Ibrexafungerp: A novel oral glucan synthase inhibitor.

Ibrexafungerp is a novel glucan synthase inhibitor currently undergoing phase II and phase III clinical trials. This compound has demonstrated in vitro activity against clinically important fungal pathogens including Candida spp. and Aspergillus spp. It is able to retain activity against many echinocandin-resistant strains of Candida due to differential avidity for the target site compared to echinocandins. In vivo animal models have demonstrated efficacy in murine models of invasive candidiasis, aspergillosis, and pneumocystis. Due to high bioavailability, it can be administered both orally and intravenously. A favorable drug interaction and tolerability profile is observed with this compound. This review summarizes existing data that have either been published or presented at international symposia.

Ryanodine receptor type 3 (RYR3) as a novel gene associated with a myopathy with nemaline bodies.

BACKGROUND AND PURPOSE: Nemaline myopathy (NEM) has been associated with mutations in 12 genes to date. However, for some patients diagnosed with NEM, definitive mutations are not identified in the known genes, suggesting that there are other genes involved. This study describes compound heterozygosity for rare variants in ryanodine receptor type 3 (RYR3) gene in one such patient. METHODS AND RESULTS: Clinical examination of the patient at 22 years of age revealed a long narrow face, high arched palate and bilateral facial weakness. She had proximal weakness in all four limbs, mild scapular winging but no scoliosis. Muscle biopsy revealed wide variation in fibre size with type 1 fibre predominance and atrophy. Abundant nemaline bodies were located in perinuclear and subsarcolemmal areas, and within the cytoplasm. No likely pathogenic mutations in known NEM genes were identified. Copy number variation in known NEM genes was excluded by NEM-targeted comparative genomic hybridization array. Next-generation sequencing revealed compound heterozygous missense variants in the RYR3 gene. RYR3 transcripts are expressed in human fetal and adult skeletal muscle as well as in human brain and cauda equina samples. Immunofluorescence of human skeletal muscle revealed a 'single-row' appearance of RYR3, interspersed between the 'double rows' of ryanodine receptor type 1 (RYR1) at each A-I junction. CONCLUSION: The results suggest that variants in RYR3 may cause a recessive muscle disease with pathological features including nemaline bodies. We characterize the expression pattern of RYR3 in human skeletal muscle and brain, and the subcellular localization of RYR1 and RYR3 in human skeletal muscle.

Epithelioid trophoblastic tumor: A single institution case series at the New England Trophoblastic Disease Center.

OBJECTIVE: Epithelioid trophoblastic tumor (ETT) is a rare form of gestational trophoblastic neoplasm which is distinct based on its development from intermediate trophoblast cells and nodular growth pattern. The aim of this study is to describe a case series from a single institution with a review of the literature to better understand the clinical characteristics and outcomes for patients with ETT. METHODS: A retrospective review was performed using the IRB approved New England Trophoblastic Disease Center (NETDC) database from 1998 to 2014. Eight patients were identified of which seven had complete records. Follow-up data was obtained from the longitudinal medical records. RESULTS: Four (57.1%) patients presented with vaginal bleeding and two (28.6%) patients were asymptomatic at presentation. Three (42.9%) patients had extrauterine disease. All three patients with extrauterine disease who received chemotherapy had stable or progressive disease at follow-up. Only two (29%) patients who presented with non-metastatic disease and underwent hysterectomy were alive with no evidence of disease. The mean interval following antecedent pregnancy was 104months. All patients with an interval >4years demonstrated stable or progressive disease despite intensive chemotherapy. Two patients with non-metastatic disease who declined hysterectomy developed stable or progressive disease despite chemotherapy. CONCLUSIONS: This series highlights several features of ETT including the potential for asymptomatic presentation of extrauterine disease. The series also demonstrates chemoresistance, even with multi-agent therapy and a poor prognosis with extrauterine disease and an interval greater than 4years following the antecedent pregnancy suggesting that surgery remains critical in disease control.

Experimental validation of a novel compact focusing scheme for future energy-frontier linear lepton colliders.

A novel scheme for the focusing of high-energy leptons in future linear colliders was proposed in 2001 [P. Raimondi and A. Seryi, Phys. Rev. Lett. 86, 3779 (2001)]. This scheme has many advantageous properties over previously studied focusing schemes, including being significantly shorter for a given energy and having a significantly better energy bandwidth. Experimental results from the ATF2 accelerator at KEK are presented that validate the operating principle of such a scheme by demonstrating the demagnification of a 1.3 GeV electron beam down to below 65 nm in height using an energy-scaled version of the compact focusing optics designed for the ILC collider.

