Lyophilized and gamma-sterilized allogeneic bone implant used as a spacer for advancement of a modified tibial tuberosity in the treatment of cranial cruciate ligament disease in dogs.

The objective of this study was to evaluate the use of a lyophilized and gamma-sterilized allogeneic freeze-dried bone wedge as a spacer for advancement of a modified tibial tuberosity (mTTA) in 16 knees that were clinically diagnosed with cranial cruciate ligament disease. Patients underwent radiography before the surgical procedure as well as immediately after surgery and at 30, 60, 90 and 120 days post-surgery, and their locomotion was evaluated at the same time points except for the immediate postoperative period. The surgical wounds were evaluated for signs of infection and rejection of the bone implant. Locomotion was graded on a scale of 0-5, with 0 indicating no limping and 5 indicating limb functional impotence. The "tibial-tibial bone-tibial implant" interfaces were evaluated radiographically, and each interface was assigned scores of 0-3, with 0 indicating no contact between the implant and adjacent bone and 3 indicating a bone bridge throughout the interface. The patients showed good clinical and radiographic recovery. The lyophilized bone spacer allowed for easy storage and transport and rapid and satisfactory execution of mTTA while showing resistance to drilling and fixation with screws in 87.5% of cases and a mean surgical time of 45.9 minutes. No immunogenic reactions were observed in 93.7% of the cases. One patient presented infection of the surgical focus, which showed remission after antimicrobial therapy. All patients showed functional recovery of the operated limb, with the number of clinically healthy patients being higher than those with claudication at 120 days (p ≤ 0.05). In all patients, it was possible to verify the incorporation of the bone implant into the tibia. Bone union occurred progressively, and the degrees of bone union observed on radiographs at postoperative days 60, 90, and 120 were significantly greater (p < 0.05) than those observed in the immediate postoperative period and at 30 days.

Retinal dysplasia in the crab-eating fox (Cerdocyon thous) / Displasia retiniana em cachorro-do-mato (Cerdocyon thous)

Previously described in humans and domestic animals, retinal dysplasia has three clinical forms: focal/multifocal, geographic and total. A young orphan crab-eating fox (Cerdocyon thous) from wildlife, male, approximately 45 days old referred to the Wildlife Medicine and Ophthalmology Services of the "Governador Laudo Natel" Veterinary Hospital of the Universidade Estadual Paulista, Jaboticabal Campus, SP, Brazil, where it received primary outpatient care. The patient was in good general health condition, without hematological, biochemistry or serological alterations and no signs of visual impairment. Indirect binocular ophthalmoscopy showed retinal changes in the left eye, distributed over the tapetal area in the form of grayish folds and rosettes. In the affected areas, tapetal reflectivity was reduced. No other ophthalmic abnormalities were observed. This is the first report of retinal dysplasia in the crab-eating fox (Cerdocyon thous) from wildlife.(AU)

Descrita no homem e em animais domésticos, a displasia de retina, se apresenta nas formas focal/multifocal, geográfica e completa. Um espécime de cachorro-do-mato (Cerdocyon thous) de vida livre, macho, com 45 dias de vida foi capturado e encaminhado aos Serviços de Medicina de Animais Selvagens e de Oftalmologia do Hospital Veterinário "Governador Laudo Natel" da Universidade Estadual Paulista ­ Unesp, Câmpus Jaboticabal-SP, Brasil, onde recebeu atendimento primário ambulatorial. O paciente apresentava-se em bom estado geral, sem alterações hematológicas e sorológicas, e não havia sinais de déficit visual. A oftalmoscopia binocular indireta mostrou alterações retinianas no olho esquerdo, distribuídas na área tapetal na forma de pregas e de rosetas de coloração acinzentada. Nas áreas acometidas, a reflectividade tapetal estava reduzida. Não foram observadas outras alterações oftálmicas. Trata-se do primeiro relato de literatura sobre displasia retiniana em cachorrodo-mato (Cerdocyon thous).(AU)

Single-port laparoscopic ovariectomy using a pre-tied loop ligature in Santa Ines ewes / Ovariectomia por um portal laparoscópico com aplicação de ligaduras pré-montadas em ovelhas Santa Inês

