Expression of B-cell translocation gene 2 protein in normal human tissues.

The antiproliferative B-cell translocation gene 2 (BTG2(TIS21/PC3)) is emerging as an important regulator of cell cycle dynamics. BTG2(TIS21/PC3) expression increases in response to the induction of DNA damage, cell differentiation, cell quiescence, cell contact, and as part of a positive feedback mechanism in response to growth stimulation. The objective of the present study was to provide further insight into the biological function of BTG2(TIS21/PC3) by determining the expression levels and cellular localization of BTG2(TIS21/PC3) in a spectrum of normal human tissues and to determine the proliferative indices (based on Ki-67 staining) and apoptotic indices (based on TUNEL assay) in those cell populations where BTG2(TIS21/PC3) was differentially expressed. Highest levels of BTG2(TIS21/PC3) expression were seen in kidney proximal tubules, lung alveolar bronchial epithelium and in the basal cell layer of prostate acini. BTG2(TIS21/PC3) was expressed at significantly different levels within the different epithelial populations of the kidney (proximal vs distal tubules) and prostate (acinar basal cells vs lumenal cells). Moderate levels of expression were seen in the acinar cells of breast and pancreas and in the mucosal epithelium of the intestine. Low levels of expression were seen in neurons, hepatocyctes, the zona granulosa of the ovary, round spermatids and thyroid follicles. Our results therefore indicate an imperfect correlation between the terminally differentiated phenotype and BTG2(TIS21/PC3) expression, but no correlation between basal cellular proliferative or apoptotic indices and BTG2(TIS21/PC3) expression levels.

Antiproliferative B cell translocation gene 2 protein is down-regulated post-transcriptionally as an early event in prostate carcinogenesis.

B cell translocation gene 2 (BTG2) is a p53 target that negatively regulates cell cycle progression in response to DNA damage and other stress. The objective of this study was to examine the expression, regulation and tumor suppressor properties of BTG2 in prostate cells. By immunohistochemistry BTG2 protein was detected in approximately 50% of basal cells in benign glands from the peripheral zone of the human prostate. BTG2 was expressed in all hyperproliferative atrophic peripheral zone lesions examined (simple atrophy, post-atrophic hyperplasia and proliferative inflammatory atrophy), but was undetectable or detectable at very low levels in the hyperproliferative epithelial cells of HGPIN and prostate cancer. BTG2 mRNA was detected in non-malignant prostate epithelial (PE) cells and in LNCaP cells, but not in PC-3 cells, consistent with p53-dependent regulation. In PE cells BTG2 protein was detected in areas of cell confluence by immunohistochemistry. BTG2 protein in LNCaP cells was undetectable by immunohistochemistry but was detected by immunoblotting at 8- to 9-fold lower levels than in PE cells. BTG2 protein levels were shown to be regulated by the ubiquitin-proteosome system. Forced expression of BTG2 in PC-3 cells was accompanied by a decreased rate of cell proliferation and decreased tumorigenicity of these cells in vivo. Taken together, these findings suggest that BTG2 functions as a tumor suppressor in prostate cells that is activated by cell quiescence, cell growth stimuli as part of a positive feedback mechanism and in response to DNA damage or other cell stress. The low steady-state levels of BTG2 protein in HGPIN and prostate cancer, a potential consequence of increased proteosomal degradation, may have important implications in the initiation and progression of malignant prostate lesions. Furthermore, these findings suggest that a significant component of the p53 G(1) arrest pathway might be inactivated in prostate cancer even in the absence of genetic mutations in p53.

Expression of the ET(A) and ET(B) endothelin receptor subtype mRNA in human detrusor cultured smooth muscle cells.

