Microbial oxidation of protoporhydrinogen, an intermediate in heme and chlorphyll biosynthesis.

The oxidation of protoporphyrinogen to protoporphyrin, a late step in heme and chlorophyll synthesis, is catalyzed aerobically by a particulate fraction of Escherichia coli at a rate significantly higher than the rate of autooxidation. This activity is heat labile and is markedly inhibited by addition of respiratory substrates such as NADH. NADH is oxidized at a rate 100-fold higher than protoporphyrinogen. Particles from a cytochrome-less mutant of E. coli were markedly deficient in protoporphyrinogen oxidizing activity. Particles from a quinone-deficient mutant were also deficient. These findings suggest a possible role for the electron transport system in aerobic protoporphyrinogen oxidation. This activity was also examined in a variety of other bacteria. Particles from Streptococcus faecalis, which does not synthesize heme, were unable to oxidize protoporphyrinogen, confirming the specificity of this activity. Particles from aerobically grown Staphylococcus aureus exhibited protoporphyrinogen oxidizing activity, but particles from anaerobically grown cells had no activity above that of the nonenzymatic control. This indicates the repressible nature of this activity, and may also explain why Staphylococci synthesize cytochromes during aerobic, but not during anaerobic growth. Particles from photosynthetically grown Rhodopseudomonas spheroides, which contain both chlorophyll and heme, oxidized protoporphyrinogen at a rate no higher than the nonenzymic control. However, particles from cells grown aerobically, when bacteriochlorophyll synthesis is markedly repressed, readily exhibited protoporphyrinogen oxidizing activity. These initial findings suggest that this activity is detectable in cells primarily synthesizing heme, but not in cells primarily synthesizing bacteriochlorophyll, and could have implications both for the mechanism and regulation of the heme and bacteriochlorophyll pathways.     

Alterations in labeling of cell-surface glycoproteins from normal and diabetic rat intestinal microvillous membranes

The effect of chronic streptozotocin-induced diabetes was studied on intestinal microvillous membrane surface carbohydrate groups. After 7 weeks of diabetes, purified microvillous membranes were prepared from rat small intestine and surface galactoproteins identified by labeling with galactose oxidase/sodium boro[³H]hydride. Membrane surface sialic acid residues were labeled using the sodium metaperiodate/sodium boro[³H]hydride technique. Membranes were solubilized in SDS and protein labeling analyzed by acrylamide electrophoresis. Membranes from diabetic rats showed an 81% increase in galactoprotein labeling ($\text{P< 0.02}$) while labeling of sialic acid residues was unchanged. The greatest increase in galactoprotein labeling occurred in protein monomers of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 116 000–200 000, where there was a 155% increase in labeling ($\text{P< 0.005}$). These results indicate that intestinal microvillous membrane protein glycosylation is altered in chronic diabetes. This increase in surface membrane carbohydrates could explain the decreased rates of proteolytic degradation previously described for at least one microvillous protein. An increase in membrane galactose groups has also been noted in hepatocyte and kidney glomerular basement membranes, which suggests the presence of a systematic change in membrane protein glycosylation occurring as a result of the diabetic state.     

A high-pressure sample cell for circular dichroism studies.

The construction and operation of a preparative polyacrylamide-gel electrophoresis system is described. Emerging protein bands are collected by an intermittent pumping system which is based on the design of Brownstone ((1969) Anal. Biochem.27, 25–46). The original pressure-sensitive operation was, however, simplified to time-volume operation. Cooling of the gel by a central cooling finger, essentially according to Jovin et al. ((1964) Anal. Biochem.9, 351–369), has also been added. To accomodate the polyethylene tubing needed for intermittent collection of protein and also the central cooling finger, it is necessary to polymerize the gel in a mold before it is installed in the gel housing compartment of the electrophoresis cell. Gel concentrations of 5% and higher can be used in this system. Dilution of emerging protein samples by the intermittent collection system is kept to a minimum. This fact, together with simplicity of design makes it suitable for general preparative work with polyacrylamide-gel electrophoresis. Operation of the apparatus and resolution of protein bands are demonstrated by separation of bovine serum albumin polymers and thyroxine-binding proteins in human serum.     

The mobile receptor hypothesis and "cooperativity" of hormone binding. Application to insulin.

