Posthumous sperm utilization in men presenting for sperm banking: an analysis of patient choice.

The question remains as to whether or not men would agree to posthumous sperm use for pregnancy initiation. Often, these individuals' lives are suddenly interrupted and prior consent is rarely given. Therefore, post-mortem retrieval or use of these spermatozoa remains controversial and the incidence of consent for post-mortem sperm use is not clear. Men who bank spermatozoa, however, represent a cohort that can be examined for frequency of consent for post-mortem sperm use. We performed a retrospective chart review for 364 patients presenting for sperm banking at a single institution from 2009 to 2011. Banked specimens represented either ejaculated or surgically retrieved spermatozoa. Demographic information was obtained for each patient and men were grouped by reason for sperm banking, relationship and paternity status, and consent for post-mortem sperm use. The frequency of post-mortem consent was determined within each group. Men were grouped based on reason for banking, including infertility ('Infertility') or malignancy prior to treatment ('Cancer'). Mean ± SD age of the infertility and cancer groups were 40.1 ± 9.9 years and 27.1 ± 9.6 years, respectively. Of the 364 men, 85.9% provided consent for post-mortem sperm use. In the infertility group, 87.4% of men consented. Of these, 92.9% men in a relationship and 62.5% single men consented. Regarding paternity status, 64.7% men with and 56.6% men without children consented. Within the cancer cohort, 83.8% men consented. Of men <18 years old and ≥18 years old, 65.2 and 85.8% consented, respectively. Relationship status yielded 93.2% men in relationships and 79.4% single men consenting. Paternity status in the cancer group yielded 95.8% with and 82.4% men without children consenting. In summary, most men presenting for sperm banking provided consent for post-mortem sperm use, irrespective of reason for banking. Men who are in a relationship or who are fathers were more likely to agree to post-mortem sperm use.

Akt1 deficiency modulates reward learning and reward prediction error in mice.

In contemporary reinforcement learning models, reward prediction error (RPE), the difference between the expected and actual reward, is thought to guide action value learning through the firing activity of dopaminergic neurons. Given the importance of dopamine in reward learning and the involvement of Akt1 in dopamine-dependent behaviors, the aim of this study was to investigate whether Akt1 deficiency modulates reward learning and the magnitude of RPE using Akt1 mutant mice as a model. In comparison to wild-type littermate controls, the expression of Akt1 proteins in mouse brains occurred in a gene-dosage-dependent manner and Akt1 heterozygous (HET) mice exhibited impaired striatal Akt1 activity under methamphetamine challenge. No genotypic difference was found in the basal levels of dopamine and its metabolites. In a series of reward-related learning tasks, HET mice displayed a relatively efficient method of updating reward information from the environment during the acquisition phase of the two natural reward tasks and in the reverse section of the dynamic foraging T-maze but not in methamphetamine-induced or aversive-related reward learning. The implementation of a standard reinforcement learning model and the Bayesian hierarchical parameter estimation show that HET mice have higher RPE magnitudes and that their action values are updated more rapidly among all three test sections in T-maze. These results indicate that Akt1 deficiency modulates natural reward learning and RPE. This study showed a promising avenue for investigating RPE in mutant mice and provided evidence for the potential link from genetic deficiency, to neurobiological abnormalities, to impairment in higher-order cognitive functioning.

Behavioral phenotyping of v-akt murine thymoma viral oncogene homolog 1-deficient mice reveals a sex-specific prepulse inhibition deficit in females that can be partially alleviated by glycogen synthase kinase-3 inhibitors but not by antipsychotics.

