Impacts of section 404 permits requiring compensatory mitigation on wetlands in California (USA)

We analyzed data from Section 404 permits issued in California from January 1971 through November 1987 that involved impacts to wetlands and required compensatory mitigation (wetland creation, restoration, or preservation). The purpose of this study was to determine patterns and trends in permitting activity and to document cumulative effects of associated management decisions on the California wetland resource. The 324 permits examined documented that 387 compensatory wetlands (1255.9 ha) were required as mitigation for impacts to 368 wetlands (1176.3 ha). The utility of the data on wetland area was limited, however, since 38.0% of the impacted wetlands and 41.6% of the compensatory wetlands lacked acreage data. The wetland type most frequently impacted (37.8% of impacted wetlands) and used in compensation (38.2% of compensatory wetlands) was palustrine forested wetlands. Estuarine intertidal emergent wetlands had the most area impacted (52.3%) and compensated (62.5%). The majority of the wetlands were small (less than or equal to 4.0 ha in size). Wildlife habitat was the most frequently listed function of impacted wetlands (90.7% of the permits) and objective of compensatory wetlands (83.3%). Endangered species were listed as affected in 20.4% of impacted and 21.0% of compensatory projects. The number of permits requiring compensatory mitigation and the number of impacted and compensatory wetlands increased from 1971 to 1986.Documentation of the details of Section 404 permit decisions was inadequate for the permits we examined. Area information and specific locations of impacted and compensatory wetlands were lacking or of poor quality. Follow-up information was also inadequate. For example, project completion dates were specified in the permit for only 2.2% of compensatory wetlands. Furthermore, less than one-third (31.5%) of the permits required the compensatory wetland to be monitored by at least one site visit. We recommend improved documentation, regular reporting, and increased monitoring for better evaluation of the Section 404 permitting system.     

Determination of aldosterone secretion rate utilizing mixed mode and high performance liquid chromatography

A simpler method for determining aldosterone secretion rate (ASR) has several applications. High performance liquid chromatography (HPLC) has several advantages over traditional chromatographic methods for purification to constant specific activity of aldosterone liberated from its 18-glucuronide by acid hydrolysis. We found it necessary to introduce several modifications to remove urochromes before HPLC. Two methods for determining ASR were developed. With Method A a more traditional initial procedure was followed, and Sephadex LH-20 chromatography allowed removal of considerable urochromes before HPLC. However, aldosterone recovery was improved with Method B, which employed several bonded phase silica derivatives (Sepralytes) and a PBE 94 column to remove urochromes before HPLC. With this procedure the Sephadex LH-20 chromatography was not required. Aldosterone purification to constant specific activity was achieved by HPLC on a diol column with a normal phase system, and quantification was performed by RIA. ASR determinations were equivalent with both methods. This methodology should be applicable to other steroid secretory rate determinations and to applications involving purification of steroid conjugates.     

The rat liver asialoglycoprotein receptor polypeptide must be inserted into a microsome to achieve its active conformation

Affinity chromatography on galactose-Sepharose has been utilized to demonstrate that rat liver asialoglycoprotein receptor synthesized in vitro in a reticulocyte lysate system is capable of binding carbohydrate ligand only when dog pancreas microsomes are present during translation. Analysis of receptor isolated from tunicamycin-treated rat hepatocytes indicates that glycosylation is not necessary for receptor activity. Genetically engineered receptor derivatives in which the natural membrane anchor is either deleted entirely or replaced with a cleavable signal sequence derived from dog preproinsulin have been used to demonstrate that: (a) inactive receptor made in the absence of membranes does not result from incorrect nucleation of folding around the hydrophobic portion of the polypeptide which is normally buried in the membrane and (b) the carbohydrate-binding domain of the receptor does not need to be tethered to the luminal side of the membrane to fold correctly. These results suggest that factors within the lumen of the microsomes are essential to establish the native conformation of the binding domain.     

von Willebrand factor. A reduced and alkylated 52/48-kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein Ib

