The G1138A mutation rate in the fibroblast growth factor receptor 3 (FGFR3) gene is increased in cells carrying the t (4; 14) translocation.

Spontaneous mutations are a common phenomenon, occurring in both germ-line and somatic genomes. They may have deleterious consequences including the development of genetic disorders or, when occurring in somatic tissues, may participate in the process of carcinogenesis. Similar to many mutational hotspots, the G1138A mutation in the fibroblast growth factor receptor 3 (FGFR3) gene occurs at a CpG site. In germ-line tissues, the G1138A mutation results in achondroplasia and has one of the highest spontaneous mutation rates in the human genome. Although not at the G1138A site, there are increased rates of other somatic mutations in the FGFR3 gene that have been reported in multiple myeloma cases associated with a translocation, t (4; 14). The chromosome-4 break points in this translocation are clustered in a 70-kb region centromeric to the FGFR3 gene. We hypothesized that this translocation may impact the mutation rate at the G1138A site. We employed a semi-quantitative polymerase chain reaction-based assay to measure the frequency of this mutation in multiple myeloma cell lines carrying t (4; 14) translocation. Analysis of these cell lines varied from no change to a 10-fold increase in the mutation frequency compared with normal controls. In general, there was an increase in the G1138A mutational frequency suggesting that chromosomal rearrangement can affect the stability of the CpG hotspots.

Evaluation of arterial stiffness in nondiabetic chronic kidney disease patients.

Chronic kidney disease (CKD) is a growing problem worldwide. Clinical and epidemiologic studies have shown that structural and functional changes that occur in major arteries are a major contributing factor to the high mortality in uremic patients. Recent studies have shown a stepwise increase of the carotid-femoral pulse wave velocity (cfPWV) from CKD Stage 1 to Stage 5. We evaluated the cfPWV and augmentation index (AIx), as indirect markers of arterial stiffness in patients with nondiabetic CKD and compared the values with normal population; we also evaluated the relationship between various stages of CKD and arterial stiffness markers. This cross-sectional study was carried out in the Department of Nephrology for a duration of two years from January 15, 2012, to January 14, 2014. Fifty patients with nondiabetic CKD were studied along with 50 healthy volunteers who did not have CKD, who served as controls. Assessment of arterial stiffness (blood pressure, PWV, heart rate, aortic augmentation pressure, and AIx) was performed using the PeriScope device. PWV positively correlated with systolic and diastolic blood pressure, mean aortic arterial pressure, serum creatinine, and serum uric acid and negatively correlated with estimated glomerular filtration rate. Arterial stiffness increased as CKD stage increased and was higher in nondiabetic CKD group than in the general population. Arterial stiffness progressed gradually from CKD Stage 2 to 5, and then abruptly, in dialysis patients. Measures to decrease the arterial stiffness and its influence on decreasing cardiovascular events need further evaluation.

Strobilocercus fasciolaris infection with hepatic sarcoma and gastroenteropathy in a Wistar colony.

Tapeworm cysts were identified in liver of Wistar rats and it induced fibrosarcoma in liver and gastroenteropathy in stomach and intestine. The tapeworm larva was confirmed as Strobilocercus fasciolaris by PCR linked mitochondrial DNA sequencing. Light microscopy, special staining (masson trichrome) and immunoflouresence supported the diagnosis of fibrosarcoma. Infiltration of plasma cells, macrophages and eosinophils were observed in the liver section. Gastric mucosal hyperplasia, dilation of gastric glands with secretion, intestinal mucosal cell hyperplasia, proliferation of duodenal submucosal glands were confirmed by light microscopy and supported by PAS, AB staining. The concomitant development of hepatic sarcoma and gastroenteropathy by larvae of Taenia taeniaeformis (S. fasciolaris) infection is very rare and is the first reported case in Wistar rats to our knowledge.

CSF amine metabolites in depression.

