Solar energy for village development.

The National Academy of Sciences held a joint workshop with the Government of Tanzania last August on the potential of solar energy for the villages of that country. Costs of five solar technologies (mini-hydroelectric generators, wind, methane generation from organic wastes, photovoltaic cells, and flat-plate solar collectors) were compared with costs of diesel-generated electricity and with electricity from the national grid. Each of the five technologies is either now competitive with diesel or will be in a few years. Although the figures presented are not conclusive since they are derived from calculations rather than an actual test, the results are encouraging enough to warrant serious testing in Third World villages.

Food science in developing countries.

It is particularly important for us not to lose sight of the fact that people have been around for a long time and that they achieved remarkable technical skills long before Western science was developed. An anonymous writer from the Food and Agriculture Organization has observed: "It is a commonplace that the fundamental discoveries which made civilization possible-fire- making, tool-making, agriculture, building, calculating, writing, money-were all apparently made outside the area which has given us the marvels of modern science" (19). The writer might well have added that it is also commonly overlooked that food technology was not suddenly developed in the 20th century but has been very much a part of the lives of people everywhere ever since they began doing more to their food than gathering it and eating it raw. Lamb's "Essay on Roast Pig" may not be an accurate account of the first conjunction of fire and food, but cooking is a rather ancient practice. Fermentation is another complicated processing technology which is a traditional part of most cultures, particularly those in warm climates-beer, yogurt, cheese, the fish pastes and sauces of Asia, the palm wine of Africa, and soy sauce, are butsome examples. Native Americans, besides accomplishing marvels in plant genetics and crop development, also developed water extraction methods for treating acorns to render the flour palatable and edible, and the alkali method of processing maize. Furthermore, they developed a cure for scurvy-by making a water extraction of pine needles which are rich in ascorbic acid-long before it was first reported by Jacques Cartier in the 16th century. Similarly, calcium-deficient diets of pregnant and nursing women were traditionally successfully supplemented by calcium-rich powdered deer antlers in northern China. Among the Chinese and Greeks, goiter was cured by eating certain kinds of seaweed centuries before the disease was traced to a lack of iodine, and Kenyans learned to suck salt-rich earth to avoid salt depletion symptoms after arduous exertion in tropical heat long before "modern science" learned why (20). The enumeration of examples could go on, but this was not meant to be an essay in folklore. The point is that all so-called primitive societies developed technologies, techniques, and a store of practical knowledge of a wide range of sophistication, by what must be admitted to be the scientific method, and neither their accomplishments and skills nor those of societies "en voie de développement" should be ignored or discounted. We are confident that modern food science and technology has much to contribute to helping the food-deficit nations eat adequately. First, we must find a way of using the best of Western technology without losing sight of the reality of the situation in the third world and without failing to take into account, better than we have done so far, the secondary and tertiary implications of the changes suggested. Second, we must encourage the examination of local problems in terms of the use and improvement of local technologies which are often quite sophisticated and the result of centuries of development. And third, we must inject a greater component of cultural awareness in the education of students to make them more creative in their application of scientific knowledge to local problems and more adaptable to the conditions that exist in developing countries. We should not lose sight of the fact that because of the precarious nature of their food supply, very often developing countries have much more rigid rules governing the production, preparation, and consumption of food than usually is the case in food-surplus societies, and disturbing these rules is a very serious matter. The time is past when "West is best" can be taken for granted; "adapt and adopt" is surely less offensively arrogant and much more to the point.

DNA of Myxococcus phage MX-1. Pyrimidine isostichs and the recognition of a minor pyrimidine.

