Identification of aromatic dihydroxy acids in biological fluids

3,5-Dihydroxyphenylpropionic acid, 3,5-dihydroxycinnamic acid and 2,3-dihydroxycinnamic acid were detected for the first time to be components of human urine. In the course of this investigation all constitutional isomers of dihydroxy-benzoic, -phenylpropionic, -phenylacetic and -cinnamic acid were synthesized. Mass spectra and retention indices of methyl and trimethylsilyl (TMS) derivatives were determined. In contrast to many other substituted aromatic compounds the mass spectra of methyl and TMS derivatives of dihydroxy aromatic acids often allow a firm distinction to be made between constitutional isomers: TMS derivatives of aromatic acids containing two hydroxy groups located in the ortho position to each other can be recognized by ions resulting from a primary cleavage reaction mainly in the side chain or ester group, followed by loss of tetramethylsilane. In methyl derivatives of 1,2,3-trisubstituted isomers, methoxy groups are lost much more easily from the ions corresponding to the benzylic cleavage than in other isomers. Methyl derivatives of dihydroxycinnamic acids containing at least one methoxy group in the ortho position to the side chain are characterized by a fragmentation reaction, corresponding to the loss of dimethyl ether. TMS and methyl derivatives of 3,5-dihydroxy aromatic acids show unique structure-specific fragmentation reactions.     

Mapping of brain activity in mesencephalic and diencephalic structures of toads during presentation of visual key stimuli: a computer assisted analysis of (14C)2DG autoradiographs

Summary The (¹⁴C)2DG autoradiographic technique has been employed to quantitatively map glucose utilization in the mesencephalon, the diencephalon and the cerebellum, of toads in response to configurational moving visual stimuli: (i) a 0.4 cm × 2.8 cm worm-like stripe (W) which elicited prey catching responses, (ii) a 8.4 cm × 8.4 cm square (S) that released predator avoidance responses, and (iii) a 2.8 cm × 0.4 cm antiworm-like stripe (A) which elicited no motor activity.For various brain nuclei different relationships were obtained: The optic tectum showed statistical significant higher 2DG uptake during worm-stimulation (¯X _W) than during antiworm stimulation (¯X _A), i.e.¯X _W>¯X _A. The latter visual pattern led to a 2DG utilization that was statistically significant stronger than during stimulation with a square (¯X _S), i.e.¯X _A>¯X _S. Thus, in comparison between right and left hemisphere as well as between brains the following ratios were obtained:Optic tectum:¯X _W>¯X _A>¯X _S; nucleus isthmi:¯X _W>¯X _A-¯X _s; posterodorsal lateral thalamic nucleus:¯X _S>¯X _A>¯X _W; posteroventral lateral thalamic nucleus:¯X _S>¯X _A¯X _W; posterior thalamic nucleus:¯X _W>¯X _A¯X _S; anteripr division of the lateral thalamic nucleus:¯X _W>¯X _A¯X _S; anterior thalamic nucleus:¯X _A>¯X _S>¯X _W; nucleus of Bellonci and dorsal division of the ventrolateral thalamic nucleus:¯X _W¯X _A¯X _S; cerebellum:¯X _S¯X _W>¯X _A.Abbreviations A anterior thalamic nucleus - Cb cerebellum - Hyp hypothalamus - Ist nucleus isthmi - cl. Ist contralateral Ist - La lateral thalamic nucleus, anterior division - Lpd lateral thalamic nucleus, posterodorsal division - Lpv lateral thalamic nucleus, posteroventral division - MP medial pallium - NB/VLd nucleus of Bellonci and ventrolateral thalamic nucleus, dorsal division - P posterior thalamic nucleus - PO preoptic area - Sna snapping evoking area=ventrolateral tectum - Str striatum - Tec tectum opticum     

Reverse effect of gram-positive bacteria vs. gram-negative bacteria on adjuvant-induced arthritis in germfree rats

Germfree (GF) F344 rats developed severe adjuvant-induced arthritis with a 100% incidence after a single intradermal injection of heat-killed Mycobacterium bovis (BCG). Specific pathogene-free (SPF) rats developed less severe arthritis with a lower incidence. The rats colonized with Escherichia coli or Bacteroides developed mild disease comparable to that in SPF rats. The rats colonized with Bifidobacterium, Propionibacterium acnes, Lactobacillus casei, L. fermentum, L. murini, and L. acidophilus developed more severe disease than that in GF rats. Furthermore, the rats colonized with a mixture of E. coli and the above lactobacilli developed very mild disease similar to that in SPF rats. These results suggest that gram-negative bacteria, such as E. coli and Bacteroides, may suppress the disease, possibly through their lipopolysaccharides, and may be responsible for the lower susceptibility of SPF rats; gram-positive bacteria, such as Bifidobacterium, P. acnes, and lactobacilli, may enhance the disease, possibly through their peptidoglycans; and E. coli may play a dominant role in modulating the development of adjuvant-induced arthritis.     

