The state of the guanosine nucleotide allosterically affects the interfaces of tubulin in protofilament.

The dynamics of microtubules is essential for many microtubule-dependent cellular functions such as the mitosis. It has been recognized for a long time that GTP hydrolysis in αß-tubulin polymers plays a critical role in this dynamics. However, the effects of the changes in the nature of the guanosine nucleotide at the E-site in ß-tubulin on microtubule structure and stability are still not well understood. In the present work, we performed all-atom molecular dynamics simulations of a αßα-tubulin heterotrimer harboring a guanosine nucleotide in three different states at the E-site: GTP, GDP-Pi and GDP. We found that changes in the nucleotide state is associated with significant conformational variations at the α-tubulin N- and ß-tubulin M-loops which impact the interactions between tubulin protofilaments. The results also show that GTP hydrolysis reduces αß-tubulin interdimer contacts in favor of intradimer interface. From an atomistic point view, we propose a role for α-tubulin glutamate residue 254 in catalytic magnesium coordination and identified a water molecule in the nucleotide binding pocket which is most probably required for nucleotide hydrolysis. Finally, the results are discussed with reference to the role of taxol in microtubule stability and the recent tubulin-sT2R crystal structures.

Paneth cell α-defensins in enteric innate immunity.

Paneth cells at the base of small intestinal crypts of Lieberkühn secrete high levels of α-defensins in response to cholinergic and microbial stimuli. Paneth cell α-defensins are broad spectrum microbicides that function in the extracellular environment of the intestinal lumen, and they are responsible for the majority of secreted bactericidal peptide activity. Paneth cell α-defensins confer immunity to oral infection by Salmonella enterica serovar Typhimurium, and they are major determinants of the composition of the small intestinal microbiome. In addition to host defense molecules such as α-defensins, lysozyme, and Pla2g2a, Paneth cells also produce and release proinflammatory mediators as components of secretory granules. Disruption of Paneth cell homeostasis, with subsequent induction of endoplasmic reticulum stress, autophagy, or apoptosis, contributes to inflammation in diverse genetic and experimental mouse models.

HIV-1 IN alternative molecular recognition of DNA induced by raltegravir resistance mutations.

Virologic failure during treatment with raltegravir, the first effective drug targeting HIV integrase, is associated with two exclusive pathways involving either Q148H/R/K, G140S/A or N155H mutations. We carried out a detailed analysis of the molecular and structural effects of these mutations. We observed no topological change in the integrase core domain, with conservation of a newly identified Omega-shaped hairpin containing the Q148 residue, in particular. In contrast, the mutations greatly altered the specificity of DNA recognition by integrase. The native residues displayed a clear preference for adenine, whereas the mutant residues strongly favored pyrimidines. Raltegravir may bind to N155 and/or Q148 residues as an adenine bioisoster. This may account for the selected mutations impairing raltegravir binding while allowing alternative DNA recognition by integrase. This study opens up new opportunities for the design of integrase inhibitors active against raltegravir-resistant viruses.

A new binding site involving the C-terminal domain to design specific inhibitors of PepX.

PepX is a X-prolyl dipeptidyl aminopeptidase of S15 family that cleaves dipeptides from the N-terminus of polypeptides having a proline or alanine residue at the second position. Involved in bacterial nutrition and in peptide maturation, this serine exopeptidase, counterpart of the mammalian DDP-4, has been proposed to play a role in pathogenicity for Streptococci and to be a promising target against trypanosomes. Searching for specific inhibitors, we undertook docking simulations on the whole surface of PepX from Lactococcus lactis, type example of the S15 family, which revealed a new putative binding site in connection with the active site and involving the C-terminal domain. Accordingly to the results of the computations, we synthesized two peptidomimetics of low molecular weight: the valinephenylpiperazine and the valine-isopropylpiperazine that can accommodate to this putative binding site. Experiments revealed that the valine-phenylpiperazine was an uncompetitive inhibitor whereas the valine-isopropylpiperazine showed to be an activator of the enzyme activity. The valine-phenylpiperazine is interacting with ASN 379, GLU 383, GLU 474, residues in connection with the specificity and active sites, and with the residues from the C-terminal domain LEU 693, GLU 710 and GLN 712. These results point out a role of the C-terminal domain in controlling access to the active site of enzymes of the S15 family, like PepX, the cocaine esterase or the alpha-amino acid ester hydrolase, and could have applications in human health giving new perspectives to struggle against streptococci or trypanosomes by designing inhibitors specific to the S15 family of enzymes.

