Immunomodulation by 'Immuplus (AquaImmu)' in giant freshwater prawn, Macrobrachium rosenbergii (De Man)

ImmuPlus, a polyherbal commercial formulation was used to modulate the immune system of commercially important giant freshwater prawn M. rosenbergii. The prawns were fed with basal diet supplemented with ImmuPlus at 1g/kg feed for 4 weeks. Results showed that the phenoloxidase activity (PO), haemagglutination and lysozyme activities were significantly elevated in ImmuPlus-fed prawn up to 3 weeks of feeding and declined after 4 weeks of feeding. The total protein level in ImmuPlus-fed prawn raised up to 2nd week of feeding. Incorporation of ImmuPlus at the rate of 1g/kg feed in the diet of prawn for 3 weeks may be beneficial in raising the immune status of prawn.     

Effect of simultaneous exposure to lead and cadmium on gonadotropin binding and steroidogenesis on granulosa cells: an in vitro study

Effects of lead (Pb) and cadmium (Cd) both alone or in combination on the binding of LH and FSH on isolated granulosa cells were studied. Granulosa cells isolated from proestrous rats were incubated (in vitro) with lead acetate and/or cadmium acetate (0.03 microM of Pb or Cd) for 1 hr. LH binding was dropped to 84% in Pb treated cells, 72.5% in Cd treated cells and 74.8% in combined metal treated cells compared to control. FSH binding dropped to 85.5% in Pb treated cells, 71.16% in Cd treated cells and 72.5% in combined metal treated cells compared to control. Activity of 17beta Hydroxy Steroid Dehydrogenase (17betaHSDH), a key steroidogenic enzyme was reduced by 52% in Cd and 37% in combined metal exposed cells whereas Pb exposed cells showed 31% reduction in the enzyme activity. Pretreatment with SH groups protectants (glutathione [GSH], dithiothretol [DTT]) and zinc caused an ameriolation in enzyme activity whereas Zn pretreatment showed an increase in gonadotropin binding in metal exposed cells. These results suggest that both Pb and Cd can cause a reduction in LH and FSH binding, which significantly alters steroid production in vitro and exerts a direct influence on granulosa cell function.     

A perikinetochoric ring defined by MCAK and Aurora-B as a novel centromere domain

Mitotic Centromere-Associated Kinesin (MCAK) is a member of the kinesin-13 subfamily of kinesin-related proteins. In mitosis, this microtubule-depolymerising kinesin seems to be implicated in chromosome segregation and in the correction of improper kinetochore-microtubule interactions, and its activity is regulated by the Aurora-B kinase. However, there are no published data on its behaviour and function during mammalian meiosis. We have analysed by immunofluorescence in squashed mouse spermatocytes, the distribution and possible function of MCAK, together with Aurora-B, during both meiotic divisions. Our results demonstrate that MCAK and Aurora-B colocalise at the inner domain of metaphase I centromeres. Thus, MCAK shows a “cone”-like three-dimensional distribution beneath and surrounding the closely associated sister kinetochores. During the second meiotic division, MCAK and Aurora-B also colocalise at the inner centromere domain as a band that joins sister kinetochores, but only during prometaphase II in unattached chromosomes. During chromosome congression to the metaphase II plate, MCAK relocalises and appears as a ring below each sister kinetochore. Aurora-B also relocalises to appear as a ring surrounding and beneath kinetochores but during late metaphase II. Our results demonstrate that the redistribution of MCAK at prometaphase II/metaphase II centromeres depends on tension across the centromere and/or on the interaction of microtubules with kinetochores. We propose that the perikinetochoric rings of MCAK and Aurora-B define a novel transient centromere domain at least in mouse chromosomes during meiosis. We discuss the possible functions of MCAK at the inner centromere domain and at the perikinetochoric ring during both meiotic divisions.     

Poly(ADP-ribose) polymerase-1-deficient mice are protected from angiotensin II-induced cardiac hypertrophy

Poly(ADP-ribose) polymerase-1 (PARP), a chromatin-bound enzyme, is activated by cell oxidative stress. Because oxidative stress is also considered a main component of angiotensin II-mediated cell signaling, it was postulated that PARP could be a downstream target of angiotensin II-induced signaling leading to cardiac hypertrophy. To determine a role of PARP in angiotensin II-induced hypertrophy, we infused angiotensin II into wild-type (PARP(+/+)) and PARP-deficient mice. Angiotensin II infusion significantly increased heart weight-to-tibia length ratio, myocyte cross-sectional area, and interstitial fibrosis in PARP(+/+) but not in PARP(-/-) mice. To confirm these results, we analyzed the effect of angiotensin II in primary cultures of cardiomyocytes. When compared with PARP(-/-) cardiomyocytes, angiotensin II (1 microM) treatment significantly increased protein synthesis in PARP(+/+) myocytes, as measured by (3)H-leucine incorporation into total cell protein. Angiotensin II-mediated hypertrophy of myocytes was accompanied with increased poly-ADP-ribosylation of nuclear proteins and depletion of cellular NAD content. When cells were treated with cell death-inducing doses of angiotensin II (10-20 microM), robust myocyte cell death was observed in PARP(+/+) but not in PARP(-/-) myocytes. This type of cell death was blocked by repletion of cellular NAD levels as well as by activation of the longevity factor Sir2alpha deacetylase, indicating that PARP induction and subsequent depletion of NAD levels are the sequence of events causing angiotensin II-mediated cardiomyocyte cell death. In conclusion, these results demonstrate that PARP is a nuclear integrator of angiotensin II-mediated cell signaling contributing to cardiac hypertrophy and suggest that this could be a novel therapeutic target for the management of heart failure.     