The mobile receptor hypothesis has been proposed to describe the process by which hormone receptor binding initiates a biological response; it states that receptors, which can diffuse independently in the plane of the membrane, reversibly associate with effectors to regulate their activity. The affinity for effector is greater when the receptor is occupied by hormone. A mathematical expression of the mobile receptor hypothesis is used to show that: (1) The predicted kinetics of hormone receptor binding may be indistinguishable from "negative cooperativity." (2) Receptor occupancy and biological response may be coupled in a non-linear fashion. By choosing specific parameters, most of the existing data on insulin binding and biological responses can be explained in terms of the mobile receptor hypothesis. Thus, the following are easily explained: (1) A single homogeneous receptor may appear kinetically to be composed of two classes (of high and low affinity) of receptors. (2) Occupancy of the apparent class of high affinity receptors is related linearly to the biological response. (3) The same receptor in different tissues may appear to have different affinity. (4) The binding of different biologically active insulin analogues may exhibit different degrees of "cooperativity." These considerations may also be pertinent to interpretations of other hormone-receptor systems and of various ligand-macromolecule interactions.     

Nitrate,fumarate, and oxygen as electron acceptors for a late step in microbial heme synthesis

Nitrate can serve as anaerobic electron acceptor for the oxidation of protoporphyrinogen to protoporphyrin in cell-free extracts of Escherichia coli grown anaerobically in the presence of nitrate. Two kinds of experiments indicated this: anaerobic protoporphyrin formation from protoporphyrinogen, followed spectrophotometrically, was markedly stimulated by addition of nitrate; and anaerobic protoheme formation from protoporphyrinogen, determined by extraction procedures, was markedly stimulated by addition of nitrate. In contrast, anaerobic protoheme formation from protoporphyrin was not dependent upon addition of nitrate. This was the first demonstration of the ability of nitrate to serve as electron acceptor for this late step of heme synthesis. Previous studies with mammalian and yeast mitochondria had indicated an obligatory requirement for molecular oxygen at this step.In confirmation of our previous preliminary report, fumarate was also shown to be an electron acceptor for anaerobic protoporphyrinogen oxidation in extracts of E. coli grown anaerobically on fumarate. For the first time, anaerobic protoheme formation from protoporphyrinogen, but not from protoporphyrin, was shown to be dependent upon the addition of fumarate.The importance of these findings is 2-fold. First, they establish that enzymatic protoporphyrinogen oxidation can occur in the absence of molecular oxygen, in contrast to previous observations using mammalian and yeast mitochondria. Secondly, these findings help explain the ability of some facultative and anaerobic bacteria to form very large amounts of heme compounds, such as cytochrome pigments, when grown anaerobically in the presence of nitrate or fumarate. In fact, denitrifying bacteria are known to form more cytochromes when grown anaerobically than during aerobic growth.An unexpected finding was that extracts of another bacterium, Staphylococcus epidermidis, exhibited very little ability to oxidize protoporphyrinogen to protoporphyrin as compared to E. coli extracts. This finding suggests some fundamental differences in these two organisms in this key step in heme synthesis. It is known that these two facultative organisms also differ in that E. coli synthesizes cytochrome during both aerobic and anaerobic growth, while Staphylococcus only synthesizes cytochromes when grown aerobically.     

Electrophysiological evidence for rod and cone-based vision in the nocturnal flying squirrel

Summary The flying squirrel (Glaucomys volans) is a strongly nocturnal rodent. Previous anatomical observations suggested that the retina of this animal contains some cone-like receptors in addition to large numbers of rods. Evidence for duplicity of function in this visual system was obtained from an examination of three indices of visual activity: the electroretinogram (ERG), the isolated PIII retinal response, and the visually evoked cortical potential (VECP). The spectral sensitivity of the dark-adapted flying squirrel is similar to that of other mammals — it has a 500 nm peak (Figs. 3, 8). Responses of the ERG and isolated PIII to flickering light indicate the operation of two processes (Figs. 4, 7), one of which is unable to follow flickering light at repetition rates above 10–15 Hz. Spectral sensitivity measurements reveal that these two processes have different spectral sensitivities. The photopic mechanism in the flying squirrel visual system has peak sensitivity at about 520 nm (Figs. 5, 7, 9). The effects of steady light adaptation are much more obvious in the cortical potentials than they are in the retinal potentials.We thank David Birch for his advice and assistance. This research was supported by a Grant from the National Eye Institute (EY-00105).     

