Temporal and Depolarization-Induced Changes in the Absolute Amounts of mRNAs Encoding Metabotropic Glutamate Receptors in Cerebellar Granule Neurons In Vitro

Abstract: Cerebellar granule neurons in primary culture express metabotropic glutamate receptors (mGluRs) coupled to the stimulation of phosphoinositide hydrolysis and to the inhibition of cyclic AMP (cAMP) formation. To evaluate which mGluR mRNAs are expressed in granule neurons under different depolarizing conditions, we measured the absolute amounts of selected receptor mRNAs in neurons cultured for 3–13 days in the presence of either 10 or 25 m M KCl. mGluR-specific primer pairs and internal standards, corresponding to unique regions of mGluR1a, mGluR2, mGluR3, mGluR4, and mGluR5, were constructed and used in a competitive PCR-derived assay to quantify the corresponding mRNA levels. For phosphoinositide-coupled receptors, the absolute content of mGluR1a mRNA was three to 10 times higher than the content of mGluR5 mRNA. The expression of mGluR5 mRNA increased up to 9 days in vitro and was much higher in 10 m M than in 25 m M KCl. For the cAMP-coupled receptors, there was a large amount of mGluR4 mRNA and a much smaller content of the mGluR3 and mGluR2 mRNAs. Maintaining the granule neurons in vitro in 10 m M KCl increased the absolute amount of mRNAs encoding mGluR2 and mGluR4 at 9 and 13 days in vitro. In contrast, the content of the mGluR3 mRNA was consistently higher in neurons cultured in 25 m M KCl. These data are consistent with the possibility that in primary cultures of cerebellar neurons, phosphoinositide responses may be predominantly mediated by mGluR1a, rather than mGluR5, and that cAMP inhibition involves preferentially mGluR4 and mGluR3.     

Wheat-germ aspartate transcarbamoylase. Purification and cold-lability.

1. Aspartate transcarbamoylase was purified approx. 3000-fold from wheat (Triticum vulgare) germ in 15-20% yield. The product has a specific activity of 14 mumol/min per mg of protein and is approx. 90% pure. The purification scheme includes the use of biospecific "imphilyte" chromatography as described by Yon [Biochem.J.(1977) 161, 233-237]. The enzyme was passed successively through columns of CPAD [N-(3-carboxypropionyl)aminodecyl]-Sepharose in the absence and presence respectively of the ligands UMP and L-aspartate. In the second passage the enzyme was specifically displaced away from impurities with which it co-migrated in the first passage. These two steps contributed a factor of 80 to the overall purification. 2. The enzyme is slowly inactivated on dilution at 0 degrees C and pH 7.0, the inactivation being partially reversible. A detailed investigation of the temperature- and pH-dependence of the cold-inactivation suggested that it was initiated by the perturbation of the pKa values of groups with a moderately high and positive heat of ionization, which were tentatively identified as histidine residues. These findings support a new concept of cold-lability proposed by Bock, Gilbert & Frieden [Biochem. Biophys. Res. Commun. (1975) 66, 564-569].     

Further freeze-fracture studies on the uterine epithelium of Salamandra salamandra (L.) (Amphibia,Urodela) using the antibiotic filipin

Summary The apical portion of the uterine lining of the ovoviviparous fire salamander, Salamandra salamandra, was studied by the freeze-fracture technique in conjunction with the polyene antibiotic filipin. Filipin-sterol complexes were found in the luminal plasmalemma and in the membranes limiting the mucous secretory granules typical of this epithelium. In all females, but particularly in non-pregnant females, more or less discrete clusters of filipinsterol complexes were occasionally found overlying heavily affected secretory granules. The findings are discussed with regard to comparable results (Orci et al. 1980) based on the examination of collapsed and stretched urinary bladders of toads.We are indebted to Mrs. K. Ott for excellent technical assistance, to Miss E.S. MacLure for linguistic corrections and to Dr. J.E. Grady of the Upjohn Co., Kalamazoo, Michigan USA, for kindly providing the filipin     

Klinik und Genetik des Joubert-Syndroms

Joubert syndrome generally represents an autosomal recessive and rarely X-linked disorder characterized by hypotonia, an irregular breathing pattern, abnormal eye movements, ataxia, developmental delay and a complex mid-hindbrain malformation causing the molar tooth sign on magnetic resonance imaging (MRI). Many patients have additional features, with nephronophthisis, retinal dystrophy, coloboma and hepatic fibrosis representing the most frequent features. Due to its clinical variability and overlap with other syndromes, the term “Joubert syndrome and related disorders” (JSRD) was proposed. To date 10 genes are known to cause JSRD. The encoded proteins are localized to cilia, linking JSRD to other human ciliopathies.     

