Enzyme inhibition activities of the extracts from rhizomes of Gloriosa superba Linn (Colchicaceae)

An alcoholic extract obtained from the rhizomes of Gloriosa superba Linn (Colchicaceae) was screened for enzyme inhibition activities. The crude extract and its subsequent fractions including chloroform, ethyl acetate, n-butanol and aqueous were screened against lipoxygenase, actylcholinesterase, butyrylcholinesterase and urease. An outstanding inhibition on lipoxygenase was observed. The highest enzyme inhibition potency was expressed by the chloroform fraction (90%) among the tested fractions on lipoxygenase. Overall 67-90% inhibition was found for lipoxygenase, 46-69% for acetylcholinesterase and 10-33% for butyrylcholinesterase, while urease was not inhibited.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.     

Tyrosine-specific MAPK phosphatases and the control of ERK signaling in PC12 cells

Background

Spatio-temporal control of extracellular signal-regulated kinase (ERK) activity, a critical determinant of the cell's response to growth factors, requires timely dephosphorylation of its regulatory tyrosine and/or threonine residue by MAPK phosphatases. We studied the physiological role of kinase interaction motif (KIM)-containing protein tyrosine phosphatases (PTPs) in the control of EGF- and NGF-induced ERK activity in neuroendocrine PC12 cells.

Results

We found a single KIM-containing PTP to be endogenously expressed in rat PC12 cells: the transmembrane PTPRR isoform termed PCPTP1. Protein knock-down of PCPTP1, or fourfold overexpression of its mouse orthologue, PTPBR7, left EGF- and NGF-induced ERK1/2 activity in PC12 cells unaltered. Ectopic expression of cytosolic PTPRR isoforms, however, resulted in reduced EGF-induced ERK1/2 activity, an effect that was dependent on the phosphatase activity and the KIM-domain of these PTPs.

Conclusion

The finding that robust changes in tyrosine-specific MAPK phosphatase expression levels have minor effects on temporal ERK1/2 activity control in PC12 cells suggests that dual-specificity MAPK phosphatases may act as major regulators of growth factor-induced ERK1/2 signaling in these cells.     

Ubiquitin crosstalk connecting cellular processes

Cullin 4 (Cul4), a member of the evolutionally conserved cullin protein family, serves as a scaffold to assemble multisubunit ubiquitin E3 ligase complexes. Cul4 interacts with the Ring finger-containing protein ROC1 through its C-terminal cullin domain and with substrate recruiting subunit(s) through its N-terminus. Previous studies have demonstrated that Cul4 E3 ligase ubiquitylates key regulators in cell cycle control and mediates their degradation through the proteasomal pathway, thus contributing to genome stability. Recent studies from several groups have revealed that Cul4 E3 ligase can target histones for ubiquitylation, and importantly, ubiquitylation of histones may facilitate the cellular response to DNA damage. Therefore, histone ubiquitylation by Cul4 E3 ligase constitutes a novel mechanism through which Cul4 regulates chromatin function and maintains genomic integrity. We outline these studies and suggest that histone ubiquitylation might play important roles in Cul4-regualted chromatin function including the cellular response to DNA damage and heterochromatin gene silencing.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Comparison of linkage disequilibrium and haplotype diversity on macro- and microchromosomes in chicken

Background

Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.

Results

The study presents association between TLR gene mutations and increased susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further in silico analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.

Conclusion

The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^th amino acid position in LRR motif (TLR1–LRR10) and 4^th residue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.     

A laser-based method for measuring thermal nociception of cattle

We describe a method for measuring nociception in cattle using a CO(2) laser aimed at the caudal aspect of the metatarsi. In Experiment 1, infrared thermography showed that calves responded by lifting their legs when skin temperatures reached 45-55 degrees C. In Experiment 2a, the validity of the method was tested by comparing the response latencies of 14 calves to two power settings (2.25 W vs. 4.5 W) with each setting being applied six times. We found that both leg-lift latencies and tail-flick latencies were lower at the higher power setting, and the calves were more likely to respond by kicking than by simply moving the leg. The standard deviations between and within calves were smaller at the higher power setting, and the large within-calf variation means that at least three tests were required to obtain reliable measures that could differentiate between calves. In Experiment 2b, application of the laser at a range of power settings (2.0, 3.0, 4.0, 4.5, 5.0 and 5.5 W) on 16 calves showed that response latencies decreased as power increased up to 4.5 W, after which no further change occurred. In Experiment 3, the repeatability of the method was evaluated on nine measures with the high power setting (4.5 W). The coefficient of variation associated with repetition of the measures was 36%. In general, we found little change in response latencies with repeated use of the laser, except that responses on the second test tended to be shorter. Experiment 4 showed that ambient temperatures between 16 degrees C and 27 degrees C did not affect response latencies, but these were longer at temperatures of 7 degrees C. We suggest that the method is a useful way of measuring cattle's sensitivity to nociception as the animals need not be restrained and the distance to the animal need not be closely controlled. However, to obtain accurate, valid and reliable measures it is necessary to use a high power setting (4.5 W) and take at least three consecutive measures of the response latency.