A novel explosive process is required for the gamma-ray burst GRB 060614.

Over the past decade, our physical understanding of gamma-ray bursts (GRBs) has progressed rapidly, thanks to the discovery and observation of their long-lived afterglow emission. Long-duration (> 2 s) GRBs are associated with the explosive deaths of massive stars ('collapsars', ref. 1), which produce accompanying supernovae; the short-duration (< or = 2 s) GRBs have a different origin, which has been argued to be the merger of two compact objects. Here we report optical observations of GRB 060614 (duration approximately 100 s, ref. 10) that rule out the presence of an associated supernova. This would seem to require a new explosive process: either a massive collapsar that powers a GRB without any associated supernova, or a new type of 'engine', as long-lived as the collapsar but without a massive star. We also show that the properties of the host galaxy (redshift z = 0.125) distinguish it from other long-duration GRB hosts and suggest that an entirely new type of GRB progenitor may be required.

Unclassified white matter disorders: A diagnostic journey requiring close collaboration between clinical and laboratory services.

BACKGROUND: Next generation sequencing studies have revealed an ever-increasing number of causes for genetic disorders of central nervous system white matter. A substantial number of disorders are identifiable from their specific pattern of biochemical and/or imaging findings for which single gene testing may be indicated. Beyond this group, the causes of genetic white matter disorders are unclear and a broader approach to genomic testing is recommended. AIM: This study aimed to identify the genetic causes for a group of individuals with unclassified white matter disorders with suspected genetic aetiology and highlight the investigations required when the initial testing is non-diagnostic. METHODS: Twenty-six individuals from 22 families with unclassified white matter disorders underwent deep phenotyping and genome sequencing performed on trio, or larger, family groups. Functional studies and transcriptomics were used to resolve variants of uncertain significance with potential clinical relevance. RESULTS: Causative or candidate variants were identified in 15/22 (68.2%) families. Six of the 15 implicated genes had been previously associated with white matter disease (COL4A1, NDUFV1, SLC17A5, TUBB4A, BOLA3, DARS2). Patients with variants in the latter two presented with an atypical phenotype. The other nine genes had not been specifically associated with white matter disease at the time of diagnosis and included genes associated with monogenic syndromes, developmental disorders, and developmental and epileptic encephalopathies (STAG2, LSS, FIG4, GLS, PMPCA, SPTBN1, AGO2, SCN2A, SCN8A). Consequently, only 46% of the diagnoses would have been made via a current leukodystrophy gene panel test. DISCUSSION: These results confirm the importance of broad genomic testing for patients with white matter disorders. The high diagnostic yield reflects the integration of deep phenotyping, whole genome sequencing, trio analysis, functional studies, and transcriptomic analyses. CONCLUSIONS: Genetic white matter disorders are genetically and phenotypically heterogeneous. Deep phenotyping together with a range of genomic technologies underpin the identification of causes of unclassified white matter disease. A molecular diagnosis is essential for prognostication, appropriate management, and accurate reproductive counseling.

Variants in ACTG2 underlie a substantial number of Australasian patients with primary chronic intestinal pseudo-obstruction.

BACKGROUND: Primary chronic intestinal pseudo-obstruction (CIPO) is a rare, potentially life-threatening disorder characterized by severely impaired gastrointestinal motility. The objective of this study was to examine the contribution of ACTG2, LMOD1, MYH11, and MYLK mutations in an Australasian cohort of patients with a diagnosis of primary CIPO associated with visceral myopathy. METHODS: Pediatric and adult patients with primary CIPO and suspected visceral myopathy were recruited from across Australia and New Zealand. Sanger sequencing of the genes encoding enteric gamma-actin (ACTG2) and smooth muscle leiomodin (LMOD1) was performed on DNA from patients, and their relatives, where available. MYH11 and MYLK were screened by next-generation sequencing. KEY RESULTS: We identified heterozygous missense variants in ACTG2 in 7 of 17 families (~41%) diagnosed with CIPO and its associated conditions. We also identified a previously unpublished missense mutation (c.443C>T, p.Arg148Leu) in one family. One case presented with megacystis-microcolon-intestinal hypoperistalsis syndrome in utero with subsequent termination of pregnancy at 28 weeks' gestation. All of the substitutions identified occurred at arginine residues. No likely pathogenic variants in LMOD1, MYH11, or MYLK were identified within our cohort. CONCLUSIONS AND INFERENCES: ACTG2 mutations represent a significant underlying cause of primary CIPO with visceral myopathy and associated phenotypes in Australasian patients. Thus, ACTG2 sequencing should be considered in cases presenting with hypoperistalsis phenotypes with suspected visceral myopathy. It is likely that variants in other genes encoding enteric smooth muscle contractile proteins will contribute further to the genetic heterogeneity of hypoperistalsis phenotypes.

