Isolation and characterization of the moxJ, moxG, moxI, and moxR genes of Paracoccus denitrificans: inactivation of moxJ, moxG, and moxR and the resultant effect on methylotrophic growth.

By using the moxF gene encoding the large fragment of methanol dehydrogenase as a probe, a downstream linked chromosomal fragment was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the fragment was determined and revealed the 3' part of moxF, four additional open reading frames, and the 5' part of a sixth one. The organization and deduced amino acid sequences of the first three frames downstream from moxF were found to be largely homologous to the moxJ, moxG, and moxI gene products of Methylobacterium extorquens AM1. Directly downstream from these three genes, a new mox gene was identified. The gene is designated moxR. By using the suicide vector pGRPd1, the moxJ, moxG, and moxR genes were inactivated by the insertion of a kanamycin resistance gene. Subsequently, suicide vector pRVS1 was used to replace the marker genes in moxJ and moxG for unmarked deletions made in vitro. As a result, the three insertion strains as well as the two unmarked mutant strains were unable to grow on methanol, even in the presence of pyrroloquinoline quinone. Growth on succinate and on methylamine was not affected. In all five mutant strains, synthesis of the large subunit of methanol dehydrogenase and of inducible cytochrome c553i was observed. The moxJ and moxG insertion mutant strains were unable to synthesize both the cytochrome c551i and the small subunit of methanol dehydrogenase, and this lack of synthesis was attended by the loss of methanol dehydrogenase activity. The moxJ deletion mutant strain partly synthesized the latter two proteins, cytochrome c551i. Partial synthesis of the small subunit of methanol dehydrogenase observed with the latter strain was attended by a corresponding extent of methanol dehydrogenase activity. The moxR insertion mutant strain was shown to synthesize cytochrome c551i as well as the large and small subunits of methanol dehydrogenase, but no methanol dehydrogenase activity was observed. The results show that periplasmic cytochrome c551i is the moxG gene product and the natural electron acceptor of methanol dehydrogenase in P. denitrificans. In contrast to earlier suggestions, this cytochrome was found to be different from membrane-bound cytochrome c552. In addition, it is demonstrated that moxI encodes the small subunit of methanol dehydrogenase. It is suggested that MoxJ is involved in the assemblage of active methanol dehydrogenase in the periplasm and, in addition, that MoxR is involved in the regulation of formation of active methanol dehydrogenase.     

Identification, characterization, and nucleotide sequence of the F17-G gene, which determines receptor binding of Escherichia coli F17 fimbriae.

Enterotoxigenic Escherichia coli strains express fimbriae which mediate binding to intestinal mucosal cells. The F17 fimbriae mediate binding to N-acetylglucosamine-containing receptors present on calf intestinal mucosal cells. These fimbriae consist of F17-A subunit peptides. Analysis of the F17 gene cluster indicated that at least the F17-A, F17-C, F17-D, and F17-G genes are indispensable to obtain adhesive F17 fimbriae (unpublished data). Genetic evidence is presented that the F17-G protein, a minor fimbrial component, is required for the binding of the F17 fimbriae to the intestinal villi. The F17-G gene was cloned and sequenced. An open reading frame of 1,032 bp encoding a polypeptide of 344 amino acids, starting with a signal sequence of 22 residues, was localized. The F17-G mutant strain produced F17 fimbriae which were morphologically identical to the fimbriae purified from strains which contained the intact F17 gene cluster. However, this F17-G mutant could no longer adhere to calf villi. The F17-G locus was shown to act in trans: transformation of the F17-G mutant strain, still expressing the genes F17-A, F17-C, and F17-D, with a vector expressing the F17-G gene restored the binding activity of this mutant strain.     

Presynaptic Mechanism of Action of 4-Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B-50 (GAP-43) Phosphorylation

Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca²⁺. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca²⁺]_i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K⁺ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K⁺ still evoked an increase in [Ca²⁺]_i, which was additive to the elevation caused by 4-AP. Several Ca²⁺ channel antagonists (CdCl₂, LaCl₃, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca²⁺]_i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na⁺ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca²⁺ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na⁺ channel activity.     

