The role of His-18 in amyloid formation by human islet amyloid polypeptide

The 37-residue islet amyloid polypeptide (IAPP) is the major protein component of the amyloid deposits found in type-II diabetes. IAPP is stored in a relatively low pH environment in the pancreatic secretory granules prior to its release to the extracellular environment. Human IAPP contains a single histidine at position 18. Aggregation of IAPP is considerably faster at a lower pH (4.0 +/- 0.3) than at high pH (8.8 +/- 0.3), as judged by turbidity and thioflavine-T fluorescence studies. The rate of aggregation at low pH increases drastically in the presence of salt. CD experiments show that the conversion of largely unstructured monomers to beta-sheet-rich structures is faster at high pH. TEM studies show that fibrils are formed at both pH values but are more prevalent at pH 8.8 (+/-0.3). Both the free N terminus of IAPP and His-18 will titrate over the pH range studied. An N-terminal acetylated fragment consisting of residues 8-37 of human IAPP was also studied to isolate contributions from the protonation of His-18. Previous studies have shown that this fragment forms protofibrils that are very similar to those formed by intact IAPP. The effects of varying the protonation state of His-18 in the 8-37 analogue indicate that the rate of aggregation and fibril formation is noticeably faster when His-18 is deprotonated, similar to the wild type. However, the pH-dependent effects are larger for full-length IAPP than for the disulfide-truncated, acetylated analogue. TEM studies indicate differences in the morphology of the deposits formed at high and low pH. These results are discussed in light of recent structural models of IAPP fibrils.     

pH-dependent interactions and the stability and folding kinetics of the N-terminal domain of L9. Electrostatic interactions are only weakly formed in the transition state for folding

The role of electrostatic interactions in the stability and the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) was investigated by determining the effects of varying the pH conditions. Urea denaturations and thermal unfolding experiments were used to measure the free energy of folding, DeltaG degrees, at 18 different pH values, ranging from pH 1.1 to pH 10.5. Folding rates were measured at 19 pH values between pH 2.1 and pH 9.5, and unfolding rates were determined at 15 pH values in this range using stopped-flow fluorescence experiments. The protein is maximally stable between pH 5.5 and 7.5 with a value of DeltaG degrees =4.45 kcal mol(-1). The folding rate reaches a maximum at pH 5.5, however the change in folding rates with pH is relatively modest. Over the pH range of 2.1 to 5.5 there is a small increase in folding rates, ln (k(f)) changes from 5.1 to 6.8. However, the change in stability is more dramatic, with a difference of 2.6 kcal mol(-1) between pH 2.0 and pH 5.4. The change in stability is largely due to the smaller barrier for unfolding at low pH values. The natural log of the unfolding rates varies by approximately four units between pH 2.1 and pH 5.5. The stability of the protein decreases above pH 7.5 and again the change is largely due to changes in the unfolding rate. ln (k(f)) varies by less than one unit between pH 5.5 and pH 9.5 while DeltaG degrees decreases by 2.4 kcal mol(-1) over the range of pH 5. 4 to pH 10.0, which corresponds to a change in ln K(eq) of 4.0. These studies show that pH-dependent interactions contribute significantly to the overall stability of the protein but have only a small effect upon the folding kinetics, indicating that electrostatic interactions are weakly formed in the transition state for folding.     

Hydrogen peroxide-induced neurotoxicity in cultured cortical cells grown in serum-free and serum-containing media

To compare different culture conditions for neuroprotection assays in cultured cortic neurons, we evaluated cell viability after H₂O₂ exposure in cells cultured with standard N2 and with the enriched B-27 developed by GIBCO, both serum-free supplements. The following additives/associations were compared: N2 (+N2), B-27 (+B-27), 10% FBS (+FBS), 1% FBS in combination with N2 (FBS/N2) or N2 supplement preceded by an 1 hour precoating with 10% FBS (N2 + precoated). Our data demonstrated that B-27 is as efficient as 10% FBS to support neuronal growth for more than a week. As shown by phase-contrast optics cells grown in N2 started degenerating within 24-48 hours although measurable absorbance was seen with MTT. The precoating procedure failed to modify substantially cell viability as compared with N2 alone. Dose-response curves for H₂O₂ to induce neuronal apoptosis were almost identical for B-27 and serum supplemented samples. Catalase (100 U/ml) or vitamin E (200 M) prevented cell death in both culture conditions. Our results indicate that DMEM/B-27 provides a serum-free cell culture environment that allows neurons to grow with optimal cell viability, comparable to that obtained with serum. We conclude that this culture condition reveals as a useful tool to test the efficacy of neuroprotectants when a serum free medium is required.     

High-Risk Cervical Human Papillomavirus Infections among Human Immunodeficiency Virus-Positive Women in the Bahamas

Background

High-risk (HR) HPV genotypes other than 16 and 18 have been detected in a significant proportion of immunocompromised females. We aim to evaluate the frequency of HR HPV genotypes in a population of HIV-positive Caribbean women.

Methods

One hundred sixty-seven consecutive, non-pregnant, HIV-positive females ≥18 years were recruited in this study. Each participant received a vaginal examination, PAP smear, and completed a questionnaire. DNA was extracted for HPV testing in 86 patients.

Results

Mean age was 39.1 years for women positive for HR HPV and 43.1 years for women negative for HR HPV (P value  = 0.040). 78% (130/167) of the women had HR HPV infections; the prevalence of abnormal cervical cytology was 38% among women who were HR HPV-positive compared to women who were HR HPV-negative (22%). Fifty-one percent of the 86 women with available genotype carried infections with HPV 16 and/or HPV 18; genotypes of unknown risk were also frequently observed. Women who had a CD4+ count of ≤200 had 7 times increased odds of carrying HR HPV infection in comparison to women with CD4+>200.

Conclusions

HR HPV infections in HIV infected females may consist of more than just HPV 16 and 18, but also HPV 52 and 58. Further studies are needed to determine whether HPV 52 and 58 play a significant role in the development of cervical cytological abnormalities in HIV+ women.     

Insight into the salivary transcriptome and proteome of Dipetalogaster maxima

Dipetalogaster maxima is a blood-sucking Hemiptera that inhabits sylvatic areas in Mexico. It usually takes its blood meal from lizards, but following human population growth, it invaded suburban areas, feeding also on humans and domestic animals. Hematophagous insect salivary glands produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain further insight into the salivary biochemical and pharmacologic complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Salivary proteins were also submitted to one- and two-dimensional gel electrophoresis (1DE and 2DE) followed by mass spectrometry analysis. We present the analysis of a set of 2728 cDNA sequences, 1375 of which coded for proteins of a putative secretory nature. The saliva 2DE proteome displayed approximately 150 spots. The mass spectrometry analysis revealed mainly lipocalins, pallidipins, antigen 5-like proteins, and apyrases. The redundancy of sequence identification of saliva-secreted proteins suggests that proteins are present in multiple isoforms or derive from gene duplications.