Innervation of the dorsal body cells of Helix aspersa: Immunocytochemical evidence for the presence of FMRF amidelike substances in nerves and synapses

In Helix aspersa, single axons or small nerves make synapse-like structures (SLS) with dorsal body (DB) cells. The present immunocytochemical study performed on the DB-containing connective tissue located around the cerebral commissure reveals the presence of FMRFamide-like substances in many nerves of this DB area. The immunogold method coupled with electron microscopy show that among the four observed axon types, only one, containing 95-110 nm granules, is positive for FMRFamide and that most of the SLS between axons and DB cells contain the same immunoreactive granule types. The results support the previously suggested role of FMRFamide-like peptides in the nervous control of DB cells.     

Method of isolating FMRF-amide-like substances in the snail, using affinity chromatography coupled with HPLC

An extraction procedure of FMRFa-like substances from brain of Helix aspersa was developed. It consists of using affinity chromatography coupled with reverse phase HPLC. Three synthetic peptides (FMRFa, pQDPFLRFa, Met-enkephalin) were used to evaluate the specificity and yield of the affinity column. Its efficiency was tested by use of snail brain extracts. The results showed that this method is efficient and reproducible.     

EXIT OF DYE FROM LIVING CELLS OF NITELLA AT DIFFERENT pH VALUES

Experiments on the exit of brilliant cresyl blue from the living cells of Nitella, in solutions of varying external pH values containing no dye, confirm the theory that the relation of the dye in the sap to that in the external solution depends on the fact that the dye exists in two forms, one of which (DB) can pass through the protoplasm while the other (DS) passes only slightly. DB increases (by transformation of DS to DB) with an increase in the pH value, and is soluble in substances like chloroform and benzene. DS increases with decrease in pH value and is insoluble (or nearly so) in chloroform and benzene. The rate of exit of the dye increases as the external pH value decreases. This may be explained on the ground that DB as it comes out of the cell is partly changed to DS, the amount transformed increasing as the pH value decreases. The rate of exit of the dye is increased when the pH value of the sap is increased by penetration of NH₃.     

Identification and characterization of a major early cytomegalovirus DNA-binding protein

We characterized a DNA-binding protein with an approximate molecular weight of 129,000 (DB129) which is present in the nuclei of cytomegalovirus- (strain Colburn) infected cells, but not in virus particles. Results of two types of experiments demonstrated that DB129 is a member of the early class of herpesviral proteins. First, time course pulse-labeling experiments showed that its synthesis begins after that of the immediate-early protein IE94, but prior to the appearance of late viral proteins, and was reduced at late times. Second, in the presence of inhibitors of viral DNA replication, DB129 continued to be made and accumulated to elevated levels. A second set of experiments showed that DB129 bound to single-stranded DNA in vitro and was eluted by a NaCl gradient in two peaks, one at about 0.2 M and the second at about 0.6 M. A similar pattern of release was observed when infected-cell nuclei were serially extracted with increasing NaCl concentrations. In addition, treatment of nuclei with DNase I selectively released DB129, along with a small but significant fraction of another DNA-binding protein, DB51. These results suggest that DB129 is associated with DNA in vivo and that it interacts directly with single-stranded DNA. It was also shown that cells infected with human cytomegalovirus (strain Towne) contain a slightly larger counterpart to DB129, which was designated DB140. Similarities between these proteins and the major DNA-binding protein of herpes simplex virus are discussed.     

Trypanosoma cruzi: Activity of heterocyclic cationic molecules in vitro

Chagas disease remains a serious public health problem in several Latin American countries. New chemotherapy is urgently needed since current drugs are limited in efficacy and exhibit undesirable side effects. Aromatic diamidines and analogs are well known anti-parasitic agents and in this study, we have evaluated the in vitro trypanocidal effect of several different heterocyclic cationic compounds, including diamidines (DB1195, DB1196 and DB1345), a monoamidine (DB824), an arylimidamide (DB613A) and a guanylhydrazone (DB1080) against amastigotes and bloodstream trypomastigotes of Trypanosoma cruzi, the etiological agent of Chagas disease. Our present findings showed that all compounds exerted, at low-micromolar doses, a trypanocidal effect upon both intracellular parasites and bloodstream trypomastigotes of T. cruzi. The activity of DB1195, DB1345, DB824 and DB1080 against bloodstream forms was reduced when these compounds were assayed in the presence of mouse blood possibly due to their association with plasma constituents and/or due to metabolic instability of the compounds. However, trypanocidal effects of DB613A and DB1196 were not affected by plasma constituents, suggesting their potential application in the prophylaxis of banked blood. In addition, potency and selectivity of DB613A, towards intracellular parasites, corroborate previous results that demonstrated the highly promising activity of arylimidamides against this parasite, which justify further studies in experimental models of T. cruzi infection.     

