Postprandial induction of chaperone gene expression is rapid in mice.

Molecular chaperones assist in the biosynthesis and processing of proteins. Most chaperones are induced by physiological stresses. We have shown that dietary energy restriction decreases the mRNA and protein levels of many endoplasmic reticulum chaperones in the livers of mice. Here, we have investigated the response of chaperone mRNA to feeding. Control and 50% energy-restricted C3B10RF1 mice were deprived of food for 24 h, fed, and killed 0, 1.5, 5 or 12 h after feeding. Chaperone mRNAs were strongly induced as early as 1.5 h after feeding in control and energy-restricted mice. The integrated levels of these mRNA over 24 h were significantly lower in energy-restricted mice. The mRNA response to energy intake was mirrored over the course of days in the level of chaperone protein. A similar but smaller response to feeding was found in kidney and muscle. Puromycin and cycloheximide failed to inhibit the feeding response, suggesting that feeding releases chaperone expression from an unstable inhibitor. Studies with dibutyryl-cAMP- and glucagon-supplemented, normal and streptozotocin-diabetic mice suggest that glucagon and insulin may be mediators of the feeding response. Adrenalectomy enhanced the feeding induction, but dexamethasone administration had no effect. Thus, postprandial changes in insulin and glucagon may link chaperone gene expression to feeding, possibly in several tissues including liver.     

Choice of an ovarian stimulation protocol according to the follicular puncture method in an in vitro fertilization programme

In order to ascertain the adequacy of ovarian stimulation protocols with a type of follicular puncture, 126 women undergoing in vitro fertilization received either combination clomiphene/hMG or hMG alone according to a randomized test protocol. Within both groups patients for whom a pelvic examination was required had laparoscopies, while others had transvaginal ultrasonically guided punctures as far as possible. Clomiphene/hMG was more efficient than hMG alone as assessed from the cleavage rate (68% vs. 54%; p less than 0.01) and the pregnancy per attempt rate (16% vs. 5%; p less than 0.05). Laparoscopic punctures were more efficient than ultrasonically guided punctures (mean number of recovered oocytes: 4.8 +/- 2.6 vs. 3 +/- 2.5; p less than 0.001), but slightly better results were achieved by this latter method in ongoing pregnancy per puncture rate (18% vs. 8%; NS). With ultrasonically guided punctures, stimulation by clomiphene/hMG allowed better oocyte recoveries (3.8 +/- 2.5 vs. 2.3 +/- 1.9, p less than 0.05). Such results constitute an argument for preferential use of the clomiphene/hMG stimulation protocol with ultrasonically guided punctures.     

Intracellular Ca2+ suppressed a transient potassium current in hippocampal neurons

The effects of intracellular Ca2+ (Ca2+i) on K+ currents in hippocampal cells were examined using acutely isolated cells obtained from adult guinea pigs. Whole-cell voltage-clamp recordings were carried out in a configuration that allowed a continuous perfusion of the intracellular medium. Recording media were made to block inward currents and allowed selective activation of K(+)-dependent outward currents. Voltage-dependent outward currents consisted of an initial rapidly decaying component followed by a sustained component. The time constant of decay of the transient current was about 25 msec, and previous studies (Numann et al., 1987) showed that the kinetic and pharmacological properties of this current closely resembled the A current recorded in invertebrate neurons (Connor and Stevens, 1971; Thompson, 1982). Intracellular perfusion of hippocampal cells with a solution containing elevated Ca2+ (about 4.5 x 10(-4) M) elicited outward currents at the holding potential (-45 to -55 mV) and produced changes in voltage-dependent K+ currents. The transient outward current (IA) activated by depolarization was suppressed with increases in Ca2+i. Delayed, sustained K+ currents were greatly potentiated. Data also showed that, among the 3 effects elicited by Ca2+i, suppression of IA was most sensitive to Ca2+i elevation. Previous results (Numann et al., 1987) showed that IA had a lower threshold (about -45 mV) than sustained currents (about -40 mV). By using low levels of depolarization (-40 mV), IA can be selectively activated, and the suppressive effect of Ca2+i on IA was confirmed on the kinetically isolated IA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene expression patterns and profile changes pre- and post-erlotinib treatment in patients with metastatic breast cancer.

PURPOSE: To delineate gene expression patterns and profile changes in metastatic tumor biopsies at baseline and 1 month after treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib in patients with metastatic breast cancer. EXPERIMENTAL DESIGN: Patients were treated with 150 mg of oral erlotinib daily. Gene expression profiles were measured with Affymetrix U133A GeneChip and immunohistochemistry was used to validate microarray findings. RESULTS: Estrogen receptor (ER) status by immunohistochemistry is nearly coincided with the two major expression clusters determined by expression of genes using unsupervised hierarchical clustering analysis. One of 10 patients had an EGFR-positive tumor detected by both microarray and immunohistochemistry. In this tumor, tissue inhibitor of metalloproteinases-3 and collagen type 1 alpha 2, which are the EGF-down-regulated growth repressors, were significantly increased by erlotinib. Gene changes in EGFR-negative tumors are those of G-protein-linked and cell surface receptor-linked signaling. Gene ontology comparison analysis pretreatment and posttreatment in EGFR-negative tumors revealed biological process categories that have more genes differentially expressed than expected by chance. Among 495 gene ontology categories, the significant differed gene ontology groups include G-protein-coupled receptor protein signaling (34 genes, P = 0.002) and cell surface receptor-linked signal transduction (74 genes, P = 0.007). CONCLUSIONS: ER status reflects the major difference in gene expression pattern in metastatic breast cancer. Erlotinib had effects on genes of EGFR signaling pathway in the EGFR-positive tumor and on gene ontology biological process categories or genes that have function in signal transduction in EGFR-negative tumors.     

