Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription.

At least eight inherited neurodegenerative diseases are caused by expanded CAG repeats encoding polyglutamine (polyQ) stretches. Although cytotoxicities of expanded polyQ stretches are implicated, the molecular mechanisms of neurodegeneration remain unclear. We found that expanded polyQ stretches preferentially bind to TAFII130, a coactivator involved in cAMP-responsive element binding protein (CREB)-dependent transcriptional activation, and strongly suppress CREB-dependent transcriptional activation. The suppression of CREB-dependent transcription and the cell death induced by polyQ stretches were restored by the co-expression of TAFII130. Our results indicate that interference of transcription by the binding of TAFII130 with expanded polyQ stretches is involved in the pathogenetic mechanisms underlying neurodegeneration.

Differential localization of protein kinase C isozymes in retinal neurons.

We report the immunohistochemical localization of protein kinase C isozymes (types I, II, and III) in the rabbit retina using the monospecific monoclonal antibodies MC-1a, MC-2a, and MC-3a. Using immunoblot analysis of partially purified protein kinase C preparations of rabbit retina, types II and III isozymes alone were detected. The activity of type III was the stronger. By light microscopic immunohistochemical analysis, retinal neurons were negative for type I and positive for type II and type III isozymes. Type II was more diffusely distributed through the retinal layers, but was distinctive in ganglion cells, bipolar cells, and outer segments. The immunoreactivity was stronger for type III isozyme, and it was observed in mop (rod) bipolar cells and amacrine cells. By using immunoelectron microscopy, the cytoplasm of the cell body, the axon, and dendrites of the mop bipolar cells were strongly immunoreactive for type III. The so-called rod bipolar cells were for the first time seen to form synapses with rod photoreceptor cells. These differential localizations of respective isozymes in retinal neurons suggest that each isozyme has a different site of function in each neuron.

Ablation of a peptidyl prolyl isomerase Pin1 from p53-null mice accelerated thymic hyperplasia by increasing the level of the intracellular form of Notch1.

Tumor suppressor p53 is essential for checkpoint control in response to a variety of genotoxic stresses. DNA damage leads to phosphorylation on the Ser/Thr-Pro motifs of p53, which facilitates interaction with Pin1, a pSer/pThr-Pro-specific peptidyl prolyl isomerase. Pin1 is required for the timely activation of p53, resulting in apoptosis or cell cycle arrest. To investigate the physiological relationship between Pin1 and p53, we created Pin1-/-p53-/- mice. These p53-deficient mice spontaneously developed lymphomas, mainly of thymic origin, as well as generalized lymphoma infiltration into other organs, including the liver, kidneys and lungs. Ablation of Pin1, in addition to p53, accelerated the thymic hyperplasia, but the thymocytes in these Pin1-/-p53-/- mice did not infiltrate other organs. The thymocytes in 12-week-old Pin1-/-p53-/- mice were CD4(-)CD8(-) (double negative) and had significantly higher levels of the intracellular form of Notch1 (NIC) than the thymocytes of p53-/- or wild-type mice. Presenilin-1, a cleavage enzyme for NIC generation from full-length Notch1 was increased in the thymocytes of Pin1-/-p53-/- mice. Pin1 depletion also inhibited the degradation of NIC by proteasomes. These results suggest that both Pin1 and p53 control the normal proliferation and differentiation of thymocytes by regulating the NIC level.

Impact of Smoking Cessation in Donor Candidates for Living Donor Liver Transplantation.

BACKGROUND: The relationship between smoking cessation and weight gain is well recognized. Examining the link between smoking cessation and weight gain in donor candidates for living donor liver transplantation (LDLT) is an important topic because of the influence of weight gain on the liver. This study assessed body weight (BW) changes after smoking cessation in donor candidates for LDLT. METHODS: The 27 donor candidates were retrospectively analyzed. The smoking status was determined based on questionnaires administered at the initial presentation, and the candidates were divided into 2 groups: recent quitters and nonsmokers. The changes in BW were compared between the groups. RESULTS: The recent quitters group included 10 (37.0%) candidates, and the nonsmokers group included 17 (63.0%). In the nonsmokers group, 1 candidate had gained weight since the initial presentation. In contrast, in the recent quitters group, 70.0% of candidates had gained weight since the initial presentation (P < .01). The change in BW from the initial presentation was greater in recent quitters than in nonsmokers (+1.6 kg [+2.4%] vs -0.5 kg [-0.9%]; P < .01). Two candidates in the recent quitters group gained ≥ 5 kg [8%] of weight. One of these 2 candidates was judged to be in a donor-inadequate status because of the appearance of fatty liver. CONCLUSIONS: Weight gain due to smoking cessation was observed in donor candidates for LDLT. The amount of weight gain after smoking cessation is highly individualized, so everyone concerned with LDLT must be alert to its potential development.