The amine metabolites, namely homovanillic acid (HVA) and 5-hydroxy indoleacetic acid (5-HIAA) were measured in cerebrospinal fluid (CSF) of depressives (n = 30) and controls (n = 30). Depressed patients had significantly lower HVA levels than controls. No significant differences were noted between the two groups in 5-HIAA levels. However, the differences between the groups for the CSF HVA/5-HIAA ratio were larger than those for the CSF HVA alone (p less than 0.01 versus p less than 0.025, respectively). HVA levels correlated positively with monoamine oxidase activity and adenosine deaminase activity.

Peripheral benzodiazepine receptors are decreased during cocaine withdrawal in humans.

In the present study, homovanillic acid in plasma (pHVA) and benzodiazepine receptors (3H-PK11195 binding) in neutrophil membranes were determined in blood obtained from cocaine-dependent (DSM-III-R) adult male inpatients at baseline-(within 72 hr of last cocaine use) and after 3 weeks of cocaine abstinence, and normal controls. The mean (+/- SEM) pHVA at baseline (10.3 ng/ml +/- 1.1) was similar to normals and did not change after 3 weeks of cocaine abstinence. Similarly, the binding indices of benzodiazepine receptors in cocaine-dependent subjects as a group were not significantly different than in normal controls. In 10 cocaine-dependent subjects, however, where both blood samples were available, the number of 3H-PK11195 binding sites was significantly (p < 0.05) decreased after 3 weeks of cocaine abstinence (mean +/- sem: Bmax = 6371 +/- 657 fmol/mg protein) compared with baseline (Bmax = 7553 +/- 925 fmol/mg protein), although there were no differences in the binding affinity (mean +/- sem: KD = 8.6 +/- 1.2 nmol/L after 3 weeks of abstinence compared with 8.1 +/- 1.0 nmol/L at baseline). These preliminary results suggest that peripheral benzodiazepine receptors may play an important role in the pathophysiology of cocaine withdrawal in cocaine-dependent human subjects.

Successful establishment of long-term bone marrow cultures in 103 patients with myelodysplastic syndromes.

We used bone marrow biopsies instead of mononuclear cells to maintain long-term cultures from 103 patients belonging to all five sub-categories of myelodysplastic syndromes (MDS), as well as 12 normal controls. By week 4, 30-50% confluency was reached and could be maintained for up to 12 weeks with 100% confluency. The four prominent cells were fibroblasts, macrophages, endothelial cells and adipocytes. Immunohistochemical and electron microscopic studies provided lineage confirmation. Normal hematopoiesis was well supported by MDS stroma. Neither the FAB nor cytogenetics was co-related with the potency of growth. MDS stroma appears to be both morphologically and functionally normal.

Effect of low pH treatment on opioid peptides binding to their receptors and functional coupling of G-proteins to adenylyl cyclase in the rat spinal cord.

Because low pH treatment is known to alter the coupling of G-proteins to brain receptors, and little is known about such an effect in the spinal cord, the present study was undertaken to examine whether preincubation of rat spinal cord membranes at low pH (pH 4.5) alters opioid receptor binding characteristics and sodium fluoride (NaF)-stimulated adenylyl cyclase (AC) activity (as a function of G, mediated). [3H][D-Ala2,MePhe4,Gly-ol]enkephalin (DAMGO) and [3H]ethylketocyclazosine (EKC) were used to label mu- and kappa-opioid receptors, respectively. AC activity was determined using ATP as substrate and cAMP formed was quantified. Low pH treatment of membranes did not affect the mu- and kappa-opioid binding characteristics in rat spinal cord. However, the low pH treatment significantly reduced the NaF-stimulated AC activity in rat spinal cord. It is concluded that low pH treatment causes selective changes in the functional coupling of Gs-proteins to AC without affecting the opioid receptor binding characteristics in the spinal cord.

Biochemical and behavioral studies on the interaction between mu- and kappa-opiate agonists in mice.

