Ras transformation results in an elevated level of cyclin D1 and acceleration of G1 progression in NIH 3T3 cells.

Ectopic overexpression of v-H-Ras protein in NIH 3T3 cells resulted in cellular transformation and an acceleration of G1 progression of these cells. A shortened G1 phase was found to be associated with an increased level of cyclin D1 but not cyclin E protein. Using an antisense blocking method, reduced synthesis of cyclin D1 in v-H-Ras transformants resulted in a slower G1 progression rate of these cells. Although constitutive overexpression of cyclin D1 in NIH 3T3 cells accelerated G1 progression, cells remained untransformed. Furthermore, inhibition of cyclin D1 synthesis greatly impaired the soft-agar cloning efficiency of v-H-Ras transformants. These results suggest that increased expression of cyclin D1 is necessary but not sufficient for the transforming activity of v-H-Ras. Similar effect on cell cycle progression was also observed in Raf-transformed cells. In addition to cyclin D1, cyclin E protein was found to be elevated in Src transformants. This may account for the further shortening of the G1 phase of these cells. Activation of an additional Ras-independent pathway was suggested to be responsible for the further acceleration of the G1 phase in Src transformants.     

Production of bacterial cellulose by Acetobacter xylinumwith an air-lift reactor

Bacterial cellulose was produced by Acetobacter xylinum subsp. surcrofermentans BPR2001 in a 50 liter air-lift reactor using fructose as the main carbon source. When air was supplied, the production of the cellulose was only 2.3 g/l in 80 h but when O -fortified air was supplied, the cellulose concentration increased to 5.63 g/l in 28 h and the productivity of the cellulose in an air-lift reactor with O -fortified air supply was comparable to that in a mechanically agitated jar fermenter.     

Kallikrein activation of a high molecular weight atrial peptide

Mammalian atrial extracts contain bioactive peptides that exert profound effects upon renal function and isolated smooth muscle preparations. Gel filtration chromatography of rat atrial extract separates the activity into two peaks having apparent molecular weights of 20,000 to 30,000 and less than 10,000. Mild proteolytic treatment (trypsin 1 U/ml) of the high molecular weight fraction enhances the smooth muscle relaxant activity of this fraction and concomitantly reduces the apparent molecular weight of this fraction to less than 10,000. In this report we show that urinary and submaxillary kallikrein enhances the activity of rat atrial extracts in a similar fashion. Pretreatment of the high molecular weight fraction with either kallikrein (1 microgram/ml) enhances the smooth muscle relaxant activity of this fraction. Similar treatment of the low molecular weight fraction had no effect. The enhancement of the bioactivity of the high molecular weight substance(s) by the kallikreins was abolished by aprotinin but was unaffected by soybean trypsin inhibitor. These results suggest that exogenous addition of tissue kallikrein activates a high molecular weight peptide by limited proteolysis. Analysis of the kallikrein-treated high molecular weight peptide fraction by gel filtration indicates that the biological activity comigrates with the low molecular weight peptides present in the original atrial extract.     

The effect of nitroimidazoles on glucose utilization and lactate accumulation in mouse brain

The radiation sensitizers misonidazole (MISO) and desmethylmisonidazole (DMM) can produce central and peripheral neuropathy in patients and laboratory animals. Behavioral and pathological investigations have indicated that in the central nervous system this primarily involves the cochlear and vestibular systems. Nitroimidazoles can also interfere with glycolysis in vitro under aerobic and anaerobic conditions. In the present work we have studied the effect of MISO or DMM on lactate production and glucose utilization in mouse brain. It is observed that these compounds result in a 25% inhibition of lactate production in brain slices relative to the control at a 10 mM level. Additionally, MISO (1.0 mg/g/day) or DMM (1.4 mg/g/day) were administered daily (oral) for 1, 4, 7, or 14 days to examine the effect of these two drugs on the regional glucose utilization in C3Hf mouse brain. Five microcuries of 2-deoxy[14C]glucose was given following the last drug dose and autoradiographs of serial brain sections were made and analyzed by a densitometer. Following a single dose of either MISO or DMM, no significant differences in glucose uptake were observed when compared with controls. However, following 4, 7, and 14 doses the rate of glucose utilization was significantly reduced in the intoxicated animals. Larger reductions were measured in specific regions including the posterior colliculus, cochlear nuclei, vestibular nuclei, and pons with increasing effects observed at later stages. These results share a degree of correspondence with the regional brain pathology produced by these nitroimidazoles.     

lac Operator nucleosomes. 2. lac Nucleosomes can change conformation to strengthen binding by lac repressor

We have shown previously that lac repressor binds specifically and quantitatively to lac operator restriction fragments which have been complexed with histones to form artificial nucleosomes (203 base pair restriction fragment) or core particles (144 base pair restriction fragment. We describe here a quantitative method for determining the equilibrium binding affinities of repressor for these lac reconstitutes. Quantitative analysis shows that the operator-histone reconstitutes may be grouped into two affinity classes: those with an affinity for repressor close to that of naked DNA and those with an affinity 2 or more orders of magnitude less than that of naked DNA. All particles in the lac nucleosome preparations bind repressor with high affinity, but the lac core particle preparations contain particles of both high and low affinities for repressor. Formaldehyde cross-linking causes all high-affinity species to suffer a 100-fold decrease in binding affinity. In contrast, there is no effect of cross-linking on species of low affinity. Therefore, the ability of a particle to be bound tightly by repressor depends on a property of the particle which is eliminated by cross-linking. Control experiments have shown that chemical damage to the operator does not accompany cross-linking. Therefore, the property sensitive to cross-linking must be the ability of the particle to change conformation. We infer that the particles of low native affinity, like cross-linked particles, are of low affinity because of an inability to facilitate repressor binding by means of this conformational change. Dimethyl suberimidate cross-linking experiments show that histone-histone cross-linking is sufficient to preclude high-affinity binding. Thus, the necessary conformational change involves a nucleosome histone core event. We find that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core. We present a general model which proposes that nucleosomes are divided into domains which function differentially to initiate conformational changes in response to physiological stimuli.     

Storage of marine fish erythrocytes in liquid nitrogen

The preservation of erythrocytes from cod ( Gadus morhua ), saithe ( Pollachius virens ) and mackerel ( Scomber scombrus ) at −196° C was studied using dimethyl sulphoxide (DMSO) as a cryoprotectant. Erythrocyte recoveries of greater than 90% were obtained from all species and cod erythrocytes were stored for eighteen months with insignificant lysis. Larger quantities of blood were stored by removal of plasma from citrated blood prior to the addition of DMSO solution, and by storage of pelleted frozen blood in aluminium canisters in liquid nitrogen. Maximum recoveries of washed intact erythrocytes required thawing of pellets in 125% DMSO solution and washing with buffer containing decreasing concentrations of DMSO. Washed erythrocytes kept at 4° for at least two days showed little haemolysis, were morphologically similar to fresh erythrocytes and equally susceptible to the δ-haemolysin of Staphylococcus aureus .     

刺激家兔中脑导水管周围灰质抑制束旁核伤害性放电的脊髓上机制

本工作比较了家兔脊髓背侧1/2进行横切前后刺激中脑中央灰质对束旁核伤害性放电的抑制率的改变。实验表明中央灰质既可通过公认的脊髓下行纤维抑制脊髓水平的痛传递,减弱束旁核单位的痛敏放电,也可通过某种脊髓上机制实现对束旁核的抑制。我们对这两种机制的相对重要性作了分析。用直11个束旁核痛敏单位所作的计算表明,在脊髓切割前,刺激中央灰质可使束旁核痛敏放电抑制67.5%,脊髓背侧1/2横切后,同样的中脑刺激只能使痛敏放电抑制42.9%。如以切割前的抑制率作为直100%,则切割可使抑制减弱36.5%,这一部分抑制应当是由被切断的脊髓下行纤维实现的,其余63.5%则可能主要通过脊髓上机制来实现。考虑到脊髓背半部横切不可能全部切断下行纤维,故实际上脊髓上机制的重要性不会有上述数值表示的那么大。     

A Brief Contact with Early Embryonic Cells Induces the Differentiation of Embryonal Carcinoma Cells

An assay system was developed to detect a switch of mouse embryonal carcinoma (EC) cells to the pathway for normal cell differentiation after a brief contact with normal embryonic cells. The system consisted of (1) the mixed aggregation of AT805 EC cells with 8-cell stage mouse embryos, (2) the stationary culture of the mixed aggregates into blastocysts and (3) the cell culture of inner cell masses isolated from chimeric blastocysts containing EC cells at 2, 3 and 4 days after the initiaion of chimeric aggregation. The number of foci of EC cells which appeared in the cultures of inner cell masses was decreased with a length of contact of EC cells with normal embryos as the mixed aggregates. After 4 days' contact, only fibroblastic and epithelial cells appeared in most cultures of inner cell masses. Examination of isozyme markers of GPI revealed that such cell cultures consisting of nonmalignant cells contained cells of tumor origin. Thus, it was concluded that a brief exposure to the environment of normal embryos can regulate the tumor cells to differentiate into non-malignant cells. This conclusion was substantiated by comparing the pattern of protein spots of the tumor cells with that of non-malignant cells of the tumor origin by two dimensional gel electrophoresis.