Glomerular filtration rate and blood pressure monitoring in awake baboons

Minimally invasive techniques were used to collect urine with an external catheter together with automated intermittent monitoring of arterial blood pressure in awake male baboons. Using endogenous creatinine, 24-hour creatinine clearances were measured for 2 to 3 consecutive days in four intact and in four uninephrectomized baboons. Despite large differences in urinary volume and sodium excretion, reproducibility of 24-hour creatinine clearances was within 15% in 15 of 19 studies obtained from 6 of 8 animals. Arterial blood pressure was monitored intermittently at 30 to 60 minute intervals over 24 hours with a Dinamap monitor and recorder. Mean blood pressure averaged 71 +/- 4.4 to 89 +/- 5.5 mm Hg in different animals. Blood pressure tended to be lower at night than during the day. In separate studies using 15 to 60 minute urine collection periods, inulin clearance was compared in awake and in anesthetized animals with endogenous or exogenous creatinine clearance measured simultaneously. The clearance of creatinine systematically exceeded the clearance of inulin, even in intact animals with a normal serum creatinine. The creatinine-to-inulin clearance ratio averaged 1.16 +/- 0.03 at a serum concentration of 0.7 to 0.8 mg/dl; 1.27 +/- 0.03 at a serum creatinine of 1.0 to 1.1 mg/dl and 1.56 +/- 0.04 at a serum creatinine greater than 10 mg/dl. All values exceed unity significantly (p less than 0.001). Thus, renal function, including inulin clearance, can be measured in awake baboons. Duplicate or triplicate 24-hour urine collections are needed to assess the reliability of creatinine excretion. However, creatinine clearance overestimates glomerular filtration rate, as it does in humans.     

Epstein-Barr virus nuclear antigen forms a complex that binds with high concentration dependence to a single DNA-binding site.

A bacterially synthesized 28-kilodalton carboxyl-terminal fragment (28K-EBNA of Epstein-Barr virus nuclear antigen shows highly concentration dependent binding to monomer, dimer, and trimer copies of synthetic DNA-binding site 5' GATCTAGGATAGCATATGCTACCCCGGGG 3' 3' ATCCTATCGTATACGATGGGGCCCCCTAG 5' in bacterial plasmids. The rate of the binding reaction is independent of the number of sites, but dependent upon the length of the DNA containing the sites. These data are consistent with 28K-EBNA locating its binding sites by a process of facilitated transfer or sliding along the DNA. The highly concentration dependent binding suggests that multiple 28K-EBNA monomer polypeptides form a complex before or during binding. Binding occurs equally well at 24 and 37 degrees C, but not at 0 degrees C. A 28K-EBNA complex bound to a single site has unoccupied binding sites capable of interacting with additional DNA molecules. Such interaction is confirmed by agarose gel electrophoresis of protein-DNA complexes which indicate that a 28K-EBNA complex forms bridges between two DNA molecules. A bridge between the two binding regions in the Epstein-Barr virus origin of plasmid replication (oriP) would form a loop structure which could be an important feature for the regulatory function of authentic Epstein-Barr virus nuclear antigen.     

云南山楂茎尖的离体培养及其无性系快速繁殖

用云南山楂(Crataegus scabrifolia(Franch.)Rehd.)成年树茎尖和实生芽两种不同发育阶段的材料为外殖体,诱导它们休眠芽萌动,丛生芽条并诱导芽条生根。实验结果如下:1.以成年态的云南山楂侧芽为外植体,培养在附加IAA 0.1—0.5mg/l+6-BA 1-2mg/l的MS培养基上可诱导芽的萌发;将芽继代培养在附加0.5—1mg/l 6-BA的SH或MS培养基上,40天后芽数增殖4—6倍;将芽条截下置于1/2MS培养基上,附加不同浓度的IAA或IBA,可得到50—80％的生根率。2.以实生芽为外殖体,在相同条件下,则20天后芽数增殖便可获4—6倍;98％以上生根。结果表明:云南山楂的幼年态要比成年态易脱分化和再分化。     