Purification of RNA-core induced streptolysin S, and isolation and haemolytic characteristics of the carrier-free toxin

RNA-core (RNAase-resistant fraction of yeast RNA) induced streptolysin S (SLS) was purified (40% recovery) to apparent electrophoretic homogeneity by hydroxylapatite chromatography followed by gel filtration on Sephadex G-100 in the presence of 6 M-guanidine. HCl. The specific activity of the purified toxin was 3 X 10(6) haemolytic units (mg protein)-1. The Mr of the toxin was below 4000 on the basis of SDS-PAGE and 20 000 by gel filtration in guanidine. HCl. High-voltage isoelectric focusing of the purified toxin allowed the isolation of the carrier-free SLS peptide for the first time. This peptide was basic (pI 9.2) as compared to native SLS (pI 3.6). The native toxin and the peptide had similar haemolytic properties except for the high lability of the peptide, which was stabilized by RNA-core. The Mr of the denatured peptide was about 1800, as estimated by gel filtration.     

Endophyte-assisted promotion of biomass production and metal-uptake of energy crop sweet sorghum by plant-growth-promoting endophyte Bacillus sp. SLS18

The effects of Bacillus sp. SLS18, a plant-growth-promoting endophyte, on the biomass production and Mn/Cd uptake of sweet sorghum (Sorghum bicolor L.), Phytolacca acinosa Roxb., and Solanum nigrum L. were investigated. SLS18 displayed multiple heavy metals and antibiotics resistances. The strain also exhibited the capacity of producing indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylic acid deaminase. In pot experiments, SLS18 could not only infect plants effectively but also significantly increase the biomass of the three tested plants in the presence of Mn/Cd. The promoting effect order of SLS18 on the biomass of the tested plants was sweet sorghum > P. acinosa > S. nigrum L. In the presence of Mn (2,000 mg kg(-1)) and Cd (50 mg kg(-1)) in vermiculite, the total Mn/Cd uptakes in the aerial parts of sweet sorghum, P. acinosa, and S. nigrum L. were increased by 65.2%/40.0%, 55.2%/31.1%, and 18.6%/25.6%, respectively, compared to the uninoculated controls. This demonstrates that the symbiont of SLS18 and sweet sorghum has the potential of improving sweet sorghum biomass production and its total metal uptake on heavy metal-polluted marginal land. It offers the potential that heavy metal-polluted marginal land could be utilized in planting sweet sorghum as biofuel feedstock for ethanol production, which not only gives a promising phytoremediation strategy but also eases the competition for limited fertile farmland between energy crops and food crops.     

Induction of alkaline phosphatase by cyclic AMP or its dibutyryl derivative in a hybrid line between mouse and Chinese hamster in culture

Cyclic AMP (c-AMP) and its derivative, dibutyryl-cyclic AMP (DB c-AMP) induced the activity of alkaline phosphatase (ALP) in a hybrid line. Theophylline was also effective and greatly potentiated the action of c-AMP when given along with the nucleotide. This effect was attributable neither to a direct activation of the enzyme nor to the formation of activators in the cells grown in DB c-AMP. In the presence of actinomycin S₃ or cycloheximide, the induction by DB c-AMP did not occur or was reduced very much. It is therefore suggested that c-AMP and DB c-AMP induce alkaline phosphatase activity through a new synthesis of both RNA and protein in the cells.     

Sjögren-Larsson syndrome: accumulation of free fatty alcohols in cultured fibroblasts and plasma

Sj?gren-Larsson syndrome (SLS) is an inherited disorder associated with deficient oxidation of long-chain aliphatic alcohols. Previous studies have reported modest elevations in total (free + esterified) fatty alcohols in SLS, but free fatty alcohols have not been selectively measured, in part because of their low concentrations in most tissues and the presence of trace fatty alcohol contaminants in some solvents used for their analysis. We adapted methods to measure free fatty alcohols in cultured cells and plasma that minimize exogenous alcohol contamination. Fatty alcohols were analyzed as acetate derivatives, using capillary column gas chromatography. By this method, cultured skin fibroblasts from SLS patients were found to have 7- and 8-fold elevations in the mean content of hexadecanol (16:0-OH) and octadecanol (18:0-OH), respectively. The mean plasma 16:0-OH and 18:0-OH concentrations in SLS patients (n = 11) were 9- and 22-fold higher than in normal controls, respectively. In SLS fibroblasts, most of the fatty alcohol (59%) that accumulated was free rather than esterified alcohol, whereas free alcohol accounted for 23% of the total alcohol in normal cells. These results indicate that elevations in free fatty alcohols provide a sensitive marker for the enzymatic defect in SLS. The ability to measure free fatty alcohols in cultured cells and plasma should prove useful for investigations of normal fatty alcohol metabolism and the deranged metabolism in SLS.     

