The complex folding behavior of HIV-1-protease monomer revealed by optical-tweezer single-molecule experiments and molecular dynamics simulations.

We have used optical tweezers and molecular dynamics simulations to investigate the unfolding and refolding process of a stable monomeric form of HIV-1-protease (PR). We have characterized the behavior under tension of the native state (N), and that of the ensemble of partially folded (PF) conformations the protein visits en route to N, which collectively act as a long-lived state controlling the slow kinetic phase of the folding process. Our results reveal a rich network of unfolding events, where the native state unfolds either in a two-state manner or by populating an intermediate state I, while the PF state unravels through a multitude of pathways, underscoring its structural heterogeneity. Refolding of mechanically denatured HIV-1-PR monomers is also a multiple-pathway process. Molecular dynamics simulations allowed us to gain insight into possible conformations the protein adopts along the unfolding pathways, and provide information regarding possible structural features of the PF state.

Identification and characterization of folding inhibitors of hen egg lysozyme: an example of a new paradigm of drug design.

Studies of protein folding indicate the presence of native contacts in the denatured state, giving rise to folding elements which contribute to the accomplishment of the native state. The possibility of finding molecules which can interact with specific folding elements of a target protein preventing it from reaching its native state, and hence from becoming biologically active, is particularly attractive. The notion that folding elements not only provide molecular recognition directing the folding process, but also have conserved sequence, implies that targeting such elements will make protein folding inhibitors less susceptible to mutations which, in many cases, abrogate drug effects. The folding-inhibition strategy can lead to a truly novel and rational approach to drug design, aside from providing new insight into folding. This is illustrated in the case of hen egg lysozyme.

Abnormal fecal flora in a patient with short bowel syndrome. An in vitro study on effect of pH on D-lactic acid production.

D-Lactic acidosis associated with encephalopathy is a clinical condition that occurs in patients with short bowel syndrome. We studied the fecal flora and the composition of fecal water of a child who developed this unusual disorder. Bacteriological studies showed that the patient's stool contained a marked predominance of gram-positive anaerobes. Two strains were identified, Lactobacillus plantarum and Lactobacillus salivarius, as the main bacteria isolated. Fecal water showed pH 4.8 and total lactic acid (sum of L- and D-lactic acids) was the principal organic anion found in the feces. We also incubated the patient's stool in a continuous culture with a view to determining the effect of the pH on the production of volatile fatty acids (VFA) and L- and D-lactic acids. The culture was maintained at pH 5.0, 5.5, 6.0, and 6.5 for four consecutive periods of four days each. We then studied the culture for a further four days at pH 5.0 once again. This study showed that with the progressive rise of the pH from 5.0 to 6.5 L- and D-lactic acids decreased and VFA production increased. D-Lactic acid formation was inhibited at pH 6.5, but when the culture was returned to pH 5.0, it increased to a high level again. These results suggest that the pH plays an important role in the ecological changes in the colonic bacteria that result in D-lactic acid production.

Fecal lactate and short bowel syndrome.

In patients with short bowel syndrome (SBS), the carbohydrate overload to the colon may disturb the normal pattern of colonic fermentation with production of D-lactic acid and subsequent development of a metabolic D-lactic acidosis. We measured D-lactic acid in blood, urine, and feces, as well as the composition of fecal water and fecal reducing substances from 11 patients with SBS, comparing the results with those from normal subjects. The fecal water from patients with SBS was characterized by low pH, potassium, and volatile fatty acids, high osmotic gap, and high concentration of L- and D-lactic acid. Five of 11 had abnormal amounts of fecal reducing substances. Fecal D-lactic acid was increased in nine of 11 patients. However, none of these patients showed D-lactic acid in urine, and only one had a very low concentration in plasma. These results show that D-lactic acid was overproduced in the colon of most of the patients with SBS. However, other factors such as absorption or impaired D-lactic acid metabolism may be necessary for a plasmatic increase of D-lactic acid.

