessels and other pathologic smooth muscle systems. In these systems, dedifferentiation is also associated with significant alterations in extracellular matrix expression. It seems likely that obstruction in the bladder induces dedifferentiation of the smooth muscle cell which alters contractility as well as extracellular matrix expression, leading to altered bladder performance and decreased compliance.

Alterations in tRNA isoaccepting species during erythroid differentiation of the Friend leukemia cell.

The chromatographic profiles of isoaccepting tRNAs were analyzed at five time points during the 96 hr, dimethylsulfoxide induced, erythroid-like differentiation of Friend leukemia cells. Sixty-four isoaccepting species of tRNA for 16 amino acids were resolved by RPC-5 chromatography. The relative amounts of tRNAphe, tRNAile, and tRNAval species were maintained by the cells during differentiation; whereas the relative amounts of some of the isoacceptor tRNAs for the other 13 amino acids changed significantly. Fluctuations in amounts of isoacceptors occurred between 36 and 72 hr after addition of dimethysulfoxide, corresponding to globin mRNA appearance and hemoglobin synthesis, respectively. In most cases, thepredominant tRNA isoacceptors of uninduced cells were retained throughout differentiation. Notable exceptions were tRNA species for threonine, proline, and methionine. Some of the isoacceptors occurring in relatively smaller amounts were not expressed at all times. These changes possibly reflect the cell's functional adaptation of tRNA in differentiation for hemoglobin synthesis.

Effects of obstruction on bladder contractile proteins.

In an animal model of obstruction, increasing load induces significant smooth muscle hypertrophy which is associated with a down-regulation of myosin heavy chain expression. This undoubtedly contributes to the decreased smooth muscle contractility seen in this model. Moreover, obstruction-induced hypertrophy leads to the development of a dedifferentiated smooth muscle phenotype. Similar alterations are seen in atherosclerotic vessels and other pathologic smooth muscle systems. In these systems, dedifferentiation is also associated with significant alterations in extracellular matrix expression. It seems likely that obstruction in the bladder induces dedifferentiation of the smooth muscle cell which alters contractility as well as extracellular matrix expression, leading to altered bladder performance and decreased compliance.

Uncoupling of muscle-specific protein expression in myocyte x myoblast heterokaryons.

The inducibility of several rat skeletal muscle proteins was examined in heterokaryons formed by fusing differentiated chick myocytes to undifferentiated rat myoblasts. Chicken and rat proteins were distinguished using species-specific antibodies or by their different migrations in polyacrylamide or agarose gels. Both rat skeletal myosin light chain 1 and rat alpha-tropomyosin were induced in the heterokaryons. In contrast, neither rat acetylcholine receptors nor creatine kinase could be detected. These results suggest that chick myocytes may contain quantities of regulatory factors that are sufficient for the activation of some but not all of these rat muscle-specific proteins within the cellular context of the heterokaryon.

Competence and progression growth factors stimulate different tRNAlys modification reactions in BALB/C 3T3 cells.

Sparse cultures of Balb/C 3T3 cells were growth arrested in a medium containing 1% calf plasma. This treatment caused a decrease in tRNA4lys, a tRNA species related to cell division, and a corresponding increase in tRNA5lys. A restoration of tRNA4lys levels and an increase in cell number was obtained by the addition of either 10% calf serum or a combination of fibroblast growth factor and melanocyte stimulating activity. Neither growth factor alone evoked a proliferative response. The progression factors, insulin and multiplication stimulating activity stimulated the rapid conversion of tRNA5lys to tRNA2lys, whereas the competence factors, fibroblast growth factor and platelet-derived growth factor stimulated a slight conversion of tRNA2lys to tRNA4lys.

Specific changes in Q-ribonucleoside containing transfer RNA species during Friend leukemia cell erythroid differentiation.

