A SENSITIVE ENZYMATIC-ISOTOPIC METHOD FOR THE ANALYSIS OF TYRAMINE IN BRAIN AND OTHER TISSUES

Abstract— An enzymatic-isotopic assay for the measurement of tyramine with a sensitivity of 1.0 ng has been developed. Using this assay, the endogenous content of tyramine in various tissues from adult rats has been determined. The highest tyramine content was found in rat heart atria, followed by salivary gland, kidney, and brain. Within the brain the distribution of tyramine is heterogeneous and the highest tyramine content was localized in the striatum.     

Engineered Soluble Monomeric IgG1 CH3 Domain: GENERATION,MECHANISMS OF FUNCTION,AND IMPLICATIONS FOR DESIGN OF BIOLOGICAL THERAPEUTICS*

Most of the therapeutic antibodies approved for clinical use are full-size IgG1 molecules. The interaction of the IgG1 Fc with the neonatal Fc receptor (FcRn) plays a critical role in maintaining their long half-life. We have hypothesized that isolated Fc domains could be engineered to functionally mimic full-size IgG1 (nanoantibodies) but with decreased (10-fold) size. Here, we report for the first time the successful generation of a soluble, monomeric CH3 domain (mCH3). In contrast to the wild-type dimeric CH3, the mCH3 exhibited pH-dependent binding to FcRn similar to that of Fc. The binding free energy of mCH3 to FcRn was higher than that of isolated CH2 but lower than that of Fc. Therefore, CH3 may contribute a larger portion of the free energy of binding to FcRn than CH2. A fusion protein of mCH3 with an engineered antibody domain (m36.4) also bound to FcRn in a pH-dependent fashion and exhibited significantly higher neutralizing activity against HIV-1 than m36.4-Fc fusion proteins. The m36.4-mCH3 fusion protein was monomeric, stable, soluble, and expressed at a high level in Escherichia coli. We also found that engineering an additional disulfide bond in mCH3 remarkably increased its thermal stability, whereas the FcRn binding was not affected. These data suggest that mCH3 could not only help in the exploration of the dual mechanisms of the CH3 contribution to Fc functions (dimerization and FcRn interactions) but could also be used for the development of candidate therapeutics with optimized half-life, enhanced tissue penetration, access to sterically restricted binding sites, and increased therapeutic efficacy.     

AN ULTRASTRUCTURAL STUDY OF THE GLAND OF LEIBLEIN OF MURICID AND NASSARIID NEOGASTROPODS IN RELATION TO FUNCTION, WITH A DISCUSSION ON ITS HOMOLOGIES IN OTHER CAENOGASTROPODS

The gland of Leiblein of the muricid Nucella lapillus and thenassariid Hinia reticulata has been examined by scanning andtransmission electron microscopy. The origin and functionalsignificance of its complex organization and its relationshipwith the rest of the mid-oesophagus in Nucella are discussed.It is absorptive as well as secretory, and a mechanism is proposedby which solute-rich fluids may enter the gland. Its epitheliumis composed of occasional mucous cells and two major cell types:ciliated cells engaged in protein metabolism and unciliatedcells responsible for uptake and storage of lipids and carbohydrates,both of which show evidence of pinocytotic uptake of solutesand intracellular digestion in lysosomes. Some enzyme activitypersists in the residual bodies they shed by apocrine secretion,but they remain intact in a mucous string until they reach thestomach. Preliminary ultrastructural examination indicates thatthe gland absorbs cadmium not only from the blood but also directlyfrom its lumen and that it may have the capacity to sequestera wide range of toxins. The same types of cell occur in Hiniain which their cyclical activity has been correlated with feeding.Similar cells have been identified in the oesophageal glandsof other prosobranchs. The foregut glands of carnivorous caenogastropodsare compared with the gland of Leiblein. There is an inversecorrelation between the role of the mid-oesophagus in digestionand absorption and the complexity of the stomach. (Received 16 August 2004; accepted 24 January 2005)     

