Histochemical and microbiochemical demonstration of reduced pyruvate kinase activity in thioacetamide-induced neoplastic nodules of rat liver

A new method for the histochemical demonstration of pyruvate kinase (PK) activity was developed using a semi-permeable membrane and ATP-dependent phosphorylation of glucose coupled with tetrazolium reduction via glucose-6-phosphate dehydrogenase (G6PD) in order to investigate normal liver tissue and neoplastic hepatic nodules induced by thioacetamide (TAA). A series of control reactions and comparison with microbiochemical analysis of microdissected lyophilised material were used to determine the specificity of the reaction. In agreement with earlier reports, an activity gradient in control liver decreasing from zone 3 to zone 1 was apparent both histochemically and after biochemical analysis. Liver neoplastic nodules induced by 25 weeks dietary thioacetamide administration and characterized by increased G6PD demonstrated a clear decrease in PK activity. In contrast, epithelial cells within areas of cholangiocellular tumour development were characterized by a strong increase. Comparison of the results with immunohistochemical and biochemical data from the literature indicate that the specific histochemical method described will be of great assistance in future assessment of disease and physiological alteration in activity of this key enzyme of glycolysis.     

Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein

Transport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of amino-terminal targeting sequences in the mitochondrial matrix. We have isolated the processing activity from Neurospora crassa. The final preparation (enriched ca. 10,000-fold over cell extracts) consists of two proteins, the matrix processing peptidase (MPP, 57 kd) and a processing enhancing protein (PEP, 52 kd). The two components were isolated as monomers. PEP is about 15-fold more abundant in mitochondria than MPP. It is partly associated with the inner membrane, while MPP is soluble in the matrix. MPP alone has a low processing activity whereas PEP alone has no apparent activity. Upon recombining both, full processing activity is restored. Our data indicate that MPP contains the catalytic site and that PEP has an enhancing function. The mitochondrial processing enzyme appears to represent a new type of "signal peptidase," different from the bacterial leader peptidase and the signal peptidase of the endoplasmic reticulum.     

Cardiovascular effects of prostaglandin I2 and prostaglandin F2 alpha in the unanesthetized bullfrog, Rana catesbeiana

The cardiovascular effects of prostaglandin (PG)I2 and PGF2 alpha were compared in the unanesthetized American bullfrog (Rana catesbeiana). Control mean arterial pressure (MAP) and heart rate (HR) were 25.7 +/- 1.1 mm Hg and 35.1 +/- 1.1 beats/min, respectively. Intravenous injections of PGI2 decreased MAP and increased HR in a dose-dependent fashion over the range of concentrations tested (0.03, 0.3, 3, and 10 micrograms/kg-body weight [bw]. Neither atropine (1 mg/kg-bw) nor verapamil (1 mg/kg-bw) treatment altered the MAP or HR responses to PGI2 (3 micrograms/kg-bw). However, propranolol (5 mg/kg-bw) significantly blunted the hypotensive effects without affecting the increase in HR. Prostaglandin F2 alpha (tested at 0.3, 3, 30, and 100 micrograms/kg-bw) increased both MAP and HR. Mean arterial pressure increased with concentrations greater than 0.3 microgram/kg-bw and reached peak effects at 30 micrograms/kg-bw. Prostaglandin F2 alpha increased HR at doses greater than 0.3 microgram/kg-bw. Neither the pressor nor positive chronotropic effects of PGF2 alpha (30 micrograms/kg-bw) were affected by atropine or propranolol. However, verapamil significantly attenuated the pressor effects without affecting the increase in HR. These results demonstrate that both prostaglandins have qualitatively similar effects on HR, but opposite effects on MAP. Prostaglandin I2 is a hypotensive prostaglandin, while PGF2 alpha is hypertensive. The pressor effects of PGF2 alpha are partially dependent on calcium influx. The positive chronotropic effects of both prostaglandins are independent of the autonomic nervous system, suggesting a different mechanism of action.     