Genetics of alcohol consumption in Drosophila melanogaster.

Individual variation in alcohol consumption in human populations is determined by genetic, environmental, social and cultural factors. In contrast to humans, genetic contributions to complex behavioral phenotypes can be readily dissected in Drosophila, where both the genetic background and environment can be controlled and behaviors quantified through simple high-throughput assays. Here, we measured voluntary consumption of ethanol in ∼3000 individuals of each sex from an advanced intercross population derived from 37 lines of the Drosophila melanogaster Genetic Reference Panel. Extreme quantitative trait loci mapping identified 385 differentially segregating allelic variants located in or near 291 genes at P < 10-8 . The effects of single nucleotide polymorphisms associated with voluntary ethanol consumption are sex-specific, as found for other alcohol-related phenotypes. To assess causality, we used RNA interference knockdown or P{MiET1} mutants and their corresponding controls and functionally validated 86% of candidate genes in at least one sex. We constructed a genetic network comprised of 23 genes along with a separate trio and a pair of connected genes. Gene ontology analyses showed enrichment of developmental genes, including development of the nervous system. Furthermore, a network of human orthologs showed enrichment for signal transduction processes, protein metabolism and developmental processes, including nervous system development. Our results show that the genetic architecture that underlies variation in voluntary ethanol consumption is sexually dimorphic and partially overlaps with genetic factors that control variation in feeding behavior and alcohol sensitivity. This integrative genetic architecture is rooted in evolutionarily conserved features that can be extrapolated to human genetic interaction networks.

Anchorage-independent growth-conferring factor production by rat mammary tumor cells.

Conditioned medium from cultures of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor cells contain factors that resemble sarcoma growth factor and other transforming growth factors in biological activity but differ in their physical properties. The mammary tumor factors (MTF) are acid stable and heat and protease sensitive. They inhibit the binding of epidermal growth factor, but not insulin, to mouse embryonal carcinoma cells. MTF confers upon normal rat kidney and BALB/c-3T3 cells the ability to grow in soft agar. This effect is enhanced synergistically by high concentrations of fetal calf serum but not by epidermal growth factor. Anchorage-independent growth promotion, however, is not seen with normal mammary epithelial cells, although MTF is mitogenic for these cells as well as normal rat kidney cells, BALB/c-3T3 cells, and chick embryo fibroblasts in monolayer culture, MTF is not mitogenic for primary cultures of the tumor cells from which the factors are derived. Two major molecular weight species of MTF, eluting at Mr 6,000 and 65,000 to 70,000 on Bio-Gel P-100 columns, are present in acid-ethanol extracts of 7,12-dimethylbenz(a)anthracene- and nitrosomethylurea-induced rat mammary tumors. Transplantable tumors derived from primary 7,12-dimethylbenz(a)anthracene- or nitrosomethylurea-induced tumors have little or no MTF activity. These results demonstrate that different chemically induced rat mammary tumors contain transforming growth factor-like activities. Furthermore, it is possible that MTF is unnecessary for the maintenance of tumorigenicity, since some tumors contain no detectable MTF.

Further defects in counterregulatory responses induced by recurrent hypoglycemia in IDDM.

