Determination of kynurenine in serum by high-performance liquid chromatography after enzymatic conversion to 3-hydroxykynurenine

A method for the determination of the physiological level of kynurenine in human serum based upon conversion of kynurenine to 3-hydroxykynurenine by enzymatic reaction with the mitochondrial fraction and NADPH and analysis by reversed-phase high-performance liquid chromatography with electrochemical detection is described. Tryptophan gave no interference. For one analysis, 0.2 ml of serum was sufficient, compared with the large volume (5.0 ml) required for other methods.     

Particle Counter Determination of Bacterial Biomass in Seawater

The applicability of the Elzone particle counter to the determination of marine bacterial biomass was investigated. The biomass of bacterial pure cultures and a mixed natural population were followed by using the particle counter, a CHN analyzer, and an ATP analyzer. The particle counter showed the precise size distribution of number and volume of submicron-size particles in seawater. For the pure cultured bacterial strains, the conversion factor from volume to carbon is 0.209 mg of C per mm³, and for natural bacterial cells of >0.6 μm in diameter, it is 0.184 mg of C per mm³. It is recommended that 0.2 be used as the conversion factor for both pure cultured marine bacterial cells and natural bacteria from coastal and near-shore marine environments.     

Generation of CD8 (T8) cytotoxic cells has a preferential requirement for CD4+2H4- inducer cells

CD8 (T8) cells are capable of both suppression and cytotoxicity. However, we have found that the activation of CD8 cytotoxic cells has a preferential requirement for a different CD4 (T4) subset from that previously reported for the activation of CD8 suppressor cells. We have recently characterized two monoclonal antibodies which subdivide CD4 cells into inducers of help for antibody production (CD4+ 4B4+) and inducers of CD8 mediated suppression (CD4+2H4+). We now report that CD4+4B4+2H4- cells also preferentially induce CD8-mediated cytotoxicity. Human peripheral blood T cells were fractionated into CD8, CD4, CD4+2H4+, and CD4+2H4- populations by both the adherence to antibody-coated plates and the fluorescence-activated cell sorter. The cells were cultured 6 days with irradiated allogeneic non-T cells and a cytotoxicity assay was then performed using cryopreserved non-T cells as targets. It was found that the combination of CD4+2H4- cells and CD8 cells resulted in greater cytotoxicity than either CD4 + CD8, or CD4+2H4+ + CD8. The combination of CD4+2H4+ cells with CD8 cells resulted in minimal cytotoxicity, which was similar to that generated by CD8 cells alone. These results were confirmed using anti-4B4 to positively select the reciprocal CD4 subset. Furthermore, the cytotoxicity induced by CD4+2H4- cells was alloantigen specific and Class I major histocompatibility complex restricted. As both CD4+2H4+ and CD4+2H4- cells proliferate equally well to alloantigen and produce similar levels of interleukin 2 (IL-2), it is likely that the generation of CD8 cytotoxic cells requires a signal in addition to IL-2.     

Complete nucleotide sequence of immunogenic protein MPB70 from Mycobacterium bovis BCG

The extracellular protein MPB70 is a heat-stable immunogenic protein which was found in the culture filtrate of Mycobacterium bovis BCG Japanese. We determined the complete nt and aa sequences of MPB70 and correlated with the previously reported data. The N-terminal sequence revealed that the signal peptide (SP) consisted of 30 aa and that the mature protein had 163 aa with a molecular weight of 16,305. The SP displayed a characteristic feature of an Ala-rich property which would be efficient in a SP function.     

A new squalid species of the genusCentroscyllium from the emperor seamount chain

A new etmopterine species,Centroscyllium excelsum, is described from 21 adult specimens captured in the Emperor Seamount Chain, central North Pacific. The present species is distinguished from its congeners in having a very high and semicircular-shaped 1st dorsal fin with a developed spine, dermal denticles only on dorsal side of head and trunk, the 2nd dorsal spine arising behind pelvic fin base, a short caudal peduncle, and prefrontal wall and chondrified eye stalk on neurocranium. Ten embryos were collected from one of the female specimens, and some embryonic features are also noted.     

Control of total peripheral resistance during hyperthermia in rats

To elucidate the effect of blood volume on the circulatory adjustment to heat stress, we studied alpha-chloralose-anesthetized rats at three levels of blood volume: normovolemia (NBV), hypervolemia (HBV; +32% plasma volume by isotonic albumin solution infusion), and hypovolemia (LBV; -16% plasma volume by furosemide administration). Body surface heating was performed with an infrared lamp to raise arterial blood temperature (Tb) at the rate of approximately 0.1 degree C/min. Before heating, central venous pressure (CVP) was significantly higher in HBV (0.41 +/- 0.25 mmHg) and lower in LBV (-1.44 +/- 0.22 mmHg) than in NBV (-0.41 +/- 0.10 mmHg). The Tb at which CVP started to decrease was approximately 40 degrees C in HBV, approximately 41 degrees C in NBV, and approximately 42 degrees C in LBV, and it decreased by 1.53 +/- 0.14, 1.92 +/- 0.24, and 0.62 +/- 0.14 mmHg from 37 to 43 degrees C of Tb in HBV, NBV, and LBV, respectively. Stroke volume was closely correlated with CVP, and this relationship was not affected by Tb. Heart rate responses to the raised Tb were similar among the three groups. Mean arterial pressure (MAP) was not affected by blood volume modification or CVP and was maintained at preheating (Tb 37 degrees C) level until Tb rose to 40 degrees C. Above this Tb, MAP increased until Tb reached 43 degrees C (+30-40 mmHg) for all three groups. Total peripheral resistance (TPR) was inversely correlated with CVP, and the slope of the linear relationship between TPR and CVP in LBV was three- to fourfold steeper than in NBV or HBV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of the human HSP70 protein: distinct domains for nucleolar localization and adenosine triphosphate binding

The human HSP70 gene was modified in vitro using oligonucleotide-directed mutagenesis to add sequences encoding a peptide from the testis-specific form of human lactate dehydrogenase (LDH) to the carboxy terminus of HSP70. The peptide-tagged HSP70 can be distinguished from the endogenous HSP70 protein using an LDH peptide-specific antiserum in indirect immunofluorescence assays of cells transiently transfected with an expression vector containing the tagged HSP70 gene regulated by the human HSP70 promoter. A series of deletion mutants within the HSP70 protein coding region were generated. Using double-label indirect immunofluorescence with the LDH peptide-specific antiserum and HSP70-specific mAbs, we compared the intracellular distribution of the deletion mutants to that of endogenous HSP70. We have determined that sequences in the carboxy terminus of HSP70 are necessary for proper nucleolar localization after heat shock. In contrast, sequences in the amino terminus of HSP70 are responsible for the ATP-binding ability of the protein. Mutants that were unable to bind ATP, however, still displayed nucleolar association, indicating that ATP binding is apparently not required for interaction with substrate. Additional support that HSP70 appears to be composed of at least two domains follows from the results of trypsin digestions of wild type and mutant HSP70. Protease digestion of the mutant HSP70 proteins identified a region of HSP70 that, when deleted, affected HSP70 conformation.     

Activation of human cytolytic cells through CD2/T11. Comparison of the requirements for the induction and direction of lysis of tumor targets by T cells and NK cells

We studied the mechanisms whereby human T cells and NK cells are activated and directed to lyse tumor targets through the CD2 (T11/E-rosette) Ag. Using two cloned NK lines, we showed that these cells, as had previously been shown for T cells, could be directed to lyse an "NK-resistant" tumor target in the presence of antibody heterodimers. These heterodimers consisted of a (mAb) to CD2 (anti-T11(2) or anti-T11(3] linked to a mAb recognizing the tumor cell (J5, anti-CALLA). However, distinct differences between NK cells and T cells were observed with regard to the requirements for such directed lysis: first, only one epitope of CD2 on NK cells (either T11(2) or T11(3] needed to be recognized by the antibody heterodimer in order for directed lysis to occur, whereas for T cells both T11(2) and T11(3) epitopes had to be recognized. Second, in confirmation of previous data with monomeric anti-T11(2) or anti-T11(3) antibody, heterodimers constructed with these reagents enhanced conjugate formation between NK cells and tumor targets, whereas no such enhancement was seen with T cells. All types of heterodimer directed lysis were dependent on the adhesion molecule LFA-1, as an anti-LFA-1 antibody-blocked lysis. Third, whereas in T cells lysis mediated through CD2 appeared to be regulated by CD3 but not vice versa, all types of lysis by NK cells appeared to be regulated through CD2. Finally we showed that F(ab')2 fragments of the anti-T11(2) and anti-T11(3) antibodies could activate NK cells, but were unable to activate T cells either as cloned cytolytic lines, or in populations of PBL. The implications of our findings with regard to the role of CD2 in the activation of cytolytic cells is discussed.     

1F7, a novel cell surface molecule, involved in helper function of CD4 cells

We have developed a monoclonal antibody, anti-1F7, that inhibits soluble Ag-driven T cell proliferation as well as PWM-driven IgG synthesis. Anti-1F7 antibody reacts with approximately 57% of unfractionated T cells, 62% of CD4+ cells, and 54% of CD8+ cells. Although the 1F7 Ag is widely distributed among lymphoid cells, this Ag on CD4+ cells is preferentially expressed on the CDw29(4B4+) helper population. Moreover, anti-1F7 antibody further subdivides the CD4+CDw29+ cell subset into CDw29+1F7+ and CDw29+1F7- populations. The CD4+CDw29+1F7+ population of cells maximally proliferates to recall Ag such as tetanus toxoid, whereas helper function for PWM-driven IgG synthesis by B cells belongs to both the CD4+CDw29+1F7+ and CD4+CDw29+1F7- population of cells. The most prominent structure defined by this antibody is a 110-kDa molecule that is different from the 135-kDa, 160-kDa, and 185-kDa glycoproteins identified by anti-CDw29 antibody and the 180-kDa glycoprotein identified by UCHL-1 antibody. It is, however, related to the molecule recognized by anti-Ta1, an activation Ag on T cells. Furthermore, although the Ta1 molecule is recognized by anti-1F7 mAb, the 1F7 family of structures also includes molecules distinct from Ta1.