Caprylic acid enhances hydroxyhexylitaconic acid production in Aspergillus niger S17-5.

AIM: Aspergillus niger S17-5 produces two alkylitaconic acids, 9-hydroxyhexylitaconic acid (9-HHIA) and 10-hydroxyhexylitaconic acid (10-HHIA), which have cytotoxic and polymer building block properties. In this study, we characterized the production of 9-HHIA and 10-HHIA by addition of their expected precursor, caprylic acid, to a culture of A. niger S17-5, and demonstrated batch fermentation of 9-HHIA and 10-HHIA in a jar fermenter with DO-stat. METHODS AND RESULTS: Production titres of 9-HHIA and 10-HHIA from 3% glucose in a flask after 25 days cultivation were 0·35 and 1·01 g l-1 respectively. Addition of 0·22 g l-1 of caprylic acid to a suspension of resting cells of A. niger S17-5 led to 32% enhancement of total 9-HHIA and 10-HHIA production compared to no addition. No enhancement of the production of 9-HHIA or 10-HHIA by the addition of oxaloacetic acid was observed. Addition of caprylic acid to the culture at mid-growth phase was more suitable for 9-HHIA and 10-HHIA production due to less cell growth inhibition by caprylic acid. DO-stat batch fermentation with 3% glucose and 14·4 g l-1 of caprylic acid in a 1·5 l jar fermenter resulted in the production titres of 9-HHIA and 10-HHIA being 0·48 and 1·54 g l-1 respectively after 10 days of cultivation. CONCLUSIONS: Addition of caprylic acid to the culture of A. niger S17-5 enhances 9-HHIA and 10-HHIA production. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that 9-HHIA and 10-HHIA are synthesized with octanoyl-CoA derived from caprylic acid, and that the supply of octanoyl-CoA is a rate-limiting step in 9-HHIA and 10-HHIA production. To the best of our knowledge, this is the first report regarding the fermentation of naturally occurring itaconic acid derivatives in a jar fermenter.

Disruption of glpF gene encoding the glycerol facilitator improves 1,3-propanediol production from glucose via glycerol in Escherichia coli.

Engineered Escherichia coli has recently been applied to produce 1,3-propanediol (1,3-PDO) from glucose. A metabolic intermediate in the production pathway, glycerol, is partially secreted into the extracellular of E. coli through a glycerol facilitator encoded by glpF, and this secretion consequently decreases 1,3-PDO production. Therefore, we aimed to determine whether disrupting the glpF gene would improve 1,3-PDO production in E. coli. The intracellular glycerol concentration in a glpF-disruptant was 7·5 times higher than in a non-disruptant. The glpF-disrupted and non-disrupted E. coli strains produced 0·26 and 0·09 g l-1 of 1,3-PDO, respectively, from 1% glucose after 72 h of cultivation. The specific growth rate (µ) and the 1,3-PDO yield from glucose (YP/S ) in the disruptant were higher than those in the non-disruptant (ΔglpF, µ = 0·08 ± 0·00 h-1 , YP/S  = 0·06 mol mol-glucose-1 ; BW25113, µ = 0·06 ± 0·00 h-1 , YP/S  = 0·02 mol mol-glucose-1 ). Disruption of the glpF gene decreased the production of the by-product, acetic acid. These results indicated that disruption of glpF increased the intracellular concentration of glycerol and consequently increased 1,3-PDO production in E. coli.

Enteric opportunistic parasitic infections among HIV seropositive patients in Kathmandu, Nepal.

