Genes involved in the biosynthesis of PQQ fromAcinetobacter calcoaceticus

From a gene bank of theAcinetobacter calcoaceticus genome a plasmid was isolated that complements four different classes of PQQ^- mutants. Subclones of this plasmid revealed that the four corresponding PQQ genes are located on a fragment of 5 kilobases. The nucleotide sequence of this 5 kb fragment was determined and by means of Tn5 insertion mutants the reading frames of the PQQ genes could be identified. Three of the PQQ genes code for proteins of M_r 29700 (gene I), M_r 10800 (gene II) and M_r 43600 (gene III) respectively. In the DNA region where gene IV was mapped however the largest possible reading frame encodes for a polypeptide of only 24 amino acids. A possible role for this small polypeptide will be discussed. Finally we show that expression of the four PQQ genes inAcinetobacter lwoffi andEscherichia coli lead to the synthesis of the coenzyme in these organisms.     

Incontinentia pigmenti: Xp breakpoint is not the same in a case of r(X) and in X/autosome translocations

X-specific DNA probes were used to characterize the r(X) of a 45,X/46,X,r(X) female patient with Incontinentia pigmenti. It was found to be of maternal origin. Breakpoints were shown to be in or distal to p11.22 and between q12.2 and q13.1. When considering all known cases of Incontinentia pigmenti and X rearrangements at least four different break sites on the X have been shown.     

Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis.

A pair of vectors for expression of heterologous genes in Lactococcus lactis was constructed. In addition to an origin of replication that has a broad host range, these vectors contain a multiple cloning site flanked by gene expression signals originating from L. lactis subsp. cremoris Wg2. The two vectors, about 3.7 kilobase pairs in size, differ only in the type of antibiotic resistance they confer to their hosts. pMG36 carries a kanamycin resistance marker, which was replaced by an erythromycin resistance marker in pMG36e. As an example of the use of these vectors, the hen egg white lysozyme-coding sequence was inserted. A fusion protein of the expected size was detected in a transformed L. lactis subsp. lactis strain by using Western blotting (immunoblotting).     

Auditory evoked potentials in distinguishing between peripheral and central tinnitus

Brainstem evoked response audiometry has been performed in 164 patients complaining of tinnitus, unilateral or bilateral. The latency of every wave and the latency between the peak of every wave have been compared with those obtained in a normal population (n = 57). The latency of the first wave is increased significantly at the same side of the tinnitus. But I-V latency is shorter at the side of the tinnitus. When this increase is absent, the I-V latency is prolonged the side of the tinnitus. It is possible to differentiate between tinnitus of peripheral origin and that of central origin, although the existence of unilateral deafness is constant. The same results have been obtained in the deaf patients with bilateral symmetric deafness, in accordance with Maurizi et al., 1985.     

Immunocytochemical study of parvalbumin fibers and cell bodies in the rat hipothalamus

The distribution of parvalbumin (PA) cell bodies and fibers in the hypothalamus of the rat was studied using a monoclonal antibody and the avidin-biotin-peroxidase method. The densest clusters of immunoreactive perikarya were observed in the nuclei mamillare medialis, arcuatus and dorsomedialis hypothalami, whereas the corpus mamillare lateralis had the lowest density. The densest network of immunoreactive fibers was observed in the corpus mamillare lateralis and nucleus arcuatus. The corpus mamillare medialis contained a moderate number of PA fibers, whereas the nucleus dorsomedialis hypothalami had the lowest density of immunoreactive fibers. In addition, a large number of immunoreactive fibers was found in the tractus opticus and the tractus mamillo-thalamicus. Essentially, the distribution of PA in the rat hypothalamus after using a monoclonal antibody seems to be broader in comparison with previous studies carried out in the same diencephalic region of the rat. The presence of PA in several nuclei of the rat hypothalamus suggests that this protein could be directly or indirectly involved in neuroendocrine, limbic and visual functions.     

Magnetic separation of pinocytic vesicles of defined age from Entamoeba histolytica

We describe a rapid and simple method to isolate pinocytic vesicles of defined age (residing time within the cell) from Entamoeba histolytica. Amoebas are allowed to pinocytize for greater than 5 min a suspension of superparamagnetic iron oxide particles, washed, and resuspended for predetermined periods (up to 150 min) in iron oxide-free medium. Subsequently, the cells are homogenized and iron oxide-containing vesicles are separated magnetically. Recovery of vesicles (estimated with fluorescein isothiocyanate-dextran as a quantitative marker for pinocytosis) was 20-40%. Contamination with "older" vesicles or with plasma membrane (estimated with fluorescein isothiocyanate-dextran and with fluorescein isothiocyanate-conjugated, succinylated concanavalin A, respectively) was negligible. Using this method we obtained evidence that in E. histolytica, contrary to the situation in animal cells, pinocytic vesicles within 150 min after invagination neither shrunk nor fused with each other to any significant extent. The method should be generally applicable to protozoa for the isolation of pinocytic vesicles and digestive vacuoles.     

Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts.