Muscle-derived vascular endothelial growth factor regulates microvascular remodelling in response to increased shear stress in mice.

AIM: The source of vascular endothelial growth factor-A (VEGF-A) may influence vascular function. Exercise-induced vascular growth has been attributed to elevated metabolic demand and to increased blood flow, involving the production of VEGF-A by skeletal muscle and by endothelial cells respectively. We hypothesized that muscle-derived VEGF-A is not required for vascular adaptations to blood flow in skeletal muscle, as this remodelling stimulus originates within the capillary. METHODS: Myocyte-specific VEGF-A (mVEGF(-/-) ) deleted mice were treated for 7-21 days with the vasodilator prazosin to produce a sustained increase in skeletal muscle blood flow. RESULTS: Capillary number increased in the extensor digitorum longus (EDL) muscle in response to prazosin in wild type but not mVEGF(-/-) mice. Prazosin increased the number of smooth muscle actin-positive blood vessels in the EDL of wild-type but not mVEGF(-/-) mice. The average size of smooth muscle actin-positive blood vessels also was smaller in knockout mice after prazosin treatment. In response to prazosin treatment, VEGF-A mRNA was elevated within the EDL of wild-type but not mVEGF(-/-) mice. Ex vivo incubation of wild-type EDL with a nitric oxide donor increased VEGF-A mRNA. Likewise, we demonstrated that nitric oxide donor treatment of cultured myoblasts stimulated an increase in VEGF-A mRNA and protein. CONCLUSION: These results suggest a link through which flow-mediated endothelial-derived signals may promote myocyte production of VEGF-A. In turn, myocyte-derived VEGF-A is required for appropriate flow-mediated microvascular remodelling. This highlights the importance of the local environment and paracrine interactions in the regulation of tissue perfusion.

Involvement of CCAAT/enhancer-binding protein in regulation of the rat serine:pyruvate/alanine:glyoxylate aminotransferase gene expression.

In the rat liver, transcription of the serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) gene occurs from two sites, +1 and +66, in exon 1, resulting in the formation of two mRNAs, one for a precursor of mitochondrial SPT/AGT and the other for peroxisomal SPT/AGT, respectively. In this study, we attempted to characterize the downstream promoter responsible for generation of peroxisomal SPT/AGT. The minimal downstream promoter was confined to the +21-+90 region. We demonstrated that C/EBPalpha and C/EBPbeta bound around the downstream start site (+66) contribute to the promoter activity. The downstream promoter activity is also regulated positively by a short inverted repeat, located 20-30 bp upstream of the downstream start site, through a protein factor(s) bound to this region. On the other hand, the sequence just downstream of the start site may negatively regulate the promoter activity.

Efficacy and tolerability of moclobemide compared with fluvoxamine in depressive disorder (DSM III). A French/Swiss double-blind trial.

The efficacy and tolerability of moclobemide and fluvoxamine, two new types of antidepressant agents, were compared in a multicentre, double-blind prospective study of patients with a diagnosis of major depressive episode (DSM III). Patients were randomized to receive either moclobemide (150 mg) or fluvoxamine (50 mg) twice daily for 7 days, immediately following a washout period of at least 1 week. Dosages were increased where necessary on day 8, to a maximum of moclobemide 450 mg or fluvoxamine 200 mg and in most cases were maintained at these levels for the remainder of the study period (4-6 weeks). Both treatment groups showed a marked antidepressant effect. While both treatments were well tolerated, moclobemide showed a more favourable side-effect profile than fluvoxamine. Of the 126 patients eligible for evaluation, 34 withdrew from therapy, 22% in the moclobemide group and 30% in the fluvoxamine group. Adverse events were reported in 41.8% of patients treated with moclobemide compared to 60.3% of patients in the fluvoxamine group. Reports of dry mouth and other anticholinergic effects were more frequent among those treated with fluvoxamine. A greater number of gastrointestinal complaints, especially nausea, also occurred in the fluvoxamine-treated patients.