BACKGROUND: Enteric opportunistic parasitic infections are the major source of diarrheal disease in developing countries mainly in Human Immunodeficiency virus (HIV) infected patients. OBJECTIVE: The study was to detect enteric parasites causing diarrhea and their association with immune status in HIV-seropositive patients. METHODS: The present study was conducted in Dirgh-Jeevan Health Care Research Center and Tribhuvan University Teaching Hospital, Public Health Research Laboratory, Kathmandu, Nepal between June 2010 and May 2011 involving 146 Human Immunodeficiency virus (HIV) positive patients. Serostatus from these patients were detected by Enzyme Linked Immunosorbent assay. CD4+ T cell counts were done by flow cytometry. Stool was examined for enteric parasites by microscopy with special staining methods. RESULTS: A total of 146 HIV sero-positive patients with and without diarrhea age between 20 to 45 years were included in the study. Of the 146 patients, the protozoan parasitic infection was found in 30.13% (44/146). Out of 146 patients, 78 had diarrhea in which parasitic infection was 39 (50%) and 7.35% (5/68) protozoal parasites positive cases did not have diarrhea. A significant difference (p less than 0.05) was observed in the level of infection of intestinal protozoan between the HIV seropositive with diarrhea and HIV-seropositive without diarrhea. Out of 43 patients whose CD4+ T cells were less than 200/µl, 29 (67.4%) had opportunistic parasitic infection whereas out of 103 patients whose CD4+ T cells were =200/mcl, only 15 (14.56%) had opportunistic parasitic infection (P less than 0.05). CONCLUSION: Enteric opportunistic parasitic infections were detected in 30.1% among HIV-seropositive patients and low CD4+ T count indicated high enteric opportunistic infection. Early detection of enteric parasitic infections will help in the management and to improve the quality of life for HIV-infected individuals.

Retinoic Acid Deficiency Underlies the Etiology of Midfacial Defects.

Embryonic craniofacial development depends on the coordinated outgrowth and fusion of multiple facial primordia, which are populated with cranial neural crest cells and covered by the facial ectoderm. Any disturbance in these developmental events, their progenitor tissues, or signaling pathways can result in craniofacial deformities such as orofacial clefts, which are among the most common birth defects in humans. In the present study, we show that Rdh10 loss of function leads to a substantial reduction in retinoic acid (RA) signaling in the developing frontonasal process during early embryogenesis, which results in a variety of craniofacial anomalies, including midfacial cleft and ectopic chondrogenic nodules. Elevated apoptosis and perturbed cell proliferation in postmigratory cranial neural crest cells and a substantial reduction in Alx1 and Alx3 transcription in the developing frontonasal process were associated with midfacial cleft in Rdh10-deficient mice. More important, expanded Shh signaling in the ventral forebrain, as well as partial abrogation of midfacial defects in Rdh10 mutants via inhibition of Hh signaling, indicates that misregulation of Shh signaling underlies the pathogenesis of reduced RA signaling-associated midfacial defects. Taken together, these data illustrate the precise spatiotemporal function of Rdh10 and RA signaling during early embryogenesis and their importance in orchestrating molecular and cellular events essential for normal midfacial development.

A new in vitro cell line established from human oat cell carcinoma of the lung.

A new tissue culture line, OAT-1975, has been established from explant cultures of human oat cell carcinoma of the lung, characterized and maintained for more than 1 year. The cells grew in a monolayered sheet with 24 hr of population-doubling time and are capable of clonal growth with about 60% plating efficiency. The cells still retain unique morphological and structural characteristics of oat cell carcinoma and showed "tumor takes" when transplanted into nude mice and conditioned young Syrian Golden hamsters. The chromosome constitution of the cells is hypertriploid with a modal peak at 74. The permanent growth integrity of the present cell line made it possible to utilize it for cell biological studies. Cell inactivation experiments indicated that the cells are sensitive to X-rays and Mitomycin C when compared to the responses of HeLa S3 cells.

Microbiological and Clinical Profile of Uropathogenic Escherichia coli Isolates in Kathmandu University Hospital.