We evaluated the effect of previous experimental hypoglycemia on counterregulatory responses to hypoglycemia in 13 IDDM patients. These patients had defects in counterregulatory responses to hypoglycemia compared with 7 nondiabetic control subjects. Plasma EPI and glucagon responses to hypoglycemia in IDDM patients were approximately 60% of levels in nondiabetic subjects (P less than 0.02 and P less than 0.001, respectively). Hepatic glucose output ([3-3H]glucose) was reduced by approximately 60% of normal (P less than 0.005), and the glucose infusion rate required to maintain plasma glucose was correspondingly greater in people with IDDM (P less than 0.001). With a modified glucose clamp (plasma insulin approximately 330 pM), the diabetic subjects underwent two sequential 120-min periods of hypoglycemia (approximately 3.0 mM) with an intervening 60-min euglycemic recovery period. In the IDDM patients, there were 30-50% decreases in plasma GH (P less than 0.005) and cortisol (P less than 0.001) responses during the second hypoglycemic period compared with the first. In addition, glucose output, already defective compared with that in nondiabetic subjects, was further reduced by 33% (P = 0.03) during the second period of experimental hypoglycemia. There was no effect of repeated hypoglycemia on the responses of plasma glucagon, EPI, or NE, though plasma EPI was correlated directly with glucose output (P less than 0.001) and inversely with glucose uptake (P less than 0.05). There was no correlation between the rise in glucose output during hypoglycemia and antecedent glycemic control as measured by HbA1.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of antecedent hypoglycemia on cognitive function and on glycemic thresholds for counterregulatory hormone secretion in healthy humans.

OBJECTIVE: To determine whether reduced hormonal, symptomatic, and/or cognitive responses to hypoglycemia are caused by an increase in the plasma glucose concentration required to stimulate these counterregulatory parameters after antecedent hypoglycemia. RESEARCH DESIGN AND METHODS: We studied nine healthy volunteers during stepped hypoglycemia clamps (plasma glucose targets from 80 to 50 mg/dl in 10 mg/dl steps) on two separate days. The study was preceded either by a 2-h period of hypoglycemia (plasma glucose 58 +/- 2 mg/dl) or a 2-h period of euglycemia (plasma glucose 94 +/- 2 mg/dl) for 90 min. RESULTS: The plasma glucose that triggered secretion of plasma norepinephrine (NE) was lower after antecedent hypoglycemia (control = 74 +/- 2 and experimental = 67 +/- 2 mg/dl, respectively, P < 0.005). In contrast, a relatively higher plasma glucose stimulated secretion of other counterregulatory hormones after antecedent hypoglycemia: growth hormone (GH) (65 +/- 2 to 72 +/- 2 mg/dl, P < 0.01); glucagon (63 +/- 2 to 70 +/- 2 mg/dl, P < 0.01); and epinephrine (EPI) (68 +/- 2 to 76 +/- 2 mg/dl, P < 0.01) when comparing control days with experimental days. Hypoglycemic symptoms were first observed at a plasma glucose plateau of 59 +/- 2 mg/dl. Motor function reflected by Digit Symbol Substitution deteriorated equally whether there had been antecedent hypoglycemia or euglycemia. Logical (immediate) memory deteriorated in the control study at a plasma glucose of 54 +/- 2 mg/dl but remained unchanged at equivalent hypoglycemia in the experimental study (P < 0.03). CONCLUSIONS: Our conclusions are as follows: 1) symptoms of moderate hypoglycemia occur at plasma glucose levels averaging approximately 5-15 mg/dl lower than the plasma glucose concentrations required to trigger counterregulatory hormone release; 2) after acute antecedent hypoglycemia, glucagon, EPI, and GH secretion occur at higher plasma glucose concentrations and NE is released at lower plasma glucose concentrations; and 3) there may be CNS adaptation to prior hypoglycemia reflected in preservation of logical memory function at plasma glucose levels of approximately 50 mg/dl. These findings suggest that thresholds for hormone secretion and for changes in cognitive function can be altered very acutely by foregoing hypoglycemia in healthy humans.

Counterregulatory adaptation to recurrent hypoglycemia in normal humans.

