On the role of rat liver cytosol in suppression of low-level chemiluminescence during nonenzymatic lipid peroxidation.

1. The effect of normal rat liver cytosol on the level of Fe/ADP-ascorbate-induced lipid peroxidation in the total particulate fraction (mitochondria plus microsomes) has been studied. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde (MDA) formation. 2. Dialysed cytosol significantly decreased the level of chemiluminescence, and to a much lesser extent, the rate of MDA production. 3. Gel filtration on a Sephadex G-200 column led to appearance of at least three cytosolic fractions which suppressed the low-level chemiluminescence. 4. The discovered components differed from each other by their molecular masses, kinetics of chemiluminescence inhibition and effects on intensity of MDA formation. 5. The putative functional role of antioxidative defence factors from rat liver cytosol is discussed.


Control of the cell cycle.

Cell division is arguably the most fundamental developmental process for single-celled and multicellular organisms alike. The pathway from one cell division to the next is known as the cell cycle. A conserved biochemical regulatory network controls progress along this pathway in plants, animals, and yeasts. This review is intended to serve as a primer on the current state of the eukaryotic cell cycle regulatory model, an introduction to the special roles of cell division and its control in plant development, and a review of recent progress in applying the universal mitotic control paradigm to higher plant systems.


The 29-kDa hemocyte proteinase dissociates fat body at metamorphosis of Sarcophaga.

Previously, we reported the purification of a 29-kDa proteinase from the pupal hemocytes of Sarcophaga peregrina (flesh fly). Antibody against this proteinase was found to inhibit dissociation of the fat body by pupal hemocytes in vitro. Moreover, the purified enzyme alone was shown to dissociate the fat body. This enzyme was found to be localized in granules of heterogeneous size in the hemocytes and to be released on their interaction with the fat body. From these results, we conclude that this 29-kDa proteinase participates in dissociation of the fat body at metamorphosis.


Mesenchyme of embryonic reproductive ducts directs process outgrowth of Retzius neurons in the medicinal leech.

In the two segments of the medicinal leech (Hirudo medicinalis) that contain the male (segment 5) and the female (segment 6) reproductive ducts, the paired Retzius (Rz) neurons are distinguished by several unique properties. For example, the muscles and glands of the body wall are the primary peripheral targets of Rz neurons in standard segments [Rz(X)], whereas the muscles and glands of the reproductive ducts are the primary peripheral targets of Rz neurons in the two reproductive segments [Rz(5,6)]. In this paper, we show that organogenesis and differentiation, which generate an epithelial tube surrounded by mesenchymal cells, occur in the embryonic reproductive ducts at approximately the time when Rz processes first contact these structures. The growth cones leading one branch of the posterior axon of Rz(5,6) contact the duct mesenchymal cells. Following initiation of this contact, these posterior growth cones enlarge and send out numerous filopodia. Secondarily, growth cones leading the anterior axon of each Rz(5,6) also modify their shapes and trajectories. When embryonic reproductive ducts were transplanted into posterior (nonreproductive) segments, the branch of the posterior Rz axon near the ectopic reproductive tissue produced enlarged growth cones and extended several secondary branches into the mesenchyme of the ectopic tissue. This result suggests that the reproductive mesenchyme is attractive to, and can modify the growth of, all Rz neurons. The behavior of Rz(5,6) growth cones suggests that the reproductive mesenchyme cells provide guidance cues that control the location in which Rz axons elaborate their peripheral arborization and form synapses, and that the mesenchyme may also stimulate the production of a densely branched arbor.


Structure and expression of the Xenopus retinoblastoma gene.

We have cloned a Xenopus homology (XRb1) of the human retinoblastoma susceptibility gene. DNA sequence analysis shows that the XRb1 gene product is highly conserved in many regions. The leucine repeat motif and many of the potential cdc2 phosphorylation sites, as well as potential sites for other kinases, are retained. The region of the protein homologous to the SV40 T antigen binding site and the basic region directly C-terminal to the E1A binding site are all conserved. XRb1 gene expression at the RNA level was studied by Northern blot analysis. Transcripts of 4.2 and 10-kb are present as maternal RNA stores in the oocyte. While the 4.2-kb product is stable until at least the mid-blastula stage, the 10-kb transcript is selectively degraded. Between stages 11 and 13 the 10-kb transcript reappears and also a minor product of approximately 11 kb becomes apparent. Both the 4.2- and the 10-kb transcripts remain present until later stages of development and are also present in all adult tissues examined, although at differing levels. Antibodies raised against human p105Rb which recognize the protein product of the XRb1 gene, pXRb1, detect the Xenopus 99-kDa protein prior to the mid-blastula stage, but at lower levels than at later stages in development.


Characterization of RNA polymerase II-dependent transcription in Xenopus extracts.

We examine the RNA polymerase II-dependent transcription directed by several promoters in extracts prepared from distinct developmental stages of Xenopus laevis. RNA polymerase II accurately initiates transcription from the cytomegalovirus, herpes simplex virus thymidine kinase, and Xenopus heat-shock protein (hsp) 70 promoters. The efficiency of transcription of these different promoters is dependent on whether extracts from oocytes, eggs, or somatic cells are used and on the temperature of incubation. In contrast to the viral promoters, the hsp 70 promoter is more active at heat shock temperatures in oocyte and egg extracts (31 degrees-34 degrees C) than at physiological temperatures for Xenopus (20 degrees-25 degrees C). These in vitro transcription extracts should be useful in examining the molecular mechanisms responsible for differential gene expression during Xenopus development.


Transient increase in expression of a glutamate decarboxylase (GAD) mRNA during the postnatal development of the rat striatum.

We recently reported that the mammalian brain has two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD, E.C. 4.1.1.15), which are the products of two genes. The two forms, which we call GAD65 and GAD67, differ from each other in sequence, molecular size, subcellular distribution, and interactions with the cofactor pyridoxal phosphate (PLP), with GAD65 activity more dependent than that of GAD67 on the continued presence of exogenous PLP. The existence of two GAD genes suggests that individual GABA neurons may be subject to differential regulation of GABA production. We have examined the expression of these two forms of GAD during postnatal development of the rat striatum to determine whether different classes of GABA neurons selectively express different amounts of the two GAD mRNAs. Here we present evidence for a dramatic developmental difference in the expression of the two mRNAs during postnatal development of the rat striatum. Using in situ hybridization to the two GAD mRNAs, we observed a selective increase in GAD65 mRNA during the second postnatal week, at the time when striatal matrix neurons innervate the substantia nigra (SN). PLP-dependent enzyme activity in the midbrain increases in parallel with increased expression of GAD65 mRNA in the striatum. We hypothesize that the innervation of the SN by striatal neurons triggers an increase in GAD65. The changing ratios of GAD65 and GAD67 in the striatum may contribute to the well-documented changes in seizure susceptibility that occur in early life.


Roles for the extracellular matrix in plant development and pollination: a special case of cell movement in plants.

Pattern formation in plants is now thought to be primarily dependent on positional information during development. We discuss the prevalent theories on how position is deciphered by cells in an organism and highlight the recent advances implicating molecules of the cell wall or extracellular matrix (ECM) in this process. We compare the functions of the ECM in plants and animals and describe the various cell and substrate adhesion molecules of the animal ECM which play a role in morphogenesis and cell movement. We propose that analogous molecules may occur in plants and provide evidence for the presence of a substrate adhesion molecule like vitronectin in plants and algae. We provide a model for how substrate adhesion molecules may be involved in a special case of cell movement in plants, pollination.


Evidence that metalloendoproteases are involved in gamete fusion of Ciona intestinalis, ascidia.

The use of specific inhibitors and substrates of metalloendoproteases provides evidence that in many systems these enzymes are involved in membrane fusion events. In this study, we investigated whether metalloendoproteases are involved in Ciona sperm-egg fusion. In vitro fertilization assays with the metal chelator 1,10-phenanthroline, specific metalloendoprotease substrates, and the vital stain Hoechst 33342 suggested that a Zn(2+)-dependent metalloendoprotease(s) takes part in Ciona sperm-egg fusion. Furthermore, electrophysiological recordings showed that insemination carried out in the presence of either 1,10-phenanthroline or the substrate CBZ-Gly-Phe-NH2 fails to induce fertilization potential or any other change in membrane potential. These results support the hypothesis that in Ciona intestinalis, a metalloendoprotease(s) is functional in gamete fusion.


Differential regulation of the transforming growth factor type-beta 2 gene promoter in embryonal carcinoma cells and their differentiated cells.

Previous studies have shown that EC cells do not express detectable levels of TGF-beta 2 or its mRNA until they differentiate. This suggested that differentiation influences the transcription of the TGF-beta 2 gene in this model system. To address this possibility, we have examined the activity of the TGF-beta 2 promoter in EC cells and their differentiated cells using gene constructs containing various portions of the TGF-beta 2 promoter inserted upstream of the reporter gene, chloramphenicol acetyltransferase (CAT). We determined that the level of CAT increases approximately ninefold when EC cells were induced to differentiate. Our studies also indicate that the TGF-beta 2 promoter contains at least two positive regulatory elements that are separated by a negative regulatory element. Finally, we have identified a CRE/ATF-like site that appears to be responsible for a positive regulatory element located between -77 and -40.


The maryland mammoth allele and rooting both perturb the fate of florally determined apices in Nicotiana tabacum.

The stability of the florally determined state in terminal and axillary buds of two tobacco cultivars was studied. We used Hicks and Hicks Maryland Mammoth, near-isogenic cultivars of Nicotiana tabacum differing at the recessive maryland mammoth locus which confers short-day behavior. The experimental design consisted of growing plants in short-day conditions and subjecting them to three bioassays in long-day conditions: in vitro culture of apices consisting of meristems and three to four leaf primordia; rooting of buds consisting of meristems and 8 to 12 leaves, leaf primordia, and internodes; and release from apical dominance of axillary buds in situ. Cultured terminal and axillary apices expressed floral determination, indicating that meristems can be florally determined. Two lines of evidence indicate that rooting destabilizes an already acquired florally determined state: cultured apices from both axillary and terminal buds produced fewer nodes after excision than homologous buds which were rooted; and a lower percentage of rooted axillary buds from Hicks Maryland Mammoth plants expressed floral determination than did homologous axillary buds grown out in situ in noninductive conditions. Rooted buds from the two genotypes expressed floral determination at different frequencies, but produced abnormal inflorescences at similar frequencies, indicating that roots and the maryland mammoth allele influence common as well as unique processes associated with floral determination.


Biophysical mechanisms for morphogenetic progressions at the shoot apex.