ABSTRACT:The aim of the study was to develop and assess the feasibility, postoperative pain and inflammatory response of the single-port laparoscopic ovariectomy in ewes, using a simple pre-tied loop ligature technique. Pre-tied Meltzer's knot was employed for prophylactic hemostasis of the ovarian pedicle. Slipknot was inserted within the abdominal cavity through a 14-gauge needle and tied surrounding the ovarian pedicle. Mean surgical time, manipulation, ligature and resection of each ovary and anesthesia time were 63±20, 20±10 and 91±26 minutes, respectively. No bleeding occurred during the surgeries. Ewes showed low scores pain (0.5±0.5) at all time-points. Postsurgical plasma fibrinogen was within the normal range for sheep specie at all time-points. The ewes showed a significant weight gain in comparison to the basal scaling (one day before the surgery). Single-port laparoscopic ovariectomy using a pre-tied loop ligature is feasible in the ovine specie and provided minimal postoperative distress and quick weight gain.

RESUMO:Objetivou-se com este trabalho desenvolver e descrever uma técnica de ovariectomia por videolaparoscopia utilizando um portal laparoscópico e um sistema de ligadura pré-montada, avaliando a sua viabilidade, o desconforto doloroso e o processo inflamatório provocado em ovelhas. O nó de Meltzer pré-montado foi utilizado para hemostasia profilática do pedículo ovariano. O nó corrediço foi inserido na cavidade abdominal através de uma agulha 14G e atado em torno do pedículo ovariano. O tempo médio de cirurgia foi de 63±20min, o de manipulação, ligadura e ressecção para cada ovário foi de 20±10min, e o de anestesia 91±26min. Não houve hemorragia durante as cirurgias. As ovelhas apresentaram escores de dor considerados baixos (0,5±0,5). Todos os valores do fibrinogênio plasmático estiveram dentro do padrão de normalidade, não havendo diferença estatística entre os momentos avaliados. Houve aumento significativo nas médias de peso das fêmeas, quando comparados ao momento controle (um dia anterior ao experimento). A ovariectomia por um portal laparoscópico com aplicação de ligaduras pré-montadas é factível para a espécie ovina, provocando mínimo estresse, desconforto doloroso e rápido ganho de peso nos animais.

Parâmetros ventilométricos e hemogasométricos em cadelas submetidas à ovário-histerectomia, pré-medicadas com tramadol ou morfina e anestesiadas com isofluorano / Ventilometric parameters and arterial blood gases in bitches undergoing ovariohisterectomy, pre-medicated with morphine or tramadol and anesthetized with isoflurane

Avaliaram-se os efeitos nos parâmetros ventilométricos e hemogasométricos em cadelas submetidas à ovário-histerectomia (OHE), pré-medicadas com tramadol ou morfina e anestesiadas com isofluorano. Utilizaram-se 12 cadelas sem raça definida (SRD), divididas em dois grupos (n= 6 para cada grupo), todas com indução anestésica com propofol e manutenção com isofluorano em O2 a 100%, sendo que a diferença entre os grupos se deu pela pré-medicação com tramadol (GT ­ 4mg/kg IM) ou morfina (GM ­ 0,4 mg/kg IM). As observações das variáveis iniciaram-se 30 minutos após a administração do bolus de propofol (M0). As demais colheitas dos dados foram realizadas em intervalos de 15 minutos, por um período de 60 minutos (M15, M30, M45 e M60, respectivamente). Não foram encontradas diferenças significativas entre os grupos ou momentos para as variáveis hemogasométricas, entretanto, as médias de pH, déficit de base (DB) e pressão parcial de dióxido de carbono no sangue arterial (PaCO2 ) ficaram fora da faixa normal para a espécie canina. Foram encontradas diferenças significativas para as variáveis: volume corrente (GM > GT de M0 a M60), pico de fluxo inspiratório e expiratório (GM > GT) e pressão de pico inspiratório, sendo que GM foi maior que GT em M0. Concluiu-se que ambos os opioides são seguros, não causando alterações importantes na ventilometria quando utilizados na pré-medicação em cadelas anestesiadas com isofluorano e submetidas à OHE, porém, a anestesia inalatória com isofluorano deve ser utilizada com cautela em animais com propensão à acidemia.