Changes in specific tRNA isoacceptors during Friend leukemia cell (F.L.C.) erythroid differentiation have been found to be concomitant with differences in the extent of the Q-base modification in certain species of tRNA. Transfer RNA was isolated from F.L.C. cultures after 0, 36, 48, 72, and 96 hr of DMSO induced differentiation. Changes in 17 isoacceptors of tRNAasn, tRNAasp, tRNAhis and tRNAtyr were compared by RPC-5 chromatography. Isoacceptors of these tRNA changed in relative amounts, following consistent trends throughout cell differentiation. The amount and distribution of Q-base containing tRNA isoacceptors was assayed by measuring the quanine-tRNA transferase catalyzed incorporation of [3H]-labeled guanine into tRNA species undermodified in Q-base followed by RPC-5 chormatography of the tRNA. The amount of Q-base containing tRNA species decreased in the first 48 hr after the induction, then increased again, indicating the level of Q-modification is correlated to the process of differentiation. Isoacceptors that lacked the Q-base were eluted late from RPC-5.

Alpha-blockade downregulates myosin heavy chain gene expression in human benign prostatic hyperplasia.

OBJECTIVES: Smooth muscle (SM), a major component of prostate stroma, plays an important role in the pathogenesis of benign prostatic hyperplasia. In many muscle systems, steroid hormones and alpha(1)-adrenergic neurotransmitters tightly regulate expression of contractile proteins. In this study, SM content and the expression of myosin heavy chain (MHC) in tissues from patients with benign prostatic hyperplasia treated with androgen ablation or alpha-blockade were compared with untreated controls. METHODS: Prostatic periurethral tissue specimens from patients receiving luteinizing hormone-releasing hormone analogues (n = 12), alpha-blocking agents (n = 12), and no treatment (n = 13) were examined. The samples were analyzed for SM MHC mRNA expression using competitive reverse transcription-polymerase chain reaction. SM content was measured by morphometric analysis of trichrome-stained sections. RESULTS: Stromal SM constituted 45.4% +/- 8.6%, 48.1% +/- 18.4%, and 45.9% +/- 10.8% of the total tissue in androgen ablated, alpha-blocked, and untreated tissues, respectively. No significant difference was observed among these three groups (P = 0.84, analysis of variance). However, SM MHC mRNA expression was markedly decreased in the alpha-blockade group (0.15 +/- 0.02 attomole/mg tissue) compared with the androgen-ablated (0.58 +/- 0.15 attomole/mg tissue) or control (0.44 +/- 0.10 attomole/mg tissue) groups. The relationship between SM content and expression of SM MHC significantly differed among the groups (P = 0.02, analysis of variance). CONCLUSIONS: Androgen ablation and alpha-blockade do not appear to alter the histologic characteristics of prostate stroma in men with symptomatic benign prostatic hyperplasia. However, contractile protein gene expression in stromal SM cells is significantly altered after alpha-blockade. These data suggest that, in addition to the simple relaxation of muscle tone, alpha-blocking agents may affect the phenotypic expression of contractile proteins in prostate SM cells.

Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD.

In this report, we describe the isolation, sequence, and initial characterization of the cDNA for the muscle-specific regulatory factor skeletal myogenin. Transfection of myogenin into the mesenchymal cell line C3H10T1/2 produces cells expressing muscle-specific markers. Myogenin is absent in undifferentiated cells, peaks, and then declines following a stimulus to differentiate, and is overexpressed in myoblasts selected with 5-bromodeoxyuridine for the overproduction of factors that regulate the decision to differentiate. High levels of myogenin transcripts are present in the myotomal region of somites at 8.5 days of gestation in the mouse. Although myogenin and MyoD are different genes, they share the myc homology domain. Myogenin and MyoD thus form part of a gene family regulating myogenesis, and together with myd may constitute a set of factors that interact to regulate the determination and differentiation of muscle cells.

Structural relationship between tRNALys2 and tRNALys4 from mouse lymphoma cells.

Mouse lymphoma cells have three major isoaccepting lysine tRNAs. Two of these isoacceptors, tRNALys2 and tRNALys4, were sequenced by rapid gel or chromatogram readout methods. They have the same primary sequence but differ in two modified nucleotides. tRNALys4 has an unmodified uridine replacing one dihydrouridine and an unidentified nucleotide, t6A*, replacing t6A. This unidentified nucleotide is not a hypomodified form of t6A. Thus, tRNALys4 is not a simple precursor of tRNALys2. Both tRNAs have an unidentified nucleotide, U**, in the third position of the anticodon. Also, partial sequences of minor homologs of tRNALys2 and tRNALys4 were obtained. The distinctions between tRNALys2 and tRNALys4 may be part of significant cellular roles as illustrated by the differential effects of these isoacceptors on the synthesis by lysyl-tRNA synthetase of diadenosine-5',5'''-P1,P4-tetraphosphate, a putative signal in DNA replication.

