src kinase catalyzes the phosphorylation and activation of the insulin receptor kinase

When a partially purified insulin receptor preparation immobilized on insulin-agarose is incubated with [gamma-32P]ATP, Mn2+, and Mg2+ ions, the receptor beta subunit becomes 32P-labeled. The 32P-labeling of the insulin receptor beta subunit is increased by 2-3-fold when src kinase is included in the phosphorylation reaction. In addition, the presence of src kinase results in the phosphorylation of a Mr = 125,000 species. The Mr = 93,000 receptor beta subunit and the Mr = 125,000 32P-labeled bands are absent when an insulin receptor-deficient sample, prepared by the inclusion of excess free insulin to inhibit the adsorption of the receptor to the insulin-agarose, is phosphorylated in the presence of the src kinase. These results indicate that the insulin receptor alpha and beta subunits are phosphorylated by the src kinase. The src kinase-catalyzed phosphorylation of the insulin receptor is not due to the activation of receptor autophosphorylation because a N-ethylmaleimide-treated receptor preparation devoid of receptor kinase activity is also phosphorylated by the src kinase. Conversely, the insulin receptor kinase does not catalyze phosphorylation of the active or N-ethylmaleimide-inactivated src kinase. Subsequent to src kinase-mediated tyrosine phosphorylation, the insulin receptor, either immobilized on insulin-agarose or in detergent extracts, exhibits a 2-fold increase in associated kinase activity using histone as substrate. src kinase mediates phosphorylation of predominantly tyrosine residues on both alpha and beta subunits of the insulin receptor. Tryptic peptide mapping of the 32P-labeled receptor alpha and beta subunits by high pressure liquid chromatography reveals that the src kinase-mediated phosphorylation sites on both receptor subunits exhibit elution profiles identical with those phosphorylated by the receptor kinase. Furthermore, the HPLC elution profile of the receptor auto- or src kinase-catalyzed phosphorylation sites on the receptor alpha subunit are also identical with that on the receptor beta subunit. These results indicate that: the src kinase catalyzes tyrosine phosphorylation of the insulin receptor alpha and beta subunits; and src kinase-catalyzed phosphorylation of insulin receptor can mimic the action of autophosphorylation to activate the insulin receptor kinase in vitro, although whether this occurs in intact cells remains to be determined.     

A study of genetic diversity among host-specific populations of the witchweedStriga hermonthica (Scrophulariaceae) in Africa

Striga hermonthica is a root hemiparasite that attacks onlyGramineae, includingSorghum and millet for which it is a principal cause of lowered yield. Enzyme electrophoresis was used to investigate genetic diversity inStriga hermonthica and to determine the level of differentiation between host-specialized populations. Nine genetic loci coding eight enzymes were interpreted and data obtained from three populations: oneSorghum-adapted population from Sudan and two populations from Burkina Faso, oneSorghum-adapted and the other millet-adapted. Levels of polymorphism were similarly high in all three populations (P=0.625, A=2.6–2.8, H=0.293–0.401). Genotypic frequencies at most loci conformed to Hardy-Weinberg expectations in each population, consistent with outcrossing as predicted from previous studies of floral biology. Occasional heterozygote deficiencies were probably the result of Wahlund effect. The mean value of F_ST over the three populations was 0.068, indicating a slight to moderate level of genetic differentiation among the populations. The two Burkina Faso populations were more closely related (S=0.940, D=0.006) than either was to the Sudan population, suggesting that geographic separation is more important than host specialization in contributing to population differentiation. TheSorghum-adapted population was slightly closer to the Burkina FasoSorghum-adapted population (S=0.873, D=0.047) than to Burkina Faso millet-adapted population (S=0.851, D=0.074). The absence of substantial genetic divergence between host-specific populations ofStriga could result either from recent evolution of host-specialized strains or from strong selection for physiological specialization in the face of substantial gene flow between the populations.     

