Directed mutagenesis studies of the metal binding site at the subunit interface of Escherichia coli inorganic pyrophosphatase

Recent crystallographic studies on Escherichia coli inorganic pyrophosphatase (E-PPase) have identified three Mg2+ ions/enzyme hexamer in water-filled cavities formed by Asn24, Ala25, and Asp26 at the trimer-trimer interface (Kankare, J., Salminen, T., Lahti, R., Cooperman, B., Baykov, A. A., and Goldman, A. (1996) Biochemistry 35, 4670-4677). Here we show that D26S and D26N substitutions decrease the stoichiometry of tight Mg2+ binding to E-PPase by approximately 0.5 mol/mol monomer and increase hexamer stability in acidic medium. Mg2+ markedly decelerates the dissociation of enzyme hexamer into trimers at pH 5.0 and accelerates hexamer formation from trimers at pH 7.2 with wild type E-PPase and the N24D variant, in contrast to the D26S and D26N variants, when little or no effect is seen. The catalytic parameters describing the dependences of enzyme activity on substrate and Mg2+ concentrations are of the same magnitude for wild type E-PPase and the three variants. The affinity of the intertrimer site for Mg2+ at pH 7.2 is intermediate between those of two Mg2+ binding sites found in the E-PPase active site. It is concluded that the metal ion binding site found at the trimer-trimer interface of E-PPase is a high affinity site whose occupancy by Mg2+ greatly stabilizes the enzyme hexamer but has little effect on catalysis.     

Diabetic gastroparesis: is tube feeding an alternative?

A woman aged 26 with long-standing insulin-dependent diabetes mellitus displayed recurrent periods of nausea, vomiting, abnormal blood sugar levels, weight loss and poor physical condition in spite of a diet and use of propulsive agents. Scintigraphy revealed decelerated gastric evacuation for solid and liquid nutrients. The patient recovered after insertion of a percutaneous endoscopic gastrostomy (PEG) catheter. Diabetic gastroparesis is associated with a vicious circle in which delayed gastric emptying leads to poor glucose regulation with frequent hyperglycaemia, which in its turn adversely affects gastric emptying. Treatment should be aimed at improvement of the gastric motility (for instance by propulsive agents), more accurate glucose regulation and nutritional counselling. If this fails to produce improvement, tube feeding via a permanent nasoduodenal tube or via a PEG catheter constitutes an acceptable alternative for oral nutrition.     

Current concept of structure and function of the Golgi apparatus. On the 100-anniversary of the discovery by Camillo Golgi

Early pregnancy factor (EPF) has been identified as a homologue of chaperonin 10 (cpn10) with immunosuppressive and growth factor properties. As a homologue of cpn10, it belongs to the heat shock family of proteins (hsp) but, unlike other members of this family, EPF is detected extracellularly. Early pregnancy factor was first discovered in pregnancy serum by the rosette inhibition test, and the novelty of its discovery was that its presence could diagnose pregnancy within 6-24 h of a fertile mating. As well as being a monitor of the presence of a viable embryo, it is necessary for embryonic survival. In this capacity it acts as both an immunosuppressant and growth factor. Early pregnancy factor is also a product of proliferating primary and neoplastic cells and functions as an autocrine growth factor both in vivo and in vitro. It has a modifying effect on the outcome of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Early pregnancy factor is considered to be one of the major factors involved in the modification of multiple sclerosis observed during pregnancy.     

Continuous purification of porcine lipase by rotating annular size-exclusion chromatography

Non-accidental head injury, be it shaking, impact(s) or a combination of the two, is characterised by subdural and/or subarachnoid haemorrhages with retinal haemorrhages, but minimal or absent external cranio-facial trauma. The classical assault scenario depicts the infant being gripped around the head, face, chest and abdomen and shaken or being gripped by a limb and swung. This gripping might be expected to leave physical evidence in the form of bruising. A study was undertaken to establish the prevalence, distribution and pathological association of external bruising in 24 cases of fatal non-accidental head injury in children. At autopsy, 17 cases had new external bruises, 15 old external bruises and 13, a combination of both. However, seven (29%) cases showed no fresh external bruising and five (21%) showed no external bruising at all. Thus, external bruising may be absent in children with fatal intracranial injury. The face was shown to be the commonest site of bruising followed by the forehead and buttocks. Limb, chest and abdominal bruising were found to be uncommon. Retinal haemorrhages were confirmed in 23 (96%) cases. It is hypothesised that bruising, when present, may be a result of abuse in the form of punches and slaps rather than due to gripping during the assault. We discuss why gripping does not necessarily result in external bruising.     

Pulpal response to a resin-modified glass-ionomer material on nonexposed and exposed monkey pulps

OBJECTIVE: This study evaluated the biocompatibility of a resin-modified glass-ionomer material on monkey pulps. METHOD AND MATERIALS: Standardized Class V cavities were prepared in 112 teeth distributed in six healthy adult monkeys. The resin-modified glass-ionomer cement was placed in 24 nonexposed and 36 exposed pulps according to manufacturer's instructions. ZOE was used as a control in nonexposed pulps, while calcium hydroxide was used as a control for exposed pulps. Tissues were collected at 6 to 7, 21 to 27, and 90 to 97 days. After demineralization, the teeth were serially sectioned, stained, and observed by light microscopy. RESULTS: Except for one resin-modified glass-ionomer pulp at 6 days, there were no differences between the responses of nonexposed pulps to resin-modified glass-ionomer specimens and ZOE controls. In exposed pulps, eight of 36 resin-modified glass-ionomer pulps showed various grades of inflammatory response, all associated with stained bacteria. Pulpal healing was similar in both resin-modified glass-ionomer and calcium hydroxide direct-capped exposures. Twenty-two of 26 exposed pulps restored with the resin-modified glass-ionomer cement showed dentin bridge formation at 21 and 97 days. CONCLUSION: The resin-modified glass-ionomer material exhibited acceptable biologic compatibility in exposed and nonexposed cavities.