Mitochondrial targeting signal-induced conformational change and repression of the peroxisomal targeting signal of the precursor for rat liver serine:pyruvate/alanine:glyoxylate aminotransferase.

In the rat liver, two mRNAs for serine:pyruvate (or alanine:glyoxylate) aminotransferase are generated from a single gene by alternative transcription initiation. The longer mRNA encodes a precursor of a mitochondrial enzyme that has a mitochondrial targeting signal at the N-terminus and is translocated into mitochondria. The shorter mRNA encodes a peroxisomal enzyme of mature size that is imported into peroxisomes. We have been interested in the mechanism of selective targeting to mitochondria of the precursor protein that also contains a peroxisomal targeting signal in the molecule. In this study, we examined the effect of the mitochondrial targeting signal on the conformation of the protein and on the function of the peroxisomal targeting signal in the precursor molecule. The results suggest that the mitochondrial targeting signal causes the conformation of the protein to become unfolded and that this conformational change in turn causes repression of the putative peroxisomal targeting signal contained in the precursor protein.

Store depletion by caffeine/ryanodine activates capacitative Ca(2+) entry in nonexcitable A549 cells.

Capacitative Ca(2+) entry is essential for refilling intracellular Ca(2+) stores and is thought to be regulated primarily by inositol 1, 4,5-trisphosphate (IP(3))-sensitive stores in nonexcitable cells. In nonexcitable A549 cells, the application of caffeine or ryanodine induces Ca(2+) release in the absence of extracellular Ca(2+) similar to that induced by thapsigargin (Tg), and Ca(2+) entry occurs upon the readdition of extracellular Ca(2+). The channels thus activated are also permeable to Mn(2+). The channels responsible for this effect appear to be activated by the depletion of caffeine/ryanodine-sensitive stores per se, as evidenced by the activation even in the absence of increased intracellular Ca(2+) concentration. Tg pretreatment abrogates the response to caffeine/ryanodine, whereas Tg application subsequent to caffeine/ryanodine treatment induces further Ca(2+) release. The response to caffeine/ryanodine is also abolished by initial ATP application, whereas ATP added subsequent to caffeine/ryanodine induces additional Ca(2+) release. RT-PCR analyses showed the expression of a type 1 ryanodine receptor, two human homologues of transient receptor potential protein (hTrp1 and hTrp6), as well as all three types of the IP(3) receptor. These results suggest that in A549 cells, (i) capacitative Ca(2+) entry can also be regulated by caffeine/ryanodine-sensitive stores, and (ii) the RyR-gated stores interact functionally with those sensitive to IP(3), probably via Ca(2+)-induced Ca(2+) release.

Synthesis of alpha-glucosidase inhibitors: kojibiose-type pseudo-disaccharides and a related pseudotrisaccharide.

Two kojibiose-type pseudo-disaccharides and a trisaccharide, containing a 5-amino-1,2,3,4-cyclopentanetetrol derivative or valienamine, linked by way of nitrogen bridges to the sugar residues, have been designed and synthesized as processing alpha-glucosidase I inhibitors. Synthesis of the pseudo-disaccharides was carried out starting from the coupling products of the sugar isothiocyanates and an aminocyclitol, respectively, by cyclization with mercury(II) oxide to the cyclic isoureas and subsequent deprotection. Pseudokojibiose was prepared in a poor yield by reaction of a protected valienamine and a sugar epoxide, followed by deprotection. Although the pseudooligosaccharides are all strong inhibitors of alpha-glucosidase (baker's yeast), they did not have any inhibitory potency against either sucrase isomaltase (rat intestine) or processing alpha-glucosidase (rat liver microsomes).

Protective effect by cell-free antigen obtained from culture supernatant of phase I Bordetella bronchiseptica.

The cell-free antigen (CFA), with highly hemagglutination activity, obtained from the culture supernatant of Bordetella bronchiseptica was compounded with oil adjuvant to make a component vaccine (CFAV). In the immunization trial in mice, the offsprings whose mothers were immunized with CFAV escaped from death when challenged intrapleurally with virulent strain of B. bronchiseptica. The protective indices (difference of LD50 dose of the challenge strain between immunized and control groups) of the offsprings from CFAV-immunized mothers were over 3.0 in common logarithm value. Moreover, about 90% of the offsprings from CFAV-immunized mothers were negative in nasal turbinate atrophy, while over 80% of them from non-immunized mothers showed obvious turbinate atrophy when challenged intranasally with virulent strain. On the one hand, remarkable differences in the number of bacteria recovered from nostrils were observed between both test groups. It was concluded that CFAV is a very effective vaccine against B. bronchiseptica infection in animals.