We have purified a reduced and alkylated tryptic fragment of von Willebrand factor (vWF) which migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 52/48-kDa doublet, but behaved as a single 46-kDa species after partial deglycosylation. After extensive treatment with denaturants, the 52/48-kDa polypeptide retained its ability to inhibit ristocetin-induced platelet aggregation in the presence of native vWF, as well as aggregation induced by desialylated vWF alone. Therefore, the 52/48-kDa polypeptide interacts with the platelet glycoprotein Ib receptor even in the absence of ristocetin. Both the 52/48- and the 46-kDa species inhibited ristocetin-induced binding of the intact molecule to platelets, but did not affect thrombin-induced binding. Determination of the NH2-terminal sequence of both members of the doublet gave identical results: VTLNPSDPEHCQ. This provided additional evidence that differences between the doublet constituents were only of carbohydrate composition and established the position of this peptide within the vWF polypeptide chain of approximately 2050 amino acid residues as beginning with the residue tentatively designated 449. These studies suggest that native conformation is not necessary for binding of vWF to platelets at the glycoprotein Ib receptor and that a linear amino acid sequence following residue 449 defines a domain responsible for this interaction.     

Both insulin and epidermal growth factor stimulate fatty acid synthesis and increase phosphorylation of acetyl-CoA carboxylase and ATP-citrate lyase in isolated hepatocytes

Insulin and EGF cause identical stimulation (congruent to 40%) of fatty acid synthesis in hepatocytes isolated from rats which have been starved and then refed a low-fat diet. In both cases this stimulation is associated with increased phosphorylation of ATP-citrate lyase and of a specific site on acetyl-CoA carboxylase. However, the altered phosphorylation of acetyl-CoA carboxylase is not associated with a change in kinetic parameters which is detectable in the purified enzyme. Whatever the mechanism involved, stimulation of fatty acid synthesis by growth factors may have a role in providing new phospholipid for growth of membranes.     

Mononuclear macrophage (M-CFC) colony formation in semisolid media in the absence of exogenous GM-CSF

Human fetal bone marrow (FBM) cells were examined for the ability to form colonies in the absence of exogenous colony-stimulating factor (CSF) in double layer agar, methylcellulose (MC), and in agar-MC (agar underlayer, MC overlayer) culture systems. Without exogenous CSF, macrophage colonies (M-CFC) were formed in a combined culture of agar and MC. Aggregates of 5-40 cells were observed on day 7. Gradually, large compact colonies which survived for 10-12 weeks of cultivation, were formed. They were composed of mononuclear monocytes and multinucleated cells. M-CFC progenitors were nonadherent, but their progeny became adherent during differentiation within the colony. Colony formation was cell-dose-dependent. Depletion of monocytes increased the number of colonies in agar-MC cultures and stimulated the development of some macrophage colonies in MC. Survival of monocyte progenitors was not dependent on CSF. Neither was their proliferation nor partial differentiation in agar-MC cultures. CSF increased M-CFC colony efficiency, however, if it was present when cultures were initiated. Addition of CSF to M-CFC growing for 2-5 weeks in CSF-deprived medium stimulated monocytes proliferation and transformation into macrophages. Epithelioid cells, an increase in the number of giant multinucleated cells, and granulocyte multiplication were also observed. The absolute dependence of macrophage colony formation on CSF described by others might be a result of inadequate culture conditions due to agar rather than an intrinsic physiological requirement.     

Regulation of haemolysin synthesis in E. coli determined by HLY genes of human origin

Summary We have previously reported the secretion of a 107K polypeptide into the medium from a haemolytic E. coli K12 strain (Mackman and Holland 1984a). In addition, we demonstrated that haemolysin production was correlated with the presence of this polypeptide in the growth medium in a large number of E. coli isolates of human and animal origin (Mackman and Holland 1984b).In this paper we confirm that the 107K polypeptide is indeed haemolysin: both haemolytic activity and the 107K polypeptide show a similar pattern of accumulation during the growth cycle; identical levels are produced in three different growth media; they have the same half-life in minimal medium. The results also show that the expression of haemolysin is not influenced by the growth medium or subject to catabolite repression. However, expression is apparently switched off as cells enter the late exponential phase of growth. Finally, we present data indicating that the previously reported variation in haemolysin production in different media is entirely due to the instability of the haemoolysin itself. Degradation of the 107K polypeptide in the medium was accompanied by the accumulation of a major breakdown product of 60K.     

