Quantitation of Residual Host Protein in Chicken Embryo-Derived Vaccines by Radial Immunodiffusion

Quantitation of soluble residual host protein in chicken embryo-derived vaccines was performed rapidly, economically, and accurately by radial immunodiffusion.     

Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P)

It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca²⁺. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca²⁺ is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of S-S bridges. In the absence of Ca²⁺, the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the culture fluid. In minimal medium and under conditions of enzyme stability [presence of Ca²⁺ and Ficoll (Pharmacia)], Coccus P accumulates the proteinase at a gradually reduced speed although the rate of cultural growth remains constant. It is shown that this decline in rate of accumulation is caused by the excreted proteinase itself, possibly acting on its own precursor emerging from the cell in a form susceptible to proteolytic attack and not amenable to Ca²⁺ protection. A proteinase precursor is actually demonstrable in a calciumless culture at the onset of the enzyme accumulation which follows Ca²⁺ addition. It is suggested that excreted proteins require an unfolded (or incompletely folded) structure to cross the cell envelope.     

Scanning and transmission electron microscopy evidence of the cytological effect of pyrrolnitrin on ‘Microsporon audouinii’ Gruby CBS 313-54 ‘in vitro’

Scanning and transmission electron microscopy showed that a ceiling quantity (1.56 mcg) of antifungal antibiotic Pyrrolnitrin caused heavy damage to dermathophyteMicrosporon audouinii Gruby CBS 313-54in vitro. Suitable preparation technique made it clear that the changes involved consisted of hyphal collapse on the edge of the culture, with loss of euplasmic organelles identity and cell autolysis. The cell wall, however, was apparently undamaged. These findings fit in with the suggestion that the mode of action of the antibiotic leads to generalised lipoproteic membranes damage. They must, however, be considered as representing the result of the terminal phase of cell distress.     

Pulsed-field gel analysis of human immunoglobulin heavy-chain constant region gene deletions reveals the extent of unmapped regions within the locus

The human immunoglobulin heavy-chain constant region gene locus is organized in three main gene groups, the physical distances of which are unknown. Different types of gene deletions, originated by unequal crossingover, have been found to encompass one or more genes in the locus. We have analyzed some of these deletions by means of pulsed-field gel electrophoresis, which allows resolution of large DNA fragments. By identifying a fragment containing two of the main gene groups and by observing the size reduction of this fragment in subjects with deletions, we were able to estimate the distance between the two groups and better locate the pseudogene in-between.     

Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.     

Internal desynchronization of circadian rhythms and tolerance of shift work

Fifteen male subjects including 12 shift workers (oil refinery operators) volunteered to document circadian rhythms in sleep-wake, grip strength of both hands, peak expiratory flow, heart rate, self-rated drowsiness, fatigue and attention. Each of these variables was measured 4 to 6 times/day for 2 to 3 weeks. In addition, both axillary temperature (with a shielded probe) and wrist activity were almost continuously recorded at 15 min intervals during the same time span. Individual time series were analyzed according to several statistical methods (power spectrum, cosinor, chi 2, etc.), in order to estimate the prominent circadian period tau and to evaluate both individual and subgroup differences with regard to tolerance of shift work, age, duration of shift work. The present study confirms for continuously recorded temperature and wrist activity, grip strength of both hands, heart rate and peak expiratory flow that intolerance of shift work is frequently associated with an internal desynchronization. However, this conclusion cannot be extended to circadian rhythms in self-rated drowsiness, fatigue and attention. The internal desynchronization among several circadian rhythms supports the hypothesis that these latter are driven by several oscillators, with presumable differences between right and left hemispheres as suggested by unequal values of tau in rhythms of both hand grip strength. Since an internal desynchronization can be observed in tolerant shift workers having no complaint, it is likely that symptoms of intolerance are related to the subject's sensitivity to internal desynchronization rather than to the desynchronization itself.