Effect of apolipoprotein activators on the specificity of lecithin:cholesterol acyltransferase: determination of cholesteryl esters formed in A-I/C-III deficiency.

Although it is known that plasma lecithin:cholesterol acyltransferase (LCAT) is activated by several apolipoproteins (apo) including A-I, C-I, D, A-IV, and E, it is not clear what the physiological importance of having different apolipoprotein activators is. One possible explanation is that the activation by different apolipoproteins may result in the utilization of different species of phosphatidylcholine (PC), leading to the formation of different species of cholesteryl esters (CE). In order to determine this possibility, we analyzed the molecular species composition of PC and CE in two patients with familial deficiency of apoA-I and apoC-III. The LCAT activity, assayed by three different procedures, was found to be 36-63% of the control value. The lower LCAT activity, however, was due to deficiency of the enzyme rather than the absence of apoA-I. The patients' plasma was relatively enriched with sn-2 18:2 PC species reflecting the partial deficiency of LCAT activity. The fatty acid composition of plasma CE was not significantly different from that of controls. HPLC analysis of labeled CE formed after incubation of plasma with [C14]cholesterol showed no significant difference in the species of CE synthesized by the LCAT reaction. The transfer of pre-existing as well as newly formed CE from HDL to the apoB-containing lipoproteins was accelerated compared to control plasma. These results show that the absence of apoA-I does not significantly affect either the activity or the specificity of LCAT, and that the other apolipoprotein activators can substitute adequately for it.     

Molecular species of cholesteryl esters formed in abetalipoproteinemia: effect of apoprotein B-containing lipoproteins

In order to study the effects of very low density (VLDL) and low density (LDL) lipoproteins on the activity and specificity of lecithin:cholesterol acyltransferase (LCAT), we determined the molecular species of cholesteryl esters (CE) synthesized in the plasma from three abetalipoproteinemic (ABL) patients, before and after supplementation with normal VLDL or LDL. The patients' plasma had significantly lower concentration of 18:2 CE and higher concentrations of 16:0 CE and 18:1 CE compared to normal plasma. Incubation of ABL plasma with [4-14C]cholesterol at 37 degrees C and the subsequent analysis of labeled CE formed by high performance liquid chromatography revealed that the major species formed was 16:0 CE (34% of total label), whereas similar incubation of the d greater than 1.063 g/ml fraction of normal plasma resulted in the formation of predominantly 18:2 CE (45% of total label). Addition of normal VLDL or LDL to ABL plasma stimulated the total LCAT activity by 30-80% and normalized the CE species synthesized. The LCAT activity of a normal d greater than 1.063 g/ml fraction also was stimulated by the normal VLDL or LDL, but there was no alteration in the species of CE formed. Most of the CE synthesized was found in the added VLDL or LDL with both ABL and normal plasma, indicating that the CE transfer (CET) activity was not affected in ABL plasma. These results suggest that while the VLDL and LDL are required for the maximal activity of LCAT, the species of CE formed are primarily determined by the molecular species composition of phosphatidylcholine in the plasma.     

Demonstration of enzymatic conversion of lysolecithin to lecithin in normal human plasma

An enzyme in normal human plasma that converts [1-acyl ¹⁴C] lysolecithin to lecithin is demonstrated. This enzyme is inhibited by heparin and is not derived from platelets or other blood elements. The synthesis of lecithin from labeled lysolecithin was not stimulated by ATP and CoA or by oleyl CoA and there was nearly an equal distribution of labeled fatty acid between the two positions of lecithin indicating that the enzyme may be a lysolecithin: lysolecithin acyl transferase (LLAT). The enzyme is associated with the lipoproteins of the plasma, and may have a physiological role in the formation of saturated cholesterol esters in plasma.     