We evaluated the effect of antecedent hypoglycemia on glucose counterregulation during hypoglycemia in non-diabetic human subjects. In single hypoglycemia studies, glucose production [( 3H]3-glucose) and counterregulatory hormone concentrations were measured (after a 3.5-h baseline period of euglycemia) during 120 min of hypoglycemia (glucose clamped at 3.0 mmol/L). During the final 60 min of hypoglycemia, counterregulation resulted in significant increments in glucose production (12.88 +/- 0.83 mumol/kg.min), and plasma glucagon (IRG; 185 +/- 22 ng/L), GH (29.3 +/- 7.0 micrograms/L), cortisol (630 +/- 100 nmol/L), epinephrine (3.44 +/- 0.76 nmol/L), and norepinephrine (2.02 +/- 0.21 nmol/L). In the recurrent hypoglycemia experiment, an antecedent period of identical hypoglycemia was induced. Glucose counterregulation during the second of two periods of hypoglycemia (HYPO 2) was then compared to that in single hypoglycemia studies. During HYPO 2, there were decreased responses in Ra (by 32%; P less than 0.03), GH (by 67%; P less than 0.05), F (by 41%; P less than 0.03), and norepinephrine (by 20%; P = 0.03) compared to those in the single hypoglycemia study. In contrast, plasma IRG values were similar in the single hypoglycemia studies and HYPO 2, but were reduced relative to those during the first hypoglycemic period of recurrent hypoglycemia (IRG, 263 +/- 18 ng/L; P less than 0.025 vs. HYPO 2 and P less than 0.05 vs. single hypoglycemia). Our results suggest that 1) antecedent hypoglycemia may alter glucose counterregulation during hypoglycemia; and 2) recurrent hypoglycemia may result in alterations in reduction of hepatic glucose production.

Deficient counterregulatory hormone responses during hypoglycemia in a patient with insulinoma.

Counterregulatory hormone responses were evaluated in a 37-yr-old woman before and after removal of a benign insulin-producing islet cell tumor. Counterregulatory hormone concentrations were measured during a glucose clamp with graded reductions of plasma glucose from 5.2 to 2.6 mmol/L. In the study before surgery, the increase in plasma epinephrine concentration was markedly blunted (by greater than 90%) compared to that in the study after surgery. The peak plasma norepinephrine concentration was similarly reduced by 71%, and plasma cortisol by 63%. In addition, the glycemic thresholds for secretion of the counterregulatory hormones were lower before removal of the tumor. Peak plasma GH responses were equivalent before and after surgery, but the threshold for GH secretion was 21% lower in the first hypoglycemia study. We conclude 1) that there is evidence for abnormal glucose counterregulatory hormone secretion in this patient, which may contribute to the pathogenesis of hypoglycemia seen in patients with insulinoma; 2) the reversal of reduced counterregulatory hormone secretion after tumor resection suggests that these defective hormonal responses may be related to recurrent hypoglycemia, persistent hyperinsulinemia, or both; and 3) that abnormal glucose counterregulation may exist in the absence of type 1 diabetes.

Physiologic hyperinsulinemia enhances counterregulatory hormone responses to hypoglycemia in IDDM.

We evaluated the effect of physiologic hyperinsulinemia (plasma insulin 329 +/- 62 vs 687 +/- 62 pmol/L) on counterregulatory hormone responses in 8 IDDM subjects studied during a 2-hour hypoglycemic clamp study with an equivalent degree of hypoglycemia (plasma glucose 3.1 +/- 0.1 and 3.0 +/- 0.1 mmol/L, respectively). Plasma epinephrine levels were increased by 71% during the last 60 minutes of hypoglycemia in the high insulin study (840 +/- 180 vs 1440 +/- 310 pmol/L, respectively p = 0.006). In addition, plasma cortisol and norepinephrine were also increased in the high insulin study (by 19% and 24% respectively, p < 0.01, for both). Plasma growth hormone and glucagon concentrations were not altered by high dose insulin infusion. In spite of increased epinephrine secretion, the glucose infusion rate required to maintain glucose was 2-fold greater in the high insulin study, and there was greater suppression of lipolysis in that group. We conclude that hyperinsulinemia may enhance counterregulatory hormone secretion in IDDM.

Effect of physiological hyperinsulinemia on counterregulatory hormone responses during hypoglycemia in humans.

