Capillary Gas Chromatography and Thermionic N-P-Specific Detection of 13-Bis(2-chloroethyl)-l-nitrosourea (BCNU) or l-(2-Chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) in Stability and Pharmacokinetic Studies

An expedient, rapid, and sensitive capillary gas chromatographic method for the analysis of l,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) or l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) in plasma is described. Separation of the underivatized nitrosourea compounds was performed on a 0.33-mm-i.d., 25-m fused-silica, SE-30 capillary column, and detection was carried out using a thermionic N–P-specific detector. The compounds were extracted from plasma with benzene with a yield of >87%. The assay was linear in the ranges of 0.001 to 0.5 and 0.5 to 25 µg/ml for CCNU or 0.003 to 0.50 and 0.5 to 25 µg/ml for BCNU, with correlation coefficients from 0.9914 to 0.9999 and coefficients of variation (CV) of <3.3%. Other antineoplastic agents did not interfere in the assay. The method was employed to study the pharmacokinetics of BCNU in rabbits. The plasma concentration-time curves were fit to a two-compartment model with a mean (SE) , , and total-body clearance of 2.898 (0.913) hr^–1, 0.1228 (0.0179) hr^–1, and 7.211 (2.862) liters/hr · kg, respectively. Further, the stability of BCNU and CCNU in solution was examined at different temperatures. Both compounds were stable in benzene or acetone (4 to 37°C) but labile in plasma even if refrigerated. The apparent rate constants for degradation of BCNU and CCNU were 0.09921 and 0.02853 hr^–1 at 4°C and 5.998 and 2.553 hr^–1 at 37°C, respectively.     

A reevaluation of the status of cholesterol in erythrocytes infected by Plasmodium knowlesi and P. falciparum

The cholesterol synthesis of rhesus monkey erythrocytes parasitized by Plasmodium knowlesi and human erythrocytes infected by P. falciparum, as measured by incorporation of [1-¹⁴C]acetate and ³H₂O, was almost undetectable, concordant with very low levels of measurable 3-hydroxy-3-methyl glutaryl-CoA reductase activity. In addition, both types of infected cells exchanged cholesterol with the plasma at the same rate as uninfected cells. The data do not exclude the possibility of cholesterol transfer from uninfected to infected cells.     

Changes in catecholamine concentrations in the hypothalamus and region of the midbrain raphe in male and female rats during postnatal development

To investigate possible sex differences in central catecholaminergic systems, the concentrations of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured by high-performance liquid chromatography with electrochemical detection in the hypothalamus-preoptic area (H-POA) and midbrain raphe (MR) region of male and female rats throughout postnatal development. Other than a small but significant sex difference in DA concentration in the MR at 36 h (higher in females), the results showed that there were no sex differences in the concentrations of DA or DOPAC at any age in either brain region. The developmental profiles of the concentrations of DA and DOPAC showed that DAergic systems in the H-POA are immature at birth, with concentrations increasing steadily until 80 days of age, but in the MR concentrations reach a maximum at 20 days of age; a situation which is perhaps a reflection of differing monoamine metabolism in the two areas.     

Functional aspects of serotonin transmission in the basal ganglia: a review and an in vivo approach using the push-pull cannula technique

Peripheral deafferentation of the rodent olfactory bulb results in loss of dopamine content, tyrosine hydroxylase activity and immunocytochemical staining for tyrosine hydroxylase in juxtaglomerular dopamine neurons. Reinnervation of the bulb by afferent neurons results in the return of all parameters to control levels suggesting that the dopamine neurons did not degenerate but that the expression of tyrosine hydroxylase enzyme was transneuronally regulated in a static population of juxtaglomerular cells. To evaluate this possibility, we determined the activity and immunocytochemical localization of the second enzyme in the dopamine biosynthetic pathway, DOPA decar?ylase. At a time when tyrosine hydroxylase activity was reduced to 25% of control values, DOPA decar?ylase activity in the lesioned bulb was maintained at about 65% of that in the unlesioned bulb. Immunocytochemical staining with antibodies to both enzymes, performed sequentially in the same sections, demonstrated that in the unlesioned bulb tyrosine hydroxylase and DOPA decar?ylase are co-localized in the same population of juxtaglomerular neurons. Similar results were obtained in adjacent sections each stained with one of the two antibodies. In contrast, in the deafferented bulb, about three times as many neurons were stained with DOPA decar?ylase as with tyrosine hydroxylase antibodies. The DOPA decar?ylase activity measurements and immunocytochemistry argue for the continued presence, in the lesioned olfactory bulb, of a population of tyrosine hydroxylase deficient dopamine neurons.The data suggest that olfactory receptor cell innervation transneuronally regulates the expression of tyrosine hydroxylase by mechanisms separate from those controlling the levels of DOPA decar?ylase.     

