From sequence to chromosome: the tip of the X chromosome of D. melanogaster.

One of the rewards of having a Drosophila melanogaster whole-genome sequence will be the potential to understand the molecular bases for structural features of chromosomes that have been a long-standing puzzle. Analysis of 2.6 megabases of sequence from the tip of the X chromosome of Drosophila identifies 273 genes. Cloned DNAs from the characteristic bulbous structure at the tip of the X chromosome in the region of the broad complex display an unusual pattern of in situ hybridization. Sequence analysis revealed that this region comprises 154 kilobases of DNA flanked by 1.2-kilobases of inverted repeats, each composed of a 350-base pair satellite related element. Thus, some aspects of chromosome structure appear to be revealed directly within the DNA sequence itself.

Embryonic expression and characterization of a Ptx1 homolog in Drosophila.

We describe the molecular characterization of the paired-type homeobox gene D-Ptx1 of Drosophila, a close homolog of the mouse pituitary homeobox gene Ptx1 and the unc-30 gene of C. elegans, characterized by a lysine residue at position 9 of the third alpha-helix of the homeodomain. D-Ptx1 is expressed at various restricted locations throughout embryogenesis. Initial expression of D-Ptx1 in the posterior-most region of the blastoderm embryo is controlled by fork head activity in response to the activated Ras/Raf signaling pathway. During later stages of embryonic development. D-Ptx1 transcripts and protein accumulate in the posterior portion of the midgut, in the developing Malpighian tubules, in a subset of ventral somatic muscles, and in neural cells. Phenotypic analysis of gain-of-function and lack-of-function mutant embryos show that the D-Ptx1 gene is not involved in morphologically apparent differentiation processes. We conclude that D-Ptx1 is more likely to control physiological cell functions than pattern formation during Drosophila embryogenesis.

A morphometric analysis of isolated Torpedo electric organ synaptic vesicles following stimulation.

The electric organ of Torpedo has been stimulated with 1800 pulses at 0.1 Hz to produce biochemical and morphological heterogeneity of its synaptic vesicle population. This was verified by biochemical and morphometric analyses of the synaptic vesicle population isolated by sucrose density gradient zonal separation following stimulation. Biochemical or metabolic heterogeneity was verified using 2 established criteria: the appearance of a second peak of acetylcholine (ACh) in denser fractions of the zonal gradient and a corresponding overlapping peak of incorporated radiolabelled ACh. Morphologic heterogeneity was deduced by the presence in this second peak of a subclass of synaptic vesicles having a mean diameter of 68 nm i.e., a diameter 20-25% smaller than the 90 nm subclass that represents the most prominent subclass of the intact terminal population. Despite having satisfied these 3 criteria, functionally relevant heterogeneity cannot be assumed. One reason is due to our failure to recover the 90 nm subclass of vesicle which provides the physical basis to explain the 2 ACh peaks along the gradient. Because of this, the point is raised whether the stimulation-induced ACh peak is not merely an artifact due to inadequate sampling. On the other hand, radioactive labelling of the ACh pool provides a more convincing demonstration of the existence of 2 metabolically different subclasses. We conclude that morphological heterogeneity of the ACh vesicle population has never been established and that metabolic heterogeneity, as it has been studied to date, pertains to a single-sized subclass population of vesicles measuring 68 nm in diameter.

A morphometric analysis of Torpedo synaptic vesicles isolated by iso-osmotic sucrose gradient separation.

The presynaptic terminal vesicle population of Torpedo electric organ is heterogeneous in size, consisting of two prominent subpopulations that comprise 80% of the total. The use of standard iso-osmotic sucrose gradients with zonal centrifugation to isolate vesicle fractions that co-localize with the acetylcholine (ACh) peak results in the recovery of: (1) 10% of the total estimated vesicle population; and (2) a single 68-nm diameter vesicle size class. The whereabouts of the major 90-nm subclass, which accounts for 60% of the total terminal population and which has long been considered to represent the resident ACh population, has been investigated. Assuming this subclass to have undergone severe osmotic stress, the effects of hypo- and hyper-osmotic salines, buffers and fixatives were examined and found to produce only negligible changes on vesicle size. Isolation of vesicles by hypo-osmotic shocking of synaptosomes purified on a Ficoll gradient, however, resulted in a reasonable approximation of the in situ distribution. As the iso-osmotic sucrose gradient procedure utilizes frozen blocks of electric tissue, this step is suspected of being involved in the loss, perhaps because of the slow freezing rates employed. These findings indicate that the 90 nm subclass is lost rather than transformed during isolation by sucrose gradient separation and that dimensionally, the cholinergic vesicle is a constant-sized and relatively stable structure.

