Berberine, a strong polyriboadenylic acid binding plant alkaloid: spectroscopic, viscometric, and thermodynamic study

The interaction of berberine with single stranded poly(rA) structure was investigated using a combination of spectrophotometric, spectrofluorimetric, circular dichroic, viscometric, and thermodynamic studies. The interaction process was characterized by typical hypochromic and bathochromic effects in the absorption spectrum of berberine, enhancement of fluorescence intensity of berberine, increase of viscosity, and perturbation of circular dichroic spectrum of single stranded poly(rA). Scatchard plot obtained from spectrophotometric analysis showed that berberine bound strongly to single stranded poly(rA) in a non-cooperative manner. In contrast, berberine does not show any significant effect (i) in its absorbance and fluorescence spectra on binding to double stranded poly(rA), (ii) alter the circular dichroic spectrum of double stranded poly(rA), or (iii) increase of viscosity of double stranded poly(rA) indicating that it does not bind at all to double stranded poly(rA) structure. Thermodynamic parameters indicated that the binding of the alkaloid to single stranded poly(rA) is an endothermic process and entropy driven. All these findings, taken together clearly support that berberine binds strongly to single stranded poly(rA) structure by a mechanism of partial intercalation leading to its use in gene regulation in eukaryotic cells.     

Mitochondrial DNA ligases of Trypanosoma brucei

The mitochondrial DNA of Trypanosoma brucei, termed kinetoplast DNA or kDNA, consists of thousands of minicircles and a small number of maxicircles catenated into a single network organized as a nucleoprotein disk at the base of the flagellum. Minicircles are replicated free of the network but still contain nicks and gaps after rejoining to the network. Covalent closure of remaining discontinuities in newly replicated minicircles after their rejoining to the network is delayed until all minicircles have been replicated. The DNA ligase involved in this terminal step in minicircle replication has not been identified. A search of kinetoplastid genome databases has identified two putative DNA ligase genes in tandem. These genes (LIG k alpha and LIG k beta) are highly diverged from mitochondrial and nuclear DNA ligase genes of higher eukaryotes. Expression of epitope-tagged versions of these genes shows that both LIG k alpha and LIG k beta are mitochondrial DNA ligases. Epitope-tagged LIG k alpha localizes throughout the kDNA, whereas LIG k beta shows an antipodal localization close to, but not overlapping, that of topoisomerase II, suggesting that these proteins may be contained in distinct structures or protein complexes. Knockdown of the LIG k alpha mRNA by RNA interference led to a cessation of the release of minicircles from the network and resulted in a reduction in size of the kDNA networks and rapid loss of the kDNA from the cell. Closely related pairs of mitochondrial DNA ligase genes were also identified in Leishmania major and Crithidia fasciculata.     

Level of alpha amylase activity in human saliva as a non-invasive biochemical marker of sleep deprivation

To investigate the usefulness of the enzyme salivary alpha amylase as a biochemical marker of sleep deprivation in human subjects. Total 168 healthy school-going adolescents studying in 9th grade were selected randomly from morning shift (n = 84) and dayshift (n = 84) schools. The study was undertaken longitudinally for a period of 2 years. Study encompassed administration of questionnaire and collection of saliva samples from the participants. Activity of salivary alpha amylase (sAA) activity was estimated spectrophotometrically and statistical analysis was performed to determine the association between sAA activity and sleep duration. Excessive daytime sleepiness among students was also studied in association with sAA activity. sAA activity of students was found to have a negative correlation with the duration of sleep and a positive correlation with their level of sleepiness. Morning shift students were found to have significantly less sleep and correspondingly higher sAA activity as compared to dayshift students. A significant increase in the sAA activity was noticed in the second year as the students progressed from 9th to 10th grade. Higher amylase activity was also observed in sleep deprived students suffering from excessive daytime sleepiness irrespective of school timings. Salivary alpha amylase activity increases in saliva in response to sleep deprivation. School timings may modulate sleep duration of students. Present finding reveals that sAA could be an appropriate non-invasive biochemical marker for the objective assessment of sleep deprivation among individuals as well as at population level.