A critical step for JNK activation: isomerization by the prolyl isomerase Pin1.

c-Jun N-terminal kinase (JNK) is activated by dual phosphorylation of both threonine and tyrosine residues in the phosphorylation loop of the protein in response to several stress factors. However, the precise molecular mechanisms for activation after phosphorylation remain elusive. Here we show that Pin1, a peptidyl-prolyl isomerase, has a key role in the JNK1 activation process by modulating a phospho-Thr-Pro motif in the phosphorylation loop. Pin1 overexpression in human breast cancer cell lines correlates with increased JNK activity. In addition, small interfering RNA (siRNA) analyses showed that knockdown of Pin1 in a human breast cancer cell line decreased JNK1 activity. Pin1 associates with JNK1, and then catalyzes prolyl isomerization of the phospho-Thr-Pro motif in JNK1 from trans- to cis-conformation. Furthermore, Pin1 enhances the association of JNK1 with its substrates. As a result, Pin1(-/-) cells are defective in JNK activation and resistant to oxidative stress. These results provide novel insights that, following stress-induced phosphorylation of Thr in the Thr-Pro motif of JNK1, JNK1 associates with Pin1 and undergoes conformational changes to promote the binding of JNK1 to its substrates, resulting in cellular responses from extracellular signals.

Cryptococcus: isolamento ambiental e caracterização bioquímica / Cryptococcus: environmental isolation and biochemical characterization

O gênero Cryptococcus caracteriza-se por ser uma levedura responsável por infecção sistêmica, causada pelas espécies Cryptococcus neoformans e Cryptococcus gattii. O fungo é encontrado em substratos de origem animal e vegetal, e a infecção ocorre com a inalação de basidiósporos ou leveduras desidratadas infectantes presentes no ambiente. O presente trabalho teve por objetivo pesquisar a existência de microfocos de Cryptococcussp.em amostras ambientais da cidade de Araçatuba, São Paulo, com a finalidade de minimizar os riscos de contaminação do homem e dos animais, buscando o conhecimento da ecoepidemiologia do Cryptococcus. Foram colhidas 50 amostras oriundas de ocos e troncos de árvores (Cassiasp., Ficussp., Caesalpinea peltophorides) de 10 locais representativos do perímetro urbano, as quais foram encaminhadas ao Laboratório de Bacteriologia e Micologia da Faculdade de Medicina Veterinária de Araçatuba-Unesp, onde foram processadas e semeadas em placas de Petri contendo ágar semente de Níger e Sabouraud dextrose com clorafenicol e incubadas à temperatura de 30ºC, por um período não inferior a cinco dias. Posteriormente, foram submetidas às provas bioquímicas: produção de urease, termotolerância a 37ºC e quimiotipagem em ágar CGB (L-canavanina-glicina-azul de bromotimol). A análise dos resultados revelaram que 17 (34%) dos cultivos foram positivos para o gênero Cryptococcus, sendo nove (18%) para Cryptococcus gattiie oito (16%) para Cryptococcus neoformans. Outras leveduras correlacionadas, como Rhodotorula sp. e Candida sp., também foram isoladas. Conclui-se que os basidiósporos de Cryptococcusencontram-se dispersos na natureza, constituindo microfocos ambientais, não vinculados necessariamente a um único hospedeiro.

