Attenuation of a virulent Aeromonas hydrophila with novobiocin and pathogenic characterization of the novobiocin-resistant strain.

AIM: To determine whether novobiocin resistance strategy could be used to attenuate a virulent Aeromonas hydrophila AH11P strain and to characterize the growth and pathogenic differences between the novobiocin-resistant strain and its virulent parent strain AH11P. METHODS AND RESULTS: A novobiocin-resistant strain AH11NOVO was obtained from a virulent Aer. hydrophila strain AH11P through selection of resistance to novobiocin. AH11NOVO was found to be avirulent to channel catfish (Ictalurus punctatus), whereas AH11P was virulent. When AH11NOVO vaccinated channel catfish were challenged with AH11P at 14 days postvaccination, relative per cent of survival of vaccinated fish was 100%. The cell proliferation rate of AH11NOVO was found to be significantly (P < 0.05) less than that of AH11P. In vitro motility assay revealed that AH11NOVO was nonmotile, whereas AH11P was motile. AH11NOVO had significantly (P < 0.05) lower in vitro chemotactic response to catfish mucus than that of AH11P. Although the ability of AH11NOVO to attach catfish gill cells was similar to that of AH11P, the ability of AH11NOVO to invade catfish gill cells was significantly (P < 0.05) lower than that of AH11P. CONCLUSIONS: The novobiocin-resistant AH11NOVO is attenuated and different from its parent AH11P in pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The significantly lower chemotactic response and invasion ability of AH11NOVO compared with that of its virulent parent strain AH11P might shed light on the pathogenesis of Aer. hydrophila.

Construction of a recombinant cellulolytic Escherichia coli.

A 1.2-kb DNA fragment from Bacillus subtilis CD4 encoding endo-beta-1,4-glucanase and cellobiase activities was cloned and expressed in Escherichia coli. Carboxymethylcellulase (CMCase) and cellobiase activities were detected in a cell-free lysate of recombinant (re-) E. coli (Ec). The re-enzymes were functional in Ec, as it utilized carboxymethylcellulose, soluble cellulose and cellobiose as sole carbon sources for growth.

Selective identification of new therapeutic targets of Mycobacterium tuberculosis by IVIAT approach.

The in vivo induced antigen technology (IVIAT)(1) has been used for the identification of open reading frames (ORFs) which could be possible therapeutic targets. A recombinant lambdagt11:: Mycobacterium tuberculosis H37Rv expression library was screened with pooled TB patient sera preabsorbed with in vitro grown M. tuberculosis H37Rv. Preabsorption of pooled TB patient sera allowed identification of antigens specifically expressed or upregulated during infection and growth in vivo. Six ORFs were identified, of which four (rv0287, rv2402, rv3878 and rv1045) were of hypothetical functions. Rv0287 is a probable regulatory protein. Rv3878 is present uniquely in M. tuberculosis H37Rv and is a part of RDI deletion region of M. bovis BCG, which includes esat 6 region. This could be exploited as a tool for diagnosis. Two ORFs were assigned function solely on the basis of homology, dnaQ (rv3711c) and lpdA (rv3303c). dnaQ codes for the epsilon subunit of DNA polymerase III, which is responsible for the proofreading activity of the complex. lpdA codes for dihydrolipoamide dehydrogenase, which is a part of many multienzyme complexes such as pyruvate dehydrogenase, keto-acid dehydrogenase and alpha-ketoglutarate dehydrogenase. These two enzymes appear to be potential targets for drug development.

Porphyrogenic properties of the terpenes camphor, pinene, and thujone (with a note on historic implications for absinthe and the illness of Vincent van Gogh).

Camphor, alpha-pinene (the major component of turpentine), and thujone (a constituent in the liqueur called absinthe) produced an increase in porphyrin production in primary cultures of chick embryo liver cells. In the presence of desferrioxamine (an iron chelator which inhibits heme synthesis and thereby mimics the effect of the block associated with acute porphyria), the terpenes enhanced porphyrin accumulation 5- to 20-fold. They also induced synthesis of the rate-controlling enzyme for the pathway, 5-aminolevulinic acid synthase, which was monitored both spectrophotometrically and immunochemically. These effects are shared by well-known porphyrogenic chemicals such as phenobarbital and glutethimide. Camphor and glutethimide alone led to the accumulation of mostly uro- and heptacarboxylporphyrins, whereas alpha-pinene and thujone resulted in lesser accumulations of porphyrins which were predominantly copro- and protoporphyrins. In the presence of desferrioxamine, plus any of the three terpenes, the major product that accumulated was protoporphyrin. The present results indicate that the terpenes tested are porphyrogenic and hazardous to patients with underlying defects in hepatic heme synthesis. There are also implications for the illness of Vincent van Gogh and the once popular, but now banned liqueur, called absinthe.