The aim of this study was to determine the expression of the endothelin receptor subtype mRNAs in human detrusor cultured smooth muscle cells using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). First strand cDNA was made from human detrusor cultured smooth muscle cells total RNA and used for PCR with primers designed to amplify fragments of the ET(A) and ET(B) endothelin receptor subtype cDNA sequences. Subcloned fragments of the ET(A) and ET(B) endothelin receptor cDNAs were used to synthesize digoxigenin-labeled cRNA probes by in vitro transcription. COS-7 cells transfected with the ET(A) and ET(B) receptor cDNAs were used as positive control and to confirm the absence of cross-hybridization due to sequence homology. Both ET(A) and ET(B) receptor mRNAs were detected by RT-PCR analysis. By ISH, both ET(A) and ET(B) receptor subtype mRNAs were detected. However, ET(A) signal was much more intense than ET(B) signal. These results indicate that mRNAs for both ET(A) and ET(B) receptors are expressed in detrusor smooth muscle cells of human urinary bladder. The ET(A) receptor is the predominant detrusor ET receptor.

Interactions between beta 2-syntrophin and a family of microtubule-associated serine/threonine kinases.

A screen for proteins that interact with beta 2-syntrophin led to the isolation of MAST205 (microtubule-associated serine/threonine kinase-205 kD) and a newly identified homologue, SAST (syntrophin-associated serine/threonine kinase). Binding studies showed that beta 2-syntrophin and MAST205/SAST associated via a PDZ-PDZ domain interaction. MAST205 colocalized with beta 2-syntrophin and utrophin at neuromuscular junctions. SAST colocalized with syntrophin in cerebral vasculature, spermatic acrosomes and neuronal processes. SAST and syntrophin were highly associated with purified microtubules and microtubule-associated proteins, whereas utrophin and dystrophin were only partially associated with microtubules. Our data suggest that MAST205 and SAST link the dystrophin/utrophin network with microtubule filaments via the syntrophins.

Mitogenic activation of human prostate-derived fibromuscular stromal cells by bradykinin.

Biologically active kinin peptides are released from precursor kininogens by kallikreins. Kinins act on kinin receptors to mediate diverse biological functions including smooth muscle contraction, inflammation, pain and mitogenicity. All components of the kallikrein-kinin system exist in human male genital secretions suggesting that these molecules participate in physiological and pathophysiological genitourinary function. The objective of this study was to assess the consequences of kinin action on prostate cells. Primary cultures of prostate secretory epithelial (PE) and prostate fibromuscular stromal (PS) cells were established from human prostate tissue. Transcripts encoding both the human B1 and B2 bradykinin receptor subtypes were detected in human prostate transition-zone tissue and in cultured cells by RT-PCR. In receptor binding assays, the B1 subtype predominated on PE cell membranes and the B2 subtype predominated on PS cell membranes. In PS cells, but not in PE cells, BK induced significant inositol phosphate accumulation and [3H]-thymidine uptake. These responses were mediated through the B2 receptor subtype. The use of signal transduction inhibitors indicated that mitogenic activation by BK occurred through both protein kinase C (PKC) and protein tyrosine kinase dependent mechanisms. PMA (phorbol 12-myristate 13-acetate) produced maximal [3H]-thymidine uptake by PS cells, resulted in cell elongation and caused the alpha-actin fibres present in PS smooth muscle cells to became organized into parallel arrays along the length of the elongated cells. In summary, the prostate contains a functional kallikrein-kinin system, which could be significant in physiological and pathophysiological prostate function.

Whistler technique used to reduce traumatic dislocation of the hip in the emergency department setting.

The authors appraised the effectiveness of an in-line traction technique developed to reduce posteriorly dislocated hips. We had found certain application difficulties with the Allis, or modified Allis, technique, and subsequently developed a method that was easier to implement for the physician. The dislocated hip is relocated using the physician's arm to raise and maneuver the affected leg as the physician's shoulder is raised. Patient data for the case series were collected from March 1994 to March 1998.

Localization and expression of the alpha1A-1, alpha1B and alpha1D-adrenoceptors in hyperplastic and non-hyperplastic human prostate.

