Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures.

The strength in vivo of 14 promoters was determined in a system which permits the quantitation of RNA synthesis with high accuracy. Up to 75-fold differences in promoter strength were measured and the most efficient signals are promoters from coliphages T7 and T5. Their activity approaches the strength of fully induced promoters of the rRNA operons which may be close to the functional optimum of a single sequence. By contrast, a synthetic 'consensus promoter' belongs to the less efficient signals. Our data show that optimal promoter function can be achieved by alternate structures and strongly suggest that information outside of the 'classical' promoter region contributes to promoter activity.     

Volume regulation by flounder red blood cells in anisotonic media

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.     

Purification and biochemical characterization of a soluble mouse interferon-gamma receptor produced in insect cells.

The extracellular domain of the mouse interferon gamma receptor comprising amino acids 17-243 of the protein was produced in Spodoptera frugiperda cells infected with a recombinant baculovirus. The receptor was mainly secreted into the culture medium and was purified to homogeneity in several hundred milligram amounts. The purification procedure involved four chromatography steps and delivered a soluble and active receptor with an overall recovery of 30%. From each purification run, two pools of soluble receptor with the same interferon gamma binding capacity were isolated. Under reducing electrophoretic conditions the protein of pool I migrates as two bands of molecular masses 32 and 34 kDa and of pool II as two bands of 30 and 32 kDa. The soluble receptor of both pools carries a heterogeneous glycosylation. After deglycosylation it appears as one protein band of 27 kDa. N-linked carbohydrates contribute about 6 kDa and O-linked carbohydrates 1 kDa to its molecular mass. The nonreduced protein specifically binds interferon gamma on ligand blots and in a solid-phase binding system and competes for the binding of radiolabeled interferon gamma to the cell surface receptor. The soluble mouse interferon gamma receptor exists as a monomer in physiological buffer and binds interferon gamma in its dimeric form. It is stable at room temperature and against tryptic digestion, but is very sensitive to proteinase K digestion. The soluble mouse interferon gamma receptor produced in the insect/baculovirus expression system may prove useful to study the function of interferon gamma receptor as an antagonist of endogenous interferon gamma in the treatment of immunological and inflammatory disorders.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Development of a versatile computer integrated control system for bioprocess controls

A general approach is described for the implementation of a networked multi-unit computer integrated control system. The use of data acquisition hardware and graphical programming tools alleviates tedious programming and maintains potency and flexibility. One application of the control system, the control of a mammalian cell perfusion culture based on a key nutrient glucose concentration, was demonstrated. The control system offers customized user interface for all process control parameters and allows the flexibility for continued improvement and implementation of new tailored functions. The temperature, pH, dissolved oxygen and glucose level were accurately controlled. This revised version was published online in June 2006 with corrections to the Cover Date.     

Long-term stable production of monocyte-colony inhibition factor (M-CIF) from CHO microcarrier perfusion cultures

Monocyte-colony inhibition factor (M-CIF) was produced in microcarrier perfusion cultures from engineered Chinese hamster ovary (CHO) cells. Three and fifteen liter microcarrier perfusion bioreactors equipped with internal spin filters were operated for over two months. Approximately 60 L and 300 L of culture filtrate were harvested from the 3L and 15L microcarrier perfusion bioreactors respectively. During the perfusion operation, cell density reached 2–6 × 10⁶ cells/ml. Importantly, stable expression of M-CIF from the CHO cells under non-selection condition was maintained at a level of 4–10 mg/L. Specific productivity was maintained at 1.8–3.4 mg/billion cells/day. The ability of the recombinant CHO cells to migrate from microcarrier to microcarrier under our proprietary HGS-CHO-3 medium greatly facilitated microcarrier culture scale-up and microcarrier replenishment. Future directions for microcarrier perfusion system scale-up and process development are highlighted. This revised version was published online in August 2006 with corrections to the Cover Date.     

Accelerated hand bone mineral density loss is associated with progressive joint damage in hands and feet in recent-onset rheumatoid arthritis

Introduction

To investigate whether accelerated hand bone mineral density (BMD) loss is associated with progressive joint damage in hands and feet in the first year of rheumatoid arthritis (RA) and whether it is an independent predictor of subsequent progressive total joint damage after 4 years.

Methods

In 256 recent-onset RA patients, baseline and 1-year hand BMD was measured in metacarpals 2-4 by digital X-ray radiogrammetry. Joint damage in hands and feet were scored in random order according to the Sharp-van der Heijde method at baseline and yearly up to 4 years.

Results

68% of the patients had accelerated hand BMD loss (>-0.003 g/cm²) in the first year of RA. Hand BMD loss was associated with progressive joint damage after 1 year both in hands and feet with odds ratios (OR) (95% confidence intervals [CI]) of 5.3 (1.3-20.9) and 3.1 (1.0-9.7). In univariate analysis, hand BMD loss in the first year was a predictor of subsequent progressive total joint damage after 4 years with an OR (95% CI) of 3.1 (1.3-7.6). Multivariate analysis showed that only progressive joint damage in the first year and anti-citrullinated protein antibody positivity were independent predictors of long-term progressive joint damage.

Conclusions

In the first year of RA, accelerated hand BMD loss is associated with progressive joint damage in both hands and feet. Hand BMD loss in the first year of recent-onset RA predicts subsequent progressive total joint damage, however not independent of progressive joint damage in the first year.     

Medicine and surgery of amphibians

Amphibians are most notably characterized by their glandular skin, which they shed regularly and ingest routinely. It is advisable to handle amphibians only with protective gloves to avoid damaging their skin. These animals absorb water readily across the skin as a means of maintaining hydration. They also easily absorb drugs and anesthetics that are applied directly to the skin. Investigators commonly utilize cutaneous respiration in amphibians and evaluate skin abnormalities via wet mount preparations, skin scrapes, and biopsy. The examination of blood samples can be useful in evaluating the status of ill amphibians, although the similarity in function of amphibian blood cell types and those of other species is largely unknown. If surgery is required, it is necessary to fast the animals before surgery, and to monitor their hydration. The wet environment required for amphibian surgery makes sterile technique challenging, and it is advisable to institute prophylactic antibiotic therapy before the procedure. The anesthetic of choice for amphibian surgery is tricaine methanesulfonate (MS-222). Postoperative recommendations include fluids, nutritional support if necessary, and analgesia. If euthanasia is required, MS-222 overdose or pentobarbital injection are the preferred methods.