Biomedical subjects
Publications and source records attributed to R Ray.
Stool samples from patients affected during a large epidemic of hepatitis in north India were examined for the presence of hepatitis E virus (HEV) genome by means of reverse transcription-polymerase chain reaction. 6 of 10 samples studied were positive for the HEV genome, which suggest that HEV caused this large epidemic.
In this paper, we describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitro-[3,5-3H]phenyl)amino]propyl ether (3H-25-ANE) (Ray et al., 1986, 1991). We have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D3 for the binding site of the latter in hDBP and (2) 3H-25-ANE is capable of covalently labeling the hDBP molecule when exposed to UV light. Treatment of a sample of purified hDBP, labeled with 3H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was associated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, our results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D3.
Vulnerability of 25-hydroxy-[26,27-3H]vitamin D3 3 beta-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D3 (25-OH-D3) (Ray et al., 1986) toward standard conditions of carboxymethylation promoted us to synthesize 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D3, and 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitro-[3,5-3H]phenyl)amino] propyl ether (3H-25-ANE), the radiolabeled counterpart of 25-ANE. Competitive binding assays of 25-OH-D3 and 25-ANE with rat serum demonstrated that 25-ANE competes for the 25-OH-D3 binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with 3H-25-ANE, covalently labeled the protein. However, very little covalent labeling was observed in the absence of UV light or in the presence of a large excess of 25-OH-D3. These results provide strong evidence for the covalent labeling of the 25-OH-D3 binding site in rDBP by 3H-25-ANE.
The c-myc protooncogene plays an important role in the regulation of cellular proliferation. Mithramycin, a DNA binding antibiotic which binds G-C-rich DNA, inhibits c-myc expression in both differentiating and nondifferentiating cells. The G-C-rich nature of the c-myc promoter suggests that mithramycin may act by directly inhibiting promoter function. The mithramycin binding sites in the c-myc promoter regions were determined by DNAse I footprinting. Particularly prominent mithramycin binding is noted in the regions just 5' of the P1 and P2 promoter TATA boxes. Gel retardation experiments performed in the presence of mithramycin demonstrate that drug binding can prevent the formation of discrete complexes between HeLa cell nuclear proteins and c-myc promoter DNA fragments. Mithramycin also directly blocks the binding of the transcription factor Sp1 to the P1 promoter region. In vitro run-off transcription demonstrates that mithramycin can completely inhibit the in vitro function of both the P1 and P2 promoters. These data suggest that mithramycin inhibits transcription of the c-myc protooncogene by blocking the binding of important regulatory factors, thus preventing formation of the c-myc transcription initiation complex.
Acetylcholinesterase (AChE) in the clonal NG108-15 cell line has been previously characterized. This cell line represents an in vitro system to study AChE regulation and effects of chemical compounds that may alter AChE activity. Recently, glycyl-L-glutamine (GLG) was demonstrated to function as a neurotrophic factor for maintenance of AChE content in cat denervated superior cervical ganglion cells. In the present study, regeneration of AChE activity in cultures of undifferentiated NG108-15 cells after soman inhibition was investigated in the presence and absence of GLG. Cells were treated with soman (5.5 x 10(-6) M) for 15 min and then washed to remove excess soman. Culture medium containing either GLG (10(-6), 10(-5), or 10(-4) M) or glycyl-L-glutamic acid (10(-6) M) was added to cultures after soman treatment and remained in the medium until cell harvest. Cells were physically detached at various times after soman treatment and specific AChE activity was determined. After soman, AChE activity dramatically decreased to less than 1% of untreated cellular activity at 1 hr. AChE activity gradually increased after 5 hr, while untreated cell AChE activity was regained 20 hr after soman. The t1/2 for AChE regeneration was approximately 10 hr. GLG did not increase the rate of AChE regeneration after soman inhibition. These results indicate that GLG is not a directly acting neurotrophic factor for AChE synthesis in NG108-15 cells after chemical AChE inactivation.
