The Streptomyces K15 DD-peptidase/penicillin-binding protein. Active site and sequence of the N-terminal region.

Fragments of the lipophosphoglycan of Leishmania donovani were generated by phospholipase C digestion and mild acid hydrolysis. The fragments were purified and examined for inhibitory activity on protein kinase C isolated from rat brains. On a molar basis, the 1-O-alkylglycerol portion of LPG exhibited the most inhibitory activity, whereas the carbohydrate domain was not as effective. In addition, several glycolipid antigens from L. major, which contain short carbohydrate chains attached to phosphatidylinositol, were also efficient inhibitors of the enzyme. These results are consistent with the hypothesis that protein kinase C may be a key target for the parasites to overcome within host macrophages.     

Bradykinin and thrombin effects on polyphosphoinositide hydrolysis and prostacyclin production in endothelial cells.

Prostacyclin (PGI2) production by thrombin- and bradykinin-stimulated bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) was related to the receptor-linked activation of inositide hydrolysis. Bradykinin caused a rapid and transient 3-fold increase in the formation of inositol polyphosphates in BAEC. The increase in InsP3 reflected changes mainly in the Ins(1,4,5)P3 isomer. Thrombin was less effective than bradykinin in increasing InsP3 levels and appeared to only minimally stimulate the production of PGI2 in BAEC. In HUVEC, thrombin caused a 5-fold elevation of Ins(1,4,5)P3, closely related to a rise in PGI2 production. However, bradykinin did not affect inositol phosphates and PGI2 production in HUVEC. Other inositol phosphates were also assessed to obtain information on putative metabolism of Ins(1,4,5)P3. The present study supports the notion that formation of Ins(1,4,5)P3 is linked to an increase in PGI2 production in endothelial cells and furthermore provides evidence for a large degree of heterogeneity in the responses of BAEC and HUVEC to thrombin and bradykinin.     

Studies on the structures of the normal and abnormal goat thyroglobulin genes

We have cloned the thyroglobulin (Tg) gene of normal goats and goitrous goats which have a Tg synthesis defect. At the 5'-end of the gene, we studied cosmid clones covering a region from 20 kilobases (kb) upstream from the Tg gene to 42 kb into it. Electron microscopy and restriction mapping show that this part of the gene contains 20 exons of 90-1190 bp, in total 4.9 kb of exonic information (56% of the mRNA) split by 19 introns of 150-9100 bp. The exons comprise 12% of the 5' sequences cloned. At the 3'-end, 55 kb were cloned, containing 10 kb of the gene which comprises only 3 exons of 550 bp in total. Sequence analysis of the 3'-end of the normal and abnormal Tg genes has revealed one transition mutation 3' to the reading frame in a stem-loop structure region of the last exon near the poly(A) addition site. Analysis of the promoter site and the first 5 exons has revealed only one difference between the normal and goitrous Tg genes: a Ser----Leu transition in exon 5. We also found an insertion in the fifth intron of the abnormal gene.     

Use of benzodiazepines to manipulate the circadian clock regulating behavioral and endocrine rhythms

Extensive studies have now been carried out demonstrating that the systemic administration of the short-acting benzodiazepine, triazolam, can have pronounced effects on both behavioral and endocrine circadian rhythms. For example, three daily injections of triazolam can phase-advance the circadian rhythm of pituitary luteinizing hormone release and locomotor activity by about 2-3 h in female hamsters maintained in constant light. Triazolam has also been found to facilitate the rate of reentrainment of the activity rhythm following an 8-hour advance or delay in the light-dark cycle. Limited studies with other short-acting benzodiazepines indicate that the effects of triazolam on the circadian system of hamsters can be generalized to this class of drugs. Recent studies in humans indicate that treatment with triazolam can alter the time it takes for human endocrine rhythms to become reentrained following an 8-hour delay in the sleep-wake and light-dark cycle. Such findings raise the possibility that short-acting benzodiazepines may prove useful in reducing the symptoms associated with 'jet-lag' and rotating shift-work schedules as well as in the treatment of various physical and mental illnesses that have been associated with a disorder of biological timekeeping.     

