Inbreeding and the incidence of childhood genetic disorders in Karnataka, South India.

Consanguineous marriages are strongly favoured among the populations of South India. In a study conducted on 407 infants and children, a total of 35 genetic diseases was diagnosed in 63 persons: 44 with single gene defects, 12 with polygenic disorders, and seven with Down's syndrome. The coefficient of inbreeding of the total study group, F = 0.0414, was significantly higher than that previously calculated for the general population, F = 0.0271, and autosomal recessive disorders formed the largest single disease category diagnosed. The results suggest that long term inbreeding may not have resulted in appreciable elimination of recessive lethals and sub-lethals from the gene pool.     

Analysis of genomic downsizing on the basis of region-of-difference polymorphism profiling of Mycobacterium tuberculosis patient isolates reveals geographic partitioning

Mycobacterium tuberculosis, the etiological agent of tuberculosis, has lost many coding and noncoding regions in its genome during the course of evolution. We performed region-of-difference (RD) analysis using PCR-based genotyping of 131 M. tuberculosis clinical isolates obtained from four different countries, namely, India, Peru, Libya, and Angola. Our studies revealed that RD patterns are often distinct for strains circulating in specific geographical regions and can be used to trace the descent and spread of an isolate from its original reservoir. We describe our findings, which show that no single isolate from the four countries (n = 131) had all the 15 RDs either deleted or retained. Tuberculosis-specific deletion 1 (TbD1) was found to be conserved in 23% of the Indian isolates, indicating their possible ancient origin. RD9 was the most conserved region, RD11 was predominantly deleted, and RD6 was the most variable among the isolates in our collection irrespective of their geographic region. In contrast to earlier reports, our results demonstrate that the deletion of RD1 does not correlate with a decrease in the virulence potential of M. tuberculosis, as Indian isolates (n = 30) examined by us were from diseased individuals and yet had lost the RD1 region. Our results further illustrated that the intactness of the RD5 region may be associated with increased virulence of the organism. This study highlights that the RDs in M. tuberculosis genomes are geographically distributed and specific and may possibly be associated with virulence spectrum.     

Inherited C8 beta subunit deficiency in a patient with recurrent meningococcal infections: in vivo functional kinetic analysis of C8

A 16 year old with recurrent meningococcal infections is reported. Absence of haemolytic activity in both the classical and alternative pathways resulted from an absence of functional C8. Addition of functional C8 restored hemolytic activity. Antigenically deficient C8 was present in the serum and isoelectric focusing of serum confirmed the absence of the C8 beta chain. Following the infusion of fresh frozen plasma, we followed the decay in C8 functional activity as well as total haemolytic activity. C8 activity peaked at about 3 h with a half-life survival estimated to be 28 h. The kinetics of total haemolytic activity showed a slower decay with an exponential decline over 72 h and a half-life of 55 h. Fresh frozen plasma may be of value in the treatment of patients with C8 deficiency and acute Neisserial infections.     

Lack of chemopreventive efficacy of DL-selenomethionine in colon carcinogenesis

Epidemiologic observations and laboratory research have suggested that dietary selenium reduces the risk of colon cancer. Selenium-enriched brewer's yeast as a dietary supplement reduces the incidence of and mortality from cancer of the colon in humans. It is not clear whether the observed inhibitory effect is due to selenomethionine, or to other forms of selenium, or to a mixture of the selenium compounds present in selenium-enriched brewer's yeast. Therefore, bioassay described in this study examined the chemopreventive efficacy of 10 and 15 ppm selenomethionine, equivalent to 3.6 and 5.4 ppm as selenium, against azoxymethane (AOM)-induced colon carcinogenesis. At five weeks of age, groups of male F344 rats were fed diets containing 0 (control diet), 10 or 15 ppm selenomethionine. At seven and eight weeks of age, all rats except those in vehicle-treated groups received s.c. injections of AOM at a dose rate of 15 mg/kg body wt. The rats were maintained on their respective diets for 52 weeks and were then sacrificed. Colon tumors were processed and evaluated histopathologically. Colon tumor incidence and multiplicity were analyzed statistically. No obvious toxic effects were observed following dietary administration of 10 or 15 ppm selenomethionine as indicated by body weight gain. Administration of 10 or 15 ppm selenomethionine had no significant effect on colon tumor incidence and multiplicity. This study suggests that i) selenomethionine lacks chemopreventive efficacy against AOM-induced colon carcinogenesis and ii) other forms of selenium or a mixture of selenium compounds present in selenium-enriched brewer's yeast need to be evaluated for their chemopreventive efficacy.     

Display of a PorA peptide from Neisseria meningitidis on the bacteriophage T4 capsid surface.