UNLABELLED: We evaluated the effect of continuous physiological hyperinsulinemia on counterregulatory hormone responses in seven healthy subjects, each studied on two occasions. Hormone responses were measured during identical 2-h periods of hypoglycemia (plasma glucose target 3.5 mmol/L) at insulin levels of 350 pmol/L or 640 pmol/L. During hypoglycemia, there were significant (50-1400%) increases in glucagon, epinephrine, norepinephrine, GH, and cortisol which were comparable in the two groups. We further evaluated the influence of the duration of mild hyperinsulinemia on the responses in an additional group of normal subjects (n = 7). Brief (30 min) exposure to insulin was compared to a prolonged (3.5 h) insulin infusion, each followed by identical hypoglycemia. Plasma insulin (approximately 350 pmol/L) and plasma glucose (target 3.3 mmol/L) were similar in both groups. The increases in epinephrine, norepinephrine, GH, and cortisol during hypoglycemia were virtually identical in the two groups. However, the secretion of glucagon was blunted following prolonged hyperinsulinemia, increasing to levels of 249 +/- 17 ng/L in the brief studies and to only 185 +/- 20 ng/L in the prolonged studies (P < 0.005). The insulin-induced decrement in plasma amino acids were similar in the two studies and could not account for the impaired glucagon secretory response. CONCLUSIONS: 1) Brief exposure to even high physiological levels of insulin do not alter the magnitude of counterregulatory hormone secretion during hypoglycemia; 2) prolonged hyperinsulinemia results in a selective blunting of the plasma glucagon response to hypoglycemia, perhaps due to a direct suppressive effect of insulin on alpha-cell secretion.

Sustained inhibitory actions of a potent antagonist of gonadotropin-releasing hormone in postmenopausal women.

The inhibitory time course and dose-related characteristics of a new potent GnRH antagonist peptide, [N-acetyl-D-pCl-Phe1,2-D-Trp3-D-Lys6-D-Ala10]GnRH, on gonadotropin secretion were studied in nine postmenopausal women. Effective suppression of gonadotropin secretion was correlated with increased circulating concentrations of immunoassayable GnRH antagonist. Inhibition of gonadotropin secretion was achieved by a parenteral dose of 300 micrograms/kg GnRH antagonist. This dose reduced plasma bioactive LH concentrations by 49-59%, immunoactive LH by 41-46%, and immunoactive FSH by 25-40%. Blockade of gonadotropin secretion was sustained for 10-28 h after a single injection of the synthetic decapeptide. This prolonged action was associated with significant plasma protein binding of the GnRH antagonist and mean plasma half-times of disappearance of 1.5 and 21 h for the fast and slow components, respectively. In summary, we have described the biological actions of a potent GnRH antagonist that binds avidly to serum proteins, has a prolonged plasma residence time, and exerts sustained inhibitory effects on bio- and immunoactive LH release in man. The extended duration of action of this compound may reflect in part its significant binding to circulating plasma proteins.

Acute androgen receptor blockade increases luteinizing hormone secretory activity in men.

To investigate the nature of androgen feedback mechanisms in normal men, we studied the hypothalamo-pituitary responses to administration of a potent, highly selective nonsteroidal androgen receptor antagonist, flutamide HCl (1 g/day, orally, for 3 days). The impact of reversible blockade of endogenous androgen action was assessed in 11 normal men by analyzing quantitative alterations in specific pulsatile properties of LH secretion basally (hypothalamic regulation) and after 2 (n = 6) consecutive iv pulses of exogenous GnRH (pituitary responsiveness). Androgen blockade resulted in significant increases in 1) 12-h mean and integrated serum immunoactive LH concentrations (P = 0.01), 2) LH pulse frequency (P = 0.01), and 3) mean interpulse (valley) serum LH concentrations (P = 0.02) and maximal LH peak heights (P = 0.01). Additionally, there were significant decreases in LH interpulse interval (P = 0.02), LH peak duration (P = 0.02), and interpeak valley duration (P = 0.02). The augmented LH pulsatility reflected enhanced hypothalamic activity, since 1) pituitary secretory responses to exogenous GnRH pulses were not altered, and 2) multiple parameter deconvolution disclosed an increased number of computer-resolved LH secretory bursts generated per 12 h, with no changes in the apparent half-duration of LH secretory impulses or the calculated mass of LH released per secretory burst. We conclude that endogenous androgens act selectively to modulate the number of spontaneous LH secretory bursts in man.

Hyperplasia in the spinal sensory system of the frog. I. Plasticity in the most caudal dorsal root ganglion.