Leaf primordia, first visible as small bumps, are produced in a cyclical pattern at the edges of the shoot apex, a smooth region at the top of the stem. Their formation is a biomechanical process. This review first considers hypothetical construction mechanisms and then summarizes research that provides information about how and where the primordia are made. Studies of growth at the primordium site indicate the importance of growth parallel to the surface in generating the forces for primordium emergence. The symmetry of the pattern of reinforcement by cellulose microfibrils correlates with the subsequent pattern of primordium production. Finite element models of the apex reveal that lateral bulging of the apex results in a gradient of shear stress, with high shear at the future primordium site. In contrast, tension parallel to the surface is lowest at the primordium site. Response of apical surface tissue to punctures indicates that an existing primordium can exert a pulling force tangential to its base and a compressive force perpendicular to its base. These observations lead to identification of a continuous biophysical cycle for apex morphogenesis, in which most of the steps are direct physical consequences of the previous step. Biophysical processes, subject to input from genetic, hormonal, and environmental sources, are thus involved in the construction and patterning of leaf primordia.


A conceptual framework for maize leaf development.

What is and is not known about the maize leaf is reviewed. Analysis of genetic mosaics and direct observation with the SEM have broken leaf development into three distinct phases: recruitment of cells within the meristem, cell division into the 0.6-mm tall primordium, and postprimordial division and differentiation into the mature leaf. New data are presented that imply that cell division rates in the leaf are coordinated by inductive signals from the internal cells. Leaf cells that tend to divide more are held in check by slower growing neighbors; this complicates the search for developmental compartments. Experiments with recessive mutants that remove the ligule and auricle have been important in identifying an inducer signal with the specific meaning "make ligule-auricle." We have studied many dominant mutant alleles at seven different genes. Each mutant alters the position of the ligule boundary. We conclude the following. First, the mutants act in particular domains of the primordium. Second, the dominant mutants all move the ligule boundary in the same direction. Third, the mutants all retard developmental stage transitions. Fourth, three and probably four of the seven genes for which dominant mutants have been studied specify homeodomain proteins in the wrong place. The concept of "maturation schedule" is used to explain these data. All of the dominant mutant phenotypes are seen as consequences of immature cells being in the wrong place when inductive signals pass through the leaf. Several specific questions of leaf development and especially questions as to source of inductive signals or homologies among juvenile and adult organ parts are recast in light of this "maturation schedule" hypothesis.


Development states associated with the floral transition.

Floral initiation can be analyzed from a developmental perspective by focusing upon how developmental fates are imprinted, remembered, and expressed. This is not an altogether new perspective, since people studying flowering have been concerned for a long time with the commitment of meristems to form flowers and the morphological, cellular, and molecular changes associated with this commitment. What is novel is the emphasis on developmental states as opposed to physiological processes. This developmental focus indicates that there appear to be at least three major developmental states that are acquired and expressed in the process of a meristem initiating floral morphogenesis. The meristem cells must first become competent to respond to a developmental signal that evokes them into a florally determined state. The leaves are the usual source of this signal and a specific leaf may or may not have the capacity to be inductively active. When a leaf does develop the capacity for inductive activity, this capacity is usually correlated with the ontogeny of the leaf. Inductive activity, however, may be continually expressed as in some day-neutral plants or may be latent as in plants where the photoperiod is the external cue for activity. Competent shoot apical meristems respond to inductive leaf signal by being evoked into a florally determined state. Under permissive conditions this florally determined state is expressed as the initiation of floral morphogenesis. Many mechanisms have evolved to regulate entry into and expression of these developmental states. As we learn more about the developmental states associated with flowering and how they are acquired and expressed, we will understand better how the various patterns of flowering are related to one another as well as which developmental processes are common to all angiosperms.


Cell-cell communication in plants: self-incompatibility in flower development.

Self-incompatibility, a mechanism that prevents self-fertilization in plants, is based on the ability of the pistil to discern the presence of self-pollen and on the female tissue's capacity to inhibit the growth or germination of self-related, but not of genetically unrelated, pollen. As a self-recognition system, self-incompatibility responds to specific cellular products and signals and thus offers a unique system in which to study the components of cellular communication in plants. The cytological manifestations of self-incompatibility have been well studied, and, with the cloning of cDNAs for several proteins associated with this recognition process, a detailed molecular view of self-incompatibility is emerging.


Mammalian sperm-egg fusion: the rat egg has complementary sites for a sperm protein that mediates gamete fusion.

Rat epididymal protein DE is localized on the fusogenic region of the acrosome-reacted spermatozoa and has a potential role in sperm-egg fusion. We investigated the presence of DE binding sites on the egg surface by co-incubating zona-free eggs and capacitated sperm in different concentrations of pure DE. Results indicate that DE produced a concentration-dependent decrease in egg penetration by sperm (fusion), with almost complete inhibition at 200 micrograms/ml. This inhibition was not due to an effect of DE on initial sperm binding to the egg membrane, since the presence of this protein did not affect the percentage of oocytes with bound sperm nor the number of bound sperm per egg. Those sperm that failed to penetrate the egg in the presence of DE became able to do so after transfer of the eggs to protein- and sperm-free medium, indicating a role for DE in an event subsequent to binding and leading to fusion. Indirect immunofluorescence using a polyclonal antibody against DE revealed a patchy labeling over the entire egg surface, with the exception of the area overlying the second metaphase spindle. This conclusion was supported by the disappearance of the DE-negative area on the fertilized egg. Zona-free eggs, incubated with DE at 4 degrees C or fixed before exposure to DE, displayed a uniform staining, suggesting that the patchy labeling resulted from aggregation of DE binding sites by the purified protein. The aggregation of these egg components may represent a necessary step of the fusion process. To our knowledge, this is the first study reporting the existence and localization of complementary sites to a specific sperm protein on the plasma membrane of the mammalian egg.


Patterning the zebrafish heart tube: acquisition of anteroposterior polarity.

The patterning of an internal organ, like the heart, is little understood. Central to this patterning is the formation, or the acquisition, of an anteroposterior (A-P) axis. We have approached the question of how the heart tube acquires polarity in the zebrafish, Brachydanio rerio, which offers numerous advantages for studying cardiac morphogenesis. During the early stages of organogenesis in the fish, the heart tube lies in an A-P orientation with the venous end lying anteriorly and the arterial end lying posteriorly. High doses (10(-6)-10(-5)M) of retinoic acid (RA) cause truncation of the body axis, as they do in Xenopus. Low doses of retinoic acid (10(-8)-10(-7) M), which do not appear to affect the rest of the embryo, have pronounced effects upon heart tube morphogenesis, causing it to shrink progressively along the A-P axis. To investigate this further, we identified monoclonal antibodies that distinguish between the zebrafish cardiac chambers and used them to show that the RA-induced cardiac truncation always begins at the arterial end of the heart tube. There is a continuous gradient of sensitivity from the arterial to the venous end, such that increasing RA exposure causes the progressive and sequential deletion first of the bulbus arteriosus and then, in order, of the ventricle, the atrium, and the sinus venosus. As exposure increases, parts of chambers are deleted before entire chambers; thus, the sensitivity to RA appears to be independent of chamber boundaries. The analysis of the heart tube's sensitivity to RA and its timing suggest that polarity is established during or shortly after initial commitment to the cardiac lineage.


Expression of the brain-type glucose transporter is restricted to brain and neuronal cells in mice.

Northern blot analysis of human tissues has demonstrated the expression of the brain-type glucose transporter isoform (GLUT 3) in liver, muscle and fat, raising the possibility that this transporter isoform may play a role in the regulation of glucose disposal in these tissues in response to insulin. We have raised an anti-peptide antibody against the C-terminal 13 amino acids of the murine homologue of this transporter isoform, and determined its tissue distribution in mouse tissues and murine-derived cell lines. The antibodies recognise a glycoprotein of about 50 kilodaltons, expressed at high levels in murine brain. In contrast to human tissues, the expression of GLUT 3 in mice is restricted to the brain, and no immunoreactivity was observed in either liver, fat or muscle membranes, or in murine 3T3-L1 fibroblasts or adipocytes. In contrast, high levels of expression of this isoform were observed in the NG 108 neuroblastoma x glioma cell line, a hybrid cell derived from rat glioma and mouse neuroblastoma cells. Taken together, these data suggest that the expression of GLUT 3 in rodents is restricted to non-insulin responsive neuronal cells and hence it is likely that the factors regulating the expression of this transporter in rodents differ to those in humans.


The contribution of hyperglycaemia and hypoinsulinaemia to the insulin resistance of streptozotocin-diabetic rats.

The relative contribution of hyperglycaemia and hypoinsulinaemia was evaluated in rats made diabetic by streptozotocin administration. Four groups of rats were studied: untreated normal rats; streptozotocin-diabetic; streptozotocin-diabetic treated with phlorizin (0.4 mg/kg body weight per day); streptozotocin-diabetic mildly treated with insulin (0.7 IU/day). In all groups, insulin action (responsiveness) was assessed with the euglycaemic (5.3 mmol/l) hyperinsulinaemic (524 mU/l) clamp technique combined with 3H-2-deoxy-D-glucose method, enabling determination of the glucose utilization index in various tissues. Responsiveness of the overall glucose utilization process to insulin was reduced by 28% in streptozotocin-diabetic rats (12.0 +/- 1.2 vs 16.5 +/- 0.6 mg.kg-1.min-1, p less than 0.001). This was associated with a significant reduction (p less than 0.05) in the glucose utilization index in all muscles studied (average = 17.0 vs 32.1 ng.mg of tissue-1.min-1), in the heart (19.6 vs 39.5 ng.mg-1.min-1), brown adipose tissue (98.9 vs 178.0 ng.mg-1.min-1), skin (6.4 vs 13.1 ng.mg-1.min-1). Phlorizin treatment normalized plasma glucose levels without affecting those of insulin, and restored overall glucose utilization to normal (16.6 +/- 1.0 mg.kg-1.min-1). This normalization was accompanied by a normalization of the glucose utilization index in all muscle types studied (29.2 ng.mg-1.min-1), in the heart (50.0 ng.mg-1.min-1), brown adipose tissue (157.2 ng.mg-1.min-1), and skin (10.0 ng.mg-1.min-1). White adipose tissue, brain and gut were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunocytochemical localization of heat-shock protein 60-related protein in beta-cell secretory granules and its altered distribution in non-obese diabetic mice.