Nonspecific Precipitation of Serum Proteins by Sodium Lauryl Sulfate in Agar Diffusion and Immunoelectrophoresis

The anionic detergent sodium lauryl sulfate (SLS), in a final concentration of 0.1% and greater, reacted with whole serum in agar diffusion and immunoelectrophoresis to form artifactual precipitin lines. These lines occurred when either Ionagar or agarose was used as the supporting gel and were not affected by the presence of urea and 2-mercaptoethanol. Analytic chemical tests confirmed that the precipitating agent is SLS, and staining techniques showed that the detergent precipitates both protein and lipoprotein components of whole serum. Multiple artifactual precipitin lines occurred with a wide variety of animal sera, and a single line formed with human 7S immunoglobulin. Hence, in agar diffusion studies in which SLS is present in the test system, these artifactual lines may be easily misinterpreted as true antigen-antibody precipitin reactions.     

Specific secondary structures in the capsid-coding region of giardiavirus transcript are required for its translation in Giardia lamblia

Enhanced translation of giardiavirus (GLV)-luciferase chimeric mRNA in Giardia lamblia requires the presence of the initial 264 nucleotides of the viral capsid-coding region. A 13 nt downstream box (DB) sequence within this region, complementary to a 15 nt sequence near the 3' end of G. lamblia 16 S-like ribosomal RNA (rRNA), was found to be essential for the enhanced translation. However, DB is located 64-78 nt downstream of the initiation codon, whereas an exponential increase of translation efficiency depends on a further increment of the coding region from nucleotides 111 to 264. Thus, there could be additional structural requirements for translation enhancement in the region downstream from DB. Four major stem-loop structures, designated I to IV, were identified in the MFOLD-predicted secondary structure of the 264 nt capsid-coding region with an estimated minimum free energy (DeltaG degrees ) of -77.16 kcal x mol(-1). Our chemical probing analysis of the free 264 nt RNA molecule in solution supports the predicted presence of stem-loops I, II and III, but casts doubts on stem-loop IV. It suggests, instead, the presence of a stem-loop IVA at a nearby location in the molecule. Site-directed mutagenesis designed to disrupt stem-loop structures I, II, III or IVA resulted in drastic reduction of translation efficiency, which was restored by compensatory sequence changes to regenerate individual stem-loop structures. Mutations disrupting the originally designated stem-loop IV did not exert any detectable effect on translation. However, alterations of the sequence UCUCC between nucleotides 216 and 220 in the flexible loop region of the revised secondary structure led to a precipitous drop in translation. Another stem-loop predicted by MFOLD that consists of a major portion of the DB sequence was examined by chemical probing but found little experimental support. Changes of the DB sequence without affecting the postulated stem structure led to drastic losses of translation efficiency. Thus, a simple structural basis for the enhanced translation could be that stem-loops I, II, III and IVA and the UCUCC sequence may facilitate the interaction between DB and the anti-DB in 16 S-like rRNA in initiating translation of GLV mRNA in G. lamblia.     

The downstream box: an efficient and independent translation initiation signal in Escherichia coli.

The downstream box (DB) was originally described as a translational enhancer of several Escherichia coli and bacteriophage mRNAs located just downstream of the initiation codon. Here, we introduced nucleotide substitutions into the DB and Shine-Dalgarno (SD) region of the highly active bacteriophage T7 gene 10 ribosome binding site (RBS) to examine the possibility that the DB has an independent and functionally important role. Eradication of the SD sequence in the absence of a DB abolished the translational activity of RBS fragments that were fused to a dihydrofolate reductase reporter gene. In contrast, an optimized DB at various positions downstream of the initiation codon promoted highly efficient protein synthesis despite the lack of a SD region. The DB was not functional when shifted upstream of the initiation codon to the position of the SD sequence. Nucleotides 1469-1483 of 16S rRNA ('anti-downstream box') are complementary to the DB, and optimizing this complementarity strongly enhanced translation in the absence and presence of a SD region. We propose that the stimulatory interaction between the DB and the anti-DB places the start codon in close contact with the decoding region of 16S rRNA, thereby mediating independent and efficient initiation of translation.     

