Insecticidal activity ofBacillus thuringiensis strains against the egyptian cotton leaf wormSpodoptera littoralis [Lep.: Nocutidae]

Among more than 50 isolates ofBacillus thuringiensis Berliner (B.t.) tested, 7 incited 100% mortality when 2nd instar larvae ofSpodoptera littoralis Boisduval were fed on alfalfa leaves dipped in a spore-crystal suspension of 10⁸ colony forming units/ml. Among those isolates,B.t. 24 demonstrated the highest activity. Larvae of instars 1 and 2 were the most susceptible toB.t. Susceptibility decreased with larval development. However, larvae of all instars were killed by isolateB.t. 24. Larvae that survived after feeding withB.t. 24 were retarded and fed less. Their weight relative to the controls was lower as the spore concentration on the leaves on which they fed was higher. Survival of the spores in the field dropped drastically to 2% after 4 days. Insecticidal activity of the sprayed suspension on those leaves, however, remained significant.B.t. 24 was also effective against larvae on cotton plants in the greenhouse and in a preliminary field experiment. Numbers of colony forming units recovered from leaves dipped in suspension of various spore concentrations showed significant correlation with the initial concentrations as did sprayed leaves. However, colony forming units recovered from sprayed leaves were 5–7.5 fold lower than from dipped leaves. Dipped cotton leaves showed 3.1×10^?5 ml attached to 1 mm² leaf surface whereas sprayed ones had 6×10^?6 ml. Those data are important for the determination of spore concentrations in suspensions required for spraying. The isolateB.t. 24 was serotyped byH. de Barjac as H-6B. thuringiensis entomocidus.     

Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture

Collagen phenotypes were determined for rabbit articular chondrocytes in cartilage slices and first through fifth monolayer cultures. During the first 24 hr of slice culture, chondrocytes exhibited the following collagen phenotype: 96% type II, 3% X₂Y and 1% type III. In primary monolayer culture, no other types of collagen were added to this differentiated chondrocyte phenotype; however, the synthesis per cell of each of the expressed collagens was stimulated. By the fifth day of primary culture, X₂Y synthesis increased 10 fold, and by the eighth day, a further 4 fold. In contrast, the synthesis of collagen types II and III showed no change by the fifth day, but increased 7 fold by the eighth day. These results suggest independent regulation of X₂Y in this situation. In a separate experiment, first through fifth cultures were studied. The synthesis per cell of type II collagen declined steadily and essentially ceased by the fifth culture, indicating the loss of differentiated function by these chondrocyte progeny. The loss of type II synthesis was not quantitatively replaced by the synthesis of type I trimer and type I collagen which was first detected in the third culture. While these qualitative changes in phenotype occurred, the stimulated rate of type III collagen synthesis did not change and that of X₂Y declined only slightly. Thus the termination of type II synthesis did not significantly alter the synthesis of the other collagens produced by differentiated chondrocytes. The final “de-differentiated” phenotype was 41% type I, 25% X₂Y, 20% type I trimer, 13% type III and 1% type II.     

Brain Micro vessel 12-Hydroxyeicosatetraenoic Acid Is the (S) Enantiomer and Is Lipoxygenase Derived

12-Hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid by cerebral microvessels isolated from perfused adult murine brain was reduced by the lipoxygenase inhibitors baicalein, esculetin, gossypol, nordihydroguaiaretic acid, and quercetin. Except for quercetin and gossypol, the IC50 did not exceed 10 microM. Each inhibitor, except baicalein, also decreased microvessel prostaglandin production when present in concentrations above their IC50 value for 12-HETE. In contrast, inhibitors of the cytochrome P450 monooxygenase system, clotrimazole, metyrapone, and proadifen (SKF-525A), had little effect on microvessel 12-HETE production. Chiral phase HPLC analysis revealed that only the (S) enantiomer of 12-HETE was formed. The major microvessel metabolite of eicosapentaenoic acid co-eluted with 12-hydroxyeicosapentaenoic acid (12-HEPE) on reverse-phase HPLC and the (S) enantiomer of 12-HEPE on chiral phase HPLC. Furthermore, like 12-HETE, 12-HEPE production was blocked by lipoxygenase inhibitors. These studies demonstrate that brain microvessels produce only the (S) enantiomeric 12-hydroxy derivatives of both arachidonic acid and eicosapentaenoic acid by the action of a lipoxygenase that can be selectively inhibited by baicalein. Since arachidonic acid and eicosapentaenoic acid are available to cerebral blood vessels in certain pathological settings, these 12-hydroxy acid lipoxygenase products may mediate some of the cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.     