Demonstration of the ascorbate dependence of membrane-bound dopamine beta-monooxygenase in adrenal chromaffin granule ghosts

Chromaffin granule ghosts from bovine adrenal medullae have been used to examine the ability of membrane-bound dopamine beta-monooxygenase to interact directly with intravesicular ascorbate and to investigate vectorial electron transfer from external ascorbate across the ghost membrane. Ghosts prepared by a modification of published procedures were shown to be fully active in both dopamine uptake and norepinephrine production. Dopamine uptake is dependent on the presence of a magnesium and ATP ionic complex, is abolished by reserpine, and reaches a steady-state level in the presence of dopamine beta-monooxygenase, ascorbate, catalase, and fumarate. Omission of ascorbate either inside or outside the ghosts greatly enhances dopamine accumulation, which reaches levels of approximately 30 nmol/mg under these conditions. Correspondingly, in the presence of all components, norepinephrine production reached approximately 100 nmol/mg in 30 min of incubation. Norepinephrine production was strictly magnesium-ATP-dependent, inhibited by either reserpine or dopamine beta-monooxygenase inactivation, and was markedly reduced when ascorbate was omitted from either inside or outside the ghosts. In the presence of limiting amounts of internal ascorbate, rapid norepinephrine production occurred which corresponded to the amount of initial ascorbate present, followed by a much slower endogenous norepinephrine production observable after complete depletion of internal ascorbate. The endogenous rate of norepinephrine production likely represents epinephrine-supported dopamine beta-monooxygenase turnover. Taken together, the data demonstrate that facile norepinephrine production by membrane-bound dopamine beta-monooxygenase occurs only when internal ascorbate is present, terminates upon depletion of internal ascorbate, and can only be sustained at a significant rate when reducing equivalents from external ascorbate are available.     

The tensiometric properties of expanded guinea pig skin

Our purpose in this study was to evaluate the tensile properties of expanded skin. In five guinea pigs, 29-cc ovoid tissue expanders were placed and sequentially expanded every 4 days until maximum volume was achieved. Five control and five expanded skins were harvested. Using an Instron tensile testing apparatus, skins were evaluated for stress-strain, maximum stiffness, and tensile strength, and the results were statistically compared. Centrally located expanded specimens demonstrated significantly weaker stress-strain values: 9.51 in.lb/in3 for expanded versus 30.11 in.lb/in3 for control (p less than 0.001). Maximum stiffness was similarly reduced: 4.56 lb/mm2 for expanded vs. 12.98 lb/mm2 for control (p less than 0.001). This is a 67.4 and 64.9 percent reduction, respectively, for the stress-strain and maximum stiffness. No statistically significant difference was seen in peripherally located expanded specimens relative to the controls: stress-strain expanded, 28.7 in.lb/in3 (p greater than 0.5); maximum stiffness expanded, 12.84 lb/mm2 (p greater than 0.5). Expanded skin demonstrated an average 35 percent reduction in tensile strength. We conclude that the tensile properties of expanded skin are significantly less than unexpanded skin and are a function of the degree of expansion.     

A computerized mechanical cell stimulator for tissue culture: Effects on skeletal muscle organogenesis

Summary A tissue culture system has been developed which can mechanically stimulate cells growing on a highly elastic plastic substratum in a 24-well cell growth chamber. The collagen-coated substratum to which the cells attach and grow in the Mechanical Cell Stimulator (Model I) can be repetitively stretched and relaxed by stepper motor with linear accuracy of 30 μm. The activity controlling unit is an Apple IIe computer interfaced with the cell growth chamber via optical data links and is capable of simulating many of the mechanical activity patterns that cells are subjected to in vivo. Primary avian skeletal myoblasts proliferate and fuse into multinucleated myotubes in this set-up in a manner similar to normal tissue culture dishes. Under static culture conditions, the muscle cells differentiate into networks of myotubes which show little orientation. Growing the proliferating muscle cells on a unidirectional stretching substratum causes the developing myotubes to orient parallel to the direction of movement. In contrast, growing the cells on a substratum undergoing continuous stretch-relaxation cycling orients the developing myotubes perpendicular to the direction of movement. Neither type of mechanical activity significantly affects the rate of cell proliferation of the rate of myoblast fusion into myotubes. These results indicate that during in vivo skeletal muscle organogenesis, when substantial mechanical stresses are placed on skeletal muscle cells by both continuous bone elongation and by spontaneous contractions, only bone elongation plays a significant role in proper fiber orientation for subsequent functional work. Supported by grants NS16753, AR36266, and RR05818 from the National Institutes of Health, Bethesda, MD.     