Colonic proteolysis following pancreatic duct ligation in the rat.

Luminal proteolytic activity (PA) of different colonic segments was ascertained in animals subjected to pancreatic duct ligation (PDL) and in control rats. The PDL rats revealed a significant PA reduction in the cecum, proximal colon (P < 0.01), and distal colon (P < 0.005). Proteolytic activity, trypsin, and chymotrypsin activity in control rats diminished progressively from the cecum to the distal colon. Conversely in PDL rats, we found maximal PA in distal colon. The conclusion is drawn that a significant proportion of colonic proteolytic activity can be attributed to pancreatic proteases with a maximal contribution at cecum level.

Concentración fecal de ácidos grasos volátiles y ácido láctico en pacientes con sindrome de instestino corto / Fecal concentration in volatile fatty acids and latic acids in patients with short bowel syndrome

El sindrome de intestino corto (SIC) está caracterizado por diarrea y mala absorción de nutrientes. Los carbohidratos no absorbidos en el intestino delgado pueden contribuir a la diarrea a través de un efecto osmótico mediado por los propios azúcares sin absorber o por sus productos de fermentación bacteriana. En condiciones de normalidad los ácidos grasos volátiles (AGV) son los principales metabolitos bacterianos, son parcialmente absorbidos por la mucosa colónica estimulando la absorción de sodio y agua el acido láctico esta en muy bajas concentraciones, su absorción es lenta y trabajos experimentales demuestran que puede ocasionar daño a la mucosa colónica. En este trabajo se estudió la composición del agua fecal (pH,Na+,K+, osmolaridad, AGV y ácidos D y L-láctico) de pacientes con SIC comparando los resultados con un grupo control. El agua fecal de los pacientes con SIC mostró una disminución en la concentración de K+, de la relación K+/Na+, y un incremento del gap osmótico. Los AGV fueron los principales aniones orgánicos en las heces de los sujetos controles mientras que el ácido láctico fue el anion preponderante en el agua fecal obtenida de pacientes con SIC. Estos resultados sugieren que este cambio metabólico bacteriano puede contribuir a la diarrea observada en los pacientes con SIC.

Concentración fecal de ácidos grasos volátiles y ácido láctico en pacientes con sindrome de instestino corto / Fecal concentration in volatile fatty acids and latic acids in patients with short bowel syndrome

El sindrome de intestino corto (SIC) está caracterizado por diarrea y mala absorción de nutrientes. Los carbohidratos no absorbidos en el intestino delgado pueden contribuir a la diarrea a través de un efecto osmótico mediado por los propios azúcares sin absorber o por sus productos de fermentación bacteriana. En condiciones de normalidad los ácidos grasos volátiles (AGV) son los principales metabolitos bacterianos, son parcialmente absorbidos por la mucosa colónica estimulando la absorción de sodio y agua el acido láctico esta en muy bajas concentraciones, su absorción es lenta y trabajos experimentales demuestran que puede ocasionar daño a la mucosa colónica. En este trabajo se estudió la composición del agua fecal (pH,Na+,K+, osmolaridad, AGV y ácidos D y L-láctico) de pacientes con SIC comparando los resultados con un grupo control. El agua fecal de los pacientes con SIC mostró una disminución en la concentración de K+, de la relación K+/Na+, y un incremento del gap osmótico. Los AGV fueron los principales aniones orgánicos en las heces de los sujetos controles mientras que el ácido láctico fue el anion preponderante en el agua fecal obtenida de pacientes con SIC. Estos resultados sugieren que este cambio metabólico bacteriano puede contribuir a la diarrea observada en los pacientes con SIC. (AU)

Effects of right hemicolectomy on fecal nitrogen excretion in rats.