The electron impact, chemical ionization and fast atom bombardment positive ion mass spectra of 1,2-bis(sulfonyl)methylhydrazines.

A series of bis(sulfonyl)-1-methylhydrazines were analyzed by positive ion electron impact (EI), chemical ionization (CI) and fast atom bombardment (FAB) mass spectrometry. Since these compounds showed activity against the L1210 leukemia, an understanding of their mass spectral behavior is important should the structural characterization of metabolites be required. FAB proved to be the most useful technique, generally providing abundant protonated molecule ion peaks, in contrast to the weak peaks observed with CI (ammonia or isobutane) and the total absence of molecular ion peaks in the EI mass spectra. In addition, utilizing FAB eliminated the problem of thermal decomposition, which was very difficult to control under EI and CI experimental conditions. Fragments observed in FAB and CI mass spectra were consistent with protonation at the methyl-bearing nitrogen. One can locate the R1 and R2 moieties relative to the methyl-bearing nitrogen in FAB and CI by assigning that nitrogen as the site of protonation, with subsequent elimination of R2SO2H.     

Food intake and body positional change alter the circadian rhythm of atrial natriuretic peptides excretion into human urine.

The 98 amino acid (a.a.) N-terminus of the 126 a.a. atrial natriuretic factor prohormone contains two natriuretic and vasodilatory peptides consisting of a.a. 1-30 (proANF 1-30) and a.a. 31-67 (proANF 31-67). The N-terminus and C-terminus (a.a. 99-126, i.e., ANF--also a vasodilatory peptide) circulate normally in humans with a circadian peak at 04:00 h in plasma. To determine if the N-terminus and C-terminus of the ANF prohormone are present in urine and possibly have a circadian variation in urine, six healthy volunteers had urine samples hourly while awake and every 3 h during sleep for five consecutive days obtained for radioimmunoassay. The sleep-awake pattern was varied so that after 2 days of normal sleep (supine)-awake (upright) positions, these volunteers were supine from 15:00 h on the third day until 10:00 h of the fourth day. They were then upright until 19:00 h that day when they became supine again until 02:30 h, and then were upright until 10:00 h of day 5. Three radioimmunoassays that immunologically recognize (a) the whole N-terminus (i.e., amino acids 1-98), (b) the midportion of the N-terminus (amino acids 31-67), and (c) the C-terminus of the ANF prohormone were utilized. ProANF 1-98, proANF 31-67, and the ANF radioimmunoassays each detected their respective peptides in urine. A circadian peak for each of these peptides was detected at 04:00 to 05:00 h whether the person was supine or upright during the night, which were significantly (p less than 0.001) higher than their concentrations in the afternoon of the previous days. Assuming a supine position during the day caused a significant (p less than 0.01) two- to threefold increase in these peptides in the urine. Food intake also increased the concentrations of proANF 1-98, proANF 31-67, and ANF in urine (p less than 0.001). Fluid intake when abstaining from food throughout the day lowered the concentration of these peptides in the urine. It was concluded that there is a circadian rhythm in both the N-terminus and C-terminus of the ANF prohormone excretion into urine with a peak at 04:00 h irrespective of posture, but that both posture and food and fluid intake throughout the day significantly influence the excretion of these peptides into the urine, with supine posture and food increasing their concentrations in the urine while fluid intake decreases their concentrations in the urine.     

Measurement of the exchange rates of rapidly exchanging amide protons: Application to the study of calmodulin and its complex with a myosin light chain kinase fragment

Summary A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in¹H–¹⁵N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys⁷⁵ through Thr⁷⁹ located in the central helix of calmodulin, and for the C-terminal residues Ser¹⁴⁷ and Lys¹⁴⁸. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr¹¹⁰ through Glu¹¹⁴.Istituto Guido Donegani, Novara, Italy     

Purification and further characterization of the second nitrate reductase of Escherichia coli K12

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes; it was purified 219 times with a yield of about 11%. It is an Mr-230,000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis.