Energy-dependent formation of free ATP in yeast submitochondrial particles, and its stimulation by oligomycin

Yeast submitochondrial particles, in a P_i- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5–10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.     

A gene (Bmn) controllingβ-mannosidase activity in the mouse is located in the distal part of chromosome 3

A gene (Bmn) with a major effect on -mannosidase activity in kidney and liver of the house mouse was revealed by assay with the synthetic substratep-nitrophenyl--d-mannoside. Activity is low in DBA/2J and CSB mice and high in C57BL/6J mice. By the use of the BXD series of recombinant inbred strains and by crosses between C57BL and CSB, it was possible to map the gene to the distal part of chromosome 3 by demonstration of linkage to a gene for cadmium resistance,cdm, as well as to theAdh-3 locus.This work was supported by Swedish Natural Science Research Council Project B-BU 2992-108.     

Neuropeptides in the gastrointestinal canal of Necturus maculosus

Summary The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut.Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum.Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa.The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity.It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.     

Recovery of proteins on a milligram scale from polyacrylamide electrophoresis gels,exemplified by purification of a retinol-binding protein

An apparatus suitable for the recovery of proteins from polyacrylamide gels on a milligram scale by displacement electrophoresis (isotachophoresis) is described along with a buffer system that is suitable for this purpose with most proteins. The technique is illustrated by the recovery of a protein from a 15% polyacrylamide gel. The recovery was almost quantitative and the eluted protein showed little contamination upon quantitative amino acid analysis and automatic Edman degradation.     

The ultrastructural localization of a cholinesterase in the body muscle of plaice (Pleuronectes platessa)

Summary By means of a histochemical method adapted for electron microscopy a cholinesterase in body muscle cells of plaice (Pleuronectes platessa) has been localized to the sarcolemma. The cholinesterase activity disappeared from the sarcolemma after the muscle tissue had been incubated with a bacterial enzyme, which had earlier been shown, by biochemical methods, to be able to liberate this cholinesterase activity from plaice muscle.The provision of live plaices from Kristineberg Zoological Station, Fiskebäckskil, Sweden, is gratefully acknowledged. We are greatly indebted to Dr. Åke Bovallius, FOA, who provided the starting material for the bacterial enzyme. We would like to express our sincere thanks to Prof. Lennart Nicander for valuable discussions and for placing the resources of the Department of Anatomy and Histology, Royal Veterinary College, to our disposal.     

Heterogeneity among human T cell clones recognizing an HLA-DR4,Dw4-restricted epitope from the 18-kDa antigen of Mycobacterium leprae defined by synthetic peptides.

Synthetic peptides have been used to exactly define a T cell epitope region from the immunogenic 18-kDa protein of Mycobacterium leprae. Four M. leprae reactive CD4+ T cell clones, isolated from two healthy individuals vaccinated with killed M. leprae, recognized a determinant initially defined by the peptide (38-50). However, fine mapping of the minimal sequence required for T cell recognition revealed heterogeneity among the T cell clones with regard to the N- and carboxyl-terminal boundaries of the epitopes recognized. MHC restriction analysis showed that the immunogenic peptides were presented to the T cells in an HLA-DR4,Dw4-restricted manner in all cases. The results suggest that a polyclonal T cell response representing different fine specificities is directed toward a possible immunodominant epitope from the M. leprae 18-kDa Ag in individuals carrying this MHC haplotype.     

13C NMR spectroscopy of Pyrrolizidine alkaloids

The ¹³C NMR spectra of nine pyrrolizidine alkaloids of the macrocyclic diester type, seven of the corresponding N-oxides and of the parent base retronecine have been recorded and the signals assigned. The 13C NMR signals were found to be sensitive to structural variation in both the diester moiety and the heterocyclic ring system, providing useful information for structural elucidation, particularly when the ¹H NMR spectra may be difficult to interpret.     

Neutralization of HIV-1 by anti-idiotypes to monoclonal anti-CD4. Potential for idiotype immunization against HIV.

Anti-idiotypic antibodies were raised in rabbits against a panel of 11 murine mAb directed to the human CD4 receptor. Selection of mAb for vaccination was based on inhibition studies demonstrating that these mAb recognized CD4/V1 epitopes implicated in HIV-1-gp120 binding. Purified antisera showed high titer anti-Id activity and reacted specifically with Ag-combining site-related Id of the mAb used for their generation. Anti-Id either detected a private Id of the immunizing mAb or displayed a partial cross-reactivity with Id of other mAb to CD4. Eight anti-Id to six different mAb were shown to recognize determinants of recombinant HIV-1-gp120 or of HIV-1-gp160 as shown by ELISA and radioimmunoprecipitation assay. These anti-Id were capable of inhibiting HIV infection up to 100% in a MT-4 cell assay in vitro. In addition to neutralizing infectivity of cell-free virus, anti-Id to two mAb--the mAb IOT4a and 7.3F11--were also shown to inhibit HIV-induced syncytia formation up to 100%. Anti-Id to the mAb IOT4a, 7.3F11, and to the mAb anti-Leu3a interfered with rgp120 binding to cellular CD4 as assessed by flow cytometry. These results demonstrated that mAb specific for both CDR2- and CDR3-like regions of CD4 were capable of inducing HIV-1-gp120 cross-reacting anti-Id neutralizing HIV-1 in vitro. These studies may have implications for the development of a gp120 internal image based vaccine against HIV.     

Determination and Health-Risk Evaluation of Nitroxynil Residues in the Edible Tissue of Cattle

A specific Polarographic method with a sensitivity of ≥ 2 jig/kg (ppb) has been used to determine the plasma and tissue concentrations of nitroxynil (NTX), which is used against Parafilaria bovicola in cattle. After treatment with the therapeutic dose on NTX (2 × 20 mg/kg b.w., s,c), there was an initial rapid decrease in the plasma concentration followed by a slower elimination phase. The plasma levels of NTX were 8 mg/kg (ppm) and 3 mg/kg after 6 weeks and 2 months, respectively. The muscle and other edible tissue from treated cattle contained 0.1–0.3 mg/kg NTX after 2 months, and jxg/kg amounts were still detectable 3 months after the injection. Based on available pharmacological and toxicological data, a 3-months withdrawal time for NTX in cattle is proposed.