Immuno-electron microscopy technique was employed to investigate the cellular distribution of 60 kDa heat-shock protein (HSP60) in pancreatic Beta cells of control and non-obese diabetic mice. In thin sections prepared from control mice, antibody to mammalian HSP60 cross-reacted with protein(s) located to mitochondria and secretory granules. In particular, prominent binding of the antibody was seen to the insulin core of the mature insulin-secreting granules. In comparison, very little immunoreactivity was observed with immature secretory granules or with the Golgi apparatus. No binding to secretory granules or mitochondria was observed with normal mouse serum or with unrelated sera. On Western blots, HSP60 antibody specifically interacted with a single 62 kDa islet cell protein. These results suggest the existence of an HSP60-related protein with a novel location in mature secretory granules of Beta cells. The preferential association of the HSP60-related protein with the insulin core was gradually lost in Beta cells of pre-diabetic non-obese diabetic mice, and correlated with the progression of insulitis. The decrease in the granular binding of the HSP60 antibody was accompanied by an increase in cytoplasm staining, and was concomitant with a significant expansion of the insulin core diameter. The altered distribution of the HSP60-related protein in prediabetic mice, together with our observation that immature secretory granules accumulate in these animals indicate that the presence of HSP60-related protein in secretory granules might be associated with the secretory function of Beta cells.


Long-term suppression of postprandial hyperglycaemia with acarbose retards the development of neuropathies in the BB/W-rat.

The effect of the alpha-glucosidase inhibitor acarbose on postprandial hyperglycaemia was explored in the spontaneously diabetic BB/W-rat. Acarbose-treatment (5 mg.kg body weight-1.day-1) of diabetic BB/W-rats maintained on small doses of insulin, was associated with a 40% reduction in the 24-h glucose area compared to non-treated diabetic rats. Over a 4 month treatment period this reduction in cumulative hyperglycaemia resulted in a complete prevention of autonomic polyneuropathy as indicated by R-BAR values. The development of somatic polyneuropathy in the BB/W-rat was significantly attenuated by acarbose treatment with a partial prevention of the characteristic nerve conduction velocity slowing during the first 3 months of diabetes, but no longer at 4 months. Characteristic structural abnormalities associated with diabetes in this model, such as axonal atrophy and axo-glial dysjunction, were significantly but only partially prevented in rats treated with acarbose for a diabetes duration of 4 months. These data suggest that postprandial lowering of hyperglycaemia resulting in a decrease in cumulative hyperglycaemia retards the development of diabetic polyneuropathies in the BB/W-rat.


Repetitive in vivo treatment with human recombinant interleukin-1 beta modifies beta-cell function in normal rats.

It is unknown whether interleukin-1 exerts a bimodal effect on Beta-cell function in vivo, and whether interleukin-1 has a diabetogenic action in normal animals. We therefore studied: (a) acute effects 2 h after an intraperitoneal bolus injection of 4 micrograms of recombinant human interleukin-1 beta per kg body weight on blood glucose, plasma levels of insulin, glucagon and corticosterone in Wistar Kyoto rats, either untreated or pre-treated with 4 micrograms/kg of interleukin-1 daily for 3 or 5 days; (b) the cumulative effects of repetitive intraperitoneal injections of 4 micrograms/kg interleukin-1 on blood glucose, glucose tolerance, plasma levels of insulin, glucagon and corticosterone, pancreatic insulin content and pancreatic ultrastructure; and (c) blood glucose and plasma concentrations of insulin, glucagon and corticosterone 10 h after the last of five intraperitoneal injections of interleukin-1, at which time point the inhibitory effect of short-term interleukin-1 exposure on insulin secretion reaches its nadir in vitro. A single injection of 4 micrograms/kg of interleukin-1 caused a slight, but significant lowering of blood glucose 2 h after interleukin-1 injection with no significant changes in plasma insulin and in spite of increases in plasma glucagon and corticosterone. A lowering of blood glucose 2 h after interleukin-1 administration was reproduced with 40, but not 0.4 micrograms/kg of interleukin-1, and was also seen in interleukin-1 pre-treated rats. Two hours after the fifth injection of interleukin-1, intraperitoneal glucose tolerance was impaired with elevated plasma insulin and corticosterone levels and increased pancreatic insulin content, indicating a state of insulin resistance.(ABSTRACT TRUNCATED AT 250 WORDS)


Habitual physical activity, aerobic capacity and metabolic control in patients with newly-diagnosed type 2 (non-insulin-dependent) diabetes mellitus: effect of 1-year diet and exercise intervention.

The aim of this study was to assess the effects of a 1-year intensified diet and exercise education regimen on habitual physical activity and aerobic capacity in middle-aged, obese patients with newly-diagnosed Type 2 (non-insulin-dependent) diabetes mellitus. In addition, we analysed whether the level and the changes in physical activity and aerobic capacity are related to the metabolic control of diabetes. After a 3-month basic education programme, 78 patients (45 men, 33 women) were randomly placed in an intervention or conventionally treated group. The intervention group received intensified diet education and continuous encouragement to increase physical activity which was monitored using exercise records and questionnaires. Aerobic capacity was assessed by measuring oxygen uptake at anaerobic threshold and at peak exercise. The proportion of patients with regular recreational exercise increased from 24% to 38% in the intervention men (0.10 less than p less than 0.20), remained at 54% in the conventionally treated men, increased from 53% to 70% in the intervention women (0.10 less than p less than 0.20) and from 31% to 50% (0.10 less than p less than 0.20) in the conventionally treated women. No measurable improvement was found in oxygen uptake in any of the groups. When the groups were combined, HbA1c showed an inverse correlation with oxygen uptake at anaerobic threshold (r = 0.27, p less than 0.01) and maximum oxygen uptake (r = 0.28, p less than 0.01) at 12 months.(ABSTRACT TRUNCATED AT 250 WORDS)


Metabolism of HDL apolipoprotein A-I and A-II in type 1 (insulin-dependent) diabetes mellitus.

Concentrations of HDL cholesterol and apolipoprotein A-I are commonly increased in Type 1 (insulin-dependent) diabetes mellitus but the mechanisms whereby diabetes influences HDL metabolism have not been studied. We investigated the metabolism of HDL apoproteins A-I and II in normolipidaemic Type 1 diabetic men (n = 17, HbA1 6.4-11.9%) without microalbuminuria but with a wide range of HDL cholesterol (0.85-2.10 mmol/l) and in nondiabetic men (n = 18) matched for body mass index and the range of HDL cholesterol. Input rates and fractional catabolic rates for apolipoproteins A-I and II were determined following injection of 125I-apolipoprotein A-I and 131I-apolipoprotein A-II tracers. Additional multicompartmental analysis was performed using a model to describe the kinetics of HDL particles containing only apolipoprotein A-I (Lp A-I) and apolipoprotein A-I and apolipoprotein A-II (Lp A-I/A-II). No gross differences from normal subjects were observed in the mean levels of lipids, lipoproteins, apoproteins and the lipolytic enzymes in the diabetic men as a result of the selection process. Furthermore, the relationship between apolipoprotein A kinetics and plasma HDL cholesterol levels appeared to be preserved in the diabetic group. However, some normal interrelationships were disrupted in the diabetic men. Firstly, the rate of apolipoprotein A-II synthesis was 22% lower than in control subjects (p less than 0.05). Modelling indicated that this was due to decreased input of Lp A-I/A-II particles whereas the input of Lp A-I particles was similar in the two groups. Secondly, there was no correlation between VLDL triglyceride and HDL cholesterol or VLDL triglyceride and the fractional catabolic rate of apolipoproteins A-I and A-II in diabetic men in contrast to that seen in control subjects. We conclude that there is a disruption in the normal association between VLDL and HDL metabolism in Type 1 diabetic men and postulate that the observed differences may be due to the therapeutic use of exogenous insulin.


Short-term regulation of insulin-mediated glucose utilization in four-day fasted human volunteers: role of amino acid availability.

Glucose homeostasis in men fasted for 84 h was assessed using isotopes, indirect calorimetry and forearm balance techniques during a basal period and three sequential hyperinsulinaemic euglycaemic clamps each lasting for 150 min. Two protocols (n = 12 in each) were used: subjects were either allowed to develop hypoaminoacidaemia or received a commercial solution of L-amino acids while maintaining near-basal plasma leucine levels. Insulin infusions resulted in 3-, 35- and 650-fold increases in plasma insulin levels in both protocols. The infusion of amino acids produced a rightward shift in the dose-response curve of insulin's effect on suppressing hepatic glucose production, indicating decreased sensitivity in addition to blunting of the maximal responsiveness. Total body glucose rate of disappearance was progressively increased with escalating insulin doses, but was 22% lower at the intermediate and highest insulin doses in the group that was infused with amino acids (3.44 +/- 0.53 vs 4.82 +/- 0.71 and 7.72 +/- 1.01 vs 10.36 +/- 1.08 mg.kg-1.min-1, respectively; p less than 0.05). Forearm balance data confirmed the isotopic data, since amino acid infusions blunted the insulin-mediated increase in net forearm glucose utilization (by 50-83%). Furthermore, the infusion of amino acids resulted in marked reductions in the rate of carbohydrate oxidation and storage as assessed by indirect calorimetry. The data indicate that the amino acid-mediated suppression of glucose utilization and carbohydrate oxidation is exerted on the responsive component of insulin action.


Dexamethasone treatment fails to increase arginine-induced insulin release in healthy subjects with low insulin response.

We have compared insulin responses to L-arginine before and during dexamethasone treatment in healthy subjects, previously classified as subjects with either high or low insulin response according to a standardized glucose infusion test. Arginine stimulation was administered as a 150 mg/kg bolus followed by 10 mg.kg-1.min-1 to six subjects with high insulin response and to seven subjects with low insulin response. Before dexamethasone treatment the incremental insulin level during 0-10 min of arginine was higher in subjects with high (36.5 +/- 6.8 microU/ml) than in subjects with low response (14.5 +/- 2.3 microU/ml), p less than 0.01 for difference. Dexamethasone treatment (6 mg/day for 60 h) markedly enhanced the insulin response to arginine in subjects with high response (+99% 0-30 min) but failed to affect the subjects with low response (+4% 0-30 min). The C-peptide response to arginine exhibited similar differences between groups. Decreased responsiveness to arginine in subjects with low insulin response, especially during dexamethasone treatment, suggests a Beta-cell capacity defect although a decreased potentiating-sensing effect of glucose cannot be completely ruled out.


Absence of the dawn phenomenon and abnormal lipolysis in type 1 (insulin-dependent) diabetic patients with chronic growth hormone deficiency.