Altered response to partial bladder outlet obstruction in mice lacking inducible nitric oxide synthase.

INTRODUCTION: Following prolonged partial bladder outlet obstruction (BOO) in the mouse, cholinergic mediated detrusor contractility decreases. Previous work has demonstrated an increase in the inducible form of nitric oxide synthase (iNOS) at the mRNA and protein levels soon after obstruction. Since nitric oxide (NO), the product of the action of iNOS on molecular oxygen and l-arginine, produces vasodilation and decreases platelet aggregation, we believe it is an integral part of the initial detrusor response to obstruction. These experiments evaluated the detrusor response in mice incapable of producing iNOS. MATERIALS AND METHODS: Wild type and knockout mice were partially obstructed for 1, 3, and 5 weeks. Physiologic evaluation consisted of cystometric analyses, and muscle strip studies in response to cholinergic and electrical stimulation. Strips were also relaxed with L-arginine, sodium nitroprusside, and 8-bromoguanosine 3' - 5' cyclic GMP, after precontraction. RESULTS: After 5 weeks of obstruction, both wild type (WT) and knockout (KO) mouse bladders increased significantly in weight. WT bladders obstructed for 5 weeks had the greatest capacity (increase of 42%, p = 0.022), and a decreased contractile response to carbachol (decrease of 32% at 10-5 M, p = 0.018). No differences were noted at 1 and 3 weeks of obstruction. In contrast, KO mice had a significantly larger bladder capacity at 1 week of obstruction compared with WT, and had significantly lower responses to electrical stimulation than WT at the same time (p = 0.03). Additionally, after 5 weeks of obstruction, bladder capacity and contractility returned to baseline levels in KO mice, at a time when WT mice had significantly larger capacity and decreased contractility. CONCLUSIONS: Bladder function following partial BOO in mice incapable of producing iNOS differed significantly from the normal response. Our data suggest that generation of iNOS soon after obstruction is necessary to prevent detrusor dysfunction at that time. Moreover, the enhanced function seen in KO bladders after longer periods of obstruction (5 weeks) in comparison to WT bladders suggests that reactive nitrogen species-induced protein nitrosylation may be involved in the loss of contractile function observed after more prolonged periods of obstruction.

Physiologic sequelae of partial infravesical obstruction in the mouse: role of inducible nitric oxide synthase.

PURPOSE: To develop a mouse model for partial infravesical obstruction, and determine the resultant changes in bladder function, with particular emphasis on the role of inducible nitric oxide synthase (iNOS) in the bladder response. MATERIALS AND METHODS: Wild type mice were subjected to no intervention, sham operation, and varying durations of partial outlet obstruction (1, 3, and 5 weeks). They then underwent cystometric evaluation, bladder strip stimulation studies using carbachol, and relaxation studies using l-arginine, sodium nitroprusside, and 8-bromoguanosine 3'-5' cyclic guanosine monophosphate. Bladder tissue was subjected to RT-PCR and Western analysis for iNOS. Bladders were also studied histologically using morphometric analysis. RESULTS: Bladders from mice obstructed for 5 weeks were heavier (weight increased by 110%), larger (capacity increased by 73%), and had a higher frequency of abnormal appearing cystometric curves than normal bladders. Tissue bath studies demonstrated decreased contractility in response to cholinergic stimulation at 5 weeks of obstruction (decreased by 55% at maximal stimulation). RT-PCR demonstrated iNOS in approximately 70% of bladders obstructed for 1 and 3 weeks, while the iNOS protein was apparent in 50% of the bladders from the same groups. CONCLUSIONS: This new animal model of infravesical obstruction is reliable and reproducible. Moreover, the physiologic changes noted are comparable to other models, but an added advantage is the relevance of this model with regard to studying new transgenic or knockout mice. Enhanced expression of iNOS seen early after obstruction may serve to improve oxygenation during obstruction-induced ischemia.

Transfer RNATyr of melanoma tissues and cells: relevance to melanin synthesis?