Male Swiss-Webster mice were rendered tolerant to morphine by subcutaneous implantation of a morphine pellet, each containing 75 mg morphine base, for 3 days. Mice implanted with placebo pellets served as controls. A high degree of tolerance to the analgesic effect of morphine developed as evidenced by decreased analgesic response to various doses of morphine. A selective kappa-opiate agonist, U-50,488H (8, 16 and 32 mg/kg, i.p.) produced dose-dependent analgesic and hypothermic effects in mice implanted with placebo pellets. A significant decrease in the analgesic and hypothermic effects of U-50,488H was observed in morphine tolerant mice as compared to placebo-treated mice. Mice were rendered tolerant to U-50,488H by injecting the drug (25 mg/kg, i.p.) twice daily for 4 days. Vehicle injected mice served as controls. Tolerance to the analgesic and hypothermic effects of U-50,488H in mice injected chronically with the drug was evidenced by the decreases in the intensity of these responses when compared to those observed in vehicle injected controls. Morphine produced a dose-dependent analgesic and hypothermic effects in mice injected chronically with vehicle but the intensity of these effects was significantly lower in mice injected chronically with U-50,488H. These results indicate that a substantial tolerance to analgesic and hypothermic effects of U-50,488H develops in morphine tolerant mice. The effect of chronic injections of U-50,488H on the binding of [3H]ethylketocyclazocine (EKC) and [3H]D-Ala2,MePhe4,Gly-ol5-enkephalin (DAMGO) to whole brain and spinal cord kappa- and mu-opiate receptors was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the behavioral supersensitivity of dopamine D2 receptors without receptor up-regulation in morphine-abstinent rats.

The effect of morphine tolerance-dependence and abstinence on the characteristics of dopamine D2 receptors in brain regions and spinal cord was determined in the rat. Male Sprague-Dawley rats were implanted s.c. under light ether anesthesia with 6 morphine pellets for a 7-day period, each containing 75 mg of morphine free base. Rats implanted with placebo pellets served as controls. This procedure resulted in the development of tolerance to morphine as evidenced by decreased analgesic response to a challenge dose of morphine. Similarly, the development of physical dependence was evidenced by a decrease in body weight and colonic temperature after morphine pellet removal (withdrawal). The binding characteristics (Bmax and Kd values) of [3H]spiroperidol to dopamine D2 receptors were determined in the tissues of morphine-tolerant and morphine-abstinent rats. In the tolerant rats, the pellets were left intact at the time of sacrificing, whereas, in the abstinent rats the pellets were removed 18 h prior to sacrificing. The binding of [3H]spiroperidol was determined in membranes prepared from brain regions (hypothalamus, hippocampus, cortex, pons and medulla, midbrain, corpus striatum and amygdala) and spinal cord of rats from various treatment groups. [3H]Spiroperidol bound to brain regions and spinal cord at a single high affinity site. The Bmax or the Kd values in brain regions and spinal cord of morphine-tolerant and -abstinent rats did not differ from their respective placebo controls. The behavioral responses to a selective dopamine D2 receptor agonist, 2-bromo-alpha-ergocryptine were also determine in the morphine-abstinent rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the role of nitric oxide in kappa-opiate tolerance in mice.

The hypothesis that inhibition of nitric oxide (NO) synthase with subsequent decrease in the production of NO might attenuate the development of kappa-opiate tolerance was examined. Concurrent treatment of NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA) (2-8 mg/kg, i.p.) along with U-50,488H (25 mg/kg, i.p.) twice daily for 4 days dose-dependently attenuated the development of tolerance to the analgesic and hypothermic effects of U-50,488H (25 mg/kg, i.p.). L-NMMA by itself did not modify the analgesic and hypothermic effects of acute administration of U-50,488H. A potential role for NO in the development of kappa-opiate tolerance is suggested.