To determine the role of growth hormone in overnight insulin requirements and lipolysis, five patients with chronic growth hormone deficiency and Type 1 (insulin-dependent) diabetes mellitus and six control patients with diabetes were each studied on two separate nights. Insulin was infused at a variable rate throughout one night to maintain euglycaemia and fixed at 04.00 hours on another. During the variable infusion, euglycaemia was maintained in control patients by a 36% increase in insulin infusion rate between 03.00 and 08.00 hours while a 46% decrease in the rate was required in growth hormone deficient patients (p less than 0.02). Despite this difference, mean free insulin values were equivalent. This finding is suggestive of increased insulin clearance in growth hormone sufficient patients. Glucose levels rose in control and fell in growth hormone deficient patients when insulin infusion rates were fixed at 04.00 hours. Glycerol production and non-esterified fatty acid concentrations were significantly lower in the growth hormone deficient diabetic patients, p less than 0.001, and when normalized with a heparin infusion, had no effect on insulin requirements. We conclude that: (1) growth hormone contributes to the development of the "dawn phenomenon," possibly by increasing insulin clearance (2) growth hormone helps sustain nocturnal lipolysis in Type 1 diabetes and (3) non-esterified fatty acids are not involved in the dawn phenomenon.


Binding of antibodies in sera from Type 1 (insulin-dependent) diabetic patients to glutamate decarboxylase from rat tissues. Evidence for antigenic and non-antigenic forms of the enzyme.

An islet protein of M(r) 64000, identified as the gamma-amino butyric acid (GABA)-synthesizing enzyme, glutamate decarboxylase, is a major target for antibodies in Type 1 (insulin-dependent) diabetes mellitus. This enzyme is also expressed in brain and in some other tissues and may exist in multiple forms. The aim of this study was to determine the ability of antibodies from diabetic patients to recognize glutamate decarboxylase from rat islets, brain and other normal rat tissues. Glutamate decarboxylase was detected at high activity levels in brain and at lower levels in islets, kidney, liver, pituitary gland, thyroid gland, adrenal gland, testis and ovary. The ability of antibodies in sera of diabetic patients to immunoprecipitate enzyme activity from detergent extracts of tissues was determined. Antibodies in sera from diabetic patients were found to bind the enzyme from islet and brain extracts, but bound less than 20% of the activity from other tissues. The ability of antibodies to immunoprecipitate the brain enzyme was significantly correlated with the presence of antibodies to the islet 64 kilodalton antigen. These studies show that the glutamate decarboxylase activity expressed in brain shares antigenic determinants with the islet 64 kilodalton antigen. Isoforms of the enzyme expressed in other non-neuronal tissues may be antigenically distinct and may lack determinants recognized by diabetes-associated antibodies.


Distinct cytoplasmic islet cell antibodies with different risks for type 1 (insulin-dependent) diabetes mellitus.

The cytoplasmic islet cell antibody patterns of sera from islet cell antibody positive non-diabetic and diabetic endocrine autoimmune patients, and newly-diagnosed Type 1 (insulin-dependent) diabetic patients were characterised using four layer immunofluorescence with monoclonal anti-proinsulin or anti-glucagon antibodies. Two distinct islet cell antibody types were identified. One gave a diffuse cytoplasmic staining in both Beta and Alpha cells ('whole' islet pattern), and was not affected by pre-incubation with rat brain homogenate. The other had a granular appearance with staining restricted predominantly to Beta cells ('selective' islet pattern) and was completely inhibited by pre-incubation with rat brain homogenate. Some sera appeared to have a 'mixed' islet pattern, in which glucagon-positive cells gave a weaker cytoplasmic staining than proinsulin-positive cells. The granular 'selective' pattern was found in sera from 19 (79%) of 24 non-diabetic endocrine autoimmune patients, in two (22%) endocrine autoimmune patients who developed Type 1 diabetes (p less than 0.0001 vs non-diabetic endocrine autoimmune patients), and in none of 19 newly-diagnosed diabetic patients. The 'whole' islet pattern was found only in sera from patients who had, or who subsequently progressed to, Type 1 diabetes. This study has identified a novel islet cell antibody specificity and demonstrates that in islet cell antibody positive endocrine autoimmune patients, only islet cell antibodies which stain both Beta and Alpha cells are associated with progression to Type 1 diabetes.


Hepatocarcinogenicity of chloral hydrate, 2-chloroacetaldehyde, and dichloroacetic acid in the male B6C3F1 mouse.

The chlorinated acetaldehydes, chloral hydrate (CH) and 2-chloroacetaldehyde (CAA), have been identified as chlorination by-products in finished drinking water supplies. Although both chemicals are genotoxic, their potential for carcinogenicity had not been adequately explored. The studies reported here are chronic bioassays conducted with male B6C3F1 mice exposed to levels of 1 g/liter CH and 0.1 g/liter CAA via the drinking water for 104 weeks. Distilled water (H2O) served as the untreated control and dichloroacetic acid (DCA; 0.5 g/liter), another chlorine disinfection by-product, was included. The mean daily ingested doses were approximately 166 mg/kg/day for CH, 17 mg/kg/day for CAA, and 93 mg/kg/day for DCA. Evaluations included mortality, body weight, organ weights, gross pathology, and histopathology. The primary target organ was the liver as the organ weights and pathological changes in the other organs (spleen, kidneys, and testes) were comparable between the treated groups and the H2O control group. Liver weights were increased for all three test chemicals at the terminal euthanasia with the greatest increase seen in the CH and DCA groups. Hepatocellular necrosis was induced by all three test chemicals, and it was also most prevalent and severe in the CH and DCA groups. A significant increase in the prevalence of liver tumors was seen for all three chemicals. The strongest response was with DCA, in which 63% of the 104-week survivors had hepatocellular carcinomas (carcinomas) and 42% possessed hepatocellular adenomas (adenomas) and the combined prevalence for carcinomas plus adenoma was 75%. The corresponding prevalence rate for carcinomas, adenomas, and combined tumors were 46, 29, and 71%; 31, 8, and 38%; and 10, 5, and 15% for CH, CAA, and H2O, respectively. In addition to the tumors we evaluated the prevalence of a possible preneoplastic lesion, the hepatocellular hyperplastic nodule (nodules), a lesion which occurred in all three treated groups but not in the H2O group.


Prechronic inhalation toxicity studies of isobutyl nitrite.

Isobutyl nitrite (IBN) is a volatile liquid that has become increasingly popular as an inhaled recreational drug. To investigate short-term toxic effects and establish exposure parameters for chronic inhalation studies, F344/N rats and B6C3F1 mice were exposed to IBN vapors on a 6 hr/day + t90, 5 days/week schedule. Twelve exposures were administered at concentrations of 0, 100, 200, 400, 600, and 800 ppm IBN. This exposure series resulted in mortality in rats exposed to greater than or equal to 600 ppm and mice exposed to 800 ppm. Animals exposed at the lower concentrations developed hyperplasia of the bronchiolar and nasal turbinate epithelium (rats and mice) and lymphocytic atrophy in the spleen and thymus (mice). Longer term, 13-week, subchronic exposures were conducted at concentrations of 0, 10, 25, 75, 150, and 300 ppm IBN. Exposure to 300 ppm IBN reduced the body weight gains in both sexes of rats and in female mice. IBN-related clinical pathology changes included reduced RBC counts accompanied by moderate increases in mean corpuscular volume and reticulocyte counts, increased WBC counts, and mildly increased methemoglobin concentration. Bone marrow hyperplasia was observed in all groups of IBN-exposed rats, while in mice only females at greater than or equal to 150 ppm IBN displayed this change. Excessive splenic pulp hematopoiesis was noted in mice at all IBN exposure levels.(ABSTRACT TRUNCATED AT 250 WORDS)


The developmental toxicity of 2-ethylhexanol applied dermally to pregnant Fischer 344 rats.

Undiluted 2-ethylhexanol (2-EH) was administered by occluded dermal application for 6 hr per day on Gestation Days 6 through 15 to pregnant Fischer 344 rats, in range-finding (R) and main (M) studies. The dermal route is considered to be the most relevant for human exposure. Treatment levels were (R) 0.0, 0.5, 1.0, 2.0, and 3.0 ml/kg/day (equivalent to 0, 420, 840, 1680, and 2520 mg/kg/day) and (M) 0.0, 0.3, 1.0, and 3.0 ml/kg/day (equivalent to 0, 252, 840, and 2520 mg/kg/day). Controls (0.0 ml/kg/day, sham controls) received deionized water at 3.0 ml/kg/day. Dermal-positive control groups received undiluted 2-methoxyethanol (2-ME) at (R) 0.5 and 1.5 ml/kg/day and (M) 1.0 ml/kg/day as a reference compound in a similar regimen. An oral reference compound, valproic acid, was administered by gavage in the range-finding study on Gestation Days 6 through 15 at 400 mg/kg/day. The range-finding study employed an untreated (naive) control group. Numbers of plug-positive females per group were (R) 8 and (M) 25. Maternal weight gain was reduced for 2-EH at 1680 (R) and 2520 (R and M studies) mg/kg/day. Exfoliation and encrustation were seen at the application site in both studies at 840, 1680, and 2520 mg/kg. Maternal liver, kidney, thymus, spleen, adrenal, and uterine weights, and gestational and fetal parameters were unaffected by treatment with 2-EH. There were no treatment-related increases in the incidence of individual or pooled external, visceral, and skeletal malformations or variations following the application of 2-EH. The NOAELs for the maternal toxicity of 2-EH were 252 mg/kg/day based on skin irritation and 840 mg/kg/day based on systemic toxicity. The developmental toxicity NOAEL was at least 2520 mg/kg/day, with no teratogenicity. Administration of 2-ME at 840 mg/kg/day resulted in reduced maternal weight gain and food consumption, increased postimplantation loss, reduced numbers of live fetuses per litter, and reduced fetal body weights per litter. The incidence of fetal malformations and variations was increased. Oral administration of VPA produced maternal toxicity, developmental toxicity, and teratogenicity. The Fischer 344 rat is thus susceptible to known rodent teratogens by both the dermal and oral routes. It is concluded that 2-EH is not developmentally toxic by the dermal route in the Fischer 344 rat at and below treatment levels which produce maternal toxicity.


Assessment of a short-term reproductive and developmental toxicity screen.