Aspirin blocks 5-azacytidine- and hydroxyurea-induced changes in hemoglobin proportions in adult rats.

The effects of 5-azacytidine and hydroxyurea on their independent ability to change adult hemoglobin proportions toward newborn proportions in adult rats were examined. The results revealed that both the chemotherapeutic agents were capable of switching certain hemoglobin components toward newborn values and required similar time-span to express their actions. However, the switching effect of these drugs was totally lost if aspirin was simultaneously administered into the rats, reflecting the need for concurrent prostaglandin synthesis.

Immunomodulatory effects of agents of plant origin.

The immunomodulatory properties of amla (Emblica officinalis) and shankhpushpi (Evolvulus alsinoides) were evaluated in adjuvant induced arthritic (AIA) rat model. Injecting Complete Freund's Adjuvant (CFA) in right hind paw of the animals induced inflammation. The crude extracts of both the herbs were administered intraperitonially following a repeated treatment profile. The anti-inflammatory response of both the extracts was determined by lymphocyte proliferation activity and histopathological severity of synovial hyperplasia. Both the extracts showed a marked reduction in inflammation and edema. At cellular level immunosuppression occurred during the early phase of the disease. There was mild synovial hyperplasia and infiltration of few mononuclear cells in amla or shankhpushpi treated animals. The induction of nitric oxide synthase (NOS) was significantly decreased in treated animals as compared to controls. These observations suggest that both the herbal extracts caused immunosuppression in AIA rats, indicating that they may provide an alternative approach to the treatment of arthritis.

Effect of bovine herpesvirus-1 on expression of interleukin-2 receptors and effect of interleukin-12 on lymphocyte proliferation.

Expression of the interleukin-2 receptor alpha chain (IL-2R alpha) by peripheral blood mononuclear cells (PBMC) from Holstein calves, both experimentally-infected with bovine herpesvirus-l (BHV-l) and controls, was measured by flow cytometry. Expression of IL-2R alpha was 35 percent and 23 percent higher in infected calves than controls, on days 2 and 3 postinfection (PI), respectively. Concurrent with this increase in IL-2R alpha expression, a significant decrease (P < 0.001) was observed in the PHA-induced proliferative responses of PBMC from infected compared with control calves. In vitro treatment with recombinant human (rhu) IL-12 enhanced PHA-induced proliferative responses of PBMC from both infected and control calves. This rhuIL-12 enhancement of mitogen-induced proliferative responses was significant (P < 0.001) in infected calves on day 2 PI and was sufficient to abrogate the decrease observed due to BHV-1 infection. Since the expression of the beta and gamma chains of IL-2R was not measured it is difficult to speculate as to the status of high affinity receptor expression during BHV-1 infection. However results of the present study suggest that the decrease in proliferative responses observed during infection may not be due to a decrease in IL-2R alpha expression but may possibly be due to a selective down-regulation of signal transduction through IL-2R and/or by modulation of the expression of other cytokines involved in lymphocyte activation and proliferation.

Exposure of neonatal male rats to estrogen induces abnormal morphology of the penis and loss of fertility.

The research objectives are to determine whether estrogen-induced infertility is associated with abnormal morphology of the penis and if morphological alterations can be reversed by testosterone (T). Male pups received diethylstilbestrol (DES) on alternate days from postnatal days 2 to 12. They received T or empty implants at 180 days, were tested for fertility at 188 days, and terminated at 200 days. While 5/7 control males sired pups, only 1/6 did in the DES group, and 0/8 in the DES plus T group. In addition to reductions in penile length and weight, the novel structural change induced by DES, and not reversed by T, was a replacement of cavernous spaces by fat cells in the penis body. Hence, T substitution for 8 days at adulthood did not reverse infertility in rats treated neonatally with DES and provided evidence that infertility probably resulted from absence of cavernous spaces and/or accumulation of fat cells in the penis body.

Effect of stress on interleukin-2 receptor expression by bovine mononuclear leukocytes.