     

SOME PROPERTIES OF ISOLATED NEURONAL CELL FRACTIONS

Abstract— 1. Histochemical evidence was presented illustrative of the composition of neuronal and neuropil ('glial') fractions isolated according to a previously published procedure. The neuropil refers to all cortical tissue other than neuronal perikarya.
2. On the basis of cell counts and of DNA content, an average cell mass of 100-110 pg was calculated for cells in the neuronal fraction. Eight per cent of the total DNA was recovered in the neuronal fraction.
3. Both fractions synthesized ATP in vitro. Concentrations after 60 min incubation with glucose were: neuropil, 7–36 μmoles/mg protein; neuronal, 12–31 μmoles/mg protein.
4. Osmotic shock or homogenization resulted in changes in turbidity of the cell fractions which were interpreted as indicative of loss of cell structure. The free pool amino acids glutamate, glutamine, GABA, aspartate and alanine were retained in the precipitable material through several washes with isotonic solutions. Homogenization released 72 per cent of the neuronal and 68 per cent of the neuropil amino acids into the supernatant, but only 37 per cent and 19 per cent respectively of the protein.
5. By contrast with earlier reports, K⁺ accumulation has now been demonstrated in both neuronal and neuropil fractions. After incubation with glucose, K⁺ level were calculated as being 80 per cent of slice in the neuronal, and 65 per cent in the neuropil fraction. These results, and those of the osmotic shock experiments, were taken as indicative of the retention of some cell structure.
6. By comparison, cell fractions prepared by other procedures, using acetone-glycerol-water or tetraphenylboron for tissue disaggregation, produced preparations with limited metabolic capabilities; oxygen uptake, CO₂ and lactate production were all lowered substantially.     

Canonical correlation between groups of acarine,fungal and environmental variables in bulk grain ecosystems

Interrelations among acarine, fungal, and environmental components of bulk grain ecosystems were determined by canonical correlation analyses. Twenty-seven variables were measured monthly in samples collected from 2 identical grain bulks in a granary in Winnipeg during the years 1959–67. The relationships between 9 kinds of arthropods and 6 environ mental variables, and between the same arthropods and 12 kinds of actinomycetes and fungi were examined. The maximum canonical correlation between arthropods and environmental factors was 0.35, and between arthropods and microorganisms was 0.28; both are highly significant (p<0.001). In the first analysis correlations of the variables with the canonical variates revealed that correlations of the variables with the canonical variates revealed that sampling location, depth, and temperature are the primary environmental antecedents involved, and the criterion is primarily composed of mites Tarsonemus spp.,Tydeus interruptus and the psocid, Lepinotus reticulatus. In the second analysis the fungi Nigrospora sphaerica, Aspergillus spp., and Cochliobolus sativus are involved with the mites Cheyletus eruditus and Acarus siro. Generally, the results of these analyses complement the findings of factor and regression analyses of the same data reported earlier.     

DIVARICATA AND RADIALIS INTERACTING FACTOR (DRIF) also interacts with WOX and KNOX proteins associated with wood formation in Populus trichocarpa

DIVARICATA AND RADIALIS INTERACTING FACTOR (DRIF) from snapdragon (Antirrhinum majus) is a MYB/SANT protein that interacts with related MYB/SANT proteins, RADIALIS and DIVARICATA, through its N‐terminal MYB/SANT domain. In addition to the MYB/SANT domain, DRIF contains a C‐terminal domain of unknown function (DUF3755). Here we describe novel protein–protein interactions involving a poplar (Populus trichocarpa) homolog of DRIF, PtrDRIF1. In addition to interacting with poplar homologs of RADIALIS (PtrRAD1) and DIVARICATA (PtrDIV4), PtrDRIF1 interacted with members of other families within the homeodomain‐like superfamily, including PtrWOX13c, a WUSCHEL‐RELATED HOMEOBOX protein, and PtrKNAT7, a KNOTTED1‐LIKE HOMEOBOX protein. PtrRAD1 and PtrDIV4 interacted with the MYB/SANT‐containing N‐terminal portion of PtrDRIF1, whereas DUF3755 was both necessary and sufficient for interactions with PtrWOX13c and PtrKNAT7. Of the two MYB/SANT domains present in PtrDIV4, only the N‐terminal MYB/SANT domain interacted with PtrDRIF1. GFP‐PtrDRIF1 expressed alone or with PtrRAD1 localized to the cytoplasm, whereas co‐expression of GFP‐PtrDRIF1 with PtrDIV4, PtrWOX13c or PtrKNAT7 resulted in nuclear localization of GFP‐PtrDRIF1. Modified yeast two‐hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments using PtrDRIF1 as a bridge protein revealed that PtrDRIF1 simultaneously interacted with PtrRAD1 and PtrWOX13c, but could not form a heterotrimeric complex when PtrDIV4 was substituted for PtrRAD1. Moreover, a Y2H competition assay indicated that PtrKNAT7 inhibits the interaction between PtrRAD1 and PtrDRIF1. The discovery of an additional protein–protein interaction domain in DRIF proteins, DUF3755, and its ability to form heterodimers and heterotrimers involving MYB/SANT and wood‐associated homeodomain proteins, implicates DRIF proteins as mediators of a broader array of processes than previously reported.     