Schizophrenia is a severe mental illness with a strong genetic predisposition. Accumulating evidence from human genetics and animal studies suggest v-akt murine thymoma viral oncogene homolog 1 (Akt1) might contribute to susceptibility for schizophrenia. In contrast to inconclusive findings in human genetic studies, a mutant mouse model is a simplified and alternative approach to determining the biological functions of AKT1 and its possible role in the pathogenesis of schizophrenia. In study 1, a comprehensive battery of behavioral tests was performed on both male and female mice. The results of behavioral phenotyping did not reveal significant differences between genotypes or sexes, except increased time of immobility in the tail suspension test and acoustic prepulse inhibition (PPI) deficits in Akt1-knockout females. On the basis of the observed PPI deficit, in study 2a, neuromorphological alterations were examined with morphometric analysis of green fluorescent protein (GFP)-labeled pyramidal neurons in the auditory cortex of female mice. The results indicated abnormalities in the architecture and complexity of the neurons of mutant females compared with those of the controls. In study 2b, potentially effective pharmacological treatments were explored to mitigate the observed PPI deficits in females. Antipsychotics (either raclopride (3 mg/kg) or clozapine (3 mg/kg)) did not alleviate observed PPI deficits in Akt1-knockout females but it was partially normalized by 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT, 5 mg/kg) and SB216763 (2.5 mg/kg). These findings imply the importance of AKT1 in some behavioral phenotypes and dendritic morphology in the auditory cortex of female mice, and they also suggest that subjects with Akt1 deficiency are insensitive to antipsychotic drugs, whereas glycogen synthase kinase-3 (GSK3) inhibitors could have therapeutic potential for the treatment of acoustic PPI deficits.

100% rapid rescreening for quality assurance in a quality control program in a public health cytologic laboratory.

OBJECTIVE: To verij5 the efficacy of the quality control (QC) program in a cytologic laboratwy with a rapid rescreening (RR) protocol. STUDY DESIGN: RR, according to the Turret RR method, of all samples initially screened as negative at the Laboratory of Cytology, Adolfo Lutz Institute, was performed. The slides were reviewed for 60 seconds. Suspect smears were fully checked by 2 reviewers to determine the final diagnoses. A total of 2954 sequential cytologic results were considered in this study. Of the 2954, 2568 (86.9%) were considered initially negative according to our internal QC, and these cases underwent RR. Also, 10% were randomly selected from these negative cases for full reviewing. The internal QC in our laboratory includes review of cases selected according to clinical and cytomorphologic criteria. RESULTS: Among the 2954 total cases, QC detected 386 (13%) atypias with final diagnoses reported according to The Bethesda System 2001 as follows: 82 (2.18%) low grade squamous intraepithelial lesions (LSILs), 35 (1.18%) high grade squamous intraepithelial lesions (HSILs), 2 (0.06%) squamous cell carcinomas, 105 (3.5%) atypical cells of undetermined significance (ASC-US), 4 (0.12%) atypical endocervical cells (AECs) and 158 (5.3%) unsatisfactory samples. RR of 2568 smears initially considered negative selected 194 (7.5%) slides. Of the 194, 146 (75.3%) were negative, 28 (14.4%) ASC-US, 5 (2.6%) AEC, 1 (0.5%) LSIL and 14 (7.2%) unsatisfactory. Full review of a 10% random fraction of the 2568 cases interpreted as negative did not detect lesions but did detect 5 (1.95%) unsatisfactory samples. CONCLUSION: Internal QC used in our laboratory based on clinical and cytomorphologic criteria to select cases for review proved to be an efficient method of detecting HSIL and cervical cancer. The consensus basis of this program strongly limits the false positive and false negative rates and also provides subjects with continuing education. One hundred percent RR is more efficient than 10% full reviewing in detecting cervical abnormalities.

Decreased sensitivity of tristetraprolin-deficient cells to p38 inhibitors suggests the involvement of tristetraprolin in the p38 signaling pathway.

Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor alpha secretion. However, bone marrow-derived macrophages from tristetraprolin (TTP)-deficient mice were less sensitive than normal macrophages to this effect of p38 inhibitors, despite evidence for normal p38 activation in response to lipopolysaccharide. TTP is known to cause decreased stability of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor mRNAs after binding to an AU-rich element in their 3'-untranslated regions. A recombinant TTP fusion protein could be phosphorylated by a recombinant p38 kinase in cell-free assays and was phosphorylated to the same extent by immunoprecipitated p38 derived from normal and TTP-deficient cells stimulated with lipopolysaccharide; in both cases, the enzyme activity was inhibited by the p38 inhibitors. TTP phosphorylation also was increased in intact macrophages after lipopolysaccharide stimulation, an effect that was blocked by the p38 inhibitors. Finally, TTP in mammalian cell extracts bound less well to an AU-rich element RNA probe than did the same amount of TTP following dephosphorylation. These results suggest that TTP may be a component of the signaling cascade, initiated by inflammatory stimuli and mediated in part by activation of p38, that ultimately leads to enhanced secretion of tumor necrosis factor alpha.

Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail.

The CCCH family of tandem zinc finger proteins has recently been shown to promote the turnover of certain mRNAs containing class II AU-rich elements (AREs). In the case of one member of this family, tristetraprolin (TTP), absence of the protein in knockout mice leads to stabilization of two mRNAs containing AREs of this type, those encoding tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor. To begin to decipher the mechanism by which these zinc finger proteins stimulate the breakdown of this class of mRNAs, we co-transfected TTP and its related CCCH proteins into 293 cells with vectors encoding full-length TNFalpha, granulocyte-macrophage colony-stimulating factor, and interleukin-3 mRNAs. Co-expression of the CCCH proteins caused the rapid turnover of these ARE-containing mRNAs and also promoted the accumulation of stable breakdown intermediates that were truncated at the 3'-end of the mRNA, even further 5' than the 5'-end of the poly(A) tail. To determine whether an intact poly(A) tail was necessary for TTP to promote this type of mRNA degradation, we inserted the TNFalpha ARE into a nonpolyadenylated histone mRNA and also attached a histone 3'-end-processing sequence to the 3'-end of nonpolyadenylated interleukin-3 and TNFalpha mRNAs. In all three cases, TTP stimulated the turnover of the ARE-containing mRNAs, despite the demonstrated absence of a poly(A) tail. These studies indicate that members of this class of CCCH proteins can promote class II ARE-containing mRNA turnover even in the absence of a poly(A) tail, suggesting that the processive removal of the poly(A) tail may not be required for this type of CCCH protein-stimulated mRNA turnover.

Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA.

Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA. TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha mRNA. In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish. We show here that representatives of all four family members were able to bind to the TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha mRNA in intact cells. Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays. We found that amino- and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNFalpha ARE in cell-free cross-linking and gel shift assays. In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNFalpha mRNA in intact cells. Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively.

Evidence that tristetraprolin is a physiological regulator of granulocyte-macrophage colony-stimulating factor messenger RNA deadenylation and stability.

Deficiency of tristetraprolin (TTP), the prototype of the CCCH zinc finger proteins, results in a complex inflammatory syndrome in mice. Most aspects of the syndrome are secondary to excess circulating tumor necrosis factor (TNF)-alpha, a consequence of increased stability of TNF-alpha messenger RNA (mRNA) in TTP-deficient macrophages. TTP can bind directly to the AU-rich element in TNF-alpha mRNA, increasing its lability. Here we show that TTP deficiency also results in increased cellular production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and increased stability of its mRNA, apparently secondary to decreased deadenylation. Similar findings were observed in mice also lacking both types of TNF-alpha receptors, excluding excess TNF-alpha production as a cause of the increased GM-CSF mRNA levels and stability. TTP appears to be a physiological regulator of GM-CSF mRNA deadenylation and stability. (Blood. 2000;95:1891-1899)

Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA.

Mice deficient in tristetraprolin (TTP), the prototype of a family of CCCH zinc finger proteins, develop an inflammatory syndrome mediated by excess tumor necrosis factor alpha (TNF-alpha). Macrophages derived from these mice oversecrete TNF-alpha, by a mechanism that involves stabilization of TNF-alpha mRNA, and TTP can bind directly to the AU-rich element (ARE) in TNF-alpha mRNA (E. Carballo, W. S. Lai, and P. J. Blackshear, Science 281:1001-1005, 1998). We show here that TTP binding to the TNF-alpha ARE is dependent upon the integrity of both zinc fingers, since mutation of a single cysteine residue in either zinc finger to arginine severely attenuated the binding of TTP to the TNF-alpha ARE. In intact cells, TTP at low expression levels promoted a decrease in size of the TNF-alpha mRNA as well as a decrease in its amount; at higher expression levels, the shift to a smaller TNF-alpha mRNA size persisted, while the accumulation of this smaller species increased. RNase H experiments indicated that the shift to a smaller size was due to TTP-promoted deadenylation of TNF-alpha mRNA. This CCCH protein is likely to be important in the deadenylation and degradation of TNF-alpha mRNA and perhaps other ARE-containing mRNAs, both in normal physiology and in certain pathological conditions.