Holstein calves given three consecutive i.m. injections of dexamethasone (DEX) (0.04 mg kg-1) showed lymphopenia and neutrophilia with increased numbers of mature neutrophils on post-injection Days 1 and 2, but these values returned to normal levels by post-injection Day 3. Interleukin-2 receptor (IL-2R) expression by peripheral blood mononuclear cells (PBMC) was evaluated by flow cytometry using a monoclonal antibody specific for bovine IL-2R alpha. Treatment with DEX significantly decreased expression of IL-2R alpha in Concanavalin A (Con A)-activated PBMC on Day 1 (P < 0.02) and on Day 2 (P < 0.1). On Day 3, expression of IL-2R alpha by PBMC was similar in control and DEX-treated calves. This decrease in IL-2R alpha expression correlated with decreased proliferative responses of PBMC to the T-cell mitogens, phytohemagglutinin (PHA) and Con A. Following in vitro treatment with recombinant human (rhu) interleukin-12 (IL-12) Con A-induced proliferative responses of PBMC tended to be higher in both groups. However, the rhu IL-12 induced increase of Con A activated proliferative responses were significantly greater in DEX-treated calves than in control calves. IL-2R alpha expression by PBMC was found to be less in calves transported 800 km in a truck as compared to that in PBMC from controls. These data suggest that stress-induced immunosuppression in calves may involve decreased IL-2R alpha expression and decreased IL-12 production. Serum chemistry results indicated a trend toward higher creatine kinase (CK) levels in DEX-treated calves. This may be due to the lysis of corticosteroid sensitive lymphocytes.

New experimental model for the evaluation of adaptogenic products.

A new animal model for evaluating improvement in physical work performance and endurance in an adverse environment is described. In this model, rats in restraint were exposed to 5 degrees C at 480 mmHg atmospheric pressure. Results were compared to an animal forced to swim in water at 23 degrees C. In both the models, colonic temperature (Tr) of the rats was continually monitored during exposure to adverse environment and during recovery at 32 degrees C and normal atmospheric pressure. The time and pattern for Tr fall to 23 degrees C and its recovery to 37 degrees C were used as measures of endurance. The cold-hypoxia-restraint model was found to provide more precise results compared with the cold-swimming model. Panax ginseng root and Ginkgo biloba leaf extracts were compared for their positive endurance-promoting properties using both models.

Mechanism of increased tolerance to hypothermia after composite Indian herbal preparation II administration.

OBJECTIVE: Investigation of the mechanism of increased tolerance to stress induced hypothermia after the administration of composite Indian herbal preparation II (CIHP II), a combination of several plant ingredients and minerals. DESIGN: The effect of oral CIHP II administration (1 mg/g of body weight), prior to cold (5 degrees C)-hypoxia (428 mm Hg)-restraint (C-H-R) exposure in rats on cardiac and skeletal muscle oxidation was studied in vitro by estimating conversion of glucose-U-14C and Palmitate-1-(14)C to 14CO2. In vitro adipose tissue lipolysis and incorporation of glucose-U-14C into skeletal muscle glycogen was also studied. RESULTS: A single dose of CIHP II-enhanced resistance to hypothermia (rectal temperature [T(rec)] 23 degrees C) during C-H-R exposure as evidenced by increased glucose turnover rate in heart and skeletal muscle tissue. The blood glucose and skeletal muscle glycogen were conserved. Cardiac free fatty acid oxidation was also increased. During recovery from hypothermia (T(rec) 37 degrees C) blood glucose and muscle glycogen levels were conserved. Five doses of CIHP II increased resistance to cold by increased adipose fat mobilization and cardiac oxidation. Glucose oxidation was spared. During recovery from hypothermia, the glucose turnover and oxidation in skeletal muscle was increased as was fat mobilization from adipose tissue and its oxidation by heart muscle. CONCLUSIONS: CIHP II intake prior to C-H-R exposure resulted in increased glucose turnover rate and fat utilization. This perhaps helped increase the resistance to C-H-R-induced hypothermia and speeded recovery.

Enhanced thermogenesis in rats by a composite Indian herbal preparation-I and its mechanism of action.

OBJECTIVE: A composite Indian herbal preparation-I (CIHP-I) containing ingredients derived from 7 different plants and asphalt was tested for its adaptogenic activity and its mechanism of action was investigated. DESIGN: CIHP-I was tested using the cold-hypoxia-restraint (C-H-R) animal model in which the restrained rats were exposed to 5 degrees C at 428 mm Hg atmospheric pressure. Rectal temperature (Trec) of the rats was continuously monitored during the exposure and the recovery periods. The time for fall of Trec to 23 degrees C and its recovery to 37 degrees C were used as indices of endurance and the adaptogenic activity. Carbohydrate and lipid parameters were investigated to find out the nature of fuel being used during thermogenesis. RESULTS: After 12 weeks of administration of an oral dose of 7.5 mg/kg(-1)/day(-1), CIHP-I was found to possess significant adaptogenic activity. CIHP-I helped improve resistance to C-H-R induced hypothermia (Trec 23 degrees C) in animals by increased mobilization of free fatty acids (FFA) from adipose tissue. Blood glucose and muscle glycogen levels were maintained. CIHP-I treatment restricted the release of creatine phosphokinase (CPK) into the circulation during C-H-R exposure. CONCLUSIONS: The results suggested that CIHP-I is a strong adaptogen. It improved cold resistance during C-H-R exposure and enhanced recovery from hypothermia. The energy-dependent cell membrane permeability was maintained. Stored lipids were mobilised and possibly used for thermogenesis in preference to carbohydrates.

