A repeat expansion in the gene encoding junctophilin-3 is associated with Huntington disease-like 2.

We recently described a disorder termed Huntington disease-like 2 (HDL2) that completely segregates with an unidentified CAG/CTG expansion in a large pedigree (W). We now report the cloning of this expansion and its localization to a variably spliced exon of JPH3 (encoding junctophilin-3), a gene involved in the formation of junctional membrane structures.

Isolation of 2,6-dibromophenol from the marine hemichordate, Balanoglossus biminiensis.

2,6-Dibromophenol has been isolated from a luminous marine enteropneust, Balanoglossus biminiensis, found on intertidal beach areas at Sapelo Island, Georgia. This compound, responsible for the characteristic "iodoform-like" odor of these animals, is present in relatively large amounts; the estimated quantity per organism is 10 to 15 milligrams. Identity of the isolated substance as 2,6 dibromophenol is based on analyses of ultraviolet, infrared, and nuclear magnetic resonance spectra, mass spectrometry analysis, and on melting-point data.

Characterization of the calcium response to thyrotropin-releasing hormone in lactotrophs and GH cells.

Thyrotropin-releasing hormone (TRH) acts via a G-protein-coupled receptor on lactotrophs to increase the intracellular free calcium ion concentration, [Ca(2+)](i). The [Ca(2+)](i) response depends on both TRH concentration and the duration of TRH exposure. An initial, short-lived [Ca(2+)](i) spike results from release of Ca(2+) from intracellular stores, whereas a later sustained [Ca(2+)](i) increase, often characterized by [Ca(2+)](i) oscillations, results from an influx of extracellular Ca(2+) through both voltage-gated and non-voltage-gated, store-operated Ca(2+) channels. The initial spike phase predominates at high doses of TRH, whereas the plateau phase predominates at low doses. The mechanisms underlying the complex [Ca(2+)](i) response to TRH are discussed.

Thyrotropin-releasing hormone-induced intracellular calcium responses in individual rat lactotrophs and thyrotrophs.

Calcium responses to TRH were recorded for individual cells cultured from rat anterior pituitary tissue loaded with fura-2, and cell type was subsequently identified by immunocytochemistry. At 100 nM and 1 microM, TRH stimulated a single transient spike of intracellular free calcium ([Ca2+]i) in 95-100% of lactotrophs. At a concentration of 10 nM or less, the proportion of TRH-responsive cells decreased, and the [Ca2+]i responses became more heterogeneous, consisting of a biphasic response in which an initial [Ca2+]i spike was followed by a sustained elevation of [Ca2+]i or [Ca2+]i oscillations. Initiation of TRH-induced oscillations required the release of intracellular Ca2+ from thapsigargin-sensitive stores, whereas maintenance of the oscillations required influx of extracellular Ca2+ through nimodipine-sensitive Ca2+ channels. The amplitude of the initial [Ca2+]i rise increased from 0.1-10 nM TRH and was not significantly reduced by removal of extracellular Ca2+. The duration of the initial [Ca2+]i transient was significantly shorter at 1 microM than at 1 nM TRH. When TRH was added to cells that had been treated with thapsigargin to block the agonist-induced [Ca2+]i increase, TRH often decreased [Ca2+]i, particularly in cells with high [Ca2+]i. These results suggest that TRH and elevated [Ca2+]i act as coactivators of Ca2+ efflux, which helps terminate the agonist-evoked [Ca2+]i transient. In addition, TRH caused increases in [Ca2+]i in individual rat thyrotrophs, and these responses were heterogeneous. TRH stimulated a [Ca2+]i response in a lesser proportion of thyrotrophs from euthyroid compared to hypothyroid male rats. Essentially all TRH-responsive cells stained for either PRL or TSH.

The effects of pyroglutamylglutamylprolineamide, a peptide related to thyrotrophin-releasing hormone, on rat anterior pituitary cells in culture.