PURPOSE: To determine the expression and localization of the alpha1A-1, alpha1B and alpha1D-adrenoceptor (AR) subtypes in hyperplastic and non-hyperplastic human prostate tissue. MATERIALS AND METHODS: The expression of the alpha1-AR subtypes was examined at the mRNA level by quantitative solution hybridization, and at the protein level by immunohistochemistry using subtype selective antibodies. RESULTS: While the overall level of alpha1-AR mRNA was not significantly different between hyperplastic and non-hyperplastic tissue, there were significant differences in the ratio of the alpha1-AR subtypes expressed in the two tissue types. The most significant finding from these studies was the reduced expression of the alpha1b-AR mRNA in both glandular and stromal hyperplasia. By immunohistochemistry, the alpha1A-1-AR was detected in the stroma and not in the glandular epithelium. The alpha1B-AR was localized predominantly in the epithelium and was weakly present in the stroma. Lower levels of the alpha1B-AR were detected in the hyperplastic prostatic epithelium. The alpha1D-AR was detected in areas of stroma and was abundantly present in blood vessels. CONCLUSIONS: The alpha1A-1-, alpha1B- and alpha1D-AR subtypes are differentially localized in human prostate, and the expression levels of all three subtypes are altered in BPH. Alterations in a1-AR subtype expression (particularly the alpha1B-AR) in BPH cannot be solely attributed to changes in tissue morphometry resulting from hyperplasia and may be of significance in the pathogenesis of BPH.

Identification of genes associated with stromal hyperplasia and glandular atrophy of the prostate by mRNA differential display.

Despite the well-characterized histology associated with benign prostatic hyperplasia, very little is known about the underlying etiology of the disease on a molecular basis. The objective of this study was to use the technique of mRNA differential display in order to identify genes differentially expressed in human transition zone prostate tissue with high stromal density, with high epithelial density, and with nonhyperplastic histology. The extracellular matrix chondroitin/dermatan sulfate proteoglycan (CDSP) mRNA was more abundantly expressed in tissue with high stromal density, consistent with earlier findings that dermatan and chondroitin 6-sulfate glycosaminoglycans are increased in hyperplastic prostates. Messenger RNA encoding the negative regulator of cell cycle progression, BTG2, was more abundantly expressed in tissue with high epithelial densities. CDSP mRNA was abundantly expressed in primary cultures of stromal cells but was undetectable in epithelial cells. BTG2 mRNA was expressed in primary cultures of both cell types, but more abundantly in epithelial cells. BTG2 mRNA, but not CDSP mRNA, was subject to significant growth cycle regulation in cultured stromal and epithelial cells, with maximum expression occurring in quiescent cells. Generation of specific antibodies to BTG2 revealed that this protein was expressed at low levels in stroma, nonhyperplastic glands, and in hyperplastic glands. Consistent with a role in cell-cycle regulation, BTG2 protein was abundantly expressed in atrophic glands and preatrophic glands.

Evidence that mammalian phosphatidylinositol transfer protein regulates phosphatidylcholine metabolism.

Phosphatidylinositol transfer proteins (PITPs) and their yeast counterpart (SEC14p) possess the ability to bind phosphatidylinositol (PtdIns) and transfer it between membranes in vitro. However, the biochemical function of these proteins in vivo is unclear. In the present study, the physiological role of PITP was investigated by determining the biochemical consequences of lowering the cellular content of this protein. WRK-1 rat mammary tumour cells were transfected with a plasmid containing a full-length rat PITPalpha cDNA inserted in the antisense orientation and the resultant cell clones were analysed. Three clones expressing antisense mRNA for PITPalpha were compared with three clones transfected with the expression vector lacking the insert. The three antisense clones had an average of 25% less PITPalpha protein than control clones. Two of the three antisense clones also exhibited a decreased rate of growth. All three antisense clones exhibited a significant decrease in the incorporation of labelled precursors into PtdCho during a 90-min incubation period. Under the same conditions, however, there was no change in precursor incorporation into PtdIns. Further experimentation indicated that the decrease in precursor incorporation seen in antisense clones was not due to an increased rate of turnover. When choline metabolism was analysed more extensively in one control (2-5) and one antisense (4-B) clone using equilibrium-labelling conditions (48 h of incubation), the following were observed: (1) the decrease in radioactive labelling of PtdCho seen in short-term experiments was also observed in long-term experiments, suggesting that the total amount of PtdCho was lower in antisense-transfected clones (this was confirmed by mass measurements); (2) a similar decrease was seen in cellular sphingomyelin, lysoPtdCho and glycerophosphorylcholine; (3) an average two-fold increase in cellular phosphorylcholine was observed in the antisense-transfected clone; (4) cellular choline was, on average, decreased; and (5) cellular CDPcholine was not significantly altered.

