A neurochemical study of rat brain maldevelopment induced by MAM treatment at different stages of gestation

The regional levels of several cell marker proteins in the brain and the ability of operant discrimination learning on a multiple fixed ratio (FR), fixed interval (FI) schedule were determined in rats with microencephaly induced by prenatal treatment with methylazoxymethanol (MAM), an antimitotic agent, on the 11 th to 13 th days (Group A) or on the 15 th day (Group B) of gestation. The cell marker proteins were determined with a sensitive enzyme immunoassay. Neuron-specific enolase (NSE; gamma gamma-enolase) had a significantly lowered level in the neocortex anterior in Group A. Non-neuronal enolase (NNE; alpha alpha-enolase) was significantly reduced in the superior colliculus, lateral geniculate body and optic nerve, but increased 1.5 fold in the retina in Group A. S-100b protein, a marker of astroglial cells, showed no significant change. As for the learning performance, the Group B animals showed an elevated behavioral activity and made evident discrimination between the FI and FR schedule. But Group A animals had prolonged FR components requiring responses to light on, and their spontaneous activity counts recorded by Automex showed an inhibition of behavior in light environments. These findings suggest a causative role of some developmental abnormality in the central visual system, indicated by the aberrant cell marker levels, in the disturbed learning ability of the Group A animals.     

Decrease of Atrial Natriuretic Peptide Content in Rat Superior Cervical Sympathetic Ganglion After Denervation and Axotomy

We found atrial natriuretic peptide (ANP), known as a humoral factor in regulating body fluid volume and blood pressure, in considerable quantities in rat superior cervical sympathetic ganglion (SCG) by radioimmunoassay after separation with reverse-phase HPLC. Although the ANP content of the immature rat 1 week after birth was low, it doubled at 2 weeks and then increased gradually, until it reached the adult level. Denervation caused a rapid decrease in the ANP content to half of the intact SCG level after 3 h, which then fell to 10% of the control value on day 2 after operation. The time course of ANP content reduction after denervation was similar but rather faster than that of activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, an observation suggesting that ANP may partly contribute to cholinergic synaptic transmission. On the other hand, axotomy produced a rather slower decrease in the ANP content than did denervation. Enucleation and sialoadenectomy also caused a considerable reduction of the ANP content. Thus, part of the ANP found in the ganglion is apparently transported from sympathetically innervated extraganglionic organs via retrograde axoplasmic flow.     

A neurochemical study of developmental impairment of the brain caused by the administration of cytosine arabinoside during the fetal or neonatal period of rats

Injection of pregnant rats with cytosine arabinoside (ara-C) (280 mg/kg) on day 15 of gestation caused a significant rise (about two times the control value) in monoamine concentrations (norepinephrine, dopamine, and serotonin) accompanied by a decrease (about 60% of the control) in the brain weight and DNA content in the cerebrum of the offspring at 60 days of age. When neonatal rats were injected with ara-C (30 mg/kg/day) for four consecutive days from the fourth to seventh days after birth, a decrease of DNA content per cerebellum and an elevation of monoamine concentrations in the cerebellum were found. However, the total content of each monoamine per cerebrum or cerebellum showed no difference from the control. These results suggest that monoaminergic neurons may remain intact, with normal monoaminergic synapses compressed into a small brain volume. The neonatal administration of ara-C caused an elevation of 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase) (EC 3.1.4.37) activity and myelin protein content in the cerebellum, suggesting a relative increase in myelin concentration as a result of hypoplasia of granule cells.     

Elevated Monoamine Levels in the Cerebral Hemispheres of Microencephlic Rats Treated Prenatally with Methylazoxymethanol or Cytosine Arabinoside

