SYNTHETIC ACTIVITIES DURING SPERMATOGENESIS IN THE LOCUST

Isolated testes of the locust Schistocerca gregaria were immersed in solutions of tritiated thymidine, cytidine, uridine, or arginine for short periods to study nucleic acid and protein synthesis during spermatogenesis. DNA synthesis in this tissue is completed prior to initiation of meiosis. Protein synthesis continues throughout the whole meiotic cycle as well as during spermatid development. Meiotic cells, except those in metaphase through early telophase, and early spermatids are also actively synthesizing RNA. The heteropycnotic X-chromosome does not produce RNA at any stage of spermatogenesis. The rates of protein and particularly RNA synthesis decrease as chromosome condensation progresses. Depression of RNA synthesis, however, is not always accompanied by cytologically detectable condensation of chromatin, since very little or no RNA is synthesized in spermatids in which chromatin condensation has barely begun.     

Identification, purification, and characterization of truncated forms of the human nerve growth factor receptor

We report the presence of truncated forms of the nerve growth factor receptor (NGFRt) in the conditioned medium of the human melanoma cell line A875 and in human urine and amniotic fluid. Radioiodinated nerve growth factor (125I-NGF) specifically bound to NGFRt was chemically cross-linked. After immunoprecipitation, labeled receptor species were visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NGFRts were purified from human adult male urine or a mixture of human amniotic fluid and infant urine by using a combination of either ion exchange chromatography (adult) or ammonium sulfate precipitation (infant) and immunoaffinity chromatography. Typical yields were about 1 microgram/liter of adult urine and 75 micrograms/liter of amniotic fluid/infant urine. The purified proteins, with molecular masses of 45, 40, and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%), were confirmed to be NGFRts by amino-terminal sequencing and were designated NGFRt-1, NGFRt-2, and NGFRt-3, respectively. The isoelectric points of these three species ranged from 3.3 to 3.95 and displayed intraspecies heterogeneity; subsequently, amino acid residues covalently modified with sialic acid-containing carbohydrates were documented. The binding affinities of these species for nerve growth factor were comparable to that of the low affinity cell surface receptor. The potential to isolate milligram quantities of human NGFRts allows for model studies of the physicochemical structure of the intact receptor and the generation of polyclonal antibodies to study the biological functions of the NGF receptor.     

High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid beta-protein precursor.

The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.     

Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

Responsiveness of mesolimbic, mesocortical, septal and hippocampal cholecystokinin and substance P neuronal systems to stress, in the male rat

The effects of acute and subchronic stress upon discrete cholecystokinin (CCK) and Substance P (SP) neuronal systems have been studied. Adult male rats were exposed to foot-shock stress for periods of 2, 4, 10, 30 or 60 min, immediately following which they were decapitated; brains were rapidly removed and frozen, and subsequently microdissected and extracted. CCK and SP were determined by RIA. In the olfactory tubercule, stress had no effect upon CCK content, but induced a rapid depletion of SP. In the prefrontal cortex, increased CCK concentrations were found following 30 min of stress exposure. In the medial septum, foot-shock led to a rapid increase in CCK content, and to a similar but delayed change in SP levels. A rapid rise in CCK concentrations was also seen in the lateral septum, but no stress effect whatsoever upon SP occurred in this structure. In the dentate gyrus, CCK exhibited a biphasic responsiveness to stress, while SP levels were increased only at the later time intervals. These data demonstrate that discrete CCK and SP neuronal systems are responsive to stress, and thereby support a functional role for these peptides in the processing of neural and hormonal signals by the CNS.     

The diffusional and osmotic water permeability of the hindgut cuticle in larval Sarcophaga bullata

The diffusional water permeability (P_D) and the pressure filtration coefficient (L_P) of isolated larval hindgut cuticle of the fleshfly, Sarcophaga bullata, were measured using tracer techniques coupled with a simple mathematical model system based on equations of non-equilibrium thermodynamics. Data obtained from the model system were matched to experimental tracer data by means of a mathematical optimization scheme. The following parameter values were obtained: P_D for tritiated water = 1.02 × 10^?6 cm-sec^?1, and L_P = 9.18 × 10^?11 cm³-dyn^?1-sec^?1. These results are now being used to determine reflection coefficients (σ's) and solute mobilities (ω's) for the cuticle system in an attempt to gain an understanding of the mechanisms controlling solute and water movements across the hindgut wall.     

Creatine kinase elevations in marathon runners: relationship to training and competition.

Elevation of creatine kinase (CK) in serum after exertion is a reliable marker of skeletal muscle injury. Limited data exist on CK levels in conditioned athletes after endurance training and competition. Serum CK was measured by a kinetic UV method (normal < 100 U/L) in 15 long distance runners before (pre-race), 24 hours after (post-race) and four weeks following (post-race) the 1979 Boston Marathon. CK levels were elevated throughout the study. Mean values for all runners and for those finishing before and after three hours and 30 minutes are as follows: Post-race CK was significantly elevated among the ten faster as compared to the five slower runners (p = 0.025). Elevations of creatine kinase drawn 24 hours post-marathon are inversely related to finishing times among the runners tested.     

Antimicrobial defense mechanisms in the horseshoe crab, Limulus polyphemus: preliminary observations with heat-derived extracts of Limulus amoebocyte lysate.

Heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) were found to contain potent “broad-spectrum” antimicrobial activity. Additional heating of the LAL extracts to 100°C for 30 min completely inactivated the antimicrobial activity and served as a control. Antimicrobial activity was observed over a temperature range of 0° to 37°C (higher temperatures not tested) with greatest activity at 37°C. Antimicrobial activity of LAL extracts was variable when tested against Gram-negative bacteria of the family Enterobacteriaceae. A twofold concentration of the extracts resulted in a significant decrease in antimicrobial effectiveness. Dialysis of single- and double-strength LAL extracts against deionized water produced a marked and significant enhancement of antimicrobial activity against both resistant and sensitive species, confirming the presence of a dialyzable inhibitor(s). Dialyzed LAL extracts were active against 13 of 14 species of Enterobacteriaceae tested. Two strains of Pseudomonas aeruginosa were susceptible as were two of three Gram-positive cocci tested. Highly sensitive bacterial species were rapidly killed with a greater than 90% reduction in viable counts occurring within the first 30 min of reaction time. Dialyzed LAL extracts also possessed considerable antifungal activity. The role of the Limulus polyphemus amoebocyte in defense against microbial invasion and dissemination is discussed.