Reduced cyclin D1 expression in the cerebella of nutritionally deprived rats correlates with developmental delay and decreased cellular DNA synthesis.

Nutritional deprivation in the early postnatal period severely inhibits cerebellar growth and development, which is related in part to reduced levels of growth factors. Cyclin D1 encodes a growth factor-inducible regulatory subunit of a serine/thereonine kinase that is capable of phosphorylating the tumor suppressor pRB, thereby allowing normal progression through the G1 phase of the cell-cycle. Because the abundance of cyclin D1 is rate limiting in this progression, we examined the regulation of cyclin D1 expression in vivo, using a model of nutritional deprivation. Cyclin D1 expression in cerebella of fed control rats was detected in the external granular layer and was associated with cellular proliferation within this layer. Nutritional deprivation of rats reduced cerebellar weight, as well as the thickness of the molecular layer that largely consists of cells migrating from the external granular layer. Refeeding partially restored cerebellar weight, molecular layer thickness and increased external granular layer cyclin D1 immunostaining. Since nutritional deprivation is accompanied by lower levels of circulating insulin-like growth factor-I (IGF-I), we determined whether IGF-I directly stimulated the cyclin D1 promoter. The human cyclin D1 promoter linked to the luciferase reporter gene was stably integrated into PC12 cells. IGF-I stimulated cyclin D1 promoter activity 4- to 6-fold at 6 hours (h). These findings are consistent with the notion that nutritional deprivation may affect proliferative growth by altering expression of cyclin D1 in the germinal cell layer and that regulation of cyclin D1 expression by growth factors may contribute to normal neonatal cerebellar development. The reduction in cyclin D1 expression as cells differentiate in the cerebellum is consistent with a potential role for cyclin D1 in this process.

Thyrotropin-releasing hormone (TRH): Measurements in human spinal fluid.

Levels of thyrotropin-releasing hormone (TRH) were quantitated in human lumbar spinal fluid (CSF) utilizing a sensitive and specific TRH radioimmunoassay. Endogenous TRH was sufficiently stable in CSF to permit 85% recovery of intact TRH after 48 h storage at 4 C. TRH levels in AM or PM samples obtained from 15 women and 12 men were easily detected in all CSF specimens. No significant difference between the TRH concentration in CSF of men and women was observed (44.2+/-6.8 and 38.1+/-6.5 pg/ml (mean+/-SE) respectively). TRH concentrations were 40.2+/-6.9 pg/ml (mean+/-SEM) in AM and 41.4+/-8.0 pg/ml in PM samples. By contrast, CSF cortisol levels obtained concurrently were twofold higher in AM than PM (0.68+/-0.08 vs 0.38+/-0.02 mug/100 ml (mean+/-SEM) respectively, P less than 0.001). These data are consistent with the possibility that a portion of the TRH in CSF can be derived from the central nervous system (CNS) and unrelated to the hypo-physiotropic control of thyrotropin (TSH) synthesis and secretion.

Urea biosynthesis I. The urea cycle and relationships to the citric acid cycle.

The urea cycle consist of five enzymatically controlled steps that are catalyzed by carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinase, and arginase, respectively. The complete cycle is present in physiological meaningful levels in the liver of terrestrial vertebrates, and in man represents the sole mechanism for ammonia disposal. The formation of carbamyl phosphate and the synthesis of argininosuccinate are potential limiting steps in urea biosynthesis but substrate and not enzymes levels are rate-limiting under physiological conditions. In the adult, urea cycle enzymes change as a unit, and are largely influenced by dietary protein content. The urea cycle is closely linked to the citric acid cycle deriving one of its nitrogens through transamination of oxalacetate to form asparate and returns fumarate to that cycle. The biosynthesis of urea demands the expenditure of energy but less than 20% of the energy derived from metabolism of gluconeogenic amino acids is required for ureogenesis. Embryological development of the urea cycle in the tadpole and in mammalian fetal liver therefore permits use of amino acids as new sources of energy to meet oxidative demands for continuing growth.

Glucose metabolism in uremia.

Formation of CO2 from uniformly labeled 14C-glucose was measured in liver slices from uremic and normal rats. Both CO2 formation and lactate concentration were decreased in the uremic liver slices suggesting an inhibition of glucose oxidation. In addition, a net loss of glucose from the medium in the uremic preparation and a net gain in the normal controls suggested that there was increased nonoxidative utilization in the uremic liver. Such changes could not be explained by differences in glucose availability consequent to alterations in glycogen degradation. The most likely explanation is diversion of glucose into other biosynthetic pathways such as the synthesis of amino acids. In this regard, synthesis of glutamine appeared to be enhanced in uremia. Thus, products of carbohydrate metabolism may provide a potential mechanism for disposition of ammonia and synthesis of amino acids in uremia.

