1H NMR studies of T4 gene 32 protein: effects of zinc removal and reconstitution

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol bound in a tetrahedral ligand field. 113Cd NMR studies of Cd-substituted wild-type and mutant (Cys166----Ser166) g32Ps show Cys77, Cys87, and Cys90 to provide three sulfur donor atoms as ligands to the metal ion [Giedroc, D. P., Johnson, B. A., Armitage, I. M., & Coleman, J. E. (1989) Biochemistry 28, 2410]. Proton NMR signals from the His and Trp side chains of the protein have been followed as a function of pH and metal ion removal by biosynthesizing the protein with amino acids carrying protons at specific positions in a background of perdeuteriated aromatic amino acids. Only one of the two pairs of His resonances (from His64 and His81) titrates over the pH range 8.0-5.9. The nontitrating His side chain is most likely ligated to the metal ion. Upon Zn(II) removal, 1H NMR spectra of the fully protonated g32P-(A + B) exhibit substantial signal broadening in several regions of the spectrum, while the His 2,4-1H resonances are broadened beyond detection. The 1H NMR spectral characteristics of the original protein are restored by reconstitution with stoichiometric Zn(II). The broadening of the 1H NMR signals is not due to oligomerization of the protein, since small-angle X-ray scattering experiments show that the average radius of gyration of the apo-g32P-(A + B) is 25.0 A and that of the reconstituted Zn(II)-g32P-(A + B) is 31.2 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ambient temperature cycles entrain the free-running circadian rhythms of the stripe-faced dunnart,Sminthopsis macroura

Summary We examined the effect of cycles of 12 h warm (35 ± 2 °C) and 12 h (21 ± 2 °C) ambient temperature (Ta) upon the circadian activity rhythms of stripe-faced dunnarts, Sminthopsis macroura, free-running in conditions of constant dark (DD) or constant light (LL). It was hypothesized that dunnarts would entrain to the temperature cycles (TaHLs) or show perturbations of period, and that LL would act synergistically with the TaHLs in these effects. Under DD, 2 of 6 animals showed clear entrainment to the TaHLs. Other animals exhibited changes of period () and heavy negative masking of activity during the warm fraction of the TaHLs. Under LL, 3 of 12 animals entrained to the TaHLs. It was concluded that Ta is a significant though weak Zeitgeber for S. macroura compared to light. It is possible that TaHLs entrain homeotherm activity rhythms by altering the rhythm of body temperature, which is usually tightly coupled to activity.Abbreviations TaHL a cycle of Higher and Lower ambient temperature - TaC Constant Ta - Tb body temperature     

Sodium valproate inhibits the movement of secretory vesicles in rat hepatocytes.

Sodium valproate (VPA), a simple 8-carbon branched chain fatty acid, is an effective anti-epileptic drug with an occasional serious side effect of liver damage, including the accumulation of triacylglycerols within hepatocytes, and reductions in serum protein concentrations. By investigating the effects of VPA, using biliary fistula rats and isolated perfused rat livers, we have shown that secretion of triacylglycerols and rat serum albumin at the sinusoidal pole of hepatocytes, and of phospholipids, lysosomal contents, and IgA at their biliary pole, are all reduced, to somewhat different extents, by acute VPA administration. In addition, the vesicular transcytosis of exogenous protein (i.e. bovine serum albumin) from the perfusion fluid into bile is also decreased by VPA administration. To determine whether the phenomena were specific to VPA, a control series of experiments was also performed using octanoate (a straight-chain analogue of VPA). With the biliary fistula rats, octanoate did not show inhibition of secretion as compared with the saline controls; with the isolated perfused livers, however, octanoate did show such an inhibition. These phenomena suggest that VPA inhibition of secretion may be a factor in its hepatotoxicity, as the effects are apparent in both the whole animal and the isolated perfused liver, whereas octanoate is not hepatotoxic in the whole animal. Since when octanoate is administered to the isolated liver it causes an inhibition in secretion similar to that caused by VPA, it may be that the large dose of this compound reaching the liver affects a key step in liver metabolism or vesicle transport under these circumstances. Since octanoate does not normally reach the liver in such amounts, as it will normally be metabolized by other tissues, it is not hepatotoxic in the whole animal as is VPA.     

Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase

The nucleotide sequence of the fabA gene encoding beta-hydroxydecanoyl thioester dehydrase, a key enzyme of the unsaturated fatty acid synthesis pathway of Escherichia coli, has been determined by the dideoxynucleotide sequencing technique. Most of the sequence was obtained by sequencing intragenic insertions of the transposon, Tn1000, isolated in vivo. A synthetic primer complementary to a portion of the inverted repeat sequences at the ends of the transposon was used to prime DNA synthesis into the flanking fabA sequences. The gene is composed of 516 nucleotides (171 amino acid residues) encoding a protein with a molecular weight of 18,800. Approximately half of the derived amino acid sequence was confirmed by automated Edman sequencing of peptides obtained by cyanogen bromide cleavage. The active site histidine residue (His-70) has been identified by analysis of the peptides labeled by reaction with 14C-labeled 3-decynoyl-N-acetylcysteamine, a specific mechanism-activated inhibitor. A cysteine residue (Cys-69) adjacent to the active site histidine may play the role in catalysis previously assigned to a tyrosine residue. We also report a simplified purification process for the dehydrase beginning with extracts of a brain which greatly overproduces the enzyme.     

