Investigations on Modulating Effect of Vanadium Supplementation on Growth and Metabolism Through Improved Immune Response,Antioxidative Profile and Endocrine Variables in Hariana heifers

Biological Trace Element Research - This study was conducted to investigate the effect of vanadium (V) supplementation on growth, metabolism, antioxidant, and immunological and endocrine variables...     

Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection

RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.     

Elucidation of structure–function relationships in Methanocaldococcus jannaschii RNase P,a multi-subunit catalytic ribonucleoprotein

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5′ leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae–RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein–protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.     

Dissecting functional cooperation among protein subunits in archaeal RNase P,a catalytic ribonucleoprotein complex

RNase P catalyzes the Mg²⁺-dependent 5′-maturation of precursor tRNAs. Biochemical studies on the bacterial holoenzyme, composed of one catalytic RNase P RNA (RPR) and one RNase P protein (RPP), have helped understand the pleiotropic roles (including substrate/Mg²⁺ binding) by which a protein could facilitate RNA catalysis. As a model for uncovering the functional coordination among multiple proteins that aid an RNA catalyst, we use archaeal RNase P, which comprises one catalytic RPR and at least four RPPs. Exploiting our previous finding that these archaeal RPPs function as two binary RPP complexes (POP5•RPP30 and RPP21•RPP29), we prepared recombinant RPP pairs from three archaea and established interchangeability of subunits through homologous/heterologous assemblies. Our finding that archaeal POP5•RPP30 reconstituted with bacterial and organellar RPRs suggests functional overlap of this binary complex with the bacterial RPP and highlights their shared recognition of a phylogenetically-conserved RPR catalytic core, whose minimal attributes we further defined through deletion mutagenesis. Moreover, single-turnover kinetic studies revealed that while POP5•RPP30 is solely responsible for enhancing the RPR’s rate of precursor tRNA cleavage (by 60-fold), RPP21•RPP29 contributes to increased substrate affinity (by 16-fold). Collectively, these studies provide new perspectives on the functioning and evolution of an ancient, catalytic ribonucleoprotein.     

Behavioral response of female Helicoverpa armigera (Hübner) to different plant part extracts of African marigold Tagetes erecta (L.)

The flowering stage of African marigold Tagetes erecta (L.) was the most preferred for oviposition by Helicoverpa armigera (Hübner). The lower sides of leaf margins received more eggs compared to buds, stem tip and flowers. Olfactometer studies revealed that more female moths were attracted towards the flower extracts, followed by leaf, bud and stem tip extracts of African marigold. The thin layer chromatography analysis of these extracts showed a blend of compounds including careen, ocimene, methyl benzoate, phenyl acetaldehyde and phenyl ethanol. Attempts have been made to correlate insect behavior with the presence of these compounds.     

Alterations in 5-HT(1A) receptors and adenylyl cyclase response by trazodone in regions of rat brain

The in vivo effect of trazodone on the density of [(3)H]5-HT binding sites and 5-HT(1A) receptors and adenylyl cyclase (AC) response was studied in regions of rat brain. The chronic administration of trazodone (10 mg/Kg body wt, 40 days) resulted in a significant downregulation of [(3)H]5-HT binding sites and 5-HT(1A) receptors in cortex and hippocampus. Trazodone significantly (p < 0.0001) decreased the density of [(3)H]5-HT binding sites in cortex (42.6 +/- 3.6 fmol/mg protein, 65%) and hippocampus (12.6 +/- 1.6 fmol/mg protein, 87%) when compared to control values of 121.9 +/- 5.4 and 99.3 +/- 7.5 fmol/mg protein in these regions, respectively. Similarly there was a significant (p < 0.0001) decrease in the density of 5-HT(1A) receptors in both cortex (7.2 +/- 0.5 fmol/mg protein, 70%) and hippocampus (6.3 +/- 1.2 fmol/mg protein, 79%) when compared to control values of 24.2 +/- 2.1 and 30.6 +/- 3.7 fmol/mg protein, in these regions respectively. However, the affinity of [(3)H]5-HT to 5-HT binding sites (1.83 +/- 0.26 nM, p < 0.0001) and [(3)H]8-OH-DPAT to 5-HT(1A) receptors (0.60 +/- 0.06 nM, p < 0.05) was significantly decreased only in cortex when compared to the control K(d) values of 0.88 +/- 0.04 nM and 0.47 +/- 0.02 nM in these regions, respectively.The basal AC activity did not alter in treated rats, where as, the inhibition of forskolin-stimulated AC activity by 5-HT (10 microM) was significantly (p < 0.0001) decreased both in cortex (43%) and hippocampus (40%) when compared to control levels. In conclusion, chronic treatment with trazodone results in downregulation of 5-HT(1A) receptors in cortex and hippocampus along with concomitant increased AC response, suggesting the involvement of 5-HT(1A) receptor-mediated AC response in the mechanism of action of trazodone.     