Cryptococcosis is an opportunistic fungal infection caused by Cryptococcusyeasts, especially C. neoformans and Cryptococcus gattii. The fungus is found in substrates of animal and vegetable origin, and infection occurs through inhalation and seedlings present in the environment. The present study aimed to investigate the existence of microfocus Cryptococcus sp. from the environmental samples of Araçatuba city, São Paulo, featuring new niches, by decoupling the direct relationship between fungus and host in order to minimize the risk of contamination of man and animals, understanding the ecoepidemiology of Cryptococcus. Fifty samples from hollows and tree trunks were harvested (Cassia sp., Ficus sp., Caesalpinea peltophorides) from ten representatives in the urban perimeter. The samples were immediately sent to the Laboratory of Bacteriology and Mycology, Faculty of Veterinary Medicine Araçatuba - Unesp where they were processed and plated on Petri dishes containing agar seed Niger and Sabouraud dextrose agar with chloramphenicol, incubated at 30ºC for a period of no less than 5 days. Afterwards they were subimitted to biochemical tests: urease production, thermotolerance at 37°C and quimiotipagem in CGB agar (L- Canavanine-Glycine-Bromothymol blue). The results showed that 17 (34%) cultures were positive for Cryptococcus, 9 (18%) for Cryptococcus gattii and 8 (16%) for Cryptococcus neoformans. Other yeast correlated as Rhodotorula sp. and Candida sp. were isolated. We conclude that the infectious propagules of Cryptococcus are dispersed in nature and constitute an environmental microfocus, not necessarily being bound to a single host.

Evolving strategies in manipulating VEGF/VEGFR signaling for the promotion of angiogenesis in ischemic muscle.

Peripheral artery disease is characterized by reduced blood flow to the lower limb, resulting in chronic ischemia in these muscles, which can lead to eventual amputation of the affected limb. Stimulation of angiogenesis in the ischemic region would be of therapeutic benefit; however, attempts to increase angiogenesis through delivery of vascular endothelial growth factor (VEGF) largely have been unsuccessful. Recent studies have shown that VEGF signaling through its receptors, VEGFR1 and VEGFR2, is much more complex than previously appreciated. This review will examine current research into the function of VEGFR1 and -2 signaling pathways, and evidence of cross-talk between these two receptors. The potential impact of endothelial cell co-stimulation via other growth factors/cell surface receptors (such as angiopoietins and ephrins) on angiogenesis also will be discussed. Evidence suggesting deficiencies in VEGF pathway signaling in individuals with chronic ischemia and diabetes will be discussed. Numerous pro-angiogenic therapies for ischemia have been employed. The successes and limitations of these therapies will be illustrated, emphasizing more recent angiogenesis therapies that focus on activating co-ordinated patterns of pro-angiogenic genes as the most promising direction in the treatment of ischemic muscle tissue in peripheral artery disease.

Fbw7 promotes ubiquitin-dependent degradation of c-Myb: involvement of GSK3-mediated phosphorylation of Thr-572 in mouse c-Myb.

Expression of oncoprotein c-Myb oscillates during hematopoiesis and hematological malignancies. Its quantity is not only regulated through transcriptional control but also through the ubiquitin-proteasome pathway, accompanied by phosphorylation, although the mechanisms are poorly understood. In this report, we tried to identify an E3 ubiquitin ligase, which targets c-Myb for ubiquitin-dependent degradation. We found that an F-box protein, Fbw7, interacted with c-Myb, which is mutated in numerous cancers. Fbw7 facilitated ubiquitylation and degradation of c-Myb in intact cells. Moreover, depletion of Fbw7 by RNA interference delayed turnover and increased the abundance of c-Myb in myeloid leukemia cells concomitantly, and suppressed the transcriptional level of gamma-globin, which receives transcriptional repression from c-Myb. In addition, we analysed sites required for both ubiquitylation and degradation of c-Myb. We found that Thr-572 is critical for Fbw7-mediated ubiquitylation in mouse c-Myb using site-directed mutagenesis. Fbw7 recognized the phosphorylation of Thr-572, which was mediated by glycogen synthase kinase 3 (GSK3). In consequence, the c-Myb protein was markedly stabilized by the substitution of Thr-572 to Ala. These observations suggest that SCF(Fbw7) ubiquitin ligase regulates phosphorylation-dependent degradation of c-Myb protein.

Prolyl isomerase, Pin1: new findings of post-translational modifications and physiological substrates in cancer, asthma and Alzheimer's disease.