Pyroglutamylglutamylprolineamide, which was first discovered in mammalian prostate, differs from thyrotrophin-releasing hormone (TRH) by substitution of glutamic acid for histidine at position two of the tripeptide. Recently, the newly discovered peptide has been identified in substantial concentrations in the rat anterior pituitary gland and, in this study, we have investigated the effects of the peptide on rat anterior pituitary cells in culture. GH3 cells were chosen to examine the possible effects of the new peptide, particularly in relation to its effects on the TRH receptor. This cell-type was deficient, in comparison with normal rat pituitary cells, in the new TRH-related peptide and appeared to be an ideal model cell in which to study the effects of pGlu-Glu-ProNH2. TRH (0.01-100 nM) was found to stimulate the secretion of both GH and prolactin from GH3 cells whereas pGlu-Glu-ProNH2 had no effect within the same concentration ranges. In contrast, at micromolar concentrations pGlu-Glu-ProNH2 exhibited intrinsic TRH-like activity causing stimulation of both GH and prolactin release from GH3 cells. Both TRH and pGlu-Glu-ProNH2 appeared to act through the same intracellular signalling mechanism, causing significant increases in intracellular inositol phosphate within the expected concentration ranges. However, pGlu-Glu-ProNH2 (up to 1 mM) displaced neither [3H]TRH nor [3H]MeTRH from membrane-binding sites on GH3 cells, suggesting that the effects of the new peptide were mediated through a second receptor. The physiological relevance of these effects of pGlu-Glu-ProNH2 requires further investigation.

pGlutamylglutamylprolineamide modulation of growth hormone secretion in domestic fowl: antagonism of thyrotrophin-releasing hormone action?

Pyroglutamyglutamylprolineamide (pGlu-Glu-ProNH2) is a tripeptide with structural and immunological similarities to thyrotrophin-releasing hormone (TRH; pGlu-His-ProNH2). Since TRH stimulates GH secretion in domestic fowl, the possibility that pGlu-Glu-ProNH2 may also provoke GH release was investigated. Unlike TRH, pGlu-Glu-ProNH2 alone had no effect on GH release from incubated chicken pituitary glands and did not down-regulate pituitary TRH receptors. However, pGlu-Glu-ProNH2 suppressed TRH-induced GH release from pituitary glands incubated in vitro and competitively displaced [3H]methyl3-histidine2-TRH from pituitary membranes. Systemic injections of pGlu-Glu-ProNH2 had no significant effect on basal GH concentrations in conscious birds, but promptly lowered circulating GH levels in sodium-pentobarbitone anaesthetized fowl. Submaximal GH responses of conscious and anaesthetized birds to systemic TRH challenge were, however, potentiated by prior or concomitant administration of pGlu-Glu-ProNH2. These results demonstrate, for the first time, that pGlu-Glu-ProNH2 has biological activity, with inhibitory and stimulatory actions within the avian hypothalamo-pituitary axis. These results indicate that pGlu-Glu-ProNH2 may act as a TRH receptor antagonist within this axis.

Pyroglutamylglutamylprolineamide is present in rat anterior and posterior pituitary gland.

A new TRH-like peptide pyroglutamylglutamylprolineamide (pGlu-Glu-ProNH2) has recently been purified and characterized from both the rabbit prostate complex and human semen. In this study, TRH-immunoreactive peptides were extracted from anterior pituitary, posterior pituitary and hypothalamus and subjected to gel exclusion chromatography. For each tissue, TRH was resolved from pGlu-Glu-ProNH2 by anion-exchange chromatography at pH 7.6. In the anterior pituitary, 63% of the TRH immunoreactivity was chromatographically identical to pGlu-Glu-ProNH2 whereas in the posterior pituitary the new peptide represented less than 5% of the total TRH immunoreactivity. Only trace levels of pGlu-Glu-ProNH2 were observed in hypothalamus, suggesting that the acidic TRH-related peptide found in the anterior pituitary may not be of hypothalamic origin. The new TRH-like peptide was purified from whole pituitaries by gel exclusion and ion-exchange chromatography, followed by high power liguid chromatography and was shown to have chromatographic properties identical to pGlu-Glu-ProNH2. Amino acid analysis of the purified peptide revealed glutamic acid and proline residues in the ratio Glx:2 Pro:1, which is the expected composition of pGlu-Glu-ProNH2 after acid hydrolysis.

