Plasmid expression of a peptide that selectively blocks oncogenic ras-p21-induced oocyte maturation.

PURPOSE: We have previously found that a synthetic peptide corresponding to ras-p21 residues 96 110 (PNC2) selectively blocks oncogenic (Val 12-containing) ras-p21 protein-induced oocyte maturation. With a view to introducing this peptide into ras-transformed human cells to inhibit their proliferation, we synthesized an inducible plasmid that expressed this peptide sequence. Our purpose was to test this expression system in oocytes to determine if it was capable of causing selective inhibition of oncogenic ras-p21. METHODS: We injected this plasmid and a plasmid expressing a control peptide into oocytes either together with oncogenic p21 or in the presence of insulin (that induces maturation that is dependent on normal cellular ras-p21) in the presence and absence of the inducer isopropylthioglucose (IPTG). RESULTS: Microinjection of this plasmid into oocytes together with Val 12-p21 resulted in complete inhibition of maturation in the presence of inducer. Another plasmid encoding the sequence for the unrelated control peptide, X13, was unable to inhibit Val 12-p21-induced maturation. In contrast, PNC2 plasmid had no effect on the ability of insulin-activated normal cellular or wild-type ras-p21 to induce oocyte maturation, suggesting that it is selective for blocking the mitogenic effects of oncogenic (Val 12) ras p21. CONCLUSION: We conclude that the PNC2 plasmid selectively inhibits oncogenic ras-p21 and may therefore be highly effective in blocking proliferation of ras-induced cancer cells. Also, from the patterns of inhibition, by PNC2 and other ras- and raf-related peptides, of raf- and constitutively activated MEK-induced maturation, we conclude that PNC2 peptide inhibits oncogenic ras p21 downstream of raf.

Influence of gestational age and maternal height on fetal femur length calculations.

OBJECTIVE: To determine whether current methods of detecting Down syndrome based on fetal femur length calculations are influenced by gestational age or maternal height. METHODS: Four formulas were used to calculate expected femur length (FL) based on the fetal biparietal diameter (BPD) between 15 0/7 weeks' gestation and 19 6/7 weeks' gestation. For each gestational age, the BPD:FL ratio for women shorter than one standard deviation (SD) below the mean height was compared with the ratio for women taller than one SD above the mean height. A measured:expected FL ratio of 0.91 or less and a BPD:FL ratio greater than 1.5 SD above the mean was considered abnormal. RESULTS: The four formulas used to calculate measured:expected FL ratios were significantly more likely to be abnormal at 15--16 weeks' gestation, compared with 18-19 weeks' gestation (P <.05). Maternal height correlated with femur lengths at 18 and 19 weeks' gestation (P <.05) but not at earlier gestational ages. At 18 and 19 weeks' gestation, women shorter than one SD below the mean were twice as likely to have an abnormal BPD:FL ratio compared with women taller than one SD above the mean (relative risk 2.38; 95% confidence interval 1.21, 4.69). CONCLUSION: Early gestational age increases a woman's risk of having an abnormal measured:expected FL ratio, whereas short stature increases a woman's risk of having an abnormal BPD:FL ratio at later gestational ages. These findings indicate that risk assessment for fetal Down syndrome for such patients might be inaccurate. (Obstet Gynecol 2001;97:742-6.

Induction of oocyte maturation by jun-N-terminal kinase (JNK) on the oncogenic ras-p21 pathway is dependent on the raf-MEK signal transduction pathway.

PURPOSE: We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway. METHODS: We microinjected raf dominant negative mutant mRNA (DN-raf) and the MEK-specific phosphatase, MKP-T4, either together with oncogenic p21 or c-raf mRNA, into oocytes or into oocytes incubated with insulin to determine the effects of these raf-MEK-MAP kinase pathway inhibitors. RESULTS: We found that oocyte maturation induced by both oncogenic and activated normal p21 is inhibited by both DN-raf and by MKP-T4. The latter more strongly blocks the oncogenic pathway. Also an mRNA encoding a constitutively activated MEK strongly induces oocyte maturation that is not inhibited by DN-raf or by MKP-T4. Surprisingly, we found that oocyte maturation induced by JNK is blocked both by DN-raf and MKP-T4. Furthermore, we discovered that c-raf induces oocyte maturation that is inhibited by glutathione-S-transferase (GST), which we have found to be a potent and selective inhibitor of JNK. CONCLUSION: We conclude that there is a strong reciprocal interaction between the SAP pathway involving JNK and the raf-MEK-MAP kinase pathway and that oncogenic ras-p21 can be preferentially inhibited by MEK inhibitors. The results imply that blockade of both MEK and JNK-oncogenic ras-p21 interactions may constitute selective synergistic combination chemotherapy against oncogenic ras-induced tumors.

BSAP (Pax5)-importin alpha 1 (Rch1) interaction identifies a nuclear localization sequence.

BSAP (Pax5) is an essential transcription factor for early B cell and central nervous system development. In later B cell development, BSAP has been implicated in the regulation of 3' Ig enhancers and a number of B cell-specific genes. Previous studies have suggested a role for BSAP-interacting proteins in the regulation of the function of BSAP. Using the yeast two-hybrid system, we identified importin alpha1 (Rch1) as a BSAP-interacting protein. Importin alpha proteins have been shown to escort proteins into the nucleus through interaction with a nuclear localization signal (NLS), composed of short stretches of basic amino acids. A predicted NLS in BSAP (NKRKRDE, located at amino acids 195-201 in the central domain) was confirmed to be essential for interaction with importin alpha1 by the yeast two-hybrid assay. Physical interaction between BSAP and importin alpha1 was detected in vitro by a glutathione S-transferase (GST) pulldown assay. The NLS sequence in BSAP conferred nuclear localization to green fluorescent protein (GFP)-BSAP fusion proteins. Although the N-terminal paired (DNA-binding) domain of BSAP also conferred nuclear localization when coupled to green fluorescent protein, this domain did not bind to importin alpha1 in the yeast two-hybrid assay. The NLS sequence in the central domain of BSAP binds to the C-terminal 98-amino acid fragment of importin alpha1.

Cost consequences of implementation of an early obstetrical discharge programme in a military teaching hospital.

We evaluate the cost consequence of a voluntary early obstetrical discharge programme in a military teaching hospital. The study involved a control group of routine obstetrical discharge patients with uncomplicated vaginal delivery from March 1 to August 31, 1994 and the study group of early obstetrical discharge (24-48 hours) patients with uncomplicated vaginal delivery from March 1 to August 31, 1996. There were 1,042 total control patients with routine vaginal delivery totalling 2,668 hospital days with a mean number of hospital days of 2.56 per patient. The study group of early obstetrical discharge patients with uncomplicated vaginal delivery encompassed 1,050 patients with 1,965 hospital days with mean hospital days of 1.87 per patient (p < 0.05) without an increase in postpartum clinic or emergency room visits. The total cost of admissions (cost calculation of $1,221 per hospital day) fell from $3,257,628 in the routine discharge group to $2,399,625 in the early discharge cohort showing a total cost savings of $858,003 over the 6-months study period. The average cost per obstetrical admission for routine vaginal delivery fell from $3,126 per day to $2,285 per day without an increase in the postpartum paediatric adverse outcomes. Maternal postpartum readmission rates were statistically significantly increased (p < 0.05) in the study group at 0.6% with an OR[2.32(2.17, 6.92)] but all readmissions fell outside the 48-hour early discharge window. This programme showed significant cost savings without concomitant increase in paediatric or maternal adverse outcomes.