马凡综合征两种新的原纤维蛋白-1基因突变

目的对9例马凡综合征(Marfansyndrome,MFS)患者的原纤维蛋白-1(fibrillin-1,FBN1)基因进行突变筛查,以发现新的FBN1基因突变。方法应用变性高效液相色谱法对MFS患者FBN1基因65个外显子中的35个进行突变筛查,对变性高效液相色谱图形异常的PCR扩增片段用DNA测序鉴定突变位置及性质,并用等位基因特异性PCR以及限制性片段长度多态性分析等方法进一步证实突变。结果在两例MFS患者中发现两种新的FBN1基因突变。其中一种为第34外显子4307～4308位4个碱基TCGT的插入突变(4307insTCGT),另一种为第43外显子5309位的点突变5309G>A。结论FBN1基因移码突变(4307insTCGT)与点突变(5309G>A)分别是这两例MFS患者的发病原因。     

生物工程技术制备人源抗-HBs Fab 片段

目的：用生物工程技术制备人源性抗-HBsFab。方法：将从抗体文库中筛选出的人源抗-HBsFab基因克隆入pBAD/gⅢA载体，进而转化Tpo10大肠杆菌，对重组质粒菌发酵表达后，利用Ni-NTA-Agarose螯合层析柱纯化周质腔可溶性Fab蛋白。对所得包涵体依次变性，溶解，纯化后，利用透析进行复性，用Western blot检测Fab蛋白的特异性，Dot blot测定其生物学活性。结果：经Ni-NTA-Agarose柱纯化的周质腔可溶性Fab蛋白，有较好的生物学活性，并且总量达到80mg/L。对所获包涵体进行透析复性后，也可得到少量有活性的蛋白，但比例很小。结论；用pBAD/gⅢA-Top10表达系统表达人源抗-HBsFab片段，发酵培养后，经有效纯化可得到生物学活性较好的可溶性蛋白。为人源抗-HBsFab片段的大量制备提供了有效手段。     

Calcitonin gene-related peptide in cardiovascular tissues of the rat

The distribution of calcitonin gene-related peptide immunoreactivity in the cardiovascular system of the rat was investigated by radioimmunoassay and immunocytochemistry. The nature of the immunoreactivity was studied by gel permeation and high performance liquid chromatography. Immunocytochemistry demonstrated the existence of calcitonin gene-related peptide-containing nerve fibres throughout the cardiovascular system. These were present in all regions of the heart, particularly in association with the coronary arteries, within the papillary muscles and within the sinoatrial and atrioventricular nodes. Calcitonin gene-related peptide-containing fibres were found mainly in the adventitia of the arteries and veins. Calcitonin gene-related peptide concentrations were high in major arteries and veins but comparatively low in the heart, aortic arch and thoracic aorta. Chromatography showed that approximately 70% of the total immunoreactivity was identical to synthetic calcitonin gene-related peptide. Calcitonin gene-related peptide concentrations in the blood vessels of rats treated neonatally with capsaicin were not found to be significantly different from those in control animals although capsaicin caused significant reductions of calcitonin gene-related peptide levels in certain other tissues. The results of this study suggest that calcitonin gene-related peptide-containing fibres are likely to be of importance in the innervation of vascular tissues and raise the possibility that these fibres are different in character from calcitonin gene-related peptide-containing fibres found in other tissues.     

Significance of methods for purification of oligodeoxyribonucleotide probes for the efficiency of gene diagnosis by real-time PCR

Analysis of the use of real-time PCR with fluorescent registration of results for gene diagnosis of infectious diseases showed that the sensitivity and reliability of quantitative evaluation of DNA targets directly depended on the method of purification of oligonucleotide probes. Chromatographic behavior of synthetic probes carrying various fluorophores and fluorescence quenchers was analyzed. Approaches to optimization of purification methods are proposed enabling elimination of previously undetectable admixtures. The importance of these studies is explained by the need in extending the armory of methods for the development and production of diagnosticums for detection of infectious and hereditary diseases, identification of genetically modified organisms, and for a wide spectrum of research in molecular biology and medicine. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 145, No. 3, pp. 280–284, March, 2008     

Recognition of an extensive range of IgE-reactive proteins in cod extract

Allergy to fish is one of the most common food allergies. Gad c 1 is the only fish allergen which has been purified and characterized. Other allergens have been detected by Western blot in cod extracts. We have now improved the Western-blot procedure in order to characterize fish IgE-reactive proteins from extracts prepared under different conditions: pre-rigor mortis and postrigor mortis. EDTA addition or not. and DEAE ion-exchange chromatography. Several IgE-reactive protein bands have been identified over a wide molecular-weight range. In particular, the 104- and 130-kDa IgEreactive protein bands were detected. These new bands may correspond to aggregates, as EDTA increased the relative amount of the 60-, 67-, 104-, and 130-kDa IgE-reactive protein bands in Western blot. All these bands were also detected by an antiparvalbumin monoclonal antibody, specific to the first calcium-binding site. The longer period of storage increased the relative amounts of the 41-, 80-, 104-. and 130-kDa IgE-reactive protein bands. The 18-kDa band was detected only in fish stored for several days. In conclusion, we have described IgE-reactive protein bands over a wide molecular-weight range (12–130 kDa) in Western blot of cod extract, and shown that EDTA and storage conditions may influence the relative distribution of IgE-reactive protein bands.     

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.