Effect of morphine tolerance and abstinence on the binding of [3H]MK-801 to brain regions and spinal cord of the rat.

The effect of chronic administration of morphine to rats on the N-methyl-D-aspartate (NMDA) receptors labeled with [3H]MK-801, a non-competitive antagonist, was determined in brain regions and spinal cord. Male Sprague-Dawley rats were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of 6 morphine pellets during a 7-day period. Each pellet contained 75 mg of morphine free base. Animals serving as controls were similarly implanted with placebo pellets. This procedure resulted in the development of a high degree of tolerance and physical dependence on morphine. Two sets of rats were used. In one, the pellets were left intact at the time of sacrifice (tolerant) and in the other the pellets were removed 16 h prior to sacrificing (abstinent). The binding constants, Bmax and Kd values of [3H]MK-801 were determined in cortex, hippocampus, hypothalamus, corpus striatum, midbrain and spinal cord. In the absence of glycine and glutamate, [3H]MK-801 bound to tissue membranes at a single high affinity site. The Bmax and Kd values of [3H]MK-801 were not altered in any of the tissues of the morphine abstinent rats. The Bmax value of [3H]MK-801 was significantly decreased in cerebral cortex of morphine tolerant rats as compared to their placebo controls but the Kd values did not change. In other brain regions and spinal cord of morphine tolerant rats and their placebo controls, the Bmax and Kd values of [3H]MK 801 did not differ.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of chronic administration of U-50,488H, a kappa-opioid receptor agonist, on central dopamine D2 receptors of the rat.

The effect of U-50,488H, a kappa-opiate agonist, induced tolerance and abstinence on the characteristics of dopamine D2 receptors of brain regions and spinal cord was determined in the rat. Male Sprague-Dawley rats were injected with U-50,488H (25 mg/kg i.p) or its vehicle twice a day for 4 days. This procedure resulted in the development of tolerance to the analgesic activity of U-50,488H. The binding characteristics (Bmax and Kd values) of [3H]spiroperidol to dopamine D2 receptors were determined in discrete brain regions (hypothalamus, hippocampus, cortex, pons and medulla, midbrain, corpus striatum and amygdala) and spinal cord of U-50,488H-tolerant and -abstinent rats. Rats labeled as tolerant to U-50,488H were injected with U-50,488H on day 5 and killed 1 h later, whereas those labeled abstinent were killed on day 5 without the injection of the drug. Vehicle-injected rats served as controls. [3H]Spiroperidol bound to brain regions and spinal cord membranes at a single high affinity site. The Bmax and Kd values of [3H]spiroperidol in brain regions and spinal cord of U-50,488H-tolerant and abstinent rats did not differ from their respective vehicle-injected controls. The behavioral responses (total distance travelled, horizontal activity, movement time, total number of movements, number of stereotypic movements, stereotypic time and rest time) to different doses of a selective dopamine D2 receptor agonist, 2-bromo-alpha-ergocryptine (bromocriptine) were also determined in rats treated chronically with U-50,488H.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence-based diagnosis of hemoglobinopathies in the clinical laboratory.

Electrophoresis at alkaline and acid pH values is the most widely used method for screening for hemoglobinopathies in the clinical laboratory. Nevertheless, the method does not provide definitive diagnostic information for a number of hemoglobins. New automated DNA sequencing techniques make it possible to definitively identify mutations which cause hemoglobinopathies. Moreover these techniques take about the same time as it takes to perform hemoglobin electrophoresis and globin chain electrophoresis. Although the reagent cost is somewhat higher, the results are definitive.

Alpha thalassemia and its impact on other clinical conditions.

Mild alpha thalassemia is the most prevalent genetic trait worldwide. We have recently developed a multiplex polymerase chain reaction method to screen for the most common deletions which give rise to alpha thalassemia. The authors have used this method to investigate a potential association of alpha thalassemia with hypertension. Our results show that the prevalence of hypertension in hospitalized blacks who have the -alpha 3.7 deletion is 71 percent higher than the prevalence in hospitalized blacks who do not have the deletion. The authors present a potential mechanism to explain this association.

