Type of negative feedback controlling sucrose ingestion depends on sucrose concentration

The sham intake of concentrated, but not weak, milk solutions requires up to three sham-feeding tests for intake to reach maximum (7). It is well known that the sham intake of concentrated (0.8 M) sucrose requires three or more sham-feeding tests to reach its maximum (4, 6, 17), but it is not known if this occurs with weaker sucrose solutions. We investigated this question by measuring the sham intake of seven concentrations of sucrose (0.025, 0.05, 0.1, 0. 2, 0.4, 0.6, and 0.8 M) during five sham-feeding tests. Sham intake of the three highest concentrations required up to three sham-feeding tests to reach maximum. Sham intake of the four lowest concentrations reached maximum in the first sham-feeding test. Our results show that the type of negative feedback that controls the intake of sucrose depends on its concentration. With weak solutions, intake is limited by a single direct, physiological, negative-feedback signal. When concentrated solutions are ingested, intake is controlled jointly by a direct physiological signal operating in conjunction with a labile one that loses its effectiveness with sham-feeding experience.     

Differential role of melanocortins in mediating leptin's central effects on feeding and reproduction

Leptin serves as a humoral link coupling the status of energy reserves to the functional activity of the reproductive system. Leptin is thought to act through melanocortinergic pathways in the brain to regulate ingestive behaviors; however, whether melanocortins mediate leptin's actions on the neuroendocrine-reproductive axis is unknown. We tested this hypothesis first by determining whether the effects of leptin on feeding behavior and reproduction in the ob/ob mouse could be blocked by the melanocortin receptor (MC-R) antagonist SHU9119 and second, by examining the effects of the MC-R agonist MTII on feeding and the endocrine-reproductive system. Administered by intracerebroventricular injections, leptin inhibited food intake, raised plasma gonadotropin levels, and increased seminal vesicle weights compared with controls; SHU9119 (intracerebroventricularly) attenuated leptin's effects on food intake and body weight but did not alter leptin's stimulatory effect on the reproductive axis. MTII (intracerebroventricularly and intraperitoneally) decreased food intake and increased body temperature compared with controls but had no effect on the reproductive-endocrine axis. These results suggest that although leptin acts centrally through melanocortinergic pathways to inhibit ingestive behaviors and stimulate metabolism, leptin's activational effect on the reproductive axis is likely to be mediated by other, unknown neuroendocrine circuits.     

Ubiquitination of the PEST-like endocytosis signal of the yeast a-factor receptor

A 58-residue-long, PEST-like sequence within the yeast a-factor receptor (Ste3p) specifies the ubiquitination, endocytosis, and consequent vacuolar degradation of the receptor protein (Roth, A. F., Sullivan, D. M., and Davis, N. G. (1998) J. Cell Biol. 142, 949-961). The present work investigates three lysyl residues that map within this sequence as the potential ubiquitin acceptor sites. Lys --> Arg substitution mutants were tested for effects on both ubiquitination and endocytosis. Results indicate that the three lysines function redundantly; a severe blockade to both ubiquitination and endocytosis is seen only for receptors having all three lysines replaced. Of the three, Lys(432) plays the predominant role; ubiquitination and turnover are significantly impaired for receptors having just the K432R mutation. CNBr fragmentation of the receptor protein, used for the physical mapping of the ubiquitin attachment sites, showed PEST-like sequence lysines to be modified both with single ubiquitin moieties as well with short multi-ubiquitin chains, two or three ubiquitins long. Thus, in addition to being the signal for ubiquitination, the Ste3p PEST-like sequence also provides the site for ubiquitin attachment. To test if this endocytosis signal functions solely for ubiquitination, we have asked if the requirement for the PEST-like sequence in endocytosis might be bypassed through pre-attachment of ubiquitin to the receptor protein. Indeed, Ste3-ubiquitin translational fusions that have a ubiquitin moiety fused to the receptor in place of the PEST-like signal do undergo rapid endocytosis and vacuolar turnover. We conclude that ubiquitin alone, with no required contribution from receptor sequences, provides the sufficient signal for initiating uptake. In addition, our results confirm conclusions originally drawn from studies with the alpha-factor receptor (Terrell, J., Shih, S., Dunn, R., and Hicke, L. (1998) Mol. Cell 1, 193-202), namely that mono-ubiquitin, and not multi-ubiquitin chains provide the primary recognition determinant for uptake. Although mono-ubiquitination suffices, our results indicate that multi-ubiquitination serves to augment the rate of uptake.     

Conventional resistance of experimental maize lines to corn earworm (Lepidoptera: Noctuidae), fall armyworm (Lepidoptera: Noctuidae), southwestern corn borer (Lepidoptera: Crambidae), and sugarcane borer (Lepidoptera: Crambidae)

Plant resistance is a useful component of integrated pest management for several insects that are economically damaging to maize, Zea mays L. In this study, 15 experimental lines of maize derived from a backcross breeding program were evaluated for resistance to corn earworm, Helicoverpa zea (Boddie); fall armyworm, Spodoptera frugiperda (J. E. Smith); southwestern corn borer, Diatraea grandiosella Dyar; and sugarcane borer, Diatraea saccharalis (F.). Experimental line 100-R-3 was resistant in the field to leaf feeding by fall armyworm and line 116-B-10 was resistant in the field to leaf feeding by fall armyworm and leaf and stalk feeding by southwestern corn borer. When corn earworm larvae were fed field harvested silks from experimental line 81-9-B in the laboratory, their pupal weights were significantly lower than the pupal weights of larvae that were fed silks from the resistant control, Zapalote Chico. Maysin levels lower than those commonly associated with corn earworm resistance were present in the resistant experimental line, 107-8-7, indicating a new basis confers resistance to corn earworm in this line. These resistant experimental lines will provide plant breeders with new sources of resistance to lepidopterous insects for the development of improved maize breeding populations.     