The lipid and protein content of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata purified to constant composition: implications for vesicle structure.

The lipid, protein, acetylcholine and ATP content of cholinergic synaptic vesicles isolated from the richly innervated electric organ of Torpedo marmorata and purified to constant composition has been determined. The number of vesicles present in the preparations has been estimated by quantitative electron microscopy and the mean composition of the vesicle deduced. The acetylcholine content of the purest preparations was considerably greater than that previously attained and reached a mean of 6.10 mmole/g of protein and 2.6 X 10(5) molecules/vesicle; the mean values, for all determinations, were 4.1 +/- S.E.M. 0.6 and 2.6 X 10(5) +/- S.E.M. 0.6 X 10(5) respectively. The lipid and protein content of the vesicle (about 140 and 80 ag/vesicle respectively) is relatively low, indicating a thin, lipid-rich membrane and a highly hydrated core of which not more than 1-2% can be occupied by protein. These findings are consistent with conclusions drawn from recent density determinations made at different osmotic pressures using penetrating and non-penetrating gradients.

Laboratory investigations on rotavirus in infantile gastroenteritis in Jamaica.

Between March 1982 and March 1983 rotaviruses were detected in faecal specimens from 193 (19%) of 1020 Jamaican children under 5 years old with acute gastroenteritis. The excretion of rotaviruses in a control group of non-diarrhoeal children was only 1.4%. Rotaviruses were detected more frequently in the cooler months of the year; the prevalence ranged from 1.7% in August to 46% in January. In a comparison of the ELISA technique and electron microscopy for investigating rotavirus infections, the ELISA was demonstrated to be more sensitive especially for weakly positive cases. Because of its relative simplicity, low cost and rapidity the ELISA appears to be more suitable for use in developing countries.

Symptomatic and asymptomatic chlamydial non-gonococcal urethritis in Jamaica: the potential for HIV transmission.

To determine the contribution of Chlamydia trachomatis to non-gonococcal urethritis (NGU) in men attending sexually transmitted disease (STD) clinics in Jamaica we studied men with NGU (n=339), and control groups including asymptomatic men who were STD contacts (n=61), asymptomatic men who were not STD contacts (n=32) and men with gonococcal urethritis (GU) (n=61). Urethral specimens were examined for C. trachomatis and Neisseria gonorrhoeae. Serological tests for syphilis (STS) and HIV-1 infection were also performed. C. trachomatis accounted for 63% of cases of NGU but high prevalences were also found in asymptomatic STD contacts (59%), asymptomatic STD non-contacts (78%) and men with GU (48%). The prevalence of C. trachomatis in men with GU differed significantly from that in men with NGU and asymptomatic STD non-contacts (P<0.05). C. trachomatis infection in men with NGU was associated with multiple sex partners (71% vs 58%; chi2=4.78; odds ratio (OR)=1.76; P<0.05) and previous history of gonococcal infection (83% vs 42%; chi2=59.8; OR=6.8; P<0.0001). Concomitant infection with HIV-1 occurred in 5.2% of cases of NGU and 50% and 90%, respectively, of the HIV-positive men had chlamydia or reactive STS. As a cost effective strategy in the control of STD and HIV we recommend presumptive treatment for C. trachomatis in men seeking STD treatment in Jamaica.

Antibodies to hepatitis B virus and hepatitis C virus in residential detoxification clients in Jamaica.

The role of non-injecting drug abuse in viral hepatitis has not been studied widely and is not well understood. A total of 301 substance abusers, residents of a detoxification/rehabilitation unit, were investigated for exposure to hepatitis B virus (HBV) and hepatitis C virus (HCV). Samples of serum were tested for anti-HCV and anti-HBc antibodies and HBsAg. All of the patients were non-injecting drug users (non-IDUs). The prevalence of anti-HCV was 1.7%; anti-HBc was found in 28.7% and HbsAg in 0.6% of patients. Anti-HCV positivity correlated with the presence of elevated aminotransferases (80%). Exposure to HBV correlated significantly with gender (p < 0.05); age (p < 0.05); and duration of substance abuse (p < 0.05). No significant correlations were found between HCV and/or HBV infection, the drug of abuse, HIV, HTLV-1 or syphilitic infection. Residential detoxification/rehabilitation provides an opportune moment to identify and treat HCV positive substance abusers in the attempt to avert the severe hepatic sequelae. Measures which exclude substance abusers from volunteer blood donation should be considered.

