Release of Endogenous Amino Acids from Striatal Neurons in Primary Culture

Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl-cellulose, ATP-agarose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442-fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory subunit from the type II cyclic AMP-dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G-200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 microM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity.     

Glutamate Dehydrogenase in Human Brain: Regional Distribution and Properties

Glutamate dehydrogenase (GDH) activity was studied in 17 regions of six human brains. Duration and conditions of the postmortem period did not affect enzyme activity. Specific activity ranged between 103 and 377 nmoles/min/mg protein at 25 degrees C and it was 10-fold higher than that found in leukocytes. Apart from exclusively white matter regions (corpus callosum and centrum ovale), there was a moderate regional distribution (2.5-fold variation), with highest values in the inferior olive and hypothalamus, and lowest in the cerebellum and lenticular nucleus. With alpha-ketoglutarate (alpha-KG), NADH, or NH4+ as variable substrate, the apparent Km values in human brain were Km alpha-KG = 1.9 X 10(-3) M, KmNADH = 0.21 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M, and in leukocytes they were Km alpha-KG = 1.7 X 10(-3) M, KmNADH = 0.24 X 10(-3) M, and KmNH4+ = 28 X 10(-3) M. The effects of cofactors, inhibitor, and pH were similar in brain and leukocyte GDH.     

A smooth muscle cell line suitable for the study of voltage sensitive calcium channels

A cell line originating from the fetal rat aorta has been studied with respect to 45Ca2+ uptake. Kinetic experiments showed an initial rapid uptake followed by a slow linear phase; both the initial rate and the maximum uptake were increased in the presence of 55 mM potassium chloride. The calcium channel antagonists, darodipine (PY 108-068) and verapamil, inhibited both the basal and the potassium chloride stimulated uptake. Neither tetrodotoxin nor furosemide affected either basal or depolarisation induced 45Ca2+ uptake. Blockade of the Na+/K+ ATPase by ouabain and of the Ca2+ ATPase by vanadate caused a net increase in cellular 45Ca2+ accumulation.     

Human natural killer cytotoxic factor (NKCF): role of IFN-alpha

The relationship between production of NKCF and IFN-alpha by human lymphocytes was studied. NKCF activity was generated in response to K562-inducer cells. The presence of NKCF in supernatants was always accompanied by antiviral activity, but in several experiments IFN was detected without concomitant NKCF. In no instance was NKCF activity detected in the absence of IFN. Cell lines which were good inducers of IFN-alpha were found to be good inducers of NKCF. NKCF activity of supernatants was completely adsorbed after incubation with MOLT-4 cells, whereas there was only minimal depletion of IFN-alpha activity. Most of the antiviral activity and all of the NKCF activity of preformed supernatants was neutralized by anti-IFN-alpha serum, whereas anti-IFN-gamma serum and pH2 inactivation had minimal effect on either activity. Addition of IFN-alpha to neutralized supernatants restored NKCF activity. These experiments support the hypothesis that IFN-alpha is involved in the modulation of NKCF-lytic activity. Both antiviral and NKCF activities were abrogated when anti-IFN-alpha serum was added to cultures of lymphocytes plus inducer cells (induction phase). The addition of purified IFN-alpha to such cultures was effective in allowing resumption of NKCF activity; however, addition of IFN-gamma to these cultures did not overcome this block. The addition of purified IFN-alpha directly to supernatants generated in the presence of anti-IFN-alpha serum could not restore their NKCF activity, thereby suggesting an additional requirement for IFN-alpha in the production of NKCF. The possible role of IFN-alpha in the generation of NKCF and expression of its lytic activity is discussed.     

Unusual stability of the Methanospirillum hungatei sheath.