Short-term tests for reproductive and developmental toxicity are needed to provide preliminary data on the toxicity of chemicals about which little or no data exist. An ideal design would test all aspects of reproduction and identify the target process in a short time period. One potential design has been evaluated using four chemicals of varying reproductive/developmental toxicity. Swiss mice were mated for 3 days prior to chemical exposure to produce time-mated females for gestational exposure and to ascertain fertility of the untreated males. The group of time-mated females was treated during Gestation Days 8-14 and allowed to litter for observations through Postnatal Day (PND) 4. Endpoints observed included pup number and body weights on PND 0, 1, and 4 and number of uterine implantation sites on PND 4. A second group of females was dosed daily for 19 days. After 7 days, these females (n = 10/group) were cohabited with male mice who had been treated for 5 days prior to this second mating. Daily chemical dosing continued during the 5-day cohabitation. This second group of females was killed after 19 days of treatment and the number of live and dead fetuses and implantation sites was recorded. After 17 days of dosing, male mice were killed and the reproductive system evaluated by organ weights, total epididymal sperm counts and motility, and testicular histology. All four chemicals tested, boric acid, ethylene glycol, ethylene glycol monomethyl ether, and theophylline, were found to be toxic to development or reproduction when tested previously by conventional developmental toxicity or continuous breeding protocols. This short-term (21 day) design correctly identified three of these four chemicals as reproductive and developmental toxicants and distinguished the potent toxicants from the less effective compounds. This design can be used to prioritize chemicals for further study, or to delineate the relative toxicities of structurally related chemicals, and to identify the proper dose range for subsequent toxicity studies.


Subchronic toxicity, metabolism, and pharmacokinetics of the aminobenzamide anticonvulsant ameltolide (LY201116) in rhesus monkeys.

Studies were undertaken to define the subchronic toxicologic profile of ameltolide, an aminobenzamide anticonvulsant, in young adult rhesus monkeys. Daily doses of ameltolide, dissolved in 10% aqueous acacia, were administered orally via nasogastric intubation at dosages of 5, 10, 20, 45, and 100 mg/kg. Deaths occurred in two monkeys, one each at 45 and 100 mg/kg, which were directly attributable to the effects of the compound. The exact cause of death in these monkeys was not readily apparent. A third monkey (100 mg/kg) was killed moribund on Day 82 of the study due to conditions not directly related to treatment. Clinical signs in monkeys treated with 100 mg/kg included convulsions, diarrhea, weakness, inappetance, vomition, and ataxia. Plasma concentrations of the N-acetyl metabolite of ameltolide were greater than parent drug concentrations by one to two orders of magnitude. Mean area under the plasma-time curve (AUC) values for ameltolide were larger than expected at doses of 20 mg/kg or greater, while AUC values for the metabolite were less than expected at 45 and 100 mg/kg. These findings suggest a saturation of metabolism and/or excretion at the two higher doses. Similar nonlinearity was seen with mean peak concentrations for both parent and metabolite. No specific target organ toxicity was found on histological evaluation of tissue sections. Methemoglobin concentration was increased in monkeys given 45 or 100 mg ameltolide/kg. This change was not considered to be toxicologically important as there were no corroborative clinical, gross, or histopathological findings. Ameltolide administered by nasogastric intubation at doses up to 20 mg/kg/day for 3 months did not cause any toxicologically important alterations in rhesus monkeys.


Joint actions of developmental toxicants in Xenopus embryos: binary mixtures of DNA synthesis inhibitors.

The joint toxic action for each of three binary combinations of DNA synthesis inhibitors was examined at concentrations that induce malformations in Xenopus laevis embryos. The three compounds, hydroxyurea, cytosine arabinoside, and 5-fluorouracil, which each inhibit a different enzyme involved in DNA synthesis, were selected for joint action testing to help determine whether joint action types are related to general modes of action (e.g., inhibit DNA synthesis) or to specific biochemical/molecular mechanisms. Three mixtures were tested, in 96-hr static-renewal tests, for each combination. Each combination was tested on three separate occasions. Using toxic unit analysis, the joint action for induction of malformations was generally antagonism, although response addition was observed for certain mixtures. An antagonistic joint action represents the phenomenon of interaction. Therefore, the tests were ineffective in determining whether a concentration addition joint action depicts two compounds that induce malformations by the same general mode of action or by the same specific mechanism of action.


An examination of the association between maximum-tolerated dose and carcinogenicity in 326 long-term studies in rats and mice.

The association between rodent carcinogenicity and maximum-tolerated dose (MTD) was evaluated in 326 long-term carcinogenicity studies in mice and rats. Others investigating this association have focused primarily on positive studies, but our investigation considered all experimental outcomes. We found that chemicals with low MTDs were somewhat more likely to be rodent carcinogens than chemicals with high MTDs, but this association was limited primarily to gavage studies. Overall, the MTD was not a reliable predictor of whether or not a chemical would be a rodent carcinogen. Our investigation confirms that comparisons of carcinogenic potencies based only on positive studies may result in artifactually elevated estimates of the underlying association between chemical toxicity and rodent carcinogenicity and thus may also inflate the estimated interspecies correlation in carcinogenic response. Nevertheless, the results of our study are consistent with the frequently cited 75% concordance in carcinogenicity outcome between rats and mice. This concordance is quite high, particularly since 80% is approximately the maximum level of observable interspecies concordance achievable for a set of chemicals with relatively low carcinogenic potency, because of the variability in observed tumor responses that can induce false negative or false positive outcomes in either of the two species. Thus, the underlying qualitative interspecies correlation in carcinogenic response between rats and mice may be greater than is commonly recognized.


A study of developmental toxicity of hydroquinone in the rabbit.

To obtain information on potential developmental toxicity, hydroquinone (HQ) was administered to pregnant New Zealand White rabbits (18 mated per dose group) in aqueous solution (0, 25, 75, or 150 mg HQ/kg/day) by gavage on Gestation Days (GD) 6 to 18. Caesarean sections were performed on GD 30. Doses of 75 and 150 mg/kg/day adversely affected feed consumption and/or body weights of dams during the treatment period. At these doses, however, treatment-related effects were not evident from physical observations, liver and kidney weights, premature delivery incidence, and caesarean sectioning data. The NOEL for maternal toxicity was 25 mg/kg/day. In the 150 mg/kg/day dose group, total incidences of external, visceral, and skeletal findings for fetuses did not differ statistically from controls. Slight, statistically insignificant, increases were found, however, in the incidences of ocular and minor skeletal malformations (micro-ophthalmia, vertebral/rib defects, angulated hyoid arch) on both a per fetus and a per litter basis. Under the conditions of this study, HQ at 150 mg/kg/day produced minimal developmental alterations in the presence of maternal toxicity. The NOEL for developmental toxicity was 75 mg/kg/day.


Maternal and fetal tissue distribution of L-carnitine in pregnant mice: low accumulation in the brain.

The distribution of L-carnitine was studied by whole-body autoradiography in pregnant CD-1 mice at 1, 2, 3, and 6 hr after receiving L-[14C]carnitine. Highest concentrations of carnitine were found in maternal tissues including liver, placenta, kidney, myocardium, and choroid plexus. High retention of tissue carnitine in excess of blood levels suggests the existence of concentrative uptake mechanism. Labeled carnitine was not detectable in either maternal or fetal brain. This suggests that the brain barrier systems limit the access of L-carnitine to the brain. In fetus, the level of carnitine was less than that seen in the maternal tissues, however, the tissue distribution was similar. The fetal tissue carnitine concentration increased with time. These findings suggest that relief of encephalopathy due to toxic organic anions in metabolic disorders following L-carnitine supplementation appears to be peripheral metabolic effects rather than direct access to the central nervous system. However, the physiological role for the concentrative uptake of L-carnitine by the choroid plexus remains to be determined. Transport of carnitine into fetal tissues via placenta further suggests the possibility of prenatal therapy in pregnancies at risk for certain inherited metabolic disorders.


Reproductive study of acrolein on two generations of rats.

Four groups of 30 male and 30 female rats were intubated with 70 daily doses of acrolein at levels of 0, 1, 3, or 6 mg/kg in a dosing volume of 5 ml/kg. Rats within each dosing group (F0 generation) were then assigned to a 21-day period of cohabitation and dosing for females continued through cohabitation gestation and lactation. Males were euthanized after cohabitation. F1 generation rats were chosen from pups, and a similar pretreatment, cohabitation, gestation, and lactation regimen was accomplished resulting in F2 generation pups. Reproductive parameters, body weights, food consumption, and clinical signs were recorded and necropsies were carried out on all treated animals. Histopathologic exams were accomplished on selected reproductive tissues. In addition, gross lesions, target tissues, stomachs, and lungs were examined. For the most part, reproductive parameters were unaffected by acrolein treatment with the exception of reduced pup weights in the F1 generation pups at the high-dose level (6 mg/kg/day). Gastric lesions were noted consistently in high-dose animals and some mid-dose (3 mg/kg/day) rats. Erosions of glandular mucosa and hyperplasia/hyperkeratosis of the forestomach were the most frequent stomach lesions observed. Effects on body weight gains were noted frequently for the high-dose animals and achieved statistical significance in the mid-dose animals on several occasions. Mortality in all high-dose animals was elevated relative to control animals. Acrolein, therefore, cannot be considered a selective reproductive toxin in the rat, but does produce toxicological effects down to a dosing level of 3 mg/kg/day.


The developmental toxicity of diethylene and triethylene glycol dimethyl ethers in rabbits.

Diethylene glycol dimethyl ether (diEGdiME) and triethylene glycol dimethyl ether (triEGdiME), widely used organic solvents, are structurally related to several compounds that produce reproductive and developmental toxicity, including teratogenicity in laboratory animals. In the present studies, diEGdiME (0, 25, 50, 100, or 175 mg/kg/day) or triEGdiME (0, 75, 125, 175, or 250 mg/kg/day) were administered by gavage in distilled water to timed-pregnant New Zealand white rabbits (15-25 dams/group) during major organogenesis [Gestational Days (gd) 6-19]. Treated females were euthanized on gd 30, uterine contents were examined, and live fetuses were examined for morphological alterations. In the diEGdiMe study, evidence of maternal toxicity, per se, was observed only at 175 mg/kg/day with 15% mortality among treated females compared to 4% among controls. No significant maternal toxicity was observed in the 25 mg/kg/day group, and only minimal maternal toxicity (decreased maternal weight gain during treatment) was observed at 50 and 100 mg/kg/day compared to the vehicle control group. The no-observed-adverse-effect level for developmental toxicity in rabbits for diEGdiME was 50 mg/kg/day. The incidences of prenatal mortality and malformed live fetuses were significantly above controls at 100 and 175 mg/kg/day. Malformations observed most frequently included fusion of ribs to each other and hydronephrosis; clubbing of the limbs without underlying bone deformities, a variation, was also observed. In the triEGdiME study, clinical signs of toxicity were minimal and there was no increased maternal mortality. Maternal body weight and gravid uterine weight were significantly reduced at 250 mg/kg/day, whereas maternal weight gain during treatment was significantly depressed at doses of 175 mg/kg/day and above.(ABSTRACT TRUNCATED AT 250 WORDS)


Systemic toxicity of the heavy fraction of a coal coprocessing product in male rats following subchronic dermal exposure.