National seroprevalence and trends in herpes simplex virus type 1 in the United States, 1976-1994.

OBJECTIVES: The objectives of this study were to estimate national seroprevalence of herpes simplex virus type 1 (HSV-1), describe trends in seroprevalence, and examine correlates of infection. GOAL: The goal of this study was to measure the burden of HSV-1 infection in the U.S. population. STUDY: We tested serum samples for HSV-1 antibody and analyzed questionnaire data collected for the second and third National Health and Nutrition Surveys (NHANES II, 1976-80; NHANES III, 1988-94). Seroprevalence estimates were weighted to represent the total U.S. population. RESULTS: At the time of NHANES III, two thirds (68%) of the U.S. population 12 years and older had HSV-1 antibody. Prevalence increased with age and varied by race/ethnicity; the majority of persons in all race/ethnic groups were HSV-1-seropositive by age 30. Overall, the national seroprevalence of HSV-1 decreased nonsignificantly by 2% in the years between NHANES II and III; decreases in HSV-1 seroprevalence in some population subgroups were balanced by increases in other groups. CONCLUSIONS: There was no overall change in the seroprevalence of HSV-1 in the U.S. population between NHANES II and III.

Conduta para o melanoma cutâneo maligno / Management of cutaneous melanoma

A incidência do melanoma maligno aumentou significativamente: de 1:1500, em 1935, para cerca de 1:75, no ano 2000. Contudo, atribuído a um diagnóstico cada vez mais precoce, tem-se observado uma melhora da sobrevida em cinco anos, com diminuição da taxa de mortalidade geral que tem se situado entre 70% a 80%, desde a década de 30. É o câncer mais prevalente na faixa etária de 25 a 35 anos nos EUA. O Brasil ocupa a 15ª posição com relação à incidência do tumor no mundo. O estadiamento inicial é baseado na pesquisa de sinais e sintomas que podem indicar doença metastática. Especial atenção deve ser dada à palpação de linfonodos regionais. A espessura e a ulceração são os principais fatores de risco independentes, em pacientes com melanoma primário com linfonodos livres. Já naqueles com metástases linfonodais, a presença de ulceração, de metástase detectada macroscopicamente e o número de linfonodos acometidos são os principais índices de impacto na sobrevida. Pacientes com metástases para o pulmão possuem melhor prognóstico no primeiro ano de sobrevida em comparação àqueles com metástases para outros órgãos. Muitas séries relacionam LDH como fator prognóstico poderoso em pacientes com estádio IV da doença. Devido à falta de uma padronização para o tratamento do melanoma, muitos pacientes ainda evoluem com um prognóstico reservado devido a uma conduta inicial inadequada. Os tratamentos vêm mudando significativamente e este trabalho visa apresentar uma revisão com ênfase nas condutas preconizadas para o melanoma.

The incidence of melanoma is increasing: 1:1500 in 1935, 1:75 in 2000. However, due to early diagnosis, an improvement in 5-year survival has been notified with decrease in mortality. Initial staging is based on signs and symptoms showing metastatic disease. Special care should be taken with lymphatic examination. The two most powerful independent prognostic variables are tumor thickness and ulceration. In patients with nodal metastasis, the three most powerful prognostic factors are number of positive nodes, tumor burden, and the presence or absence of ulceration of primary lesion. Great differences exist in survival between patients with melanoma metastasis in visceral sites and those with metastasis in non-visceral sites. The survival of patients with lung as the only site of metastasis was higher than those with metastasis in other visceral sites. In many large series, high LDH level has been shown to be a consistent, independent, and powerful prognostic factor in patients with stage IV disease. The lack of treatment pattern in melanoma lead to a bad prognosis due to an error in the first approach.