Identification of four CCCH zinc finger proteins in Xenopus, including a novel vertebrate protein with four zinc fingers and severely restricted expression.

Tristetraprolin (TTP), the prototype of a class of CCCH zinc finger proteins, is a phosphoprotein that is rapidly and transiently induced by growth factors and serum in fibroblasts. Recent evidence suggests that a physiological function of TTP is to inhibit tumor necrosis factor alpha secretion from macrophages by binding to and destabilizing its mRNA (Carballo, E., Lai, W.S., Blackshear, P.J., 1998. Science, 281, 1001-1005). To investigate possible functions of CCCH proteins in early development of Xenopus, we isolated four Xenopus cDNAs encoding members of this class. Based on 49% overall amino acid identity and 84% amino acid identity within the double zinc finger domain, one of the Xenopus proteins (XC3H-1) appears to be the homologue of TTP. By similar analyses, XC3H-2 and XC3H-3 are homologues of ERF-1 (cMG1, TIS11B) and ERF-2 (TIS11D). A fourth protein, XC3H-4, is a previously unidentified member of the CCCH class of vertebrate zinc finger proteins; it contains four Cx8Cx5Cx3H repeats, two of which are YKTEL Cx8Cx5Cx3H repeats that are closely related to sequences found in the other CCCH proteins. Whereas XC3H-1, XC3H-2, and XC3H-3 were widely expressed in adult tissues, XC3H-4 mRNA was not detected in any of the adult tissues studied except for the ovary. Its expression appeared to be limited to the ovary, oocyte, egg and the early embryonic stages leading up to the mid-blastula transition. Its mRNA was highly expressed in oocytes of all ages, and was enriched in the animal pole cytosol of mature oocytes. Maternal expression was also seen with the other three messages, suggesting the possibility that these proteins are involved in regulating mRNA stability in oocyte maturation and/or early embryogenesis.

Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin.

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of both acute and chronic inflammatory responses in many diseases. Tristetraprolin (TTP), the prototype of a class of Cys-Cys-Cys-His (CCCH) zinc finger proteins, inhibited TNF-alpha production from macrophages by destabilizing its messenger RNA. This effect appeared to result from direct TTP binding to the AU-rich element of the TNF-alpha messenger RNA. TTP is a cytosolic protein in these cells, and its biosynthesis was induced by the same agents that stimulate TNF-alpha production, including TNF-alpha itself. These findings identify TTP as a component of a negative feedback loop that interferes with TNF-alpha production by destabilizing its messenger RNA. This pathway represents a potential target for anti-TNF-alpha therapies.

Promoter sequence, expression, and fine chromosomal mapping of the human gene (MLP) encoding the MARCKS-like protein: identification of neighboring and linked polymorphic loci for MLP and MACS and use in the evaluation of human neural tube defects.

The MARCKS-like protein (MLP), also known as F52, MacMARCKS, or MARCKS-related protein, is a widely distributed substrate for protein kinase C (PKC). Recent studies using gene disruption in vivo have demonstrated the importance of both MARCKS and MLP to the development of the central nervous system; specifically, mice lacking either protein exhibit a high frequency of neural tube defects. We isolated a genomic clone for human MLP and discovered a directly linked polymorphism (MLP1) useful for genetic linkage analysis. The MLP promoter was 71% identical over 433 bp to that of the corresponding mouse gene, Mlp, with conservation of many putative transcription factor-binding sites; it was only 36% identical over 433 bp to the promoter of the human gene, MACS, which encodes the MLP homologue MARCKS. This 433-bp fragment drove expression of an MLP-beta-galactosidase transgene in a tissue-specific and developmental expression pattern that was similar to that observed for the endogenous gene, as shown by in situ hybridization histochemistry. In contrast to MACS, the MLP and Mlp promoters contain a TATA box approximately 40 bp 5' of the presumed transcription initiation site. MLP was localized to chromosome 1p34-->1pter by analysis of human-mouse somatic cell hybrid DNA and to 1p34 by fluorescence in situ hybridization. Radiation hybrid mapping of MLP placed it between genetic markers D1S511 (LOD > 3.0) and WI9232. MACS was localized to 6q21 between D6S266 (LOD > 3.0) and AFM268uh5 by the same technique. We tested the novel MLP1 polymorphism and the MACS flanking markers in a series of 43 Caucasian simplex families in which the affected child had a lumbosacral myelomeningocele. We found no evidence of linkage disequilibrium, suggesting that these loci were not major genes for spina bifida in these families. Nonetheless, the identification of linked and neighboring polymorphisms for MACS and MLP should permit similar genetic studies in other groups of patients with neural tube defects and other neurodevelopmental abnormalities.