A high throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of bisoprolol in human plasma using multiplexing technique

A high throughput and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of bisoprolol in human plasma using multiplexing technique (two HPLC units connected to one MS). Bisoprolol was extracted from human plasma using solid-phase extraction technique using metoprolol as internal standard. A Betabasic 8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 326.2-->116.1 for bisoprolol and m/z 268.2-->191.0 for metoprolol. The method involves a simple multiplexing, rapid solid-phase extraction, simple isocratic chromatography conditions and mass spectrometric detection which enable detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.5-70.0 ng/mL with correlation coefficient > or =0.9991. The precision and accuracy were within 10% for intra-HPLC runs and inter-HPLC runs. The overall recoveries for bisoprolol and metoprolol were 93.89% and 77.65%, respectively. Total MS run time was 0.90 min only. The developed method was applied for the determination of pharmacokinetic parameters of bisoprolol following a single oral administration of a 10mg bisoprolol tablet in 18 healthy male volunteers.     

Engineering Silkworms for Resistance to Baculovirus Through Multigene RNA Interference

Bombyx mori nucleopolyhedrovirus (BmNPV) that infects the silkworm, B. mori, accounts for >50% of silk cocoon crop losses globally. We speculated that simultaneous targeting of several BmNPV essential genes in transgenic silkworm would elicit a stable defense against the virus. We introduced into the silkworm germline the vectors carrying short sequences of four essential BmNPV genes in tandem, either in sense or antisense or in inverted-repeat arrangement. The transgenic silkworms carrying the inverted repeat-containing transgene showed stable protection against high doses of baculovirus infection. Further, the antiviral trait was incorporated to a commercially productive silkworm strain highly susceptible to BmNPV. This led to combining the high-yielding cocoon and silk traits of the parental commercial strain and a very high level of refractoriness (>75% survival rate as compared to <15% in nontransgenic lines) to baculovirus infection conferred by the transgene. We also observed impaired infectivity of the occlusion bodies derived from the transgenic lines as compared to the wild-type ones. Currently, large-scale exploitation of these transgenic lines is underway to bring about economic transformation of sericulture.     

Chemical biology approaches in plant stress research

In addition to their importance as essential agrochemicals and life-saving drugs, small molecules serve as powerful research tools to address questions at all levels of biological complexity from protein function to plant biotic interactions. In certain contexts, chemical tools are complementary or even preferred to genetic analysis, since not all experimental systems are amenable for genetic dissection. For example, mutants impaired in oxygen sensing cannot easily be recovered. Pharmacological and chemical genetics approaches have come to the rescue of biologists in unraveling such genetically intractable systems. In this review, I have discussed my own efforts to analyze oxygen deprivation signaling in plants to illustrate the validity of small molecular approaches in elucidating an essential pathway such as oxygen sensing. Chemical biology is also a potent approach to tease out genetically redundant biological processes. The recent breakthrough in identifying the elusive abscisic acid receptors has clearly demonstrated the power of chemical tools in dissecting redundant pathways and led to the blossoming of this area as a distinct discipline of plant biology research. I present a summary of this work and conclude the review with potential challenges in using chemical tools.     

Myoepithelioma within the carpal tunnel: a case report and review of the literature

Myoepitheliomas of the extremity are rare and usually benign, while a minority display malignant features. This case demonstrates the diagnosis and management of myoepithelioma within the carpal tunnel. Clinical and radiological tumour features were evaluated. Hematoxylin and eosin stained tumour sections were examined, and immunohistochemistry was performed. Histology revealed a nodular mass of epithelioid cells in clusters within a myxoid/chondroid stroma. No mitoses were noted. Cytokeratins, neuron-specific enolase, synaptophysin, glial fibrillary acidic protein, and S100 were positive on immunohistochemistry. A literature review revealed very few prior reports of myoepithelioma in the wrist, and limited data concerning any relationship between recurrence and quality of surgical margins. In this case, wide local excision would have significantly compromised dominant hand function, and therefore a marginal excision was deemed appropriate in the context of bland histological features. Surgical margins noted in future case reports will aid clinical decision making.