香荚兰种子的无菌萌发试验

香荚兰(Vanilla planifolia)是兰科、香荚兰属植物,主产于湿热地区。香荚兰的果实是国际上重要的食用香料的原料。大约在1960年,我国引入香荚兰试栽。生产上,通常用扦插法繁殖;但如用种子繁殖,虽然结实较晚(约7至8年开始结实,较扦插苗晚4—5年),但结实期长,盛产期可维持约15年。而且,在杂交育种过程中,一定要用种子繁殖。     

Molecular cloning and differential expression of somatic and testis-specific H2B histone genes during rat spermatogenesis

We have cloned cDNA of a testis-specific histone, TH2B (a variant of H2B), and rat somatic H2B gene to investigate regulation of testis-specific histone genes during rat spermatogenesis. The amino acid sequences deduced from DNA sequences show extensive sequence divergence in the N-terminal third of the two histones. The rest is highly conserved. One cysteine residue was found in TH2B. No cysteine is present in somatic histones except in H3 histone. We investigated the expression of TH2B and H2B genes using the regions of sequence divergence as hybridization probes. The TH2B gene is expressed only in the testis, and the expression of this gene is detected 14 days after birth, reaching a maximum at Day 20. The level of H2B mRNA shows a reciprocal pattern. This contrasting pattern can be explained by the gradually changing proportion of spermatogonia and spermatocytes with testicular maturation. In situ cytohybridization studies show that H2B gene is expressed primarily in proliferating spermatogonia and preleptotene spermatocytes, whereas TH2B gene is expressed exclusively in pachytene spermatocytes which first appear in testis about 14 days after birth. H2B and TH2B genes appear to be ideal markers for the study of proliferation and differentiation events in spermatogenesis and their regulatory mechanisms.     

Purification and properties of formylglutamate amidohydrolase from Pseudomonas putida.

Formylglutamate amidohydrolase (FGase) catalyzes the terminal reaction in the five-step pathway for histidine utilization in Pseudomonas putida. By this action, N-formyl-L-glutamate (FG) is hydrolyzed to produce L-glutamate plus formate. Urocanate, the first product in the pathway, induced all five enzymes, but FG was able to induce FGase alone, although less efficiently than urocanate did. This induction by FG resulted in the formation of an FGase with electrophoretic mobility identical to that of the FGase induced by urocanate. A 9.6-kilobase-pair HindIII DNA fragment containing the P. putida FGase gene was cloned into the corresponding site on plasmid pBEU1 maintained in Escherichia coli. Insertion of the fragment in either orientation on the vector resulted in expression, but a higher level was noted in one direction, suggesting that the FGase gene can be expressed from either of two vector promoters with different efficiencies or from a single vector promoter in addition to a less efficient Pseudomonas promoter. FGase was purified 1,110-fold from the higher-expression clone in a yield of 10% through six steps. Divalent metal ions stimulated activity, and among those tested (Co, Fe, Zn, Ca, Ni, Cd, Mn, and Mg), Co(II) was the best activator, followed by Fe(II). FGase exhibited a Km of 14 mM for FG and a specific activity of 100 mumol/min per mg of protein in the presence of 5 mM substrate and 0.8 mM CoCl2 at 30 degrees C. The enzyme was maximally active in the range of pH 7 to 8. FGase was found to be a monomer of molecular weight 50,000. N-Acetyl-L-glutamate was not a substrate for the enzyme, but both it and N-formyl-L-aspartate were competitive inhibitors of formylglutamate hydrolysis, exhibiting Ki values of 6 and 9 mM, respectively. The absence of FGase activity as an integral part of histidine breakdown in most other organisms and the somewhat uncoordinated regulation of FGase synthesis with that of the other hut enzymes in Pseudomonas suggest that the gene encoding its synthesis may have evolved separately from the remaining hut genes.     