DRPLA gene (atrophin-1) sequence and mRNA expression in human brain.

Dentatorubral pallidoluysian atrophy (DRPLA, Smith's disease) is one of five disorders currently known to result from expansion of a CAG trinucleotide repeat encoding glutamine. The reported full length cDNA sequence encodes a serine repeat and a region of alternating acidic and basic amino acids, as well as the glutamine repeat. We now report the nucleic acid and deduced amino acid sequences of the open reading frame of this gene, obtained from a series of independently isolated and sequenced cDNA clones. Eight nucleotide differences from the originally published sequence result in a change of 34 amino acids, most prominently in the region of alternating acidic and basic residues. Northern analysis and in situ hybridization indicate that atrophin-1 mRNA is expressed in multiple brain regions. The level of mRNA expression as determined by in situ hybridization in a DRPLA-diseased brain is indistinguishable from the level observed in a matched control brain. These results indicate that the correlation between atrophin-1 expression and regions of pathology in DRPLA is at best partial, and that the expanded allele does not cause a major loss of mRNA expression. The pathology of the disorder may therefore arise from the altered structure and function of the abnormal protein.

Algorithm-derived, computer-generated interpretive comments in the reporting of laboratory tests.

The volume of laboratory and other data that physicians encounter daily is more than most can mentally process without oversight or error. Routine use of computer-generated reminders is one solution to this problem that is now widely suggested. We report our experience with the daily use of a user-written program of algorithm-derived, computer-generated comments in the reporting of chemical profiles and routine thyroid function tests in a busy medium-sized privately owned independent clinical laboratory.

A computerized word-processing and data system for histology in a private medical laboratory.

This report describes the use of a word-processing micro-computer, the Lanier Text Editor, for preparation of surgical tissue reports. Gross and microscopic description formats are stored on floppy disks, which may easily be completed and edited on a cathode-ray tube (CRT) for rapid computerized printing. Also described is the use of a PDP 8/E mini-computer for entry, storage, and manipulation of historic, demographic and diagnostic data, printing of numeric and alphabetic listings, cross-indexing of cases by diagnosis, and the accumulation of data for direct patient or physician billing.

A computer system for cytology in a private laboratory using a PDP 8/E minicomputer.

A computer software package written in Dibol for a PDP 8/E computer is described that is designed for the needs of the cytology department of a moderate-sized private medical laboratory. Computerized accessioning of cases, report preparation and printing, historical filing, activity summary reports, and direct patient and physician billing are provided by the relatively inexpensive system. The software has been in use for over two years in our cytology department and has resulted in increased efficiency, cost-effectiveness, and quality of reports.

Buffering intracellular calcium disrupts motoneuron development in intact zebrafish embryos.

Numerous studies, performed mainly on dissociated cells, have shown that calcium signals have a role during different stages of neuronal development. However, the actions of calcium during neuronal development in vivo remain to be established. The present study has investigated the role of intracellular calcium signals during development of motoneurons in the spinal cord of intact zebrafish embryos. Loading blastomeres of early embryos with either the calcium buffer BAPTA or the calcium reporter dye Calcium Green, was shown to disrupt motoneuron development in the spinal cord of embryos at 24 h postfertilisation. Loading the calcium buffer BAPTA, at an intracellular concentration of 1 mM, into the blastomeres of early embryos did not alter the resting levels of intracellular calcium, but significantly dampened transient rises in intracellular calcium in the cells of later stage embryos. Loading cells with 1 mM BAPTA significantly decreased the number of motoneurons present in the spinal cord at 24 h, indicating that calcium signals are important for normal motoneuron differentiation. Furthermore, in those BAPTA-filled cells that did adopt a motoneuron cell fate, axogenesis was found to be inhibited, suggestive of a role for calcium signalling in neurite initiation. This work provides evidence that calcium signals are necessary at several stages of motoneuron development in vivo.

Receptors for thyrotropin-releasing hormone on rat lactotropes and thyrotropes.

