Characterization of the spontaneously forming hydrogels composed of water-soluble phospholipid polymers

Spontaneously forming hydrogels composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymers, poly(MPC-co-methacrylic acid) (PMA), and poly(MPC-co-n-butyl methacrylate) (PMB) were examined. The MPC copolymer hydrogel was observed to have a spontaneous gelation property. To determine the properties of the hydrogels and why the gelation takes place, we have studied the properties of the hydrogels by scanning electron microscopy, X-ray photoelectron spectroscopy (XPS), and differential scanning calorimetry (DSC). The morphologies of the hydrogels were spongelike with a homogeneous structure. By XPS analysis in terms of the molecular distributions in the hydrogels, it was observed that a stabilization time was required for the hydrogel to undergo chain rearrangement. DSC thermograms of the hydrogels were different from their components, PMA and PMB. For the hydrogel, a crystallization peak around -30 degrees C was observed. This result indicated that some ordered structures existed in the hydrogels. To determine the role of the MPC groups, aqueous solutions of poly(methacrylic acid) (PMAc) and PMB were mixed. The mixture of PMAc-PMB turned into a sol state, and the sol state remained for a week. When the mixture was cooled, a very weak hydrogel was prepared. This result suggested that the MPC groups were the dominant unit for spontaneously forming the hydrogels.     

Urinary homovanillic acid (HVA) and vanillymandelic acid (VMA) in workers exposed to manganese dust

The neurotoxicity of manganese (Mn) is well known, however, the neurochemical effect caused by this metal is less well investigated. In this study, urinary homovanillic acid (HVA) and vanillymandelic acid (VMA), two end products of catecholamine metabolism, were measured in 39 workers chronically exposed to Mn in a manganese smelting plant. The average duration of Mn exposure was 17.4 yr. Nineteen nonexposed workers were also studied. Concentrations of Mn in serum (MnS) and in urine (MnU) were measured by Zeeman graphite furnace atomic absorption spectrophotometry (ZAAS), and HVA and VMA determined by high performance liquid chromatography (HPLC). For Mn-exposed workers, the concentration of MnS was nearly 2.8 times (1.61 ± 0.16 mg/L vs 0.56 ± 0.16 mg/L) and MnU about 4.5 times higher (7.62 ± 0.17 mg/L vs 1.69 ± 0.16 mg/L) than the nonexposed. Although the geometric mean concentration of HVA in exposed workers was similar to that of the nonexposed (3.09 ± 1.39 mg/g ere. vs 2.99 ± 1.40 mg/g cre.), the VMA concentration was significantly higher (3.02 ± 1.43 mg/g cre. vs 2.49 ± 1.58 mg/g cre.,p = 0.033). Multiple regression analysis showed that although there were no correlations between any of these parameters with the duration of exposure to Mn, both HVA and VMA showed significant correlations with increase in MnS and MnU. These data provide evidence that exposure to Mn was associated with measurable increase in catecholamine metabolites. This finding is compatible with recent observations in laboratory animals that Mn interferes with neurochemical metabolism.     

The recovery of poly(3-hydroxybutyrate) by using dispersions of sodium hypochlorite solution and chloroform

Summary To take advantage of both differential digestion by hypochlorite and solvent extraction, we used dispersions of sodium hypochlorite solution and chloroform in the recovery of microbial PHB. The treatment with hypochlorite alone caused such severe degradation and the molecular weight decreased drastically with increasing hypochlorite concentration. However, using the dispersion, the degradation of PHB was markedly diminished owing to theshielding effect of chloroform. In this case, we could obtain PHB of above 97% purity with a number average molecular weight of 1,000,000 comparable to the original molecular weight of 1,200,000.     

Isolation and characterization of two cDNA clones that are rapidly induced during the wound response of Arabidopsis thaliana

Differential screening of a cDNA library of Arabidopsis thaliana constructed from the plant tissues harvested 1 h after wounding resulted in isolation of 2 wound-inducible cDNA clones. Kinetic analysis revealed that the corresponding genes are rapidly induced upon wounding. Expression of these clones reached the maximum level around 1–1.5 h after wounding and then were progressively reduced. The time by which expression returned to the control level was around 3 h after wounding. Partial sequence analysis revealed that the two clones are highly homologous to the S-adenosylmethionine synthetase and the glutathione-S-transferase gene, respectively.Abbreviations SAM S-adenosylmethionine - GST glutathione-S-transferase     

Analysis of spontaneous electrical activity in cerebellar Purkinje cells acutely isolated from postnatal rats

Whole-cell patch recording techniques were used to analyze spontaneous electrical activity in cerebellar Purkinje cells acutely isolated from postnatal rats. Spontaneous activity was present in 65% of the cells examined, and it included simple and complex firing patterns which persisted under conditions that eliminated residual or reformed synaptic contacts. Under voltage clamp, both spontaneous and quiescent cells displayed similar voltage-dependent conductances. Inward current was carried by Na⁺ through tetrodotoxin (TTX)-sensitive channels and by Ca²⁺ through P-type and T-type Ca channels. P-type current was present in all cells examined. T-type current was found in <50%, and it did not correlate with spontaneous activity. We found no evidence of a transient (A-type) potassium current or hyperpolarization-activated cationic current in either spontaneous or quiescent cells. Spontaneous activity did correlate with a lower activation threshold of the Na current, resulting in substantial overlap of the activation and inactivation curves. TTX reduced the holding current of spontaneous cells clamped between −50 and −30 mV, consistent with the presence of a Na "window" current. We were unable, however, to measure a persistent component of the Na current using voltage steps, a result which may reflect the complex gating properties of Na channels. An Na window current could provide the driving force underlying spontaneous activity, as well as plateau potentials, in Purkinje cells. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 18–32, 1997     

