GJB2 mutations and genotype-phenotype correlation in 335 patients from germany with nonsyndromic sensorineural hearing loss: evidence for additional recessive mutations not detected by current methods.

We report on 335 patients (319 families) with mild-to-profound nonsyndromic sensorineural hearing loss. We identified 178 mutated GJB2 alleles representing 29 different sequence changes (including 3 novel mutations: Q7P, N14D, H100Q), and 2 alleles with the deletion del(GJB6-D13S1830) of the GJB6 gene. Eleven GJB2 mutations (119 mutated alleles) were truncating (T), and 18 mutations (59 alleles) were nontruncating (NT). Biallelic GJB2 mutations were found in 71 patients (21.2%; 67 families; 25 different genotypes). Audiograms of 62 patients (56 families) with biallelic GJB2 mutations typically indicated a profound hearing loss with T/T mutations, moderate hearing loss with T/NT mutations, and mild hearing impairment with NT/NT mutations (p < 0.01, Student's t test). From 37 patients (34 families) with biallelic GJB2 mutations, audiograms at different ages were available and indicated progressive hearing loss (>15 dB) in 10 patients (27.0%, 10 families). Interestingly, we identified an unexpectedly large subset of patients (n = 29; 8.7%) presenting with only one GJB2 mutation (n = 14 T/wild-type; n = 15 NT/wild-type). This strongly suggests the presence of additional recessive mutations that are not detected by current GJB2 mutation and GJB6 deletion analyses.

A mode of formation of tubular myelin from lamellar bodies in the lung.

A mechanism is suggested by which the membranes of lamellar bodies are converted to tubular myelin (TM) in the lung. It is argued that a simple corrugation of the membranous sheets can produce the TM formation. Such corrugation would occur in response to simple stresses acting on the lamellar body membranes. The intersections of the tubular figures are formed by fusion of adjacent corners in the corrugations. This results in a more stable hydrophobic bonding of phospholipid molecules. Strong supportive evidence for the mechanism is given by electron micrographs of TM formations.

Enzymes of the lung. I. Detection of esterase with a new cytochemical method.

The esterases of rabbit lung have been investigated from two viewpoints, the cytochemical and the biochemical. To accomplish this objective, we designed and synthesized a series of ester substrates which provide both a cytochemical indicator of the location of the enzyme and a means of following the enzymatic activity in tissue homogenates and subfractions. The substrates are p-nitrophenylthiol esters which yield, upon hydrolysis, carboxylic acid and p-nitrothiophenol. The latter can react with aurous ions to give an electron-opaque deposit; in addition, the strong absorption of p-nitrothiophenol at 410 mmicro permits continuous kinetic measurements. Thus, it is possible to correlate the intracellular site of action and the biochemical behavior of the esterases. The new substrates are the thiol analogues of the p-nitrophenyl esters frequently employed as esterase substrates. The rates of hydrolysis of the two series of esters are compared in vitro. During tissue fractionation, most of the esterase activity sediments with a particulate fraction. The effects of a number of common esterase inhibitors, such as diisopropyl phosphorofluoridate and eserine sulfate, are examined, and the effects of enzyme concentration and heat inactivation are shown with the use of the partially purified preparations. The cytochemical work shows that the esterase activity is most prominent in the lamellar bodies of the giant alveolar (type II, septal, or granular pneumatocyte) cells of the lung and to a lesser extent in squamous (type I, or membranous pneumatocyte) epithelial and endothelial cells. In both the cytochemical and biochemical studies, the enzymes are inhibited by diisopropyl phosphorofluoridate and phenyl methylsulfonyl fluoride but are insensitive to eserine sulfate.

Polyanionic agents as inhibitors of phagosome-lysosome fusion in cultured macrophages: evolution of an alternative interpretation.

Various natural and synthetic substances classified as polyanionics have been implicated in antagonizing phagosome-lysosome fusion in cultured macrophages. The phenomenon has been judged by comparing the transfer of selected markers from secondary lysosomes to phagosomes in control and in "polyanion" cells. Our earlier studies showed that use of one of the markers, the membrane-permeating acridine orange, was plagued with artifacts that were especially misleading in the presence of polyanionic agents. We now question the validity of data obtained by the alternative technique, electron microscopy. Our present evidence shows that nonionic hydrocolloids of sufficiently high molecular weight prevent the transfer of various colloidal electron-opaque markers from lysosomes to phagosomes in the same manner as does the powerful polyanionic "fusion inhibitor" dextran sulfate. Both kinds of hydrocolloids, however, allow delivery of lysosomal, low-molecular-weight highly charged non-permeant fluorescent markers to phagosomes, probably by a fusion process. We propose that neither type of hydrocolloid inhibits fusion; instead, when sufficiently concentrated, they trap particulate electron-opaque markers in a gelatinous matrix, which may move only slowly out of lysosomes. The polyanionics trap the electron-opaque markers physically and acridine orange ionically. Hence, the semblance of "fusion inhibition."

Polyanionic agents do not inhibit phagosome-lysosome fusion in cultured macrophages.