The 1986 McCollum award lecture. Fuel-mediated teratogenesis during early organogenesis: the effects of increased concentrations of glucose, ketones, or somatomedin inhibitor during rat embryo culture.

Whole rat embryos were explanted at head-fold, late pre-somite stage (day 9.5 of gestation) and cultured in rat sera varyingly supplemented with glucose (3, 6, 9, or 12 mg/mL), D,L sodium beta-hydroxybutyrate (2, 4, 8, or 16 mM), or both (6 mg/mL D-glucose plus 8 mM beta-hydroxybutyrate). During 48 h culture, increasing glucose alone or beta-hydroxybutyrate alone effected growth retardation and faulty neural and extraneural organogenesis in dose-dependent fashion. Synergistic dysmorphogenic effects occurred when minimally teratogenic concentrations of glucose and beta-hydroxybutyrate were combined. Sera from diabetic animals containing somatomedin inhibitor bioactivity were also able to produce growth retardation and major developmental lesions in presence of amounts of glucose and ketones which of themselves were not teratogenic. Thus, aberrant fuels and fuel-related products can impair growth and organogenesis in early post-implantation embryo. Such fuel-mediated teratogenesis may be multifactorial and include possibilities for synergistic and additive interactions.

Adaptation of the diphenylamine (DPA) assay to a 96-well plate tissue culture format and comparison with the MTT assay.

A simple spectrophotometric method for measuring DNA in proliferating cells is described. The method is an adaptation of the widely used diphenylamine (DPA) reaction to examine DNA in cells grown in a 96-well plate. This assay was capable of detecting as little as 10 ng DNA and could be used to measure DNA in stored as well as viable tissue samples. The DPA assay paralleled the MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay of mitochondrial reductase in A549 cells, a human lung cancer cell line; in EMT6 cells, a mouse breast cancer cell line; and in a primary cell culture of neonatal rat astrocytes. Over several days of proliferative growth in tissue culture, the ratio of MTT to DPA remained constant. Since the DPA assay and MTT assay measured different parameters of the same cells, they could be employed as complementary spectrophotometric assessments of cell growth on a 96-well plates using the same automated enzyme-linked immunosorbent assay plate reader.

Characterization of rat placental TRH-like material and the ontogeny of placental and fetal brain TRH.

TRH immunological and biological activity was characterized in rat placental extracts. Placentae were extracted sequentially with 2 N acetic acid and glacial acetic acid. The lyophilized residues were further extracted with 90 per cent methanol, and the extracts were then dried, and reextracted with distilled water. When a 100 000 g supernatant fraction of this extract was utilized for characterization, TRH extracted from the placenta was found to be similar to synthetic TRH by four criteria: parallelism of immunoassay inhibition curves, similarity of elution volumes after Sephadex G-10 chromatography, TRH biological activity (TSH release from rat pituitaries in vitro), and identity on high-pressure liquid chromatography. Between the 16th and 20th day of gestation, placental TRH activity increased nearly threefold, from 10.9 +/- 3.0 to 29.7 +/- 3.7 pg/mg protein. Changes of a similar magnitude were apparent in the fetal brain (6.0 +/- 0.5 to 18.9 +/- 2.4 pg/mg protein). Our studies suggest that TRH activity in the rat placenta increases with gestational age in a pattern similar to that described previously for certain placental protein hormones and developmental changes in fetal brain TRH.

Fetal fuels VI. Metabolism of alpha-ketoisocaproic acid in fetal rat brain.

The metabolic regulation of alpha-ketoisocaproic acid was studied in fetal brain from rats. Starvation of the mother for days 18-20 did not alter CO2 evolution from alpha-ketoisocaproic acid in fetal brain slices but significantly diminished the incorporation of the branched-chain keto acids into leucine. When fetal brain slices from starved mothers were exposed to graded concentrations of labeled alpha-ketoisocaproic acid (0.05-2.5 mM), over 70% of the labeled products were consistently represented by leucine and less than 30% by CO2. Both beta-hydroxybutyrate and pyruvate, alone and in combination, diminished the amount of 14CO2 that evolved from alpha-ketoisocaproic acid-1-14C, but had no effect on the conversion of the keto acid to labeled leucine. It is concluded that exogenous alpha-ketoisocaproic acid is preferentially converted to leucine by fetal brain slices independent of the nutritional state of the mother. During maternal starvation, beta-hydroxybutyrate, by restraining irreversible decarboxylation of alpha-ketoisocaproic acid, could act to salvage the keto acid for conversion to leucine. Thus alpha-ketoisocaproic acid metabolism in the fetal brain may be regulated in part by altered metabolic functions in this structure and in part by changing components in circulating fuel mixtures reaching the fetus from the starved mother.