Pathology of pulmonary parasitic migration: morphological and bronchoalveolar cellular responses following Nippostrongylus brasiliensis infection in rats

Nippostrongylus brasiliensis has an obligatory migratory phase through the lungs during its development in rats. This migration is associated with marked tissue damage and pronounced cellular reaction. Given that cells from the lower respiratory tract, especially alveolar macrophages, can adhere to and kill larvae of N. brasiliensis in vitro, we studied the time course of morphological changes associated with parasitic migration. Compared to a primary infection, a secondary infection resulted in significant changes in the pulmonary tissue characterized by an early acute inflammation leading to granulomatous reaction in the parenchyma and a leucocytosis in the bronchoalveolar lavage fluids with an anamnestic increase in absolute numbers of neutrophils, alveolar macrophages, eosinophils, and lymphocytes. Scanning electron microscopy showed that inflammatory cells, especially alveolar macrophages, granulocytes, lymphocytes, erythrocytes, and platelets, adhered to the larvae following secondary infection and this adhesion was associated with disruption of cuticular surface in some larvae. Secondary infection also resulted in retention of larvae in granulomatous lesions in the lungs even up to 21 days postinfection. There was mast cell and type II pneumocyte hyperplasia and these cells appeared to be activated. Thus, the histopathological changes in lungs correlated with the bronchoalveolar cellular responses and further document the inflammatory and immunological reactions during the migration of N. brasiliensis larvae.     

The action of luteinizing hormone on the testis

Luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) receptors are coupled to intracellular effector systems, most notably adenylate cyclase, through guanyl nucleotide-binding proteins or G-proteins. The molecular mechanism involved in the dynamic coupling of the LH/hCG receptor however, are not known. It has been postulated that receptor aggregation at the molecular level plays a critical role in this process. There have been attempts to understand the receptor association and dissociation phenomena at the molecular level. One of them involves the participation of the major histocompatibility complex (MHC) class I antigen in the mechanism of receptor activation and/or expression. One molecular basis for these mechanisms consists of a physical interaction between MHC proteins and receptors to form "compound receptors" able to transfer a hormonal signal to the cell. Using a photo-reactive probe we demonstrated that the LH/hCG receptors and the class I antigens are closely associated in the membrane. Thus, it is possible to form covalent complexes of hCG and class I antigens through the binding of the hormone to specific receptors. These findings imply that LH/hCG receptors and the MHC class I antigens may interact at the level of the plasma membrane in the mechanism of LH action. We also performed experiments using a single cell and limiting stimulation to a patch of membrane. The results stimulating the cell in a localized area suggested that even if all components are entirely free to float there is a constraint in the localization of the receptor, G-protein, and/or the effector, supporting the constraint dissociation model. Within a limited area subunits could dissociate, but they would not be free to diffuse throughout the membrane. Moreover the concept of compartmentalization that has been utilized to explain some inconsistencies in second-messenger action now can be proved by experimental design.     

Effects of growth conditions on exopolysaccharide production by Cyanospira capsulata

Batch cultures of Cyanospira capsulata, a heterocystous cyanobacterium possessing a thick polysaccharidic capsule, were characterized by increasing viscosity owing to the continuous release of a soluble polysaccharide (EPS) into the culture medium. Both capsulated trichomes and solubilized EPS contributed to the flow properties of whole cultures. A typical pseudoplastic behaviour, the more marked the more aged were the cultures, was evidenced.The production of EPS was investigated under different growth conditions by changing some nutritional and physical parameters known to affect the synthesis of exopolysaccharides in algae and cyanobacteria. Among the factors tested (Ca²⁺, Mg²⁺ or PO₄⁻⁴ deficiencies, salinity and pH) only Mg²⁺ shortage caused a significant enhancement of the EPS production. Under continuous illumination in open ponds, the EPS productivity of batch cultures on standard mineral medium was about 5·8 g m⁻² day⁻¹, whereas under Mg²⁺ deficiency with a consequent increase of the cultures' viscosity     

Genetic counseling of a couple presenting respectively terminal transverse defects and congenital arthrogryposis.

A 29-year-old woman consulted together with her husband for a problem of congenital joint contractures. Clinical findings in the woman were characteristic of "amyoplasia congenita". In view of the severe distal limb involvement, the possibility of a distal arthrogryposis was also considered. Unexpectedly, the husband presented terminal transverse defects, mainly of the feet, suggestive of Adams-Oliver syndrome, an autosomal dominant condition with variable expression. This discovery significantly altered the counseling given to the couple, i.e. they had to be given a high recurrence risk for congenital limb malformations in their offspring and rigorous echographic monitoring of future pregnancies was advised.     

Oculocerebral syndrome with hypopigmentation (Cross syndrome): the mixed pattern of hair pigmentation as an important diagnostic sign.

The present report describes two female siblings of mixed ancestry (Cape Coloured) with consanguineous parents as further examples of Cross oculocerebral hypopigmentation syndrome. The mixed pattern of hair pigmentation as an important diagnostic sign is emphasized.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-l-yl and substituted triazol-4-yl derivitives.