Characteristics of the intron involvement in the mitogen-induced expression of Zfp-36.

Zfp-36, the gene encoding the putative zinc finger protein tristetraprolin (TTP), is rapidly induced in fibroblasts by a variety of growth factors. Recent gene knockout experiments have shown that TTP-deficient mice developed arthritis, cachexia, and autoimmunity, all apparently mediated by an excess of tumor necrosis factor alpha. We recently showed that full serum inducibility of Zfp-36 requires elements in the promoter; in addition, removal of the single intron strikingly inhibited serum-induced TTP expression. We show here that replacement of the intron with unrelated sequences, or removal of 95% of the intron but retention of the splice sites, each resulted in the maintenance of approximately 45 and 19%, respectively, of full serum-induced expression. In addition, deletion of intron sequences base pairs 601-655 decreased the serum-induced expression of TTP by 65%. Sequence base pairs 618-626 bound specifically to the transcription factor Sp1; mutation of this binding motif decreased TTP expression by 70%, suggesting that Sp1 binding to this motif contributes to serum induction of Zfp-36. We conclude that full serum-induced expression of Zfp-36 depends on the activation of conventional promoter elements as well as elements in the single intron, and that the presence per se of the intron in its natural location also contributes significantly to the regulated expression of this gene.

Developmental expression of MARCKS and protein kinase C in mice in relation to the exencephaly resulting from MARCKS deficiency.

The roles of protein kinase C and its substrates in development are poorly understood. Recently, we disrupted the mouse gene for a major cellular substrate for protein kinase C, the MARCKS protein (Proc. Natl. Acad. Sci. USA, 92, 944-948, 1995). The resulting phenotype consisted of universal perinatal lethality, agenesis of the corpus callosum and other forebrain commissures, and neuronal ectopia and other cortical and retinal lamination disturbances. These mice also had high frequencies of exencephaly (25% overall, 35% in females). In the present study, we have examined the normal expression of MARCKS and the various isozymes of protein kinase C at the time of cranial neural tube closure, in an attempt to correlate MARCKS expression in time and anatomical location with the exencephaly characteristic of MARCKS deficiency. Failure of neural tube closure occurred at various sites in the cranial neural tube, suggesting a cellular functional defect that was not limited to a specific location. Non-exencephalic MARCKS-deficient embryos appeared to be anatomically normal on embryonic day (E) 8.5-9.5. MARCKS and PKC alpha were expressed at the plasma membrane of the neuroepithelial cells comprising the future neural tube, as well as in the surface ectoderm and underlying mesenchyme. Endogenous protein kinase C species, comprising either or both alpha and delta, were capable of phosphorylating MARCKS in intact E8.5 embryos. Thus, MARCKS is expressed at the plasma membranes of the specific cell types involved in cranial neurulation; its deficiency presumably results in a still-to-be-elucidated functional defect in these cells that leads to exencephaly in a high proportion of cases.

A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency.

Tristetraprolin (TTP) is a widely expressed potential transcription factor that contains two unusual CCCH zinc fingers and is encoded by the immediate-early response gene, Zfp-36. Mice made deficient in TTP by gene targeting appeared normal at birth, but soon manifested marked medullary and extramedullary myeloid hyperplasia associated with cachexia, erosive arthritis, dermatitis, conjunctivitis, glomerular mesangial thickening, and high titers of anti-DNA and antinuclear antibodies. Myeloid progenitors from these mice showed no increase in sensitivity to growth factors. Treatment of young TTP-deficient mice with antibodies to tumor necrosis factor alpha (TNF alpha) prevented the development of essentially all aspects of the phenotype. These results indicate a role for TTP in regulating TNF alpha synthesis, secretion, turnover, or action. TTP-deficient mice may serve as useful models of the autoimmune inflammatory state resulting from chronic effective TNF alpha excess.