We evaluated the effects on blood gas and ventilometric parameters in bitches undergoing ovariohysterectomy (OHE), pre-medicated with morphine or tramadol and anesthetized with isoflurane. We used 12 mongrel dogs, distributed into two groups (n = 6 for each group), all with induction of anesthesia with propofol and maintained with isoflurane in 100% O2, and the difference between groups was given by premedication with tramadol (GT - 4mg/kg IM) or morphine (GM - 0.4 mg / kg IM). The measurement of all variables was performed 30 minutes after propofol bolus (M0), and then, in intervals of 15 minutes, during 60 minutes (M15, M30, M45 and M60). No significant differences were found between groups or times for the gas variables, however pH, PaCO2 and DB were outside the normal range for dogs. Significant differences were found for the variables: tidal volume (GM> GT M0 to M60), peak inspiratory and expiratory flow (GM> GT) and peak inspiratory pressure (GM was higher than GT in M0). It was concluded that both opioids are safe and will not cause major changes in ventilometry when used in premedication in dogs anesthetized with isoflurane and submitted to OHE, however, inhalation anesthesia with isoflurane should be used with caution in animals with a tendency to acidemia. .

Parâmetros ventilométricos e hemogasométricos em cadelas submetidas à ovariohisterectomia, pré-medicadas com tramadol ou morfina e anestesiadas com isofluorano / Ventilometric parameters and arterial blood gases in bitches undergoingovariohisterectomy, pre-medicated with morphine or tramadol and anesthetized with isoflurane

Avaliaram-se os efeitos nos parâmetros ventilométricos e hemogasométricos em cadelas submetidas à ovário-histerectomia (OHE),pré-medicadas com tramadol ou morfina e anestesiadas com isofluorano. Utilizaram-se 12 cadelas sem raça definida (SRD), divididasem dois grupos (n= 6 para cada grupo), todas com indução anestésica com propofol e manutenção com isofluorano em O2 a 100%, sendo que a diferença entre os grupos se deu pela pré-medicação com tramadol (GT – 4mg/kg IM) ou morfina (GM – 0,4 mg/kg IM). As observações das variáveis iniciaram-se 30 minutos após a administração do bolus de propofol (M0). As demais colheitas dos dados foram realizadas em intervalos de 15 minutos, por um período de 60 minutos (M15, M30, M45 e M60, respectivamente). Não foram encontradas diferenças significativas entre os grupos ou momentos para as variáveis hemogasométricas, entretanto, as médias de pH, déficit de base (DB) e pressão parcial de dióxido de carbono no sangue arterial (PaCO2) ficaram fora da faixa normal para a espécie canina. Foram encontradas diferenças significativas para as variáveis: volume corrente (GM > GT de M0 a M60), pico de fluxo inspiratório e expiratório (GM > GT) e pressão de pico inspiratório, sendo que GM foi maior que GT em M0.Concluiu-se que ambos os opioides são seguros, não causando alterações importantes na ventilometria quando utilizados na pré-medicação em cadelas anestesiadas com isofluorano e submetidas à OHE, porém, a anestesia inalatória com isofluorano deve ser utilizada com cautela em animais com propensão à acidemia.

We evaluated the effects on blood gas and ventilometric parameters in bitches undergoing ovariohysterectomy (OHE), pre-medicatedwith morphine or tramadol and anesthetized with isoflurane. We used 12 mongrel dogs, distributed into two groups (n = 6 foreach group), all with induction of anesthesia with propofol and maintained with isoflurane in 100% O2, and the difference betweengroups was given by premedication with tramadol (GT - 4mg/kg IM) or morphine (GM - 0.4 mg / kg IM). The measurement of allvariables was performed 30 minutes after propofol bolus (M0), and then, in intervals of 15 minutes, during 60 minutes (M15, M30,M45 and M60). No significant differences were found between groups or times for the gas variables, however pH, PaCO2 and DBwere outside the normal range for dogs. Significant differences were found for the variables: tidal volume (GM> GT M0 to M60),peak inspiratory and expiratory flow (GM> GT) and peak inspiratory pressure (GM was higher than GT in M0). It was concludedthat both opioids are safe and will not cause major changes in ventilometry when used in premedication in dogs anesthetized withisoflurane and submitted to OHE, however, inhalation anesthesia with isoflurane should be used with caution in animals with atendency to acidemia.

Differential IL-21 signaling in APCs leads to disparate Th17 differentiation in diabetes-susceptible NOD and diabetes-resistant NOD.Idd3 mice.