The survival of some intracellular pathogens within macrophages may be aided by an ability of the organism to antagonize, from within the entrapping phagosome, its fusion with lysosomes. On the other hand, certain polyanionic agents have been implicated in imposing a similar block to fusion from the lysosomal domain--because the transfer of various foreign markers from lysosomes to newly formed phagosomes is remarkably inhibited in these polyanion-containing cells. Based on an analysis of various observations and our own recent data, we propose that the polyanionics do not, in fact, prevent phagosome-lysosome fusion but, instead, physically entrap the usual markers in a gelatinous matrix within the lysosomes. This view accounts for many paradoxical consequences of polyanionic accumulation and for the curiously normal behavior of macrophages that are presumed to be suffering from such a crucial intracellular dysfunction.

Macrophage effector function in pulmonary oxygen toxicity: hyperoxia damages and stimulates alveolar macrophages to make and release chemotaxins for polymorphonuclear leukocytes.

Macrophages synthesize many secretory products in vitro but the stimuli for their production and their pathophysiologic significance in vivo are largely unknown. In the present investigation, we found that hyperoxia damaged rabbit alveolar macrophages (AM) in vitro as manifested by decreased cell numbers, increased lactate dehydrogenase (LDH) release, and the development of ultrastructural abnormalities that resembled those seen in AM in situ or lavaged from lungs of rabbits exposed to hyperoxia in vivo. Hyperoxia also stimulated cultured rabbit AM to release chemotaxins for polymorphonuclear leukocytes (PMN) that were similar in molecular weight to chemotaxins obtained from lung lavages of rabbits exposed to hyperoxia in vivo. Our results suggest that alveolar macrophage secretory products may play a physiologically relevant role in recruitment of PMN to the lungs in pulmonary oxygen toxicity.

Internalization and cellular processing of somatostatin in primary culture of rat anterior pituitary cells.

Somatostatin (SRIF) binding, internalization, and intracellular processing in primary culture of anterior pituitary cells have been studied using somatostatin coupled to an electron-opaque marker, colloidal gold. Initially, after 2 min of incubation (37 C), gold-conjugated SRIF is localized on the cell surface, with 38% of the marker being found around microvilli, 10% at the junction of secretion vesicles with the plasma membrane, and 51% distributed over the remaining areas of the cell membrane. There was no internalization of SRIF at this time. After 20 min of incubation, distribution of the cell-surface bound hormone was similar to that at 2 min (40.6% at microvilli, 12% at the junction with the secretion vesicle, and 47.4% over the rest of the plasma membrane). However, 12% of the electron-opaque markers were found intracellularly in association with coated vesicles, intermediate-sized vesicles, lysosomes, and Golgi structures. SRIF did not enter pituitary cells at 4 C. To study the role of coated vesicles in internalization of SRIF, we have measured somatostatin binding to isolated coated vesicles before and after various treatments and sonication. SRIF binding to sonicated vesicles (3.46 +/- 0.36 fmol/micrograms protein), was much greater than to intact ones (0.75 +/- 0.16 fmol/micrograms protein), suggesting intraluminal localization of SRIF receptors in the coated vesicles. Approximately 80% of SRIF-binding sites were recovered on the intraluminal surface of the coated vesicles. The results of these experiments suggest that internalization of SRIF is a time- and temperature-dependent process. Within the cell, SRIF is routed to either lysosomes or the Golgi apparatus. Coated vesicles participate in intracellular translocation of SRIF-receptor complexes. It appears that the receptor for SRIF being internalized is located on the intraluminal surface of the coated vesicle.

Somatostatin receptors are biologically active before they are inserted into the plasma membrane.

We have examined the biological activity of intracellular somatostatin (SRIF) receptors in cultured rat anterior pituitary cells. We used digitonin-permeabilized cells to introduce free SRIF intracellularly and chloroquine-treated cells to promote intracellular accumulation of SRIF via a receptor-mediated pathway. At a concentration of 0.001%, digitonin (3-min incubation at 37 C) allowed [125I]SRIF to enter the cells without affecting cell viability. Autoradiography of [125I]SRIF demonstrated its association with secretion vesicles (28%), nuclei (25%), and other intracellular organelles. An acid wash technique that removes cell surface-bound ligand revealed that both digitonin-permeabilized cells and chloroquine-treated cells accumulated approximately twice as much intracellular SRIF as did control cells. The biological activities of intracellular SRIF accumulated via two different pathways, receptor mediated and through digitonin-produced pores in the plasma membrane, were different. In chloroquine-treated cells, the accumulation of intracellular SRIF did not result in its additional biological effect. SRIF inhibited GH-releasing factor-induced GH release from 578 +/- 12 to 168 +/- 9 ng/10(6) cells X 30 min, which did not differ from the control value. Cells incubated with digitonin demonstrated normal basal (160 +/- 9 ng/10(6) cells X 30 min) and GH-releasing factor-stimulated GH release (564 +/- 11 ng/10(6) cells X 30 min). However, the inhibitory action of SRIF in these cells was approximately 30% greater (98 +/- 8 ng/10(6) cells X 30 min) than that in either control or chloroquine-treated cells, suggesting that SRIF freely admitted intracellularly produces additional biological activity. These observations confirm the presence of the intracellular receptors and suggest that these receptors exist in a biologically active form.