Abstract: Methylazoxymethanol (MAM) injection to rats on day 15 of gestation caused a significant rise in monoamine concentrations (1.6, 2.0, and 2.8 times the control value for serotonin, norepinephrine, and dopamine, respectively) accompanying a decrease in the brain weight and DNA content in the cerebral hemispheres of the offspring at 3 months of age; in the brain stem, these changes were much smaller. Similar change of monoamine concentrations was observed in cytosine arabinoside-induced microencephaly. The decrease of DNA content and the elevation of monoamine levels were lower with MAM injection on day 15, 13, or 17 of gestation (in that order). Serotonin content of the MAM-treated cerebral hemispheres was already 50% higher than the control immediately after birth. The activity of tryptophan hydroxylase in the MAM-treated cerebrum was 1.6 times the control value, with no change in the brain stem, while the concentration of tryptophan in the brain and plasma was equal to the control value, suggesting an important role played by this enzyme in the elevation of serotonin content. Although the marked decrease of DNA content in the cerebral hemispheres of MAM-treated rats indicates a loss of cerebral cells due to prenatal MAM poisoning, the kind of cells destroyed remain to be studied. That the remaining neurons, axons, and oligodendroglia were intact was suggested by the normal activity of CNPase.     

Long-Duration Carbon Dioxide Anesthesia of Fish Using Ultra Fine (Nano-Scale) Bubbles

Introduction: We investigated whether adding ultrafine (nano-scale) oxygen-carrying bubbles to water concurrently with dissolved carbon-dioxide (CO₂) could result in safe, long-duration anesthesia for fish. Results: To confirm the lethal effects of CO₂ alone, fishes were anesthetized with dissolved CO₂ in 20°C seawater. Within 30 minutes, all fishes, regardless of species, died suddenly due to CO₂-induced narcosis, even when the water was saturated with oxygen. Death was attributed to respiration failure caused by hypoxemia. When ultrafine oxygen-carrying bubbles were supplied along with dissolved CO₂, five chicken grunts were able to remain anesthetized for 22 hours and awoke normally within 2–3 hours after cessation of anesthesia. Conclusions: The high internal pressures and oxygen levels of the ultrafine bubbles enabled efficient oxygen diffusion across the branchia and permitted the organismal oxygen demands of individual anesthetized fish to be met. Thus, we demonstrated a method for safe, long-duration carbon dioxide anesthesia in living fish under normal water temperatures.     

Protective immunity of gene-deleted SHIVs having an HIV-1 Env against challenge infection with a gene-intact SHIV

We constructed three simian-human immunodeficiency viruses (SHIVs) lacking regulatory gene(s) and analyzed their induction of protective immunity against challenge infection with gene-intact SHIV in rhesus macaques. Inoculation of SHIV-dn lacking nef and SHIV-drn lacking nef and vpr induced transient viremia, while that of SHIV-dxrn lacking nef, vpr, and vpx induced no viremia. The SHIVs with fewer deletions were more effective in inducing neutralizing antibodies and cytotoxic T lymphocyte responses. When these macaques were challenged with parental gene-intact SHIV-NM-3rN, all the SHIV-dn-vaccinated macaques and two of the four SHIV-drn-vaccinated macaques showed complete resistance. The other two SHIV-drn-vaccinated macaques and all SHIV-dxrn-vaccinated macaques did not show complete resistance, but they did show suppression of replication of the challenge virus. These results suggested that as more genes were deleted, protective immunity was decreased.     

A large gene cluster for the Clostridium cellulovorans cellulosome

A large gene cluster for the Clostridium cellulovorans cellulosome has been cloned and sequenced upstream and downstream of the cbpA and exgS genes (C.-C. Liu and R. H. Doi, Gene 211:39-47, 1998). Gene walking revealed that the engL gene cluster (Y. Tamaru and R. H. Doi, J. Bacteriol. 182:244-247, 2000) was located downstream of the cbpA-exgS genes. Further DNA sequencing revealed that this cluster contains the genes for the scaffolding protein CbpA, the exoglucanase ExgS, several endoglucanases of family 9, the mannanase ManA, and the hydrophobic protein HbpA containing a surface layer homology domain and a hydrophobic (or cohesin) domain. The sequence of the clustered genes is cbpA-exgS-engH-engK-hbpA-engL-man A-engM-engN and is about 22 kb in length. The engN gene did not have a complete catalytic domain, indicating that engN is a truncated gene. This large gene cluster is flanked at the 5' end by a putative noncellulosomal operon consisting of nifV-orf1-sigX-regA and at the 3' end by noncellulosomal genes with homology to transposase (trp) and malate permease (mle). Since gene clusters for the cellulosome are also found in C. cellulolyticum and C. josui, they seem to be typical of mesophilic clostridia, indicating that the large gene clusters may arise from a common ancestor with some evolutionary modifications.     