Type 1 diabetes (T1D) is an autoimmune disease that shows familial aggregation in humans and likely has genetic determinants. Disease linkage studies have revealed many susceptibility loci for T1D in mice and humans. The mouse T1D susceptibility locus insulin-dependent diabetes susceptibility 3 (Idd3), which has a homologous genetic interval in humans, encodes cytokine genes Il2 and Il21 and regulates diabetes and other autoimmune diseases; however, the cellular and molecular mechanisms of this regulation are still being elucidated. Here we show that T cells from NOD mice produce more Il21 and less Il2 and exhibit enhanced Th17 cell generation compared with T cells from NOD.Idd3 congenic mice, which carry the protective Idd3 allele from a diabetes-resistant mouse strain. Further, APCs from NOD and NOD.Idd3 mice played a central role in this differential Th17 cell development, and IL-21 signaling in APCs was pivotal to this process. Specifically, NOD-derived APCs showed increased production of pro-Th17 mediators and dysregulation of the retinoic acid (RA) signaling pathway compared with APCs from NOD.Idd3 and NOD.Il21r-deficient mice. These data suggest that the protective effect of the Idd3 locus is due, in part, to differential RA signaling in APCs and that IL-21 likely plays a role in this process. Thus, we believe APCs provide a new candidate for therapeutic intervention in autoimmune diseases.

A study of PCR inhibition mechanisms using real time PCR.

In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co-extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance.

Genetic evidence that the differential expression of the ligand-independent isoform of CTLA-4 is the molecular basis of the Idd5.1 type 1 diabetes region in nonobese diabetic mice.

Idd5.1 regulates T1D susceptibility in nonobese diabetic (NOD) mice and has two notable candidate genes, Ctla4 and Icos. Reduced expression of one of the four CTLA-4 isoforms, ligand-independent CTLA-4 (liCTLA-4), which inhibits in vitro T cell activation and cytokine production similarly to full-length CTLA-4 (flCTLA-4), has been hypothesized to increase type 1 diabetes (T1D) susceptibility. However, further support of this hypothesis is required since the Idd5.1 haplotypes of the diabetes-susceptible NOD and the resistant B10 strains differ throughout Ctla4 and Icos. Using haplotype analysis and the generation of novel Idd5.1-congenic strains that differ at the disease-associated Ctla4 exon 2 single-nucleotide polymorphism, we demonstrate that increased expression of liCTLA-4 correlates with reduced T1D susceptibility. To directly assess the ability of liCTLA-4 to modulate T1D, we generated liCTLA-4-transgenic NOD mice and compared their diabetes susceptibility to nontransgenic littermates. NOD liCTLA-4-transgenic mice were protected from T1D to the same extent as NOD.B10 Idd5.1-congenic mice, demonstrating that increased liCTLA-4 expression alone can account for disease protection. To further investigate the in vivo function of liCTLA-4, specifically whether liCTLA-4 can functionally replace flCTLA-4 in vivo, we expressed the liCTLA-4 transgene in CTLA-4(-/-) B6 mice. CTLA-4(-/-) mice expressing liCTLA-4 accumulated fewer activated effector/memory CD4(+) T cells than CTLA-4(-/-) mice and the transgenic mice were partially rescued from the multiorgan inflammation and early lethality caused by the disruption of Ctla4. These results suggest that liCTLA-4 can partially replace some functions of flCTLA-4 in vivo and that this isoform evolved to reinforce the function of flCTLA-4.

Developmental validation of reduced-size STR Miniplex primer sets.

This paper describes a developmental validation study of three Miniplex sets covering 12 of the 13 CODIS loci. As these new sets will be used for the analysis of degraded and low level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. To demonstrate the applicability of the Miniplex sets to forensic casework, these validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM). A range of tests were performed including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. Additionally, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. Our results demonstrate that Miniplex STR amplification procedures are a robust and sensitive tool for the analysis of degraded DNA.

Anti-thymocyte globulin (ATG) prevents autoimmune encephalomyelitis by expanding myelin antigen-specific Foxp3+ regulatory T cells.