Why do cytotoxic T lymphocytes fail to eliminate hepatitis C virus? Lessons from studies using major histocompatibility complex class I peptide tetramers.

Hepatitis C virus (HCV) infection is a major public health problem, affecting an estimated 3% of the world's population, and over 10% in some countries. Infection in most cases becomes persistent, and can lead to hepatic inflammation, fibrosis and liver failure. The T lymphocyte reponse, in particular that mediated by cytotoxic T lymphocytes (CTLs), is likely to be involved in determining the outcome of infection, although its overall role is not clear. The use of major histocompatibility complex (MHC) class I peptide tetrameric complexes (tetramers) to study antiviral CTL responses has revolutionized our approach to the study of human infection. We have used a panel of MHC class I tetramers to analyse immune responses in HCV-infected individuals at various stages of disease. We find that the CTL response against HCV is vigorous in its early phases but dwindles over time both in terms of lymphocyte number and function. A number of potential explanations for this 'CTL failure' are discussed.     

Multiple isoforms of porcine aromatase are encoded by three distinct genes

Cytochrome P450 aromatase, a product of the CYP 19 gene and the terminal enzyme in the estrogen biosynthetic pathway, is synthesized by the ovary, endometrium, placenta, and peri-implantation embryos in the pig and other mammals, albeit to varying levels, implying its functional role(s) in pregnancy events. The aromatase produced by the pig tissues exists as three distinct isoforms (type I - ovary, type II - placenta, and type III - embryo), with presumed differences in substrate specificities, expression levels, activity, and mode of regulation. In order to delineate the molecular mechanisms whereby estrogen synthesis is regulated in these diverse tissues, the present study examined if these aromatase isoforms represent products of multiple genes or of a single gene via complex splicing mechanisms. Porcine genomic DNA from a single animal was used as a template in the polymerase chain reaction (PCR) to amplify isoform-specific sequences corresponding to exons 4 and 7, respectively. Nucleotide sequence analysis of the generated fragments revealed the presence of only clones corresponding to the three known aromatase types. Screening a porcine Bacterial Artificial Chromosome (BAC) library for aromatase gene by PCR yielded a single clone approximately 80 kb in length. Southern blot analysis, using probes specific for exons 1A-1B, 2-3, 4-9, and 10 sequences indicated that the BAC genomic clone contains the entirety of the coding exons as well as the proximal promoter region. Sequence analysis of the fragment generated with exon 4 primers determined that this BAC clone contains only the type II gene. The presence and relative orientation of the untranslated 5'- exons 1A and 1B, previously demonstrated for the type III isoform were evaluated in the BAC clone and genomic DNA by PCR. The 265 bp fragment generated from both PCR reactions was confirmed by sequence analysis to contain exons 1A and 1B that are located contiguous to each other and separated by only three bp. A diagnostic procedure for typing aromatase isoforms was developed, based on the presence of specific restriction sites within isoform-specific exons. The use of this protocol confirmed the existence of only three aromatase isoforms in the porcine genome and indicated changes in aromatase types expressed by the uterine endometrium as a function of pregnancy stage. The presence of distinct genes encoding each of the aromatase isoform predicts important differences in the mechanisms underlying the molecular evolution and regulation of porcine aromatase, unique from those of other mammals, and suggests a critical role for P450 aromatase steroidal products in uterine functions related to pregnancy events.     

Matrix metalloproteinase activity is required for activity-induced angiogenesis in rat skeletal muscle

Proteolysis of the capillary basement membrane is a hallmark of inflammation-mediated angiogenesis, but it is undetermined whether proteolysis plays a critical role in the process of activity-induced angiogenesis. Matrix metalloproteinases (MMPs) constitute the major class of proteases responsible for degradation of basement membrane proteins. We observed significant elevations of mRNA and protein levels of both MMP-2 and membrane type 1 (MT1)-MMP (2.9 +/- 0.7- and 1.5 +/- 0.1-fold above control, respectively) after 3 days of chronic electrical stimulation of rat skeletal muscle. Inhibition of MMP activity via the inhibitor GM-6001 prevented the growth of new capillaries as assessed by the capillary-to-fiber ratio (1.34 +/- 0.08 in GM-6001-treated muscles compared with 1.69 +/- 0.03 in control 7-day-stimulated muscles). This inhibition correlated with a significant reduction in the number of capillaries with observable breaks in the basement membrane, as assessed by electron microscopy (0.27 +/- 0.27% in GM-6001-treated muscles compared with 3.72 +/- 0.65% in control stimulated muscles). Proliferation of capillary-associated cells was significantly elevated by 2 days and remained elevated throughout 14 days of stimulation. Capillary-associated cell proliferation during muscle stimulation was not affected by MMP inhibition (80.3 +/- 9.3 nuclei in control and 63.5 +/- 8.5 nuclei in GM-6001-treated animals). We conclude that MMP proteolysis of capillary basement membrane proteins is a critical component of physiological angiogenesis, and we postulate that capillary-associated proliferation precedes and occurs independently of endothelial cell sprout formation.