High throughput screening of protein formulation stability: practical considerations.

The formulation of protein drugs is a difficult and time-consuming process, mainly due to the complexity of protein structure and the very specific physical and chemical properties involved. Understanding protein degradation pathways is essential for the success of a biopharmaceutical drug. The present review concerns the application of high throughput screening techniques in protein formulation development. A protein high throughput formulation (HTF) platform is based on the use of microplates. Basically, the HTF platform consists of two parts: (i) sample preparation and (ii) sample analysis. Sample preparation involves automated systems for dispensing the drug and the formulation ingredients in both liquid and powder form. The sample analysis involves specific methods developed for each protein to investigate physical and chemical properties of the formulations in microplates. Examples are presented of the use of protein intrinsic fluorescence for the analysis of protein aqueous properties (e.g., conformation and aggregation). Different techniques suitable for HTF analysis are discussed and some of the issues concerning implementation are presented with reference to the use of microplates.     

Wounding,shock and stress lesions of the upper gastrointestinal tract

This retrospective study included 2532 wounded, of whom 354 (14 per cent) were treated in surgical intensive care units. In 32 patients, 1.3 per cent of all admissions, upper gastrointestinal bleeding was detected. It occurred on average 8.9 days (3–21 days) after the wounding or surgical procedure in severely injured patients and those treated in intensive care units, respectively (32 of 354 patients, 9.0 per cent). All patients received different analgesic drugs and 17 of a group that presented with bleeding were given psychotropic agents as well. The majority of patients (96.3 per cent) were administered H₂-receptor antagonists as prophylaxis against stress ulcer disease. There was a statistically significant difference between these patients treated with H₂-receptor antagonists and those on no prophylactic therapy. No statistically significant difference was found between cimetidine and ranitidine in terms of their efficacy. Endoscopic examination revealed multiple bleeding gastric and duodenal erosions. The lesions were most commonly located in the corpus of the stomach. In the majority of patients (56.25 per cent), the haemorrhage stopped spontaneously and rebleeding presented in four of 32 (12.5 per cent) patients. Of 354 patients treated in intensive care units, five (1.4 per cent) had to be operated on because of bleeding arrest. Despite all therapeutic and surgical procedures undertaken, five of 32 (15.6 per cent) patients died.     

The effects of phorbol esters with different biological activities on protein kinase C

The sapintoxins are a series of naturally occurring fluorescent phorbol esters with a range of selective biological activities (e.g. pro-inflammatory but non-tumour promoting). Their ability to activate protein kinase C (PKC) in vitro has been studied. Both tumour promoting and non-promoting phorbol derivatives activate the enzyme in vitro at low concentrations. 12-deoxyphorbol-13-phenylacetate-20 acetate (DOPPA) acts as a partial agonist in the activation of protein kinase C. Structurally distinct phorbol esters may therefore preferentially activate different forms of protein kinase C. α-sapinine, a biologically inactive compound, binds to protein kinase C without stimulating the enzyme and prevents subsequent activation by phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA).     

Clinical and radiographic features of simple and hydatid cysts of the liver

The advances of hydatid chemotherapy and the non-operative management of simple (epithelial) hepatic cysts make a correct diagnosis of increasing importance. Twenty-six patients with hepatic hydatid cysts and eleven with simple cysts were reviewed. In both groups clinical presentation was most frequently due to pain. Sex, age and size of the cysts were similar. Hydatid serology was negative in six of the hydatid patients (23 per cent). None of the simple cyst patients had positive serology but one had a borderline titre. Ultrasound and computerized tomography identified daughter cysts within the main cyst in only 17 hydatid cysts (65 per cent) and considerable intra-cyst debris was also present in five of the simple cysts. Seven of the simple cysts were deroofed surgically and the remainder underwent percutaneous aspiration. Sixteen of the hydatid cysts were found to have a biliary communication whereas this was not found with any simple cyst. The difficulties in making a precise diagnosis in some patients with a liver cyst should deter the interventional radiologist and restrain the hydatid chemotherapist.     

Medullary control of the pontine swallowing neurones in sheep

Summary The origin of the inputs from the medullary swallowing centre (dorsal region including the nucleus of the solitary tract, or ventral region corresponding to the reticular formation surrounding the nucleus ambigous) to the pontine swallowing neurones (PSNs) was studied in sheep anaesthetized with halothane.Out of 101 PSNs located in the posterior part of the trigeminal (Vth) motor nucleus, 46 were activated by stimulating either the dorsal (21 neurones) or the ventral (25 neurones) region of the ipsilateral medullary swallowing centre, 3–4 mm rostral from the obex. Thirty-one neurones out of the 46 were identified as a motoneurones supplying swallowing muscles (mylohyoïd, anterior body of digastric and medial pterygoïd). Their average activation latency through stimulation of the dorsal medullary region was about 1 ms longer than through stimulation of the ventral region (3.63 ms±0.81 versus 2.72 ms±0.32).To determine the origin of the medullary input to the PSNs, we tried to activate the medullary swallowing neurones (MSNs) antidromically through stimulating the posterior part of the Vth motor nucleus, which contains the swallowing motoneurones. Seventy-three MSNs were tested (25 located in the dorsal and 48 in the ventral region). None of the dorsal neurones tested could be antidromically activated by pontine stimulation: 15 ventral neurones showed a clear antidromic response (collision test) with an average latency of 2.5 ms±0.73. These neurones, which send their axons into the pons, were all located in the reticular formation, above the nucleus ambiguus, 3–4 mm rostral from the obex.These results suggest that MSNs in the ventral reticular formation connect the medullary swallowing centre to the Vth motor nucleus. They also suggest that during swallowing, inputs originating from the dorsal region of the medullary centre (interneurones programming the motor sequence) are relayed in the ventral region (reticular formation adjacent to the nucleus ambiguus) before reaching the PSNs.This work was supported, in part, by grants from CNRS (LA 205), INRA and M.R.I. (82 E 0685)     

Size of the Streptococcus mutans GS-5 chromosome as determined by pulsed-field gel electrophoresis.

Rare cutting restriction endonucleases were used to cut the Streptococcus mutans chromosome into large fragments. Restriction enzymes utilizing recognition sites containing 6-, 7-, or 8-base-pair sequences with only G and C nucleotides produced few fragments, most of which were greater than 100 kilobase pairs in size. Addition of the fragments from digests of SmaI, NotI, ApaI, RsrII, and EagI yielded a molecular size for the S. mutans GS-5 genome of 2,819 +/- 60 kilobase pairs.