Isolation and preliminary characterization of auxotrophs of a filamentous Cyanobacterium.

Auxotrophic mutants of the filamentous cyanobacterium Anabaena variabilis were isolated by a method in which, after mutagenesis and before penicllin enrichment, mutant and wild-type cells were separated by cavitation. Auxotrophs were identified by their inability to grow on minimal medium, and they were partially characterized by replica plating to media supplemented with single nutrients or specific groups of nutrients. Of the 83 auxotrophs isolated, 65 required an inorganic source of nitrogen for growth. In addition, auxotrophs were isolated that required methionine (six), uracil (two), adenine (one), biotin (two), and nicotinic acid (two). (The number of isolates of each type is indicated in parentheses.) The nutrient requirements of five auxotrophs appeared complex and were not determined. A large proportion of the mutants requiring inorgainic fixed nitrogen was altered in the differentiation of heterocysts. The following morphological aberrancies were observed: abnormally high and abnormally low frequencies of heterocysts; thick, uneven heterocyst envelopes; incompletely developed pore regions; very distinct pore regions; and protoplasts separated from the envelope of the heterocyst. Spontaneously occurring, N2-fixing, prototrophic revertants of mutants with aberrant heterocysts have been isolated at a frequency of 2 X 10(-8) to 4 X 10(-8) of the cells plated. That most such revertants produced morphologically normal heterocysts is consisten with the idea that heterocysts play an essential role in aerobic N2 fixation.     

Forms and activity of high molecular weight adrenocorticotropin in guinea pig

High molecular weight forms of adrenocorticotropin (ACTH) and endorphin were identified in extracts of guinea pig anterior and intermediate/posterior pituitary. Extracts of anterior pituitary contained ACTH immunoactive material with apparent molecular weights of 36,000, 24,000 and 4,500 daltons. The highest molecular weight form the ACTH co-migrated with a peak of endorphin immunoactive material. No material the size of glycosylated ACTH(1--39) was detected. Separated forms of high molecular weight ACTH prepared from mouse tumor cell culture medium stimulated the same maximal production of steroid as ACTH(1--39) in the guinea pig adrenal cell bioassay. Pro-ACTH/endorphin and ACTH biosynthetic intermediate were two orders of magnitude less potent than synthetic human ACTH(1--39); glycosylated ACTH(1--39) was equipotent to ACTH(1--39) although no similar material was detected in guinea pig pituitary extracts. Isolated guinea pig adrenal cortical cells were incubated with the various separated form of mouse tumor cell ACTH and products synthesized from (3H)pregnenolone were analyzed by two-dimensional thin-layer chromatography. The ratio of cortisol-related to corticosterone-related products was the same in response in glycosylated and nonglycosylated ACTH.     

Fluorescence staining of living cells with fluorescamine

Summary The interaction of fluorescamine with living plant and animal cells was investigated to determine which subcellular structures and molecular species might react with the dye and to assess its effects on cell viability and function.Plasma and nuclear membranes ofXenopus erythrocytes, mitochondria of sea urchin sperm, growing apices of Timothy root hairs, and various organelles ofNitella andEuglena were labelled as judged by fluorescence microscopy. Cytoplasmic fluorescence was particulate inNitella and easily displaced by moderate centrifugal fields in sea urchin eggs. Chloroplasts and nuclei isolated from cells labelledin vivo exhibited fluorescamine dependent fluorescence.Reaction seemed to have little or no effect on cell viability (Euglena) photoautotrophic growth (Euglena), cell motility (sperm), fertilizability (sperm or egg), embryonic development (sea urchin), or cytoplasmic streaming (Nitella, Timothy).Quantitative fluorometric analysis of thein vivo reactants in sperm indicated a reaction preference for phospholipid over protein compared to control cells dissociated in SDS prior to labelling. The bulk of labelled lipid was phosphatidylethanolamine.These results suggest that fluorescamine is a true vital dye which can label the cell surface as well as penetrate deeply within cells to label a variety of organelles. The distribution of fluorescence and results of chemical analysis suggest thatin vivo the dye may preferentially react with membrane.     

Translocations of chromosome 12

Summary Human chromosome 12 has been used as a model for studying the distributions of sites of induced and spontaneous breaks. The breakpoints were determined from (1) translocations involving chromosome 12, (2) spontaneous breaks in untreated cultures, (3) radiation-induced breaks, and (4) spontaneous breaks in Fanconi's anaemia.Statistical analysis showed discordance in the results both between the eleven individual bands and between the four assessments. Also, the distribution of breaks for all bands was significantly diferent from random in each assessment. Certain bands added considerable bias to the results, and when analysed individually, only four bands (p11.1, q13, q24, and p13) showed distributions over the four assessments that were significantly different from random. These four bands are Giemsa-negative bands, and two (p13 and q24) are adjacent to telomeres, while p11.4 is adjacent to the centromere. The fourth band, q13, is a known fragile site.It is concluded that bands adjacent to centromeres, which are not C-banded, are peculiarly sensitive to breakage. Telomeric bands are variable in their response to different conditions of breakage, and both the physical structure of the telomere and the specific gene sequences of individual telomeres are probably of importance in determining this response. The fragile site q13 responds as if breakage at this site is due to the base composition of the DNA.     