Fetal fuels. I. Utilization of ketones by isolated tissues at various stages of maturation and maternal nutrition during late gestation.

The availability and utilization of B-hydroxybutyrate as an alternate oxidative fuel during fasting hypoglycemia has been examined in the rat conceptus at 18 and 20 days gestation. A 48-hr maternal fast between days 16 and 18 or 18 and 20 resulted in a 50% fall in fetal glucose levels and a marked rise in B-hydroxybutyrate, i.e., 30-fold at 18 and 60-fold at 20 days. Tissue concentrations of B-hydroxybutyrate or acetoacetate did not exceed extracellular levels. Placenta, fetal brain, carcass, and liver all oxidized 14C-labeled B-hydroxybutyrate to 14CO2 when incubated in vitro in the presence of B-hydroxybutyrate. Highest rates of oxidation were apparent in the placenta, followed by brain, liver, and carcass. The D isomer of B-hydroxybutyrate appeared to be oxidized preferentially by all tissues studied. Despite levels of 3-ketoacid CoA transferase and acetoacetyl CoA thiolase lower at 18 than at 20 days, rates of oxidation in individual tissues incubated under identical concentrations of substrate were similar at both times. In liver and brain, increasing rates of 14CO2 generation proportionate to graded concentrations of B-hydroxybutyrate in vitro indicated that such rates were probably determined by substrate availability. B-hydroxybutyrate oxidation in extrahepatic fetal tissues was unaffected by maternal fasting. By contrast, fetal liver derived from fasted mothers generated significantly less 14CO2 from B-hydroxybutyrate than livers from fed mothers. It has been suggested that capabilities for ketone utilization are widespread in tissues of the conceptus, and that such utilization may fulfill in part the oxidative demands for continued anabolic growth during fasting hypoglycemia in the mother.

Immunohistochemical examination of the INK4 and Cip inhibitors in the rat neonatal cerebellum: cellular localization and the impact of protein calorie malnutrition.

Expression of the cyclin-dependent kinase inhibitors (CKIs) has been linked to the inhibition of cellular proliferation and the induction of differentiation. Based on structure function analysis, two distinct families of CDKIs, the INK4 and the Cip/Kip family have been identified. The INK4 family member p16(Ink4), and the Cip/Kip protein p27(Kip1) have been implicated in normal development of the CNS and cerebellum. Recent studies have suggested a functional inter-dependence between the CKI and the abundance of cyclin D1 in orchestrating growth factor-induced cellular proliferation. The neonatal rat cerebellum undergoes proliferative growth and differentiation, localized to distinct topographical regions and cell types. The cell type and the temporal profile of CKI expression during postnatal cerebellar development had not been described. The current studies determined the specific cerebellar cell types in which the CKIs were expressed during post natal development by co-staining for cell-type specific markers. p16(Ink4a) and p27(Kip1) immunostaining was identified in both neurons and glial cells, increasing progressively between postnatal days 6 to 13 into adulthood. By contrast, neuronal and glial cell p21(Cip1) staining was prominent at days 6-11 and decreased thereafter. Cyclin D1 was expressed in the proliferating external granular cells, with occassional staining in the molecular, and internal granular layers. Dual immunostaining demonstrated cyclin D1 within cells expressing CKI (p16(Ink4a), p21(Cip1),p27(Kip1)). Cerebellar cellular growth arrest, induced by protein-calorie malnutrition, inhibited cyclin D1 protein levels without affecting CKI immunostaining suggesting CKI do not mediate the developmental arrest. These results demonstrate that the CKIs are induced by differentiation cues in specific cell types with distinct kinetics in the developing cerebellum in vivo.

Regulation of cyclin dependent kinase inhibitor proteins during neonatal cerebella development.