Adenine nucleotides in the secretory granule fraction of rat islets.

Concomitant with glucose-induced insulin release, there occurred an increase of ATP from 4.40 plus or minus 0.21 to 23.16 plus or minus 0.52 pmol/100 islets/min (P less than 0.001) in the effluent from perifused rat islets. There is a linear relationship between circulating ATP and insulin levels both in the stimulated and basal state (r = 0.689, P less than 0.01). Islets incubated with labeled adenine for a short period of time (37.5 min) showed no release of radioactivity upon subsequent glucose-induced insulin release. Islets incubated for a prolonged interval with labeled adenine (150 min) showed an increase in acid soluble radioactivity in the effluent during glucose-induced insulin release. Following incubation of the islets with labeled adenine for 150 min, approximately 5% of the homogenate radioactivity was found in the secretory granules. Using column chromatography to separate the adenine nucleotides, the distribution of radioactivity among the various nucleotides in the secretory granule fraction was found to be: AMP 54.42 plus or minus 4.96%, ADP 14.20 plus or minus 1.63%, ATP 15.39 plus or minus 3.84%, and cAMP 16.07 plus or minus 2.11%. The distribution of radioactivity in the effluent adenine nucleotides after glucose-induced insulin release was: AMP 32.83 plus or minus 4.62%, ADP 24.52 plus or minus 2.77%, ATP 28.13 plus or minus 5.45%, and cAMP 26.01 plus or minus 3.34%. The absolute levels of adenine nucleotides in the secretory granules were ATP 4.19 plus or minus 0.88, ATP madp 7.94 plus or minus 2.20 and cAMP 4.46 plus or minus 1.74 pmol/ug prot. The levels in the islet effluent were ATP, 15.30 plus or minus 2.70, ATP qDP, 29.43 plus or minus 3.49 and cAMP 7.66 plus or minus 1.93 pmol/100 islets/min for the first ten min of glucose-stimulated insulin release. Thereafter there was a rapid decline in effluent cAMP while ATP and ADP remained in essentially equivalent amounts. The distribution of radioactivity and absolute levels of the adenine nucleotides in the effluent reflects that found in the secretory granules, confirming previous observations that insulin release is occurring by exocytosis.

Brain tumors in children: long-term survival after radiation treatment.

PURPOSE: To determine the cause of death in children who survive more than 5 years after radiation treatment of a brain tumor. METHODS AND MATERIAL: Nine hundred and twelve consecutive children with a primary brain tumor irradiated at the Princess Margaret Hospital or Toronto-Bayview Regional Cancer Center from 1958 to 1991, were evaluated for long-term outcome. RESULTS: Overall 10- and 20-year survival rates were 44% and 37%. Subsequent survival of 377 5-year survivors was, at an additional 10 and 20 years, 78% and 67%. Most (83%) deaths that occurred more than 5 years from diagnosis were a result of relapse of the original tumor. The 10-year survival rate subsequent to relapse was 9% when the first relapse occurred less than one year from diagnosis, 17% for 1-2 years, and 31% when the time to relapse was 3 years or greater. The cumulative actuarial incidence of, and death from, second malignant tumors at 30 years from diagnosis was 18% and 13%, respectively. CONCLUSIONS: Death later than 5 years from diagnosis of a brain tumor in children is common and is usually due to progressive disease in slowly evolving low grade tumors. Death from a second malignant tumor becomes more frequent than death from the original tumor after 15 years from diagnosis.

Interaction of bradykinin with sodium dodecyl sulfate and certain acidic lipids.

The striking change in the circular dichroism (CD) of bradykinin (BK) occasioned by its interaction with sodium dodecyl sulfate (SDS) is evidently due in large part to a change in the conformation of the C-terminal tetrapeptide moiety of the hormone. The full change in CD is induced by the binding of two molecules of monomeric SDS per peptide molecule, the complex being aggregated. Formation of the 1:2 BK-SDS complex apparently proceeds via intermediates of stoichiometry 1:1 and 2:1. The cooperative nature of the interaction is attributed to the SDS-promoted aggregation of BK. Electrostatic interactions with the Arg residues appear important for the binding reaction per se. CD reveals that BK also interacts with acidic lipids which bear a net electrical charge (e.g., cerebroside sulfate and phosphatidyl inositol) but not with lipids bearing no net charge (e.g., cerebroside and phosphatidyl choline). The interactions are with particular mixed micelles of the lipid and the nonionic surfactant used for their solubilization, micellar size and structure being examined by surface tensiometry and electron microscopy.

Fimbriae of Actinomyces viscosus t14v: their relationship to the virulence-associated antigen and to coaggregation with Streptococcus sanguis 34.

1) Fimbriae from A. viscosus T14V may be similar to those found on other bacteria. 2) The antigenic difference between virulent and avirulent A. viscosus T14 appears to be of a quantitative rather than a qualitative nature and is related to fimbriae and not to the cell wall polysaccharide. 3) Coaggregation between A. viscosus T14V and S. sanguis 34 is mediated by fimbriae on the former which have specificity for beta-linked galactosyl residues.