Diffuse reflectance infrared Fourier transform spectroscopic characterization of a silica-immobilized N-hydroxysuccinimide active ester crosslinking agent and its precursors

Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy was used to characterize the product of each step in the preparation of a silica-immobilized N-hydroxysuccinimide (NHS) active ester. The preparation of this NHS active ester linkage was based on a literature procedure for the immobilization of proteins. The DRIFT method was used to guide modification of this literature procedure. The DRIFT method also was used to indicate an impurity entrapped in the 60-A diameter pores of the silica support during the formation of the immobilized active ester. Degradation of the immobilized NHS active ester, stored under either argon or dioxane, can be followed by the DRIFT method. Myoglobin and glycine were allowed to react with the active ester, and the result for this silica support was evaluated by the DRIFT method. Elemental analysis was used to provide information on the loading of the silica-immobilized moieties that were presented for DRIFT analysis.     

Non-C-G recognition sequences of DNA cytosine-5-methyltransferase from rat liver

The eukaryotic DNA cytosine-5-methyltransferase (E.C.2.1.1.37) is known to methylate cytosine in DNA mainly, but not exclusively in C-G. In the present study the minor, non-C-G recognition sequences of a rat DNA methyltransferase were analyzed by Maxam-Gilbert sequencing of in vitro methylated SV40 DNA. The enzyme methylates C-A and C-T at a 50-fold lower initial rate than C-G. Methylation of C-C at the 5'C was not observed in the piece of DNA sequenced. The methylation of C-A is very low in the trinucleotides ACA and CAC, the other C-A containing trinucleotides in DNA are much better methylacceptors. C-T was found methylated predominantly in the sequences CCTAA, ACTAA, and ACTGT. A comparison of the activity with different substrates is in favour of the enzyme making its recognition in the major groove of the DNA.     

Cloning and expression of alpha-amylase from Bacillus amyloliquefaciens in a stable plasmid vector in Lactobacillus plantarum

Lactobacillus plantarum is used in a wide range of agricultural and food fermentations. In this paper we report the introduction of alpha-amylase into the organism from Bacillus amyloliquefaciens on a stable recombinant plasmid. The genetically manipulated organism grew on MRSB medium supplemented with starch and it may be a prototype for the development of lactobacilli able to use an increased range of substrates in commercial fermentations.     

Albumin — vitamin D-binding protein haplotypes in Asian-Pacific populations

Summary We have determined the various haplotypic combinations between alleles as well as restriction fragment length polymorphisms of two linked genetic markers, albumin and vitamin D-binding protein or group-specific component, in a number of Asian-Pacific populations. Using the partial maximum likelihood method, we constructed a phylogenetic network from the haplotype frequencies to assess relationships among the populations sampled. No systematic linkage disequilibrium was detected between most of the combinations, suggesting a lack of operation of any selection pressure at the two loci. The phylogenetic analysis confirmed the known interrelationships among various populations in the Asian-Pacific region. The Australian aborigines clustered closely with the non-Austronesian-speaking highlanders from Papua New Guinea, as expected. Similarly, the Austronesian-speaking Polynesians, Micronesians, and the Southeast Asians branched off together as a separate group. The position of the Austronesian-speaking Tolais from New Britain with respect to other populations from the Southwest Pacific was anomalous. The Tolais revealed a strong affinity with the Australian aborigines, which is inexplicable. The populations from China formed a tight cluster with other populations from the Asian-Pacific region. Genetic interrelationships of these populations with the white Australians were remote, which is in accordance with the known affinities of various human racial groups.     

Light-evoked walking in crayfish: Behavioral and neuronal responses triggered by the caudal photoreceptor

The caudal photoreceptors (CPRs) of crayfish (Procambarus clarkii) can trigger walking and abdominal movements by their response to light.

Developmental regulation of expression of the lactate dehydrogenase (LDH) multigene family during mouse spermatogenesis

Expression of the Lactate Dehydrogenase (LDH) genes during various stages of spermatogenesis was studied by using a combination of Northern blot analyses and in situ hybridization techniques. These studies have indicated that developmentally programmed expression of all three functional LDH genes occurs during differentiation of germ cells. The LDH/C (ldh-3) gene was expressed exclusively during meiosis and spermiogenesis, beginning in leptotene/zygotene spermatocytes and continuing through to the elongated spermatids. LDH/C (ldh-3) gene expression was accompanied by transient expression of the LDH/A (ldh-1) gene in pachytene spermatocytes and round spermatids. The LDH/B (ldh-2) gene was expressed mainly in Sertoli and spermatogonial cells. By using somatic cell hybrids, the LDH/C (ldh-3) gene has been mapped to mouse chromosome 7, establishing that it is syntenic with the LDH/A (ldh-1) gene locus. Experimental observations made in this study provide new insight into the order and sequence of events involved in the regulation of gene expression of the LDH gene family during spermatogenesis.     

Structure and expression of ubiquitin genes of Drosophila melanogaster.

We isolated and characterized two related ubiquitin genes from Drosophila melanogaster, polyubiquitin and UB3-D. The polyubiquitin gene contained 18 repeats of the 228-base-pair monomeric ubiquitin-encoding unit arranged in tandem. This gene was localized to a minor heat shock puff site, 63F, and it encoded a constitutively expressed 4.4-kilobase polyubiquitin-encoding mRNA, whose level was induced threefold by heat shock. To investigate the pattern of expression of the polyubiquitin gene in developing animals, a polyubiquitin-lacZ fusion gene was introduced into the Drosophila genome by germ line transformation. The fusion gene was expressed at high levels in a tissue-general manner at all life stages assayed. The ubiquitin-encoding gene, UB3-D, consisted of one ubiquitin-encoding unit directly fused, in frame, to a nonhomologous tail sequence. The amino acid sequence of the tail portion of the protein had 65% positional identity with that of yeast UBI3 protein, including a region that contained a potential nucleic acid-binding motif. The Drosophila UB3-D gene hybridized to a 0.9-kilobase mRNA that was constitutively expressed, and in contrast to the polyubiquitin gene, it was not inducible by heat shock.     

Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans

Summary By use of antisera raised against purified moultinhibiting (MIH) and crustacean hyperglycemic hormone (CHH) from Carcinus maenas, complete and distinct neurosecretory pathways for both hormones were demonstrated with the PAP and immunofluorescence technique. By double staining, employing a combination of silver-enhanced immunogold labelling and PAP, both antigens could be visualized in the same section. Immunoreactive structures were studied in Carcinus maenas, Liocarcinus puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of MIH-positive perikarya, which were dispersed among the more numerous CHH-positive perikarya of the medulla terminalis X-organ (XO). The MIH-positive neurons form branching collateral plexuses adjacent to the XO and axons that are arranged around the CHH-positive central axon bundle of the principal XO-SG tract. In the SG, MIH-positive axon profiles and terminals, clustered around hemolymph lacunae, are distributed between the more abundant CHH-positive axon profiles and terminals. Colocalisation of MIH and CHH was never observed. The gross morphology of both neurosecretory systems was similar in all species examined, however, in U. pugilator and M. squinado immunostaining for MIH was relatively faint unless higher concentrations of antiserum were used. Possible reasons for this phenomenon as well as observed moult cycle-related differences in immunostaining are discussed.     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1