Microtubules are required for cytokeratin aggresome (Mallory body) formation in hepatocytes: an in vitro study

Mallory bodies are cytokeratin-ubiquitin aggresomes that form in hepatocytes in many different chronic liver diseases. One of the key components in aggresome formation, not yet investigated in Mallory body formation, is the role of microtubules. An in vitro tissue culture assay is required to test for microtubule involvement in Mallory body formation so that Mallory body formation can be observed in the presence or absence of microtubule-disrupting agents. In this report, a new model of in vitro Mallory body formation was developed, which uses cultured hepatocytes isolated from drug-primed mice. When hepatocytes were incubated in the presence of antimicrotubule agents, they failed to form Mallory bodies. It is concluded that intact microtubules are required for Mallory body formation.     

Reduced virulence of HWP1-deficient mutants of Candida albicans and their interactions with host cells

The Candida albicans gene HWP1 encodes a surface protein that is required for normal hyphal development in vitro. We used mutants lacking one or both alleles of HWP1 to investigate the role of this gene in virulence. Mice infected intravenously with the homozygous hwp1 null mutant, CAL3, survived a median of >14 days, whereas mice infected with a control strain containing two functional alleles of HWP1 survived only 3.5 days. After 1 day of infection, all strains produced similar levels of infection in the kidneys, spleen, and blood. However, after 2 and 3 days, there was a significant decrease in the number of organisms in the kidneys of the mice infected with CAL3. This finding suggests that the hwp1 homozygous null mutant is normal in its ability to initiate infection but deficient in its capacity to maintain infection. CAL3 also germinated minimally in the kidneys. The ability of the heterozygous null mutant to germinate and cause mortality in mice was intermediate to CAL3, suggesting a gene dosage effect. To investigate potential mechanisms for the diminished virulence of CAL3, we examined its interactions with endothelial cells and neutrophils in vitro. CAL3 caused less endothelial cell injury than the heterozygous hwp1 mutant. We conclude that the HWP1 gene product is important for both in vivo hyphal development and pathogenicity of C. albicans. Also, the ability to form filaments may be critical for candidal virulence by enabling the fungus to induce cellular injury and maintain a deep-seated infection.     

Partial androgen insensitivity with phenotypic variation caused by androgen receptor mutations that disrupt activation function 2 and the NH(2)- and carboxyl-terminal interaction

Partial androgen insensitivity with sex phenotype variation in two unrelated families was associated with missense mutations in the androgen receptor (AR) gene that disrupted the AR NH(2)-terminal/carboxy terminal interaction. Each mutation caused a single amino acid change within the region of the ligand-binding domain that forms activation function 2 (AF2). In one family, the mutation I737T was in alpha helix 4 and in the other F725L was between helices 3 and 4. Neither mutation altered androgen binding as determined by assays of mutant AR in the patient's cultured genital skin fibroblasts or of recombinant mutant receptors transfected into COS cells. In transient cotransfection assays in CV1 cells, transactivation with the AR mutants at low concentrations of DHT was reduced several fold compared with wild-type AR but increased at higher concentrations. Defects in NH(2)-terminal/carboxy terminal interactions were identified in mammalian two hybrid assays. In similar assays, there was reduced binding of the p160 coactivators TIF2/SRC2 and SRC1 to the mutant AR ligand binding domains (LBD). In the family with AR I737T, sex phenotype varied from severely defective masculinization in the proband to a maternal great uncle whose only manifestation of AIS was severe gynecomastia. He was fertile and passed the mutation to two daughters. The proband of the F725L family was also incompletely masculinized but was raised as a male while his half-sibling by a different father was affected more severely and reared as a female. These studies indicate that the function of an AR AF2 mutant in male development can vary greatly depending on the genetic background.     

