Experimental alterations of the spores of lycopodium clavatum as related to diagenesis

Lycopodium clavatum spores have been heated to different temperatures at atmospheric pressure, at room temperature with 0.5 kbar pressure and at different temperatures with 1 kbar pressure, The effects of heat, pressure, and heat and pressure together on the spore have been examined in detail using different microscope techniques. Effects of some chemicals on these spores have also been observed.It is known that temperature and pressure change the colour of spore and pollen grain walls, mainly the exine (outer wall of the spore). Changes to the intine and the matter present in the cytoplasmic cavity (= inner contents), however, have not been taken into account by very many workers. In most of the previous works the inner contents were extracted before the experimental treatment began.In the present work, unextracted spores are used for the experiments which show two types of alterations of the spores with rising temperature at atmospheric pressure: (1) alteration of the inner contents, i.e. gradual colour change of the inner contents and their ultimate exudation from the spore through the exine at about 300°C; (2) gradual shrinkage of the exine due to the exudation of the inner contents which also causes an overall size reduction of the spore from 200° to 350°C. The exine does not change its colour up to 325°C; and this temperature, its starts to change its colour slowly, amalgamates with other exines of the empty spores, becomes amorphous, and ultimately deteriorates into unidentifiable organic matter.The process of colour change of the inner contents of spores and the general deformation are much slower when the spores are subjected to 1 kbar pressure with rising temperatures. Spores at room temperature with 0.5 kbar pressure show no colour change but only physical deformation.     

Evolution of the immunodominant domain of the circumsporozoite protein gene from Plasmodium vivax. Implications for vaccines

Recent work directed toward the development of a malarial vaccine has focused on the identification and production of the immunodominant repeating peptide of the circumsporozoite protein of the human malaria parasites as an antigen. An important factor which relates to the usefulness of this antigen in a vaccine is the rate at which the molecule changes in sequence. We have determined the sequence and arrangement of the repeating epitope of the circumsporozoite protein gene from a Plasmodium vivax isolate from La Paz, El Salvador (Sal-I). This is compared with a portion of the previously published sequence of the circumsporozoite protein gene from a P. vivax isolate from Belém, Brazil. The genes appear to be very similar in the repeat region. There are 20 similar repeating units in the El Salvador strain and only 19 units are conserved in the Brazilian strain. Following this there are degenerate repeats in both strains. Even the pattern of silent mutations in the repeat area are similar; however, they are not necessarily in the identical location and appear to have shifted. The data suggest that the repeat region of these genes may be evolving by an accelerated mechanism(s). Such a phenomenon could severely decrease the long-term efficacy of a repeat-based anti-sporozoite vaccine.     

Microbial production of d-amino acids

The production of d-aminoacylase by Alcaligenes denitrificans and Alcaligenes faecalis has been studied. The enzyme was inducibly produced and N-acetyl-d-leucine and N-acetyl-d-valine were the most effective inducers. d-methionine, d-valine, d-phenylalamine and d-leucine were produced by the enzymic hydrolysis of the appropriate N-acetyl-d-amino-acids with whole cell biomass. The hydrolysis of N-acetyl-d-methionine by A. denitrificans and N-acetyl-d-valine by A. faecalis was preferential. Maximum yields of d-methionine and d-valine were 94.3 and 84.7% at a specific product formation rate of 20.10 and 19.19 μmol min⁻¹ mg⁻¹ of wet cells at 20 mM substrate concentration and 5 mg ml⁻¹ of cell density.     

Metabolic Syndrome Is Associated with Increased Oxo-Nitrative Stress and Asthma-Like Changes in Lungs

Epidemiological studies have shown an increased obesity-related risk of asthma. In support, obese mice develop airway hyperresponsiveness (AHR). However, it remains unclear whether the increased risk is a consequence of obesity, adipogenic diet, or the metabolic syndrome (MetS). Altered L-arginine and nitric oxide (NO) metabolism is a common feature between asthma and metabolic syndrome that appears independent of body mass. Increased asthma risk resulting from such metabolic changes would have important consequences in global health. Since high-sugar diets can induce MetS, without necessarily causing obesity, studies of their effect on arginine/NO metabolism and airway function could clarify this aspect. We investigated whether normal-weight mice with MetS, due to high-fructose diet, had dysfunctional arginine/NO metabolism and features of asthma. Mice were fed chow-diet, high-fat-diet, or high-fructose-diet for 18 weeks. Only the high-fat-diet group developed obesity or adiposity. Hyperinsulinemia, hyperglycaemia, and hyperlipidaemia were common to both high-fat-diet and high-fructose-diet groups and the high-fructose-diet group additionally developed hypertension. At 18 weeks, airway hyperresponsiveness (AHR) could be seen in obese high-fat-diet mice as well as non-obese high-fructose-diet mice, when compared to standard chow-diet mice. No inflammatory cell infiltrate or goblet cell metaplasia was seen in either high-fat-diet or high-fructose-diet mice. Exhaled NO was reduced in both these groups. This reduction in exhaled NO correlated with reduced arginine bioavailability in lungs. In summary, mice with normal weight but metabolic obesity show reduced arginine bioavailability, reduced NO production, and asthma-like features. Reduced NO related bronchodilation and increased oxo-nitrosative stress may contribute to the pathogenesis.     