The tRNA present in swine melanoma tumor tissue and normal gray skin tissue were compared by aminoacylation of the unfractionated tRNA preparations. Of the seventeen amino acids studied, seven showed differences in rate of acceptance to tRNAs from normal and tumor tissues; the tRNAs of two amino acids, tyrosine and glycine, showed dramatic three fold increases in melanoma tumor. As melanin biosynthesis proceeds from tyrosine oxidation the investigations focused on the increase in tyrosine tRNA. Kinetic analysis of tyrosine aminoacylation to normal and melanoma tRNAs revealed no differences. Analysis of the isoaccepting species of tRNATyr from normal skin and melanoma tumor tissues identified three isoacceptors; tRNATyr, represented the predominant species in normal gray skin, while tRNA2Tyr predominated in melanoma tumor tissue. The tyrosine acceptances by tRNAs from three human melanoma cell lines were analyzed and found to be variable, but isoaccepting species analysis of the tRNATyr of these three cell lines still showed a correlation between the preponderance of tRNA2Tyr and extent of tyrosine acceptance. Additionally the enzymatic activity for the oxidation of tyrosine was found to be related to tyrosine acceptance and tRNA2Tyr predominance..

Lysine tRNA and cell division: a G1 cell cycle mutant is temperature sensitive for the modification of tRNA5Lys to tRNA4Lys.

Ts-694 is a temperature sensitive mutant of hamster cells which is blocked in the G1 phase of the cell cycle at the restrictive temperature of 39 degrees. A comparison of the Lys-tRNA isoacceptors by RPC-5 chromatography showed a decrease in tRNA5Lys and an increase in tRNA4Lys at 39 degrees. This was identical to the changes seen in confluent cultures at the permissive temperature of 33 degrees. These Lys-tRNA changes were not seen in ts-694 cells blocked in G1 by isoleucine deficiency, nor in two other G1 ts mutants at the restrictive temperature. Cells trapped in S phase by a thymidine block also contained decreased levels of tRNA4Lys when raised to 39 degrees. Both tRNA4Lys levels and cell division increased when the cells were returned to the permissive temperature. An in vitro assay was established for the modification of tRNA5Lys to tRNA4Lys with tRNA6Lys and tRNA2Lys as intermediates. The first reaction is the synthesis of tRNA6Lys which involves the introduction of a modified uridine at the third position of the anticodon. Extracts of 694 cells grown at 33 degrees were able to modify rat liver [3H] tRNA5Lys to tRNA6Lys and tRNA4Lys in vitro when assayed at 25 degrees but not at 39 degrees. Extracts of Balb/c 3T3 cells, however, were more active at 39 degrees than at 25 degrees showing that the normal enzyme is not temperature sensitive. Ts-694 cell tRNA, isolated from cells grown at 33 degrees was aminoacylated at both 25 degrees and 39 degrees with rat liver synthetases. tRNA4Lys was present at both temperatures indicating that ts-694 cells do not contain a temperature sensitive tRNA4Lys.

Contractile protein expression in bladder smooth muscle is a marker of phenotypic modulation after outlet obstruction in the rabbit model.

PURPOSE: We determined changes in contractile protein expression before and after the relief of partial bladder outlet obstruction in the rabbit model and assessed their potential role as predictors of recovery. MATERIALS AND METHODS: We examined the ratio of the smooth muscle myosin heavy chain isoforms SM2-to-SM1, caldesmon isoform expression and bladder function in obstructed and unobstructed adult rabbit bladders. Cystometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were done to determine changes in bladder function and contractile protein expression. RESULTS: Overall we observed significant correlation of bladder weight with the SM2-to-SM1 ratio (p <0.05). Regardless of the duration of obstruction (up to 10 weeks) the ratio appeared to stabilize around a value comparable to that in fetal rabbit smooth muscle cells, suggesting a reversal of SM2 and SM1 expression to a level similar to that at the fetal stage. The pattern of h and l-caldesmon isoform expression showed an increase in l-caldesmon expression in obstructed bladders. Except for decreased leak point pressure in the obstructed group we noted no statistically significant urodynamic changes in bladder capacity or compliance. CONCLUSIONS: There is significant correlation of bladder weight, which is the best known marker of obstruction, with the SM2-to-SM1 ratio. The myosin heavy chain isoform expression ratio appears to be an indicator of phenotypic modulation in bladder smooth muscle before and after the relief of bladder outlet obstruction. Thus, it may be useful as a marker of bladder dysfunction and predictor of functional recovery. Regression to a fetal pattern of protein expression may suggest irreversible damage to smooth muscle cells, possibly limiting recovery.