The nucleotide sequence of the human parainfluenza type 1 (Pl1) virus nucleoprotein (NP) gene was determined from cDNA clones of the mRNA. A cDNA clone, 4-31, containing a 1.7-kb insert, was identified as a Pl1 NP-specific clone by partial nucleotide sequence analysis and sequence comparison with Sendai virus (SV) NP. Using a vaccinia virus transient expression system, a polypeptide with electrophoretic mobility similar to that of Pl1 NP was synthesized from this clone, which reacted specifically with both polyclonal and monoclonal antibodies against Pl1 virus and Pl1 NP, respectively. The complete nucleotide sequence of this clone was determined and was found to contain a single open reading frame that can encode a protein of 524 amino acids with a predicted molecular weight of 57,547. Comparison of the amino acid sequence of Pl1 NP with that of other paramyxoviruses showed that two conserved amino acid sequences found within other paramyxoviruses are also present in Pl1 NP. Although Pl1 and SV showed a high sequence homology, approximately 100 amino acids at the C-terminal region were highly divergent as found also among other paramyxoviruses.
Studies conducted by Fisons Pharmaceuticals and the Antiepileptic Drug Development Program (ADD Program) of the Epilepsy Branch (NINDS, NIH) revealed that 'remacemide' (FPL 12924, formerly PR 934-423) was effective orally in the prevention of maximal electroshock seizures (MES) in rats. In this context (-)stereoisomer (FPL 14145) was of equal potency to the racemate (remacemide), while the (+)stereoisomer (FPL 14144) was 54% less potent. With respect to neurotoxicity, remacemide and its enantiomers possessed more favorable therapeutic indices than phenobarbital and valproate and less favorable indices than phenytoin and carbamazepine. The duration of protection of rats in the MES test at the ED50 or 3 x ED50 of remacemide and the (+)isomer was better or on par with the best reference compounds, phenytoin and phenobarbital. After subchronic administration of either the ED50 or the ED97 of remacemide, no tolerance developed in the hexobarbital sleep test, however, the activities of 3 hepatic microsomal enzymes were elevated. In naive rats high doses of remacemide or its (-)isomer and low doses of phenobarbital caused an increase in spontaneous motor activity. Alternatively, motor activity was depressed subsequent to high doses of phenobarbital and phenytoin. Remacemide was inactive against pentylenetetrazol and 'kindling' seizures. It was without effect in 5 electrophysiological tests (evoked responses, recurrent inhibition, long-term potentiation, penicillin-induced discharge rate and veratridine-induced depolarization) employing the in vitro hippocampal slice technique. Moreover, remacemide failed to demonstrate potent binding in vitro to neuronal L-glutamate, gamma-amino-butyrate A, adenosine A1, benzodiazepine, N-methyl-D-aspartate (strychnine-insensitive glycine and ion channel subsites) or muscarinic receptors. In conclusion, remacemide specifically prevents seizures elicited by MES, an action predicting utility in patients with generalized tonic/clonic convulsions.
The main subject of this hospital-based case-control study was the possible relationship between use of combined oral contraceptives (OCs) containing chlormadinone acetate and breast cancer. Analyses were based on data from 490 cases with newly diagnosed breast cancer and 1,223 controls and were separately performed for combined OCs with and without chlormadinone. For either of the combined OCs, risk was not elevated in ever users, did not increase with duration of use and did not change with time since initial exposure or with time since most recent use. However, the relative risk was increased in current users: RR = 1.72 (0.88, 3.36) for combined OCs with chlormadinone and RR = 1.42 (1.01, 2.00) for combined OCs without chlormadinone, which is, however, explained as a screening effect. These results show that chlormadinone as a constituent of combined OCs does not influence breast cancer risk.
The nucleotide sequence of the P gene of human parainfluenza virus type 1 (PIV1) was determined from cloned cDNA copies of the mRNA. By analogy with the gene organization of Sendai virus, two open reading frames in the mRNA sense of the gene were identified as coding sequences for the P protein (568 amino acids with an estimated molecular weight of 64,655) and the C protein (204 amino acids with an estimated molecular weight of 24,108). Comparison of the deduced amino acid sequences of the P and C proteins of PIV1 with those of Sendai virus showed a high degree of homology. However, a sequence for the cysteine-rich V protein, which was considered a common feature of other paramyxoviruses, was interrupted by the presence of multiple stop codons. The sequence analysis of three P-gene-specific cDNA clones generated from genomic RNA by polymerase chain reaction and one additional clone generated from mRNA confirmed that the coding sequence for the cysteine-rich region is silent in the PIV1 gene and thus is not translated into protein. Two potential editing sites with the consensus sequence 3'UUYUCCC were found in the PIV1 P gene at positions 564 to 570 and 1430 to 1436. However, examination of the PIV1 mRNA population by a primer extension method indicated that neither of these sites is utilized. These results indicate that the PIV1 P gene has a coding strategy different from those of other paramyxovirus P genes.