ISOLATION AND REGENERATION OF HAPLOID PROTOPLASTS FROM BANGIA ATROPURPUREA (RHODOPHYTA) WITH MARINE BACTERIAL ENZYMES1

Three kinds of enzymes, agarase, β-1,4-mannanase, and β-1,3-xylanase, required for isolation of protoplasts from the red alga Bangia atropurpurea (Roth) C. Ag. were prepared from bacterial culture fluids of Vibrio sp. PO-303, Vibrio sp. MA-138, and Alcaligenes sp. XY-234, respectively, isolated from the sea environment. The optimal pH of all enzymes was around 7.5. Suitable conditions for protoplast isolation from B. atropurpurea were examined. The pretreatment of the fronds with pa-pain solution (20 mM Mes buffer, pH 7.5, containing 2% papain and 0.5 M mannitol) contributed to successful protoplast isolation. When razor-cut fragments of the fronds (about 200 mg in fresh weight) immersed in 20 mM Mes buffer, 7.5, containing 0.5 M mannitol and one unit each of agarase, β-1,4-mannanase, and β-1,3-xylanase were incubated at 22°C for 90 min with gentle agitation, 5.7 × 10⁶ protoplasts were released from them. Many protoplasts regenerated into fronds of regular or irregular shape.     

Effects of dietary soybean peptides on hepatic production of ketone bodies and secretion of triglyceride by perfused rat liver

Soybean protein isolate (SPI) was digested with protease to produce a peptides containing the low-molecular fraction (LD3) or a mixture of high- and low-molecular fractions (HD1). Rats were fed a diets containing SPI, LD3, or HD1 at a protein level equivalent to the 20% casein diet for 4 weeks. The serum triglyceride concentration was lower in rats fed SPI, LD3, and HD1 diets than in rats fed the casein diet, and the differences were significant for the cholesterol-enriched diet. The value for the LD3 group was the lowest among all groups for both the cholesterol-free and -enriched diets. The level of triglyceride in the post-perfused liver was significantly lower in the LD3 and HD1 groups and the SPI group than in the casein group irrespective of the presence of cholesterol in the diet. In the cholesterol-free diet, LD3 feeding as compared to casein feeding caused a reduction in triglyceride secretion from the liver to perfusate and an increment of hepatic ketone body production. The addition of cholesterol to the diets somewhat attenuated these effects of LD3. These results suggest that the low-molecular fraction in soybean peptides causes triglyceride-lowering activity through a reduction in triglyceride secretion from the liver to the blood circulation and the stimulation of fatty acid oxidation in the liver. There is a possibility that soybean peptides modulate triglyceride metabolism by changes in the hepatic contribution.     

Interactions of root and leaf herbivores on purple loosestrife (<Emphasis Type="Italic">Lythrum salicaria</Emphasis>)

Interspecific interactions of herbivores sharing a host plant may be important in structuring herbivore communities. We investigated host plant-mediated interactions of root (Hylobius transversovittatus) and leaf herbivores (Galerucella calmariensis), released to control purple loosestrife (Lythrum salicaria) in North America, in field and potted plant experiments. In the potted plant experiments, leaf herbivory by G. calmariensis reduced H. transversovittatus larval survival (but not larval development) but did not affect oviposition preference. Root herbivory by H. transversovittatus did not affect either G. calmariensis fitness or oviposition preference. In field cage experiments, we found no evidence of interspecific competition between root and leaf herbivores over a 4-year period. Our data suggest that large populations of leaf beetles can negatively affect root-feeding larvae when high intensity of leaf damage results in partial or complete death of belowground tissue. Such events may be rare occurrences (or affected by experimental venue) since field data differed from data obtained from potted plant experiments, particularly at high leaf beetle densities. Interspecific interactions between G. calmariensis and H. transversovittatus are possible and may negatively affect either species, but this is unlikely to occur unless heavy feeding damage results in partial or complete plant death.