The human pronuclear ovum: Fine strcture of monospermic and polyspermic fertilization in vitro

The fine structure of pronuclear ova (monospermy and polyspermy) and one-cell embryos has been investigated in our IVF programme. Sixteen oocytes were collected at laparoscopy after appropriate hormonal stimulation and were matured and fertilized in vitro by methods that have given rise to normal pregnancies. Pronuclear ova showing monospermic fertilization had two vesicular pronuclei surrounded by aggregations of cellular organelles. The male pronucleus was closely associated with a sperm axoneme, while the female pronucleus was dismantling its envelope and condensing its chromatin ahead of its counterpart in late pronuclear ova. Each pronucleus had dispersed chromatin, dense compact nucleoli, and intranuclear annulate lamellae. Smooth endoplasmic reticulum, annulate lamellae, Golgi complexes, and mitochondria formed a conspicuous part of the perinuclear ooplasm. The one-cell embryos were either in syngamy or in the process of undergoing first cleavage. Positive evidence of cortical granule release and second polar bodies were detected in the perivitelline space. A block to polyspermy seemed to operate at the level of the inner zona. Dispermic and polyspermic ova had 3–16 pronuclei resembling those of monospermic ova and had sperm tails in the ooplasm. Sperm were also seen penetrating the inner zona and were occasionally found in the perivitelline space. Incomplete cortical granule release and early signs of cytoplasmic fragmentation were noted in polyspermic ova. Both normal and abnormal features of these ova are reported and compared with pronuclear structure in vivo and in vitro.     

Metabolism of 4''-phosphopantetheine in Escherichia coli.

Coenzyme A (CoA) and acyl carrier protein (ACP) contain 4'-phosphopantetheine moieties that are metabolically derived from the vitamin pantothenate. The utilization of metabolites in the biosynthetic pathway during growth was investigated by using an Escherichia coli beta-alanine auxotroph to specifically and uniformly label the pathway intermediates. Pantothenate and 4'-phosphopantetheine were the two intermediates detected in the highest concentration, both intracellularly and extracellularly. The specific cellular content of CoA and ACP was not constant during growth of strain SJ16 (panD) on 4 microM beta-[3-3H]alanine, and alterations in the utilization of 4'-phosphopantetheine and pantothenate correlated with the observed fluctuations of the intracellular pool sizes of CoA and ACP. Double-label experiments indicated that extracellular 4'-phosphopantetheine was derived from the degradation of ACP, and the extent that this intermediate was utilized by 4'-phosphopantetheine adenylyltransferase exerted control over the degradative aspect of the pathway. Control over the biosynthetic aspect of the biochemical pathway was exerted at the level of pantothenate utilization by pantothenate kinase. Reduction in the specific cellular content of CoA and ACP by 4'-phosphopantetheine excretion was irreversible since, in contrast to pantothenate, strain SJ16 was unable to assimilate exogenous 4'-phosphopantetheine into CoA or ACP.     

Effect of topically applied steroidal and nonsteroidal anti-inflammatory agents on skin repair and regeneration

We studied the effects of topically applied steroidal and nonsteroidal anti-inflammatory agents on dermal and epidermal wound healing. Superficial wounds (0.3 mm deep) on the skin of domestic pigs were treated daily with either 0.1% triamcinolone acetonide (TA), 1% hydrocortisone (HC), 1% nandrolone decanoate (ND), 1% ND + 0.1% TA, 10 mg ibuprofen, 10 mg meclofenamate sodium, 3 mg indomethacin, vehicle (USP petrolatum or 70% ethanol), or control (untreated). Wounds were excised on days 2-7 after wounding and the epidermis was separated from the dermis. The dermis was assayed for collagen biosynthesis and the epidermis was evaluated for reepithelialization. A significant decrease (P less than 0.01) in relative collagen synthesis was observed in the wounded dermis in both HC- and TA-treated groups on day 3 after wounding, but there were no significant differences on days 4-7. Depressed collagen and noncollagenous protein production was also noted in vehicle-treated wounds on day 3. Topical application of ND did not affect collagen synthesis, but when combined with TA it eliminated the inhibitory effect observed as a result of TA alone. Topical ND accelerated wound reepithelialization by 12.5% compared with vehicle and by 26% compared with untreated controls. TA delayed epidermal resurfacing by 22%, but when combined with ND (ND + TA) the rate of reepithelialization was similar to vehicle-treated wounds. HC enhanced resurfacing when compared with untreated wounds but did not differ markedly from its vehicle. The nonsteroidal anti-inflammatory drugs when topically applied markedly reduced inflammation (erythema, heat, and edema) but did not influence the healing process.     