The peptidyl prolyl cis/trans isomerase Pin1 specifically binds phosphorylated Ser/Thr-Pro protein motifs and catalyzes the cis/trans isomerization of the peptide bond. Accumulating studies have revealed that Pin1 isomerase activity is regulated by its post-translational modifications, including phosphorylation and oxidation. Various transcription factors and regulators have been identified as substrates for Pin1. It enhances AP-1 activity via isomerization of both c-Jun and c-Fos for cellular proliferation and stabilizes the oncosuppressors p53 and p73 against DNA damage at the checkpoint. We demonstrated the association between the intracellular form of Notch1 (NIC) and Pin1 by analyzing Pin1/p53 double-knockout mice. Pin1 also regulates the post-transcriptional level of some cytokines, associated with asthma, that possess 3' untranslated region AU-rich elements (AREs) via interaction withAUF1, the nucleoprotein in the ARE-binding complex. Pin1 has been identified as the molecular partner of tau and amyloid precursor protein (APP), the key factors of Alzheimer's disease (AD). It interacts with the phosphorylated Thr-231 of tau and regulates its activity to bind microtubules. It further interacts with the phosphorylated Thr-668 of APP and affects its metabolism. Thus, Pin1 is probably involved in the pathogenesis of human diseases, including cancer, asthma, and AD, presenting an attractive target for future therapeutical drugs.

Ubiquitin-dependent degradation of SnoN and Ski is increased in renal fibrosis induced by obstructive injury.

Transforming growth factor-beta (TGF-beta) plays a critical role in the progression of renal fibrosis. The activity of TGF-beta is tightly controlled by various mechanisms, among which antagonizing Smad-mediated gene transcription by co-repressors represents one of the important components. We investigated the expression, degradation, and ubiquitination of Smad transcriptional co-repressors SnoN (ski-related novel gene N) and Ski (Sloan-Kettering Institute proto-oncogene) in renal fibrogenesis. We also studied the involvement of Smad-ubiquitination regulatory factor 2 (Smurf2) in ubiquitination of SnoN protein. The kidneys of mice with unilateral ureteral obstruction (UUO) and those of sham-operated mice were used. Renal lesions and the expression of TGF-beta1, type I collagen, SnoN, Ski, and Smurf2 were examined by immunohistochemistry, Western blot, and/or real-time reverse transcriptase-polymerase chain reaction. Degradation and ubiquitination of SnoN/Ski proteins were also investigated. The obstructed kidneys of UUO mice showed progressive tubulointerstitial fibrosis, high expression levels of TGF-beta1, type I collagen, SnoN and Ski mRNAs, and low levels of SnoN and Ski proteins. Both degradation and ubiquitination of SnoN/Ski proteins were markedly increased in the obstructed kidneys, in which Smurf2 expression was increased. Smurf2 immunodepletion in extracts of obstructed kidneys resulted in reduced ubiquitination of SnoN. Our results suggest that the reduction of SnoN/Ski proteins resulting from increased ubiquitin-dependent degradation is involved in the progression of tubulointerstitial fibrosis.

Synthesis and inhibitory activity of glycosidase inhibitors, glycosylamino-oxazolines.

In connection with structural modification of the trehalase inhibitor trehazolin (1), as a new-type of glycohydrolase inhibitor, some glycosylamino-oxazolines were designed and synthesized. Among three oxazolines beta-galacto (3), beta-gluco (5) and alpha-manno-types (6) obtained in stable form, the latter 6 has been shown to possess a moderate inhibitory activity against alpha-mannosidase.

Ca(2+)-independent, phospholipid-activated protein kinase in 3Y1 cells.

We obtained a Ca(2+)-independent but 12-O-tetradecanoyl phorbol ester (TPA).phospholipid-activated protein kinase from rat embryo fibroblast 3Y1 cells by succeeding steps of DEAE-cellulose, H-9 affinity, and hydroxylapatite chromatography. This kinase was separated chromatography. This kinase was separated from a conventional PKC (Type III), by H-9 affinity column chromatography. The major peak from H-9 affinity column was eluted at 0.4 M of arginine and on the following step of hydroxylapatite column chromatography, at the KPO4 concentration of 0.1 M. The enzyme could be stimulated by phospholipids and by the tumor promoter TPA, but did not respond to calcium. The Ca(2+)-independent, phospholipid-activated protein kinase activity was susceptible to the protein kinase C inhibitors H-7 and K252a, but showed a phospholipid dependency and substrate specificity distinct from the conventional types of PKC. This protein kinase did not react with monoclonal antibodies against Types I, II, and III PKC. The activity of this enzyme was specifically reduced by immunoprecipitation, depending on the concentration of the polyclonal antibody, PC-delta, which was raised against a peptide synthesized according to a sequence of rat brain nPKC delta. The enzyme had a Mr of 76,000 as estimated by Western blotting. These results provide evidence for a unique type of Ca(2+)-independent, phospholipid-activated kinase, as expressed in 3Y1 cells.