DNA was isolated from bacteriophage MX-1 and was hydrolysed with acids. Hydrolysis with 90% formic acid produced several interconvertable fluorescent artefacts. These substances were produced in greater quantity following hydrolysis with H2SO4. In the course of the course of the study it was found that DNA extracted from phage by 4-aminosalicylate and phenol contained 4-aminosalicylate and breakdown products derived from 4-aminosalicylate following acid hydrolysis. Pyrimidine oligonucleotides released by formic acid-diphenylamine hydrolysis were fractionated by two-dimensional paper chromatography and by ion-exchange chromatography. In this way, the pyrimidine isostichs were obtained and further fractionated. The distribution and with isostich patterns from a variety of DNA sources. MX-1 DNA was found to contain an unusually high frequency of cytosine-rich isostichs 5 and 6 and very low occurrence of the sequence R-Y-R. Based on its absorption spectrum and the fact that it can be labelled in vivo with [14C] orotic acid, we suggest that H-base is a pyrimidine.

Molecular characterization of the gor gene encoding glutathione reductase from Pseudomonas aeruginosa: determinants of substrate specificity among pyridine nucleotide-disulphide oxidoreductases.

In order to investigate the basis of functional diversity among the pyridine nucleotide-oxidoreductases the gor gene from Pseudomonas aeruginosa PAO, which encodes glutathione reductase, was analysed. The P. aeruginosa gor gene was identified by hybridization with a short DNA sequence from the gene encoding mercuric reductase in transposon Tn501. The gene was cloned, sequenced and overexpressed in Escherichia coli. Expression of the gene enabled rescue of an E. coli gor- mutant, confirming the identity of the cloned gene. The predicted sequence of the gene product showed homology with other members of the pyridine nucleotide-disulphide oxidoreductase family, and allowed determination of positions that may be involved in substrate specificity. These predictions provided information on the relationship of sequence to function, independently of structural data used in previous studies.

Regulation of transcription in Escherichia coli from the mer and merR promoters in the transposon Tn501.

We report studies on deletion mutants of the regulatory region of the mercuric ion resistance (mer) genes of transposon Tn501, isolated from Pseudomonas aeruginosa. Transcription of the mer genes in Escherichia coli from the promoter Pmer is regulated both positively (in the presence of mercuric salts) and negatively (in their absence) by the product of the merR gene. The merR gene is transcribed divergently with respect to the other mer genes, and negatively regulates its own synthesis. The experiments described here suggest that both positive and negative regulation by MerR, as well as its autoregulation, are largely mediated by MerR binding to a single site on DNA. This site contains a hyphenated dyad symmetrical sequence centred 24 base-pairs before the start of the mer transcript. Additional sites may be involved in full repression of the mer and merR promoters. Studies on deletions of the Pmer promoter show that the -35 sequence is not required for constitutive activity. An alternative -10 sequence may be used in the absence of the -35 and normal -10 sequences, but the properties of a point mutation indicate that, in the presence of the -35 sequence, the normal -10 sequence is required for promoter activity. A model for the regulation of expression of the mercury resistance genes by mercuric ions and the MerR protein is discussed.

RNA polymerase-DNA interactions in Streptomyces. In vitro studies of a S. lividans plasmid promoter with S. coelicolor RNA polymerase.

DNA fragments of the Streptomyces lividans plasmid pIJ101 have been tested for their ability to bind Streptomyces coelicolor RNA polymerase in vitro or to promote transcription in Streptomyces in vivo. One DNA fragment which does both was shown to encode a transcript which was expressed at low cell-density in cultures of pIJ101-containing cells. The transcript start was located on the DNA sequence of the fragment by nucleotide-primed RNA polymerase binding experiments and by S1 nuclease mapping. The pattern of DNase I protection, the sites of enhanced DNase I cleavage and the DNA sequence of the fragment suggest that the RNA polymerase holoenzyme form, which recognizes this promoter, is similar in its interaction with DNA to the major RNA polymerase of Escherichia coli. Regions showing 3/6 nucleotide homology with each of the -35 and -10 regions of the consensus sequence of E. coli promoters are present in the same positions relative to the transcript start. Symmetrical sequences which may be involved in the regulation of expression of the promoter and a potential polypeptide coding sequence can be identified.