Localization of P2Y1 purinoceptor transcripts in the rat penis and urinary bladder.

PURPOSE: The aim of this study was to determine the expression and localization of the P2Y1 purinoceptor mRNA in rat penis and urinary bladder using reverse transcription polymerase chain reaction (RT-PCR), northern blotting and in situ hybridization (ISH). MATERIALS AND METHODS: RT-PCR: First strand cDNA was prepared from rat penis and urinary bladder dome total RNA and used for PCR with primers designed to amplify fragments of the P2Y1 purinoceptor cDNA sequence. Northern blotting: PCR products were subcloned into the pGEM-5Zf(+) plasmid vector, sequenced and random primer labeled using 32p. Labeled probe was hybridized. ISH: Digoxigenin labeled cRNA probes were synthesized by in vitro transcription. RESULTS: P2Y1 purinoceptor mRNA was detected by RT-PCR analysis in both rat penis and urinary bladder. RNA blotting using a P2Y1 purinoceptor cDNA probe revealed a single transcript of 4.2kb in both tissues. This band was the same size as that expressed by the heart, which contains high levels of P2Y1 purinoceptor (Burnstock, G.: Physiological and pathological roles of purines: an update. Drug. Dev. Res., 28: 195, 1993). By ISH, P2Y1 purinoceptor mRNA was localized in detrusor smooth muscle cells and blood vessels in urinary bladder. In penis, positive signals were detected in endothelial cells which line the lacunar space and blood vessels. No hybridization was seen in corpus cavernosum smooth muscle cells and urethra. CONCLUSION: These results indicate that mRNAs for P2Y1 purinoceptor are expressed in detrusor smooth muscle cells and blood vessels of rat urinary bladder. However, in penis, this receptor is expressed in endothelial cells which lines the lacunar space and blood vessels, but not expressed in corpus cavernosum smooth muscle cells and urethra.

Endothelin-1 production and agonist activities in cultured prostate-derived cells: implications for regulation of endothelin bioactivity and bioavailability in prostatic hyperplasia.

BACKGROUND: Endothelin-1 (ET-1) interacts with specific G-protein-coupled receptors to initiate short-term (contraction) and long-term (mitogenesis) events in target cells. ET-1 is an abundant prostate secretory protein that, in its biologically active form, elicits prostatic smooth muscle contraction. The present study was designed to determine the effects of ET-1 on prostate cell growth and to examine the regulation of endogenous ET-1 activity and bioavailability. METHODS: Primary cultures of prostate secretory epithelial (PE) and prostate fibromuscular stromal (PS) cells were established from benign human prostate tissue. RESULTS: In culture, PE cells secrete immunoreactive ET-1 (38.5 +/- 1.6 pg/ml/10(6) cells/24 hr) into the conditioned medium. Levels of immunoreactive ET-1 produced by PS cells were more than 10-fold lower. Endothelin-converting enzyme-1 (ECE-1) mRNA was detected in PE cells and not in PS cells; however, big ET-1 was the predominant immunoreactive ET-1 secretory product of PE cells. The ET(B) endothelin receptor was the predominant subtype in both PE and PS cells. In PS cells, but not PE cells, ET-1 induced significant inositol phosphate accumulation and [3H]-thymidine uptake. Agonist activity was inhibited by the ET(B) receptor selective antagonist, BQ 788. Intact PE cell monolayers secrete ET-1 through the apical surface, consistent with secretion of ET-1 into the glandular lumen in vivo. CONCLUSIONS: On the basis of these findings, regulation of ET-1 activity and bioavailability appears to be tightly regulated. Such findings have important implications in the pathophysiology of prostate disease.