Interleukin 2 up-regulates its own production

It has been previously reported that a combination pair of anti-CD2 monoclonal antibodies (mAb) T11(2)+T11(3) induces a strong proliferation of T cells, which does not require the involvement of accessory cells and exogenous interleukin 2 (IL-2). More recently, we have shown that the requirement for optimal T cell proliferation depends on the combination pairs of anti-CD2 mAb used. Among them, anti-GT2+T11(1) mAb do not allow optimal proliferation of TA4 helper cloned T cells due, at least in part, to a low level of IL-2 production. This observation offered us the opportunity to study the effect of IL-2 on its own production. We show here that stimulation of cloned TA4 cells with anti-GT2+T11(1) mAb induces only a marginal level of IL-2 production. By contrast, significantly higher levels of IL-2 activity are detected in the culture supernatant of TA4 cells preincubated with recombinant IL-2 (rIL-2) before stimulation with anti-GT2+T11(1) mAb. This effect is dose-dependent over a wide range (5 to 50 IU/ml) of rIL-2 concentrations added during preincubation time. In addition, it is not due to carryover of rIL-2 bound during the preincubation time, or to lesser IL-2 consumption by these cells, or to increasing numbers of IL-2-producing cells induced by exogenous IL-2. Moreover, the observation was confirmed with IL-2 mRNA. Although neither rIL-2 nor anti-GT2+T11(1) mAb alone could induce a significant production of IL-2, rIL-2 appears to up-regulate its own production when the TA4 cells are activated by the anti-CD2 mAb-mediated second signal.     

Formation of lymphocyte colonies under serum-free culture conditions in normal individuals and patients with chronic lymphocytic leukemia

This paper describes a culture system which supports the formation of B cell and some T cell colonies under serum-free conditions in peripheral blood samples of normal individuals and patients with chronic lymphocytic leukemia (CLL) of B cell type. In this system, serum is replaced by bovine serum albumin, transferrin, cholesterol, insulin and catalase or horseradish peroxidase. In addition, it is necessary to add staphylococcus protein A, mitomycin-treated T cells as feeders and phytohemagglutinin leukocyte-conditioned medium as a source of growth factors. The plating efficiency is greatly enhanced when normal cells are incubated with galactose oxidase prior to plating and when CLL cells are exposed sequentially to neuraminidase and galactose oxidase.     

Semiquantitative calculations of catalytic free energies in genetically modified enzymes

The catalytic free energy and binding free energies of the native and the Asn-155----Thr, Asn-155----Leu, and Asn-155----Ala mutants of subtilisin are calculated by the empirical valence bond method and a free energy perturbation method. Two simple procedures are used; one "mutates" the substrate, and the other "mutates" the enzyme. The calculated changes in free energies (delta delta G not equal to cat and delta delta Gbind) between the mutant and native enzymes are within 1 kcal/mol of the corresponding observed values. This indicates that we are approaching a quantitative structure-function correlation. The calculated changes in catalytic free energies are almost entirely due to the electrostatic interaction between the enzyme-water system and the charges of the reacting system. This supports the idea that the electrostatic free energy associated with the changes of charges of the reacting system is the key factor in enzyme catalysis.     

Estrogen influences dolichyl phosphate distribution among glycolipid pools in mouse uteri

The steroid hormone 17 beta-estradiol dramatically induces uterine N-linked glycoprotein assembly [Dutt, A., Tang, J.-P., Welply, J. K., & Carson, D. D. (1986) Endocrinology (Baltimore) 118, 661-673]. To determine the role that dolichyl phosphate availability plays in this induction, we studied the effects of estrogen priming on the content of dolichyl phosphate and the distribution of dolichyl phosphate among various glycolipids in uteri. Dolichol-linked saccharides were metabolically labeled to equilibrium with either [3H]glucosamine or [3H]mannose and extracted from primary explants of uterine tissue. The amount of dolichol-linked saccharide was calculated from the specific radioactivity determined for the corresponding sugar nucleotides extracted from the tissues. The major dolichol-linked saccharides identified were mannosylphosphoryldolichol (MPD), oligosaccharylpyrophosphoryldolichol (OSL), and N,N'-diacetylchitobiosylpyrophosphoryldolichol (CBL). Estrogen increased the levels of MPD and OSL 4-fold; however, CBL levels did not change. After 3 days of treatment, the levels of these glycolipids were very similar to those in uteri from pregnant mice. Remarkably, MPD constituted 90-95% of dolichol-linked saccharides detected under all conditions. The tissue contents of total dolichyl phosphate and alkali-labile dolichyl phosphate, presumably MPD, were estimated by liquid chromatography. The levels of alkali-labile dolichyl phosphate determined in this way were in good agreement with the values estimated for MPD by metabolic labeling; moreover, alkali-labile dolichyl phosphate constituted 50-98% of the total dolichyl phosphate pool. The variations in MPD content depended upon the steroid hormone influence, most notably that of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)