Genetic and Biochemical Characterization of Mutations Affecting the Ability of the Yeast Pachysolen tannophilus To Metabolize d-Xylose

Induced mutants, selected for their defective growth on d-xylose while retaining the ability to grow normally on d-glucose, were studied in Pachysolen tannophilus, a yeast capable of converting d-xylose to ethanol. Fourteen of the mutations were found to occur at nine distinct loci, and data indicated that many more loci remain to be detected. Most of the mutations were pleiotropic in character, and the expression of some of them was much affected by nutritional conditions and by genetic background. Mutations at several loci resulted in poor growth on at least one compound that was either an intermediate of the tricarboxylic acid cycle, succinate or alpha-ketoglutarate, or on compounds metabolizable via this cycle, ethanol or glycerol. An initial biochemical characterization of the mutants was undertaken. Analysis for xylose reductase, xylitol dehydrogenase, and xylulose kinase activity showed that one or more of these activities was affected in 12 of 13 mutants. However, drastic reduction in activity of a single enzyme was confined to that of xylitol dehydrogenase by mutations at three different loci and to that of d-xylose reductase by mutation at another locus. Growth of these latter four mutants was normal on all carbon sources tested that were not five-carbon sugars.     

Growth dynamics of a ctenophore (Mnemiopsis) in relation to variable food supply. II. Carbon budgets and growth model

Our goal was to test our understanding of ingestion, assimilationefficiency and metabolism for Mnemiopsis mccradyi by formulatingand validating a simulation model of growth under differentconditions of food availability. The model was based on a carbonbudget approach using formulations derived from empirical results,including how each process was affected by food availabilityand ctenophore size. An experimentally measured carbon budgetfor pulsed food availability indicated that, relative to totalingestion, growth was high (17–48%), respiration plusorganic release was relatively low (24–48%) and little(<10%) of the ingested carbon was unaccounted for. New laboratoryinvestigations of feeding and assimilation efficiency were necessaryto refine the formulations so that model predictions comparedfavorably with a variety of laboratory measurements of growth,and growth efficiency, as well as the complete experimentallymeasured carbon budget. The refined model predicted a high ratioof growth to metabolism (>2) and a high gross growth efficiency(>30%) for smaller ctenophores at high food concentrations(>20 prey l^–1). Both growth rates and growth efficiencieswere predicted to decrease for larger ctenophores. Model predictionswere generally consistent with experimental results, includinginvestigations using pulsed food availability to simulate environmentalpatchiness. Although the model underpredicted ctenophore growthin some experiments at low food densities, the model predictionof a minimum prey concentration of about 8 l^–1 (24 µgC l^–1) for sustaining a ctenophore population of reproductivesize agreed with field observations.     

Growth dynamics of a ctenophore (Mnemiopsis) in relation to variable food supply. I. Carbon biomass, feeding, egg production, growth and assimilation efficiency

This paper contains new experimental data on the growth dynamicsof a lobate coastal ctenophore, Mnenmiopsis mccradyi, whichadd significantly to our understanding of the nutritional ecologyof ctenophores and their role as opportunistic predators. Theseexperimental observations were necessary to refine the dynamiccarbon budget presented as a simulation model in another report.The ratio of carbon biomass to dry weight may vary several-folddepending on the nutritional state and size from >12% inwell-fed larvae to <1% in starved adults. Feeding effort(clearance rate) is higher for previously starved animals, fallingsharply within a few hours after re-exposure to food. Directvisual observations of feeding activity of animals confirmedthis. Assimilation efficiency can be high (72%) in these animalsbut if they continue to feed at high food concentrations, incomingfood displaces material which is only partially digested andassimilation efficiency decreases substantially. Except at verylow food concentrations, growth efficiency ranges between 20and 45%. Mnemiopsis, begins to produce eggs at a size much lessthan its maximum. Egg production is very sensitive to food supply,and somatic growth is favored over egg production at low fooddensities. Even though several thousand eggs may be producedover a few days, they represent <2% day^–1 of the carbonbiomass of the ctenophore and several-fold less than respiratorycarbon.     

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.     

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.     

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.     

Identification and sequence of a Na+-linked gene from the marine bacterium Alteromonas haloplanktis which functionally complements the dagA gene of Escherichia coli

A 4.0 kb fragment from a plasmid genomic DNA library of the marine bacterium Alteromonas haloplanktis ATCC 19855 was found in the presence of Na+ to complement the dagA gene of Escherichia coli. We have completely sequenced this fragment and the position of the Na(+)-linked D-alanine glycine permease gene (dagA) on the fragment has been determined by complementation. The predicted carrier protein consists of 542 amino acid residues (M(r) 58,955). Its hydropathy profile suggests it is composed of eight transmembrane segments with a long hydrophilic region between segments six and seven. Significant similarity has been found between this Na(+)-linked permease and the Na+/proline permeases of E. coli and Salmonella typhimurium and the human and rabbit intestinal Na+/glucose cotransporters.     

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.