Isolation of a yeast vector marked by the mutation of multiple resistance to antibiotics

The plasmid mutation AntR determining multiple resistance to antibiotics--tetracycline and cycloheximide in Saccharomyces cerevisiae was earlier obtained and genetically characterized. In this work we describe experiments on cytoduction and transformation, proving the localization of this mutation in the yeast 2 mu DNA. As a result of cotransformation of the sensitive cells carrying a double mutation in the gene LEU2 with the yeast vector marked by LEU2 and 2 mu DNA obtained from the yeast AntR mutant, the Leu+ AntR clones were selected. Though the primary co-transformans contain both plasmids in an unlinked state, we managed to get clones in which the markers AntR and LEU2 were linked. The putative recombinant molecules were cloned in Escherichia coli and then introduced into the yeast recipient cells, differing by the presence of the endogenous 2 mu DNA. Retransformation of cir0 cells results in the appearance of the clones in which LEU2 and AntR markers segregate together. Thus, the result of cotransformation and selection in vivo is that the mutation of multiple resistance was included into the yeast vector plasmid, presumably, in its 2 mu part.     

Isolation of acid-resistant urinary trypsin inhibitors by high performance liquid chromatography and their characterization by N-terminal amino-acid sequence determination

Two crude fractions of acid-resistant trypsin inhibitors (apparent molecular masses 44 and 20 kDa, respectively) were prepared from human urine by gel permeation chromatography. From both preparations the pure inhibitors were isolated by high performance liquid chromatography (HPLC). Their N-terminal amino-acid sequences were determined and compared with those of HI-30 and HI-14 as isolated by reversible binding to either immobilized trypsin or immobilized chymotrypsin. The N-terminal amino-acid sequence of the high-molecular mass inhibitor UI-I isolated by HPLC was identical with those of HI-30 and UI-C-I isolated via immobilized trypsin or chymotrypsin, respectively. The low-molecular mass inhibitors UI-II and UI-C-II differ from HI-14 by the N-terminal extension Glu-Val-Thr-Lys-when obtained by HPLC or by the extension Thr-Lys-when obtained via immobilized chymotrypsin, respectively. The comparison of these N-termini with the amino-acid sequence of HI-30 (Ala1-...-Val16-Thr-Glu-Val-Thr-Lys-HI-14) defines the low molecular urinary trypsin inhibitors as proteolytic degradation products of the high-molecular urinary inhibitor. Proteolysis may occur at different bonds. The existing discrepancies in molecular architecture and in molecular masses of the urinary trypsin inhibitors are discussed.     

Deamination and reductive deamination of (2-amino-5, 6-dimethylbenzimidazolyl)cobamide. Preparation of (2-hydroxy-5, 6-dimethylbenzimidazolyl)cobamide and (5, 6-dimethyl-[2(-14)C]-benzimidazolyl)cobamide

(2-Amino-5, 6-dimethylbenzimidazolyl)-cobamide (III) is transformed to (2-hydroxy-5, 6-dimethylbenzimidazolyl) cobamide (IV) by nitrous acid. Exchange of the NH2-group by hydrogen with nitrous acid/hypophosphorous acid yields vitamin B12 (I). This reaction completes a cycle vitamin B12 (I)----[carboxy(2-cyanoamino-4,5-dimethylphenyl)amino]cobamide+ ++ (II)----(2-amino-5,6-dimethylbenzimidazolyl)cobamide (III)----vitamin B12 (I), which allows chemical 14C-labelling of vitamin B12. In this procedure cyanogen bromide, which is necessary for the first step, was labelled with [14C] cyanide. By the following reactions a vitamin B12 was formed in which C-2 of the 5, 6-dimethylbenzimidazole moiety is labelled.