The effects of intrahippocampal ibotenic acid and their blockade by (−) 2-amino-7-phosphonoheptanoic acid: Morphological and electroencephalographical analysis

Intrahippocampal injections of the excitotoxin ibotenic acid caused a biphasic behavioral pattern in rats. Both the immediate induction of hyperactivity and stereotypical behaviors and a secondary phase of progressively decreasing behavioral activities beginning 15-20 min after ibotenate administration, were accompanied by characteristic seizures measured by bilateral recordings in hippocampus and cortex. Typically, EEG changes consisted of high voltage spiking in all leads. The phase of behavioral depression was accompanied by episodic, short (5-30 s) seizures. In addition, cortical slow waves very similar to those elicited by intrahippocampal muscimol were observed during this period. Attempts were made to correlate occurrence and severity of seizures with the extent of neuronal degeneration. While a trend in favor of such a correlation existed, a firm causal relationship could not be established; some animals could display seizure activity with only marginal concomitant nerve cell loss and vice versa. Intrahippocampal co- and post-administration of ibotenate with (-) 2-amino-7-phosphonoheptanoic acid entirely prevented or stopped the occurrence of both EEG changes and neuronal degeneration. Ibotenate-induced seizures and lesions and their specific blockade by a selective antagonist may constitute valuable experimental paradigms for the investigation of seizure disorders.     

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.      

Intensity of aggressive interactions modulates testosterone in male marmosets

Androgen is associated with the expression of male-typical behavior, including aggressive behavior, but high levels of androgen may be incompatible with other behavioral systems, such as paternal care. In a variety of species of birds that display paternal care, testosterone (T) levels in males are maintained at low levels, and these levels rise only in response to direct agonistic challenges. This idea has not been thoroughly studied in mammals with biparental care, and we exposed male marmosets (Callithrix kuhlii), a monogamous and biparental primate to aggressive interactions with unfamiliar intruders. Urinary levels of T and cortisol (CORT) were monitored prior to and following these interactions. Baseline T was not correlated with variation in aggression in either residents or intruders, and CORT was not affected by the encounters. However, males responded to an encounter with male intruders with changes in T that correlated with the level of aggression displayed by the resident male during the trial. Encounters with male intruders that elicited high frequencies of aggressive displays by the male resident were associated with increased T 2-6 h and 24 h following the encounter, and encounters that had few aggressive displays resulted in no change or a decrease in T concentrations. Intruders did not demonstrate a significant relationship between T and aggression. Thus, the magnitude of the hormonal response is dependent on the intensity of aggression during a male-male encounter, suggesting that elevated androgens are likely to be a consequence, rather than a cause, of aggressive interactions in marmosets.     

Low-dose citrate continuous veno-venous hemofiltration (CVVH) and acid-base balance

OBJECTIVE: To evaluate the acid-base effect of low-dose regional citrate anticoagulation (RCA) during continuous veno-venous hemofiltration (CVVH). DESIGN: Prospective observational study. SETTING: ICUs of tertiary public and private hospitals. SUBJECTS: Thirty critically ill patients with acute renal failure at risk of bleeding or with a major contraindication to heparin-CVVH and/or short filter life. METHODS: We used a commercial citrate-based fluid (11 mmol/L, sodium: 140 mmol/L, chloride: 108 mmol/L and 1 mol/L of potassium) as pre-dilution replacement fluid during CVVH. Further potassium was added according to serum potassium levels. We measured all relevant variables for acid-base analysis according to the Stewart-Figge methodology. RESULTS: Before treatment, study patients had a slight metabolic acidosis, which worsened over 6 hours of RCA-CVVH (pH from 7.39 to 7.38, p < 0.005; bicarbonate from 23.2 to 21.6 mmol/L, p < 0.0001 and base excess from -2.0 to -3.0 mEq/L, p < 0.0001) due to a significant increase in SIG (from 5.8 to 6.6 mEq/L, p < 0.05) and a decrease in SIDa (from 37.5 to 36.6 mEq/L, p < 0.05). These acidifying effects were attenuated by hypoalbuminemia and a decrease in lactate (from 1.48 to 1.34 mmol/L, p < 0.005) and did not lead to progressive acidosis. On cessation of treatment, this acidifying effect rapidly self-corrected within six hours. CONCLUSIONS: Low dose RCA-CVVH induces a mild acidosis secondary to an increased strong ion gap and decreased SIDa which fully self-corrects at cessation of therapy. Clinicians need to be aware of these effects to correctly interpret changes in acid-base status in such patients.