BACKGROUND: Treatment of patients infected by multidrug resistant bacteria is a major challenge. Immunocompromised status, prolonged hospital stay, malignancy, diabetes are some of the risk factors for emergence of multidrug resistance. Our study focused on microbiological and clinical profile of multidrug resistant uropathogenic Escherichia coli. METHODS: This was a cross-sectional study conducted between June 2014-May 2015 in Kathmandu University Hospital. Urine sample from outpatients and inpatients from which Escherichia coli isolated was included. Specimen collection, culture, identification tests were done following guidelines given by American Society for Microbiology. RESULTS: Total number of urine samples received during the study were 3,554. Escherichia coli isolates were 645(18.14%) and 245(37.98%) were Extended Spectrum Beta-Lactamase producer. Extended Spectrum Beta-Lactamase producers were found more among inpatients 148(60.41%) [p<0.001], patients with underlying urological abnormalities 38 (15.51%) [p=0.0039], pregnant ladies 46(18.77%) [p=0.0028], diabetic patients 27 (11.02%) [p=0.0084], patients who received prior antibiotic therapy 155 (63.26%) [p=0.0043] than Extended Spectrum Beta-Lactamase non-producer. Malignancy was seen more among Extended Spectrum Beta-Lactamase producer having patients 5 (2.04%) [p=0.031] and all these isolates were more resistant to fluoroquinolones 168(68.57%), Trimethoprim-sulfamethoxazole 239 (97.55%) [p=0.0633], aminoglycosides [p=0.0001] but only 2(0.80%) were resistant to carbapenems. CONCLUSIONS: Diabetes, pregnancy, malignancy, prior antibiotic therapy, underlying urological abnormalities were found associated with emergence of Extended Spectrum Beta-Lactamase producer in urine samples. Proper antibiotic usage may help to overcome the problem of emergence of antibiotic resistance.

Emergence of Aminoglycoside Resistance Due to armA methylase in Multi-drug Resistant Acinetobacter Baumannii Isolates in a University Hospital in Nepal.

BACKGROUND: The emergence of multidrug-resistant Acinetobacter baumannii associated with hospital-acquired infections has been increasingly reported worldwide. 16S rRNA methylase producing Gram-negative bacteria are highly resistant to all clinically important aminoglycosides. We analyzed A. baumannii clinical isolates resistant to aminoglycosides from hospitalized patients. The objective of this study was to investigate the emergence of armA in A.baumannii species associated with nosocomial infection in a university hospital in Nepal. METHODS: This was a cross-sectional study conducted at the department of Clinical Microbiology, Tribhuvan University Teaching Hospital (TUTH), from December 2013 to December 2014. A total of 246 Acinetobacter species were isolated from different patients were screened for MDR A. baumannii. Identification at the species level was confirmed by 16S rRNA sequencing. Drug susceptibility testing was performed by Kirby- Bauer disc diffusion method and minimum inhibitory concentrations (MICs) were determined using the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Screening for 16S rRNA methylase-production was done for the isolates resistant to gentamicin and amikacin. Detection of 16S rRNA methylase gene was done by PCR. RESULTS: All 122 multidrug-resistant A. baumanniiisolates were resistant to majority of the antibiotics used except polymyxin and tigecycline. Ninty-six MDR A. baumannii isolates had MICs of > 512 mg/L to amikacin and arbekacin indicating their high resistance to aminoglycosides.Of the 96 pan-aminoglycoside resistant isolates, 75 isolates had 16SrRNAmethylasewith all isolates harboring armA gene. CONCLUSIONS: This is the first report describing multidrug-resistant A. baumannii strains harboring armA from hospitalized patients in Nepal. A methylase gene (armA), conferring high level of resistance to aminoglycosides, was detected in majority of our isolates.

Evidence that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-kappaB.