Acute pseudogout in chronic renal failure.

Acute pseudogout (calcium pyrophosphate dihydrate deposition disease [CPPD disease]) developed in two patients with chronic renal failure. The disease had atypical features. The calcification of the involved joints was more diffuse than the usual linear stippled calcification. The first patient, age 39, was young to have pseudogout. The second patient had pseudogout and chondrocalcinosis limited to the elbow. Review of wrist roentgenograms of 82 patients (mean age, 49.0 years), undergoing hemodialysis for chronic renal failure revealed three patients (a 3.7% incidence) with chondrocalcinosis. The incidence increased to three of 19 (15.8%) in the patients over the age of 60. Although considered uncommon, pseudogout may cause acute arthritis in chronic renal failure more often than previously suspected. Joint aspiration and identification of CPPD cystals with compensated polarized light microscopy will establish the diagnosis of pseudogout.

A prospective evaluation of a simplified captopril test for the detection of renovascular hypertension.

Renovascular hypertension is potentially curable but of low prevalence. A previous retrospective study has demonstrated the use of a potentiated increase in plasma renin activity after captopril administration as a diagnostic test for renovascular hypertension; this requires two blood samples for plasma renin activity determination and three inclusive criteria for a positive test result. We applied this test prospectively to screen 100 hypertensive patients for renovascular hypertension. We evaluated 29 patients with renovascular hypertension; the remainder were diagnosed as having essential hypertension. In our patient population, a postcaptopril plasma renin activity of 5.7 ng of angiotensin per milliliter per hour (ngAl.mL-1.h-1) or greater had a 100% sensitivity and an 80% specificity for renovascular hypertension. An absolute increase in plasma renin activity with captopril of 4.7 ngAl.mL-1.h-1 or greater had a lower sensitivity of 90% and a specificity of 87%, whereas a fractional increase in plasma renin activity after captopril of 150% or higher had the lowest sensitivity of 69% and a specificity of 86%. A subgroup analysis of 38 patients who were receiving diuretic therapy demonstrated that the test sensitivity was unchanged but the specificity was reduced. In conclusion, a single postcaptopril plasma renin activity value of 5.7 ngAl.mL-1.h-1 or greater is a simplified screening test for renovascular hypertension, with excellent sensitivity and acceptable specificity. This test is well tolerated, inexpensive, and easy to perform.

Luteinizing hormone receptor knockout (LuRKO) mice and transgenic human chorionic gonadotropin (hCG)-overexpressing mice (hCG alphabeta+) have bone phenotypes.

Considerable attention has been paid to the role of sex steroids during periods of major skeletal turnover, but the interaction of the gonadotropic hormones, which include LH, FSH, and human chorionic gonadotropin (hCG), within bone tissue have been overlooked. The question is pertinent due to the recent detection of extragonadal expression of gonadotropin receptors. Western blotting, immunolocalization, and RT-PCR supported the presence of osteoblast LH receptors. However, osteoblast cells failed to bind [(125)I]hCG and treatment with hCG failed to generate either cAMP or phosphorylated ERK 1/2. Bone mineral density (BMD) and bone histomorphometry were examined in the following models: 1) LH receptor null mutant (LuRKO) mice; 2) transgenic mice overexpressing hCG (hCG alphabeta+); and 3) ovariectomized (OVX) hCG alphabeta+ model. Male LuRKO mice showed a decrease in BMD after 5 months, apparently secondary to suppressed gonadal steroid production. Similarly, 9- to 10-wk-old female LuRKO mice exhibited decreases in histomorphometric parameters tested. The data indicate that loss of LH signaling results in a reduction in bone formation or an increase in bone resorption. By contrast, there were significant increases in BMD and histomorphometric indices for female, but not male, hCG alphabeta+ mice, indicating that chronic exposure to hCG results in bone formation or a decrease in bone resorption. However, OVX of the hCG alphabeta+ mice resulted in a significant reduction in BMD comparable to OVX WT controls. Although gonadotropin levels are tightly linked to sex steroid titers, it appears that their effects on the skeleton are indirect.