The T cell-depleting polyclonal antibody, anti-thymocyte globulin (ATG) has long been used in organ transplantation to treat acute rejection episodes. More recently, it is also being used as part of an induction regimen to protect allografts. It has been proposed that ATG might deplete effector T cells (T-effs) while sparing regulatory T cells (T-regs). In order to test whether ATG is effective in autoimmune disease, we used Foxp3gfp 'knock-in' mice in combination with a myelin oligodendrocyte glycoprotein (MOG)(35-55)/IA(b) tetramer to study more closely the effect of ATG treatment on antigen-specific T cell responses in vivo during MOG-induced experimental autoimmune encephalomyelitis (EAE), an animal model for Multiple Sclerosis. ATG treatment enhanced the expansion of MOG-specific T-regs (CD4(+)Foxp3(+)) in MOG-immunized mice. T-effs were depleted, but on a single-cell basis, the effector function of residual T-effs was not compromised by ATG. Thus, ATG tipped the balance of T-effs and T-regs and skewed an auto-antigen-specific immune reaction from a pathogenic T cell response to a potentially protective T-reg response. In both acute and relapsing remitting disease models, ATG treatment resulted in the attenuation from EAE, both in a preventive and early therapeutic setting. We conclude that ATG treatment enforces the development of a dominant immunoregulatory environment which may be advantageous for the treatment of T cell-driven autoimmune diseases.

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains.

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.

Specificity of inhibition of ras-p21 signal transduction by peptides from GTPase activating protein (GAP) and the son-of sevenless (SOS) ras-specific guanine nucleotide exchange protein.

In previous studies, involving molecular modeling of wild-type and oncogenic forms of the ras-p21 protein bound to GTPase activating protein GAP and the ras-specific guanine nucleotide exchange-promoting protein, SOS, we identified specific domains of GAP and SOS proteins that differ in conformation when the computed average structures of the corresponding wild-type and oncogenic complexes are superimposed. Additionally, in these previous studies, we have synthesized peptides corresponding to these domains and found that all of them inhibit either or both oncogenic (Val 12-containing) p21- and insulin-activated wild-type p21-induced oocyte maturation. To document further the specificity of the inhibition of these peptides for the ras signal transduction pathway, we have now tested their effects on progesterone-induced maturation that occurs by a ras-independent pathway. None of these peptides, including a peptide corresponding to residues 980-989 of SOS that completely blocks oncogenic p21-induced maturation and also causes extensive inhibition of insulin-induced maturation, affects progesterone-induced maturation, suggesting that all of these peptides are specific for the ras pathway. Since our approach to the design of peptides that can inhibit oncogenic ras-p21 selectively is based on identifying domains that differ in conformation between oncogenic and wild-type complexes, we have now further synthesized peptides that correspond to domains of GAP (residues 903-910) and SOS (residues 792-804) that do not differ in conformation when the average structures are superimposed. These peptides do not inhibit either oncogenic p21- or insulin-induced oocyte maturation, supporting the overall strategy of using peptides from domains that change conformation as the ones most likely to inhibit oncogenic and/or wild-type ras-p21. These results further support the specificity of inhibition of the GAP and SOS peptides from the conformationally distinct domains of both proteins.

Functional interactions of Raf and MEK with Jun-N-terminal kinase (JNK) result in a positive feedback loop on the oncogenic Ras signaling pathway.

In previous studies we have found that oncogenic (Val 12)-ras-p21 induces Xenopus laevis oocyte maturation that is selectively blocked by two ras-p21 peptides, 35-47, also called PNC-7, that blocks its interaction with raf, and 96-110, also called PNC-2, that blocks its interaction with jun-N-terminal kinase (JNK). Each peptide blocks activation of both JNK and MAP kinase (MAPK or ERK) suggesting interaction between the raf-MEK-ERK and JNK-jun pathways. We further found that dominant negative raf blocks JNK induction of oocyte maturation, again suggesting cross-talk between pathways. In this study, we have undertaken to determine where these points of cross-talk occur. First, we have immunoprecipitated injected Val 12-Ha-ras-p21 from oocytes and found that a complex forms between ras-p21 raf, MEK, MAPK, and JNK. Co-injection of either peptide, but not a control peptide, causes diminished binding of ras-p21, raf, and JNK. Thus, one site of interaction is cooperative binding of Val 12-ras-p21 to raf and JNK. Second, we have injected JNK, c-raf, and MEK into oocytes alone and in the presence of raf and MEK inhibitors and found that JNK activation is independent of the raf-MEK-MAPK pathway but that activated JNK activates raf, allowing for activation of ERK. Furthermore, we have found that constitutively activated MEK activates JNK. We have corroborated these findings in studies with isolated protein components from a human astrocyte (U-251) cell line; that is, JNK phosphorylates raf but not the reverse; MEK phosphorylates JNK but not the reverse. We further have found that JNK does not phosphorylate MAPK and that MAPK does not phosphorylate JNK. The stress-inducing agent, anisomycin, causes activation of JNK, raf, MEK, and ERK in this cell line; activation of JNK is not inhibitable by the MEK inhibitor, U0126, while activation of raf, MEK, and ERK are blocked by this agent. These results suggest that activated JNK can, in turn, activate not only jun but also raf that, in turn, activates MEK that can then cross-activate JNK in a positive feedback loop.