Demineralized water was seeded with controlled numbers of oocysts of Cryptosporidium parvum purified from fresh calf feces and subjected to different treatments with ozone or chlorine dioxide. The disinfectants were neutralized by sodium thiosulfate, and neonatal mice were inoculated intragastrically and sacrificed 7 days later for enumeration of oocyst production. Preliminary trials indicated that a minimum infection level of 1,000 oocysts (0.1-ml inoculum) per mouse was necessary to induce 100% infection. Treatment of water containing 10(4) oocysts per ml with 1.11 mg of ozone per liter (concentration at time zero [C0]) for 6 min totally eliminated the infectivity of the oocysts for neonatal mice. A level of 2.27 mg of ozone per liter (C0) was necessary to inactivate water containing 5 x 10(5) oocysts per ml within 8 min. Also, 0.4 mg of chlorine dioxide per liter (C0) significantly reduced infectivity within 15 min of contact, although some oocysts remained viable.     

Shifts in Methanogenic Subpopulations Measured with Antibody Probes in a Fixed-Bed Loop Anaerobic Bioreactor Treating Sulfite Evaporator Condensate

A fixed-bed loop, high-rate anaerobic bioreactor treating sulfite evaporator condensate was sampled when it reached steady state and afterwards following perturbations during a 14-month period. By using immunotechnology, it was observed that shifts in methanogenic subpopulations occurred in association with perturbations, such as restarting and relocating the biomass into a different tank. Methanogens related to Methanobacterium bryantii MoHG and Methanobrevibacter smithii ALI were numerous throughout the observation period, while Methanosarcina mazei S6 and Methanosarcina thermophila TM1 were found in the early and late samples, respectively. Also, Methanobacterium formicicum was more numerous at the top portion of the bioreactor, while Methanobrevibacter arboriphilus AZ and DC were at the bottom. Sample formalinization required for prolonged storage proved suitable for antigen preservation.     

Four different b-type cytochromes in the halophilic archaebacterium, Halobacterium halobium

The halophilic archaebacterium, Halobacterium halobium has been found to contain four different b-type cytochromes. The four components were recognized by their potentiometric characteristics in situ in their functional environment in the membrane of H. halobium. Oxidation-reduction midpoint potentials of these four b-type cytochromes were determined to be +261, +160, +30, and -153 mV, respectively. We also demonstrate that the pathway involved in the transport of reducing equivalents from succinate to oxygen proceeds through the b-type cytochromes with oxidation-reduction midpoint potentials of +261 and +161 mV. The cytochrome with oxidation-reduction midpoint potential of -153 mV was not substrate reducible by NADH but was chemically reducible by dithionite. Antimycin inhibits reduction of b-type cytochrome in the succinate pathway, but has no effect on b-type cytochrome reduction when reducing equivalents are provided by NADH. The carbon monoxide difference spectrum of H. halobium membranes shows at least one carbon monoxide-binding b-type cytochrome, indicating a terminal oxidase. A scheme for electron transport in H.halobium involving the b-type cytochromes and terminal oxidase is suggested.     

Selective oxidation of mitochondrial glutathione in cultured rat adrenal cells and its relation to polycyclic aromatic hydrocarbon-induced cytotoxicity

Primary cultures of rat adrenal cells, as well as rat adrenals in vivo, are sensitive to the potent carcinogen 7,12-dimethylbenz[a]anthracene and its liver metabolite 7-hydroxymethyl-12-methylbenz[a]anthracene, whereas unmethylated polycyclic aromatic hydrocarbons like benzo[a]pyrene or benzo[a]anthracene are ineffective. The adrenocorticolytic potencies of the hydrocarbons are affected by adrenocorticotrophic hormone and various steroids, cytochrome P450 inhibitors, and antioxidants. In the present investigation digitonin was used to fractionate cultured rat adrenal cells. It was found that the mitochondria and cytosol of the cells contained 3-5 nmol/10(6) cells (approximately 15%) and 20-30 nmol/10(6) cells (approximately 85%) of the total soluble cellular glutathione equivalents, respectively. After exposing the cells to 7-hydroxymethyl-12-methylbenz[a]anthracene in the culture medium, a time- and concentration-dependent selective oxidation of mitochondrial glutathione was observed, whereas the effect on the cytosolic glutathione was negligible. Under the same conditions, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene were unable to alter the redox levels of the subcellular pools of glutathione. Omission of adrenocorticotrophic hormone lowered the oxidation of mitochondrial glutathione induced by 7-hydroxymethyl-12-methylbenz[a]anthracene about twofold. The results suggest that rat adrenal cells contain two separate pools of glutathione, one cytosolic and one mitochondrial, of which the latter is selectively influenced by 7-hydroxymethyl-12-methylbenz[a]anthracene. Moreover, it is concluded that rat adrenal cells offer a unique model system for general studies of the effects of a selective oxidation of mitochondrial glutathione on various cell functions. These effects may constitute early changes in cytotoxicity, preceding, e.g., membrane damage and loss of cytosolic components.