Functional studies on the EGF receptor with an antibody that recognizes the intracellular portion of the receptor

An antibody against the human epidermal growth factor receptor (EGF), capable of activating its tyrosine kinase has been produced. Antibody 2913 recognizes only the cytoplasmic portion of the EGF receptor in A431 carcinoma cells, in normal human fibroblasts, and in a variety of other human tumor cell lines (Xu, Y.-A., Richert, N., Ito, S., Merlino, G. T., and Pastan, I. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7308-7313). Indirect immunofluorescence and electron microscopy show that the antibody binds to intact cells only after membrane permeabilization. Moreover the antibody immunoprecipitates the v-erb-B gene product in avian myeloblastosis virus-infected cells but does not recognize the secreted form (105 kDa) of the A431 cell EGF receptor which lacks the cytoplasmic domain. Antibody 2913 activates the EGF receptor kinase in solubilized A431 membranes causing autophosphorylation on tyrosine residues only. Tryptic peptide maps suggest that antibody 2913 and EGF stimulate phosphorylation of the same amino acid residues. By electron microscopy, the cytoplasmic portion of the receptor was followed throughout its endocytotic pathway. The results show that the kinase domain is rapidly degraded in lysosomes with no accumulation in the cytoplasm or in the nucleus.     

3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli fumarate reductase by site-directed mutagenesis.

Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys204, Cys210, and Cys214 were individually replaced by Ser and in which Val207 was replaced by Cys were constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters were investigated. The FrdB Cys204Ser, Cys210Ser, and Cys214Ser mutations result in enzymes with negligible activity that have dissociated from the membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters, centers 3 and 2, respectively. In contrast, the FrdB Val207Cys mutation results in a functional membrane-bound enzyme that is able to support growth under anaerobic and aerobic conditions. However, EPR studies indicate that the indigenous [3Fe-4S]+,0 cluster (Em = -70 mV), center 3, has been replaced by a much lower potential [4Fe-4S]2+,+ cluster (Em = -350 mV), indicating that the primary sequence of the polypeptide determines the type of clusters assembled. The results of these studies afford new insights into the role of centers 2 and 3 in mediating electron transfer from menaquinol, the residues that ligate these clusters, and the intercluster magnetic interactions in the wild-type enzyme.     

Steady-state amounts of alpha- and luteinizing hormone (LH) beta-subunit messenger ribonucleic acids are uncoupled from pulsatility of LH secretion during sexual maturation of the heifer.

Our primary objective for this study was to determine whether steady-state amounts of alpha- and LH beta-subunit mRNAs in the anterior pituitary are altered during sexual maturation in the bovine female. A secondary objective was to determine whether 17 beta-estradiol (E2) alters amounts of LH subunit mRNAs before onset of puberty. Heifers (7 mo old) were assigned to one of three treatments: 1) ovariectomized (OVX, n = 16); 2) OVX and administered E2 (OVXE, n = 16); or 3) ovary-intact (INTACT, n = 20). Pituitaries were collected at an estimated 120 days before onset of puberty (prepuberty) or 25 days before onset of puberty (peripuberty). Six INTACT heifers were used to determine time of puberty during the experimental period, and their pituitaries were collected 40 h after administration of prostaglandin F2 alpha (postpubertal INTACT group). Relative amounts of mRNAs for LH subunits in each pituitary were determined by Northern analysis and scanning densitometry. Amounts of alpha- and LH beta-subunit mRNAs were lower in pituitaries of INTACT heifers and OVXE heifers, regardless of stage of sexual maturation, than in those of OVX heifers. Amounts of alpha-subunit mRNA were similar in OVXE and INTACT heifers regardless of stage of sexual maturation. Amounts of LH beta-subunit mRNA did not change during sexual maturation in heifers in the INTACT group. Concentrations of E2 were higher and LH beta-subunit mRNA were lower in heifers from the prepubertal OVXE group than in heifers in all other treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

A similar distribution of gonadotropin isohormones is maintained in the pituitary throughout sexual maturation in the heifer.

Our working hypotheses for this study were that 1) the profile of intrapituitary LH and FSH isoforms would be shifted toward acidic forms as sexual maturation progresses in the bovine female; and 2) concentration of 17 beta-estradiol (E2) in circulation during sexual maturation would be a major factor modulating the percentage of the more acidic isoforms. In addition, the biological-immunoreactive (B:I) ratios of each isoform of LH were evaluated at selected stages of sexual maturation. Heifers (7 mo of age) were assigned to one of three treatment groups: 1) ovariectomized (OVX; n = 16); 2) OVX and administered E2 (OVXE; n = 16); or 3) ovary-intact (INTACT; n = 14). Pituitaries were collected from heifers in each group at an estimated 120 days (prepubertal) of 25 days before puberty (peripubertal). A fourth group of 6 heifers remained intact (postpubertal INTACT) to determine time of puberty during the experimental period. Pituitaries of heifers assigned to the postpubertal INTACT group were collected during the follicular phase of the first or second estrous cycle postpuberty. Pituitaries were used for determination of relative amounts of gonadotropin isohormones. Tissue extracts of the pituitaries were chromatofocused on pH 10.5-4.0 gradients. The LH of all pituitaries resolved into thirteen isoforms that were designated isoforms A-L, and S, with isoform A the most basic form. Isoforms F and G (basic pH range) were the predominant isoforms of each chromatofocusing profile and comprised 50-60% of the immunoreactive LH. Isoforms J and K were the major isoforms eluting in the acidic pH range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of Different Polymeric Organic Materials for Creating Conditions That Favor Reductive Processes in Groundwater

