Anti-heparan sulfate peptides that block herpes simplex virus infection in vivo

Heparan sulfate (HS) and its highly modified form, 3-O-sulfated heparan sulfate (3-OS HS), contribute strongly to herpes simplex virus type-1 (HSV-1) infection in vitro. Here we report results from a random M13-phage display library screening to isolate 12-mer peptides that bind specifically to HS, 3-OS HS, and block HSV-1 entry. The screening identified representative candidates from two-different groups of anti-HS peptides with high positive charge densities. Group 1, represented by G1 peptide (LRSRTKIIRIRH), belongs to a class with alternating charges (XRXRXKXXRXRX), and group 2, represented by G2 peptide (MPRRRRIRRRQK), shows repetitive charges (XXRRRRXRRRXK). Viral entry and glycoprotein D binding assays together with fluorescent microscopy data indicated that both G1 and G2 were potent in blocking HSV-1 entry into primary cultures of human corneal fibroblasts and CHO-K1 cells transiently expressing different glycoprotein D receptors. Interestingly, G2 peptide isolated against 3-OS HS displayed wider ability to inhibit entry of clinically relevant strains of HSV-1 and some divergent members of herpesvirus family including cytomegalovirus and human herpesvirus-8. To identify functional residues within G1 and G2, we performed point mutations and alanine-scanning mutagenesis. Several arginine and a lysine residues were needed for anti-HSV-1 activity, suggesting the importance of the positively charged residues in virus-cell binding and virus-induced membrane fusion. In vivo administration of G1 or G2 peptide as a prophylactic eye drop completely blocked HSV-1 spread in the mouse cornea as evident by immunohistochemistry. This result also highlights an in vivo significance of HS and 3-OS HS during ocular herpes infection.     

Processing and trafficking of Leishmania mexicana GP63. Analysis using GP18 mutants deficient in glycosylphosphatidylinositol protein anchoring

GPI8 is a clan CD, family C13 cysteine protease and the catalytic core of the GPI-protein transamidase complex. In Leishmania mexicana, GPI8 is nonessential, and Deltagpi8 mutants lack the GPI-anchored metalloprotease GP63, which is the major surface protein of promastigotes. We have identified the active site histidine and cysteine residues of leishmanial GPI8 and generated Deltagpi8 lines expressing modified GPI8 proteins. This has allowed us to study the processing and trafficking of GP63 in wild type and Deltagpi8 mutants. We show using pulse-chase labeling that in Deltagpi8 non-GPI-anchored GP63 was glycosylated and secreted without further processing from the cell with a t(12) of 120 min. This secretion was prevented by growth of cells in the presence of tunicamycin, indicating that glycosylation is necessary for secretion of non-GPI-anchored proteins. In contrast, in wild type cells the majority of GP63 was rapidly glycosylated, GPI-anchored, and trafficked to the surface with defined processing intermediate forms. Tunicamycin inhibited glycosylation but did not prevent GPI anchor addition or trafficking. These results show that GPI-anchored and unanchored GP63 are trafficked via different pathways. In addition, the balance between GPI anchor addition and secretion of GP63 in Leishmania can vary depending on the activity of the GPI-protein transamidase, which has implications for the host-parasite interaction.     

Free radical scavenging activity, metal chelation and antioxidant power of some of the Indian spices

Food constituents are the major source of various phytochemicals and micronutrients. The importance of these dietary constituents has been stressed in recent years due to their antioxidant and anticarcinogenic potential. Spices used in Indian foods such as cloves (Syzygium aromaticum), licorice (Glycyrrhiza glabra), mace (aril of Myristica fragans), and greater cardamom (Amomum subulatum) were tested for their antioxidant properties in vitro. The metal chelating activity, bleomycin dependent DNA oxidation, diphenyl-p-picryl hydrazyl (DPPH) radical scavenging activity and the ferric reducing /antioxidant power (FRAP) were measured in rat liver homogenate in presence of spices. Metal chelating activity was significantly high with all the spice extracts except mace. The spices due to higher reducing potential (in presence of bleomycin-FeCl_{3}) showed increased DNA oxidation. Cloves showed the highest DPPH radical scavenging activity, followed by licorice, mace and cardamom. FRAP values for cloves were also the highest, while other spices showed comparatively lesser FRAP values. The results show that the spices tested are strong antioxidants and may have beneficial effects on human health.     