Characterization of immunodominant epitopes of gag and pol gene-encoded proteins of human T-cell lymphotropic virus type I.

A series of synthetic peptides derived from the corresponding regions of the gag, pol, and env proteins of human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) were used in an enzyme immunoassay to map the immunodominant epitopes of HTLV. Serum specimens from 79 of 87 (91%) HTLV-I-infected patients reacted with the synthetic peptide Gag-1a (amino acids [a.a.] 102 to 117) derived from the C terminus of the p19gag protein of HTLV-I. Minimal cross-reactivity (11%) was observed with serum specimens from HTLV-II-infected patients. Peptide Pol-3, encoded by the pol region of HTLV-I (a.a. 487 to 502), reacted with serum specimens from both HTLV-I- and HTLV-II-infected patients (94 and 86%, respectively). The antibody levels to Pol-3 were significantly higher (P less than 0.01) in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis than in either adult T-cell leukemia patients or HTLV-I-positive asymptomatic carriers. None of the other peptides studied demonstrated significant binding to serum specimens obtained from HTLV-I- or HTLV-II-infected individuals. While Gag-1a did not react with serum specimens from normal controls, Pol-3 demonstrated some reaction with specimens from seronegative individuals (11.4%). The antibodies to Gag-1a and Pol-3 in serum specimens from HTLV-I-infected patients could be specifically inhibited by the corresponding synthetic peptides and by a crude HTLV-I antigen preparation, indicating that these peptides mimic native epitopes present in HTLV-I proteins that are recognized by serum antibodies from HTLV-I- and -II-infected individuals.     

Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei

A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387). This plasmid could be isolated from liquid culture as well as mycelium from agar plates by a modified procedure. Plasmid pA387 is about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment. Hybridization experiments showed that this plasmid is present in free form and does not integrate into the chromosome. A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the Escherichia coli vector pDM10. This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei and A. orientalis by electroporation. A transformation frequency of 2.2 x 10(3) transformants per micrograms of DNA at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A. mediterranei, whereas 1.1 x 10(5) transformants per microgram of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms in A. orientalis. Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the transformation of A. mediterranei. The plasmid has a rather high copy number, is genetically stable, and can be easily reisolated from A. mediterranei. Plasmid pRL1 will be useful for further construction of a shuttle vector for E. coli and A. mediterranei and becomes the basis for the development of gene cloning techniques in Amycolatopsis spp.     

Study of properties of cholinephosphotransferase from fetal guinea pig lung mitochondria and microsomes

Summary We have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, K_m values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on K_m and V_max indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID₅₀ values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.     

Endogenous phosphorylation of retinal photoreceptor outer segment proteins by calcium phospholipid-dependent protein kinase

A calcium phospholipid-dependent protein kinase (C-kinase) activity was detected in the soluble fraction of rod outer segments (ROS) of the bovine retina. The enzyme required calcium, phosphatidylserine (PS) and diacylglycerol for maximal activity. In the presence of calcium and PS, C-kinase endogenously phosphorylated proteins with molecular weights of 95,000, 91,000, 31,000, 21,000, 19,000, 18,000, 16,000, 14,000 and 11,000. Addition of diolein in the reaction mixture further enhanced the endogenous phosphorylation of these proteins. Retinal was found to inhibit the phosphorylation of endogenous proteins by C-kinase in a concentration dependent manner. Half-maximal inhibition of enzyme activity was obtained at a retinal concentration of about 12μM. These results suggest that calcium, phospholipids and the C-kinase enzyme may play an important role in the functional regulation of rod photoreceptors and, with retinal, perhaps in the visual process as well.