Brain and spinal cord kappa opiate receptors and pharmacological responses to U-50,488H in rats of differing ages.

The analgesic and hypothermic responses to U-50,488H (25 mg/kg IP), a kappa opiate receptor agonist, were determined in male Sprague-Dawley rats aged 4, 8, and 24 weeks. In addition, the characteristics of the binding of [3H]ethylketocylazocine (EKC) to kappa opiate receptors in whole brain and spinal cord of rats of three age groups were also determined. Administration of U-50,488H produced an age-related increase in the analgesic response in the rat, i.e., the older rats exhibited a higher intensity of analgesic response than the younger rats. U-50,488H also produced a hypothermic response. The response in 4- and 24-week-old rats was similar, but that in 8-week-old rats was smaller than the rats in the other two age groups. [3H]EKC bound to whole brain and spinal cord membranes of rats at a single high affinity site. The Bmax value of [3H]EKC in the brain and spinal cord of 24-week-old rats was significantly lower than in 4- and 8-week-old rats; however, the Kd values did not differ. It is concluded that kappa opiate receptor agonist produces age-related increase in its analgesic response and that such effects are not related to the characteristics of kappa receptors in the brain and spinal cord.

Effect of systemically administered nitric oxide donor, sodium nitroprusside on picrotoxin-induced convulsions in rats.

Nitric oxide (NO), the gaseous neurotransmitter has been reported to have an endogenous anticonvulsant property. This has prompted proposals to develop NO donors as anticonvulsant drugs. In the present study, the effect of NO donor, sodium nitroprusside (SNP) on picrotoxin (PCT)-induced convulsions was investigated. A convulsant dose of PCT (5 mg/kg) was administered 5, 10, 15 and 30 min after intraperitoneal injection of graded doses (0.7, 1.25 and 2.5 mg/kg) of SNP. SNP at doses 0.7 and 1.25 mg/kg increased dose dependently the severity of PCT-induced convulsions. But, pretreatment with the higher dose (2.5 mg/kg) of SNP was protective against PCT-induced convulsions. However, post treatment (5 and 10 min) with the same dose exacerbated convulsions and caused death of the animals. These results indicate that the vasodilator effect of SNP and an increased perfusion of PCT into brain may be responsible for the proconvulsant action of SNP. A decreased entry of PCT because of marked vasodilation and hypotension has been speculated for an inhibition of convulsions in animals pretreated with a higher dose of SNP. In conclusion, the results reveal the non-suitability of SNP to be developed as an anticonvulsant.

Calcium unresponsive hypocalcemic tetany: gitelman syndrome with hypocalcemia.

Introduction. Gitelman's syndrome (GS) is autosomal recessive renal tubular disorder characterized by hypokalemia, hypomagnesemia, hypocalciuria, metabolic alkalosis, and hyperreninemic hyperaldosteronism. It is usually associated with normal serum calcium. We report a patient presented with hypocalcemic tetany, and evaluation showed Gitelman's syndrome with hypocalcemia. Case Report. A 28-year-old woman presented with cramps of the arms, legs, fatigue, and carpal spasms of one week duration. She has history of similar episodes on and off for the past two years. Her blood pressure was 98/66 mmHg. Chvostek's sign and Trousseau's sign were positive. Evaluation showed hypokalemia, hypocalcemia, hypomagnesemia, metabolic alkalosis, and hypocalciuria. Self-medication, diuretic use, laxative abuse, persistent vomiting, and diarrhoea were ruled out. Urinary prostaglandins and genetic testing could not be done because of nonavailability. To differentiate Gitelman syndrome from Bartter's syndrome (BS), thiazide loading test was done. It showed blunted fractional chloride excretion. GS was confirmed and patient was treated with spironolactone along with magnesium, calcium, and potassium supplementation. Symptomatically, she improved and did not develop episodes of tetany again. Conclusion. In tetany patient along with serum calcium measurement, serum magnesium, serum potassium, and arterial blood gases should be measured. Even though hypocalcemia in Gitelman syndrome is rare, it still can occur.

