Biological characterization of Drosophila Rapgap1, a GTPase activating protein for Rap1

The activity of Ras family proteins is modulated in vivo by the function of GTPase activating proteins, which increase their intrinsic rate of GTP hydrolysis. We have isolated cDNAs encoding a GAP for the Drosophila Rap1 GTPase. Drosophila Rapgap1 encodes an 850-amino acid protein with a central region that displays substantial sequence similarity to human RapGAP. This domain, when expressed in Escherichia coli, potently stimulates Rap1 GTPase activity in vitro. Unlike Rap1, which is ubiquitously expressed, Rapgap1 expression is highly restricted. Rapgap1 is expressed at high levels in the developing photoreceptor cells and in the optic lobe. Rapgap1 mRNA is also localized in the pole plasm in an oskar-dependent manner. Although mutations that completely abolish Rapgap1 function display no obvious phenotypic abnormalities, overexpression of Rapgap1 induces a rough eye phenotype that is exacerbated by reducing Rap1 gene dosage. Thus, Rapgap1 can function as a negative regulator of Rap1-mediated signaling in vivo.     

The early recognition of dementia in the elderly: A preliminary communication about a longitudinal study using the gms–agecat package (community version)

A random community sample of 1070 subjects aged over 65 was interviewed by trained non-medical interviewers using the Geriatric Mental State, community version (GMSA). A sub-sample of 126 subjects was selected so as to contain possible early cases of dementia, pseudo-dementia, and normal subjects; and re-interviewed, a mean 1 year and 23 weeks later, by a group of psychiatrists in training. The computer diagnosis AGECAT, based on GMSA applied by non-medical raters, had predicted at initial interview, nine out of twelve cases of dementia at follow-up and five out of nine borderline cases. An Organic/Depression Index may prove useful in predicting which of those cases with early organic levels will eventually develop dementia, depression or recover.     

Mediastinal teratoma in a neonate

A neonate with severe respiratory distress due to a benign mediastinal teratoma (MT) is reported. Despite early and easy surgical excision of the tumor, the child died due to poor cardiac function. Only ten cases of MT in neonates have been reported in the literature so far. While the tumor has been known to interfere with lung development in utero, postnatal myocardial dysfunction due to poor heart development has not been previously documented. Accepted: 22 September 1997     

Comparative cytological study of lymph node tuberculosis in HIV-infected individuals and in patients with diabetes in a developing country

Tuberculosis (TB) is a common infection affecting patients with human immunodeficiency virus (HIV) and diabetes mellitus (DM). With the increasing incidence of HIV infection and DM in a developing country like India, TB is definitely on the rise. In a given population, one expects to see these three diseases in varying combinations, such as HIV and TB, DM and TB, HIV and DM with TB. In such combinations TB may lack the characteristic clinical and histological picture due to the associated depressed cell-mediated immunity seen in both diseases and TB may have an unusual clinical presentation and cytology picture. In this retrospective study of 36 months, from January 1997 to December 1999, 109 cases diagnosed cytologically as tuberculous lymphadenitis and tested for HIV infection and investigated as well for DM were selected. Forty-six (42%) were nondiabetic HIV patients, 13 (12%) were non-HIV DM patients, and 50 (46%) had TB without HIV infection or DM. The coexistence of both HIV and DM was not noted. The cytomorphological characteristics supplemented by culture studies of each of these three groups were compared in detail and based on these four cytological patterns, Pattern 1, Pattern 2, Pattern 3, and Pattern 4 emerged and were characterized. This study highlights the usefulness of cytomorphology of the lymph nodes to characterize the cytopathological profile of TB in both HIV and DM, which have many clinical and immunological similarities, and indirectly postulate the extent of immune suppression and evolve effective strategies in the management of coexisting diseases. Such a comparative study has not been carried out in the past.     

Novel porous aortic elastin and collagen scaffolds for tissue engineering

Decellularized vascular matrices are used as scaffolds in cardiovascular tissue engineering because they retain their natural biological composition and three-dimensional (3-D) architecture suitable for cell adhesion and proliferation. However, cell infiltration and subsequent repopulation of these scaffolds was shown to be unsatisfactory due to their dense collagen and elastic fiber networks. In an attempt to create more porous structures for cell repopulation, we selectively removed matrix components from decellularized porcine aorta to obtain two types of scaffolds, namely elastin and collagen scaffolds. Histology and scanning electron microscopy examination of the two scaffolds revealed a well-oriented porous decellularized structure that maintained natural architecture of the aorta. Quantitative DNA analysis confirmed that both scaffolds were completely decellularized. Stress-strain analysis demonstrated adequate mechanical properties for both elastin and collagen scaffolds. In vitro enzyme digestion of the scaffolds suggested that they were highly biodegradable. Furthermore, the biodegradability of collagen scaffolds could be controlled by crosslinking with carbodiimides. Cell culture studies showed that fibroblasts adhered to and proliferated on the scaffold surfaces with excellent cell viability. Fibroblasts infiltrated about 120 microm into elastin scaffolds and about 40 microm into collagen scaffolds after 4 weeks of rotary cell culture. These results indicated that our novel aortic elastin and collagen matrices have the potential to serve as scaffolds for cardiovascular tissue engineering.     

Specificity and mechanism of the histone methyltransferase Pr-Set7

Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. We present here the crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. We provide evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor and develop a model for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. We also show how a highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and probably also how this part of the structure ensures that Pr-Set7 is nucleosome specific.     

A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin

Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. Here we show that H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, we identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, the Suv4-20 homolog is a novel PEV modifier to regulate position-effect variegation. Together, our data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin.     

Immune reconstitution inflammatory syndrome in association with HIV/AIDS and tuberculosis: Views over hidden possibilities

Background  

CD4+ T lymphocyte (CD4) cell count testing is the standard method for determining eligibility for antiretroviral therapy (ART), but is not widely available in sub-Saharan Africa. Total lymphocyte counts (TLCs) have not proven sufficiently accurate in identifying subjects with low CD4 counts. We developed clinical algorithms using TLCs, hemoglobin (Hb), and body mass index (BMI) to identify patients who require ART.