Brain stem death and organ donation.

Organs for donation are in short supply in the United Kingdom, resulting in allegations that relatives of potential donors are not being asked for consent. Legislation on "required request" has been proposed to overcome this. The incidence, causes, complications, and patterns of organ donation in brain stem dead patients in one referral centre were studied over 12 months. Data were collected on all patients fulfilling criteria for brain stem death or considered suitable for donating organs after circulatory arrest. Forty two patients fulfilled the criteria for brain stem death, and in 10 further patients circulatory arrest occurred before formal testing was finished. The major causes of brain stem death were head injury (28) and intracranial haemorrhage (17). Consent to organ donation was obtained for 24 potential donors, and organs were donated by 23 of them. Twenty nine patients did not donate organs. The commonest reasons for failure to donate were medical unsuitability (13) and the coroner not releasing the body (eight). Consent was not sought in three cases, and the relatives refused consent in the remaining five. This study suggests that required request will not considerably increase the supply of donor organs.     

The effects of antibodies and complement in macrophage-mediated cytotoxicity on metacercariae of the lung fluke, Paragonimus westermani

Paragonimus westermani is a tissue migrating parasite in the early stage until arriving at lung, and most of the parasites spend their life spans there. Considerable immune responses including activation of macrophages are taken place during the residence of parasites in the host. However, concerning the immunologic defense mechanisms of the host against this parasite, only a few document is available so far. In this study, the cytotoxic effect of peritoneal macrophages under the presence of antibody and/or complement against metacercariae of P. westermani was investigated in vitro. Metacercariae were collected from the crayfish, Cambaroides similis and hatched out in Tyrode solution (pH 7.4). Plastic adherent cells from normal or infected rat (Wistar) peritoneal exudates were used as experimental macrophages. Polyclonal antibodies were obtained from infected rats and a cat. Cat IgG was fractioned with ion exchange chromatography. Fresh rabbit complement was used according to experimental scheme. Various combinations of peritoneal macrophages, normal or infected rat serum, complement and cat IgG were incubated at 36 degrees C in 5% CO2 incubator for 6, 14, 24 and 48 hours. The results obtained were as follows: 1. P. westermani infection activated peritoneal macrophages non-specifically and this activation induced increases of cell adherence and cytotoxicity on metacercariae. 2. In the presence of infected rat serum the antibody-dependent cell-mediated cytotoxicity of peritoneal macrophages on metacercariae was significantly increased and showed a peak at 6-hour incubation. But the cytotoxic effect was markedly reduced after inactivation of complement and heat-labile IgE antibody by the heating of infected serum at 56 degrees C for 30 minutes. 3. The highest cytotoxic effect (100%) of concomitant incubation with IgG and complement showed 24 hours after incubation, although cell adherence was relatively low at 6-hour incubation and 0% at 24-hour incubation. 4. Coordinative functions of complement with serum and IgG were effective in cell adherence and in cytotoxicity, but it is not clear the independent role of complement on the macrophage-mediated cytotoxicity in this study. With these results it is assumed that P. westermani infection can induce the non-specific activation of peritoneal macrophages, and serum antibodies including IgE antibody might enhance the cytotoxicity by macrophages.     

Salmonella typhimurium metC operator-constitutive mutations

We used an Escherichia coli lac deletion strain lysogenized with a metC-lacZ fusion phage (lambda Clac) to select operator-constitutive mutations in the Salmonella typhimurium metC gene control region. The mutations were located in a region containing 2 tandemly repeated 8 bp palindromes previously proposed to be the MetJ repressor binding site. Lysogens carrying lambda Clac mutant phage exhibit high beta-galactosidase levels that are only partially repressible by methionine. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product.     

The essential role of hypusine in eukaryotic translation initiation factor 4D (eIF-4D). Purification of eIF-4D and its precursors and comparison of their activities

Eukaryotic translation initiation factor 4D (eIF-4D) is the only protein known to contain the amino acid, hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. This unusual amino acid is formed post-translationally by modification of a single specific lysine residue in an eIF-4D precursor protein. Two separate eIF-4D precursors, each of which contains a lysine residue in place of the hypusine residue and each of which thereby serves as a protein substrate for the hypusine modification, were purified from DL-2-difluoromethylornithine-treated Chinese hamster ovary cells by means of a five-step procedure. These two precursors termed PI and PII both have apparent molecular masses of approximately 17 kDa, indistinguishable from that of eIF-4D, but exhibit more acidic isoelectric points (5.1 and 5.25 for PI and PII, respectively, compared with 5.37 for eIF-4D). These physical characteristics, together with other properties, indicate that eIF-4D differs from PII only in possessing the hypusine residue in place of a lysine residue, whereas an additional structural difference exists between PI and eIF-4D. eIF-4D from CHO cells provides a significant enhancement of methionyl-puromycin synthesis, a model assay for translation initiation. Neither PI nor PII stimulates this in vitro system. These findings are the first direct evidence that hypusine is essential for the biological activity of eIF-4D.     

Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization

The N-nitrosodimethylamine demethylase (P450I-IE1) is induced severalfold in liver by giving rats ethanol, acetone, pyrazole, and other related small molecular weight compounds. This induction is not the result of an increase in IIE1 mRNA, but could be due to either an increase in translation rate or a decrease in protein degradation. To determine the mechanism of induction, we measured IIE1 synthesis and degradation rates in untreated and acetone-treated rats. This was accomplished by immunopurification of radiolabeled IIE1 protein using a specific monoclonal antibody subsequent to in vivo labeling of total cellular protein with either NaH14CO3 or [3H]leucine. We found that in rats fed acetone, the rate of IIE1 synthesis was not changed; however, IIE1 degradation was markedly altered. In untreated rats, IIE1 protein was degraded via a biphasic pathway consisting of both a rapid and slow component with approximate half-lives of 7 and 37 h, respectively. However, in acetone-treated rats, only a monophasic curve with a half-life of 37 h was observed. The abolition of the rapid degradation component of the IIE1 turnover cycle indicates that induction of IIE1 by acetone is primarily due to specific stabilization of IIE1 protein. Since acetone is also metabolized by IIE1, we believe that this may be a substrate-induced enzyme stabilization.     

Light Activation of Pyruvate, Orthophosphate Dikinase in Maize Mesophyll Chloroplasts: A Role for Adenylate Energy Charge

Pyruvate, orthophosphate dikinase (EC 2.7.9.1 [EC] ) was activatedin the light and inactivated following a dark treatment in intactmaize mesophyll chloroplasts. Addition of catalase (100–250units/ml) to the assay medium was necessary to obtain good activationand to keep the enzyme in an active state during illumination.Arsenate and carbonyl cyanide m-chlorophenyl-hydrazone, uncouplersof photophosphorylation, inhibited the activation. Pyruvate,which has been proposed to have a critical role in supportingthe light activation of pyruvate, orthophosphate dikinase, actuallyinhibited the activation. The pyruvate level in the chloroplastsuspension decreased when the enzyme was light-activated. Measurementsof adenylates and pyruvate in the chloroplasts indicated thatthe energy state of the chloroplasts was more important forthe light activation than was the level of pyruvate. ¹Present address: Department of Biochemistry, Faculty of Science,Saitama University, 255, Shimo-Okubo, Urawa, 338 Japan ²Present address: National Institute of Agrobiological Resources,Yatabe, Tsukuba, Ibaraki, 305 Japan (Received May 2, 1989; Accepted October 2, 1989)     

Senescence and stomatal aperture as affected by antibiotics in darkness and light

In leaves of barley (Hordeum vulgare), as previously found with oats (Avena sativa), a group of six antibiotics that interfere in different ways with the sequence DNA → mRNA → protein all delay senescence in the dark, acting to conserve chlorophyll (Chl) and protein and also to open the stomata. Among the active compounds is chloramphenicol, which had previously been reported to act only on procaryotes. It is now shown that all these compounds with senescence-delaying action in darkness have the opposite effect in light, accelerating Chl destruction and partially or completely closing the stomata. Leaves of the dicot Tropaeolum majus show most of the same responses, though the changes in protein and amino acids are more variable. The data as a whole support the previous conclusion that the synthesis of one or more proteins controls both the opening and the closing of the stomata. An additional compound, kanamycin, acts in the same way as the other six compounds on oats and barley, though its action on proteolysis is unclear. On Tropaeolum, however, it opens the stomata in both light and darkness. Anisomycin and ethidium bromide have comparably atypical effects. Thus, although changes in stomatal opening or closing in the majority of cases are closely linked to the breakdown or preservation of Chl, the occasional exception shows that the biochemical phenomena of senescence cannot be under the direct control of changes in stomatal aperture.     

The effects of gamma irradiation on the survival and development of Clonorchis sinensis metacercariae

The effects of gamma irradiation on the survival and development of C. sinensis metacercariae were studied to evaluate the feasibility of irradiation as a control measure for clonorchiasis. Pseudorasbora parva were collected at an endemic river of clonorchiasis and were used for irradiation of the fluke in three schemes. The first (Scheme 1) was irradiation of the isolated metacercariae from the fish followed by infection to experimental rats. The second (Scheme 2) was irradiation of the fish, and then the metacercariae were isolated and infected to rats. The third (Scheme 3) was irradiation on the rat livers after infection with normal metacercariae. Irradiation doses varied from 5 to 100 Gy for Schemes 1 and 2, and 10 to 25 Gy for Scheme 3. The rats were sacrificed 2 to 6 weeks after infection. In Scheme 1, the metacercariae irradiated at 50 Gy failed to survive in the rats after 2 or 6 weeks. However, 1 to 44% of the metacercariae irradiated at 5-30 Gy survived. The estimated LD50 of Scheme 1 was 16.5 Gy. The flukes irradiated in Scheme 2 survived better than those in Scheme 1. The average worm recovery rate in 50 Gy was 28%(7-39% individually). Increasing the dose up to 100 Gy brought a remarkably low survival rate of an average 1%(0-3% individually). The LD50 of Scheme 2 was 47.5 Gy. Worm recovery rates in the 10 Gy group of Scheme 3 were 21-39%, and those in the 25 Gy group were 2% and 34%. Although the metacercariae were irradiated, all of the recovered worms were morphologically normal.(ABSTRACT TRUNCATED AT 250 WORDS)