A gene fusion at a homeobox locus: alterations in leaf shape and implications for morphological evolution.

Compound leaves are seen in many angiosperm genera and are thought to be either fundamentally different from simple leaves or elaborations of simple leaves. The knotted1-like homeobox (knox) genes are known to regulate plant development. When overexpressed in homologous or heterologous species, this family of genes can cause changes in leaf morphology, including excessive leaf compounding in tomato. We describe here an instance of a spontaneously arisen fusion between a gene encoding a metabolic enzyme and a homeodomain protein. We show that the fusion results in overexpression of the homeodomain protein and a change in morphology that approximates the changes caused by overexpression of the same gene under the control of the cauliflower mosaic virus 35S promoter in transgenic plants. Exon-shuffling events can account for the modularity of proteins. If the shuffled exons are associated with altered promoters, changes in gene expression patterns can result. Our results show that gene fusions of this nature can cause changes in expression patterns that lead to altered morphology. We suggest that such phenomena may have played a role in the evolution of form.     

Assignments, secondary structure and dynamics of the inhibitor-free catalytic fragment of human fibroblast collagenase

Fibroblast collagenase (MMP-1), a 169-residue protein with amolecular mass of 18.7 kDa, is a matrix metalloproteinase which has beenassociated with pathologies such as arthritis and cancer. The assignments ofthe ¹H, ¹⁵N, ¹³CO and¹³C resonances, determination of the secondary structure andanalysis of ¹⁵N relaxation data of the inhibitor-freecatalytic fragment of recombinant human fibroblast collagenase (MMP-1) arepresented. It is shown that MMP-1 is composed of a -sheet consistingof five -strands in a mixed parallel and antiparallel arrangement(residues 13–19, 48–53, 59–65, 82–85 and94–99) and three -helices (residues 27–43, 112–124and 150–160). This is nearly identical to the secondary structuredetermined from the refined X-ray crystal structures of inhibited MMP-1. Themajor difference observed between the NMR solution structure ofinhibitor-free MMP-1 and the X-ray structures of inhibited MMP-1 is thedynamics of the active site. The 2D ¹⁵N-¹H HSQCspectra, the lack of information in the ¹⁵N-edited NOESYspectra, and the generalized order parameters (S²) determinedfrom ¹⁵N T₁, T₂ and NOE datasuggest a slow conformational exchange for residues comprising the activesite (helix B, zinc ligated histidines and the nearby loop region) and ahigh mobility for residues Pro¹³⁸-Gly¹⁴⁴ in thevicinity of the active site for inhibitor-free collagenase. In contrast tothe X-ray structures, only the slow conformational exchange is lost in thepresence of an inhibitor.     

Comparative aerobiology,allergenicity and biochemistry of three palm pollen grains in Calcutta,India

A Burkard volumetric trap was used at Salt Lake City, Calcutta, to record the occurrence and frequency of three common palm pollen, namely,Areca catechu, Borassus flabellifer andPhoenix sylvestris for two consecutive years (July 1988–June 1990). The meteorological factors responsible for the frequency of relevant airborne pollen grains were analysed. The allergenic potential of these pollen types was investigated by skin-prick tests on adult respiratory allergic patients. These were also chemically analysed in terms of total carbohydrate, lipid and soluble protein. Total soluble protein of the above types was used in 11% SDS-polyacrylamide gel electrophoresis to study the range of molecular components.     

Effect of cryoprotectants on release of various enzymes from buck spermatozoa during freezing

Semen samples from 12 bucks Were extended with 10 different extenders containing glycerol, DMSO, glycerol + DMSO, and glycerol + lactose in varying concentrations as cryoprotective agents. The activities of acrosin, hyaluronidase, alkaline phosphatase (AKP), aspartate aminotransferase (AST), alanine amino transferase (ALT) and lactic dehydrogenase (LDH) were assayed in equilibrated (Prefreeze) and frozen thawed (Postfreeze) semen samples. Significantly (P < 0.01) higher intracellular activity of acrosin was recorded in semen samples extended with lactose than with the other extenders, with the maximum being with Tris yolk glycerol lactose (TYGL(180)). Effects of extenders on acrosin activity were significant (P < 0.01) at both of the pre-and postfreeze stages. However, extracellular activities of hyaluronidase, alkaline phosphatase, transaminases (AST and ALT), and lactic dehydrogenase were significantly higher in extenders containing DMSO than lactose. Leakage of these enzymes was found to increase from the prefreeze to the post freeze stage.