The transcription factor NF-kappaB is a positive transcription factor for a number of genes and has been recognized as an anti-apoptotic regulator. However, the mechanism by which NF-kappaB blocks apoptosis is still controversial. Here, we demonstrate the evidence that NF-kappaB could attenuate the TNF-alpha-induced apoptosis without de novo protein synthesis using human pancreatic cancer cell lines, MIA PaCa-2 and Capan-2. The TNF-alpha-induced apoptosis was blocked by IL-1beta, a potent inducer of NF-kappaB activation. This inhibitory effect of IL-1beta was evident when cells were treated with protein synthesis inhibitors such as cycloheximide (CHX). Moreover, NF-kappaB decoy oligonucleotides could not block the anti-apoptotic effect of IL-1beta at doses sufficient to block the NF-kappaB-dependent transcription induced by IL-1beta. To confirm the role of NF-kappaB in blocking apoptosis, we generated stable cell lines expressing IkappaBdeltaN, a highly stable form of IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB. In these stable transfectants, the antiapoptotic effect of IL-1beta was totally abolished, indicating that the anti-apoptotic action of IL-1beta could be ascribed to the NF-kappaB action. These findings show that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-kappaB and support the possibility that NF-kappaB could exert its anti-apoptotic action through protein-protein interaction.

Reduction of prostaglandin D2 to 9 alpha,11 beta-prostaglandin F2 by a human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme.

Prostaglandin (PG) specificity of two 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozymes, DD2 and DD4, of human liver was examined. DD2 exhibited NADPH-linked reductase activity for 9-,11- and 15-ketoprostaglandins at a pH optimum of 6.0, whereas DD4 reduced only 15-ketoprostaglandin F2 alpha. DD2 showed the highest Vmax/Km value for PGD2 of the PG substrates, and the reduced product of PGD2 was identified to 9 alpha,11 beta-PGF2 by gas chromatography-mass spectrometry. In the reverse reaction with NADP+ as a cofactor, the two enzymes slowly oxidized several PGs with 9-, 11- and/or 15-hydroxy groups, except that DD2 showed high activity for 9 alpha,11 beta-PGF2 at a pH optimum of 10.0. The Km and Vmax values of DD2 for PGD2 were 57 microM and 250 nmol/min per mg, respectively, at pH 7.0 and 37 degrees C, and the respective values for 9 alpha,11 beta-PGF2 were 72 microM and 10 nmol/min per mg. PGD2 11-ketoreductase activity in human liver cytosol was recovered in 30-75% saturated ammonium sulfate fraction. More than 77% of the PGD2 11-ketoreductase activity in the ammonium sulfate fraction was immunoprecipitated by antibodies against DD2, and inhibited by known inhibitors of the enzyme. These results suggest that DD2 is a major soluble PGD2 11-ketoreductase species in human liver.

Efficacy of famotidine and omeprazole in healing symptoms of non-erosive gastro-oesophageal reflux disease: randomized-controlled study of gastro-oesophageal reflux disease.

BACKGROUND: The epidemiology and pathophysiology of non-erosive gastro-oesophageal reflux disease differs from erosive gastro-oesophageal reflux disease. There is a possibility that non-erosive gastro-oesophageal reflux disease treatment requires a different regimen/approach but it is not yet acknowledged. AIM: To investigate the efficacy of famotidine and omeprazole in the treatment of gastro-oesophageal reflux disease, especially non-erosive gastro-oesophageal reflux disease. PATIENTS AND METHODS: A randomized, open-label trial was conducted. Fifty-four gastro-oesophageal reflux disease patients were assigned to treatment with famotidine at a dosage of 20 mg twice daily; or omeprazole, 20 mg once daily, for a period of 8 weeks. The Short Form-36 Health Survey and Gastrointestinal Symptom Rating Scale administered at baseline and after 8 weeks of treatment as well as a symptom questionnaire were conducted daily. RESULTS: Short Form-36 revealed that gastro-oesophageal reflux disease has severe impact on health-related quality of life. Thirty-nine subjects (77%) were endoscopically diagnosed as non-erosive gastro-oesophageal reflux disease. The mean Gastrointestinal Symptom Rating Scale abdominal pain, and indigestion score of non-erosive gastro-oesophageal reflux disease significantly improved in famotidine-treated patients (P < 0.05), but not in the omeprazole. There was no significant change regarding improved heartburn symptoms of non-erosive gastro-oesophageal reflux disease between treatments in the daytime or night-time. CONCLUSION: Famotidine and omeprazole were both effective in improving symptoms of gastro-oesophageal reflux disease, particularly non-erosive gastro-oesophageal reflux disease.