Mechanism of transient inhibition of DNA synthesis in ultraviolet-irradiated E. coli: inhibition is independent of recA whilst recovery requires RecA protein itself and an additional, inducible SOS function

Summary The mechanism of the inhibition and of the recovery of DNA synthesis in E. coli following UV-irradiation was analysed in several mutants defective in repair or in the regulation of the RecA-LexA dependent SOS response. Several lines of evidence indicated that inhibition is not an inducible function and is probably due to the direct effect of lesions in the template blocking replisome movement.Recovery of DNA synthesis after UV was largely unaffected by mutations in the uvrA, recB or umuC genes. Resumption of DNA synthesis does however require protein synthesis and the regulatory action of recA. Experiments with a recA constitutive mutant and recA 200 (temperature sensitive RecA) demonstrated that RecA protein itself is directly required but is not sufficient for recovery of DNA synthesis. We therefore propose that recovery of DNA synthesis depends upon the concerted activity of RecA and the synthesis of an inducible Irr (induced replisome reactivation) factor under RecA control. We suggest that the mechanism of recovery involves the action of Irr and RecA to promote movement of replisomes past non-instructive lesions, uncoupled from polymerisation and/or that Irr and RecA are required to promote re-initiation of a stalled replication complex downstream of a UV-lesion subsequent to such an uncoupling step.     

An inner membrane protein N-terminal signal sequence is able to promote efficient localisation of an outer membrane protein in Escherichia coli

To test the importance of N-terminal pre-sequences in translocation of different classes of membrane proteins, we exchanged the normal signal sequence of an Escherichia coli outer membrane protein, OmpF, for the pre-sequence of the inner membrane protein, DacA. The DacA-OmpF hybrid was efficiently assembled into the outer membrane in a functionally active form. Thus the pre-sequence of DacA, despite its relatively low hydrophobicity compared with that of OmpF, contains all the essential information necessary to initiate the translocation of OmpF to the outer membrane. Since processing of DacA was also shown to be dependent upon SecA we conclude that the initiation of translocation of this inner membrane polypeptide across the envelope occurs by the same mechanism as outer membrane and periplasmic proteins. The N-terminal 11 amino acids of mature OmpF, which in the hybrid are replaced by the N-terminal nine amino acids of DacA, carry no essential assembly signals since the hybrid protein is apparently assembled with equal efficiency to OmpF.     

Evidence that glucagon-mediated inhibition of acetyl-CoA carboxylase in isolated adipocytes involves increased phosphorylation of the enzyme by cyclic AMP-dependent protein kinase.

The kinetic parameters and phosphorylation state of acetyl-CoA carboxylase were analysed after purification of the enzyme by avidin--Sepharose chromatography from extracts of isolated adipocytes treated with glucagon or adrenaline. The results provide evidence that the mechanism of inhibition of acetyl-CoA carboxylase in adipocytes treated with glucagon [Zammit & Corstorphine (1982) Biochem. J. 208, 783-788] involves increased phosphorylation of the enzyme. Hormone treatment had effects on the kinetic parameters of the enzyme similar to those of phosphorylation of the enzyme in vitro by cyclic AMP-dependent protein kinase. Glucagon treatment of adipocytes led to increased phosphorylation of acetyl-CoA carboxylase in the same chymotryptic peptide as that containing the major site phosphorylated on the enzyme by purified cyclic AMP-dependent protein kinase in vitro [Munday & Hardie (1984) Eur. J. Biochem. 141, 617-627]. The dose--response curves for inhibition of enzyme activity and increased phosphorylation of the enzyme were very similar, with half-maximal effects occurring at concentrations of glucagon (0.5-1 nM) which are close to the physiological range. In general, the patterns of increased 32P-labelling of chymotryptic peptides induced by glucagon or adrenaline were similar, although there were quantitative differences between the effects of the two hormones on individual peptides. The results are discussed in terms of the possible roles of cyclic AMP-dependent and -independent protein kinases in the regulation of acetyl-CoA carboxylase activity and of lipogenesis in white adipose tissue.