The systemic toxicity of a coal coprocessing product [heavy gas oil II (HGOII)] following subchronic, dermal exposure in male Sprague-Dawley rats was investigated. HGOII was applied to the dorsal skin daily at doses of 8.7, 20.8, 50.0, or 120.0 mg/kg body weight (bw) for 13 weeks. Another group of rats treated with a medium boiling coal liquefaction product (CLP) served as positive controls. Growth suppression and decreased food consumption were noted in the groups exposed to HGOII at 20.8 mg/kg and higher, and to CLP starting at the third week of treatment. Relative liver, kidney, and brain weights in the 20.8 mg/kg HGOII group and up were higher than those of the control. Increased spleen weight was observed in all HGOII-treated groups. CLP treatment also caused increased relative kidney and brain weights. Serum cholesterol was elevated in the HGOII-treated groups starting at 8.7 mg/kg while increased uric acid and lactate dehydrogenase were observed at 20.8 mg/kg and up. Decreased erythrocyte, hemoglobin, and platelet counts were observed at 20.8 mg/kg and higher. All HGOII-treated groups had elevated reticulocytes. These biochemical and hematological changes were not observed in the CLP-treated group. Mild to marked histological changes were observed in the thyroid, thymus, liver, spleen, and bone marrow of HGOII groups. In contrast, morphological changes were relatively mild in CLP-treated animals. Data from the present study demonstrated that the hematological endpoints were sensitive to the liquid fuels and that HGOII was more toxic than CLP.(ABSTRACT TRUNCATED AT 250 WORDS)


Age-related percutaneous penetration of 2-sec-butyl-4,6-dinitrophenol (dinoseb) in rats.

[14C]Dinoseb was applied to previously clipped back skin of 33- and 82-day-old female Fischer 344 rats at a dosage range of 210-2680 nmol/cm2. Radioactivity in the treated skin, tissues, urine, and feces was determined at 1, 6, 24, 48, 72, and 120 hr following dermal application. In vitro dermal absorption of [14C]dinoseb was also measured in rats of the same age by static and flow-through methods. In vivo dermal absorption in both young and adults appeared biphasic with 55.6 and 82.7% of the recovered dose, respectively, penetrating in 72 hr. In vitro measurements of skin absorption at 72 hr with static cells showed higher values in young and lower values in the adult compared to in vivo dermal absorption values. In vitro flow-through measurements at 72 hr gave lower dermal absorption values for both young and adult rats, compared to in vivo values. Following in vivo application, adults excreted about 70% of the total recovered dose in urine, 16% in feces, and retained 7% in the body at 120 hr. HPLC analysis of urine collected at 24 hr from adults administered [14C]dinoseb showed extensive metabolism of parent. Excretion and retention results for young were about 80% of the adult values, which also was the young to adult ratio of dermal penetration. Blood had the highest concentration of dinoseb-derived radioactivity of the tissues examined. The kidney to blood ratio averaged 0.60 in young and 0.41 in adults, while the liver and carcass to blood ratio averaged 0.18 in young and 0.11 in adult. Dermal absorption in young rats was slightly less than that in adults, and the subsequent kinetics of retention and excretion appeared different. In vitro dermal penetration of dinoseb was usually lower than in vivo absorption.


Toxicology of maternally ingested trichloroethylene (TCE) on embryonal and fetal development in mice and of TCE metabolites on in vitro fertilization.

Trichloroethylene (TCE), an industrial solvent, is a soil and ground water contaminant found across the United States. The metabolism and carcinogenic potential of TCE have been studied extensively in the past 15 years yet there is little information on the chemical's possible effects on reproduction. No reference to the reproductive effects in mice of TCE by oral administration exists in the literature. In this study, female B6D2F1 mice were gavaged from Days 1 to 5, 6 to 10, or 11 to 15 (Day 1 = vaginal plug) with TCE in corn oil at 0, 1/10, and 1/100 of the oral LD50. Weights of mice were recorded and the livers and kidneys were weighed and preserved in 10% buffered formalin. Litters were counted, sexed, weighed, and measured for crown-rump length until weaning on Day 21 and some animals were allowed to develop to 6 weeks of age. At this time, a minimum of two litters from each dose were killed and gonads removed, weighed, and preserved in Bouin's fixative. Litters were also assessed for developmental abnormalities. No maternal or reproductive effect of TCE was seen at either dose level. TCE, administered consecutively on Days 1 to 5, 6 to 10, and 11 to 15 of pregnancy, does not appear to be a potential reproductive toxicant up to 1/10 the oral LD50. In a second series of studies, TCE and its metabolites dichloroacetic acid (DCA), trichloroacetic acid (TCAA), and trichloroethanol (TCOH) were added to culture media to assess the toxic effects on in vitro fertilization (IVF) in mice.(ABSTRACT TRUNCATED AT 250 WORDS)


Accumulation of Cd(II) in the CNS depending on the route of administration: intraperitoneal, intratracheal, or intranasal.

The uptake and subsequent neuronal transport of certain heavy metals in the olfactory mucosa may be a major means by which these compounds gain access to the CNS. To contrast olfactory versus blood-borne routes of exposure, three groups (n = 4) of adult Long-Evans rats were exposed to solutions of radiolabeled CdCl2. Exposure was by one of three routes: unilateral intranasal instillation (IN), intratracheal lavage (IT), or intraperitoneal injection (ip). The dose level for the intranasal route was 30 microliters of 1 microM CdCl2 labeled with 1 microCi 109Cd. For IT and ip, the dose was 30 microliters of 1 microM CdCl2 diluted to 300 microliters in saline and labeled with 1 microCi 109Cd. Rats were euthanized 24 hr after exposure, tissue samples were taken, and radioactivity was counted. Cd levels were low in the olfactory bulbs of rats exposed either intratracheally or intraperitoneally. However, in rats intranasally exposed, Cd levels were nearly 40 x higher in olfactory bulbs ipsilateral to the exposed side than in those on the contralateral side. With all routes of exposure, Cd levels in brain samples were only slightly elevated. These results suggest that for certain airborne toxicants, especially those that are excluded from the CNS by the blood-brain barrier, the olfactory system may provide a direct route of entry into the CNS.


Pulmonary bioavailability and fine particle enrichment of 2,3,7,8-tetrachlorodibenzo-p-dioxin in respirable soil particles.

The pulmonary bioavailability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the enrichment of polychlorinated dioxins (PCDDs) and furans (PCDFs) in fine particles were evaluated to assess the implications that these factors have on risk and exposure assessments. Respirable subfractions of PCDD-contaminated soil from a former 2,4,5-trichlorophenoxyacetic acid manufacturing site were isolated by chemical dispersion and gravity sedimentation. Analysis of the subfractions revealed that there was a size-dependent enrichment of PCDDs and PCDFs, with smaller particles more highly contaminated. TCDD was enriched up to 33-fold as compared to unfractionated soil. Soil and laboratory-recontaminated gallium oxide, which served as the positive control, were administered by intratracheal instillation to female Sprague-Dawley rats. Animals were terminated up to 28 days following treatment and pulmonary bioavailability of TCDD was assessed by hepatic enzyme induction and TCDD concentration. Enzyme induction was dependent on the duration of exposure with up to 56 and 918% increases in cytochrome P450 and aryl hydrocarbon hydroxylase (AHH) activity, respectively, following exposure to PCDD-contaminated soil. There was no significant difference in AHH induction between animals which received contaminated soil and those treated with the positive control. Hepatic concentration of TCDD in soil-exposed rats was 115, 101, and 179% of positive controls at 1, 7, and 28 days post-treatment, suggesting that the soil or cocontaminants influenced retention of TCDD in the liver. These data indicate that the relative pulmonary bioavailability of TCDD on respirable soil particles is 100% as compared to laboratory-recontaminated gallium oxide and that PCDDs and PCDFs are highly enriched on respirable particles.(ABSTRACT TRUNCATED AT 250 WORDS)


Developmental toxicity of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). I. Multireplicated dose-response studies in four inbred strains and one outbred stock of mice.

A large-scaled multireplicated developmental toxicity study was conducted in various strains/stocks of mice with the herbicide, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), by gavage on Gestational Days 6 through 14. The most important attributes of the study design were replicated test groups, a minimum of four dose levels per replicate, use of multiple stocks/strains of animals to obtain an estimate of the range in sensitivities due to genotype, complete pathological evaluation of maternal animals, and histopathological as well as teratological evaluation of the fetuses. Developmental toxicity was observed at doses below those producing discernible or measurable maternal toxicity. Regression and/or probit analyses were conducted to determine whether a dose-response relationship existed. Reduced fetal weight and increased incidence of cleft palate and embryolethality were the most significant prenatal effects of 2,4,5-T exposure observed in this study. Each strain/stock exhibited a dose-related decrease in fetal weight with the CD-1 mice having the steepest slope and the A/J mice having the shallowest slope. There was a striking similarity among the slopes of the dose-response curves for the various strains/stocks. The mean incidence of embryolethality in the A/J strain was significantly greater than that of the other strains or stocks. There was substantial variation among replicates within strains. The use of the replicated study design was logistically necessary due to the magnitude of the study and it also served to increase the statistical power of the study.


Developmental toxicity of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). II. Multireplicated dose-response studies with technical and analytical grades of 2,4,5-T in four-way outcross mice.

A series of multireplicated developmental toxicity studies were conducted in four-way outcross mice and CD-1 outbred mice administered either analytical or technical grades of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by gavage on Gestational Days 6 through 14. The formulations of 2,4,5-T differed by a factor of 10-fold in 2,3,7,8-tetrachlorodibenzo-p-dioxin levels. Reduced fetal weight and increased incidences of cleft palate and embryolethality were the most significant prenatal effects of both formulations of 2,4,5-T observed in all strains/stocks of mice. Both the outcross and outbred mice exhibited a dose-response relationship with each of the above endpoints and the dose-response curves were parallel. There were no embryotoxic or fetotoxic differences between the technical and analytical grades of 2,4,5-T with regard to extent of fetal weight reduction, resorption rate, or cleft palate incidence. There was little difference in the results between the four-way outcross mouse and the CD-1 outbred mouse.


One-year dietary toxicity study with methidathion in beagle dogs.

