A rapid method for the determination of the base composition of bacterial DNA

A rapid method to evaluate the (G+C)-content of bacterial DNA is described. About 2 × 10⁸ cells are lysed by the combined action of detergents and enzymes and put into a CsCl-gradient without any purification. Up to five samples may be run at once. The high background absorption of cell-derived material other than DNA is overcome by novel collimating optics.     

Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma.

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero-complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins.     

Heligmosomoides polygyrus: CD4+ but not CD8+ T cells regulate the IgE response and protective immunity in mice.

Oral inoculation of BALB/c mice with infective larvae of Heligmosomoides polygyrus resulted in chronic infection characterized by the release of parasite eggs in the feces for several months. The actual number of eggs per gram of feces was dependent on the dose of the inoculum. Serum IgE in infected mice peaked at a level of greater than 70 micrograms/ml during Weeks 3 through 6 following inoculation, and high levels of IgE (greater than 40 micrograms/ml) persisted for over 14 weeks. Protective immune responses resulted in reduced egg production and the development of markedly fewer adult worms in the small intestines following a challenge inoculation. The role of CD4+ and CD8+ T cells in these responses was examined by depletion in vivo of either T cell subpopulation with rat mAb specific for the appropriate determinants. Mice treated with anti-CD4 during a primary infection had increased EPG which was due primarily to an increase in worm fecundity (eggs produced per adult female). A challenge inoculation of mice that had been cleared of the primary infection with an anthelmintic drug induced a protective response that reduced development of new adult worms by 70-80% and their fecundity by greater than 90%. This protective response was abrogated by injection of mice with anti-CD4. Serum IgE diminished when adult worms were removed after anthelmintic treatment. A more precipitous drop in serum IgE followed successive treatments of mice with an anthelmintic and anti-CD4. In addition, the anamnestic serum IgE response to a challenge inoculation was reduced by over 80% in anti-CD4-treated mice. Anti-CD8 treatment had no appreciable effect on the immunological or parasitological parameters measured following a challenge inoculation with H. polygyrus. Thus, CD4+ T cells regulate host protective immunity, worm fecundity, and IgE levels in an H. polygyrus infection. This experimental system may be particularly suitable for analysis of chronic nematode infections of humans and livestock because of the responsiveness of the parasite in vivo to changes in host immune function.     

Effects of Intranigral Substance P and Neurokinin A Injections on Extracellular Dopamine Levels Measured with Microdialysis in the Striatum and Frontoparietal Cortex of Rats

Extracellular levels of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), in the striatum and frontoparietal (sensorimotor) cortex in halothane-anesthetized rats were analyzed simultaneously using in vivo microdialysis. Basal DA levels, measured from the microdialysis perfusate, were 6.4 +/- 0.8 nM (n = 15) in the striatum and 0.9 +/- 0.1 nM (n = 15) in the frontoparietal cortex. Subcutaneous injections of d-amphetamine (2 mg/kg) increased DA levels 10-fold in the striatum and fivefold in the cortex. Injections of substance P (0.07 nmol/0.2 microliters) into the substantia nigra pars reticulata (SNR) increased DA and DOPAC levels approximately 30% in the ipsilateral striatum and approximately 50% in the ipsilateral frontoparietal cortex. Injections of neurokinin A (0.09 nmol/0.2 microliter) into the SNR increased DA and DOPAC levels approximately 30% in the ipsilateral striatum but did not significantly affect DA levels in the ipsilateral frontoparietal cortex, although DOPAC levels were increased by approximately 50%. It is suggested that striatal and cortical DA release is regulated differently by nigral substance P and neurokinin A terminals.     

Enhanced Rate of Expression and Biosynthesis of Neuropeptide Y After Kainic Acid-Induced Seizures

Recent studies have shown marked increases in brain content of neuropeptide Y (NPY) after seizures induced by intraperitoneal injection of kainic acid and after pentylenetetrazole kindling in the rat. We have now investigated possible changes in the rate of biosynthesis of NPY after kainic acid treatment, by using pulse-labeling of the peptide and by determining prepro-NPY mRNA concentrations. For pulse labeling experiments, [3H]tyrosine was injected into the frontal cortex, and the incorporation of the amino acid into NPY was determined after purifying the peptide by gel filtration chromatography, antibody affinity chromatography, and reversed-phase HPLC. At 2 and 30 days after kainic acid treatment, the rate of tyrosine incorporation was enhanced by approximately 380% in the cortex. In addition, concentrations of pre-pro-NPY mRNA were determined in four different brain areas by hybridization of Northern blots with a complementary 32P-labeled RNA probe 2, 10, 30, and 60 days after kainic acid treatment. Marked increases were observed in the frontal cortex (by up to 350% of controls), in the dorsal hippocampus (by 750%), and in the amygdala/pyriform cortex (by 280%) at all intervals investigated. In the striatum only a small, transient increase was observed. The data demonstrate increased expression of prepro-NPY mRNA and an enhanced rate of in vivo synthesis of NPY as a result of seizures induced by the neurotoxin kainic acid.     

Eight newRubus species described from Czech Republic

Eight newRubus species are described from the area of Czech Republic (western Czechoslovakia), four of them belonging to ser.Discolores, and one by one to subsect.Rubus, ser.Rhamnifolii, ser.Micantes and ser.Hystrices. *** DIRECT SUPPORT *** AAY02002 00005     

Grayanotoxin-I-modified eel electroplax sodium channels. Correlation with batrachotoxin and veratridine modifications.