A study on the effects of degradation and template concentration on the amplification efficiency of the STR Miniplex primer sets.

In forensic DNA analysis, the samples recovered from the crime scene are often highly degraded leading to poor PCR amplification of the larger sized STR loci. To avoid this problem, we have developed STR markers with redesigned primer sequences called "Miniplexes" to produce smaller amplicons. To assess the effectiveness of these kits, we have tested these primer sets with enzymatically degraded DNA and compared the amplifications to a commercial kit. We also conducted sensitivity and peak balance studies of three Miniplex sets. Lastly, we report a case study on two human skeletal remain samples collected from different environmental conditions. In both types of degraded DNA, the Miniplex primer sets were capable of producing more complete profiles when compared to the larger sized amplicons from the commercial kit. Correct genotypes were obtained at template concentrations as low as 31 pg/25 microL. Overall, our data confirm that our redesigned primers can increase the probability of obtaining a usable profile in situations where standard kits fail.

An effector peptide from glutathione-S-transferase-pi strongly and selectively blocks mitotic signaling by oncogenic ras-p21.

Oncogenic ras-p21 directly activates jun-N-terminal kinase (JNK) and its substrate, jun as a unique step on its mitogenic signal transduction pathway. This activation is blocked by the specific JNK-jun inhibitor, glutathione-S-transferase-pi (GST-pi). Four domains of GST-pi have been implicated in this regulatory function: 34-50, 99-121, 165-182, and 194-201. The 34-50 domain is unique in that it does not affect GST-pi binding to JNK-jun but blocks jun phosphorylation by JNK. We now find that it completely blocks oncogenic (Val 12-) ras-p21-induced oocyte maturation but has no effect on insulin-induced oocyte maturation. Because the latter process requires activation of wild-type ras-p21, this peptide appears to be specific for inhibiting only the oncogenic form of ras-p21, suggesting its use in treating ras-induced tumors.

Loop domain peptides from the SOS ras-guanine nucleotide exchange protein, identified from molecular dynamics calculations, strongly inhibit ras signaling.

In the accompanying paper, we found, using molecular dynamics calculations, four domains of the ras-specific SOS guanine nucleotide exchange protein (residues 589-601, 654-675, 746-761, and 980-989) that differ markedly in conformation when SOS is complexed with either oncogenic (Val 12-) ras-p21 or wild-type ras-p21. Three of these domains contain three crystallographically undefined loops that we modeled in these calculations, and one is a newly identified non-loop domain containing SOS residues 980-989. We have now synthesized peptides corresponding to these four domains and find that all of them block Val 12-ras-p21-induced oocyte maturation. All of them also block insulin-induced oocyte maturation, but two of these peptides, corresponding to SOS residues 589-601 and 980-989, block oncogenic ras to a significantly greater extent. These results suggest that SOS contains domains, including the three loop domains, that are important for ras signaling and that several of these domains can activate different pathways specific to oncogenic or wild-type ras-p21.

A protein methyl transferase, PRMT5, selectively blocks oncogenic ras-p21 mitogenic signal transduction.

A Janus-2 (JAK-2) binding protein, JBP1, has been found to function as an arginine methyl transferase and is now designated PRMT5. Co-injection of plasmids encoding this protein together with oncogenic (Val 12-containing) ras-p21 protein into Xenopus leavis oocytes results in strong inhibition of oncogenic p21-induced oocyte maturation. This inhibition appears to be dependent on the methyl transferase function since a partially active R368A mutant shows diminished ability to inhibit Val 12-p21-induced oocyte maturation, and an almost totally inactive GAGRG (365-369) deletion mutant fails to inhibit Val 12-p21-induced maturation. In contrast, PRMT5 (JBP1) does not inhibit insulin-induced oocyte maturation. Since insulin-induced maturation depends on activation of cellular ras-p21, PRMT5 does not appear to inhibit the wild-type p21 protein. We also find that arginine methyl transferase inhibitors strongly block oncogenic ras-p21-activated, but not insulin-activated, wild-type ras-p21-induced oocyte maturation. Thus signaling by oncogenic p21 appears to involve methyltransferases uniquely. Surprisingly, the active site peptide, Gly-Arg-Gly, strongly suppresses insulin-induced maturation but has no effect on Val 12-p21-induced maturation. This peptide may therefore be useful in defining steps in the wild-type ras pathway.