Human chromosome-specific DNA libraries: construction and purity analysis

We report the construction of eight human chromosome-specific DNA libraries. Metaphase chromosomes were purified by flow-sorting, and the extracted DNA was cleaved with HindIII before cloning into lamba Charon 21A. There is now a complete digest HindIII library containing greater than five chromosome equivalents for each human chromosome. These are available to the scientific community through the American Type Culture Collection in Rockville, MD. The amount of hamster DNA in libraries in which the chromosome was sorted from human x hamster hybrid cells was estimated by species-specific hybridization. It ranged from 5% to 39%. The sorted chromosomes were examined by fluorescence in situ hybridization with species-specific DNA, and the main source of the hamster DNA contamination was found to be intact hamster chromosomes. In addition, we examined a chromosome 21 library, LL21NS02, for clones that fail to grow on the rec+ host LE392. Less than 0.6% of the recombinant phage exhibited the rec+-inhibited phenotype.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.     

Dietary zinc deficiency has no effect on auditory brainstem responses in the rat

Zinc has been shown to effect--in vitro--a number of processes associated with neurotransmission. We have tested whether the rate of impulse conduction--in vivo--as measured from the latencies of auditory brainstem responses (ABR), is influenced by dietary zinc deficiency in the rat. Dietary zinc deficiency for up to 26 wk had no effect on the wave I-IV interval compared to zinc-adequate fed animals. The results are discussed in relation to the observed constancy of brain overall and extracellular fluid zinc concentrations under conditions of dietary zinc deficiency.     

Primary photosynthetic processes in Heliobacterium chlorum at 15K

Absorbance changes induced by 25-ps laser flashes were measured in membranes of Heliobacterium chlorum at 15 K. Absorbance difference spectra, measured at various times after the flash showed negative bands in the Q_y region at 812, 793 and 665 nm. The first of these bands was attributed to the formation of excited singlet states of a long-wavelength form of antenna bacteriochlorophyll g (BChl g 808). Absorbance changes of shorter wavelength absorbing antenna BChls g were at least an order of magnitude smaller, indicating rapid excitation energy transfer (i.e. within the time resolution of the apparatus) from these BChls to BChl g 808. Excited BChl g 808 showed a bi-exponential decay with time constants of 50 and 200 ps. The bands at 793 and 665 nm may be attributed to the primary charge separation and reflect the photooxidation of the primary electron donor P-798 and photoreduction of a primary electron acceptor absorbing near 670 nm, presumably a BChl c or Chl a-like pigment. The bleaching of this pigment reversed with a time constant of 300 ps at 15 K and of 800 ps at 300 K. This indicates that electron transfer from the primary to the secondary electron acceptor is approximately 2.5 times faster at 15 K than at room temperature.Abbreviations BChl bacteriochlorophyll - FWHM full width at half maximum - P-798 primary electron donor - Tris tris(hydroxymethyl)amino methane     

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.     

Formation of zeatin allylic phosphate by the microsomal fractions of bulb disks of Iris × hollandica Tub. and tubers of Helianthus tuberosus L.

It is shown that in bulbous Iris zeatin originates from a nucleotide. This nucleotide is probably zeatin-allylic-phosphate, in which a phosphate group is attached to the isoprenoid side-chain of zeatin. The formation of zeatin-allylic-phosphate from t-zeatin and 8-[¹⁴C]-zeatin by the microsomal fractions of Iris bulb disks and Helianthus tubers was demonstrated. The responsible enzyme was partially purified. 5-AMP was found to be a phosphate group delivering substrate. Adenosine and adenine inhibited the enzyme reaction. The significance of the results is discussed in relation to cytokinin biosynthesis and the occurrence of bud blast in Iris.