The Pro33 isoform of integrin beta3 enhances outside-in signaling in human platelets by regulating the activation of serine/threonine phosphatases

Integrin beta(3) is polymorphic at residue 33 (Leu(33) or Pro(33)), and the Pro(33)-positive platelets display enhanced aggregation, P-selectin secretion, and shorter bleeding times. Because outside-in signaling is critical for platelet function, we hypothesized that the Pro(33) variant provides a more efficient signaling than the Leu(33) isoform. When compared with Pro(33)-negative platelets, Pro(33)-positive platelets demonstrated significantly greater serine/threonine phosphorylation of extracellular signal-regulated kinase (ERK2) and myosin light chain (MLC) but not cytoplasmic phospholipase A2 upon thrombin-induced aggregation. Tyrosine phosphorylation of integrin beta(3) and the adaptor protein Shc was no different in the fibrinogen-engaged platelets from both genotypes. The addition of Integrilin (alpha(IIb)beta(3)-fibrinogen blocker) or okadaic acid (serine/threonine phosphatase inhibitor) dramatically enhanced ERK2 and MLC phosphorylation in the Pro(33)-negative platelets when compared with Pro(33)-positive platelets, suggesting that integrin engagement during platelet aggregation activates serine/threonine phosphatases. The phosphatase activity of myosin phosphatase (MP) that dephosphorylates MLC is inactivated by phosphorylation of the myosin binding subunit of MP at Thr(696), and aggregating Pro(33)-positive platelets exhibited an increased Thr(696) phosphorylation of MP. These studies highlight a role for the dephosphorylation events via the serine/threonine phosphatases during the integrin outside-in signaling mechanism, and the Leu(33) --> Pro polymorphism regulates this process. Furthermore, these findings support a mechanism whereby the reported enhanced alpha granule secretion in the Pro(33)-positive platelets could be mediated by an increased phosphorylation of MLC, which in turn is caused by an increased phosphorylation and subsequent inactivation of myosin phosphatase.     

Two new bacterial DNA primase inhibitors from the plant Polygonum cuspidatum

The 70% aqueous methanolic extract of the Peruvian plant Polygonum cuspidatum sp. was found to contain two novel phenolic saccharides 1 and 2, which were identified as inhibitors of the bacterial DNA primase enzyme. Structures of these two compounds were established based on high resolution NMR studies. Compound 1 and 2 inhibited the primase enzyme with an IC(50) of 4 and 5 microM, respectively.     

Optimization of an immunostaining protocol for the rapid intraoperative evaluation of melanoma sentinel lymph node imprint smears with the 'MCW melanoma cocktail'

BACKGROUND: In the management of cutaneous melanoma, it is desirable to complete the regional lymphadenectomy during the initial surgical procedure for wide excision of biopsy site and sentinel lymph node (SLN) biopsy. In this study, we optimized and evaluated a rapid 17 minutes immunostaining protocol. The discriminatory immunostaining pattern associated with the 'MCW Melanoma Cocktail' (mixture of Melan- A, MART- 1, and tyrosinase) facilitated the feasibility of intraoperative evaluation of imprint smears of SLNs for melanoma metastases. METHODS: Imprint smears of 51 lymph nodes from 25 cases (48 SLNs and 3 non-SLNs, 1 to 4 SLNs/case) of cutaneous melanoma were evaluated. RESULTS: Sixteen percent, 8/51 lymph nodes (28%, 7/25 cases) were positive for melanoma metastases in immunostained permanent sections with the 'MCW melanoma cocktail'. All of these melanoma metastases, except 1 SLN from 1 case, were also detected in rapidly immunostained wet-fixed and air-dried smears (rehydrated in saline and postfixed in alcoholic formalin). The cytomorphology was superior in air-dried smears, which were rehydrated in saline and postfixed in alcoholic formalin. Wet-fixed smears frequently showed air-drying artifacts, which lead to the focal loss of immunostaining. None of the 5 SLNs from 5 cases exhibiting capsular nevi showed a false positive result with immunostained imprint smears. CONCLUSIONS: Melanoma metastases can be detected intraoperatively in both air-dried smears and wet-fixed smears immunostained with the MCW Melanoma cocktail. Air-dried smears rehydrated in saline and postfixed in alcoholic formalin provide superior results and many practical benefits.