Topology of the porin MspA in the outer membrane of Mycobacterium smegmatis

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the outer membrane (OM). It is the prototype of a new family of octameric porins with a single central channel of 9.6 nm in length and consists of two hydrophobic beta-barrels of 3.7 nm in length and a more hydrophilic, globular rim domain. The length of the hydrophobic domain of MspA does not match the thicknesses of mycobacterial OMs of 5-12 nm as derived from electron micrographs. Further, the membrane topology of MspA is unknown as it is for any other mycobacterial OM protein. We used MspA as a molecular ruler to define the boundaries of the OM of M. smegmatis by surface labeling of single cysteine mutants. Seventeen mutants covered the surface of the rim domain and were biotinylated with a membrane-impermeable reagent. The label efficiencies in vitro were remarkably similar to the predicted accessibilities of the cysteines. By contrast, six of these mutants were protected from biotinylation in M. smegmatis cells. Tryptophan 21 defines a horizontal plane that dissects the surface-exposed versus the membrane-protected residues of MspA. The 8 phenylalanines at position 99 form a ring at the periplasmic end of the hydrophobic beta-barrel domain. These results indicated that (i) the membrane boundaries of MspA are defined by aromatic girdles as in porins of Gram-negative bacteria and (ii) loops and a 3.4-nm long part of the hydrophilic rim domain are embedded into the OM of M. smegmatis. This is the first report suggesting that elements other than hydrophobic alpha-helices or beta-sheets are integrated into a lipid membrane.     

Dietary administration of bovine lactoferrin influences the immune ability of the giant freshwater prawn Macrobrachium rosenbergii (de Man) and its resistance against Aeromonas hydrophila infection and nitrite stress

Haemocyte count, phenoloxidase activity, agglutinin levels, total protein content, bacterial clearance efficiency, resistance to the pathogen Aeromonas hydrophila and nitrite stress were measured in the giant freshwater inter-moult sub-adult prawn Macrobrachium rosenbergii (15-20 g) which had been fed diets containing bovine lactoferrin (Lf) at 50, 100, 200mg kg(-1) feed for 7 or 14 days. M. rosenbergii fed a diet containing 100mg Lf kg(-1) diet for 7 days showed significant (P<0.05) increase in total protein levels, agglutination titres against bacteria A. hydrophila and rabbit RBC, phenoloxidase activity, bacterial clearance (as observed through reduced number of circulating bacteria) as well as survival against A. hydrophila challenge. Increased bacterial clearance was also noticed in prawns fed Lf at 50 or 200mg kg(-1) for 14 days compared to control. Feeding of Lf at 50mg kg(-1) diet for 7 or 14 days was able to enhance only PO activity and reduce percent mortality against A. hydrophila challenge compared to its control. Total haemocyte count was higher in the lowest dose of Lf feeding, i.e. 50mg kg(-1) for 7 days. However, there was no significant alteration in the differential haemocyte population with respect to graded levels of Lf feeding for 7 or 14 days. A notable reduction in mortality percent after 120 h of nitrite stress was observed in prawn fed Lf at 100mg kg(-1) diet for 14 days. On the contrary, feeding of the highest dose of Lf, i.e. 200mg kg(-1) diet for 14 days failed to stimulate most of the innate immune parameters or reduce the percent mortality against A. hydrophila challenge or nitrite stress. It is therefore concluded that administration of Lf in the diet at 100mg kg(-1) for 7 days could enhance the immune ability of M. rosenbergii and increase its resistance to A. hydrophila infection or nitrite stress.     

Morphometric sex determination of Milky and Painted Storks in captivity

Logistic regression was applied to develop a morphometric sexing method of two closely related stork species that were previously sexed through amplification of the CHD gene. Tarsus length (TL) and bill length (BL) measurements were recorded from captive populations of adult Milky Stork (Mycteria cinerea) (n = 60) and Painted Stork (Mycteria leucocephala) (n = 58) at Zoo Negara Malaysia. Despite having monomorphic plumages, both stork species exhibited normal sexual size dimorphism in which males were significantly larger than females in the tested variables. Based on logistic regression analysis, BL correctly classified the sex of sampled individuals from Painted and Milky stork with an overall predicted accuracy of 94.8 and 90.0%, respectively. However, TL measurements generated a lower predicted accuracy level of 86.2% and a same accuracy level of 90% on the sex classification of individuals from Painted and Milky stork, respectively. By comparing the measurements of both species, only the average BL measurements of the Milky storks were significantly lower than that of Painted storks (t-test, P80.001). The logistic regression equation in this study may serve as a simple and more practical option for sexing Milky and Painted storks for their breeding and conservation programmes.