Chlamydial infections in selected populations in Jamaica.

The importance of Chlamydia trachomatis as a cause of genital tract infection in Caribbean countries is largely unknown. Two hundred and sixteen cervical and urethral specimens were collected from patients with cervicitis and urethritis attending a Sexually Transmitted Diseases (STD) Clinic, a Gynaecology Clinic and a Family Planning Clinic in Kingston. Specimens were tested for chlamydiae by direct immunofluorescence and cell culture methods. They were also investigated for N. gonorrhoeae infections. Seventy-two of 138 patients (52.2%) at the STD Clinic were positive for chlamydiae; 28 of 50 (56.0%) from the Gynaecology Clinic and 11 of 28 (39.3%) from the Family Planning Clinic were positive. The prevalence of C. trachomatis (52.3%) was higher than that of N. gonorrhoeae (11.1%) in all groups. The results indicate that C. trachomatis is a common cause of genital tract infections in Jamaica.

Risky behaviour in Jamaican adolescent patients attending a sexually transmitted disease clinic.

The prevalence of sexually transmitted diseases (STD) and risky behaviour patterns were studied in 165 adolescents attending a STD clinic in Jamaica. A standardised structured questionnaire, clinical algorithms for STD and serological tests for HIV and syphilitic infection were applied. High prevalences of risky behaviour including young age at onset of sexual activity, especially in boys, (mean age 12.5 +/- 2.5 years); unprotected sexual intercourse (only 4% used condoms consistently); multiple sexual partners (mean 3.8 +/- 2.4 and 1.8 +/- 1.2 for boys and girls, respectively) were found. Marijuana, used by 60% of the boys, was an independent risk factor for dysuria (adjusted Odds Ratio (OR), 2.0; 95% CI, 1.6-3.4). Repeated episodes of STD (33%), coinfection with HIV (1.2%), syphilis (1.2%) and teenage pregnancy (13%) were prominent findings. Educational strategies which promote behaviour intervention at an early age, frequent and consistent use of condoms, abstinence or delayed onset of sexual activity are essential to reducing the HIV/AIDS and STD risk in adolescents in Jamaica.

Antibiotic sensitivity of Helicobacter pylori in Jamaica.

Helicobacter pylori infection is one of the commonest chronic infections worldwide. Eradication regimes usually contain two antibiotics, however resistance is increasing and this decreases treatment success. This study reports on the sensitivity and resistance of H pylori to several antibiotics in patients undergoing upper gastrointestinal endoscopy in Jamaica. The rapid urease test (CLO) was positive in 128 (38%) of 336 patients. Fifty patients (39%; 50/128) with positive CLO tests had positive cultures for H pylori. Two-thirds (32/48) of islolates were sensitive to metronidazole and one-third (16/48) were resistant. Ninety-seven per cent of isolates (31/32) were sensitive to erythromycin. The sensitivity for clarithromycin was 92% (11/12) with one isolate (8%) resistant. All strains of H pylori (48/48) were sensitive to ampicillin and amoxicillin - clavulanate. Metronidazole resistance is present in one-third of H pylori isolates and resistance to macrolides is relatively low in Jamaican patients. It is important to monitor antibiotic resistance in order to provide clinicians with data on the most appropriate and cost effective eradication regimes for H pylori.

Prevalence of viral and bacterial sexually transmitted pathogens in Jamaican pregnant women.

In this study we investigated the prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, human immunodeficiency virus type I (HIV-I), human T cell lymphotropic virus type 1 (HTLV-1) and hepatitis B virus (HBV) infections in 200 pregnant women attending antenatal clinics at the University Hospital of the West Indies. 19% of the women had at least one pathogen: C. trachomatis was found in 16%, HTLV-1 in 2%, HIV-1, HBV and N. gonorrhoeae each in 0.5% C. trachomatis infection was more prevalent in women less than 20 years of age (31%) than in those 20 years and older (16%; OR = 0.43; chi 2 = 5.66; p < 0.05). The study demonstrates the need for identification of sexually transmitted pathogens in antenatal women for syndromic management of genital infections as part of the strategy for prevention and control of HIV/AIDS (acquired immunodeficiency syndrome) in Jamaica.

Autoantibodies, human T lymphotropic virus type 1 and type 1 diabetes mellitus in Jamaicans.