Two promoters from the Streptomyces plasmid pIJ101 and their expression in Escherichia coli.

An RNA polymerase-binding restriction fragment from the small, high-copy-number Streptomyces plasmid pIJ101 has been shown to have promoter activity in vivo using a promoter-probe vector. The nucleotide sequence of the promoter (the pIJ101B promoter) and the approximate position of the transcription start point as identified by in vitro run-off transcription are presented. Both the pIJ101B promoter and the previously characterised pIJ101A promoter were found to promote transcription in Escherichia coli. The transcription start point in E. coli for the pIJ101A promoter has been determined using high-resolution S1 mapping. Initiation occurs at the same point or within 1 or 2 nucleotides of the transcription start point previously identified in Streptomyces lividans, indicating that the same transcriptional signals are recognised in both genera. The data support the idea that one type of RNA polymerase holoenzyme in Streptomyces recognises a class of promoters similar to the major consensus promoters of E. coli, and that the manner of promoter recognition is similar in both genera.

Role of the merT and merP gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions.

The bacterial transposon Tn501 carries, in addition to the genetic information for its own transposition, the genes of the mer operon (in the order merRTPAD), which code for resistance to Hg2+ ions. The basis for the resistance to Hg2+ is the enzymatic reduction of Hg2+ to Hg0 by mercuric reductase, the product of the Tn501 merA gene. We show here that deletion of the merT and merP genes from Tn501 leads to almost complete loss of the resistance phenotype, even if mercuric reductase is still present in the cytoplasm. Expression of the merT and merP genes in the absence of mercuric reductase gives a mercury-supersensitive phenotype. Mercury-dependent induction of transcription of the mer operon can occur in the absence of the merT and merP gene products. However, this induction is reduced by the presence of mercuric reductase in the cell. We conclude that one or both of the merT and merP genes of Tn501 are required for the expression of the mercury-resistance phenotype. They may in addition have a role in maximising the induction of the mer operon in the presence of Hg2+ ions. This is consistent with their proposed role in transport of Hg2+ into the cytoplasm.

Investigation of sequence homology in a group of type-II restriction/modification isoschizomers.

We have dissected the cloned PstI M and R genes to make DNA hybridization probes spanning most of the sequence. These subclones, and also the intact sequence, were used to search for nucleic acid homology by Southern blot in the DNA from twelve organisms which produce PstI isoschizomers. One of these probes, a 206-bp fragment from the N-terminal domain of the endonuclease, showed significant hybridisation in four strains (Escherichia coli strains RFL48, RFL49 and RFL83, and Streptomyces albus P). No significant hybridisation was detected with other parts of the PstI sequences. We have used computer similarity searches to look for homology between the PstI proteins and the known sequences of other type-II systems that recognise different sites. We postulate a possible recognition domain within the M.PstI methyltransferase based on similarity to the M.PaeR7 and M.TaqI methyltransferases.

HgiAI: a restriction endonuclease from Herpetosiphon giganteus HP1023.

A new class II restriction endonuclease, HgiAI has been partially purified from Herpetosiphon giganteus HP1023. The enzyme activity has been characterized and shown to recognize the family of related hexanucleotide sequences (Formula: see text) where the second and fifth nucleotide pairs are A:T pairs in either orientation. Cleavage occurs as shown, to give DNA fragments with 3'-terminal tetranucleotide extensions. The recognition sites of the enzymes SacI and SstI (Formula: see text) form a subset of the recognition site of HgiAI. One of the four possible tetranucleotide 3'-extensions (cohesive ends), generated by HgiAI is identical with those generated by SacI and SstI, another is identical with that of PstI. HgiAI should be useful for molecular cloning.