Phytochrome mediates germination responses to multiple seasonal cues

We identified a new role of phytochrome in mediating germination responses to seasonal cues and thereby identified for the first time a gene involved in maternal environmental effects on germination. We examined the germination responses of a mutant, hy2-1, which is deficient in the phytochrome chromophore. The background genotype, Landsberg erecta (Ler), lacked dormancy in most treatments, while hy2-1 required cold stratification for germination in a manner that resembled a more dormant ecotype, Columbia (Col). Unlike Col, hy2-1 was not induced into dormancy by warm stratification. Therefore, the down-regulation of phytochrome-mediated germination pathways results in sensitivity to cold, but we found no evidence that reduced phytochrome activity enables the warm-induction of dormancy. Cool temperatures during seed maturation induced dormancy. The hy2-1 mutants did not overcome this dormancy, indicating that phytochrome-mediated pathways are required to break cold-induced dormancy. Ler did not respond to post-stratification temperature, but hy2-1 did respond, suggesting phytochrome pathways are involved in germination responses to temperature. In summary, phytochromes mediate dormancy and germination responses to seasonal cues experienced both during seed maturation and after dispersal. Phytochromes therefore appear to be involved in mediating seasonal germination timing, a trait of great ecological importance and one that is under strong natural selection.     

Two-step wiring plasticity--a mechanism for estrogen-induced rewiring of cortical circuits

Estrogens have been shown to exert powerful effects on cognitive behaviors mediated by several areas of the brain including the cortex. Remodeling of spiny synapses is a key step in the rewiring of neuronal circuitry thought to underlie the processing and storage of information in the forebrain. Whereas estrogen has been shown to regulate synapse structure and function, we are only just starting to understand the molecular and cellular underpinnings of how estrogens can modulate neuronal circuits. Here I will review recent molecular and cellular work that offers a potential mechanism of how estrogen may modulate synapse structure and function of cortical neurons. This mechanism allows cortical neurons to respond to activity-dependent stimuli with greater efficacy in a cellular model termed "Two-Step Wiring Plasticity". This novel form of spine plasticity thus provides insight into how estrogens may modulate the rewiring of neuronal circuits, underlying its ability to influencing cortically based behaviors. This article is part of a Special Issue entitled 'Neurosteroids'.     

13C NMR chemical shifts can predict disulfide bond formation

The presence of disulfide bonds can be detected unambiguously only by X-ray crystallography, and otherwise must be inferred by chemical methods. In this study we demonstrate that ¹³C NMR chemical shifts are diagnostic of disulfide bond formation, and can discriminate between cysteine in the reduced (free) and oxidized (disulfide bonded) state. A database of cysteine ¹³C C and C chemical shifts was constructed from the BMRB and Sheffield databases, and published journals. Statistical analysis indicated that the C shift is extremely sensitive to the redox state, and can predict the disulfide-bonded state. Further, chemical shifts in both states occupy distinct clusters as a function of secondary structure in the C/C chemical shift map. On the basis of these results, we provide simple ground rules for predicting the redox state of cysteines; these rules could be used effectively in NMR structure determination, predicting new folds, and in protein folding studies.     

The C-terminal domain of the MutL homolog from Neisseria gonorrhoeae forms an inverted homodimer

The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage.     

Evaluation of the influence of strain rate on Colles' fracture load

Colles' fracture, a transverse fracture of the distal radius bone, is one of the most frequently observed osteoporotic fractures resulting from low energy or traumatic events, associated with low and high strain rates, respectively. Although experimental studies on Colles' fracture were carried out at various loading rates ranging from static to impact loadings, there is no systematic study in the literature that isolates the influence of strain rate on Colles' fracture load. In order to provide a better understanding of fracture risk, the current study combines experimental material property measurements under varying strain rates with computational modeling and presents new information on the effect of strain rate on Colles' fracture. The simulation results showed that Colles' fracture load decreased with increasing strain rate with a steeper change in lower strain rates. Specifically, strain rate values (0.29s(-1)) associated with controlled falling without fracture corresponded to a 3.7% reduction in the fracture load. On the other hand, the reduction in the fracture load was 34% for strain rate of 3.7s(-1) reported in fracture inducing impact cadaver experiments. Further increase in the strain rate up to 18s(-1) led to an additional 22% reduction. The most drastic reduction in fracture load occurs at strain rates corresponding to the transition from controlled to impact falling. These results are particularly important for the improvement of fracture risk assessment in the elderly because they identify a critical range of loading rates (10-50mm/s) that can dramatically increase the risk of Colles' fracture.