Meiofauna communities along a depth transect off Halley Bay (Weddell Sea-Antarctica)

Summary Meiofauna communities from 10 stations along a depth transect from approximately 500 to 2,000 m off the Halley Bay Station (Weddell Sea) are investigated. Representatives of about 30 smallsized taxa of higher category are found, most of them belonging to the meiofauna. Loricifera are recorded for the first time for the Southern Ocean. At one of the stations a maximum of 22 taxa occur, the mean number of taxa ranges from 7 to 16. Nematoda, Harpacticoida, Ostracoda, Polychaeta and Bivalvia are present at all sampling sites. Nematodes are always dominant representing more than 90% of the individuals per sample, followed by harpacticoids (3%) and kinorhynchs (1.2%). Important fractions of the meiofauna (an average of more than 50%) occur in strata below the top 0–1 cm layer. Maximal density is 3,800 individuals (10 cm^–2), the mean abundance per station ranges from 790 to 3,720 individuals (10 cm^–2) and the overall mean is 1,700 individuals (10 cm^–2). Multivariate analysis (TWINSPAN, Cluster analysis, DCA) discriminates between three communities which are correlated with depth and sediment characteristics: the near shelf-ice, the slope and the deep-sea communities.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Theory of molecular machines. II. Energy dissipation from molecular machines

Single molecules perform a variety of tasks in cells, from replicating, controlling and translating the genetic material to sensing the outside environment. These operations all require that specific actions take place. In a sense, each molecule must make tiny decisions. To make a decision, each "molecular machine" must dissipate an energy Py in the presence of thermal noise Ny. The number of binary decisions that can be made by a machine which has dspace independently moving parts is the "machine capacity" Cy = dspace log2 [(Py + Ny)/Ny]. This formula is closely related to Shannon's channel capacity for communications systems, C = W log2 [(P + N)/N]. This paper shows that the minimum amount of energy that a molecular machine must dissipate in order to gain one bit of information is epsilon min = kB T ln (2) joules/bit. This equation is derived in two distinct ways. The first derivation begins with the Second Law of Thermodynamics, which shows that the statement that there is a minimum energy dissipation is a restatement of the Second Law of Thermodynamics. The second derivation begins with the machine capacity formula, which shows that the machine capacity is also related to the Second Law of Thermodynamics. One of Shannon's theorems for communications channels is that as long as the channel capacity is not exceeded, the error rate may be made as small as desired by a sufficiently involved coding. This result also applies to the dissipation formula for molecular machines. So there is a precise upper bound on the number of choices a molecular machine can make for a given amount of energy loss. This result will be important for the design and construction of molecular computers.     

Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells.

Genes that act inside the cell to negatively regulate proliferation are of great interest because of their implications for such processes as development and cancer, but these genes have been difficult to clone. This report details the cloning and analysis of cDNA for prohibitin, a novel mammalian antiproliferative protein. Microinjection of synthetic prohibitin mRNA blocks entry into S phase in both normal fibroblasts and HeLa cells. Microinjection of an antisense oligonucleotide stimulates entry into S phase. By sequence comparison, the prohibitin gene appears to be the mammalian analog of Cc, a Drosophila gene that is vital for normal development.