The proteinaceous sheath of Methanospirillum hungatei was isolated by lysing cells in 50 mM dithiothreitol, separating the sheath from other cellular material by discontinuous sucrose density centrifugation, and removing the "cell spacers" with dilute NaOH. The isolated sheath material consisted of hollow tubes which had a highly ordered surface array. The stability of the sheath to treatment with denaturants and to enzymatic digestion was examined by a turbidimetric assay in conjunction with electron microscopy and optical or electron diffraction. The sheath was resistant to a range of proteases and also was not digested by peptidoglycan-degrading enzymes, a lipase, a cellulase, a glucosidase, or Rhozyme (a mixture of galactosidases, acetylglucosaminidase, acetylgalactosaminidase, fucosidase, and mannosidases). In addition to being unaffected by common salts, thiol-reducing agents, and EDTA, the layer was resistant to powerful denaturants such as 6 M urea, 6 M guanidinium hydrochloride, 10 M LiSCN, cyanogen bromide, sodium periodate, and 1% sodium dodecyl sulfate. Strong bases, boiling 3 N HCl, and performic acid did attack the sheath; in these cases, the array was systematically disassembled in a progressive manner, which was followed by electron microscopy. The layer was slightly modified by N-bromosuccinimide in urea, but the array remained intact. The stability of the sheath was remarkable, not only as compared to other bacterial surface arrays, but also as compared to proteins generally, and possibly indicated the presence of covalent cross-links between protein subunits.     

Cellular location of origin and terminus of replication in Bacillus subtilis.

The origin of replication of Bacillus subtilis 168 trp thy dna-1 (temperature-sensitive initiation mutant) was labeled with [3H]thymidine. Analysis of labeled cells by autoradiography revealed that most of the radioactivity was associated with cell pole areas. To label the terminus, cells that had initiated were treated with chloramphenicol to inhibit cell growth and division but to allow continued DNA synthesis. These cells were then labeled with [3H]thymidine at a time when chromosome replication was nearly complete. The distribution of radioactivity was similar to that observed in origin-labeled cells. In contrast, exponentially growing cells that were labeled for a brief time at the permissive temperature showed a random distribution of radioactivity. These data indicate that the origin and terminus of replication are located at cell poles.     

The combination of photogrammetry and finite elements for a fine grained functional analysis of anatomical structures

Summary A new method of functional morphological analysis is presented. Combining stereophotogrammetry with the finite element technique, a new approach, permits a three-dimensional numerical stress analysis of arbitrarily shaped bodies to be performed. The stereophotogrammetric method which originated for three-dimensional calculations in the study of surfaces in land surveying is well suited for the determination of the nodal co-ordinates required for the finite element method, an engineering technique developed for behavioural analysis of solids and fluids responding to external forces. This approach was tested in a study of the functional morphology of the bill of an African wading bird, the shoebill Balaeniceps rex. A few findings of that study are given here in order to demonstrate the method. Advantages of the finite element method compared with other techniques for stress analysis of anatomical structures are also discussed. The method presents exciting possibilities for predicting displacement and stress responses more accurately and in much greater detail. The scope of this powerful computerized stress analysis technique is greatly enhanced with the introduction of stereophotogrammetry for determining the three-dimensional co-ordinates of complex anatomical structures. With the finite element method, the properties of the bone structure can be modelled as they occur in the life of the animal. This is not possible with physical models. Furthermore, rare specimens can be analysed non-destructively.     

Acute post-irradiation canine intestinal blood flow

Radiation-induced early transient incapacitation (ETI) is accompanied by severe systemic hypotension, during which arterial blood pressure often decreases to less than 50 per cent of normal. One haemodynamic compensatory mechanism is increased peripheral resistance due to vasoconstriction. This vasoconstriction in the small intestine of dogs is disproportionately increased during haemorrhagic or endotoxic shock, and intestinal ischaemia is frequent. In an attempt to elucidate mechanisms underlying radiation-induced ETI and the gastrointestinal radiation syndrome, canine intestinal submucosal blood flow was measured by the hydrogen polarographic technique, both before and after exposure to gamma radiation. Systemic blood pressures, blood gases and haematocrits were determined simultaneously. Data obtained from 12 sham-irradiated dogs and 12 irradiated dogs indicated that 90 Gy, whole-body, gamma radiation produced a 31 per cent decrease in systemic mean blood pressure beginning within 20 min post-irradiation and lasting for at least 90 min. However, the intestinal submucosal blood flow did not decrease as anticipated, but it exhibited an actual post-irradiation increase. This increase in post-irradiation intestinal submucosal blood flow began within 5 min after irradiation and lasted for at least 90 min. Post-irradiation haematocrits were 10.5 per cent higher than those obtained before irradiation and those obtained from sham-irradiated subjects. Histopathological examination of ileal mucosa revealed significant pathologic lesions in some irradiated animals within two hours after exposure.     