The exterior of bacteriophage T4 capsid is coated with two outer capsid proteins, Hoc (highly antigenic outer capsid protein; molecular mass, 40 kDa) and Soc (small outer capsid protein; molecular mass, 9 kDa), at symmetrical positions on the icosahedron (160 copies of Hoc and 960 copies of Soc per capsid particle). Both these proteins are nonessential for phage infectivity and viability and assemble onto the capsid surface after completion of capsid assembly. We developed a phage display system which allowed in-frame fusions of foreign DNA at a unique cloning site in the 5' end of hoc or soc. A DNA fragment corresponding to the 36-amino-acid PorA peptide from Neisseria meningitidis was cloned into the display vectors to generate fusions at the N terminus of Hoc or Soc. The PorA-Hoc and PorA-Soc fusion proteins retained the ability to bind to the capsid surface, and the bound peptide was displayed in an accessible form as shown by its reactivity with specific monoclonal antibodies in an enzyme-linked immunosorbent assay. By employing T4 genetic strategies, we show that more than one subtype-specific PorA peptide can be displayed on the capsid surface and that the peptide can also be displayed on a DNA-free empty capsid. Both the PorA-Hoc and PorA-Soc recombinant phages are highly immunogenic in mice and elicit strong antipeptide antibody titers even with a weak adjuvant such as Alhydrogel or no adjuvant at all. The data suggest that the phage T4 hoc-soc system is an attractive system for display of peptides on an icosahedral capsid surface and may emerge as a powerful system for construction of the next generation multicomponent vaccines.     

Serotonergic inhibition of action potential evoked calcium transients in NOS-containing mesopontine cholinergic neurons

Nitric oxide synthase (NOS)-containing mesopontine cholinergic (MPCh) neurons of the laterodorsal tegmental nucleus (LDT) are hypothesized to drive the behavioral states of waking and REM sleep through a tonic increase in firing rate which begins before and is maintained throughout these states. In principle, increased firing could elevate intracellular calcium levels and regulate numerous cellular processes including excitability, gene expression, and the activity of neuronal NOS in a state-dependent manner. We investigated whether repetitive firing, evoked by current injection and N-methyl-D-aspartate (NMDA) receptor activation, produces somatic and proximal dendritic [Ca(2+)](i) transients and whether these transients are modulated by serotonin, a transmitter thought to play a critical role in regulating the state-dependent firing of MPCh neurons. [Ca(2+)](i) was monitored optically from neurons filled with Ca(2+) indicators in guinea pig brain slices while measuring membrane potential with sharp microelectrodes or patch pipettes. Neither hyperpolarizing current steps nor subthreshold depolarizing steps altered [Ca(2+)](i). In contrast, suprathreshold currents caused large and rapid increases in [Ca(2+)](i) that were related to firing rate. TTX (1 microM) strongly attenuated this relation. Addition of tetraethylammonium (TEA, 20 mM), which resulted in Ca(2+) spiking on depolarization, restored the change in [Ca(2+)](i) to pre-TTX levels. Suprathreshold doses of NMDA also produced increases in [Ca(2+)](i) that were reduced by up to 60% by TTX. Application of 5-HT, which hyperpolarized LDT neurons without detectable changes in [Ca(2+)](i), suppressed both current- and NMDA-evoked increases in [Ca(2+)](i) by reducing the number of evoked spikes and by inhibiting spike-evoked Ca(2+) transients by approximately 40% in the soma and proximal dendrites. This inhibition was accompanied by a subtle increase in the spike repolarization rate and a decrease in spike width, as expected for inhibition of high-threshold Ca(2+) currents in these neurons. NADPH-diaphorase histochemistry confirmed that recorded cells were NOS-containing. These findings indicate the prime role of action potentials in elevating [Ca(2+)](i) in NOS-containing MPCh neurons. Moreover, they demonstrate that serotonin can inhibit somatic and proximal dendritic [Ca(2+)](i) increases both indirectly by reducing firing rate and directly by decreasing the spike-evoked transients. Functionally, these data suggest that spike-evoked Ca(2+) signals in MPCh neurons should be largest during REM sleep when serotonin inputs are expected to be lowest even if equivalent firing rates are reached during waking. Such Ca(2+) signals may function to trigger Ca(2+)-dependent processes including cfos expression and nitric oxide production in a REM-specific manner.     

Immune reactions in human filariasis.

Sera from cases of elephantiasis due to Wuchereria bancrofti infection promoted an intense adhesion of peripheral blood leukocytes to W. bancrofti microfilariae in vitro. A similar adhesion was also seen using sera from some normal persons living for several years in areas where filariasis is endemic. No such adhesion was evident with sera from microfilaria carriers or from normal subjects from nonendemic areas. The adhesion was complement independent and was associated with the immunoglobulin G fraction of serum. 51Cr release studies suggested the occurrence of cell-mediated cytotoxicity to W. bancrofti microfilariae in the presence of elephantiasis serum. Microfilariae of Litomosoides carinii could be isolated free of blood cells, from the blood of infected rats. In the presence of serum, or its immunoglobulin G fraction, from patients with elephantiasis, L. carinii microfilariae adhered to human peripheral blood leukocytes or rat spleen cells.