The quantity and quality of overwintered larvae in five species of Myrmica (Hymenoptera: Formicidae)

The numbers of queens, workers and larvae were recorded for a sample of hibernating colonies from five different species of Myrmica. The larvae were divided into three size classes. The frequencies and distribution of larvae within these size classes have been compared between species and between queened and queenless colonies. A sample of each larval class was reared to the pupal stage and the resulting castes were recorded. The Discussion compares all the species with Myrmica rubra and attempts to explain the differences observed for the different species.     

Interactions of lanthanum with purified and intact cell acetylcholinesterase of electrophorus electricus

Summary The effects of lanthanum on the activity of purified preparations of acetylcholinesterase (AChE) from the electric organ ofE. electricus and on the activity of AChE in intact electro-plaques from the same species were studied. 0.1mm LaCl₃ produced an initial inhibition of purified AChE which was followed by a delayed activation of the enzyme. Upon pretreatment of purified enzyme with LaCl₃, initial activity was markedly increased. LaCl₃ exerted a marked, concentration-dependent inhibition of intact cell AChE.La³⁺ and Ca²⁺ appear to interact competitively. In the presence of both 10mm CaCl₂ and 0.1mm LaCl₃, the initial activity of purified AChE was increased at lower ACh concentrations and inhibited at ACh concentrations greater than 3 × 10^–4 m. Inhibition of intact cell enzyme by 0.1mm LaCl₃ was relieved by increasing the CaCl₂ concentration to 10mm at ACh concentrations less than 2 × 10^–4 m.The data were analyzed assuming Michaelis-Menten kinetics and interpreted with reference to the differential binding of divalent and trivalent cations to regulatory anionic sites which are separate and distinct from the anionic site of the active center of the enzyme.     

Receptor-mediated phagocytosis of zymosan is unaffected by some conditions which reduce macrophage lysosomal enzyme secretion

We have previously shown that several agents which interfere with binding of ligands to the mannose-glycoprotein receptor on macrophages can inhibit zymosan-induced lysosomal enzyme secretion. Here we show that mannose only reduces the association of zymosan with macrophages during the first hour of exposure; after longer periods of uptake no effect is detectable. We have previously shown that mannose reduces surface binding of zymosan, probably by interfering selectively with binding to the mannose receptor. The present inhibition of association of zymosan with macrophages during short exposures can be entirely explained by this reduction of binding. Macrophages must therefore internalize zymosan at sites in addition to the mannose receptor. In contrast to macrophages the murine macrophage-like cell line P388D1 is lacking the mannose-glycoprotein receptor. Accordingly we find that binding of zymosan to P388D1 is much slighter than to macrophages and is unaffected by mannose or mannose-6-phosphate. The spontaneous lysosomal enzyme secretion of P388D1 is also unaffected by mannose. The data on macrophages confirm our previous suggestion that agents interfering with the mannose receptor inhibit the induction of lysosomal enzyme secretion by acting directly on the receptor. The data on P388D1 cells support this assertion by excluding effects at later steps in the secretory pathway.     

Development and characterization of monoclonal antibodies to human type III procollagen

Hybridomas which secrete monoclonal antibodies against human type III procollagen have been developed. By an enzyme-linked immunosorbent assay, three of the monoclonal antibodies have been determined to be against non-helical extensions of the molecules while two of the antibodies are against helical portion of the molecules which is sensitive to bacterial collagenase action. These findings have been further confirmed by carrying out immuno-reaction of the pro α-chains transferred on nitrocellulose paper from sodium dodecyl sulfate polyacrylamide gels. These monoclonal antibodies have been found to be suitable reagents for immunohistochemical studies as well as for immunoassays of type III procollagen and collagen.     

The effect of temperature, water activity and sorbic acid on ketone rancidity produced by Penicillium crustosum Thom in coconut and palm kernel oils

In a model system medium-chain fatty acids (MCFA) C₆–C₁₂ in coconut and palm kernel oil are converted to methyl ketones, one carbon atom less than the parent fatty acids, by two strains of Penicillium crustosum Thom. Conversion rates of up to 32% were seen for decanoic acid at 25°C. The optimum temperature for ketone production was 25°C in liquid suspension culture. Coconut oil contains 3.23 mmol/g MCFAs compared with 2.34 mmol/g for palm kernel oil. Coconut oil is more prone to fungal spoilage (growth and ketone production) than palm kernel oil. The main end product of fermentation was 2-undecanone reflecting the high concentration of dodecanoic acid in the substrates. Ketonic rancidity is fungal engendered. The reaction can be controlled by reducing the temperature (4°C), reducing the water activity (0.91) or by addition of sorbic acid (20 mmol/l).     

Sex Determination in Caenorhabditis elegans

In Caenorhabditis elegans, the decision to develop as a hermaphrodite or male is controlled by a cascade of regulatory genes. These genes and other tissue-specific regulatory genes also control sexual fate in the hermaphrodite germline, which makes sperm first and then oocytes. In this review, we summarize the genetic and molecular characterization of these genes and speculate how they mutually interact to specify sexual fate.