Phenotypic Characterization of Multidrug-resistant Escherichia Coli with Special Reference to Extended-spectrum-beta-lactamases and Metallo-beta-lactamases in a Tertiary Care Center.

INTRODUCTION: The increasing reports on extended-spectrum-beta-lactamase and metallo-beta-lactamase producing Escherichia coli have addressed a potential threat to global health since it is found to be highly resistance to most of the currently available antibiotics including carbapenems. The present study was aimed to determine the antibiogram of extended-spectrum-beta-lactamase and metallo-beta-lactamase producing MDR E. coli isolates from various clinical samples. METHODS: This was a cross-sectional study conducted over a period of seven months from December 2013 to July 2014 at bacteriology laboratory of Tribhuvan University Teaching Hospital. A total of 250 clinical specimens (urine, pus, sputum, blood, body fluid, bile, tissue and central venous pressure line tip) were processed from inpatients, with multidrug-resistant Escherichia coli infections. Standard microbiological techniques were used for isolation and identification of the isolates. The presence of extended-spectrum-beta-lactamase was detected by phenotypic confirmatory test recommended by Clinical and Laboratory Standards Institute and imipenem (IMP) /EDTA combined disc method was performed to detect metallo-beta-lactamase mediated resistance mechanism. RESULTS: We found high level of beta lactamase mediated resistance mechanism as part of multidrug resistance. Among 250 MDR isolates, 60% isolates were extended-spectrum-beta-lactamase producers and 17.2% isolates were metallo-beta-lactamase producers. Co-existence of extended-spectrum-beta-lactamase and metallo-beta-lactamase identified in 6.8% isolates. CONCLUSIONS: Beta-lactamase mediated resistance mechanisms are accounting very high in the multidrug resistant isolates of E. coli. Therefore, early detection of beta lactamase mediated resistant strains and their current antibiotic susceptibility pattern is necessary to avoid treatment failure and prevent the spread of MDR.

Reduction of drug ketones by dihydrodiol dehydrogenases, carbonyl reductase and aldehyde reductase of human liver.

In this study, we compared the enzymatic reduction of 10 drugs with a ketone group by homogeneous carbonyl reductase, aldehyde reductase and three dihydrodiol dehydrogenases of human liver cytosol. At least one and in some cases all of the three dihydrodiol dehydrogenases reduced each of the ten drugs. Among these naloxone, naltrexone, befunolol, ethacrynic acid and ketoprofen were substrates specific for the dehydrogenases. The other drugs--haloperidol, metyrapone, loxoprofen, daunorubicin and acetohexamide--were highly reduced by carbonyl reductase and/or aldehyde reductase. The dihydrodiol dehydrogenases also showed lower Km values for haloperidol and loxoprofen than did carbonyl reductase. The results indicate that the three dihydrodiol dehydrogenases, as well as the two reductases, are implicated in the reduction of ketone-containing drugs in human liver cytosol.

Injurious effect of Helicobacter pylori culture fluid to gastroduodenal mucosa, and its detoxification by sucralfate in the rat.