Phosphorylation of tristetraprolin, a potential zinc finger transcription factor, by mitogen stimulation in intact cells and by mitogen-activated protein kinase in vitro.

Tristetraprolin (TTP) is a potential transcription factor that contains three PPPPG repeats and two putative CCCH zinc fingers. TTP is encoded by the early response gene Zfp-36, which is highly expressed in response to growth factors and in several hematopoietic cell lines. In the present studies, we investigated the possibility that TTP is phosphorylated in intact cells. In NIH/3T3 cells that were made to overexpress TTP constitutively, we found that the protein was phosphorylated on serine residues, and that this phosphorylation was rapidly (within 10 min) stimulated by several mitogens. In cell-free assays, recombinant mouse TTP was a substrate for the mitogen-activated protein (MAP) kinase. By a combination of protease digestion experiments and site-directed mutagenesis strategies, we found that serine 220 was phosphorylated by p42 MAP kinase in vitro. Expression of mutant TTP in fibroblasts confirmed that serine 220 was one of the major, mitogen-stimulated phosphorylation sites on the protein in intact cells. These results suggest that TTP may be phosphorylated by MAP kinases in vivo and that this phosphorylation may regulate its function.

Rapid insulin-stimulated accumulation of an mRNA encoding a proline-rich protein.

By differential hybridization screening of a cDNA library derived from insulin-stimulated cells, we selected a clone which hybridized to an mRNA species that rapidly accumulated in response to insulin. The insert from this clone encoded a putative polypeptide of Mr 33,600, pI 11.2; because the protein was enriched in proline residues (14.4 mol %) and contained three Pro-Pro-Pro-Pro repeats, we have tentatively labeled it tris-tetraprolin (TTP). The function of this protein is not known, but it contains two regions very rich in proline (30-40 mol %); similar proline-rich regions have been shown to be involved in transcriptional activation by other proteins. The mRNA (2.0 kilobases) encoding the TTP protein was essentially undetectable in serum-deprived HIR 3.5 cells, but accumulated dramatically within 10 min of stimulation by insulin. This effect appeared to be due to insulin acting through the intrinsic protein-tyrosine kinase activity of its own receptor. Insulin induction of TTP mRNA accumulation was prevented by actinomycin D and superinduced by cycloheximide. Accumulation of TTP mRNA was also stimulated by a variety of growth factors and active phorbol esters; however, the insulin effect was virtually normal in cells depleted of protein kinase C. A single TTP gene appeared to be present in the mouse genome. This gene joins the group of genes whose members are rapidly transcribed in response to insulin and other mitogens.

Protein kinase C is involved in desensitization of muscarinic receptors induced by phorbol esters but not by receptor agonists.

Preincubation with receptor agonists or phorbol esters desensitized muscarinic-receptor-mediated [3H]cyclic GMP responses in mouse neuroblastoma N1E-115 cells. However, desensitization mediated by phorbol esters was heterologous, whereas that effected by receptor agonist was specific towards the muscarinic receptors. In addition, there was no loss of cell surface muscarinic receptors, as measured by the binding of the hydrophilic ligand [3H]N-methylscopolamine, when cells were treated with phorbol esters, but receptor-agonist-induced desensitization was accompanied by a decrease in cell surface receptor density. We examined the role of protein kinase C (PKC) in the desensitization of muscarinic receptors by employing a kinase inhibitor and by down-regulation of PKC by long-term incubation of cells with phorbol esters. Whereas these manoeuvres had marked effects on phorbol-ester-induced desensitization of muscarinic responses, they did not block agonist-induced down-regulation and desensitization of muscarinic receptors. In addition, when phosphoinositide hydrolysis was suppressed, the muscarinic agonist was still capable of mediating receptor sequestration and desensitization. These results suggest that the mechanisms for regulating muscarinic receptor sensitivity could be both PKC-dependent and PKC-independent, being mediated by phorbol esters and receptor agonists respectively.