Mercuric reductase structural genes from plasmid R100 and transposon Tn501: functional domains of the enzyme.

The nucleotide sequence for the 2240 bp of plasmid R100 following the merC gene of the mercuric resistance operon has been determined and compared with the homologous sequence of transposon Tn501. The sequences following merC and preceding the next structural gene merA are unrelated between R100 and Tn501 and differ in length, with 72 bp in Tn501 and 509 bp in R100. The R100 sequence has a potential open reading frame (ORF) for a 140 amino acid polypeptide with a reasonable translational start signal preceding it. The merA genes contain 1686 (Tn501) and 1695 (R100) bp respectively. When optimally aligned, the merA sequences differ in 18% of their positions. These differences were clustered in specific regions. In addition, there was one nucleotide triplet in the Tn501 sequence which has no counterpart in the R100 sequence and one dodecyl-nucleotide sequence in the R100 sequence without counterpart in Tn501. Thus the predicted merA polypeptide of Tn501 contains 561 amino acids and the R100 counterpart contains 564 amino acids. Comparison of the R100 mercuric reductase sequences with that for human glutathione reductase [Krauth-Siegel et al.: Eur. J. Biochem. 121 (1982) 259-267], for which there is a 2 A resolution electron density map [Thieme et al.: J. Mol. Biol. 152 (1981) 763-782] shows a strong homology, with 26% identical amino acids and many conservative substitutions. This homology allows the conclusion that the active site of these enzymes and the contact positions for flavin adenine dinucleotide (FAD) and NADPH are highly conserved, while the amino- and carboxyl-terminal sequences differ.

A complex family of class-II restriction endonucleases, DsaI-VI, in Dactylococcopsis salina.

A series of class-II restriction endonucleases (ENases) was discovered in the halophilic, phototrophic, gas-vacuolated cyanobacterium Dactylococcopsis salina sp. nov. The six novel enzymes are characterized by the following recognition sequences and cut positions: 5'-C decreases CRYGG-3' (DsaI); 5'-GG decreases CC-3' (DsaII); 5'-R decreases GATCY-3' (DsaIII); 5'-G decreases GWCC-3' (DsaIV); 5'-decreases CCNGG-3' (DsaV); and 5'-GTMKAC-3' (DsaVI), where W = A or T, M = A or C, K = G or T, and N = A, G, C or T. In addition, traces of further possible activity were detected. DsaI has a novel sequence specificity and DsaV is an isoschizomer of ScrFI, but with a novel cut specificity. A purification procedure was established to separate all six ENases, resulting in their isolation free of contaminating nuclease activities. DsaI cleavage is influenced by N6-methyladenine residues [derived from the Escherichia coli-encoded DNA methyltransferase (MTase) M.Eco damI] within the overlapping sequence, 5'-CCRYMGGATC-3'; DsaV hydrolysis is inhibited by a C-5-methylcytosine residue in its recognition sequence (5'-CMCNGG-3'), generated in some DsaV sites by the E. coli-encoded MTase, M.Eco dcmI.

EaeI: a restriction endonuclease from Enterobacter aerogenes.

We describe the isolation and characterization of a type II restriction endonuclease from Enterobacter aerogenes. This recognises and cleaves the family of related sequences: 5'-Py-G-G-C-C-Pu-3' to generate DNA fragments with 5'-tetranucleotide extensions. EaeI may be useful in molecular cloning experiments, especially in conjunction with other enzymes which generate the same terminal extensions. Potential problems in the methods used to determine the cleavage specificity are discussed.

MerF is a mercury transport protein: different structures but a common mechanism for mercuric ion transporters?