Peptides designed from molecular modeling studies of the ras-p21 protein induce phenotypic reversion of a pancreatic carcinoma cell line but have no effect on normal pancreatic acinar cell growth.

PURPOSE: From molecular modeling studies we found that two ras-p21 peptides, corresponding to p21 residues 35-47 (PNC-7) and 96-110 (PNC-2), selectively block oncogenic (Val 12-p21), but not insulin-activated wild-type, p21-induced oocyte maturation. Our purpose was to determine if these peptides block the growth of mammalian cancer cells but not their normal counterpart cells. METHODS: Since oncogenic ras has been implicated as a causative factor in over 90% of human pancreatic cancers, we have established a normal pancreatic acinar cell line (BMRPA1) and the corresponding ras-transformed pancreatic cancer cell line (TUC-3). We treated both cell lines with PNC-7 and PNC-2 and the unrelated negative control peptide, X13, attached to the penetratin sequence that allows membrane penetration and also transfected these cell lines with plasmids encoding all three peptides. RESULTS: Treatment of TUC-3 cells with each peptide resulted in their complete phenotypic reversion to the untransformed phenotype as revealed by the lack of tumor formation of these revertant cells implanted in the peritoneal cavities of nude mice. In contrast, treatment with X13-leader resulted in no inhibition of cell growth. Identical results were obtained when TUC-3 cells were transfected with plasmids expressing PNC-2, PNC-7 and X13. None of these peptides affected the normal growth of BMRPA1 cells. CONCLUSIONS: PNC-2 and PNC-7 peptides induce phenotypic reversion of ras-induced pancreatic cancer cells and have no effect on normal pancreatic cell growth. Since the plasmid encoding PNC-2 without penetratin also had the same effect on the TUC-3 cell line, we conclude that the penetratin sequence has no effect on the activity of this peptide. Since X13 attached to penetratin had no effect on TUC-3 cells, the effect is specific for PNC-2 and PNC-7 and further confirms that the effect is not caused by the penetratin sequence. PNC-2- and PNC-7-penetratin may therefore be useful in the treatment of ras-induced pancreatic carcinomas.

Oncogenic and activated wild-type ras-p21 proteins induce different isoforms of protein kinase C in mitogenic signal transduction.

We have previously found that the protein kinase C (PKC) inhibitor, CGP 41 251, blocks oncogenic ras-p21 protein- and beta-PKC-induced oocyte maturation, but only weakly inhibits insulin-induced oocyte maturation (which requires activation of wild-type endogenous ras-p21). Because the dose-response curves for inhibition of oncogenic p21- and beta-PKC-induced oocyte maturation by CGP 41 251 superimpose and because the ras-p21-inactivating antibody, Y13-259, does not inhibit beta-PKC-induced oocyte maturation, we concluded that the oncogenic, but not wild-type, protein requires beta-PKC as a downstream target. Because multiple isoforms of PKC exist and several of these, such as epsilon-PKC, have been found to be important on ras signal transduction pathways, we have investigated which PKC isoforms are critical to each ras protein. For this purpose, we used PKC-isoform-specific inhibitors, which have been shown to inhibit selectively the function and translocation of PKC isoforms in vitro and in vivo. Specifically, the peptides KLFIMN, QEVIRN, and EAVSLKPT each inhibit beta-1, beta-2, and epsilon-PKC, respectively, but do not cross-inhibit other PKC isoforms. We find that the epsilon-PKC inhibitory peptide strongly blocks insulin- but not oncogenic ras-p21-induced oocyte maturation whereas the beta-2 inhibitory peptide more strongly inhibits oncogenic ras-p21-induced oocyte maturation, corroborating our previous studies. The beta-1 inhibitory peptide has little effect on either protein. We conclude that selective inhibition of individual PKC isoforms permits the distinction between signal transduction initiated by oncogenic and activated wild-type p21 proteins and implicate different specific PKC isoforms in mitogenic signal transduction by each of these proteins. The ability to dissect the role of individual PKC isozymes in this regulation is of therapeutic significance.