Evidence that the mammary fat pad mediates the action of growth hormone in mammary gland development.

Recent evidence from our laboratory suggests that GH and insulin-like growth factor I (IGF-I) mediate glandular mammary development together with estrogen. It has also been well established that both stromal and epithelial elements must interact for mammary glandular development to occur. To determine whether the effect of GH is mediated by the stromal or epithelial tissue, we set up the following experiment. Bovine GH (bGH; 100 microg) or BSA (as a control), without or with estradiol (E2), was injected i.p. into sexually immature female rats that were hypophysectomized and oophorectomized. Mammary glands and subscapular fat pads were removed from the animals. The mammary glands were divided into two parts: a gland-free fat pad and remaining glandular tissue. The end point of bGH activity was induction of IGF-I messenger RNA (mRNA). This was determined quantitatively by solution hybridization and also by RT-PCR. We found that the effects of GH on stimulation of IGF-I mRNA in the gland-free mammary fat pad and the remainder of the mammary gland were similar (3.6- vs. 3.9-fold, respectively; P < 0.001). In both sorts of mammary tissue, bGH was found to synergize with E2 in the induction of IGF-I mRNA (5.8- vs. 5.3-fold; P < 0.001). There was also an increase in IGF-I mRNA in subscapular fat pads in response to 100 microg bGH (5.3-fold; P < 0.001); however, no synergism between bGH and E2 was found. These data indicate that bGH works as well on mammary stromal tissue as on tissue with glands and suggests that GH acts on the stromal compartment of the mammary gland to induce IGF-I mRNA and possibly IGF-I itself, which, in turn, causes differentiation of epithelial ducts into terminal end buds. These data also might explain why mammary epithelium is also able to differentiate in nonmammary fat pads when transplanted there.

Evaluation of the effect of endothelin-1 and characterization of the selective endothelin a receptor antagonist PD155080 in the prostate.

PURPOSE: To evaluate the contractile effect of endothelin-1 (ET-1) on prostatic urethral pressure and to characterize the effect of the selective ETA receptor antagonist PD155080 on ET-1 mediated prostatic urethral pressure. MATERIALS AND METHODS: The effect of intravenous ET-1 administration on canine urethral pressure was determined in the presence and absence of PD155080. The affinity of PD155080 for endothelin-mediated contraction was determined using antagonist dissociation studies. Saturation and competition binding studies were performed using [125I] ET-1 in both human and canine prostate. RESULTS: ET-1 bolus injection elicited shallow and prolonged increases the prostatic urethral pressure. Pretreatment with PD155080 totally abolished the urethral contractile response to ET-1. Specific [125I] ET-1 binding was saturable and of high affinity. Two ET receptor subtypes (ETA receptor, ETB receptor) have been identified in human prostate. The ratio of ETA to ETB receptors was approximately 1.5:1 in both human and canine prostates. Isometric tension studies revealed that PD155080 shifted the ET-1 dose-response curves to the right and exhibited no effect on the ETB receptor selective agonist sarafotoxin dose-response curves. CONCLUSION: ET-1 mediates prostate smooth muscle tone and may play a role in the pathophysiology and treatment of benign prostatic hyperplasia (BPH).

Localization of mRNA and receptor binding sites for the alpha 1a-adrenoceptor subtype in the rat, monkey and human urinary bladder and prostate.