Mercury resistance determinants are widespread in Gram-negative bacteria, but vary in the number and identity of genes present. We have shown that the merF gene from plasmid pMER327/419 encodes a 8.7 kDa mercury transport protein, by determining in vivo mercury volatilisation when MerF is expressed in the presence of mercuric reductase. We have confirmed that MerC of Tn21 is also a mercuric ion transporter. We have been able to detect interaction of the periplasmic protein MerP only with the MerT transporter, and not with MerF or MerC. Hydropathy analysis led to the prediction of models for MerT, MerC and MerF having three, four and two transmembrane regions respectively. In all three cases one pair of cysteine residues is predicted to be within the inner membrane with a second pair of cysteine residues on the cytoplasmic face, and the second helix contains a proline and at least one charged residue. The mechanisms of mercuric ion transport may be similar in these transporters even though their structures in the membrane differ.

Generation of vector-insensitive dig-labeled probes from large cosmid library inserts using PCR.

We have devised a general method for producing vector-insensitive probes from clones in which insert DNA (ca. 40 kb) could not be amplified in one piece nor be excised from the vector sequence. The method involves PCR and vector-specific primers in combination with restriction digestion and ligation. It yields specific PCR products that could subsequently be labeled using DIG-11-dUTP in a single-cycle PCR. In colony blot hybridization, the probes were specific for the insert DNA from which the probe was derived and, importantly, did not detect vector sequences.

The venous antithrombotic effect of LF 1351 in the rat following oral administration.

The venous antithrombotic effects of a novel chemical entity, LF 1351, were investigated in rats following single oral administration, in comparison with i.v. administered heparin. LF 1351 demonstrated a dose-related antithrombotic effect in three models of venous thrombosis. The compound was approximately equipotent in two models involving complete stasis of the vena cava and administration of factor Xa or porcine serum, giving respective ED50 values of 48.7 mg/kg and 36.7 mg/kg. LF 1351 was less effective in a model involving partial stasis in the presence of an endothelial lesion. In this case, the antithrombotic effect did not exceed 60-65%, the ED50 being 150 mg/kg. Heparin (50-300 micrograms/kg; 7.5-45.0 U/kg) was effective in all three models. At the approximate ED80 value against factor Xa-induced thrombosis (75 mg/kg) the antithrombotic effect of LF 1351 persisted for 6 h. The antithrombotic effect of LF 1351 (300 mg/kg) occurred without significant changes in APTT or thrombin time.

The venous antithrombotic profile of naroparcil in the rabbit.

The venous antithrombotic profile of naroparcil or (4-[4-cyanobenzoyl]-phenyl)-1.5-dithio-beta-D-xylopyranoside was investigated in the rabbit following single i.v. and oral administration. Naroparcil attenuated thrombus development in a Wessler stasis model of venous thrombosis (jugular vein) employing bovine factor Xa as a thrombogenic stimulus giving ED50 values of 21.9 mg/kg and 36.0 mg/kg after respectively i.v. and oral administration. Venous antithrombotic activity was maximal 2-3 h after i.v. administration and 4-8 h after oral administration. Four hours after the oral administration of maximal antithrombotic (Wessler model, factor Xa) doses (100 and 400 mg/kg), naroparcil had no significant effect on bleeding time. In platelet poor plasma obtained from animals treated 4 h previously with various doses (25 to 400 mg/kg) of naroparcil, there was no detectable anti-factor Xa nor antithrombin activity. Similarly, naroparcil had no effect on APTT nor on thrombin time. A sensitized thrombin time (to about 35 s) was modestly but significantly increased following oral administration of the compound at 400 mg/kg. However, thrombin generation by the intrinsic pathway was reduced in a dose-related manner, maximal reduction being 65% at 400 mg/kg. The same dose of naroparcil enhanced the formation of thrombin/heparin cofactor II complexes at the expense of thrombin/antithrombin III complexes in plasma incubated with (125I)-human alpha-thrombin and induced the appearance of dermatan sulfate-like material in the plasma of treated rabbits, as measured by a heparin cofactor II-mediated thrombin inhibition assay.(ABSTRACT TRUNCATED AT 250 WORDS)