Increases in the amount of periphery available for innervation have been achieved by the unilateral removal of hindlimb dorsal root ganglion (DRGs) in Rana pipiens, a procedure which generally results in a compensatory cell number increase (hyperplasia) in the DRGs which remain. We have found that the hyperplastic response is extremely variable, and we have investigated various factors which might control its production. Our findings indicate, however, that the pattern of DRGs removed, the animal's age at the time of removal, and the survival period are not strictly related to the production of hyperplasia in hindlimb DRGs. Special emphasis has been placed on DRG 10, the caudalmost DRG which normally innervates the cloaca and sends a small projection to the hindlimb. This DRG displayed dramatic cell number increases of up to 564%. In addition, several unique features of the hyperplastic response have been observed in DRG 10. This DRG showed increases in cell number on both the operated and the unoperated sides. It showed hyperplasias in animals subjected to ganglionectomy past metamorphosis as well as during larval development. Finally the production of DRG 10 hyperplasias exclusively occurred in male pre- and postmetamorphic animals. To account for these distinctive features of DRG 10 hyperplasia, baseline studies of the normal course of proliferation and cell death in DRG 10 were undertaken. They reveal no fundamental developmental differences between DRG 10 and other hindlimb DRGs. Other mechanisms responsible for these unusual features of developmental plasticity in DRG 10 are discussed.

Hyperplasia in the spinal sensory system of the frog. II. Central and peripheral connectivity patterns.

Central and peripheral connectivity patterns of hyperplastic dorsal root ganglia (DRGs) in Rana pipiens are examined in order to determine the relative roles of peripheral and central contacts in the production of DRG hyperplasias. The hyperplasias are produced in the intact hindlimb DRGs after the removal in tadpoles and young postmetamorphic frogs of neighboring DRGs (Davis and Constantine-Paton, '83). The peripheral target zones of the hyperplastic DRGs, determined by physiological recordings of sensory receptive fields, are found to undergo a significant degree of expansion relative to controls. Peripheral expansion is most pronounced in caudalmost DRG 10, and this effect occurs in experimental animals operated during larval and postmetamorphic stages. Further, anatomical labelling of peripheral sensory fibers coursing to the hindlimb reveals that the hyperplastic DRG 10 actually contains additional fibers projecting to the denervated regions. The central projection of the hyperplastic DRG 10 does not show corresponding increases in longitudinal arborization after the application of horseradish peroxidase to the appropriate dorsal roots. These observations are made on some of the same experimental animals in which peripheral fields are shown to have vastly expanded. We conclude that the peripheral processes of the hyperplastic DRGs are less rigidly specified than the central terminations, and that it is the periphery which plays the primary role in controlling the cell numbers increases. A second aim of this investigation is to identify whether sexually dimorphic connectivity patterns in normal frogs explain the production of DRG 10 hyperplasias exclusively in male experimental animals (Davis and Constantine-Paton, '83). We apply the same techniques used in our connectivity studies of hyperplastic DRGs to the investigation of connectivity patterns of DRG 10s in normal males and females. No sex-dependent differences in peripheral and central connectivity are found. Thus, since normal male and female frogs possess an equivalent amount of target space for DRG 10, the unique production of hyperplasias in male experimental animals cannot be explained solely on the basis of connectivity. We speculate on what other factors may be involved.

A novel ryanodine receptor gene mutation causing both cores and rods in congenital myopathy.

BACKGROUND: Central core disease (CCD) and nemaline rod myopathy are generally considered two genetically and histologically distinct disorders. CCD is defined by the presence of well-demarcated round cores within most myofibers. Nemaline rod myopathy is distinguished by the presence of characteristic nemaline bodies within myofibers. The simultaneous occurrence of both cores and rods in the same muscle biopsy has been described, but no gene mutations have been reported yet for this condition. OBJECTIVE: To describe a family containing 16 affected individuals in six generations with an autosomal dominant congenital myopathy that shows clinical and histologic features of both CCD and nemaline myopathy, and to determine the genetic etiology and protein composition of the cores/rods in this family. METHODS AND RESULTS: The results of linkage analyses excluded involvement of the two autosomal dominant nemaline myopathy loci on chromosome 1, but were consistent with a localization of the disease gene at the CCD locus on chromosome 19q13.1 (ryanodine receptor). SSCP analysis and DNA sequencing identified a novel Thr4637Ala mutation in the transmembrane region of the ryanodine receptor protein. Immunofluorescence studies of patient muscle biopsies showed the central cores to stain for ryanodine receptor. CONCLUSIONS: These data suggest that the occurrence of nemaline bodies can be a secondary feature of CCD, and that genetic studies on previously reported core/rod families should be targeted to the ryanodine receptor locus. The results of the immunofluorescence studies suggest that the cores contain excess abnormal ryanodine receptor protein.