Myosin heavy chain gene expression in normal and hyperplastic human prostate tissue.

BACKGROUND: Benign prostatic hyperplasia (BPH) is common among aging men. Over 80% of males 50-60 years and older have various degrees of bladder outlet obstruction secondary to BPH. Despite the tremendous medical impact of BPH, its molecular pathophysiology remains unclear. Current BPH research focuses on steroid hormonal effects, stromal-epithelial cell interaction, and oncogenes and growth factors. But little is known about the potential prostatic smooth muscle (SM) alterations that may occur during stromal hyperplasia. METHODS: To study SM phenotypic modulation in hyperplastic prostatic growth, we isolated and characterized the 3' end of human SM myosin heavy chain (SMMHC) cDNA as a molecular probe. Expression of SMMHC and nonmuscle myosin heavy chain (NMMHC) in human prostates was analyzed using Western blot, Northern blot, and in situ hybridization to determine if BPH tissue expresses significantly less SMMHC and more NMMHC than a normal prostate. In addition, a competitive, reverse transcription (RT) polymerase chain reaction (PCR) method was adapted to quantify SMMHC and NMMHC mRNA expression at the sensitivity level of 10(-21) mole per mg of wet tissue. RESULTS: Western blot, Northern blot, and in situ hybridization results reveal that both SMMHC and NMMHC are expressed in the human prostate, while SMMHC is the predominant form found in normal prostate stroma. Results from competitive RT-PCR analysis indicate that NMMHC mRNA expression is approximately 10(-20) mole/mg of tissue. The SMMHC mRNA expressed is approximately 10(-18) mole/mg. No significant difference was found when NMMHC mRNA expression was compared between normal and BPH periurethral tissues. However, SMMHC expression was reduced almost fivefold in BPH compared to normal prostate, despite an increase in prostatic stromal mass. CONCLUSIONS: Our results suggest the pathogenesis of BPH is associated with a unique type of SM proliferation. Such proliferation is characterized by downregulation of SMMHC mRNA expression but without upregulation of NMMHC mRNA expression, the pattern seen in proliferating SM cells in culture and in other pathologic forms of SM hyperplasia (e.g., atherosclerosis). These findings support a model of BPH typified by active smooth muscle proliferation early in the disease process, and supports clinical observations that suggest ongoing prostate growth of the prostate is minimal in older men. Therapeutic strategies to prevent disease progression should therefore focus on early phases of prostatic growth.

Up-regulation of a gene homologous to the human tumor necrosis factor receptor associated factor 6 gene in the obstructed rabbit bladder determined by differential display polymerase chain reaction.

PURPOSE: We identified differentially expressed genes in the rabbit bladder after partial outlet obstruction. MATERIALS AND METHODS: Differential display polymerase chain reaction (PCR) was performed on smooth muscle tissue from normal, 2 and 6-week obstructed rabbit bladders. Semiquantitative reverse transcriptase PCR, Western and RNA blot analysis were done to confirm messenger RNA and protein up-regulation. RESULTS: A signal transducing protein human tumor necrosis factor receptor associated factor 6 (TRAF6)-like protein was identified on differential display PCR. TRAF6-like protein was up-regulated in rabbit bladders after 2 weeks of partial outlet obstruction. Reverse transcriptase PCR demonstrated TRAF6-like protein in bladder muscle tissue and semiquantitative analysis confirmed up-regulation in 2-week obstructed tissue. These findings were confirmed by RNA and Western blot analysis. CONCLUSIONS: TRAF6-like protein is up-regulated during the early phase of bladder outlet obstruction in rabbits. To our knowledge involvement of this gene in bladder outlet obstruction has not been described previously. TRAF6 may have a role in the regulation of molecular changes during the early bladder response to outlet obstruction, such as the up-regulation of growth factors and proto-oncogenes. Further understanding of this signaling pathway and its role in bladder outlet obstruction may open new avenues for treating detrusor dysfunction.