The Effect of Unheated Bovine Serum on the Complement-Fixing Activity of Heat-Inactivated Bovine Antiserum with Homologous Antigen. IV. Chromatographic Studies

Unheated, non-dialyzed, normal bovine sera were fractionated by column chromatography on the cross-linked dextran, Sephadex G-25, and the fraction tested for "supplementing" properties, that is for complement-fixation augmenting activities when added to mixtures of heated bovine antiserum and homologous antigen. Supplementing activity was shown by precipitated fractions from earlier eluates with pH values below 7.2 and also by both supernatant and precipitated fractions of the later eluates with pH values from 7.6 to 8.1. The possibility is briefly discussed that certain alkaline protein substances of relatively lower molecular weight may be involved in the supplementing activities of the later fractions. Heating at 56 degrees C. for 30 min. destroyed the supplementing activity of each of these fractions. Some of the supplementing fractions proved to be anti-complementary, others were not or only slightly so. First component of complement, C(1)1, was detected in the precipitated fractions of certain of the earlier eluates with pH values below 6.5; second component of complement, C(1)2, was found exclusively in supernatant fractions of earlier eluates with pH values less than 6.2. Conglutinin was not separated from C(1)1 by this method.     

The effects of hemorrhagic shock on thyrotropin-releasing hormone and its receptors in discrete regions of rat brain

The effects of hemorrhagic shock on thyrotropin-releasing hormone (TRH) levels and its receptors were studied in different regions of the rat brain. Rats were bled for 30 min from the left femoral artery, and their mean arterial pressure was kept at 40 mmHg for the following hour. The rats were killed by decapitation. Rat brains were immediately removed and dissected into 7 regions. Hemorrhagic shock decreased TRH significantly in the frontal cortex, septum, hippocampus, and hindbrain but TRH was not changed in the striatum, hypothalamus, and midbrain. Hemorrhagic shock significantly decreased TRH receptor binding in the septum and hindbrain. Scatchard analysis of saturation isotherms of specific TRH binding showed that the decreased specific TRH binding in the hindbrain resulted not from an increase of the dissociation constant (Kd), but from a decrease in the maximum number of binding sites (Bmax). In the septum, the decrease in specific binding was due both to a decrease in Bmax and an increase in Kd. The findings indicate that TRH plays a role in the physiological response to hemorrhagic shock.     

Characterization of antibody against human liver guanase by immunoblotting and immunohistochemical staining of human liver guanase by a direct labeling antibody-enzyme method

Human liver guanase was purified and a specific antibody against it was raised in rabbits. The antiserum formed a single precipitin line with human liver extract, and also completely inhibited the activity of the liver enzyme. An immunoblotting study showed that the antibody bound specifically to one band of protein with guanase activity and not to other proteins. Therefore, we concluded that this antiserum against the liver enzyme was suitable for use in immunohistochemical demonstration of guanase. In tissue sections, the immunohistochemical reaction with this antibody was positive in the same locations as the histochemical guanase reaction with DAB (3,3'-diaminobenzidine tetrahydrochloride).     

A Novel Class of Herbicides (Specific Inhibitors of Imidazoleglycerol Phosphate Dehydratase)

A new mode of herbicidal action was established by finding specific inhibitors of imidazoleglycerol phosphate dehydratase, an enzyme of histidine (His) biosynthesis. Three triazole phosphonates inhibited the reaction of the enzyme with Ki values of 40 [plus or minus] 6.5, 10 [plus or minus] 1.6, and 8.5 [plus or minus] 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. This effect was completely reversed by the addition of His, proving that the cytotoxicity was primarily caused by the inhibition of His biosynthesis. These inhibitors showed wide-spectrum, postemergent herbicidal activity at application rates ranging from 0.05 to 2 kg/ha.     

Host nest usurpation and colony foundation in the European amazon ant,Polyergus rufescens Latr. (Hymenoptera: Formicidae)

Summary The socially parasitic mode of founding new colonies by queens of the European amazon antPolyergus rufescens was analysed in the laboratory. Newly-mated females of this obligatory slave-maker were individually introduced into queenright and queenless artificially established colonies of bothFormica cunicularia (the slave present in the natal dulotic nest) andF. rufibarbis (another potentialServiformica host). Particular attention was devoted to the behavioural patterns displayed by these young queens during the usurpation phases. Our observations, supported also by video-taping, show that the slave-making female, before laying her eggs, must penetrate the host colony, kill the resident queen, become accepted by the adult workers and appropriate the host brood. The parasite was almost always adopted in the colonies ofF. cunicularia, whereas in the presence ofF. rufibarbis it was generally killed in a short time. The failure in the attempt of usurping the colonies ofF. rufibarbis is discussed in relation to the host specificity typical of this slave-maker. Finally, egg-laying byPolyergus successful usurpers, the subsequent eclosion of the brood, and its complete social integration in the newly-established mixed colonies were also recorded.     

Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product.

We attempted to clone an inosine kinase gene of Escherichia coli. A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning. A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine. The fragment was sequenced, and one protein of 434 amino acids long was found. This protein was overexpressed. The overexpressed protein was purified and characterized. The enzyme had both inosine and guanosine kinase activity. The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively. The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively. This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate. These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine. The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W. Harlow, P. Nygaard, and B. Hove-Jensen, J. Bacteriol. 177:2236-2240, 1995).     

Origin of 33 kDa protein of vitelline membrane of quail egg: Immunological studies

The inner layer of vitelline membrane is an investment of avian ovum at the time of ovulation, but its formation is poorly understood. In order to elucidate the origin of the inner layer of vitelline membrane, a 33 kDa protein, one of the components of the inner layer, was purified from quail eggs and polyclonal antibody was raised against this protein. The tissue distribution of protein interacted with the antibody was studied by Western blotting technique. No immunoreactive component could be observed in extracts of liver, kidney, heart, lung, small intestine, brain, infundibulum, albumen-secreting region of oviduct, uterus, and wall of small white follicles. The intensive band was detected in the granulosa layer, which was isolated from the large preovulatory follicles as a monolayer of granulosa cells sandwiched between the inner layer of vitelline membrane and the basal lamina. The granulosa cells isolated from the granulosa layer also reacted with this antibody. Theca layer had no immunoreactive components. The position of the band of the 33 kDa protein on SDS-PAGE was sifted to higher molecular weight in follicular tissues as compared with that in the laid eggs, indicating that the structural change of the protein occurs after ovulation. These studies indicate that the material reactive to the antibody raised against a 33 kDa protein of quail vitelline membrane is synthesized by the granulosa cells.     

Immunological characterization of a 36 kDa Fe deficiency specific peptide in barley roots

In a previous paper we reported that an acidic 36 kDa peptide is the most strongly induced peptide among several peptides induced by Fe deficiency in barley roots. In this paper, polyclonal antibodies were raised against the 36 kDa peptide. This peptide appeared in the roots of all the graminaceous species tested (barley, rye, wheat, oat, maize, sorghum and rice) in response to Fe deficiency. More of the peptide was found in the roots of graminaceous species which secrete higher amounts of mugineic acids (MAs) under Fe deficient nutrition status. Induction of the 36 kDa peptide was first observed on the third day of Fe deficiency, rising to a maximum value on the seventh day. The trend has a positive correlation with secretion of MAs during Fe deficiency. Further, resupply of Fe resulted in a decrease in peptide production on the second day, reaching a control level on the seventh day. The rate of decrease in peptide production was observed to be slower than that of MA secretion. Other nutrient stresses such as B excess, B deficiency, Cu excess, Cu deficiency, Mn excess, Mn deficiency, Zn excess and Zn deficiency induced far less of the peptide. The specific expression of the 36 kDa peptide in roots of graminaceous species under Fe deficiency suggested the positive association of the peptide with a specific Fe deficiency tolerance mechanism in graminaceous plants.     

Post-ischemic administration of the acetylcholinesterase inhibitor ENA-713 prevents delayed neuronal death in the gerbil hippocampus

We examined by morphological methodology the effect of (S)-N-ethyl-3-[(1-dimethyl-amino)ethyl]-N-methyl-phenylcarbamate hydrogentartrate (ENA-713), an acetylcholinesterase (AChE) inhibitor, on ischemia-induced neuronal death in the gerbil hippocampus due to a 5-min ligation of bilateral common carotid arteries after light ether anesthesia. Pyramidal cells had been decreased to 27% of sham-operated controls and the number of hypertrophic astrocytes expressing glial fibrillary acidic protein (GFAP) markedly increased in the hippocampal CA1 subfield 14 days after ischemia. However, post-ischemic administration of ENA-713 (three times 0.2 mg/kg, i.p.) significantly ameliorated this ischemia-induced decrease in the number of pyramidal cells by 47% of sham-operated controls, furthermore, it reduced the ischemia-induced accumulation of GFAP-positive astrocyte in the CA1 region. Together with previous results showing that ENA-713 protected against the ischemia-induced cholinergic abnormalities in the gerbil brain and improved cholinergic dysfunctions in the senescent rat brain, our present findings suggest that ENA-713 prove to be useful for treatment with senile dementia such as cerebrovascular dementia.