Primary cultures of rat pituitary cells were stained with an antibody to the native thyrotropin-releasing hormone (TRH) receptor and with a bioactive, fluorescent analogue of TRH, Rhod-TRH. Rhod-TRH specifically stained 86% of lactotropes and 21% of nonlactotropes from primary pituitary cell cultures. Lactotropes and thyrotropes accounted for 90% of cells that stained with Rhod-TRH, but there were occasional lactotropes and thyrotropes that did not show detectable staining with antireceptor antibodies or with Rhod-TRH. The intensity of staining was generally higher in the GH3 line of tumor cells than in normal pituicytes, and 100% of the tumor cells stained with Rhod-TRH. To determine whether the TRH receptor undergoes ligand-directed endocytosis in normal cells, TRH receptor immunocytochemistry was performed before and after TRH binding. TRH receptors were localized on the surface of cells prior to TRH exposure, and Rhod-TRH fluorescence was confined to the plasma membrane when TRH binding was performed at 0 degrees C, where endocytosis is blocked. When cells were incubated with TRH at 37 degrees C, receptors were found in intracellular vesicles in both lactotropes and thyrotropes, and Rhod-TRH was rapidly internalized into endosomes at elevated temperatures. Internalization of Rhod-TRH was inhibited by hypertonic sucrose, indicating that it occurs through clathrin-coated pits. These findings show that some of the heterogeneity in the secretory and calcium responses of pituicytes to TRH occurs at the level of the TRH receptor.

The form and function of retractile claws in the Felidae and other representative carnivorans.

Recent behavioral studies have shown the primary organ of prehension used in capturing prey to be the claw equipped forelimbs. In light of its functional importance, the claw retractile mechanism for 15 felid species is described and its function studied . The anatomy of the claw retractile mechanism for felids is then compared to that of other carnivorans. For felids,claw retraction is mechanically possible due to the unique shape of the middle and distal phalanges. Claw retraction, however, is a function of the dorsal elastic ligaments and not of the forearm extensor muscles. The resistance provided by these ligaments allows for flexion of the wrist and digital joints without claw protrusion. Moreover, co-contraction of both forearm flexor and extensor muscles is necessary to produce claw protrusion. The functional anatomy of claw retraction for felids differs considerably from that of most other carnivorans. However, the claw retractile mechanism for some advanced viverrids is structurally similar to that of the felids. For these viverrids prey seizing, as in the felids, has become a function of the forelimbs. For the other families of carnivorans, the jaws and not the forelimbs are used as the primary organ of prehension and the anatomy of the claw retractile mechanism reflects functional demands placed on it other than grasping and holding prey.

Four-laboratory validation study of the determination of chloramphenicol in veal calf urine.

A four-laboratory validation study of a method for the quantitation and confirmation of low part-per-billion levels of chloramphenicol extracted from veal calf urine was done. With this method, chloramphenicol, derivatized to the bis(trimethylsilyl)ether, was quantitated by gas chromatography with electron capture detection (GC-ECD) and confirmed by selected-ion monitoring in a negative ion chemical ionization gas chromatograph-mass spectrometer (GC-NICI-MS). Four analysts from 4 laboratories participated in the portion of the study devoted to chloramphenicol quantitation. Every analyst analyzed 5 sets, one set per day, on 5 different days. Each set included 6 samples consisting of a blank, 2 fortified samples, 2 incurred urine samples, and one duplicate. Thus, each analyst worked on a total of 30 samples. Chloramphenicol concentrations ranged from 0 to 9.7 ppb. All data were reported to 0.1 ppb. Coefficients of variation for distribution, CVd, ranged from 8.82 to 14.14% and for precision, CVr, ranged from 9.15 to 14.80%. Three analysts from 3 laboratories also participated in the confirmatory portion of the study, which was carried out to test whether the NICI-MS method developed earlier for higher concentrations of chloramphenicol and for an extract of muscle tissue could be applied to lower levels of chloramphenicol and to an extract prepared from urine. The extracts of 10 of the 30 samples were designated for confirmation only, but were obtained by the same procedure as extracts used for the quantitation part of the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue sulfonamide concentration and correlation in turkeys.