Comparison of hamstring and quadriceps femoris electromyographic activity between men and women during a single-limb squat on both a stable and labile surface

The purpose of this study was to determine if women are quadriceps dominant and men are hamstring dominant during the performance of a partial single-leg squat (SLS) on both a stable and labile ground surface against body weight resistance. Thirty healthy participants (15 men and 15 women) performed an SLS on both a stable surface and a 6.4-cm-thick vinyl pad. Surface electromyographic (EMG) recordings were obtained from the quadriceps femoris and hamstring muscles during the extension phase of the SLS. Statistical analysis revealed that women produced 14% more EMG activity (p = 0.04) in their quadriceps than the men during the SLS on a stable surface, whereas the men generated 18% more EMG activity (p = 0.04) in their hamstrings than the women during the SLS on a labile surface. Additionally, we found a statistically significant sex effect (p = 0.048) for the hamstring/quadriceps (H/Q) EMG ratio, which was 2.25 and 0.62, respectively, for men and women on the stable surface and 2.52 and 0.71, respectively, on the labile surface. We concluded that women are quadriceps dominant and men are hamstring dominant during the performance of SLS against body weight resistance on either a stable or labile surface condition. During an SLS, men showed an H/Q ratio approximately 3.5 times larger than their female counterparts, suggesting that men activate their hamstrings more effectively than women during an SLS. According to our data, the SLS may not be an ideal exercise for activating the hamstring muscles in women without additional neuromuscular training techniques, because women are quadriceps dominant during the SLS.     

Molecular determinants for DNA minor groove recognition: design of a bis-guanidinium derivative of ethidium that is highly selective for AT-rich DNA sequences

The phenanthridinium dye ethidium bromide is a prototypical DNA intercalating agent. For decades, this anti-trypanosomal agent has been known to intercalate into nucleic acids, with little preference for particular sequences. Only polydA-polydT tracts are relatively refractory to ethidium intercalation. In an effort to tune the sequence selectivity of known DNA binding agents, we report here the synthesis and detailed characterization of the mode of binding to DNA of a novel ethidium derivative possessing two guanidinium groups at positions 3 and 8. This compound, DB950, binds to DNA much more tightly than ethidium and exhibits distinct DNA-dependent absorption and fluorescence properties. The study of the mode of binding to DNA by means of circular and electric linear dichroism revealed that, unlike ethidium, DB950 forms minor groove complexes with AT sequences. Accurate quantification of binding affinities by surface plasmon resonance using A(n)T(n) hairpin oligomer indicated that the interaction of DB950 is over 10-50 times stronger than that of ethidium and comparable to that of the known minor groove binder furamidine. DB950 interacts weakly with GC sites by intercalation. DNase I footprinting experiments performed with different DNA fragments established that DB950 presents a pronounced selectivity for AT-rich sites, identical with that of furamidine. The replacement of the amino groups of ethidium with guanidinium groups has resulted in a marked gain of both affinity and sequence selectivity. DB950 provides protection against DNase I cleavage at AT-containing sites which frequently correspond to regions of enhanced cleavage in the presence of ethidium. Although DB950 maintains a planar phenanthridinium chromophore, the compound no longer intercalates at AT sites. The guanidinium groups of DB950, just like the amidinium group of furamidine (DB75), are the critical determinants for recognition of AT binding sites in DNA. The chemical modulation of the ethidium exocyclic amines is a profitable option to tune the nucleic acid recognition properties of phenylphenanthridinium dyes.     

Immunofluorescent localization, by use of anti-idiotypic antibody, of monoclonal anti-progesterone antibody in the mouse uterus before implantation

Mouse monoclonal anti-progesterone IgG1 antibody designated DB3 has an anti-fertility effect when injected into female mice shortly after mating. In BALB/c mice, pregnancy is blocked, probably as a result of progesterone withdrawal with inhibition of implantation. Rabbit polyclonal anti-idiotype raised against DB3 has been used in an indirect immunofluorescence method on frozen tissue sections to demonstrate the presence of DB3 on the surface of uterine luminal and glandular epithelia before implantation. DB3 was only detectable 30-60 h after a single parenteral injection (9 nmol antibody per mouse i.p. or i.v. at 32 h post coitum). Immunolocalization was both pregnancy-dependent and anti-progesterone antibody-specific, as it was not seen in pseudopregnant mice or mice treated with P3 (mouse myeloma IgG1 protein, using polyclonal anti-P3 anti-idiotype as a probe) or saline. The immunofluorescent reaction was completely blocked by addition of DB3 idiotype in vitro. The results indicate that anti-progesterone antibody binds to an antigen associated with luminal and glandular epithelia which may locally inhibit the uterine uptake of progesterone and disrupt the process of implantation.     