A human cDNA clone encoding a c-myc promoter-binding protein was detected by screening a HeLa cell lambda phage expression cDNA library. The library was screened by using an XhoI-NaeI human c-myc P2 promoter fragment as a probe. The recombinant phage encoded a fusion protein, myc-binding protein 1 (MBP-1), which had an apparent molecular size of 40 kDa. A corresponding protein with a molecular size of 35 kDa was present in a HeLa cell extract. Sequence analysis of the cloned gene reveals an open reading frame of 1,038 bp with a 3' untranslated region of 378 bp. The predicted protein sequence contains a proline-rich region in the amino terminus but does not demonstrate a known DNA-binding domain. DNase I footprint analysis demonstrates that MBP-1 binds to the sequence just 5' of the TATA box sequence of the human c-myc P2 promoter. MBP-1 cDNA hybridizes to a 1.4-kb mRNA from HeLa and HL-60 cells, indicating that the cDNA insert (1,416 bp) is a full-length clone. Coexpression of the MBP-1 protein repress transcription from the human c-myc promoter, suggesting that MBP-1 may act as a negative regulatory factor for the human c-myc gene.
The promoter of the human dihydrofolate reductase (DHFR) gene contains two consensus binding sites for the DNA binding protein Sp1. DNAse protection and gel mobility shift assays demonstrate binding of recombinant Sp1 to both decanucleotide Sp1 binding sequences which are located 49 and 14 base pairs upstream of the transcription start site. The more distal of the two binding sites exhibits a somewhat higher affinity for Sp1. The G-C specific DNA binding drug, mithramycin, binds to both consensus sequences and prevents subsequent Sp1 binding. Promoter-dependent in vitro transcription of a DHFR template is selectively inhibited by mithramycin when compared to the human H2b histone gene. A similar effect is also noted in vivo. Mithramycin treatment of MCF-7 human breast carcinoma cells containing an amplified DHFR gene induces selective inhibition of DHFR transcription initiation, resulting in a decline in DHFR mRNA level and enzyme activity. This selective inhibition of DHFR expression suggests that it is possible to modulate the overexpression of the DHFR gene in methotrexate resistant cells.
Utilizing the PCR, we have devised a detection and typing system for the human papillomaviruses (HPVs) that are commonly associated with preneoplastic and cancerous lesions of the genital and aero-digestive tracts: HPV 6, 11, 16 and 18. Utilizing computer sequence analysis, we designed a single, "consensus" set of oligomeric nucleotide primers capable of amplifying a 571-594-bp region of the E1 open reading frame of all of these HPVs. Detection via PCR amplification is followed by restriction endonuclease digestion of the resultant products that yield distinctive and reproducible banding patterns by polyacrylamide gel electrophoresis because of their internal sequence diversity. The system is sensitive, does not require sophisticated molecular biology expertise or radioisotopes and can be modified as new information on HPV types and their relationship to diseases becomes available.
Out of a total of 1,224 subjects aged 55 years and above who attended the Senior Citizens' Health Care Centres for health screening, new medical conditions were picked up for the first time in 472 subjects, giving an overall pick-up rate of 38.6%. This rate increased with age rising from 29.4% among those aged 55-64, to 45.8% among those aged 65-74 and to 51.4% in the 75 years and above. This trend was found to be of statistical significance (p greater than 0.001). The objective of the health screening programme in the Senior Citizens' Health Care Centres is to detect geriatric problems early in order to take adequate steps to prevent disability and dependency states, so that the elderly may continue to live at home. In this paper, we studied the 472 subjects with abnormal medical conditions. The pick-up rates (per 100) of medical conditions were as follows: cataract 13.8, other eye conditions 6.5, deafness 4.5, other ear conditions 2.7, diabetes mellitus 2.5, hypertension 2.2 and osteoarthritis 1.6. The socio demographic profile and the medical conditions of the 472 subjects were also studied, with implications on their quality of life in future. We recommend that physicians specifically screen for diabetes mellitus, hypertension, eye and ear abnormalities in all asymptomatic elderly, during periodic health check ups.
Serum levels of luteinizing hormone (LH), testosterone and hepatic functions were measured in 38 male alcoholics and in 24 male control subjects. Significant elevation of serum LH and a trend of low serum testosterone were noted with scant evidence of advanced liver disease. None of the alcoholics had clinical stigmata of hypogonadism and feminization.