LIGHT-REGULATED WD1 and PSEUDO-RESPONSE REGULATOR9 form a positive feedback regulatory loop in the Arabidopsis circadian clock

In Arabidopsis thaliana, central circadian clock genes constitute several feedback loops. These interlocking loops generate an ~24-h oscillation that enables plants to anticipate the daily diurnal environment. The identification of additional clock proteins can help dissect the complex nature of the circadian clock. Previously, LIGHT-REGULATED WD1 (LWD1) and LWD2 were identified as two clock proteins regulating circadian period length and photoperiodic flowering. Here, we systematically studied the function of LWD1/2 in the Arabidopsis circadian clock. Analysis of the lwd1 lwd2 double mutant revealed that LWD1/2 plays dual functions in the light input pathway and the regulation of the central oscillator. Promoter:luciferase fusion studies showed that activities of LWD1/2 promoters are rhythmic and depend on functional PSEUDO-RESPONSE REGULATOR9 (PRR9) and PRR7. LWD1/2 is also needed for the expression of PRR9, PRR7, and PRR5. LWD1 is preferentially localized within the nucleus and associates with promoters of PRR9, PRR5, and TOC1 in vivo. Our results support the existence of a positive feedback loop within the Arabidopsis circadian clock. Further mechanistic studies of this positive feedback loop and its regulatory effects on the other clock components will further elucidate the complex nature of the Arabidopsis circadian clock.     

Cloning of Japanese flounder Paralichthys olivaceus CD3 cDNA and gene, and analysis of its expression

Two distinct CD3 homologue cDNAs, CD3-1 and CD3-2, were isolated from a Japanese flounder leukocyte cDNA library. CD3-1 consisted of 961 bp encoding 178 amino acid residues, and CD3-2 consisted of 927 bp encoding 182 amino acid residues. The two deduced amino acid sequences had an identity of 95.1%, and neither had N-linked glycosylation sites. The identities between the Japanese flounder CD3s and previously reported CD3s (CD3 epsilon, CD3 gamma, or CD3 delta) of Xenopus laevis, chicken, and various mammals were approximately 25%. The Japanese flounder CD3s had an extracellular domain, a CXXCXE motif, and an immunoreceptor tyrosine-based activation motif (ITAM), each of which are important characteristics of CD3 chains. Furthermore, the positions of four cysteine residues in the extracellular domain were preserved in both of the Japanese flounder CD3s. A phylogenetic tree based on the amino acid sequences confirmed that the Japanese flounder CD3s are closer to CD3 epsilon than to CD3 gamma and CD3 delta. However, the gene structure of Japanese flounder CD3 is identical to the chicken and Xenopus CD3 gamma/delta genes and the mammalian CD3 delta gene. Southern blot hybridization and the DNA sequence of the CD3 gene of homocloned Japanese flounder indicated that the CD3 gene exists as a single copy. Southern blot hybridization also showed the presence of a polymorphic variant of Japanese flounder CD3. An RT-PCR analysis detected Japanese flounder CD3 mRNA in several organs that contained lymphocytes. The proportion of CD3-positive cells in the peripheral blood leukocytes was 34.9%.     

Targeted cDNA differential display (TcDD)

Targeted cDNA differential display (TcDD) was developed to study expression of a different selected gene families especially those at low copy numbers per cell. This method is an adaptation of our previously described targeted genomic differential display method (TGDD). In TcDD, the expression of genes containing target sequences such as CAG repeating sequences or genes encoding for zinc-finger binding proteins were followed in an experimental rat model with salt-induced hypertension. DNA sequencing experiments demonstrated that the effectiveness of targeting was greater than 99%.     

In Vivo Readout of CFTR Function: Ratiometric Measurement of CFTR-Dependent Secretion by Individual,Identifiable Human Sweat Glands

To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (∼50) individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat) was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ∼0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with ‘CFTR-related’ conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.     

Lymphonodular cryptococcosis diagnosed by fine needle aspiration cytology in hyper-IgM syndrome. A case report

BACKGROUND: Most cases of cryptococcosis are diagnosed when signs of meningitis have appeared. We report a case of lymphonodular cryptococcosis that was diagnosed by fine needle aspiration cytology (FNAC), excisional biopsy of a cervical lymph node and culture of aspirated material. CASE: An 11-year-old boy presented with a history of fever and enlarged bilateral cervical lymph nodes of two weeks' duration. Past medical history included immunoglobulin replacement for hyper-IgM syndrome for the previous eight years. FNAC smears from a cervical lymph node showed numerous yeasts of various sizes, ranging from 5 to 15 microns in diameter, located in the cytoplasm of multinucleated giant cells and in the background. In air-dried, Diff-Quik-stained slides, the yeasts stained blue and were surrounded by clear halos. Aspirated material collected in the syringe was cultured, and Cryptococcus neoformans was isolated. CONCLUSION: This case report suggests that a combination of FNAC and culture is a simple and useful method of diagnosing fungal infections. Early diagnosis by FNAC makes possible the early initiation of treatment.