The purpose of this study was to determine the chronic toxicity of methidathion, an organophosphate insecticide, in dogs. Groups of beagle dogs, four/sex/dose, were fed methidathion at constant dietary concentrations of 0, 0.5, 2, 4, 40, or 140 ppm for 1 year. The equivalent daily dosages were approximately 0, 0.02, 0.07, 0.15, 1.4, and 4.7 mg/kg. There were no deaths or adverse clinical signs associated with the treatment. Weekly body weights and weight gains were not affected. Mean daily food consumption was reduced in male dogs given the 140-ppm diet. Major treatment-related effects were cholestasis, chronic inflammation in the liver, and cholinesterase (ChE) inhibition. The liver effects were indicated by gross and microscopic pathologic findings as well as moderate increases in serum bile acids and enzyme activities (alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, and alkaline phosphatase) in all dogs receiving greater than or equal to 40 ppm. RBC ChE was inhibited in males at greater than or equal to 40 ppm and in females and 140 ppm. Brain ChE was inhibited in both sexes at 140 ppm; the magnitude of inhibition relative to control was slightly greater with the cerebellar fraction than with the cerebral fraction. Serum ChE was not affected at any dose level. In conclusion, liver was the target organ in beagle dogs given greater than or equal to 40 ppm (equivalent to 1.4 mg/kg/day) methidathion in diet for 1 year. The no-observable-effect level was 4 ppm (0.15 mg/kg/day) for both liver cholestasis and ChE inhibition.


[Ultrastructural changes in lysosomal apparatus of human platelets in pathologic states].

The author has presented the results of electron-microscopic and electron-histochemical investigations of the lysosomal apparatus in human platelets under the action of certain agents (acetylsalicylic acid, vincristine) and in pathologic states (acute leukemia, myeloproliferative diseases, autoimmune thrombocytopenic purpura). Interrelation has been recorded between ultrastructural changes in the lysosomal apparatus and parameters of platelet functional activity. Biogenesis of lysosomal enzymes has been considered basing on molecular biology of the cell.


[Dynamics of blood lymphocyte blast-transformation parameters during hemosorption in patients with gastric ulcer].

The influence of autotransfusions of hemosorbent-treated blood on parameters of DNA-synthetic activity of blood lymphocytes was studied in 114 patients with gastric ulcer. A statistically significant increase in parameters of DNA-synthetic activity of lymphocytes was recorded simultaneously with the appearance of morphological signs of blast transformation on electrograms. A conclusion has been made on association of lymphocyte blastogenesis under the influence of autotransfusions with the process of ulcerous sanagenesis acceleration.


[Changes in the organ of vision in beta-thalassemia: clinical and morphologic parallels].

Ophthalmologic examination and morphologic investigations of the spleen were conducted in 10 patients with beta-thalassemia. Pathologic changes of vascular and dystrophic character were detected in the organ of vision, symptoms of angiopathy, hemosiderosis and spleno-cirrhosis were recorded in the spleen tissue. Clinical changes in the organ of vision were correlated with morphological shifts in the spleen tissue. A conclusion has been made that ophthalmologic examination of thalassemia patients could be useful for evaluation of severity of the main disease and effectiveness of the treatment conducted.


[Changes in the parameters of functional systems of donors undergoing apparatus plasmacytapheresis].

Apparatus plasmacytapheresis resulted in the lowering of total protein level and serum iron content in the blood as compared to the initial values. At the same time these parameters were within the range of reference standards and reached the initial levels 3-4 weeks after the completion of the plasmacytapheresis cycle.


[Iron metabolism, hemopoiesis, prevention and treatment of iron deficiency anemia in multiparous women].

A total of 160 multiparous women with iron deficiency anemia (IDA) who have 4 and more children were under study. Hemoglobin parameters in patients with non-severe anemia (n = 64) fluctuated from 119 to 90 g/l, in patients with moderate severity (n = 60) from 89 to 70 g/l, in those with severe stage of anemia--69 g/l and lower. The latent anemia group consisted of women whose hemoglobin values were 120 g/l and higher, however, the figures characterizing serum iron, transferrin and ferritin content evidenced iron deficiency. Parameters of serum iron, transferrin and ferritin metabolism were studied in all the patients. Decreased levels of serum iron and ferritin and a compensatory increase of transferrin content were recorded. Manifest signs of acquired dyserythropoiesis and a significant increase of ineffective erythropoiesis (34.96 +/- 0.89%) were demonstrated in myelograms. It has been recommended that routine measures used for the treatment of IDA in multiparous women should be supplemented by anabolic agents and blood substitutes for protein parenteral nutrition.


[Morphometric parameters of malignant cells in non-leukemic hemoblastosis with primary lymph node involvement].

Morphometric studies were conducted to distinguish reactive and malignant lymphoid processes and to obtain a more objective and reproducible grading system for non-Hodgkin's lymphomas (NHL). Twenty-three untreated patients (3 with reactive lymphadenitis, 2 with diffuse lymphomas, 18 with lymphosarcomas) were studied. The sections of lymph nodes were stained with 50% aqueous silver nitrate solution and then counter-stained with methylene blue. MOP-Videoplan (Reichert, Austria) image analyzer was used for morphometry. The size and shape of nucleus, number and position of nucleoli and the number of silver grains (K1) and relative size (K2) of each grain were investigated. The computer classification of lymphoid cells based on a stepwise linear discriminant analysis resulted in 63% correctly classified cells. Among seven most discriminating parameters, the nucleoli area and the number and size of silver grains demonstrated the highest correlation with malignancy. The data obtained have evidenced that nucleolar parameters can be applied to computer classification of lymphoid neoplastic cells and for objective grading of NHL.


Cloning of a Bacteroides fragilis chromosomal determinant coding for 5-nitroimidazole resistance.

Strain BF8 is a plasmid-free Bacteroides fragilis, resistant to 5-nitroimidazole (5-Ni) antibiotics (metronidazole, ornidazole and tinidazole). The resistance was transferable by conjugation into Bacteroides fragilis BF638R. The total DNA of a Nir transconjugant was used for the construction of a Sau3A genomic library in a B. fragilis cloning vector pFK707 delta H1 (4.2 kb). By electrotransformation of strain BF638R, a recombinant plasmid containing an insert of 5.4 kb was obtained which conferred to the host strain the resistance to 5-Ni. The physical map of the insert was established. After deletion analysis of the insert, the Nir determinant was localized on a HpaII-HincII fragment of 1.6 kb in size. This Nir determinant has been compared by Southern-blot analysis with other Bacteroides Nir determinants of plasmid origin.


OmpF channel permeability of quinolones and their comparison with beta-lactams.

The diffusion rates of nalidixic acid, ofloxacin and ofloxacin's two optically active isomers through OmpF channels were measured in proteoliposomes and compared with the rates of beta-lactams. The four quinolones showed high diffusion rates, exceeding that of cephaloridine and being comparable to imipenem. There was no significant difference in diffusion rate between nalidixic acid and ofloxacin, or between the two optically active isomers. The diffusion rates of enoxacin and norfloxacin were also estimated to be higher than many beta-lactams.


Effect of restrictive conditions on the growth and morphology of a temperature-sensitive mannose-requiring mutant of Aspergillus nidulans.

When incubated at 45 degrees C in the absence of added mannose, pregrown hyphae of a temperature-sensitive, mannose-relief mutant (mnrA455) of Aspergillus nidulans grew normally for a short time (4-5 h) before exhibiting an abnormal morphology consisting of the production by hyphae of discrete spherical swellings called balloons. These swellings could be up to 10 microns in diameter and were produced either at or behind the hyphal apex. Often only one swelling was produced in association with each hyphal tip, but in a significant minority of cases (approximately 19.6%) a second balloon was produced in close association with the first. Hyphal tip extension slowed before and during balloon formation, but growth at individual tips did not usually stop when a balloon began to be formed in the same hypha. All tip extension ceased after approximately 8 h in cultures maintained at 45 degrees C. However, normal growth resumed 45-60 min after transfer of such a culture to the permissive temperature of 37 degrees C even after 48 h at 45 degrees C. Electron microscopic examination indicated that balloons consistently had thicker walls than the surrounding hyphae but that no accumulation of cytoplasmic vesicles was apparent within them. This indicates that a modification of wall structure, probably including deposition of new wall material, was caused by a mannose deficiency, but that this altered wall synthesis and attendant hyphal swelling was not due to diversion of the normal vesicle-mediated tip-extension system to the side walls of hyphae.


Mycobacterium avium and Mycobacterium intracellulare chromatotypes defined by curvilinear gradient HPLC of mycolic acids.

Seventy-nine strains of Mycobacterium avium complex bacteria (MAC), previously characterized by genetic probe analysis, were assayed using two methods of reverse phase high-performance liquid chromatography (HPLC) that employed curvilinear gradients. Although different in column length and cycle time, the methods produced equivalent results, yielding seven distinct chromatographic patterns (chromatotypes) of M. avium and M. intracellulare based on the ratio of mycolate concentrations in the late vs. the middle of three peak clusters (L:M ratio). The M. avium strains (n = 36) were assigned to chromatotypes 1 through 4 (L:M ratios less than 3), and the M. intracellulare strains (n = 25) to chromatotypes 5 through 7 (L:M ratios greater than 4). Of 18 Mycobacterium 'X' strains, seven resembled M. avium, seven others resembled M. intracellulare, and four were intermediate between M. avium and M. intracellulare.


Re-examination of the metabolic potentials of the acetogens Clostridium aceticum and Clostridium formicoaceticum: chemolithoautotrophic and aromatic-dependent growth.

Both Clostridium formicoaceticum and Clostridium aceticum grew chemolithoautotrophically on carbon monoxide plus CO2 in defined medium in the absence of carbohydrates, amino acids, or other carbon and energy sources. Formate supported the growth of both organisms as well in both defined and undefined media (both of which also contained CO2). Hydrogen was stimulatory to the growth of C. formicoaceticum upon first transfer into H2-enriched formate medium; however, neither chemolithoautotrophic growth at the expense of H2 plus CO2 nor hydrogenase could be demonstrated with this acetogen. Consistent with recent findings with other acetogens, numerous aromatic compounds were utilized by C. aceticum and C. formicoaceticum: (i) aromatic methoxyl groups were O-demethylated; (ii) aromatic acrylates were reduced; and (iii) aromatic aldehydes were oxidized. These findings demonstrate that the metabolic potentials of these two acetogens are greater than previously recognized.


Rapid identification of Aerococcus viridans using the polymerase chain reaction and an oligonucleotide probe.

A polymerase chain reaction/oligonucleotide probe method was developed for the specific identification of the Gram-positive bacterium Aerococcus viridans. Primers for the enzymatic amplification reaction were designed from specific sequences within the 16S rRNA. The method was also highly sensitive and 10 cfu of A. viridans could be detected in 5 h although the reliability of detection was poor in mixed cultures with Escherichia coli.


The DNA polymerases of Leishmania mexicana.