The influence of right hemicolectomy (RH) on fecal nitrogen excretion was determined with selected protein levels up to 25%. The endogenous fecal N was determined by extrapolating protein intake to zero. Fecal N was higher in RH than in control rats at all protein levels used. However, the slope of regression curves describing fecal nitrogen excretion was greater for RH compared with the control group. The endogenous fecal nitrogen was not significantly different between the two groups of rats. The feces from rats fed with 25% of protein were partitioned into individual fractions by physical separation and a study was made of the distribution of nitrogen in the bacterial, soluble and fiber fractions of the stool. RH decreased the N excreted in the bacterial fraction by 33% (from 1.71 +/- 0.32 to 1.15 +/- 0.18 mmol/day) and increased the N excreted in the soluble fraction by 280% (from 1.60 +/- 0.30 to 6.08 +/- 1.16 mmol/day). These results show that the RH increased the fecal N excretion and that this N is mainly in the soluble fraction.

Effect of total food deprivation on the proteoglycans of rat hyaline cartilage and bone.

Existing hypotheses suggest that the effect of food deprivation on bone occurs via alterations in the synthesis of the organic matrix. Thus, this work was carried out to characterize the modifications of the physico-chemical properties of the proteoglycans (PG) of rat hyaline cartilage and femur. Male Wistar rats were assigned at random to a control group which was fed a standard pellet diet or to an experimental group which was given water "ad libitum" and starved over the experimental period. On day 4 or 8 the animals were administered a dose of 35S, weighed and killed. PG and glycosaminoglycans (GAG) were isolated from femurs and xyphoid cartilages. Uptake of 35S, GAG distribution patterns, PG molecular weight, molecular size of the side chains and the PG density gradient were determined. The aforementioned parameters decreased significantly after 4 and 8 days of total starvation. The GAG distribution pattern only exhibited a reduction of the Chondroitin-4-Sulphate fraction. These changes could alter the binding properties of PG to other macromolecules such as collagen which plays an important role in the ossification process.

Effect of total food deprivation on the proteoglycans of rat hyaline cartilage and bone.

Existing hypotheses suggest that the effect of food deprivation on bone occurs via alterations in the synthesis of the organic matrix. Thus, this work was carried out to characterize the modifications of the physico-chemical properties of the proteoglycans (PG) of rat hyaline cartilage and femur. Male Wistar rats were assigned at random to a control group which was fed a standard pellet diet or to an experimental group which was given water [quot ]ad libitum[quot ] and starved over the experimental period. On day 4 or 8 the animals were administered a dose of 35S, weighed and killed. PG and glycosaminoglycans (GAG) were isolated from femurs and xyphoid cartilages. Uptake of 35S, GAG distribution patterns, PG molecular weight, molecular size of the side chains and the PG density gradient were determined. The aforementioned parameters decreased significantly after 4 and 8 days of total starvation. The GAG distribution pattern only exhibited a reduction of the Chondroitin-4-Sulphate fraction. These changes could alter the binding properties of PG to other macromolecules such as collagen which plays an important role in the ossification process.

Alteraciones en la matriz organica del hueso y cartilago de rata por el uso prolongado de fluoruno de sodio / Alterations in the organic matrix of bone and cartilage after prolonged administration of sodium fluoride in the rat