Microstructural and peripheral-layer viscosity effects on peristaltic transport of seminal fluid.

Most of the physiological fluids are transported through peristalsis of the walls which is the inherent neuromuscular property of tube smooth muscle structure. The present paper deals with the transport of seminal fluid introducing the microstructural effects and peripheral-layer viscosity effects on the flow rate and the drag for the system. It has been observed that the flow rate increases tremendously with the decrease of the peripheral layer viscosity whereas a reverse trend has been noted for the frictional drag. Further other sub-structural effects i.e. the effects of relative rotational viscosity and viscosity gradients have also been broughtout and discussed.

Effect of simulated hypoxia (7620 m, 6 h) on hepatic microsomal drug metabolising enzymes activity in rats given ethanol and vitamin A.

The hepatic MFO activity has been studied in rats exposed to acute hypoxia given single dose of ethanol (3 g/kg body weight) alone or along with 10,000 I.U. of vitamin A. The cytochrome P-450 was found to be depressed on ethanol administration in rats. Hypoxic exposure of rats given ethanol with or without vitamin A, however, restored the depressed cytochrome P-450 activity. Cytochrome-c-reductase, on the other hand, was increased on administration of ethanol with or without vitamin A supplementation. Exposure to hypoxia did not have any additional effect on cytochrome c-reductase activity. Aminopyrine-N-demethylase was depressed on ethanol administration. Vitamin A administration further depressed this enzyme. Hypoxic exposure, however, did not restore this enzyme level. Aniline hydroxylase was not affected by ethanol administration, vitamin A supplementation and hypoxic exposure. Thus, mixed function oxidase system appears to be differently affected on ethanol administration, vitamin A supplementation and hypoxic exposure suggesting that these effectors have different loci of interaction in microsomal structure and/or in its micromileau of cofactors.

Interaction of ethanol and acute hypoxia (7620 m) on vitamin A distribution in rat organs.

The effect of a moderate dose of ethanol (3 g/kg body weight) and acute hypoxia (282 mmHg pressure) for 6 h on vitamin A content of rat tissue has been investigated. In addition, to find the effect of ethanol administration and hypoxic exposure on freshly absorbed vitamin A, a tracer dose of 3H-retinyl acetate was administered prior to hypoxic exposure. The results indicate that ethanol and hypoxia interfere in the transfer of vitamin A stored as well as freshly absorbed, from intestine to liver. When ethanol administered rats were exposed to hypoxia, the secretion of vitamin A from liver is also disturbed. The vitamin A metabolism in the tissues like kidney and lung are affected adversely on ethanol administration and/or hypoxic exposure. This could be due to interference in the transfer of vitamin A from intestine to liver and secretion of vitamin A from liver into plasma.

Effect of vitamin A on hepatic microsomal drug metabolising enzymes activity in rats exposed to acute hypoxia.

The hepatic drug metabolising enzymes activity has been studied in rats exposed to acute hypoxia alone or along with supplementation of single dose of 10 000 I.U. Vitamin A. Hypoxic stress reduces the activities of cytochrome P-450, cytochrome c-reductase and aniline hydroxylase. Administration of single dose of 10 000 I.U. of Vitamin A to the rats 12 h before hypoxia reverses the effect of hypoxia on these enzymes. Thus Vitamin A seems to play a protective role in rats exposed to hypoxia on the activity of MFO system.

Unusual lesions of the patella.

We have reported 13 cases illustrating various rare lesions of the patells and discussed the incidence and treatment and reviewed other reported cases of each type of lesion.

Unusual lesions of the clavicle.

Eight types of rare clavicular lesions comprising 41 cases with their treatment and results are presented. Total claviclectomy was performed in ten cases of pyogenic osteomyelitis with no resultant functional disability to the patient. We advocate this as the treatment of choice.

Volvulus of stomach in childhood.

Six children, aged upto one year, with volvulus of the stomach are reported. Vomiting and regurgitation of feeds were the main presenting symptoms. Eventration of the left hemidiaphragm was present in four cases. Barium meal confirmed the diagnosis. Five cases were operated on through an abdominal approach. Plication of the diaphragm (3 cases), colonic displacement (2) and gastrostomy (3) were resorted to. One child needed reoperation for a missed Ladd's band. There was no operative mortality.

Diploic dermoids.

A diploic dermoid involving the orbital plate of the frontal bone is described in a 52-year-old man. It presented proptosis, downward displacement of the globe and limitation of its movement while looking up. Its extension into the anterior cranial fossa was detected by injecting a radio-opaque material into the cyst.