Removal of mandibular tooth follicles before distraction osteogenesis

Distraction osteogenesis is an innovative technique that has transformed the treatment of craniofacial malformations in young children. Bone generation obviates the need for graft material, which is in short supply in young patients, thus making possible surgical procedures on the craniofacial skeleton in young children. Sufficient mandibular volume is required for the osteotomy and placement of the device screws and/or pins. To have adequate bone stock and to facilitate distraction, the authors preoperatively examined all patients radiographically and selected those with tooth follicles that precluded successful osteotomy and pin placement for planned mandibular distraction. This report is of the first 13 children, aged 9 months to 6 years, who underwent predistraction enucleation. The osteotomy and device placement were performed successfully at least 4 months after enucleation. The described procedure has minimal morbidity and has resulted in successful subsequent distraction. The advantages, disadvantages, and cost-benefit issues are discussed.     

The effect of an early Le Fort III surgery on permanent molar eruption

The purpose of this retrospective study was to evaluate the extent to which an early Le Fort III osteotomy affects the position and eruption of the permanent maxillary first and second molars. To test the null hypothesis that there are no changes in eruption patterns, 31 patients diagnosed with craniosynostoses (13 with Crouzon's syndrome, nine with Apert's syndrome, eight with Pfeiffer's syndrome, and one with Carpenter's syndrome) with a mean age at the time of surgery of 5.3 +/- 1.3 years were studied. All patients underwent a Le Fort III osteotomy performed by a single surgeon to correct the anatomical deformity for functional and psychosocial reasons. Eighteen patients with craniosynostoses who had not been operated on (11 with Crouzon's syndrome, four with Apert's syndrome, and three with Pfeiffer's syndrome) served as controls; they had a mean age of 21.2 +/- 9.5 years. First and second molar positions and eruption patterns were assessed separately on panoramic radiographs by three observers. For the patients who underwent surgery, long-term evaluation showed that although 79 percent of all first molars erupted compared with 100 percent for the control group (p < 0.001), only 18 percent of all second permanent molars erupted compared with 89 percent for the control group (p < 0.0001). The authors conclude that in a significant minority of cases, early Le Fort III osteotomy affects first molar eruption, whereas the probability of second molar eruption is significantly decreased in the majority of cases. Therefore, Le Fort III osteotomy sites should be positioned distal to the second molar tooth buds. If this is not possible, patients, parents, and dental professionals should be made aware of these early postosteotomy sequelae so that later treatment planning can be enhanced.     

Differential mortality and the Donner Party disaster

The story of the Donner Party combines powerful American themes. The beginning of the tale is immersed in the romance of the western frontier and in the American dream of improving life by taking bold steps. The end of the tale, in contrast, is awash in death and cannibalism. No wonder Americans learn the story as children; no Grimm's fairy tale can match it. However, detailed analyses of the patterning of Donner Party deaths suggest that the story is even better read as a piece of biology than as a piece of history. Virtually all aspects of Donner Party mortality can now be explained by our knowledge of the factors that cause differential mortality in human societies. The differential fates of these emigrants show us natural selection in action.     

Lipid content and metabolism of human keratinocyte cultures grown at the air-medium interface

The differentiation of human keratinocytes in most culture systems is incomplete; e.g., lamellar bodies, the characteristic lipid-delivery organelles of epidermis, are not present. Moreover, their lipid profile does not reflect the distinctive composition found in cornifying epidermis. In contrast, keratinocytes that grow at an air-medium interface exhibit more complete differentiation. In this study, we compared the elaboration of lamellar bodies, the lipid content, and the lipid metabolism of human keratinocytes, cultured both under standard immersed conditions and after lifting to an air-medium interface. Whereas submerged cultures neither elaborated lamellar bodies nor displayed a lipid distribution characteristic of cornifying epidermis, lifted cultures displayed advanced cornification, elaborated lamellar bodies which were deposited in intercellular domains, and a lipid profile more typical of cornifying epidermis. Moreover, lipid biosynthesis was 5-10-fold more active in lifted than in immersed cultures, and was not inhibited by exogenous lipoproteins. These findings are consistent with recent studies that demonstrate both high rates of lipogenesis in differentiating layers of the epidermis as well as autonomy of lipogenesis from the influence of circulating lipoproteins. Thus, the lipid content and metabolism of human keratinocyte cultures, grown at an air-medium interface, demonstrate features that simulate the epidermis.