Los fluoruros, por mecanismos aun no aclarados, estimulan la formación ósea y son, en consecuencia, usados en el tratamiento de las osteoporosis. Desde un punto de vista terapéutico, uno de los efectos más esperados por la ingesta de fluoruro en pacientes osteoporóticos es la disminución en el índice de fracturas. Si bien esto ocurre, observaciones clínicas sugieren que este efecto es menor que el esperado por el aumento de la masa ósea (ej.: la resistencia por unidad de tejido estaría disminuida en el hueso fluorótico). El o los mecanismos por los cuales el fluoruro conduce a las alteraciones mencionadas, todavía no son bien conocidos, pero como el componente inorgánico ha sido extensamente estudiado, hemos llevado a cabo este trabajo con el objeto de caracterizar cuali y cuantitativamente a los GAG y el colágeno de hueso y cartílafo de rata, en función de la ingesta prolongada de fluoruro de sodio. Las variaciones producidas por la ingesta de fluoruro implican un aumento significativo en la concentracicón de GAG, después de dos meses de tratamiento, debidas a un incremento en las fracciones correspondentes al condroitín-6-sulfato y dermatán sulfato. Esta modificación en el patrón de distribución de los GAG no es atribuible a variaciones en el peso de las moléculas. Aunque otros estudios han informado que no se observan efectos sobre la síntesis de colágeno o de DNA, como consecuencia de la ingesta de fluoruro, nuestros resultados muestran que el contenido de OH-Pro se halla aumentado significativamente luego de 2 meses de tratamiento. Los datos presentados sugieren que las alteraciones óseas inducidas por el fluoruro, podrían se, al menos en parte, debidas a cambios en la concentración y distribución de los GAG y el colágeno en la matriz calcificable de hueso y cartílago de rata

Alteraciones en la matriz organica del hueso y cartilago de rata por el uso prolongado de fluoruno de sodio / Alterations in the organic matrix of bone and cartilage after prolonged administration of sodium fluoride in the rat

Los fluoruros, por mecanismos aun no aclarados, estimulan la formación ósea y son, en consecuencia, usados en el tratamiento de las osteoporosis. Desde un punto de vista terapéutico, uno de los efectos más esperados por la ingesta de fluoruro en pacientes osteoporóticos es la disminución en el índice de fracturas. Si bien esto ocurre, observaciones clínicas sugieren que este efecto es menor que el esperado por el aumento de la masa ósea (ej.: la resistencia por unidad de tejido estaría disminuida en el hueso fluorótico). El o los mecanismos por los cuales el fluoruro conduce a las alteraciones mencionadas, todavía no son bien conocidos, pero como el componente inorgánico ha sido extensamente estudiado, hemos llevado a cabo este trabajo con el objeto de caracterizar cuali y cuantitativamente a los GAG y el colágeno de hueso y cartílafo de rata, en función de la ingesta prolongada de fluoruro de sodio. Las variaciones producidas por la ingesta de fluoruro implican un aumento significativo en la concentracicón de GAG, después de dos meses de tratamiento, debidas a un incremento en las fracciones correspondentes al condroitín-6-sulfato y dermatán sulfato. Esta modificación en el patrón de distribución de los GAG no es atribuible a variaciones en el peso de las moléculas. Aunque otros estudios han informado que no se observan efectos sobre la síntesis de colágeno o de DNA, como consecuencia de la ingesta de fluoruro, nuestros resultados muestran que el contenido de OH-Pro se halla aumentado significativamente luego de 2 meses de tratamiento. Los datos presentados sugieren que las alteraciones óseas inducidas por el fluoruro, podrían se, al menos en parte, debidas a cambios en la concentración y distribución de los GAG y el colágeno en la matriz calcificable de hueso y cartílago de rata (AU)

Does secretin influence rat colonic absorption and secretion?

In rats, a secretin (Jorpes) intravenous infusion superimposed on an intracolonic sodium acetate perfusion elicits, with respect to control values, a significant depression of Na+ absorption (0.16 mEq./h-0.00 mEq./h.) and mucus secretion (230-40 mg.). When the hormone is superimposed upon an intracolonic infusion of acetic acid, mucus secretion is also significantly inhibited (790-340 mg.). The influence of secretin on organic anion movement was pH related. At pH values of 7.0, absorption was unchanged (0.34--0.33 mEq./h.), at pH values of 2.9, absorption was significantly reduced (0.67-0.41 mEq./h). The secretin impairment of colonic mucus secretion could influence the transport of watersoluble (Na+) and lipid soluble (acetic acid) substances, probably through changes at the "unstirred layer" level.