A murine IgM monoclonal antibody binds an antigenic determinant in outer surface protein A, an immunodominant basic protein of the Lyme disease spirochete

A hybridoma cell line formed by the fusion of the P3x63-Ag8.653 myeloma cell line with splenocytes from BALB/c mice immunized with Borrelia burgdorferi produced an IgM monoclonal antibody (mAb-11G1) with kappa-light chains which detected an antigenic determinant in a major spirochetal protein of m.w. approximately 31,000, also known as outer surface protein A (OSP-A). Apparent saturation was reached in approximately 35 min with 34 ng of mAb-11G1 binding to 5 X 10(7) spirochetes giving an estimated 4.8 X 10(2) IgM molecules per spirochete and thus a minimum of 480 binding sites per organism. Enzymatic digestion studies suggest that the antigenic determinant to mAb-11G1 is contained within the peptide chain of OSP-A as binding could be eliminated by treatment of the spirochetes with proteinase K, Pronase and pepsin (100 to 200 micrograms/ml of enzyme) but not by trypsin or bromelain treatment. Periodate oxidation as well as mixed and endoglycosidase treatment of the spirochetes did not alter the binding of mAb-11G1. Two-dimensional gel electrophoresis of whole spirochetal cell lysates disclosed that OSP-A is a heterogeneously charged basic protein with an apparent isoelectric point range from 8.5 to 9.0. Amino acid analysis of OSP-A showed a 10% lysine component which could provide the basic nature to the protein. OSP-A with the intact antigenic determinant for mAb-11G1 can be found in the urine of hamsters experimentally infected with B. burgdorferi.     

Patents and patent office resources in biotechnology

Patents play an increasingly important role in the dissemination of information in many fast moving fields such as biotechnology and semiconductors. Quite a few new developments are introduced as patents, and only later, if at all, do they find their way into the scientific literature. In spite of this, patents lack wide acceptance as a source of information among scientists in academia and, to a lesser degree, industry. Patents share many similarities with scientific papers. They both are organized in a similar way and are carefully reviewed by experts in the field. Both can be effective and timely sources of information. Patents can be accessed through data bases, library collections, the "Official Gazette of the Patent and Trademark Office," or directly in the Patent and Trademark Office. This article is designed to serve as a guide to the type of information which can be found in patents, and alternatives for obtaining this information.     

Photoperiod control of poplar bark storage protein accumulation

Bark storage proteins (BSPs) accumulate in the inner bark parenchyma of many woody plants during autumn and winter. We investigated the effect of a short-day (SD) photoperiod on the accumulation of the 32-kilodalton bark storage protein of poplar (Populus deltoides Bart. ex Marsh.) under controlled environmental and natural growing conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein gel blot analysis revealed that 10 days of SD exposure (8 hours of light) resulted in a 20% increase in the relative abundance of the 32-kilodalton bark storage protein of poplar. After 17 days of SD exposure, the 32-kilodalton bark storage protein accounted for nearly one-half of the soluble bark proteins. In natural field conditions, accumulation of the 32-kilodalton bark storage protein was observed to start by August 18 (daylength 14.1 hours). Immunoprecipitation of in vitro translation products with anti-BSP serum revealed that the SD protein accumulation was correlated with changes in the pool of translatable mRNA. A survey of poplar clones from different geographic origins revealed the presence of the 32-kilodalton BSP in the dormant bark of all the clones tested. These results demonstrate that a SD photoperiod induces, whether directly or indirectly, rapid changes in woody plant gene expression, leading to the accumulation of BSP.     

Experimental effects of elevated salinity on three benthic invertebrates in Pyramid Lake,Nevada

Salinity of Pyramid Lake increased from 3.7 to 5.5 between 1933 and 1980. Concern over future reductions in overall species richness prompted experiments to assess responses of dominant lake organisms to elevated salinity. Salinity tolerances of three important benthic invertebrates, Hyalella aztecta, Chironomus utahensis, and Heterocypris sp., were tested in controlled laboratory bioassays and also in a semi-natural environment consisting of large (47 m³) mesocosms.Densities of H. azteca in mesocosms were significantly lower at salinities of 8.0 and 11.0 compared with 5.6 controls in year one, but not in 8.5 salinity mesocosms in year two. The 96-h LC₅₀ for H. azteca was high at 19.5. Short-term mortalities of C. utahensis were 100% at salinities of 13.3 and greater. Fifty-seven percent fewer larvae matured from third to fourth instar at 8.9 than at 5.5 salinity in 17 day subacute bioassays. Furthermore, larval chironomid densities and emergence of adults from mesocosms were significantly reduced at salinities of 8.0 and higher compared with controls. Mortality of Heterocypris sp. was 50% at a salinity of 18.6 in laboratory bioassays and populations in mesocosms ranged between 40 and 100% lower at salinities of 8.0 and 11.0 than in controls.Multiple generation mesocosm experiments indicated all three invertebrates were more sensitive to elevated salinity than results of short-term bioassays. Our studies suggest populations of these invertebrates may be reduced from present levels if Pyramid Lake's salinity were to double, although none are expected to be extirpated. Food habit shifts and reduced production of lake fishes are likely consequences of salinity-induced disruption in the benthic invertebrate forage base.