To probe the structure-function relationships of voltage-dependent sodium channels, we have been examining the mechanisms of channel modification by batrachotoxin (BTX), veratridine (VTD), and grayanotoxin-I (GTX), investigating the unifying mechanisms that underlie the diverse modifications of this class of neurotoxins. In this paper, highly purified sodium channel polypeptides from the electric organ of the electric eel were incorporated into planar lipid bilayers in the presence of GTX for comparison with our previous studies of BTX (Recio-Pinto, E., D. S. Duch, S. R. Levinson, and B. W. Urban. 1987. J. Gen. Physiol. 90:375-395) and VTD (Duch, D. S., E. Recio-Pinto, C. Frenkel, S. R. Levinson, and B. W. Urban. 1989. J. Gen. Physiol. 94:813-831) modifications. GTX-modified channels had a single channel conductance of 16 pS. An additional large GTX-modified open state (40-55 pS) was found which occurred in bursts correlated with channel openings and closings. Two voltage-dependent processes controlling the open time of these modified channels were characterized: (a) a concentration-dependent removal of inactivation analogous to VTD-modified channels, and (b) activation gating similar to BTX-modified channels, but occurring at more hyperpolarized potentials. The voltage dependence of removal of inactivation correlated with parallel voltage-dependent changes in the estimated K1/2 of VTD and GTX modifications. Ranking either the single channel conductances or the depolarization required for 50% activation, the same sequence is obtained: unmodified > BTX > GTX > VTD. The efficacy of the toxins as activators follows the same ranking (Catterall, W. A. 1977. J. Biol. Chem. 252:8669-8676).     

Effect of capsaicin and resiniferatoxin on peptidergic neurons in cultured dorsal root ganglion.

The neurotoxic effect of capsaicin has been shown to be selective on a subpopulation of small dorsal root ganglion neurons in newborn animals. The aim of this study was to provide evidence of the long lasting effect of capsaicin and its ultrapotent analog resiniferatoxin (RTX) on sensory peptidergic neurons maintained in organotypic cultures. The effects of the two irritants were examined on neurons that contained substance P (SP) and calcitonin gene-related peptide (CGRP). Exposure of the cultures to 10 microM capsaicin and 100 nM RTX for periods of 2 days or longer resulted in almost complete elimination of SP-immunoreactive (IR) neurites and reduction, but not elimination, of CGRP-IR neurites. In addition, both 10 microM capsaicin and 100 nM RTX significantly reduced the number of SP- and CGRP-IR cell bodies within DRG explants. Capsaicin in 100 microM concentration produced complete elimination of SP-IR fibers and a greater decrease in the number of CGRP-IR fibers, but failed to completely eliminate IR cell bodies. Exposure of the cultures to the irritants in the same concentrations for 90 min did not produce a measurable effect on SP- or CGRP-IR in neurites or cell bodies. It is important to establish that the effect of capsaicin and RTX on cultured neurons was of long duration (longer than 4 days) and is therefore different from depletion of peptides. These findings demonstrate that processes of cultured sensory neurons are much more sensitive to capsaicin and RTX than cell bodies. Furthermore, our results show that SP-IR neuronal elements are more sensitive to capsaicin than CGRP-IR elements. These data suggest that cultured sensory neurons express the functional properties of differentiated sensory neurons in vivo.     

Cytokines: making the right choice

Fred Finkelmon and Joseph Urban propose that optimal host defense against different classes of parasite depends upon induction of different sets of immune effector mechanisms, which are, in turn, dependent upon secretion of different sets of cytokines. The authors suggest that hosts identify characteristics common to parasites of a given type as those triggers that stimulate secretion of the proper cytokine set.     

Comparison of 4-hydroxynzoate decarboxylase and phenol carboxylase activities in cell-free extracts of a defined, 4-hydroxybenzoate and phenol-degrading anaerobic consortium

Summary Two 4-hydroxybenzoate decarboxylase activities and a phenol carboxylase activity were found in cell-free extracts of a defined, 4-hydroxybenzoate- or phenol-grown consortium. Both decarboxylase activities were loosely membrane-associated and required K⁺ but a different pH and ion strength. Loss of activity of both decarboxylases by EDTA could be compensated by Zn²⁺ ions. The K _m values for 4-hydroxybenzoate and K⁺ of the decarboxylase activities with pH optima at 6.4 or 7.8 were 0.02 and 2.5 or 0.004 and 0.5 mm, respectively. 3,4-Dihydroxybenzoate, 3,4,5-tridydroxybenzoate, 3,5-dimethoxy-4-hydroxybenzoate and 3-chloro-4-hydroxybenzoate were also decarboxylated by both enzyme activities. The phenol carboxylase was a soluble enzyme with its pH optimum at 6.5. It required K⁺, Rb⁺ or NH _inf4 ^sup+ as monovalent, Zn²⁺, Mg²⁺, Mn²⁺ or Ni²⁺ as divalent cations and catalysed the carboxylation of phenol if 2,4-,2,3,4- or 2,4,6-hydroxybezoates were absent. The three enzyme activities were not influenced by Avidin and thus were probably not biotin-dependent enzymes. Offprint requests to: J. Winter