BACKGROUND: Helicobacter pylori plays an important role in the pathogenesis of peptic ulcer. Although several cytotoxins related to H. pylori have been reported, their effects on gastroduodenal mucosa have not been well evaluated in vivo. AIM: To investigate the effects of the combination of acid and toxic substances derived from H. pylori on gastroduodenal mucosa, and to observe the effect of sucralfate on such factors in the rat. METHODS: Male Sprague-Dawley rats were fasted overnight and anaesthetized. The pylorus was ligated, and a double-lumen cannula was inserted into the forestomach for gastric luminal perfusion. In other animals, a cannula was inserted to perfuse the proximal duodenum. 51Cr-EDTA was administered intravenously and mucosal integrity was monitored by measuring the blood-to-lumen 51Cr-EDTA clearance. After 72 h of culture of H. pylori (NCTC11637 and Sydney strain 1), Brucella broth containing 3% FBS was filtered to remove the bacteria (supernate of H. pylori culture fluid; HPsup). HPsup was acidified (pH=2.0) with HCl, and tested for its injurious action on gastric or duodenal mucosa by luminal perfusion. HPsup was incubated with sucralfate for 30 min. The supernate was collected by centrifugation and the pH was readjusted to 2.0. This sucralfate-treated HPsup was used to test the effect of sucralfate against H. pylori-related mucosal injurious factors. RESULTS: Non-acidified and acidified HPsup did not cause any detectable injury to the gastric mucosa. Non-acidified HPsup did not cause injury in the duodenal mucosa. However, acidified HPsup induced a significantly greater increase in 51Cr-EDTA clearance and greater histological damage than in controls. Sucralfate completely reversed this. CONCLUSION: These results suggest that an H. pylori-related toxic substance may aggravate duodenal acid injury by acting on luminal surfaces, and that the detoxification of this substance by sucralfate may contribute to its anti-ulcer action.

Helicobacter pylori eradication decreases blood neutrophil and monocyte counts.

BACKGROUND: The effect of Helicobacter pylori infection on systemic disorders is not well understood. AIM: The purpose of this study was to elucidate the systemic effects of H. pylori infection by comparing differential counts of leukocytes and platelets in peripheral blood before and after eradication of H. pylori. METHODS: A total of 164 H. pylori-positive patients underwent eradication therapy, and populations of peripheral blood leukocytes and platelets before and 0 (just after therapy), 1, 3 and 12 months after eradication were retrospectively analysed. RESULTS: In the eradicated group (n = 138), blood leukocytes, neutrophils and monocytes decreased significantly after eradication, but there was no significant change in eosinophils, basophils, lymphocytes or platelets. In the non-eradicated group (n = 26), there was no significant change in any studied parameter. With regard to smoking status, although leukocytes and neutrophils did not decrease after eradication in the smoking group, they significantly decreased after eradication in the nonsmoking group. CONCLUSIONS: These findings suggest that: (1) H. pylori infection increases neutrophil and monocyte counts in the peripheral blood, which indicates a significant role of H. pylori infection in systemic disorders; and (2) Smoking may mask the effect of H. pylori eradication on peripheral leukocytes, which would explain the controversy in previous reports concerning H. pylori infection and peripheral leukocytes.

The protective effect of rebamipide on paracellular permeability of rat gastric epithelial cells.

BACKGROUND: Barrier function in gastric epithelial cells is essential for the gastric defence mechanism against acid back-diffusion into the mucosal layer. Our previous study indicated that trans-epithelial resistance (TER) of rat gastric epithelial cells was rapidly increased when the cells were exposed to acid. This response to acid was diminished by indometacin. AIM: Evaluate the effects of a mucoprotective agent, rebamipide, on the nonsteroidal anti-inflammatory drug (NSAID)-induced increase of gastric epithelial permeability. METHODS: Rat gastric epithelial cells were plated on tissue culture inserts. Cells were exposed to a NSAID (indometacin, 10-7 M). Trans-epithelial permeability was measured by TER and diffusion rate of 14C-mannitol. The effect of rebamipide was evaluated by measuring TER. Endogenous prostaglandin E2 (PGE2) production in culture medium was also measured. RESULTS: Indometacin gradually and significantly decreased TER and increased 14C-manitol permeability. Rebamipide reversed the indometacin-induced changes in epithelial permeability and induced PGE2 synthesis. This induction was blocked by either indometacin or a Cyclooxygenase (COX)-2 specific inhibitor. CONCLUSIONS: COX inhibitors such as indometacin inhibit regulation of epithelial permeability by reducing PGE2. COX-1 has an important role in the gastric defense mechanism. Rebamipide suppressed an indometacin-induced increase in gastric epithelial permeability by increasing PGE2 levels in a COX-2 dependent manner.