High-level expression of the tick-borne encephalitis virus NS1 protein by using an adenovirus-based vector: protection elicited in a murine model.

Tick-borne encephalitis virus (TBEV) encodes an abundant, highly immunogenic nonstructural glycoprotein, NS1. The function of this protein has yet to be determined. We have cloned the NS1 gene from the Neudorfl strain of TBEV under the control of the powerful constitutive cytomegalovirus major immediate-early promoter into an adenovirus E1 deletion mutant. The novel combination of the cytomegalovirus immediate-early promoter and the adenovirus vector produced extremely high levels of NS1 expression in cells which do not support replication of the adenovirus deletion mutant. The recombinant protein was shown to be indistinguishable from authentic TBEV NS1 in its (i) apparent molecular weight by polyacrylamide gel electrophoresis, (ii) glycosylation pattern, (iii) ability to form high-molecular-weight complexes, and (iv) ability to be secreted from cells. Appropriate processing of NS1 expressed by the adenovirus recombinant occurred independently of any additional TBEV-encoded gene function. When directly inoculated into mice, the recombinant adenovirus RAd51 was shown to elicit an antibody response to the TBEV NS1 protein. Immunization with RAd51 conferred protection against challenge with TBEV.     

A biostratigraphic zonation of the middle and upper siwaliks of the potwar plateau of Northern Pakistan

Two biostratigraphic reference sections are designated for the middle and upper Siwalik formations of northern Pakistan. The stratigraphic ranges of 41 mammalian taxa are established in the reference sections and the two sections are correlated to each other and to the standard Magnetic Polarity Time Scale by the magnetic-polarity stratigraphy. The resulting stratigraphic and magnetostratigraphic framework is used to define four contiguous biostratigraphic interval-zones. The proposed interval-zones are bounded by five important faunal events which are identified in the reference sections. The four proposed interval-zones and the approximate ages in the reference sections of their lower and upper boundaries are: (1) the “Hipparion s.l.” Interval-Zone 7.4–9.5 m.y. B.P.; (2) the Selenoportax lydekkeri Interval-Zone 5.3–7.4 m.y. B.P.; (3) the Hexaprotodon sivalensis Interval-Zone 5.3-2.9 m.y. B.P.; and (4) the Elephas planifrons Interval-Zone 2.9-1.5 m.y. B.P.The paleomagnetic and biostratigraphic evidence indicates that the Tatrot Beds, from which Pilgrim developed his “Tatrot Faunal Zone”, are between 2.5 and 3.4 m.y. B.P., with a preferred maximum age of 2.9 m.y. B.P. The biostratigraphic evidence indicates the Sethi Nagri locality (Y311)—the primary source of Pilgrim's “Nagri Faunal Zone”— is in the “Hipparion s.l.” Interval-Zone. The paleomagnetic evidence suggests an approximate age of 9 m.y. B.P. A major faunal event at 9.5 m.y. B.P. introduced equids, suids, and large giraffes from Eurasia into the Indain subcontinent. A second, more diffuse faunal event between 5.3 and 2.9 m.y. B.P. introduced several African taxa as well as Equus and cervids from Eurasia.     

Historical biogeography of tamarins,genus Saguinus: the molecular phylogenetic evidence

Hypotheses of the historical biogeography of tamarins (genus Saguinus) based on variation in coat colors and body size are tested using phylogenetic relationships inferred from mitochondrial DNA (mtDNA) sequence data. Samples from all 12 species of Saguinus and several subspecies are included in the analysis. Approximately 1,200 bases of mtDNA sequence from the cytochrome b and D-loop regions are reported for the tamarins and several outgroup taxa. Parsimony analysis of the mtDNA sequence data reveals Saguinus to be a monophyletic taxon composed of two major clades: one, the Small-bodied clade, contains S. nigricollis, S. tripartitus, and S. fuscicollis, and the other, the Large-bodied clade, contains the other nine species. The phylogenetic relationships among tamarins inferred from the mtDNA sequence data reject previous hypotheses for the historical biogeography of tamarins and suggest different dispersal routes for this group of New World monkeys. The molecular data suggest that tamarins dispersed across South America in two major waves from an origin somewhere south of the Amazon. One wave moved in a westerly direction, whereas the other moved in a northeastern direction toward the Amazon delta and then west along the northern portion of the continent into northern Colombia and Panama. Am J Phys Anthropol 108:65–89, 1999. © 1999 Wiley-Liss, Inc.