USE OF ULTRASONIC SPECTROSCOPY AND VISCOSIMETRY FOR THE CHARACTERIZATION OF CHICKEN SKIN COLLAGEN IN COMPARISON WITH COLLAGENS FROM OTHER ANIMAL TISSUES

Use of traditional sources of collagen such as pork, bovine, and carp has some limitations. Chicken skin can be valuable alternative. In this work collagen was isolated from chicken skin using a modified procedure. Molecular properties of chicken collagen were analyzed and compared to collagen from other animal skins. Acid-soluble collagen type I was obtained with a yield of 25% and water content around 67%. Viscosimetry and ultrasonic spectroscopy were newly used for molecular characterization. By ultrasonic attenuation measurements, a pre-aggregation phase in the interval from 20°C to 27°C was observed, which is a proof of disaggregation and liquefaction. From 40°C upward, the liquefaction process finishes and aggregation continues. In a bovine sample this phenomenon starts at 40°C, in chicken at 50°C, and continues until 70°C. By viscosimetry, the denaturation temperature was confirmed as 40°C for bovine and 50°C for chicken collagen. Chicken collagen has a two times higher lysine level than bovine, which provides molecular stability side-chain interactions. With regard to higher thermal stability and favorable amino acid composition, waste chicken skin has the potential to be an excellent alternative source of raw collagen with applications in the food industry and biomedicine.     

上皮性卵巢肿瘤组织MKP-1及p-ERK1/2蛋白表达的研究

研究丝裂原活化蛋白激酶（mitogen activated protein kinase, MAPK） 相关蛋白丝裂原活化蛋白激酶磷酸酶（mitogen activated protein kinase phosphatase-1, MKP-1）和磷酸化细胞外信号调节激酶（phosphorylation extracellular signal-regulated kinases, p-ERK1/2）曲在上皮性卵巢肿瘤组织及正常卵巢组织中的表达差异,并探讨其在卵巢癌发生、发展中的作用,为卵巢癌的治疗提供新的思路及实验依据。选取64例上皮性卵巢癌、35例卵巢上皮性交界瘤及32例卵巢上皮性良性肿瘤患者的组织,另选取26例正常卵巢组织作对照,进行MKP-1及p-ERK1/2的免疫组化分析,并同时对其中部分病例进行上述蛋白的Western—blot研究。结果显示正常卵巢、良性肿瘤、交界瘤及卵巢癌组织MKP—1的表达依次递减,各组之间进行两两比较均有显著性差异（P〈0．01）,FIGOⅢ期与Ⅳ期卵巢癌组织MKP-1的表达显著低于Ⅰ期与Ⅱ期卵巢癌组织（P〈0．01）;而p-ERK1/2在正常卵巢、良性肿瘤、交界瘤及卵巢癌组织的表达依次递增．各组之间进行两两比较也均有显著性差异（P〈0．01）。FIGOⅢ期与Ⅳ期卵巢癌组织P～ERK1/2的表达显著高于Ⅰ期与Ⅱ期卵巢癌组织（P〈0．01）。且免疫组化及western—blot均显示MKP-1与p—ERK1/2在卵巢癌组织中的表达存在显著的负相关性,（r=-0．90,P〈0．01及r=-0．78,P〈0．01）。本研究结果表明MKP-1与p—ERK1/2的异常表达可能跟上皮性卵巢肿瘤的发生、发展有关,它们之间的表达失衡可能是卵巢癌发生、发展的原因之一．     

THE FINE STRUCTURE AND FUNCTION OF THE ANAL GLAND OF THE MURICID NUCELLA LAPILLUS (NEOGASTROPODA) (AND A COMPARISON WITH THAT OF THE TROCHID GIBBULA CINERARIA)