Two previously isolated DNA polymerases from the parasitic protozoan Leishmania mexicana were further characterized by exposure to inhibitors of mammalian DNA polymerases. DNA polymerase A, a high molecular mass enzyme, and DNA polymerase B, a beta-like DNA polymerase were compared to each other and to their mammalian counterparts regarding pH optimum, utilization of templates, and response to various inhibitors and ionic strengths. The results suggest that the DNA polymerases from L. mexicana differ from the host enzymes and may offer a target for chemotherapeutic intervention.


Constitutive transcription of the gene for metallothionein in a cadmium-resistant yeast.

A cadmium-resistant strain of Saccharomyces cerevisiae produces a cadmium metallothionein encoded by the CUP1 gene as does a copper-resistant strain. The mechanism of expression of the gene is inducible by copper ions in the copper-resistant strain. However, assays of CUP1-specific mRNA revealed that the transcription of the CUP1 gene in the cadmium-resistant strain is constitutive and the rate of transcription is further increased by exposure to cadmium or copper ions. This result was confirmed by the appearance of constitutive-expression segregants from diploid crosses between the cadmium-resistant strain and a strain with a reporter gene having the promoter of CUP1.


Nitrosating activity in Escherichia coli.

Nitrosation activity was measured in Escherichia coli isolates and a range of nitrite reductase (nir) mutants. Activity was only detected in intact cells and could be inhibited by a number of treatments such as sonication and osmotic shock. Aerobically-grown cells had highest nitrosation activity compared to oxygen-limited ones. Inclusion of nitrite in growth media induced high activities of nitrite reductase and for some isolates, nitrosation. Analysis of nir mutants identified two which were unable to nitrosate. This result suggested that NADH-dependent nitrite reductase was implicated either directly or indirectly in nitrosation.


Dual effect of monovalent cations on the glucose-induced transient increase in the rate of DMP influx into Saccharomyces cerevisiae.

The transient increase in the rate of 2-(4-di-methyl-aminostyryl)-1-ethylpyridinium (DMP) influx in Saccharomyces cerevisiae caused by addition of glucose to a suspension of non-metabolizing cells at relatively high pH can be prevented by monovalent cations. Their concentrations for half-maximum inhibition of DMP uptake are of the same order of magnitude as the corresponding Km values for their uptake into yeast cells. It is argued that the inhibition of DMP uptake by monovalent cations is caused by a fast depolarization of the cell membrane and a second further decrease in the rate of DMP uptake. The latter effect develops slowly with time and depends upon the extent of accumulation of the monovalent cations in the cells.


Genetic recombination in bacteriophage T4: single-burst analysis of cosegregants and evidence in favor of a splice/patch coupling model.

To reveal the structure of penultimate DNA intermediates in T4 bacteriophage recombination, resolution of which produces free recombinant molecules, a single-burst analysis of the recombinant progeny was made in multifactor crosses, enabling one to determine quantitatively the different recombinants generated by one or two exchanges within the same chromosome segment. It was found that double and single exchanges are highly correlated in T4 recombination. These results were interpreted as evidence for simultaneous formation of a splice/patch pair as the primary recombination products. A recombination model called here the "splice/patch coupling model" is presented according to which resolution of a single DNA intermediate results in two linear heterozygous molecules containing a patch and a splice, respectively, in homologous positions.


Mechanisms of directed mutation.

Spontaneous mutants arise among nondividing populations of Escherichia coli in apparent response to selective conditions. In this report we investigate several hypotheses to account for the role of selection in the production of these "directed" or "adaptive" mutations. We found that the Lac+ phenotypes of some mutants that arise late after lactose selection are due to suppressor mutations that are unlinked to the mutant lacZ allele; thus the production of these Lac+ mutants does not require an information flow from successful proteins back to the DNA that encodes them. Transcriptional induction of the lac operon, even in the presence of another, utilizable carbon source, did not stimulate the occurrence of Lac+ mutants in the absence of lactose, indicating that the role of the selective agent is not merely to induce transcription. The absence of two DNA repair pathways-methyl-directed mismatch repair and alkylation repair-also did not result in an accumulation of Lac+ mutants in the absence of lactose, suggesting that these repair pathways are not normally responsible for correcting transient variants that might arise in the absence of selection. However, in one case the Lac+ mutation is likely to be due to a miscoding lesion occurring on the nontranscribed DNA strand, indicating that, at least in this instance, DNA replication is required before directed mutations can arise.


Expression of the maize MnSod (Sod3) gene in MnSOD-deficient yeast rescues the mutant yeast under oxidative stress.

Superoxide dismutases (SOD) are ubiquitous in aerobic organisms and are believed to play a significant role in protecting cells against the toxic, often lethal, effect of oxygen free radicals. However, direct evidence that SOD does in fact participate in such a protective role is scant. The MnSOD-deficient yeast strain (Sod2d) offered an opportunity to test the functional role of one of several SOD isozymes from the higher plant maize in hopes of establishing a functional bioassay for other SODs. Herein, we present evidence that MnSOD functions to protect cells from oxidative stress and that this function is conserved between species. The maize Sod3 gene was introduced into the yeast strain Sod2d where it was properly expressed and its product processed into the yeast mitochondrial matrix and assembled into the functional homotetramer. Most significantly, expression of the maize Sod3 transgene in yeast rendered the transformed yeast cells resistant to paraquat-induced oxidative stress by complementing the MnSOD deficiency. Furthermore, analyses with various deletion mutants of the maize SOD-3 transit peptide in the MnSOD-deficient yeast strain indicate that the initial portion (about 8 amino acids) of the maize transit peptide is required to direct the protein into the yeast mitochondrial matrix in vivo to function properly. These findings indicate that the functional role of maize MnSOD is conserved and dependent on its proper subcellular location in the mitochondria of a heterologous system.


Isolation of crt mutants constitutive for transcription of the DNA damage inducible gene RNR3 in Saccharomyces cerevisiae.

Ribonucleotide reductase is an essential enzyme that catalyzes the rate limiting step for production of the deoxyribonucleotides required for DNA synthesis. It is encoded by three genes, RNR1, RNR2 and RNR3, each of which is inducible by agents that damage DNA or block DNA replication. To probe the signaling pathway mediating this DNA damage response, we have designed a general selection system for isolating spontaneous trans-acting mutations that alter RNR3 expression using a chromosomal RNR3-URA3 transcriptional fusion and an RNR3-lacZ reporter plasmid. Using this system, we have isolated 202 independent trans-acting crt (constitutive RNR3 transcription) mutants that express high levels of RNR3 in the absence of DNA damaging agents. Of these, 200 are recessive and fall into 9 complementation groups. In some crt groups, the expression of RNR1 and RNR2 are also elevated, suggesting that all three RNR genes share a common regulatory pathway. Mutations in most CRT genes confer additional phenotypes, among these are clumpiness, hydroxyurea sensitivity, temperature sensitivity and slow growth. Five of the CRT genes have been identified as previously cloned genes; CRT4 is TUP1, CRT5 is POL1/CDC17, CRT6 is RNR2, CRT7 is RNR1, and CRT8 is SSN6. crt6-68 and crt7-240 are the first ts alleles of RNR2 and RNR1, respectively, and arrest with a large budded, cdc terminal phenotype at the nonpermissive temperature. The isolation of crt5-262, an additional cdc allele of POL1/CDC17, suggests for the first time that directly blocking DNA replication can provide a signal to induce the DNA damage response. crt2 mutants show a defect in basal level expression of RNR1-lacZ reporter constructs. These are the first mutants isolated in yeast that alter the regulation of DNA damage inducible genes and the identification of their functions sheds light on the DNA damage sensory network.


Characterization of Caenorhabditis elegans lectin-binding mutants.

We have identified 45 mutants of Caenorhabditis elegans that show ectopic surface binding of the lectins wheat germ agglutinin (WGA) and soybean agglutinin (SBA). These mutations are all recessive and define six genes: srf-2, srf-3, srf-4, srf-5, srf-8 and srf-9. Mutations in these genes fall into two phenotypic classes: srf-2, -3, -5 mutants are grossly wild-type, except for their lectin-binding phenotype; srf-4, -8, -9 mutants have a suite of defects, including uncoordinated movement, abnormal egg laying, and defective copulatory bursae morphogenesis. Characterization of these pleiotropic mutants at the cellular level reveals defects in the migration of the gonadal distal tip cell and in axon morphology. Unexpectedly, the pleiotropic mutations also interact with mutations in the lin-12 gene, which encodes a putative cell surface receptor involved in the control of cell fate. We propose that the underlying defect in the pleiotropic mutations may be in the general processing or secretion of extracellular proteins.


Mosaic analysis gives an estimate of the extent of genomic involvement in the development of the visual system in Drosophila melanogaster.

To investigate the role of vital loci in the development of the visual system of Drosophila, we induced mitotic recombination in individuals heterozygous for recessive organismal lethals and selected for analysis the resulting mosaics with homozygous mutant eye clones. Heads bearing clones were serially sectioned, silver-stained and examined for aberrations in the ommatidia and the neural structures to which they project. In our screen of 68 lines bearing diepoxybutane-induced X-linked lethals, 26 yielded few or no homozygous mutant clones (putative cell-lethals). Of the rest, 20 lines produced individuals with morphologically abnormal eye clones showing various degrees of aberrations in the ommatidial architecture. In 14 of these 20, the laminar cartridges innervated by the mutant clones were also disorganized. Clones with normal structure were found in 18 of the lines, and three lines were resistant to the induction of mitotic recombination. In a single line, comparatively normal clones in the eye projected to a lamina with subtle but consistent abnormalities. To the extent that we have a representative sample, these results suggest that about two-thirds of all vital genes may be essential for the normal assembly and neural connectivity of the eye. This points to a high degree of pleiotropy in the manner in which information in the genome of the fly is used in development.


Mutations in the second-largest subunit of Drosophila RNA polymerase II interact with Ubx.

Specific mutations in the gene encoding the largest subunit of RNA polymerase II (RpII215) cause a partial transformation of a structure of the third thoracic segment, the capitellum, into the analogous structure of the second thoracic segment, the wing. This mutant phenotype is also caused by genetically reducing the cellular concentration of the transcription factor Ultrabithorax (Ubx). To recover mutations in the 140,000-D second-largest subunit of RNA polymerase II (RpII140) and determine whether any can cause a mutant phenotype similar to Ubx we attempted to identify all recessive-lethal mutable loci in a 340-kilobase deletion including this and other loci. One of the 13 complementation groups in this region encodes RpII140. Three RpII140 alleles cause a transformation of capitellum to wing but unlike RpII215 alleles, only when the concentration of Ubx protein is reduced by mutations in Ubx.