PURPOSE: To localize the mRNAs and receptor binding sites for the alpha 1a/A, alpha 1b/B and alpha 1d/D- adrenoceptor (AR) subtypes in the rat, monkey and human urinary bladder and prostate. MATERIALS AND METHODS: alpha 1-AR mRNAs were localized on slide mounted tissue sections by in situ hybridization using [35S]-labeled subtype specific oligonucleotide probes. alpha 1-AR receptor binding sites were localized on slide mounted tissue sections by competitive displacement of [3H]-prazosin using subtype selective ligands. RESULTS: Only the alpha 1a-AR subtype mRNA was discernible by in situ hybridization. The alpha 1a-AR mRNA was localized in all smooth muscle areas of the rat, monkey and human urinary bladder and prostate. High levels of alpha 1a mRNA were detected in bladder dome and bladder base urothelium. Competitive displacement studies using the alpha 1A-AR selective ligand SNAP 5272 revealed that the alpha 1A-AR represented over 80% of the total alpha 1-AR in monkey bladder and prostate. In general, localization of the alpha 1A-AR corresponded to the alpha 1a-AR mRNA localization, that is, receptor protein was localized to smooth muscle areas of the bladder dome, trigone and base and prostate. One notable exception was the bladder urothelium, which contained high levels of alpha 1a-AR mRNA, but undetectable levels of alpha 1A-AR protein. The alpha 1a-AR mRNA appeared to be transcribed but not translated in bladder urothelium. CONCLUSIONS: The alpha 1A-AR represents the major subtype in the smooth muscle of rat, monkey and human urinary systems. Selective alpha 1A-AR agents are therefore potentially useful in the treatment of multiple urinary smooth muscle related disorders.

Increased activity associated with the MAST205 protein kinase complex during mammalian spermiogenesis.

The morphological and biochemical changes that occur in the haploid male germ cell during spermiogenesis facilitate the natural delivery of the paternally imprinted chromosomes into oocytes. Despite the obvious morphological changes, little is known about the molecular events underlying spermiogenesis. We recently cloned a novel 205-kDa manchette microtubule-associated serine/threonine protein kinase (MAST205) from mouse testis. The objective of this study was to further delineate the role of MAST205 in mammalian spermiogenesis. While MAST205 RNA levels were similar in pachytene spermatocytes, round spermatids, and residual bodies, MAST205 protein could be detected only in round spermatids and residual bodies. Kinase activity associated with MAST205 immunoprecipitates was low in pachytene spermatocytes, high in round spermatids, and maximal in residual bodies, indicating that MAST205-associated kinase activity is modified during spermatid maturation. Furthermore, MAST205 protein and the associated kinase activity were not detected in epididymal spermatozoa, indicating that MAST205 protein is either excluded from, or degraded in, the latter cell type. Multiple heterologous protein species were seen in immunoprecipitates from 35S-labeled mouse seminiferous tubules using an affinity-purified MAST205 antiserum. Consistent with this observation, MAST205 eluted as part of a 1-2 x 10(6) dalton protein complex when extracts of mouse testis were fractionated by Superose 6 column chromatography. MAST205 mRNA was detected in human testis indicative of conservation in other mammalian species. Taken together, these results indicate that the MAST205 complex functions in spermatid maturation in mammals.

The alpha-adrenoceptor antagonist properties of the enantiomers of doxazosin in the human prostate.

