Chromosome Spreading Induced by Vegetable Oils

Seeds soaked in the oil extracted from castor beans (Ricinus communis) for 2 hr were germinated in petri dishes on moist filter papers. Root tips were fixed in acetic alcohol (1:3) at 10-14°C, for 24 hr, washed successively with 70% alcohol (15 min) and water (10 min), hydrolysed in 1 N HCl at 60°C for 15 min and stained in leucobasic fuchsin for 30 min. The stained tip was squashed under a cover glass in a drop of acetocarmine and sealed with paraffin wax. The slides were made permanent by separating the cover glass in a mixture of acetic acid and n-butyl alcohol (1:1), passing through 2 changes of n-butyl alcohol and mounting in balsam. Such a method leads to contraction and spreading of chromosomes, without affecting either the clarity of the constriction regions or the anaphase separation of chromosomes.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

Are the proteinase inhibitory activities in lenticular tissues real?

The possibility of proteinase inhibitory activities in lenses measured with synthetic substrates being spurious, due to the effective competition of lens proteins as substrates for the target enzymes, was investigated. Goat, sheep and human cataractous lens proteins were found to be poor substrates for trypsin, elastase and papain compared to casein or bovine serum albumin. Further, the inhibition of elastase catalyzed hydrolysis of succinyl trialanyl p-nitroanilide by casein (500 μg, 53%) and albumin (500 μg, 49%) and of trypsin-catalyzed hydrolysis of benzoyl argininep-nitroanilide by albumin (1 mg, 24%) were significant only at high protein concentrations. These data indicated that the relatively high antielastase and antitryptic activities observed in human cataractous lenses were real. On the other hand, coincident lens protein hydrolysis elevating the true antitryptic and antielastase activities in goat and sheep lenses (that have low activities) could not be ruled out The lesser papain inhibitory activities observed in lenses when albumin was used as substrate compared to activities with benzoyl arginine p-nitroanilide as substrate, appeared to be partly due to lens protein hydrolysis masking the actual inhibition in the former method. Preincubation of goat, sheep and human lens extracts with trypsin for 1 h resulted in complete loss of antitryptic and antielastase activity except in the case of human lens antielastase activity which underwent 50% loss. Papain inhibitory activity was fully stable. Similar papain treatment caused loss of 80–100% of antielastase activity and 45–55% loss of antitryptic activity.     

Nucleotide sequence analysis of two virulence-associated genes in Listeria monocytogenes serotype 1/2b and comparison with the same genes in other serotypes important in human disease

The nucleotide sequences of the hly and plc A genes (encoding listeriolysin, a thiol-activated cytolysin and a non-specific phospholipase C, respectively) were determined for two strains of Listeria monocytogenes serotype 1/2b. The deduced amino acid sequences for listeriolysin were identical for the two strains and identical to that of listeriolysin from serotype 4b. The two serotype 1/2b strains differed in one amino acid (histidine vs /asparagine) in the deduced amino acid sequence for phosopholipase C.     

Flexibility of NS5 Methyltransferase-Polymerase Linker Region Is Essential for Dengue Virus Replication

We examined the function of the conserved Val/Ile residue within the dengue virus NS5 interdomain linker (residues 263 to 272) by site-directed mutagenesis. Gly substitution or Gly/Pro insertion after the conserved residue increased the linker flexibility and created slightly attenuated viruses. In contrast, Pro substitution abolished virus replication by imposing rigidity in the linker and restricting NS5''s conformational plasticity. Our biochemical and reverse genetics experiments demonstrate that NS5 utilizes conformational regulation to achieve optimum viral replication.     

MicroRNA 320a Functions as a Novel Endogenous Modulator of Aquaporins 1 and 4 as Well as a Potential Therapeutic Target in Cerebral Ischemia

Aquaporins facilitate efficient diffusion of water across cellular membranes, and water homeostasis is critically important in conditions such as cerebral edema. Changes in aquaporin 1 and 4 expression in the brain are associated with cerebral edema, and the lack of water channel modulators is often highlighted. Here we present evidence of an endogenous modulator of aquaporin 1 and 4. We identify miR-320a as a potential modulator of aquaporin 1 and 4 and explore the possibility of using miR-320a to alter the expression of aquaporin 1 and 4 in normal and ischemic conditions. We show that precursor miR-320a can function as an inhibitor, whereas anti-miR-320a can act as an activator of aquaporin 1 and 4 expressions. We have also shown that anti-miR-320a could bring about a reduction of infarct volume in cerebral ischemia with a concomitant increase in aquaporins 1 and 4 mRNA and protein expression.