The effects of sodium and magnesium-ion interactions on chromatin structure and solubility

The effects of sodium and magnesium-ion interactions on chromatin structure and solubility were examined in isolated mouse liver nuclei. To facilitate this study, a simple assay of chromatin structure was developed, based on the absorbances at 260 nm (A260) and 320 nm (A320) of nuclei in test solutions. By subtracting the A320 from the A260, a single "spectral index" was obtained which served as a useful, but not absolute, indicator of chromatin structure. Electron microscopy verified the validity of this approach. The results indicate that either 200 mM NaCl or 0.5 mM MgCl2 were capable of preserving the native 20 to 30 nm chromatin fiber structure. Below 200 mM NaCl, the native fiber progressively uncoiled to the 10 nm unit fiber. The presence of 0.5 mM MgCl2 inhibited this uncoiling. Only divalent cations stabilized condensed chromatin (heterochromatin) within the nucleus. Monovalent and divalent cations interacted with one another at critical concentrations and modified their individual effects on chromatin structure; e.g., 10 to 25 mM NaCl interfered with the action of 0.5 to 1.5 mM MgCl2, causing a complete loss of condensed chromatin. Maximum solubility of micrococcal nuclease-digested chromatin occurred at 10 mM NaCl, which treatment allowed the chromatin to unfold to the 10 nm fiber. However, ionic conditions that disrupted condensed chromatin but maintained the native chromatin fiber morphology still resulted in relatively high yields of soluble chromatin. Minimum solubility occurred under conditions which preserved the structure of condensed chromatin.     

Versatile shuttle cloning vectors for the unicellular cyanobacterium Anacystis nidulans R2

Abstract 3 new shuttle cloning vectors for gene transfer into Escherichia coli and Anacystis nidulans have been constructed by utilizing the cyanobacterial origin of replication of the small plasmid pANS from A. nidulans . 2 of these new vectors, pXB7 (pDPL13 derivative) and pECAN8 (pUC8 derivative), convey ampicillin resistance, and transform A. nidulans with relatively high frequencies. Vector pXB7 has 10 unique cloning sites; pECAN8 contains 4 cloning sites within the lacZ gene permitting rapid detection of DNA inserts in the presence of Xgal. The third vector, pKBX, has a lower transformation frequency but adds kanamycin resistance as a selectable gene for shuttle vectors of cyanobacteria.     

Obesity: new insight into the anthropometric classification of fat distribution shown by computed tomography.

Twenty eight women presenting for routine computed tomography had their waist, hip, and thigh circumferences measured. The ratio of the area of intra-abdominal fat to the area of subcutaneous fat shown in the computed tomogram taken at the umbilical level was calculated and found to correlate highly significantly with the ratio of waist to hip circumference. The correlation between these two ratios remained significant after allowing for the degree of obesity (weight (kg)/height (m)2) and age. In contrast, there was no significant correlation between the ratio of intra-abdominal to subcutaneous fat and degree of obesity. A high ratio of waist to hip circumference has been shown to be associated with a high proportion of intra-abdominal fat. Thus women with a centralised distribution of fat (high waist to hip ratio: "apples") tend to have a greater proportion of their fat in the intra-abdominal depot than do women with a peripheral fat distribution (low waist to hip ratio: "pears"). The metabolic complications of obesity, which are associated with a high ratio of waist to hip circumference, may therefore relate specifically to the amount of intra-abdominal fat.     

Fatal myocarditis in mice fed rancid purified feed

An outbreak of disease characterized by diarrhea, severe myocarditis, and high mortality occurred in a group of 800 male B6C3F1/ Har mice fed powdered purified diets. A total of 292 animals died. No evidence of an infectious agent was found, and the disease was reproduced in healthy mice by feeding the purified diets, suggesting a nutritional deficiency or toxicity. Analysis of the feed revealed adequate levels of vitamin E, reduced levels of thiamine, and elevated levels of lipoperoxide. It was concluded that mortality was due to myocarditis associated with the ingestion of rancid feed.     