Total one-stage repair of interrupted aortic arch associated with aortic septal defect and patent ductus arteriosus.

Successful total repair in one stage was performed in a 3-year-old girl who had interrupted aortic arch associated with aortic septal defect and patent ductus arteriosus. Surface-induced deep hypothermia and interrupted perfusion were used. The results of postoperative catheterization and angiocardiographic studies are analyzed, and the literature and results of previous surgical attempts at correction are reviewed.

Interrelations between sympathoadrenal system and hypothalamo-pituitary-adrenocortical/thyroid systems in rats exposed to cold stress.

The interrelations between sympathoadrenal (SA) system and hypothalamo-pituitary-adrenocortical (HPA) or hypothalamo-pituitary-thyroid (HPT) system during cold stress were examined by measuring plasma levels of dihydroxyphenylalanine (DOPA), catecholamine and their metabolites in adrenalectomized (ADX) and thyroidectomized (TX) rats exposed to cold stress (-3 degrees C). Plasma levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), thyroid-stimulating hormone (TSH) and thyroid hormones in cold-stressed rats were measured also. Plasma ACTH levels were increased transiently after 1 h of cold exposure, after which the circadian rhythm and plasma levels of ACTH were similar to those of normal rats. Plasma CORT levels were also elevated after 1 h of cold exposure; the increased levels of CORT tended to return to normal levels after 9 h of cold, but remained higher than those of normal rats during at least 24 h of cold exposure. Plasma ACTH levels of 5 day cold-stressed rats were no longer elevated above those of control rats and plasma CORT levels were only slightly higher than in control animals. However, plasma levels of TSH and free thyroid hormones were elevated after 1 day and remained elevated after 5 days of cold exposure. Thus, cold stress appears to activate chronically the HPT system, but only transiently activates the HPA system. ADX rats had higher basal plasma levels of dihydroxyphenylglycol (DHPG), methoxyhydroxyphenylglycol (MHPG), DOPA and homovanillic acid (HVA) than those of sham-operated (SHAM) rats, but norepinephrine (NE) levels were not significantly greater than in SHAM animals. TX rats had higher basal plasma levels of NE, epinephrine (EPI) and dopamine (DA), as well as much higher plasma levels of the metabolites. Exposure to cold increased plasma NE levels in both ADX and TX rats, but the increments in TX rats were much greater than in SHAM and ADX groups. Plasma EPI levels were not significantly elevated during cold exposure in SHAM rats, but were highly elevated in TX rats exposed to cold. TX rats had much larger increments in plasma levels of DHPG, MHPG, DA, dihydroxyphenylacetic acid (DOPAC) and HVA during cold exposure than those of SHAM and ADX rats. These results are consistent with the view that endogenous glucocorticoids restrain responses of catecholamine synthesis, release, reuptake, and metabolism in sympathetic nervous system of cold-stressed animals, but that in the absence of an effective HPT system, there is enhanced sympathoadrenal medullary function and augmentation of their responses to cold as a means for maintaining body temperature when the HPT thermogenesis system is impaired.

Effects of continuous and intermittent cold (SART) stress on sympathoadrenal system activity in rats.