Down-regulation of N-methyl-D-aspartate (NMDA) receptors of brain regions and spinal cord of rats treated chronically with morphine.

1. The effects of morphine tolerance and abstinence on the characteristics of N-methyl-D-aspartate (NMDA) receptors, labeled with [3H]MK-801, were determined in the brain regions and spinal cord of the rat. 2. Male Sprague-Dawley rats were rendered tolerant to and physically dependent on morphine by subcutaneous implantation of six morphine pellets during a 7-day period. In tolerant (non-abstinent) rats, the pellets were left intact at the time of sacrificing, whereas in the abstinent rats the pellets were removed 16 hr prior to sacrificing. 3. The binding of [3H]MK-801, an NMDA receptor antagonist, to membranes prepared from spinal cord and brain regions (cortex, striatum, amygdala, hippocampus, hypothalamus, midbrain and pons-medulla) was determined using 5 nM concentration of the ligand in the presence of 30 microM glycine and 50 microM of glutamate. 4. In non-abstinent morphine tolerant rats, the binding of [3H]MK-801 was decreased by 40 and 33% in the midbrain and spinal cord, respectively, in comparison with their placebo controls. In morphine abstinent rats, the binding of [3H]MK-801 was decreased by 42, 29 and 50% in hypothalamus, midbrain and spinal cord, respectively, in comparison with their placebo controls. The binding of [3H]MK-801 to other brain regions and spinal cord of morphine tolerant and abstinent rats did not differ from their respective placebo controls. 5. Thus, these studies demonstrate, for the first time, that in the presence of glutamate and glycine, NMDA receptors of selected brain regions and spinal cord are down-regulated in rats treated chronically with morphine.

Effects of naltrexone on the binding of [3H]D-Ala2, MePhe4, Gly-ol5-enkephalin to brain regions and spinal cord and pharmacological responses to morphine in the rat.

1. The effects of naltrexone pellet implantation and removal on the analgesic and hypothermic effects of morphine and the binding of 3H-D-Ala2, MePhe4, Gly-ol5-enkephalin (DAMGO) to mu-opiate receptors in rat brain regions and spinal cord were determined. 2. Male Sprague-Dawley rats were implanted subcutaneously with a pellet containing 10 mg of naltrexone for 7 days. Placebo pellet implanted rats served as controls. The pellets were removed on day 8, and the analgesic and hyperthermic effects were determined in the rat 24 hr later. Morphine produced a dose-dependent analgesic and hyperthermic responses in rats implanted with placebo pellets. Enhanced analgesic and hyperthermic responses to morphine were produced in rats implanted with naltrexone pellets. 3. The binding constants (Bmax and Kd values) of [3H]DAMGO in regions of the brain (amygdala, hypothalamus, striatum, midbrain, hippocampus, pons + medulla and cortex), and spinal cord of rats with naltrexone pellet left intact or removed were determined. The Bmax values of [3H]DAMGO were increased in all brain regions and spinal cord of rats in which the naltrexone pellets were left in place or removed prior to sacrificing. However, the Kd values of [3H]DAMGO were unaffected by naltrexone treatment. 4. It is concluded that enhanced analgesic and hyperthermic response to morphine is produced in rats implanted with naltrexone pellets and such alterations in the pharmacological responses are due to up-regulation of mu-opiate receptors in all the brain regions and spinal cord. Additionally whether the pellets were left intact (receptors blocked) or removed (receptors not blocked), the mu-opiate receptors were up-regulated in spinal cord and all the regions of the brain.