Persistent reversal of diabetes by transplantation of fetal pig proislets into nude mice

Facing the limited availability of human adult and fetal pancreases, fetal pig proislets (pancreatic islet precursors) were investigated in view of several inherent advantages. Six litters of fetuses of mean +/- SE gestational age 75 +/- 3 days were obtained from commercially available farm pigs. Pancreatic tissue was gently digested with collagenase, then a 10-day culture was performed. During culture, fetal proislets showed no insulin response to glucose alone but a significant response to glucose plus theophylline. The insulin content per microgram of DNA in the cultured proislets continuously increased. Histological examination by immunoperoxidase staining showed that, apart from single insulin- and glucagon-positive cells, there were no discrete islets in the pancreatic tissue and the cultured proislets. Diabetes was induced with streptozocin (STZ) in eight nude mice 3-4 wk after proislet transplantation and in another eight nude mice without transplantation. During the initial week, blood glucose levels of mice in both groups increased rapidly. The mean +/- SE peak value of blood glucose levels in the transplanted group was 20.4 +/- 2.0 mM and was 20.1 +/- 1.3 mM in the group without transplantation. Simultaneously, body weight decreased from 29.5 +/- 0.7 to 21.5 +/- 0.9 g and from 27.9 +/- 0.7 to 19 +/- 1 g in the groups, respectively. Afterward, blood glucose levels of mice in the transplanted group gradually decreased, and normoglycemia was achieved in all mice within 50 +/- 13 days after injection of STZ, i.e., 74 +/- 13 days after transplantation. The group without transplantation persistently maintained blood glucose levels greater than 16.7 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential changes of retina dopamine binding sites and adenylyl cyclase responses following 6-hydroxydopamine treatment.

Both dopamine D1 and D2 receptors are present in rat retina. D1 receptors are positively coupled to adenylyl cyclase, while D2 receptors are negatively coupled. After intraocular administration of the neurotoxin 6-hydroxydopamine (6-OHDA) there is depletion of retinal dopamine by about 90%, as well as a decrease of the number of D1 and D2 receptor binding sites. Following the 6-OHDA lesion, there is an enhancement of the D1 receptor-stimulated accumulation of cyclic-AMP and a loss of D2 receptor-inhibited accumulation of cyclic-AMP. Our results suggest that some of the retinal D2 receptors are coupled to adenylyl cyclase and some are not.     

c-myc is required for osteoclast differentiation.

The role of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL)-a tumor necrosis factor (TNF)-related cytokine-in osteoclast formation has been established clearly. However, the downstream signaling pathways activated by this cytokine remain largely unknown. To identify genes that play a role in osteoclastogenesis, we used RAW 264.7 mouse monocytes as a model system for the differentiation of multinucleated osteoclasts from mononucleated precursors. RAW 264.7 cells were induced with RANKL to form multinucleated giant osteoclast-like cells (OCLs) that expressed a number of osteoclast-specific markers and were able to form resorption pits on both calcium phosphate films and bone slices. This system was used to identify genes that are regulated by RANKL and may play a role in osteoclast differentiation. The proto-oncogene c-myc was strongly up-regulated in RANKL-induced OCLs but was absent in undifferentiated cells. Expression of Myc partners Max and Mad, on the other hand, was constant during OCL differentiation. We expressed a dominant negative Myc in RAW 264.7 cells and were able to block RANKL-induced OCL formation. Northern Blot analysis revealed a delay and a significant reduction in the level of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K. We conclude that c-myc is a downstream target of RANKL and its expression is required for RANKL-induced osteoclastogenesis.     

Slowly progressive hemiparesis in childhood as a consequence of Rasmussen encephalitis without or with delayed-onset seizures

Five young children developed slowly progressive hemiparesis as the initial manifestation of Rasmussen encephalitis (RE). Three have remained seizure free over an observational period of 1.3–1.9 years. In the remaining two patients, seizures occurred after 0.5 and 0.6 years respectively. We suggest that RE might be presently underdiagnosed and should be suspected in cases of new onset hemiparesis. In this series, three out of five patients showed oligoclonal bands on examination of cerebrospinal fluid (CSF) which represented additional diagnostic hints towards an immune-mediated condition. According to recently published formal diagnostic criteria, evidence of progressive cerebral hemiatrophy or bioptic identification of RE-typical inflammation confirms the diagnosis in such cases. Long-term immunotherapy is recommended in order to prevent further tissue loss and functional decline.