Insertion and fate of the cell wall in Bacillus subtilis

Cell wall assembly was studied in autolysin-deficient and -sufficient strains of Bacillus subtilis. Two independent probes, one for peptidoglycan and the other for surface-accessible teichoic acid, were employed to monitor cell surface changes during growth. Cell walls were specifically labeled with N-acetyl-D-[3H]glucosamine, and after growth, autoradiographs were prepared for both cell types. The locations of silver grains revealed that label was progressively lost from numerous sites on the cell cylinders, whereas label was retained on the cell poles, even after several generations. In the autolysin-deficient and chain-forming strain, it was found that the distance between densely labeled poles approximately doubled after each generation of growth. In the autolysin-sufficient strain, it was found that the numbers of labeled cell poles remained nearly constant for several generations, supporting the premise that completed septa and poles are largely conserved during growth. Fluorescein-conjugated concanavalin A was also used to determine the distribution of alpha-D-glucosylated teichoic acid on the surfaces of growing cells. Strains with temperature-sensitive phosphoglucomutase were used because in these mutants, glycosylation of cell wall teichoic acids can be controlled by temperature shifts. When the bacteria were grown at 45 degrees C, which stops the glucosylation of teichoic acid, the cells gradually lost their ability to bind concanavalin A on their cylindrical surfaces, but they retained concanavalin A-reactive sites on their poles. Discrete areas on the cylinder, defined by the binding of fluorescent concanavalin A, were absent when the synthesis of glucosylated teichoic acid was inhibited during growth for several generations at the nonpermissive temperature. When the mutant was shifted from a nonpermissive to a permissive temperature, all areas of the cylinder became able to bind the labeled concanavalin A after about one-half generation. Old cell poles were able to bind the lectin after nearly one generation at the permissive temperature, showing that new wall synthesis does occur in the cell poles, although it occurs slowly. These data, based on both qualitative and quantitative experiments, support a model for cell wall assembly in B. subtilis, in which cylinders elongate by inside-to-outside growth, with degradation of the stress-bearing old wall in wild-type organisms. Loss of wall material, by turnover, from many sites on the cylinder may be necessary for intercalation of new wall and normal length extension. Poles tend to retain their wall components during division and are turned over much more slowly.     

Canine postradiation plasma glucose variations

One of the observations of endotoxic or septic shock in canines is the report of concurrent hypoglycemia. Canines exposed to supralethal gamma radiation also develop acute systemic hypotension. This study was performed in order to determine if hypoglycemia develops in the canine concurrent with radiation-induced hypotension. Systemic arterial mean blood pressure (MBP) was measured via femoral arterial catheter. Blood for plasma glucose determinations was obtained from the systemic arterial circulation at the level of the abdominal aorta and from the hepatic portal vein. Plasma glucose levels were determined on a Beckman Glucose Analyzer which employs the enzymatic reaction of β-D-glucose and oxygen. Glucose levels and MBP were monitored for one hour before and for one hour after exposure to 100 Gy, whole-body, gamma radiation or sham radiation for the control animals. Concurrent with postradiation hypotension, we measured a significant decrease in plasma glucose levels in both the systemic arterial circulation and in the hepatic portal vein. Arterial glucose levels in the sham radiated animals showed a slight rise two minutes after sham radiation, falling back to pretreatment, base line levels four minutes later and remaining at that level for the remainder of the hour. Arterial levels in the radiated animals showed a sharp decline two minutes postradiation, falling even further to twenty percent below preradiation levels by one hour postradiation. Venous blood glucose levels in sham radiated animals showed an initial increase and a gradual decrease to five percent below pretreatment base line levels; while glucose levels in radiated animals showed an immediate postradiation decrease continuing to twenty percent below preradiation levels by one hour postradiation. These findings suggest impaired hepatic gluconeogenesis, resulting in postradiation hypoglycemia.