Dimeric bisbenzimidazoles inhibit the DNA methylation catalyzed by the murine Dnmt3a catalytic domain

When located in the DNA minor groove, dimeric bisbenzimidazoles DB(n) effectively inhibited in vitro the Dnmt3a catalytic domain (IC₅₀ 5–77 μM). The lowest IC₅₀ value was observed for compound DB(11) with an 11-unit methylene linker joining the bisbenzimidazole fragments. Increased time of incubation of DNA with DB(n) as well as the presence of AT-clusters in DNA enhances the inhibitory effect.     

Abnormal fatty alcohol metabolism in cultured keratinocytes from patients with Sjögren-Larsson syndrome

Sj?gren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder characterized by ichthyosis, mental retardation, spasticity, and deficient activity of fatty aldehyde dehydrogenase (FALDH). FALDH is an enzyme component of fatty alcohol:NAD oxidoreductase (FAO), which is necessary for fatty alcohol metabolism. To better understand the biochemical basis for the cutaneous symptoms in this disease, we investigated lipid metabolism in cultured keratinocytes from SLS patients. Enzyme activities of FALDH and FAO in SLS cells were <10% of normal. SLS keratinocytes accumulated 45-fold more fatty alcohol (hexadecanol, octadecanol, and octadecenol) than normal, whereas wax esters and 1-O-alkyl-2,3-diacylglycerols were increased by 5.6-fold and 7.5-fold, respectively. SLS keratinocytes showed a reduced incorporation of radioactive octadecanol into fatty acid (24% of normal) and triglyceride (13% of normal), but incorporation into wax esters and 1-O-alkyl-2,3-diacylglycerol was increased by 2.5-fold and 2.8-fold, respectively. Our results indicate that FALDH deficiency in SLS keratinocytes causes the accumulation and diversion of fatty alcohol into alternative biosynthetic pathways. The striking lipid abnormalities in cultured SLS keratinocytes are distinct from those seen in fibroblasts and may be related to the stratum corneum dysfunction and ichthyosis in SLS.     

Solution-Mediated Phase Transformation of Haloperidol Mesylate in the Presence of Sodium Lauryl Sulfate

Forming a salt is a common way to increase the solubility of a poorly soluble compound. However, the solubility enhancement gained by salt formation may be lost due to solution-mediated phase transformation (SMPT) during dissolution. The SMPT of a salt can occur due to a supersaturated solution near the dissolving surface caused by pH or other solution conditions. In addition to changes in pH, surfactants are also known to affect SMPT. In this study, SMPT of a highly soluble salt, haloperidol mesylate, at pH 7 in the presence of a commonly used surfactant, sodium lauryl sulfate (SLS), was investigated. Dissolution experiments were performed using a flow-through dissolution apparatus with solutions containing various concentrations of SLS. Compacts of haloperidol mesylate were observed during dissolution in the flow-through apparatus using a stereomicroscope. Raman microscopy was used to characterize solids. The dissolution of haloperidol mesylate was significantly influenced by the addition of sodium lauryl sulfate. In conditions where SMPT was expected, the addition of SLS at low concentrations (0.1–0.2 mM) reduced the dissolution of haloperidol mesylate. In solutions containing concentrations of SLS above the critical micelle concentration (CMC) (10–15 mM), the dissolution of haloperidol mesylate increased compared to below the CMC. The solids recovered from solubility experiments of haloperidol mesylate indicated that haloperidol free base precipitated at all concentrations of SLS. Above 5 mM of SLS, Raman microscopy suggested a new form, perhaps the estolate salt. The addition of surfactant in solids that undergo solution-mediated phase transformation can add complexity to the dissolution profiles and conversion.     

A Sequence Downstream of the Initiation Codon Is Essential for Cold Shock Induction of cspB of Escherichia coli

Cold shock induction of cspB has been shown to be primarily regulated at the mRNA level. Here, we demonstrate that the induction of cspB at low temperature also requires the translational cis-acting element called the downstream box (DB). Full induction of cspB at low temperature is achieved in the presence of both the Shine-Dalgarno sequence and DB. We propose that the DB sequence functions as a translational enhancer for the biosynthesis of CspB to bypass the inhibitory effect in translation caused by cold shock.