The fine structure of the anal gland of Nucella lapillus isdescribed, and compared with that of Gibbula cineraria. It isconcluded that the glands are not analogous in function andare unlikely to be homologous. The anal gland of Gibbula isa small rectal diverticu-lum near the anal papilla, lined bymucus-secreting cells. The evidence suggests that the largeanal gland of Nucella extracts macromolecules and cations fromthe blood, metabolizes or sequesters them in lyso-somes andultimately expels the resultant residual bodies by apocrinesecretion. It has large reserves of lipid and glycogen, andthe abundance of melanin indicates that it is a major site oftyrosine degradation. The invariable presence of bacteria inthe lumen, and of pits in the epithelium to house them, impliesa symbiotic relationship in which the bacteria metabolize anddegrade the cell debris, some of which is resorbed. The lossof the anal gland in bucci-nids and nassariids is accompaniedby great reduction in size and importance of the rectal sinus,and increase in complexity of the kidney. (Received 31 December 1991; accepted 23 January 1992)     

PHYSIOLOGICAL ONTOGENY : A. CHICKEN EMBRYOS. IX. THE IODINE REACTION FOR THE QUANTITATIVE DETERMINATION OF GLUTATHIONE IN THE TISSUES AS A FUNCTION OF AGE.

The iodine reaction to determine—SH groups in tissues according to the technique of Tunnicliffe showed that the percentage concentration of such compounds in the dried substance of chicken embryos declines with age chiefly during the third quarter of the incubation period.     

THE PATTERNS OF ARYLSULPHATASES A AND B IN HUMAN NORMAL AND METACHROMATIC LEUCODYSTROPHY TISSUES AND THEIR RELATIONSHIP TO THE CEREBROSIDE SULPHATASE ACTIVITY

Abstract— The arylsulphatase A and B patterns of human tissues and leucocytes have been established by isoelectric focussing. Assay conditions, which enable an evaluation of these patterns as quantitatively as possible, have been studied. The dependences of the enzyme patterns on the origin of the tissues and on the storage conditions have been determined. The arylsulphatase A obtained by isoelectric focussing exhibits cerebroside sulphatase activity in the presence of detergents. A purified preparation of the arylsulphatase B likewise shows a significant, although low, cerebroside sulphatase activity. In cases of the conventional types of metachromatic leucodystrophy the arylsulphatase A activity is missing, while in an atypical form of this disease ('ML Variant' according to A ustin et al . (1965) the arylsulphatase A, B and C activities are deficient. In both forms, however, residual activities of the deficient enzymes could be detected which showed isoelectric points identical to those of the normal enzymes.
The following nomenclature is proposed: 'Variant B' for the conventional type, in which the arylsulphatase B activity is present, and 'Variant O' for the exceptional cases, in which all arylsulphatase activities are deficient. The significance of the cerebroside sulphatase activity of arylsulphatase B for a possible residual turnover of cerebroside sulphates in the conventional type of the disease is discussed.     

PHOTOSYNTHETIC PIGMENTS OF PSEUDOSCOURFIELDIA MARINA AND SELECT GREEN FLAGELLATES AND COCCOID ULTRAPHYTOPLANKTON: IMPLICATIONS FOR THE SYSTEMATICS OF THE MICROMONADOPHYCEAE (CHLOROPHYTA)1

Photosynthetic pigments of the green flagellate Pseudoscourfieldia marina (Throndsen) Manton (Micromonadophyceae) are similar to those of the coccoid Pycnococcus provasolii Guillard; prasinoxanthin is the predominant carotenoid. Other organisms that possess prasinoxanthin also possess additional pigments not found in either P. marina or P. provasolii. Uriolide, a xanthophyll previously described from the coccoid done URI 266G, was also found in Mantoniella squamata (Manton et Parke) Desikachary, Micromonas pusilla Manton et Parke and Mamiella gilva (Parks et Rayns) Moestrup, all flagellate members of the Mamiellales, and the coccoid clone IV E5G. Other unidentified carotenoids were also present in M. squamata, M. pusilla, and M. gilva. These results suggest that P. marina and the coccoid organisms URI 266G and IV E5G may be related to the Mamiellales, and that P. provasolii may be more closely related to P. marina than to M. squamata, M. pusilla, and M. gilva.     