Appearance of a State 1 – State 2 transition during chloroplast development in the wheat leaf: Energetic and structural considerations

The ability of developing chloroplasts to dynamically regulate the distribution of excitation energy between photosystem 1 and photosystem 2, and thus perform a State 1 – State 2 transition, was examined from analyses of chlorophyll fluorescence kinetics in 4- and 8-day-old Triticum aestivum L. cv. Maris Dove leaves grown under a diurnal light regime. Chloroplasts at all stages of development in the two leaf systems could undergo a State 1 – State 2 transition, except those found in the basal 0.5 cm of the 4-day-old leaf. The ability to physiologically modify the excitation energy distribution between the chlorophyll matrices of the two photosystems developed after the development of mature, fully photochemically competent photosystem 2 units and the appearance of excitation energy transfer between photosystem 2 and photosystem 1. Also, changes in the degree of energetic interaction between the two photosystems, in vivo rates of electron transport and the chlorophyll a/b ratio could not be correlated with the appearance of a State 1 – State 2 transition. Ultrastructural studies demonstrated a 32% increase in the degree of thylakoid appression in chloroplasts at the base of the 8-day-old leaf compared to the situation in the basal 0.5 cm of the 4-day-old leaf. This difference in thylakoid stacking can account for the differing abilities of these two tissues to perform a State 1 – State 2 transition when considered in the context of the distribution of the two photosystems within appressed and non-appressed regions of thylakoid membranes.     

Secretogogue-stimulated phosphatidylinositol breakdown in the exocrine pancreas liberates arachidonic acid, stearic acid, and glycerol by sequential actions of phospholipase C and diglyceride lipase

When mouse pancreatic "minilobules" prelabeled with either [14C]arachidonic acid (AA), [14C]stearic acid (SA), or [3H]glycerol were stimulated with the secretogogue, caerulein, there was a 60-70% loss in radioactivity in phosphatidylinositol (PI) at 30 min. This loss was accompanied by the formation of [14C] phosphatidic acid (PA), [14C]diacylglycerol (DG), [14C] triacylglycerol (TG), and free [14C]AA, [14C]SA, and [3H]glycerol. The loss in radioactive PI was the same as the loss in chemically measured PI-phosphorus. Thirty to fifty per cent of the caerulein-induced loss of prelabeled PI could be accounted for as free [14C]AA, [14C]SA, or [3H]glycerol. Increased incorporation of fatty acid or glycerol residues into DG, PA, and TG accounted for the balance of the loss in PI. The specific DG-lipase inhibitor, RHC 80267, markedly inhibited the caerulein-stimulated release of [14C]AA, [14C]SA, and [3H]glycerol and roughly doubled the caerulein-induced increment in [14C]AA-, [14C]SA-, or [3H]glycerol-labeled DG, showing that the source of the caerulein-induced increment in fatty acids and glycerol was DG. When the PI was prelabeled with either [32P] orthophosphate, [3H]myoinositol, or [3H]glycerol, only 1% or less of the radioactivity in PI was in lysophosphatidylinositol (LPI), and there was no increase in radioactivity in LPI on stimulation with caerulein. These observations, taken together, argue strongly for a phospholipase C-catalyzed breakdown of PI followed by DG-lipase and argue against any significant involvement of phospholipase A2 in PI degradation in mouse pancreas. The formation of substantial amounts of free [14C]AA on stimulation supports the view that, among other things, the phosphoinositide effect in the exocrine pancreas serves to generate arachidonate (and its metabolites). The release of appreciable amounts of free fatty acids and glycerol shows that a significant portion of the DG formed as a result of caerulein-stimulated PI breakdown is not conserved in the phosphoinositide cycle.     

Structural investigation of the capsular polysaccharide produced by a novel Klebsiella serotype (SK1). Location of O-acetyl substituents using NMR and MS techniques

The capsular polysaccharide of Klebsiella SK1 was investigated by methylation analysis, Smith degradation, and ¹H NMR spectroscopy. The oligosaccharides (P1 and P2) obtained by bacteriophage ΦSK1 degradation of the polymer were studied by methylation analysis, and 1D- and 2D-NMR spectroscopy. The resulting data showed that the patent repeating unit is a branched pentasaccharide having a structure identical to the revised structure recently proposed for Klebsiella serotype K8 capsular polysaccharide.

The 2D-NMR data showed that one third of the glucuronic acid residues in the SK1 polymer are acetylated at O-2, O-3, or O-4. FABMS studies confirmed the presence of monoacetylated glucuronic acid residues. Thus, the relationship between the Klebsiella K8 and SK1 polymers is akin to that found for Klebsiella polysaccharides K30 and K33, which have been typed as serologically distinct yet their structures differ only in the degree of acetylation.