The ability of three different polymeric organic materials (POMs) to create redox conditions favorable for reductive processes such as dechlorination was evaluated over a period of 70 days. Corn crop residue, unrefined chitin, and wood shavings were mixed with sand, packed into columns, and flushed with fresh groundwater once per day. Extracted groundwater was evaluated for pH, hydrogen, methane, volatile fatty acids (VFAs), alcohols, dissolved organic carbon (DOC), and chemical oxygen demand (COD). The pH measured for all POMs was between 5 and 8.5, with values after 30 days generally between 6 and 8. Hydrogen and methane concentrations indicated that all columns remained anaerobic during the entire experiment. Acetate was the dominant VFA in all columns, although propionate, butyrate, isobutyrate, and isovalerate were detected at much lower concentrations. Also, acetate concentrations were greater at all times in columns containing unrefined chitin (4 to 43 mM) than in those containing either corn crop residue (1 to 7 mM) or wood shavings (0 to 3 mM). Total electron donating capacities for dechlorination (expressed as total chloride removal capacities) were estimated from total COD and hydrogen concentrations. This capacity was greater in the column containing unrefined chitin than in those containing either corn crop residue or wood shavings. Overall the electron donating capacities remaining after 70 days were 40%, 11%, and 44% of the initial values for the corn crop residue, the unrefined chitin, and the wood shavings, respectively. The results indicate that over a 70-day period, anaerobic and nutrient-rich conditions were sustained for all POMs, but that the amount of electron donor available for reductive terminal electron accepting processes varies between POMs and is greatest for unrefined chitin.     

Magnetic circular dichroism and electron paramagnetic resonance studies of iron(II)-metallothionein

The electronic and magnetic properties of the Fe(II)-thiolate centers in Fe(II)-metallothionein have been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance spectroscopies at various levels of Fe(II) incorporation. In agreement with previous results [Good, M., & Vasák, M. (1986) Biochemistry 25, 8353-8356], rabbit liver metallothionein was found to bind a maximum of seven Fe(II) ions, with cluster formation occurring when more than four Fe(II) ions are bound at pH 8.5. The results indicate that all the iron in fully loaded Fe(II)-metallothionein is accommodated in Fe(II)-thiolate clusters that have either S = 0 or S = 2 ground states as a result of antiferromagnetic coupling between high-spin Fe(II) ions. By analogy with the cluster composition and mechanism of assembly that have been established for other divalent metal ions, the clusters with S = 0 and S = 2 ground states are attributed to tetranuclear and trinuclear centers, respectively. EPR signals indicative of S = 2 species were observed for samples containing monomeric tetrathiolate-Fe(II) centers and trinuclear Fe(II)-thiolate clusters. However, the nature of the zero-field splitting of the S = 2 ground states that is indicated by the EPR signals is not consistent with that deduced from M?ssbauer and magnetic circular dichroism studies, suggesting heterogeneity in both types of center.     

New views of humpback whale flow dynamics and oral morphology during prey engulfment

The rise of inexpensive, user‐friendly cameras and editing software promises to revolutionize data collection with minimal disturbance to marine mammals. Video sequences recorded by aerial drones and GoPro cameras provided close‐up views and unique perspectives of humpback whales engulfing juvenile salmon at or just below the water surface in Southeast Alaska and Prince William Sound. Although humpback feeding is famous for its flexibility, several stereotyped events were noted in the 47 lunges we analyzed. Engulfment was rapid (mean 2.07 s), and the entrance through which the tongue inverts into the ventral pouch was seen as water rushes in. Cranial elevation was a major contributor to gape, and pouch contraction sometimes began before full gape closure, with reverberating waves indicating rebounding flow of water within the expanded pouch. Expulsion of filtered water began with a small splash at the anterior of the mouth, followed by sustained excurrent flow in the mouth's central or posterior regions. Apart from a splash of rebounding water, water within the mouth was surprisingly turbulence‐free during engulfment, but submersion of the whale's head created visible surface whirlpools and vortices which may aggregate prey for subsequent engulfment.