The alpha-adrenoceptor antagonist properties of doxazosin and its enantiomers were characterized using human prostate tissue and cell membranes isolated from rat-1 fibroblast expressing each of the cloned human alpha 1-adrenoceptor subtypes. In the alpha 1-adrenoceptor-binding studies on the human prostate with [3H]doxazosin and 2-{[beta-(3-[125I],4-hydroxyphenyl)ethyl]aminomethyl}-l-tetralone ([125I]HEAT), no significant differences were observed between racemic doxazosin, R-doxazosin and S-doxazosin (mean -log Ki (pKi) values were 8.60-8.63, 8.47-8.55 and 8.61-8.65, respectively), whereas the alpha 2-adrenoceptor-binding studies with [3H]rauwolscine and [3H]clonidine revealed that the alpha 2-adrenoceptor-binding affinity of S-doxazosin (pKi = 5.91-5.94) was slightly (3- or 4-fold), but significantly lower than that of R-doxazosin (pKi = 6.47-6.54). Studies in phenylephrine-contracted prostatic tissue showed no significant difference in alpha 1-adrenoceptor antagonist potency between racemic doxazosin, R-doxazosin and S-doxazosin (pA2 values were 8.43 +/- 0.28, 8.64 +/- 0.56 and 8.75 +/- 0.38, respectively). In the binding studies with cloned alpha 1-adrenoceptor subtypes using [3H]prazosin and [125I]HEAT, racemic doxazosin, R-doxazosin and S-doxazosin showed no selectivity for the alpha 1-adrenoceptor subtypes. The present study demonstrated that doxazosin and its enantiomers are highly selective alpha 1-adrenoceptor antagonists and that there is no evidence suggesting differential alpha 1-adrenoceptor antagonist effects of doxazosin and its enantiomers in the human prostate. Doxazosin, therefore, could be described as displaying balanced activity across all three alpha 1-adrenoceptor subtypes.

A novel cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin.

The folding of alpha- and beta-tubulin requires three proteins: the heteromeric TCP-1-containing cytoplasmic chaperonin and two additional protein cofactors (A and B). We show that these cofactors participate in the folding process and do not merely trigger release, since in the presence of Mg-ATP alone, alpha- and beta-tubulin target proteins are discharged from cytoplasmic chaperonin in a nonnative form. Like the prokaryotic cochaperonin GroES, which interacts with the prototypical Escherichia coli chaperonin GroEL and regulates its ATPase activity, cofactor A modulates the ATPase activity of its cognate chaperonin. However, the sequence of cofactor A derived from a cloned cDNA defines a 13-kD polypeptide with no significant homology to other known proteins. Moreover, while GroES functions as a heptameric ring, cofactor A behaves as a dimer. Thus, cofactor A is a novel cochaperonin that is structurally unrelated to GroES.

A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette.

To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules.

Recognition of specific Physarum alpha-tubulin isotypes by a monoclonal antibody. Sequence heterogeneity around the acetylation site at lysine 40.

The monoclonal antibody 6-11B-1 recognises specifically the acetylated form of alpha-tubulin. The acetylation event occurs on a unique lysine residue, lysine 40. Using 6-11B-1, acetylated alpha-tubulin was detected in myxamoebae but not plasmodia of Physarum polycephalum. Following chemical acetylation plasmodial alpha-tubulin was detected by 6-11B-1. The monoclonal antibody KMP-1 recognises certain Physarum alpha-tubulin isotypes but only in non-acetylated form. Whilst recognising all the non-acetylated fraction of myxamoebal alpha-tubulin only a proportion of plasmodial alpha-tubulin was recognised by KMP-1. Peptides were synthesised corresponding to the acetylation domains (containing lysine 40) of myxamoebal alpha-tubulin and the inferred acetylation domains of two plasmodial-specific alpha-tubulin isotypes. The only difference between the two peptides was at a single residue corresponding to amino acid 44 in the polypeptide. Tyrosine was present in myxamoebal alpha-tubulin and glycine was present in the plasmodial specific peptides; the peptides are referred to as the Tyr44 and Gly44 peptides respectively. Both peptides in acetylated form blocked 6-11B-1 reactivity towards acetylated myxamoebal alpha-tubulin. The Tyr44 but not the Gly44 peptide blocked KMP-1 reactivity towards non-acetylated myxamoebal alpha-tubulin. Tyrosine at position 44 is not found in any other known alpha-tubulin. Thus a unique antigenic determinant exists in certain Physarum alpha-tubulin isotypes, close to the acetylation site at lysine 40. This antigenic determinant forms part of the KMP-1 recognition epitope and explains the unique isotype selectivity of this monoclonal antibody.