The clinical characteristics, autoantibody profiles and seroprevalence of human T lymphotropic virus Type 1 (HTLV-1) were assessed in 30 Jamaican patients with Type 1 diabetes mellitus. Two hundred and fifty-two blood donors and 108 patients with Graves' disease were included as controls for the HTLV-1 component of the study. The mean age of onset of diabetes mellitus was 20.5 +/- 9.2 years and the mean duration of diabetes mellitus was 10.5 +/- 6.1 years. The remarkable clinical data included an absence of other associated organ-specific autoimmune diseases, and clinical evidence and history of congenital rubella in one patient. Islet cell cytoplasmic antibodies (ICA) were absent but 17% (5/30) of the diabetic patients tested positive for glutamic acid decarboxylase (GAD) antibodies. No other organ-specific autoantibodies were detected but non-organ-specific autoantibodies were present in 9 (30%) of the sera of diabetic patients. The seroprevalence of HTLV-1 in the patients with diabetes mellitus was significantly higher than that in the healthy controls (17% (5/30) versus 4% (11/252), p = 0.05). Autoantibodies were found in the sera of 4/5 (80%) of the diabetic patients who were positive for HTLV-1. None of the patients with onset of diabetes mellitus below age 15 years was HTLV-1 positive. The likely polyaetiological nature of Type 1 diabetes mellitus in Jamaicans is being further investigated at the molecular level.

A serosurvey of hepatitis B virus, hepatitis C virus, human T lymphotropic virus type-1 and syphilis in HIV-1-infected patients in Jamaica.

The seroprevalences of hepatitis B virus (HBV), hepatitis C virus (HCV), human T lymphotropic virus type-1 (HTLV-1) and syphyilis were determined in 129 HIV-1-infected patients using commercially prepared reagents. The seroprevalences were HCV, 0% (0/129); HBV, 37% (48/129); HTLV-1, 5% (6/129) and syphilis, 20% (26/129). Fifteen per cent (19/129) of the patients had active/chronic HBV infection. The seroprevalence of HBV was statistically significantly higher in HIV-1 infected men (24/49, 50% versus 17/80, 21%; p = 0.005), while the seroprevalence of syphilis was statistically significantly increased in HIV-1 infected patients in the over-40 age group (10/31, 32% versus 6/53, 11%; p = 0.05). These findings throw the spotlight on HBV infection and syphilis and suggest that these two sexually transmitted infections should be carefully surveyed in patients with HIV/AIDS in Jamaica. It is essential for management protocols in Jamaica to include screening for evidence of these co-infections.

Identification of a heparan sulphate-containing proteoglycan as a specific core component of cholinergic synaptic vesicles from Torpedo marmorata.

Cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata were found to contain a proteoglycan in their core. The glycosaminoglycan part co-migrates upon thin layer electrophoresis with heparan sulphate and shows a chemical composition characteristic for this carbohydrate. [35S]Sulphate injected into the electric lobes of Torpedo, which contain the perikarya of the electromotor neurons innervating the electric organs, appeared 48 h later in covalently bound form in the synaptic vesicle fraction. The radiolabel had been incorporated into the vesicular heparan sulphate. Upon SDS-polyacrylamide gel electrophoresis fluorography of labelled vesicles a major and a minor band are formed both migrating above a protein standard of mol. wt. 200 000. Similarly, a major peak in the void volume and a minor peak in the included volume are seen upon gel filtration in Ultrogel AcA 34 in the presence of SDS. We interpret the minor fraction as being formed by the loss of glycosaminoglycan from the major fraction. The proteoglycan is located inside the vesicle since antibodies directed against it form immunoprecipitates only with vesicles lysed by detergent treatment. The experiments show that it is possible to label a synaptic organelle specifically by axonal transport.

The isolation of pure cholinergic synaptic vesicles from the electric organs of elasmobranch fish of the family Torpedinidae.

1. Zonal centrifuging permitted the separation, on the milligram scale and in a form largely free from contamination by soluble cytoplasmic protein or membrane fragments derived from other structures, of synaptic vesicles from the purely cholinergic terminals of the electric organ of Torpedo. Up to 100g of tissue could be processed in a single run. 2. As much as 46% of the bound acetylcholine from the original tissue preparation was recovered as a single peak of density equivalent to 0.38m-sucrose-0.21m-NaCl and with a concentration of up to 680nmol of acetylcholine/mg of protein. 3. The limiting concentration of acetylcholine in isolated vesicles when allowance had been made for non-vesicular protein appeared to be about 600nmol/mg of protein. 4. Vesicle counts by a ;bead-tagging' procedure indicated an acetylcholine content of about 360mumol/ml of vesicles; thus the vesicle protein content would be about 60% (w/v). 5. Calculations showed that the core of the vesicle, accounting for about 55% of the vesicle volume, could be largely filled with acetylcholine and protein.