The cyclin dependent kinase holoenzymes (CDKs), composed of catalytic (cdk) and regulatory (cyclin) subunits, promote cellular proliferation and are inhibited by cyclin dependent kinase inhibitor proteins (CDKIs). The CDKIs include the Ink4 family (p15Ink4b, p16Ink4a, p18Ink4c, p19Ink4d) and the KIP family (p21Cip1 and p27Kip1). The sustained induction of p21 and p18 during myogenesis implicates these CDKI in maintaining cellular differentiation. Herein we examined the CDK (cyclin D1, cdk5) and CDKI expression profiles during the first 24 days of postnatal rat cerebella development. Cdk5 abundance increased and cyclin D1 decreased from day 9 through to adulthood. The CDKIs increased transiently during differentiation. p27 increased 20-fold between days 4 and 24, whereas p21 rose twofold between 6 to 11 days. p19, p18 and p16 increased approximately two- to threefold, falling to low levels in the adult. Immunostaining of cyclin D1 was localized in the external granular cells, whereas p27, was found primarily in the Purkinje cells. The period of maximal differentiation between days 9 to 13 was associated with a change in p21 and p16 staining from the external granular and Purkinje cells to a primarily Purkinje cell distribution. Protein-calorie malnutrition, which was previously shown to arrest rat cerebella development, reduced cyclin D1 kinase activity and p27 levels. However, p16 and p21 levels were unchanged. We conclude that the CDKIs are induced with distinct kinetics in specific cell types and respond differentially to growth factors during cerebella development, suggesting discrete roles for these proteins in normal cerebella development.

Otospongiosis (otosclerosis): polytomographic and histologic correlation.

The Temporal Bone Laboratory of Northwestern University Medical School has three sets of temporal bones from patients who had antemortem polytomographic examinations resulting in a diagnosis of otospongiotic involvement of the cochlea. One of these cases was thought to have been an example of pure cochlear otospongiosis. The other two cases were patients with clinical (stapedial) otospongiosis, and their polytomograms were interpreted as unilateral otospongiosis with involvement of the basal turn of the cochlea. In the first set of temporal bones, no otospongiosis was present. In the other two sets, the otospongiotic lesion did not involve the cochlea, and a contralateral otospongiotic lesion was present that had not been seen on the polytomograms. Caution must be exercised in the interpretation of subtle polytomographic changes noted in the cochlear capsule and restraint used in the X-ray diagnosis of pure cochlear otospongiosis until there is evidence of correlation with pathological material.

An holistic approach to Ménière's disease. Medical and surgical management.

A review of the medical and surgical management of 195 patients with Meniere's disease is presented. In order to avoid a piecemeal approach to the problem of Meniere's disease, the "whole" patient must be treated. Psychological counseling, medical management, and when indicated selective surgical management is advocated. The diagnosis of Meniere's must be confirmed through careful history, physical, neurological evaluation and selective testing. The majority of patients can be controlled medically; however, in this study 52 (26%) patients underwent surgical therapy. Twenty-eight patients underwent some type of saccus surgery for Meniere's disease with overall relief of 71%; however, better relief of vertigo was seen with labyrinthectomy and nerve section. The most common indication for surgery was disabling vertigo. However, fluctuating progressive sensorineural loss, may be an important reason to advise surgery. Surgical techniques for Meniere's disease continue to evolve. A graduated approach is preferred, starting with endolymphatic-mastoid shunt, proceeding to middle fossa vestibular nerve section when medical status and hearing and adequate. When hearing is socially inadequate, labyrinthectomy with or without vestibular nerve section is preferred. A good working relationship with a neurosurgeon is advised for otolaryngologists performing middle fossa surgery.

Intestinal absorption of fluoride preparations.

Gastric intestinal absorption of five different preparations of sodium fluoride was measured by means of ten hour urinary excretion. Best absorption was by nonenteric coated sodium fluoride. All three enteric coated preparations showed poorer absorption, with marked individual variations. Florical, the currently available preparation, showed better absorption than any enteric coated tablets and only slightly less than sodium fluoride alone. For active cochlear otospongiosis two capsules three times a day should be prescribed.

Endolymphatic sac valve implant surgery. I: The technique.

A resurgence of interest in surgery on the endolymphatic sac (ELS), not only for relief of vertigo but also for stabilization or improvement of hearing, has emerged. With the concept of operating early in the course of the disease after the diagnosis is well established, various modifications of existing procedures on the ELS have been utilized in an attempt to improve the overall results. One such major modification is the unidirectional inner ear valve implant modified from the eye valve for glaucoma. This valve implant has produced impressive one-year results for both hearing improvements and relief of vertigo in patients who were scheduled for labyrinthectomy. Subsequent preliminary reports by several investigators show good to excellent results for the hearing (AAOO--Class B or A) as well as the vertigo without consideration for the stage of the disease. This study presents primarily the step by step technique with drawings and a color atlas to facilitate the correct performance of this surgery.

Tumor involvement of the facial nerve.

Tumors involving the facial nerve are rare and challenging in both diagnosis and treatment. In this paper we report 18 cases of benign and malignant neoplasms involving the temporal portion of the facial nerve. The selection of those patients with facial paralysis who require detailed evaluation is discussed. Often, despite thorough evaluation of these patients, a preoperative diagnosis is unavailable or erroneous. An occasional patient may require surgical exposure of the nerve from the middle cranial fossa to the parotid gland.