Cerebrovascular disease resulting in stroke had shown a rising trend in the last decade among the elderly population in Singapore. To prevent dependency states, a home stroke rehabilitation programme was implemented in 1988 with the help of staff nurses trained to provide rehabilitation for stroke patients at home. These nurses were specially trained by experienced physiotherapists. A simple format was used to identify patients with good rehabilitation potential, while they were in the acute hospitals. The 244 stroke patients were studied by their age, sex, the status of motivation to go through rehabilitation and the level of family support. Out of the 224, 139 patients who had good and fair rehabilitation potential were referred to the Senior Citizens' Health Care Centres (SCHCCs) directly, as they already had sitting and standing balance. Twenty-two patients with poor rehabilitation potential were provided home nursing care. The rest of 83 patients with good and fair rehabilitation potential needed home rehabilitation before being referred to the SCHCC, as they had no sitting and standing balance. These 83 patients followed-up at home, were studied by their age, sex, rehabilitation potential, family support, number of nurse home visits and duration of rehabilitation required to gain independence in mobility. By 12 weeks, 84% of the patients improved in their goals towards independence irrespective of age. Marked improvement were shown in all the ten areas studied namely concentration, head control, return of tone, sitting balance, standing balance, mobility, power of upper and lower limbs, co-ordination and continence.(ABSTRACT TRUNCATED AT 250 WORDS)
A systematic health screening programme for the elderly aged 55 years and above was implemented in the first Senior Citizens' Health Care Centre (SCHCC) in 1986. This programme was expanded to two more SCHCCs by 1989. The first 1,224 clients aged 55 years and above, who attended this programme in the first three SCHCCs were studied in this paper. The characteristics of the elderly who voluntarily attended the SCHCCs for health screening were studied with respect to their referrals, socio demographic status, whether they had regular physician care, the existing medical problems and the new medical problems picked up at health screening. Of those screened, 49.6% were referred for the programme by relatives and self, and 28.2% by community centres, thus, showing that community and family supported the programme. Only 7.2% of referrals to the screening programme came from general practitioners and 4% from polyclinic doctors. It is interesting to note that 40% of clients had no regular physician care. About two thirds (57.2%) had one or more existing chronic medical conditions or disability at the time of screening. The majority with existing conditions had hypertension (24.8%) and diabetes (15.5%). Other existing conditions were ischaemic heart disease (6.2%), cataract (3.9%) and asthma (3.6%). The pick-up rate of medical conditions among the clients at the time of health screening was 38.6% for the whole group. This showed a rising trend from the younger to the older age groups being 29.4% in the age group 55 to 64, to 45.8% in the age group 65 to 74, and 51.4% in the age group 75 years and above (p less than 0.001). Some of the topical issues related to a health screening programme for the elderly have been discussed.
In an effort to develop an effective centrally acting pretreatment compound against organophosphorus poisons, the tertiary pyridostigmine (Pyr) derivative 3-(N,N-dimethyl-carbamyloxy)-1-methyl-delta 3-tetrahydropyridine (THP) was synthesized and studied for its anticholinesterase properties, as well as its efficacy against soman intoxication in guinea pigs. Injection of THP (262 micrograms/kg, im) into adult male guinea pigs caused inhibition of blood (30%) and brain (25%) acetylcholinesterase (AChE), showing that THP penetrates the blood-brain barrier. Pyr (131 micrograms/kg, im) caused AChE inhibition in the blood (59%), but not in the brain. The inhibitory potencies of THP and Pyr were compared by determining their IC50 values for in vitro inhibition of both AChE (brain, erythrocyte) and pseudo-cholinesterase (plasma) in three mammalian species (guinea pig, rat, rabbit). THP, although effective in inhibiting both types of cholinesterase, was in general less potent than Pyr. Pretreatment of guinea pigs with THP (262 micrograms/kg, im) plus Pyr (131 micrograms/kg, im), 30 min prior to subcutaneous soman challenge, with no antimuscarinic or oxime treatment, protected 60% of the animals against 2 X LD50 of soman. Neither THP nor Pyr alone was effective. The protective pretreatment regimen did not prevent convulsions, but shortened the recovery time in surviving animals (median recovery time 1.6 hr, compared to 24 hr in control and other groups of animals pretreated with THP or Pyr alone). A combination of THP and Pyr thus appears to provide a means of evaluating the relative importance of selective peripheral plus central vs peripheral AChE protection against soman.
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