Nineteen hen turkeys (10 to 12 kg each) were used in a feeding study to determine sulfadimethoxine and sulfaquinoxaline concentrations in blood serum, liver, and skeletal muscle, as well as the respective ratios at selected withdrawal intervals. Two feeds were prepared by use of premixes to achieve 60 mg of sulfadimethoxine/kg and 100 mg of sulfaquinoxaline/kg, respectively. Each of the medicated feeds was given to 9 turkeys for 7 days. The turkeys were then fed nonmedicated feed at intervals from 24 to 56 hours and were slaughtered. One turkey was used as control. The serum/liver and serum/muscle ratios for sulfaquinoxaline were 60 to 70% higher than for sulfadimethoxine. However, the liver/muscle ratio for both sulfonamides was equivalent, approximately 3. Disposition of both sulfonamides approximated first-order pharmacokinetics. The calculated half-life of sulfadimethoxine was half that of sulfaquinoxaline, approximately 16 vs 30 hours. The coefficients of variation in the serum/tissue ratios for both sulfonamides were between 13% and 25% for serum/liver and less than 15% for serum/muscle, indicating excellent potential for using serum as a predictor of actionable concentrations of sulfonamide residues.

Chloramphenicol concentrations in calf muscle tissue.

Twenty-five 9- to 11-week-old calves were administered 2 doses of chloramphenicol prepared in propylene glycol (13.6 mg/kg of body weight IV; 6.8 mg/kg IM; or 13.6 mg/kg IM) at 24-hour intervals. Calves were euthanatized at designated times from 2 to 72 hours after the last dose was administered. Muscle tissues were collected immediately after euthanasia, and chloramphenicol concentrations in the tissues were determined.

Sulfamethazine blood/tissue correlation study in swine.

Seventy market-weight hogs (90 to 113 kg) were used in a feeding study to determine the correlation of serum sulfamethazine concentrations with sulfamethazine concentrations in liver and muscle at time of slaughter. Test groups were fed medicated feeds prepared from commercial medicated premixes containing 110 g of sulfamethazine/metric ton for 30 days. Fifteen days before hogs were slaughtered, test groups were given maintenance feeds containing 1.1 to 13.9 g of sulfamethazine/metric ton and were fed these diets until slaughtered. Comparison of data from positive- and negative-control groups indicated that total withdrawal of sulfamethazine in the feed was not necessary for the liver to contain less than the allowed tolerance of 0.1 mg of sulfamethazine/kg of liver at slaughter. Feed concentrations of up to 2 g of sulfamethazine/metric ton could be tolerated in withdrawal feeds before liver sulfamethazine values exceeded 0.1 mg/kg of liver. Serum/tissue sulfamethazine ratios were erratic in hogs given 1.1 to 2.7 g of sulfamethazine/metric ton, but became less variable in hogs given greater than 5.7 g/metric ton. Feed concentrations greater than 8 g of sulfamethazine/metric ton produced values greater than 0.1 mg/kg of muscle and values of about 0.4 mg/kg of liver. When serum sulfamethazine concentrations alone were used as a predictor for tissue sulfamethazine values, 100% of the liver values exceeded 0.10 mg/kg of liver when sulfamethazine in serum was greater than 0.45 mg/L. However, 57.4% of samples having serum concentrations between 0.10 and 0.45 mg/L had associated sulfamethazine values greater than 0.1 mg/kg of liver. All hogs having serum sulfamethazine concentrations less than 0.1 mg/L had sulfamethazine concentrations less than 0.1 mg/kg of liver.

A high performance liquid chromatographic system for the analysis of tetracycline drug standards, analogs, degradation products and other impurities.

This paper describes the high performance liquid chromatographic (HPLC) analysis of eight parent tetracycline standards: tetracycline, chlortetracycline, rolitetracycline, oxitetracycline, minocycline, doxycycline, democlocycline, methacycline; and three tetracycline epimers: epitetracycline, epianhydrotetracycline, and anhydrotetracycline. The HPLC system employs an octadecylsilane reverse phase column and an isopropanol-diethanolamine-phosphate-ammonium EDTA-water mobile phase. This system produced at least partial resolution of all eight parent compounds and many of their degradation products.