We compared sympathoadrenal responses to intermittent cold (SART) stress (in which cold exposure is interrupted by 4-hourly intervals daily at room temperature) with those to continuous cold (-3 degrees C) stress. Plasma levels of dihydroxyphenylalanine (DOPA), catecholamines and their metabolites as well as tyrosine hydroxylase (TH) activities in sympathetically innervated tissues were examined in rats exposed to each stressor for 1 day or for 5 days. Neither SART nor continuous exposure to cold for 1 day or 5 days altered plasma epinephrine (EPI) levels. However, norepinephrine (NE) and dihydroxyphenylglycol (DHPG) levels increased markedly during exposure to these stressors. On the first day of SART or continuous cold stress, NE levels were increased similarly, but the increments in DHPG levels were greater during SART stress. Since DHPG is formed in neurons, neural reuptake of NE may be more enhanced on the first day of SART stress than on the first day of continuous cold stress. After 5 days of SART stress plasma NE levels were significantly higher than those found after 5 days of continuous cold exposure. Plasma levels of DHPG were elevated to the same extent in both 5 days SART- and continuously cold-stressed rats, whereas plasma levels of methoxyhydroxyphenylglycol (MHPG) increased only by 5 days SART stress. Even at 1 h after the removal from 5 days SART stress, increased plasma levels of NE, DHPG and MHPG were still evident. These results suggest that 5 days SART stress elevates extraneuronal O-methylation of DHPG, and that NE turnover is more greatly increased by SART stress than by continuous cold stress. Plasma levels of DOPA, dopamine, dihydroxyphenylacetic acid and homovanillic acid also increased after either SART or continuous cold stress for 1 day and 5 days. Adrenal TH activities were significantly increased in rats exposed to SART or continuous cold stress for 1 day and 5 days, but in brown fat TH activity was elevated only in rats exposed to 5 days of continuous cold. Both SART and continuous cold stress are selective and potent stimuli for activation of the sympathoneural system, apparently without significant adrenomedullary EPI release. The increase of TH activity in the brown fat pad as well as of plasma NE and its metabolites is probably a result of adaptation to cold. It appears that even short intervals of return to a normal environmental temperature, as in SART, are sufficient to diminish sympathetic adaptation to cold.

Beta-glucuronidase of rat preputial gland. Crystallization, properties, carbohydrate composition, and subunits.

Rat preputial gland beta-glucuronidase [ED 3.2.1.31] was purified by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G-200 and crystallization. The purified enzyme appeared homogeneous on electrophoresis in polyacrylamide gel, and on analytical ultracentrifugation and had a molecular weight of approximately 320,000, and a sedimentation coefficient of 12S. SDS polyacrylamide gel electrophoresis indicated that the enzyme consisted of subunits with molecular weight of 79,000, so the native enzyme appeared to be a tetramer. The Km with p-nitrophenyl beta-D-glucosiduronic acid as substrate was about 0.53 mM. The enzyme had a single pH optimum at 4.5. The enzyme had a very low content of sulphur-containing amino acid and contained 5.7 per cent carbohydrate, consisting of mannose, glucose, fucose, galactose, and glucosamine in a ratio of 44;9;6;2;41. Sialic acid was not detected in the crystallized enzyme.

Irradiation of mixed beam and design of spread-out Bragg peak for heavy-ion radiotherapy.

Data on cellular inactivation resulting from mixed irradiation with charged-particle beams of different linear energy transfer (LET) are needed to design a spread-out Bragg peak (SOBP) for heavy-ion radiotherapy. The present study was designed to study the relationship between the physical (LET) and biological (cell killing) properties by using different monoenergetic beams of 3He, 4He and 12C ions (12 and 18.5 MeV/nucleon) and to attempt to apply the experimental data in the design of the SOBP (3 cm width) with a 135 MeV/nucleon carbon beam. Experimental studies of the physical and biological measurements using sequentially combined irradiation were carried out to establish a close relationship between LET and cell inactivation. The results indicated that the dose-cell survival relationship for the combined high- and low-LET beams could be described by a linear-quadratic (LQ) model, in which new coefficients alpha and beta for the combined irradiation were obtained in terms of dose-averaged alpha and square root of beta for the single irradiation with monoenergetic beams. Based on the relationship obtained, the actual SOBP designed for giving a uniform biological effect at 3 cm depth was tested with the 135 MeV/nucleon carbon beam. The results of measurements of both physical (LET) and biological (90% level of cell killing, etc.) properties clearly demonstrated that the SOBP successfully and satisfactorily retained its high dose localization and uniform depth distribution of the biological effect. Based on the application of these results, more useful refinement and development can be expected for the heavy-ion radiotherapy currently under way at the National Institute of Radiological Sciences, Japan.