SEASONAL FLUCTUATIONS IN POPULATION DENSITES OF BULINUS SENEGALENSIS AND B. TRUNCATUS (PLANORBIDAE) IN TEMPORARY POOLS IN A FOCUS OF SCHISTOSOMA HAEMATOBIUM IN NIGER: IMPLICATIONS FOR CONTROL

Annual and seasonal changes in population densities of Bulinussenegalensis Müller 1781 and B. truncatus (Audouin, 1827)were studied in temporary pools of the west sahelian zone inNiger in relation to environmental factors. B. senegalensisis present in the temporary pools whatever their duration, whereasB. truncatus inhabits only the more persistent pools. B. senegalensispopulations develop mostly during the rainy season when thewater temperature is warm. B. truncatus is most abundant afterthe rainy season when the water temperature is cooler, but thissnail may increase when the water temperature is warmer duringunusual rainy seasons with low and intermittent rainfall. Fieldobservations suggest that interspecific competition is exceptionallyoccurring in the pool colonized by both B. senegalensis andB. truncatus. The implications of these observations on treatmentand retreatment schedules for schistosome infection in peopleand on snail control in temporary pools are discussed. (Received 4 May 1993; accepted 22 July 1994)     

SEASONAL PERIODICITY OF CHRYSOPHYCEAE AND SYNUROPHYCEAE IN A SMALL NEW ENGLAND LAKE: IMPLICATIONS FOR PALEOLIMNOLOGICAL RESEARCH1

The seasonal periodicity of taxa of Chrysophyceae and Synurophyceae from a small New England lake is described for the period September 1983 through June 1988. We found 51 taxa, including 29 that accounted for over 10% of the total in at least one collection. The taxa were fitted into one of five seasonal patterns. Patterns I and II represented taxa restricted to warm (pattern I) or cold (pattern II) months, respectively. Pattern HI represented organisms that began growth in the summer, persisted through autumn and disappeared with the onset of an ice cover. Pattern IV was an extension of pattern III, in which the taxon remained in the plankton throughout the winter and disappeared soon after ice out. Species without a clear seasonal pattern were grouped as pattern V. The seasonal periodicity of the flora, as examined with ordination analyses, was found to remain remarkedly similar during the 58–month study. Except for episodes of low pH during spring snow melt and unseasonally warm or cold weather, sample scores followed a fairly consistent pattern along the first and second primary axes. Water temperature, specific conductance, and pH were important variables that controlled changes in the species composition during the course of a given year. The flora was used to develop an inference model for water temperature. According to the analyses, the remains of a surface sediment sample represented a flora that grew primarily during the late autumn period at 7.6 ° C. Ways in which seasonal data could be utilized to improve paleolimnological inference work are discussed.     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

THE VARIABILITY OF NUCLEAR DNA AND ITS IMPLICATIONS FOR POLYPLOIDY IN WHITE ASH (FRAXINUS AMERICANA L.: OLEACEAE)

Cytophotometric studies of both mature and embryonic tissues of white ash (Fraxinus americana L.) revealed considerable variability of nuclear DNA content in this dioecious polyploid species (2n = 46, 92, 138). Triploid and pentaploid embryos may be produced, and mature individuals of these odd ploidy levels, in turn, may occur in natural populations. Polyploidy may have originated in this species through the union of unreduced gametes.     

A SIMPLE METHOD FOR THE ISOLATION AND ENUMERATION OF SPARSE POPULATIONS OF CYANOBACTERIA IN ESTUARINE WATERS1

A simple, sensitive assay method for the isolation and enumeration of sparse populations of cyanobacteria in an estuarine system is described. The method, based on the standard membrane-filter plate count technique, differentiates between viable and nonviable cells. It was found that an estuarine water-based agar medium was the most suitable medium for isolation of cyanobacteria. Because of the restricted nature of colony development, isolation of individual species is easily accomplished.